mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Abstracts

Table 1
Poster Presentations
P001

Susceptibility of Candida albicans biofilms to caspofungin

and anidulafungin is not affected by metabolic activity
L. J. Marcos-Zambrano,1 P. Escribano,2 E. Bouza1 and J. Guinea1
1
Gregorio Maran~on Hospital, Madrid, Spain and 2Hospital
General Universitario Gregorio Maran~on, Madrid, Spain

Objectives Biofilm formation allows Candida albicans to attach to

plastic surfaces such as catheters and bioprosthetic devices, thus
complicating the management of infected patients. Caspofungin,
anidulafungin, and micafungin have potent antibiofilm activity
in vitro. We previously observed that the antibiofilm activity of mica- activity of caspofungin and anidulafungin was not dependent on the
fungin against C. albicans clinical isolates was dependent on the metabolic activity of the biofilm. These observations were confirmed
metabolic activity of the biofilm and that biofilm with high metabolic by scanning electron microscopy (Figure).
activity was more susceptible to micafungin than biofilm with low Conclusions Micafungin was the most active echinocandin against
metabolic activity. It remains unknown whether this observation C. albicans biofilm. In contrast to that observed for micafungin, the
applies to caspofungin and anidulafungin. We compared the antifun- antifungal activity of anidulafungin and caspofungin against C. albi-
gal activity of anidulafungin, caspofungin, and micafungin against cans biofilms in vitro was not dependent on metabolic activity.
preformed Candida albicans biofilms with different degrees of metabolic
activity from 301 isolates causing fungemia in patients admitted to
Gregorio Mara~ non Hospital (January 2007 to September 2014).
Methods Biofilm was formed and, based on our previously reported P002
score, each strain was tested in triplicate using the XTT reduction
assay and classified as forming biofilm with low metabolic activity
(LMA), moderate metabolic activity (MMA), or high metabolic activ- Trailing effect of fluconazole against Candida spp. Which
ity (HMA). Echinocandin MICs for planktonic and sessile cells were is the frequency when using EUCAST?
measured using the EUCAST E.Def 7.2 procedure and XTT reduction L. J. Marcos-Zambrano,1 P. Escribano,2 C. Sanchez-Carrillo,3
assay, respectively. Preformed biofilms were treated with increasing E. Bouza1 and J. Guinea1
concentrations of the three drugs (0.015 mg L
1 to 16 mg L
1);
after incubation at 37°C for 24 h, the metabolic activity was mea-
1
Gregorio Maran~on Hospital, Madrid, Spain; 2Hospital General
sured by the XTT reduction assay. The sessile MIC (SMIC80) was Universitario Gregorio Maran~on, Madrid, Spain and 3Gregorio
defined as 80% reduction in the metabolic activity of the biofilm trea- Maran~on Hospital, Madrid, Spain
ted with the antifungal compared with the control well. Three C.
albicans isolates were selected for scanning electronic microscopy Objectives Trailing is a well-known phenomenon defined as a
(one each for LMA, MMA, and HMA biofilms) to study the effect of reduced but persistent visible growth of Candida spp. at fluconazole
caspofungin, anidulafungin, and micafungin against the biofilm concentrations above the MIC obtained by broth microdilution proce-
structure. dures. Although the clinical impact of trailing is unkown it may
Results The three echinocandins were uniformly active against the interfere with the MIC endpoint determination. Trailing has been
isolates in planktonic form, regardless of the metabolic activity of commonly detected when using CLSI M27-A3 method but its fre-
their respective sessile form. All three drugs were active against the quency when using EUCAST EDef 7.2. method is unkown. We ass-
preformed C. albicans biofilms with micafungin showing the highest esed the frequency and degree of fluconazole trailing observed after
activity (P < 0.001). HMA biofilms were significantly more suscepti- studying the antifungal susceptibility to fluconazole by EUCAST
ble to micafungin than MMA and LMA biofilms (P < 0.001); anidu- against a large number of Candida spp. isolates from patients with
lafungin was slightly more active against HMA biofilms than LMA candidemia.
and MMA, and caspofungin showed the lowest activity (Table). The Methods We studied 639 fluconazole-susceptible non-krusei Candida
spp. isolates from patients with candidemia (n = 570) admitted to
Gregorio Mara~ n Hospital (January 2007 to March 2015). Isolates
no
were identified after amplification and sequencing of the ITS1-5.8S-
ITS2 region and further tested for in vitro fluconazole susceptibility
according to the EUCAST EDef 7.2 procedure. The MIC was defined
as a reduction ≥50% compared to the growth control after 24 hours
of incubation; trailing was defined as the presence of residual growth
(0.1–49%) in wells containing two-fold fluconazole concentrations
above MIC compared to growth control. For each isolate, mean trail-
ing was defined as the mean percentage of residual growth observed
in the wells after the MIC. The percentage of isolates showing differ-
ent degrees of trailing [0.1–5% (residual trailing), 6–10%, 11–15%,
and >15% (prominent trailing)] was studied.
Results The species distribution of isolates, the mean fluconazole
MICs, and the mean trailing is shown in the Table. The mean per-
centage of trailing of all isolates was 6.79%. Differences reaching sta-
tistical significance were found among species: C. albicans and C.
tropicalis showed the highest trailing (9.75% and 9.29%, respectively;
P < 0.001). Figure shows the proportion of isolates within a species
showing different degree of trailing. Most of C. albicans and C. tropi-
calis showed trailing >5% (90% and 81% of isolates, respectively),
Figure 1

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 doi:10.1111/myc.12380
Poster Presentations

voriconazole (VRZ) and flucytosine (FC) using the broth microdilu-

tion method previously described (Zaragoza O., Antimicrob. Agents
Chemother. 2011): growth medium yeast nitrogen base (YNB) added
of 0.5% glucose, pH 7.0, final inoculum of 104 CFU ml
1, shaking
(14 000 rpm) every 30 min incubation at 30°C. FC was tested in
YNB 2% glucose, pH 5.6 (Viviani M.A., J. Chemother. 2003). Read-
ing was performed spectrophotometrically at 492 nm after 48 h of
incubation. The MIC was defined as the lowest drug concentration
giving 50% growth inhibition compared to growth in control well.
The geometric mean (GM) and the ECV, defined as the MIC (mg/L)
that encompassed at least 95% of isolates in the distribution (Pfaller
M.A., Drug Resist Update 2010), were calculated.
Results The MIC distribution of the tested antifungals had the fol-
lowing ranges: 0.12–16 for FLZ, 0.03–0.5 for ITZ, 0.03–0.25 for
VRZ and 0.12–64 for FC. The ECVs and the % of isolates that had
MIC≤ECV were the following: FLZ 8, 97.7%, ITZ 0.25, 98.8%, VRZ
0.12, 96.5%, FC 1, 96.5%. Different patterns of susceptibility have
been observed in the three genotypes. VNIV isolates had a pattern of
susceptibility to all azoles lower than the other two genotypes: FLZ
Figure 1 GM 1 vs 2.5 for VNIII and 2.2 for VNI, VRZ GM 0.03 vs 0.06 for
VNIII and 0.07 for VNI, and ITZ GM 0.04 vs 0.11 for VNIII and
0.12 for VNI. The two isolates with FLZ MIC values higher than ECV
were both VNIII hybrids, the same two isolates plus one VNI isolate
Table 1 had also VRZ MIC>ECV. Only one isolate (VNI genotype) had a MIC
value one twofold-dilution above the ECV for ITZ. All the three iso-
lates with FC MIC>ECV were VNI genotype.
Conclusions The results of this study on C. neoformans wild-type iso-
lates confirm the low frequency of reduced antifungal susceptibility.
The establishment of MIC distributions of wild type C. neoformans iso-
lates and ECVs will be useful in resistance surveillance and in the
development of clinical breakpoints for the antifungals agents and C.
neoformans.

P004

Pulmonary infection due to an Aspergillus fumigatus with

whereas less than a half of C. parapsilosis and Candida spp. exhibit a peculiar pattern of azole susceptibility
trailing greater than 5%. C. albicans and C. tropicalis were the species A. M. Tortorano,1 M. C. Esposto,1 A. Prigitano,2 G. de Lorenzis,1
with the highest percentage of isolates producing prominent trailing E. de Carolis,3 A. Grancini4 and S. Antinori1
(>15%).
Conclusions C. albicans and C. tropicalis were the species showing
1
Universita degli Studi di Milano, Milano, Italy; 2University of
the highest production of fluconazole trailing. The clinical impact of Milan, Milano, Italy; 3Catholic University of Sacred Heart, Roma,
this finding should be evaluated in future studies. Italy and 4IRCCS Ospedale Maggiore Policlinico, Milano, Italy
This work was supported by grant numbers PI11/00167 and
PI14/00740 from Fondo de Investigaci on Sanitaria (FIS). P. Escrib- Objective To report a case of aspergillosis caused by Aspergillus fumi-
ano is contracted by Instituto de Investigaci on Sanitaria Gregorio gatus isolates with a peculiar pattern of antifungal susceptibility.
Mara~ non (grant number PI14/00740). J. Guinea is supported by a Case report The patient with a history of tuberculosis presented a
Miguel Servet contract (CP09/00055) from FIS. LJ Marcos-Zambrano cavity with an inclusion in the apical right lung and a nodulation
is supported by a predoctoral PFIS grant (FI12/00265) from FIS. widespread the upper field of the left lung. Alternation of resolution
and progression of the infection at the left lung required a prolonged
antifungal treatment with different drugs (itraconazole, voriconazole,
posaconazole, caspofungin) to obtain a complete resolution of the
P003 infection before the right pneumonectomy.
From a fine-needle biopsy several colonies of A. fumigatus showing
different features grew in culture: some colonies had the typical
Antifungal susceptibility of Cryptococcus neoformans heavily sporulated pattern and other, characterized by slower
clinical isolates from Italy growth, a flocculent lightly sporulated Appearance The typical iso-
A. M. Tortorano,1 A. Prigitano,2 M. Cogliati,2 M. C. Esposto,1 lates were susceptible to azole compounds when tested with E-test,
C. Lazzarini2 and A. Zani2 namely MICs of itraconazole, posaconazole and voriconazole were
0.25, 0.12 and 0.12 mg L
1, respectively. On the contrary the
1
Universita degli Studi di Milano, Milano, Italy and 2University of lightly sporulated isolates showed a peculiar pattern of susceptibility
Milan, Milano, Italy with absence of growth at the lower concentrations of azoles and
growth only in presence of the higher antifungal concentrations
Objectives to determine the in vitro antifungal susceptibility of Cryp- (paradoxical effect with azole requirement to grow). Tubulin and
tococcus neoformans clinical isolates collected during a prospective sur- cyp51A genes of the anomalous isolates were sequenced. The isolates
vey carried out in Italy from 2010 to 2014 and to calculate the were identified as A. fumigatus sensu stricto and M220I mutation
epidemiological cutoff values (ECVs). was found in cyp51A gene. Six isolates were genotyped by
Methods Eighty-three C. neoformans isolates (37 VNI, 14 VNIV and microsatellite analysis and two different allelic profiles were identified,
32 intervarietal hybrids VNIII), each representative of a single case, one of the typical isolates and another of the lightly sporulated
were tested for susceptibility to fluconazole (FLZ), itraconazole (ITZ), isolates.

ª 2015 The Authors

52 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Clinical improvement and radiological clearance of the left lung,

obtained with a 3-month voriconazole treatment (200 mg 9
2 day
1), allowed the right pneumonectomy. Unfortunately, only
fixed tissues were available avoiding culture and antifungal suscepti-
bility testing. However molecular methods performed on the bioptic
sample allowed the detection of the M220I mutation.
Conclusions The patient resulted infected by two A. fumigatus geno-
types with different patterns of azole susceptibility. Probably the azole
susceptible isolates were responsible of the infection in the left lung
that resolved with voriconazole treatment and the resistant ones of
the aspergilloma of the right lung as demonstrated by the detection
of the mutation M220I in the cyp51A gene directly in the bioptic
sample.
Probably other mutations than that found in the cyp51A gene
could be responsible of the inability to grow in absence of azoles
shown by these last isolates.

Figure 1. The incidence of candidaemias in the University Hospitals

Leuven for the period 2004–2014
P005

Trends in candidaemia and antifungal consumption in

Belgium
B. Goemaere,1 P. T. Becker,1 A. Ingenbleek,1 M. Goossens,1
B. Catry,1 M. Hendrickx1 and K. Lagrou2
1
Scientific Institute of Public Health, Brussels, Belgium and
2
UZ Leuven, Leuven, Belgium

Objectives The incidence of Candida species causing bloodstream

infection was investigated in the University Hospitals Leuven (Bel-
gium) over the period 2004–2014. Antifungal resistance in Candida
glabrata was also evaluated as this species is known to display
decreased susceptibilities to azoles and echinocandins. Clustering Figure 2. Antifungal consumption in Belgian hospitals for the period
between resistant C. glabrata isolates was analysed by strain typing 2007–2012
and the molecular mechanisms underlying the resistance were identi-
fied. In addition, antifungal consumption in hospitals over the period
2007–2012 was analysed on a national level.
Methods Candida blodstream isolates of the University Hospitals Leu- species was not observed in our study. In addition, fluconazole and
ven were collected and identified between 2004 and 2014. The num- echinocandin resistance remained relatively low in C. glabrata in
ber of patient days was used to calculate incidence rates. Sequencing comparison with other centres in Europe.
and qRT-PCR analyses were performed on selected genes implicated Fluconazole consumption in Belgian hospitals decreased but
in the azole or echinocandin resistance. Antifungal consumption for remained the most prescribed antifungal drug. Antifungal consump-
the period 2007–2012 in all Belgian acute and chronic care hospi- tion is much higher and less stable in teaching hospitals compared to
tals (>150 beds) was provided by the national surveillance of antimi- non-teaching hospitals. In teaching hospitals, amphotericin B,
crobial consumption, stratified by teaching activities. Results were voriconazole and caspofungin are relatively more prescribed, which
calculated at the ATC-level 5 (chemical substance, WHO classifica- could be related to the more specialized cares provided in these
tion system) and displayed in defined daily dose (DDD)/1000 patient settings.
days.
Results Between 2004 and 2014, 734 episodes of candidaemia
occurred in the University Hospitals Leuven. The highest incidence
was caused by C. albicans while C. glabrata and C. parapsilosis ranked P006
respectively the 2nd and the 3rd most common agent each year (Fig-
ure 1). Analysis of the antifungal resistance in 172 C. glabrata iso-
lates revealed resistance to fluconazole and echinocandins in 12.2% Detection of clinical Aspergillus fumigatus isolates
and 1.2%, respectively. A minimum-spanning tree did not show any resistant to triazoles
correlation between resistant strains, nor within hospital units. E. M. Kurzyk,1 U. Nawrot,2 M. Mroczynska,1 K. Wlodarczyk,2
Antifungal consumption at the national level was much lower in M. Ussowicz,2 P. Zdziarski,3 M. C. Arendrup4 and A. Brillowska-
non-teaching hospitals compared to teaching hospitals (Figure 2). Dabrowska1
Fluconazole was the drug prescribed most frequently but its usage 1
Gdansk University of Technology, Gdansk, Poland; 2Wroclaw
declined between 2007 and 2012 (from 73.6% to 59.7% and from
85.6% to 79% in teaching and non-teaching hospitals, respectively).
Medical University, Wroclaw, Poland; 3Hospital Infections Control
Among the mould active triazoles, DDDs were in general highest for Team, Wroclaw, Poland and 4Statens Serum Institut,
voriconazole, followed by itraconazole and posaconazole. Ampho- Copenhagen, Denmark
tericin B was the 2nd most common antifungal drug prescribed in
teaching hospitals (21.2% of all antifungals in 2012). The consump- Objectives The aim of this study was to investigate the presence
tion of caspofungin showed a peak in 2010 while anidulafungin and molecular mechanisms of resistance to triazoles within clinical
remained below 2% since its introduction in 2006 (Figure 2). Aspergillus fumigatus isolates collected in Wrocław Medical University,
Conclusion Results indicate that C. albicans is the most common Poland.
cause of candidaemia in the University Hospitals Leuven, followed by Methods The study included 89 clinical isolates of A. fumigatus col-
C. glabrata and C. parapsilosis. This distribution is similar to other lected during routine diagnostic procedures from several Wroclaw
Northwestern European centres although an increase in non-albicans hospitals in the period of 2006–2014. The isolates were identified

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 53
Poster Presentations

based on classical morphological criteria followed by molecular con-

firmation of the species identification (sequencing of ITS1, 5,8S, ITS2
region). Minimal Inhibitory Concentration (MIC) was tested for itra-
conazole (ITR), voriconazole (VOR) and posaconazole (POS) using
broth microdilution methods, according to European Committee for
Antimicrobial Susceptibility Testing E.DEF 1.9. The cyp51A gene
with promoter region of all isolates was sequenced.
Results The sequencing results of the fragment of rDNA region con-
firmed identification of all examined isolates as A. fumigatus. Accord-
ing to the EUCAST breakpoints for the interpretation of antifungal
susceptibility testing 87/89 (97.75%) isolates were susceptible to
ITR, VOR, and POS with median MIC values of 0.5 mg L
1 (ITR),
0.25 mg L
1 (VOR), and 0.03 mg L
1 (POS). Two isolates were
resistant to ITR with MIC values 32 mg L
1, one of which were
cross resistant to VOR, and POS (4 mg L
1 and 0.5 mg L
1 respec-
tively), whereas the second were classified as intermediate susceptible
(MIC for VOR: 2 mg L
1 and POS: 0.25 mg L
1). The sequencing of
the cyp51A with the promoter region revealed mutation TR34/L98H
in both resistant strains.
Conclusion Triazole resistant A. fumigatus strains harboring muta- Conclusions Combination of fluconazole and lactoferrin has a
tion TR34/L98H are the emerging pathogens in many European and potent synergistic effect against fluconazole-resistant Candida albicans
Asian countries. The presented study shows that such a resistance and Candida glabrata.
exist also in Poland. The prevalence of triazole resistance among clin- Funding: This study was financed in part by Fundaci on Jesus de
ical A. fumigatus isolates collected in Wrocław was low (2.25%), nev- Gangoiti Barrera, Fundaci on ONCE ‘Oportunidad al Talento’and pro-
ertheless it indicates the necessity to include susceptibility testing in jects GIC12 210-IT-696-13, UFI 11/25 (UPV/EHU).
routine diagnostic procedures.
Acknowledgments This work was supported by grant 2013/11/B/
NZ7/04935 from the National Science Centre of Poland.
P008

Synergistic antifungal activity of fluconazole combined

P007
with cyclosporine A
J. D. Carton, C. Trobajo-Sanmartın, C. Marcos-Arias, E. Eraso
Increased efficacy of fluconazol combined with lactoferrin and G. Quindo s
against azole-resistant Candida isolates
Univ. Paıs Vasco UPV/EHU Facultad de Medicina y Odontologıa,
J. D. Carton, C. Trobajo-Sanmartın, C. Marcos-Arias, E. Eraso
Bilbao, Spain
and G. Quindo s
Univ. Paıs Vasco UPV/EHU Facultad de Medicina y Odontologıa, Objective To evaluate the in vitro activity of fluconazole in combina-
Bilbao, Spain tion with the calcineurin inhibitorcyclosporine A against fluconazole-
resistant Candida.
Candidiasis caused by fluconazole-resistant Candida are related to Methods A total of 15 clinical azole resistant isolates of Candida
increased morbidity and mortality. The use of new antifungal drugs were included in this study: 7 Candida albicans, 3 Candida glabrata, 2
and/or the combination of fluconazole with different compounds, Candida tropicalis, 1 Candida krusei and 1 strain of Candida parapsilosis.
such as lactoferrin, with antimicrobial and antiinflammatory proper- Candida krusei ATCC 6258 was the quality control. The minimum
ties, can help to solve this important clinical problem. inhibitory concentrations (MIC) were determined according to Clini-
Objective To assess the in vitro synergistic effect of fluconazole and cal and Laboratory Standards Institute guidelines for yeasts (M27-
lactoferrin combinations against fluconazole-resistant Candida clinical A3), and defined as the lowest drug concentrations that produced a
isolates. 50% inhibition of visible fungal growth after 24 h of incubation.
Methods Seven fluconazole-resistant Candida albicans, three Candida Drug interactions were assessed by the checkerboard microdilution
glabrata, two Candida tropicalis, one Candida krusei and one Candida method. Different fluconazole and cyclosporin A combinations were
parapsilosis clinical isolates and the quality control Candida krusei studied: the final concentration of fluconazole ranged from 1 to
ATCC 6258 were tested. Different fluconazole and lactoferrin combi- 64 lg ml
1 and of cyclosporine A ranged from 0.0625 to
nations were studied: fluconazole concentrations (from 1 to
64 lg ml
1) were selected according to the minimum inhibitory con-
centration (MIC) determined by Clinical and Laboratory Standards
Institute (CLSI) M27-A3 document. MIC was defined as the lowest
drug concentration showing 50% growth reduction after 24 h incu-
bation. Lactoferrin concentrations ranged from 0.0625 to
32 lg ml
1. The interaction between fluconazole and lactoferrin was
assessed by a checkerboard microdilution method. The fractional
inhibitory concentration index (FICI) classified drug interaction as
synergistic (FICI ≤0.5), indifferent (0.5< FICI ≤4), and antagonistic
(FICI >4).
Results Table 1 summarizes MICs for lactoferrin and fluconazole
treatment alone and in combination against different species of Can-
dida. Lactoferrin MIC range was6- > 32 lg ml
1 and fluconazole
range8- > 64 lg ml
1. However, combination of both drugs reduced
the concentrations necessary for inducing 50% reduction in the
growth of fluconazole-resistant isolates. This combination flucona-
zole-lactoferrin was synergistic in most of cases. No antagonism was
found.

ª 2015 The Authors

54 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

32 lg ml
1. The in vitro interaction of the drug combination was Conclusions Point mutations previously associated with azole resis-
interpreted in terms of the fractional inhibitory concentration index tance were present in many of the A. fumigatus isolates in this U.S.
(FICI) as follows: FICI ≤ 0.5, synergistic; 0.5 < FICI ≤ 4, indifferent; collection. In addition, 1 TR34/L98H isolate and 2 with TR46/Y121F/
and FICI >4, antagonistic. T289A mutations were found, which have not previously described in
Results Results of the checkerboard analysis are summarized in the the U.S. However, approximately half of these azole resistant isolates
table. The addition of cyclosporine A resulted in a high decrease in did not have Cyp51A associated mechanisms of resistance.
the MIC of fluconazole from 16–64 to 1 lg ml
1 against all isolates
of Candida albicans, Candida tropicalis and Candida parapsilosis.
Although this combination was less effective against Candida glabrata,
the effect was synergistic against two out of three isolates. P010
Conclusions Cyclosporine A enhanced the effect of fluconazole
against fluconazole-resistant isolates. The current results underline
the potential use of calcineurin inhibitors as strengtheners of antifun- Influence of microtitre tray plastic type on azole minimum
gal therapy for recalcitrant candidiasis. inhibitory concentrations against Candida and Aspergillus
Funding This study was financed in part by projects PI11/00203 Species
(FIS, Gobierno de Espa~ na), GIC12 210-IT-696-13 (Gobierno Vasco- N. P. Wiederhold, S. Trippy and A. W. Fothergill
Eusko Jaurlaritza) and UFI 11/25 (UPV/EHU).
UT Health Science Center San Antonio, San Antonio, USA

Objective Antifungal susceptibility testing provides evidence of

P009 in vitro activity that is used to classify fungal isolates as susceptible
or resistant to individual drugs. Clinicians may then use this infor-
mation to make treatment decisions in patients with invasive fungal
Evaluation of Cyp51A Mechanisms of Azole Resistance in infections. The CLSI and EUCAST broth microdilution reference
Aspergillus fumigatus Isolates from the United States methods for antifungal susceptibility testing both use cell culture
N. P. Wiederhold,1 V. Garcia Gil,1 J. Lindner,1 C. Sanders,1 microtitre plates for in vitro susceptibility testing. However, the type
H. Fan,1 D. A. Sutton2 and A. W. Fothergill1 of plastic that is used (treated vs. untreated) is not specified. Our
1 objective was to evaluate the influence of the plastic type on the
UT Health Science Center San Antonio, San Antonio, USA and in vitro activity of azole antifungals.
2
The University of Texas Health Center, San Antonio, USA Methods Candida spp. (C. albicans and C. glabrata; N = 46 total iso-
lates) and Aspergillus spp. (A. flavus, A. fumigatus, A. niger, and A. ter-
Objective The azole antifungals itraconazole, posaconazole, reus; N = 50 total isolates) received by our clinical reference
voriconazole, and isavuconazole are important therapeutic agents laboratory were utilized. Antifungal susceptibility testing was per-
against invasive aspergillosis. However, increased resistance in Asper- formed with fluconazole, itraconazole, posaconazole, and voricona-
gillus fumigatus to these clinically available antifungal agents has zole according to the methods in the CLSI M27-A3 and M38-A2
been reported, especially in parts of Europe. Resistance to these standards. Testing was performed using both cell culture treated and
agents in clinical isolates has primarily been associated with muta- untreated plastic microtitre plates from the same manufacturer. Geo-
tions within the CYP51A gene that lead to amino acid substitutions metric mean (GM) MIC values between treated and untreated plastic
within the Cyp51A enzyme, as well as tandem repeats (TR) in the for each drug between were compared by paired t-test.
promoter region of CYP51A. Our objective was to evaluate the Results The type of plastic in the microtitre trays significantly influ-
Cyp51A associated mechanisms of azole resistance in a U.S. collec- enced the in vitro potencies of itraconazole and posaconazole, while
tion of A. fumigatus isolates. the activities of fluconazole and voriconazole appeared to be less
Methods The antifungal susceptibility database in the Fungus Test- affected. Against C. glabrata, the GM MIC values for itraconazole and
ing Laboratory at the UT Health Science Center San Antonio was posaconazole were both 2.5 times higher when measured using
queried for itraconazole, voriconazole, and posaconazole MIC data untreated plastic compared to treated plastic plates (P < 0.0001 for
against A. fumigatus isolates from 2001 through 2014. This database each comparison). Similarly, against C. albicans, the posaconazole GM
is populated with antifungal MIC data against fungal isolates sent to MIC value was 2 times higher when in vitro activity was measured
our mycology reference laboratory from institutions across the U.S. using untreated versus treated plastic. Against Aspergillus spp., the
Susceptibility testing was performed according to methods in the type of plastic affected the in vitro activity of posaconazole the most.
CLSI M38-A2 standard. Isolates were classified as resistant based on Posaconazole GM MICs measured using untreated plastic were signifi-
the EUCAST clinical breakpoints (voriconazole & itraconazole >4 mg/ cantly higher against all Aspergillus spp. compared to treated plastic
L, posaconazole >0.5 mg/L). Isolates with elevated azole MICs were (fold increase GM MIC of 2.7–3.3 times; P < 0.001 for all compar-
confirmed to be A. fumigatus by morphology and growth at 50°C. isons). In addition, significantly more posaconazole MICs were
Isavuconazole MICs were also measured against the confirmed A. >0.5 mg l
1 (EUCAST clinical breakpoint for posaconazole resistance
fumigatus isolates. The CYP51A gene and its promoter region were against A. fumigatus and A. terreus) when measured using untreated
sequenced to evaluate for mutations associated with azole resistance. plastic compared to treated plastic (33 of 50 vs. 5 of 50, respectively;
Results A total of 220 isolates sent to our laboratory with elevated P < 0.0001 by Fisher’s exact test). In contrast, the in vitro activities
azole MICs and a preliminary identification of A. fumigatus were of itraconazole and voriconazole against Aspergillus spp. were less
screened, and 37 were confirmed to be this species by morphology affected by the type of plastic.
and growth at 50°C. Of the 37 isolates, 16 had mutations in the Conclusions The type of plastic used for broth microdilution suscep-
CYP51A gene resulting in amino acid substitutions in the Cyp51A tibility testing may influence the results of these assays, and this may
enzyme associated with azole resistance. One isolate was found to be drug specific. Against Candida and Aspergillus spp., the in vitro
have the TR34/L98H resistance mechanism not previously found potency of posaconazole was significantly reduced when untreated
within the U.S. and was pan-azole resistant. Two additional isolates plastic was used. This may have clinical implications in terms of the
contained the TR46/Y121F/T289A resistance mechanism. Both of classification of isolates as being susceptible or resistant to this anti-
these isolates were highly resistant to voriconazole and isavuconazole fungal agent.
(MIC >16 mg/L) but had lower itraconazole and posaconazole MICs
(range 1–4 mg/L). No CYP51A mutations or tandem repeats in the
promoter region of this gene were found in the remaining 18 iso-
lates. Strong correlations were found between itraconazole and
posaconazole MICs (Spearman r-value 0.73) and between voricona-
zole and isavuconazole MICs (Spearman r-value 0.93).

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 55
Poster Presentations

P011 P012

Characterization of Aspergillus fumigatus in COPD patients Patients with Candida glabrata candidemia due to
and their homes reveals frequent coexistence of isolates caspofungin-susceptible and -intermediate strains have
with various azole-resistance profiles superior outcomes to patients infected with caspofungin-
E. Frealle,1 C. Dauchy,2 N. Bautin,2 S. Nseir,2 G. Reboux,3 resistant strains
R. Wintjens,4 O. Le Rouzic,2 B. Sendid,2 E. Viscogliosi,5 E. Dei- C. Clancy, M. H. Nguyen, E. Press and R. K. Shields
Cas5 and S. Fry2 University of Pittsburgh, Pittsburgh, USA
1
Pasteur Institute of Lille, Lille, France; 2Lille University Hospital
Center, Lille, France; 3University Hospital J. Minjoz, Besanc
on, Background In a recent multi-center study, we showed that caspo-
France; 4Universite Libre de Bruxelles, Bruxelles, Belgium and fungin (CSP) minimum inhibitory concentrations (MICs) against Can-
5
Center for Infection and Immunity of Lille, Pasteur Institute of dida glabrata (Cg) measured by Sensititre YeastOne (SYO) cluster
Lille, Lille, France tightly, suggesting this method overcomes inter-laboratory variability
seen with broth microdilution reference methods. Overall, 18% of Cg
strains were defined as intermediate-resistant (I) to caspofungin (CSP)
Objectives Increasing reports on Aspergillus fumigatus azole-resis-
by CLSI criteria (susceptible (S)≤0.12 lg ml
1, I = 0.25, resistant
tance make detection of resistant isolates in respiratory samples a
(R)≥0.5). Almost all of these strains were S to other echinocandins
challenging issue in clinical practice. Moreover, detection in environ-
(EC), indicating that breakpoints (BPs) may over-call CSP non-sus-
mental reservoirs could be helpful to identify risk factors for acquisi-
ceptibility. In this study, we evaluated correlations between CSP MICs
tion of A. fumigatus resistant isolates and to improve the prevention
and outcomes of patients (pts) with Cg candidemia.
of aspergillosis. In this study, A. fumigatus clinical and environmental
Methods We performed a retrospective cohort study of pts with Cg
isolates obtained from azole-naive COPD patients and their homes
candidemia treated with an EC for ≥3 days as initial therapy. Pts
were characterized in order to determine the prevalence of azole-re-
with candidemia due to >1 Candida spp. were excluded. Treatment
sistance and cyp51A mutations, and to clarify the circulation of A.
(Tx) success was defined as survival and sterilization of blood cul-
fumigatus azole-resistant or mutated isolates between clinical and
tures at 14 days. EC MICs were determined by SYO panels, which we
environmental reservoirs.
previously showed to be more sensitive than broth microdilution.
Methods Seventy-five respiratory samples from 41 COPD patients
PCR and DNA sequencing were used to detect mutations in hot spots
(56 sputa and 19 oropharyngeal washes) and environmental samples
of FKS1 and FKS2.
from 36 homes of these patients (mainly electrostatic dust collectors
Results 106 cases were included. 46% of pts were men; median age
exposed for 10 weeks in the patient’s bedroom) were prospectively
was 59 years (range: 21–89). 56% of pts were in the ICU and 25%
collected in the Lille University Hospital between August 2011 and
had septic shock. Median CSP MIC was 0.12 (range: 0.03–16). 6% of
February 2015. Clinical and environmental A. fumigatus isolates
strains harbored FKS mutations. 54%, 35%, and 11% of strains were
obtained by culture on Sabouraud agar medium were further grown
CSP-S, I and R, respectively. All CSP-I strains were S to anidula
in medium containing itraconazole (ITZ) for selection of azole-resis-
(MIC≤0.12) and micafungin (MIC≤0.06), and none harbored FKS
tant isolates. A. fumigatus identification was confirmed by culture at
mutations. 29% of strains were collected from pts with prior EC expo-
50°C, as well as ITS rDNA regions and beta-tubulin genes sequenc-
sure (≥3 days of EC before candidemia). Rates of EC exposure were
ing. Then, the detection of mutations in the cyp51A gene was per-
similar for S and I strains (28% vs. 19%; P = 0.34), but higher for R
formed by sequencingfor all A. fumigatus isolates. Finally, ITZ,
strains (67%; P = 0.005). The overall rate of Tx success was 69%.
voriconazole and posaconazole Minimum Inhibitory Concentrations
Success rates for candidemia due to S, I and R strains were 68%,
(MIC) were determined for isolates with positive growth on ITZ-med-
81%, and 33%, respectively (I vs. R, P = 0.004; I vs. S, P = 0.23).
ium and/or cyp51A mutation.
The combined success rate for candidemia due to S and I strains was
Results A. fumigatus detection was positive in clinical samples for
74%, compared to only 33% for R (P = 0.008). Likewise, mortality
11/41 patients (26.8%) and in 15/36 patient’s homes (41.7%), yield-
rates at 30 days were 22%, 25%, and 42% for CSP-S, I and R Cg
ing 68 clinical and 48 environmental isolates. Growth on ITZ med-
strains. Mortality rates were higher among pts infected with CSP-R
ium was positive for 4 clinical isolates from 2/41 patients (4.9%) and
strains than CSP-S or -I strains (23%; P = 0.004).
3 environmental isolates from 2/36 patient’s home (5.6%). Among
Conclusions Identification ofCg strains as CSP-R by SYO and CLSI
clinical isolates, 1 had no cyp51A mutation and 3 others from 1
criteria is predictive of Tx failures and death among pts with Cg can-
patient had A284T mutation. Two environmental isolates from 2 dif-
didemia. CSP-I strains are not associated with FKS mutations, prior
ferent patients had TR34/L98H mutation, and 1 had the H285Y
EC exposure, or worse pt outcomes, suggesting that they should be
mutation, which was found to be localized by 3D modeling near the
re-classified as CSP-S.
heme-binding domain of CYP51A, at the entry of channel 1. Growth
on ITZ medium was negative for the remaining 109 isolates, but 4
environmental isolates had the F46Y/M172V/N248T/D255E/E427K
(n = 3) or the F46Y/M172V/E427K (n = 1) mutation. Coexistence of
different cyp51A genotypes and/or azole-resistance profiles was P013
detected in 3/8 respiratory and 2/10 environmental samples where
more than 1 A. fumigatus isolate had been detected (<i.e. 37.5% and Epidemiology of FKS mutations among Candida strains at
20.0%, respectively). Both clinical and environmental isolates were
high-risk for echinocandin resistance
obtained from 3 patients, but all were azole-sensitive and had no
cyp51A mutation. C. Clancy, M. H. Nguyen, E. Press and R. K. Shields
Conclusion The high frequency of azole-resistant A. fumigatus iso- University of Pittsburgh, Pittsburgh, USA
lates in COPD patient’s homes suggests that domestic exposure could
play a key role in the emergence and spread of azole-resistance in Background Echinocandin (EC) breakpoints (BP) proposed by CLSI
patients. Coexistence of azole-sensitive and resistant isolates in clini- for Candida spp. are not validated and may overstate caspofungin
cal samples indicates that picking a single colony for MIC determina- (CSP) resistance among C. glabrata (Cg). The significance of dis-
tion is not sufficient to exclude the presence of azole-resistant isolates crepant EC susceptibility is unknown, as are FKS mutation rates
in clinical practice, and confirms that MIC determination from all among various spp. The objective of this study was to determine FKS
colonies or screening using azole-supplemented medium should be mutation rates by systematic screening of high-risk Candida strains,
performed when patients are to be treated. including those with discrepant EC susceptibility.
Methods EC MICs were determined by YeastOne among consecutive
strains causing candidemia from 2009 to 14. Hot spots in FKS1 and

ª 2015 The Authors

56 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

FKS2 (Cg only) were sequenced for strains from patients receiving ≥ respectively. Rates were inversely related to total d of AF exposure;
3 days of prior EC therapy or those non-susceptible (NS) to any EC success rates were 44%, 58%, and 79% among pts exposed to >100,
by CLSI criteria. 31–100, or <30 d of AFs, respectively.
Results 453 strains from 384 pts were included. C. albicans (Ca, Conclusion Prior AF exposure is associated with a shift from Ca to
37%), Cg (37%), C. parapsilosis (Cp, 16%) and C. tropicalis (Ct, 8%) Cg and Cp as causes of candidemia. Our data suggest that stress
were most common. 16% of strains were EC NS. NS rates were imposed by AF exposure, regardless of specific agent, lowers the
higher for CSP (15%) than anidula (ANF; 2%) or micafungin (MCF; threshold for development of EC resistance among Cg. Total and tim-
2%, P < 0.0001 for both). EC NS was higher for Cg (34%) than other ing of AF exposure predict tx responses, underscoring the importance
spp (5%, P < 0.001). 27% of EC NS strains were resistant to flucona- of prior exposure when treating pts with candidemia.
zole. 21% of strains were from pts with prior EC exposure, including
3% classified as breakthrough (BT) during EC therapy. Prior exposure
was more common among Cp (40%) and Cg (25%) than Ca (12%;
P < 0.001 and 0.003, respectively), and among NS strains (24% vs P015
13%; P = 0.02). 32% of strains met criteria for sequencing, among
which the FKS mutation rate was 6% (overall 2% [8/453]). Muta-
tions occurred only in prior exposure Ca (2) and Cg (6), for spp.- FKS Mutation Rates of Candida glabrata (Cg) Vary by
specific rates of 10% and 15%, respectively. FKS mutation rates were Echinocandin (EC)
71%, 33%, 2%, and 0.3% in strains NS to 3, 2, 1, and 0 ECs, respec- C. Clancy, M. H. Nguyen, E. Press and R. K. Shields
tively. 67% of BT Cg were mutants. No Cg with CSP
University of Pittsburgh, Pittsburgh, USA
MIC = 0.25 lg ml
1 (i.e., intermediate) harbored a mutation. Using
a NS cutoff ≥0.5 lg ml
1, the rate of CSP NS Cg was reduced from
34% to 8% and the number of high-risk strains from 32% to 25% Background EC resistance in Cg is mediated by point mutations in
(P < 0.001 for both). With the revised cutoff, 58% of NS strains from FKS1 and FKS2 hot spots (HS). The objective of this study was to
pts with prior exposure harbored mutations. measure breakthrough mutant frequencies (MF) and FKS mutation
Conclusions FKS mutations are only encountered among high-risk rates among Cg exposed to anidulafungin (ANF), caspofungin (CSP),
Cg and Ca, but are rare with the exception of BT Cg strains. Strains and micafungin (MCF).
NS to a single EC are almost never FKS mutants, suggesting such Methods 20 FKS wild-type (WT) Cg with varying EC MICs from pts
classifications are CLSI BP artifacts and not biologically-driven. Based with or without prior EC exposure were selected. MF was calculated
on our cumulative experience, CSP-intermediate Cg should be consid- as ratio of CFU on SDA plates with or without ECs at 39 MIC. Break-
ered susceptible and the NS breakpoint increased to ≥0.5 lg ml
1. through mutant prevention concentration (MPC) was the lowest EC
concentration inhibiting >99% growth on SDA agar w/vs w/o drug.
MICs and FKS genotypes were determined for colonies growing at ≥
MPC for each EC.
Results 50% of strains had CSP MICs > or ≤ the CLSI breakpoint
P014 (≤0.12 lg ml
1), of which 50% in each group were from pts with
prior EC exposure. Overall median ANF, CSP, and MCF MICs were
Prior Azole or Echinocandin (EC) Exposure Increases the 0.06 (range: 0.03–0.12), 0.12 (0.03–1), and 0.03 (0.015–0.03),
Risk for EC Resistance and Antifungal (AF) Treatment (Tx) respectively. Corresponding median MPCs were 0.5 (0.25–4), 2 (1 to
Failure among Patients (pts) with Candidemia >8), and 0.25 (0.25–1), respectively. CSP MPCs were higher than
C. Clancy, M. H. Nguyen, E. Press and R. K. Shields other ECs (P < 0.0001 for each). Median Cg MF rates in ascending
order were 2.8E-9 (0 to 1.6E-7), 4.2E-7 (1.2E-8 to 5.9E-6), and
University of Pittsburgh, Pittsburgh, USA 4.8E-6 (1.2E-7 to 5.2E-3) for MCF, ANF, and CSP, respectively
(P < 0.0001 for each 2-way comparison). MF rates did not vary by
Background Prior EC exposure is an important determinant of EC CSP MIC or prior EC exposure. 81, 17, and 7 strains growing at ≥
resistance among Candida spp. We hypothesized that azole exposure MPC of CSP, ANF, and MCF, respectively, were collected. Compared
may also influence EC resistance. Our objective was to determine the to parent strains, CSP, ANF, and MCF MICs against 63%, 29%, and
impact of prior AF exposure on candidemia epidemiology, AF suscep- 25% of MPC mutants, respectively, were increased by ≥2-fold. Over-
tibility, and tx outcomes. all, 28% had FKS mutations (90% were in FKS2, HS2). 88% of
Methods Consecutive pts with candidemia from 2009 to 14, includ- strains with ≥2-fold MIC increase to all ECs had FKS mutations (vs
ing up to 1 recurrent episode per pt were evaluated. Prior AF expo- 14% w/MIC increase to 1 EC; P < 0.0001). Rate of FKS mutations
sure was defined as ≥ 3 days (d) of AFs. Tx success was defined as among strains recovered from CSP, ANF, and MCF-containing agar
survival and sterilization of blood cultures at 14 d. Fluconazole (FLC) were 20%, 47%, and 71% (P = 0.03 and 0.008, comparing CSP to
and caspofungin (CSP) MICs were determined by YeastOne. ANF and MCF, respectively). 76% of mutations occurred at position
Results 444 strains from 395 pts were included; C. albicans (Ca; F659 (88% of CSP-associated mutants vs 38% of ANF/MCF muta-
37%) and C. glabrata (Cg; 36.5%) were most common, followed by C. tions; P = 0.19). Mutations at S645 (FKS1) or S663 (FKS2) occurred
parapsilosis (Cp; 14%), C. tropicalis (Ct; 8.5%), and other spp (4%). Cg only among strains exposed to ANF or MCF (31% of mutants com-
(32 to 45%, P = 0.02) and Cp (10 to 21%, P = 0.003) were more pared to 0% for CSP, P = 0.03).
common among pts with prior AF exposure, and Ca was less com- Conclusions MF and FKS mutation rates are inversely related for
mon(45–21%, P < 0.001); data were consistent for prior azole and each EC against Cg. Specific FKS mutations also differ by agent. In
EC exposure individually. CSP and FLC MICs varied by exposure for rank order, MF rates are CSP>ANF>MCF, but FKS mutation rates are
Cg, but not other spp. Geometric mean CSP MICs increased stepwise MCF>ANF>CSP. Mutations at F659 are more common for CSP,
among Cg strains not exposed to AFs (0.11 lg ml
1), exposed to whereas S645 and S663 mutations are more likely when Cg are
azole alone (0.15, P = 0.02), EC alone (0.22, P = 0.04), or both exposed to ANF or MCF.
(0.30, P = 0.0002). FLC MICs against Cg were increased if exposed
to azoles (P = 0.03), but not ECs (P = 0.21). 357 cases of can-
didemia were treated w/≥3 days of AFs. Rates of tx success were
higher for pts receiving an EC (70%) vs azole (58%, P = 0.02), and
those not exposed to AFs (71%) vs exposed (52%, P = 0.0005).
Among pts exposed to an AF, success was significantly lower when
treated with an EC (52 vs 76%, P = 0.007) or azole (32 vs 66%,
P = 0.0003). Excluding breakthrough candidemia (defined as failure
a priori), rates of success were 65%, 76%, and 87% for pts exposed to
AFs w/in 30 d, 31–100 d, or >100 d prior to candidemia,

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 57
Poster Presentations

P016 P017

Activity of Isavuconazole and other antifungal agents Candida glabrata mutator phenotype promotes resistance
against clinical strains of mucorales to multiple antifungal drugs
A. Alastruey-Izquierdo,1 A. Santerre-Henriksen,2 M. Jones2 and K. R. Healey,1 S. R. Lockhart,2 J. D. Sobel,3 D. Farmakiotis,4
M. Cuenca-Estrella3 D. P. Kontoyiannis,4 D. Sanglard,5 E. Shor1 and D. S. Perlin6
1 1
Spanish National Centre for Microbiology, Instituto de Salud Public Health Research Institute, New Jersey Medical School,
Carlos III, Majadahonda, Spain; 2Basilea Pharmaceutica Rutgers, Newark, USA; 2Centers for Disease Control and
International Ltd, Basel, Switzerland and 3Spanish National Prevention, Atlanta, Georgia, USA; 3Wayne State University
Center for Microbiology, Madrid, Spain School of Medicine, Detroit, MI, USA; 4The University of Texas
MD Anderson Cancer Center, Houston, TX, USA; 5Institute of
Objectives Isavuconazole (ISAV) is a new triazole antifungal agent Microbiology of the University Hospital of Lausanne, Lausanne,
that was granted approval by the U.S. Food and Drug Administration Switzerland and 6New Jersey Medical School-Rutgers, Newark,
on March 6, 2015 for the treatment of invasive aspergillosis and USA
mucormycosis. The aim of this study was to evaluate the activity of
isavuconazole and other antifungal agents against mucorales isolated Objectives Both the incidence of invasive fungal infections and rates
from clinical samples. of multi-drug resistance associated with Candida glabrata have
Methods Eighty-two mucorales strains were tested for antifungal increased in recent years. The cellular mechanisms involved in the
susceptibility following EUCAST and CLSI methodologies. All strains proclivity of C. glabrata to rapidly develop resistance to multiple drug
were obtained from clinical samples and identified to species level by classes, specifically triazoles and echinocandins, are unknown. Others
sequencing the Internal Transcribed Spacer Region of the rDNA. Spe- have shown that defects in bacterial DNA repair can cause resistance
cies included in the study were: Lichtheimia corymbifera, Lichtheimia to multiple antibiotics. Here, we investigate DNA repair in relation to
ramosa, Mucor circinelloides, Rhizomucor miehei, Rhizomucor pusillus, C. glabrata antifungal resistance.
Rhizopus arrhizus and Rhizopus microsporus. The antifungal suscepti- Methods DNA mismatch repair genes, MSH2 and PMS1, and dou-
bility testing was performed following EUCAST reference method 9.1 ble-strand break repair gene, RAD50, were disrupted and deletion
and CLSI M38-A2. The antifungals used in the study were: ampho- strains selected on multiple antifungals to determine mutational fre-
tericin B (AMB), ISAV, voriconazole (VCZ) and posaconazole (PCZ). quencies. Drug target genes, e.g. FKS1/2, were sequenced in resistant
All antifungals tested ranged from 0.03 to 16 mg l
1. Aspergillus fla- mutants. MSH2 was subsequently sequenced in 234 diverse, clinical
vus ATCC 204304 and Aspergillus fumigatus ATCC 204305 were strains with varying susceptibility profiles. Identified mutations were
used as quality control strains in all test performed for both methods. expressed in the msh2 deletion strain and frequencies of 5-fluoroan-
Minimal Inhibitory Concentrations (MICs) were read after 24 and thranilic acid (5-FAA)- and echinocandin-resistant mutants mea-
48 hours of incubation. sured. To determine if altered mismatch repair activity also promotes
Results With CLSI methodology over 23% of the strains did not the development of mutations in vivo, we developed a mouse model of
grow at 24 h (the recommended incubation time) in contrast with gastrointestinal (GI) colonization. Mice were orally inoculated with
EUCAST that showed good growth at 24 h for all mucorales strains. either a wild-type or msh2D strain followed by daily, sub-inhibitory
Comparable results were found between CLSI 48 h and EUCAST caspofungin (0.5 mg kg
1 i.p.) or vehicle (PBS i.p.) treatments for
24 h. Amphotericin B was the most active compound against all spe- 21 days. GI burdens were measured for 30 days through fecal yeast
cies with MICs90 (MICs causing the inhibition of 90% of the isolates) colony counts and resistance was examined through replica plating
< 2 mg l
1 for all species but M. circinelloides with CLSI methodology on caspofungin and Fks1/2 hotspot sequencing.
(MIC90 = 16 mg l
1 at 48 h). Isavuconazole showed good activity, Results Disruption of MSH2 or PMS1, but not RAD50, led to a
with geometric means of MICs (GM) and MICs50 < 2 mg/L to all hyper-mutable phenotype and a significant increase in the emergence
species but M. circinelloides (GM = 3.49 for EUCAST 24 h and 2.08 of echinocandin-, triazole-, and amphotericin B-resistant mutants. As
for CLSI 48 h) and moderate activity to Rh. pusillus with expected, the echinocandin-resistant mutants contained Fks1 or Fks2
MICs50< 2 mg/L but MIC90 = 4 mg l
1. Voriconazole showed no hotspot mutations. Out of the 234 clinical strains analyzed, 48%
activity against the species tested. Posaconazole was active (MIC50< (77/161) of susceptible isolates demonstrated a nonsynonymous
0.5 mg l
1) for L. corymbifera, L. ramosa, R. arrhizus and R. micro- mutation within Msh2, while a mutation was discovered in 67%
sporus, but M. circinelloides and Rh. pusillus showed high MICs to this (24/36; P < 0.05 vs. susceptible) of fluconazole-resistant isolates and
compound (MICs90 > 2 mg l
1). 68% (15/22; P = 0.07 vs. susceptible) of multi-drug resistant iso-
Conclusions CLSI showed poor growth at 24 h. Amphotericin B lates. When transferred to a lab strain, the msh2 mutations from
was the most active antifungal. Voriconazole showed no activity clinical isolates maintained the hyper-mutable phenotype. Mice were
against the species tested. Isavuconazole and posaconazole showed effectively colonized (2–3 9 106 CFU/fecal pellet) with both the wild-
good activity to all species but M. circinelloides and Rh. pusillus. type and msh2D strains for 30 days. Fks1 mutations (625delF;
S629P) were identified in yeast recovered from caspofungin-exposed,
msh2D-colonized mice (mutants recovered from 3 of 3 mice ana-
lyzed). No Fks1/2 mutants were recovered from caspofungin-exposed,
wild-type-inoculated mice (4 mice analyzed).
Conclusion Overall, we show that strains containing loss-of-func-
tion msh2 mutations exhibit a higher propensity to breakthrough
antifungal treatment in vitro and in vivo, and that such strains are
recovered at high frequencies from patients. These data suggest that
defects in mismatch repair represent a key, underlying cellular mech-
anism that facilitates emergence of resistance to multiple antifungals
in C. glabrata.

ª 2015 The Authors

58 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P018 to triazoles, the first line therapy, has been reported in azole naive
patients. This resistance has been linked to fungicide-driven muta-
tions in the CYP51A gene and its promoter (TR34/L98H, TR46/
In vitro killing activities of two major antifungals against Y121F/T289A). This particular mechanism of resistance has been
clinically relevant Mucorales using minimal fungicidal reported in several European countries, Asia, Africa and the United
concentrations of amphotericin B and posaconazole. States. By contrast, no resistance was demonstrated in the few
R. Caramalho, C. Lass-Flo € rl and M. Lackner strains investigated in South America (Lockart et al., AAC, 2011;
Van de Linden et al., EID, 2015). Colombia is the fourth country in
Medical University of Innsbruck, Innsbruck, Austria
the world in use of pesticides (14.5 tons/1000 hectares) (http://
www.fao.org), of which 30% are fungicides. The main objective of
Objectives Mucorales are filamentous fungi causing invasive infec- our study was to investigate the existence of azole-resistant Aspergil-
tions, which are particularly aggressive and often lethal in immuno- lus fumigatus in soil samples related to the use of fungicides.
compromised hosts. There is a rising interest in antifungal Materials After analysis of the fungicides used in Colombia as well
susceptibility testing (AST) of amphotericin B (AMB) and posacona- as their use in agriculture, the horticulture sector was selected. Soil
zole (PSC), mainly due to the increasing incidence of breakthrough samples were collected in ornamental plant beds and flower fields
invasive mucormycosis (IM). AMB has a good in vitro activity against from the outskirts, suburbs and downtown areas of the city of
Mucorales, and a minor efficacy for PSC is commonly observed. An Bogot a. The primary culture isolates were sent to Nantes University
antifungal agent with cidal activity is therapeutically more promising for molecular identification and resistance study. To detect azole-re-
than an agent with fungistatic activity. Hence, the main goal of the sistant Aspergillus fumigatus, all the strains isolated on Sabouraud
present work was to evaluate a) in vitro killing activities of AMB and agar at 43 °c were screened on agar supplemented with itraconazole
PSC by using minimum fungicidal concentrations (MFCs), and b) dif- or voriconazole. For resistant strains, the CYP51A gene and its pro-
ferences in MFCs on both standard medium (RPMI) and alternative moter were PCR amplified and subsequently DNA sequenced.
supplementary minimal medium (SUP). Results From the 60 soil samples, 12 were positive for Aspergillus fumi-
Methods A strain collection comprising 131 Mucorales strains was gatus and 6 exhibited strains (n = 38) that grew on agar supplemented
catalogued for species identification using the internal transcribed with itraconazole or voriconazole. Most triazole-resistant strains were
spacer region (ITS) direct sequencing analysis. In vitro antifungal sus- isolated in soil samples from the flower fields and from greenhouses. Of
ceptibilities against AMB and PSC were tested according to EUCAST the 38 resistant strains, 20 were studied for resistance mechanisms.
standard method. In parallel, a modified EUCAST was performed, Sequence analysis of the CYP51 gene and its promoter indicated great
where RPMI was replaced by SUP to provide optimal growth condi- polymorphism with the presence of TR46/Y121F/T289A (n = 17 iso-
tions for Mucorales (1). The MFCs were determined as previously lates), TR34/L98H (n = 1), TR53 (n = 1) and no mutations (n = 1).
described (2) and evaluated using the EUCAST method with either Difenoconazole and tebuconazole were the azole fungicides found to be
RPMI or with SUP medium. MFC: MIC ratio was calculated for each use in both places, flowers fields and greenhouses.
species and antifungal compound. Both antifungals were categorized Conclusion This is the first study describing A. fumigatus harboring
as either cidal or static. fungicide-driven alterations in Colombia and South America. These
Results A clear killing activity of AMB was observed in Rhizopus results underline the need for extensive monitoring of Aspergillus
arrhizus and Mucor genus. However, in all other four studied species resistance in azole exposed as well as naive patients and the impor-
(Lichtheimia species, R. microsporus and Rhizomucor pusillus), the drug tance of continuing research of the environmental factors in other
appeared to be fungistatic, as MFCs were consistently higher than regions of South America.
respective MICs (≥ 4 dilution steps). When assessing AMB MFCs in
SUP, a static effect of the drug was seen in all species. For PSC MFCs
in standard RPMI, the drug was in vitro fungistatic against all Muco-
rales. The same effect was seen when the drug was tested using SUP P020
medium, with the exception of L. corymbifera.
Conclusion The killing activity of AMB is species- and medium-de-
pendent, ranging from static to cidal. Posaconazole was clearly fungi- Patient’s home: a clinical relevant source of Aspergillus
static under standard procedure. Higher MICs were obtained when fumigatus harboring the TR46/Y121F/T289A alteration
both drugs were tested in modified methods using SUP. This may be R. Lavergne,1 T. Chouaki,2 B. Toublanc,3 H. Dupont,2
associated to their generally higher growth ability in this medium. V. Jounieaux,3 F. Morio4 and P. le Pape5
The exception was L. corymbifera since the species was able to grow 1
Universite de Nantes, Nantes, France; 2Amiens University
better in RPMI than in SUP.
Hospital, Amiens, France; 3CHU Amiens-Picardie, Amiens, France;
References: 4
1. J. W€ ostemeyer. 1985. Strain-dependent variation in ribosomal
CHU de Nantes, Nantes, France and 5CHU de Nantes, Nantes,
DNA arrangement in Absidia glauca. FEBS J. 146:443–448. France
2. C. Lass-Fl€
orl, D. Fuchs, M. Ledochowski, C. Speth, M. P. Dierich,
and R. W€ urzner. 2003. Antifungal properties of 5-hydrox- Azole resistance in Aspergillus fumigatus is an emerging worrisome
ytryptamine (serotonin) against Candida species in vitro. J. Med. problem. Environmental resistance has been highlighted relatively
Microbiol. 52:169–171. recently in the Netherlands (Verweij, N Engl J Med 2007; Mellado,
Antimicrob Agents Chemother 2007). While strains bearing the
TR34/L98H alteration have been found in many countries, TR46/
Y121F/T289A strains are currently less frequently isolated. We
P019 recently described the first TR46/Y121F/T289A clinical strains in
France in a cystic fibrosis patient (Lavergne, Antimicrob Agents Che-
mother 2015). However, environmental investigations failed to detect
First description of fungicide-driven alterations in azole- TR46/Y121F/T289A strains near the patient’s residence. Here, we
resistant Aspergillus fumigatus in Colombia, South report the case of a 66-year-old patient, treated for a rheumatoid
America arthritis (methotrexate and infliximab) who was admitted in inten-
C. Alvarez,1 R. Lavergne,2 F. Morio3 and P. Le Pape3 sive care united because of bilateral pneumopathy. BAL fluid grew
with A. fumigatus with antifungal susceptibility testing using E-test
1
Universidad Nacional, Bogota, Bogota, Colombia; 2Universite de
method evidencing high resistance to voriconazole (>32 lg ml
1)
Nantes, Nantes, France and 3CHU de Nantes, Nantes, France together with resistance to itraconazole (6 lg ml
1) and posacona-
zole (1 lg ml
1). Seventeen days after admission, the patient died
Objective Aspergillus fumigatus causes a variety of diseases in because of cerebral hemorrhage and multivisceral failure in the con-
humans leading to high morbidity and mortality. Recently, resistance text of probable invasive aspergillosis.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 59
Poster Presentations

Objectives We investigated the A. fumigatus isolate from the patient Results Over a 1–4 month period, each center aimed to collected
and looked for the presence of azole-resistant A. fumigatus strains 50 A. fumigatus isolates (n = 470). The number of azole-resistant iso-
bearing environmental molecular alterations at the patient’s home lates was n = 76 (16%). Resistance frequency varied from 0% to
and environment. 49% per center and the time to collect isolates varied from 1 month
Methods A total of thirty-four surface or soil samples were per- to 4 months. The majority of resistant isolates were recovered from
formed: home (n = 10), garden (n = 11) and cultivated fields near respiratory material (n = 444). The remaining isolates originated
the home of our patient (n = 13). All samples that yield A. fumigatus from ear swabs. All except one were from patients without previous
were screened on both 4 mg l
1 itraconazole and voriconazole-con- azole exposure. Of the cyp51A mutations (n = 35), TR34/L98H was
taining media. Azole-resistant strains were subjected to nucleotide the most common (n = 28) followed by TR46/Y121F/T289A
sequencing of the cyp51A gene and its promoter. (n = 4), TR46/Y121F/T289A/M172I/G448S (n = 1), F46Y/M172V/
Results As expected from MIC testing, the clinical isolate harbored E427K (n = 1) and N248K (n = 1). The remaining resistant isolates
the TR46/Y121F/T289A alteration. Samples from the home (n = 3), had no mutation in the Cyp51A (n = 41). In two centers we found
the garden (n = 3) and one sample of a field crops grew on azole- clusters of 4 and 5 patients with similar isolates.
containing media. Thirty-seven representative isolates from these Conclusions The prevalence of resistance increased significantly
samples were sequenced. Thirty-two isolates from the home and the compared to a previous surveillance study in 2006. The number of
garden harbored the TR46/Y121F/T289A alteration while the 5 iso- non-cyp51A mediated resistant isolates was higher than found in
lates from the soil of field crops only harbored the TR34/L98H alter- Dutch University Centers and comparable to the level found in the
ation. Microsatellite genotyping using a panel of nine short tandem UK. Regular surveillance is warranted to monitor trends in azole
repeats is ongoing. resistance at a national scale.
Conclusion This observation confirms the presence of TR46/Y121F/
T289A strains in France and is the first description of this fungicide-
driven mutation in a patient’s home in our country. Taken together,
this study highlights the need for regular antifungal susceptibility
P022
testing of A. fumigatus clinical isolates and underlines the importance
of indoor and outdoor environment as a potential source of azole-re- Effect of haloperidol, promethazine and cyclosporine A on
sistant A. fumigatus isolates. the fluconazole susceptibility of Malassezia spp.
Correction added on 19 October 2015, after online publication. C. Cafarchia, R. Iatta, M. R. Puttilli, D. Immediato and D. Otranto
Author V. Letsher Bru was replaced with P. le Pape and his corre-
sponding affiliation to CHU de Nantes, Nantes, France Universita degli Studi di Bari, Valenzano Bari, Italy

Objectives Malassezia pachydermatis and Malassezia furfur cause vari-

ous forms of dermatitis in both animals and humans, as well sys-
P021 temic infections in immunocompromised patients (Velegraki et al.,
PLoS Pathog 2015). Clinical data as well as the in vitro antifungal
susceptibility studies suggest that M. pachydermatis and M. furfur are
Multicenter surveillance of azole resistant Aspergillus low susceptible or resistant to fluconazole (FLZ) (Iatta et al., J Med
fumigatus isolates in non-academic hospitals in the Microbiol 2014; Cafarchia et al., Med Mycol 2015). The defence
Netherlands mechanisms against azoles as well as the mechanisms of azole resis-
J. F. Meis,1 R. de Lorijn,2 I. Curfs-Breuker,2 W. Rozemeijer,3 tance have never been investigated in Malassezia spp. The increased
B. Ridwan,4 M. Tersmette,5 M. L. van Ogtrop,6 A. Muller,7 expression of drug efflux pumps in the cell plasma membrane is
widely recognized to be the major cause of high-level minimal inhibi-
E. de Brauwer,8 A. Bielefeld,9 C. Timmerman,10 M. Wolfhagen,11
tory concentration (MIC) values of azoles in Candida spp. (Pfaller
C. M. Verduin,12 F. Hagen2 and P. E. Verweij13 et al., Am J Med 2012). It has been shown that haloperidol (HAL),
1
Canisius Wilhelmina Hospital and Radboud University Hospital, promethazine (PTZ) and cyclosporine A (CYS) act as modulators or
Nijmegen, the Netherlands; 2Canisius Wilhelmina Hospital, inhibitors of drug efflux pumps in Candida spp., therefore causing a
Nijmegen, the Netherlands; 3Medical Center Alkmaar, Alkmaar, significant azoles MIC reductions in Candida spp. (Castelo-Branco
the Netherlands; 4COMICRO, Hoorn/Zaandam, the Netherlands; et al., Med Mycol 2013; Stylianou et al., Antimicrob Agents Che-
5
St. Antonius Hospital, Nieuwegein, the Netherlands; 6Onze mother 2014; Wibawa et al., Trop Biomed 2015). This study aims to
Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands; 7Medical evaluate the effects of HAL, PTZ and CYS on FLZ MIC values of M.
Center Haaglanden, The Hague, the Netherlands; 8Atrium furfur and M. pachydermatis strains, in order to assess the role of drug
Medical Center, Heerlen, the Netherlands; 9Certe Laboratory, efflux pumps in the defence mechanism or in resistance phenomena
against FLZ in Malassezia spp.
Groningen, the Netherlands; 10Tergooi Hospital, Hilversum, the
Methods A total of 33 Malassezia strains, 21 M. furfur from humans
Netherlands; 11Isala Hospital, Zwolle, the Netherlands; 12Amphia with bloodstream infections and 12 M. pachydermatis from dogs with
Hospital, Breda, the Netherlands and 13Radboudumc, Nijmegen, skin lesions, were tested. The vitro efficacy of FLZ in combination
the Netherlands with HAL or PTZ or CYS was performed using a broth microdilution
checkerboard analysis. Data were analysed using the model-frac-
Objectives Aspergillus fumigatus is a common cause of invasive and tional inhibitory concentration index (FICI) method (Odds, J Antimi-
allergic diseases. Limited data are available on the prevalence of azole crob Chemother 2003).
resistance outside university medical centers in the Netherlands. We Results The MIC values of FLZ of M. furfur and M. pachydermatis
aimed to determine the burden of azole resistance in medical centers decreased in presence of sub-inhibitory concentrations of HAL (from 4-
outside universities. fold to >16-fold reduction) and PTZ (from 0 to >4-fold reduction) but
Methods Investigators from 10 non-academic community hospitals in not of CYS. Synergism (FICI <0.5) was observed only with HAL and
the Netherlands prospectively collected up to 50 Aspergillus fumiga- FLZ in 12 M. furfur and 8 M. pachydermatis strains presenting high FLZ
tus isolates. Isolates were identified morphologically and by growth at MIC value (i.e., MIC≥128 lg ml
1 and ≥64 lg ml
1, respectively).
48°C and were subsequently tested for azole resistance on screening Conclusion The present study demonstrated for the first time that
agar plates with itraconazole, voriconazole and posaconazole. Isolates HAL and PTZ could enhance the antifungal effect of FLC in M. furfur
which grew on azole containing agar where molecularly identified and M. pachydermatis strains, thus indicating that drug efflux pumps
with b- tubulin and ITS sequencing. Antifungal susceptibility testing are involved in defence mechanism to azole drugs. The synergistic
was performed with both CLSI and EUCAST reference methods. Resis- effect of FLZ and HAL in Malassezia strains with high FLZ MIC values
tant isolates were further analyzed by sequence analysis of might be related to an increased expression of efflux pumps genes,
the Cyp51A gene. All isolates were typed with microsatellite typing. eventually resulting in a possible FLZ resistance as in Candida spp. (in
Origin of isolates, age of patients, and prior azole use was documented. Pfaller et al., Am J Med 2012). The FLZ MIC≥128 lg ml
1 for

ª 2015 The Authors

60 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

M. furfur and MIC≥64 lg ml
1 for M. pachydermatis might be consid- Table 1. Susceptibility in vitro of Candida parapsilosis against anidu-
ered as cut-off values for separating susceptible and resistant strains, lafungin before and after induction of resistance.
respectively. Further efflux pumps expression analyses could be per-
formed in order to confirm our results.

P023

Aspergillus spp. antifungal susceptibility to triazoles and

Amphotericin B
R. Iatta,1 M. R. Puttilli,1 D. Immediato,1 L. Fracchia,2 D. Otranto1
and C. Cafarchia1 the second Candida species most commonly isolated from blood cul-
tures in Latin America. C. parapsilosis has emerged as an important
1
Universita degli Studi di Bari, Bari, Italy and 2Universita del nosocomial pathogen, causing invasive candidiasis in hospitalized
Piemonte Orientale, A. Avogadro, Novara, Italy, Novara, Italy patients in critical condition and in low-weight newborns, as well as,
in immunocompromised individuals, transplant and patients receiv-
Objectives The emergence of resistance to antifungal agents among ing parenteral nutrition. These diseases show high rates of morbidity
Aspergillus species isolated from different sources is dramatically and mortality and are of difficult prevention and treatment. The
increasing. The aim of this study was to evaluatethe in vitro antifun- echinocandins are recommended as the first-line treatment for inva-
gal susceptibility to itraconazole (ITZ), posaconazole (POS), voricona- sive candidemia, however, these drugs have shown reduced activity
zole (VOR) and amphotericin B (AMB) against Aspergillus spp. in vitro and treatment failures against other Candida species. This
isolated from different sources. study aimed to evaluate the in vitro induction of resistance to anidu-
Methods A total of 102 Aspergillus strains (63 of A. fumigatus, 19 of A. lafungin of different clinical isolates of C. parapsilosis considered sus-
oryzae,16 of A. tubingensis and 4 of A. niger), identified phenotypically ceptible to anidulafungin.
and molecularly by sequencing the b-tubulin gene, were tested. Isolates Methods The resistance was induced in the 30 clinical isolates of C.
were divided into three groups: group I consisting of 29 strains from parapsilosis susceptible to anidulafungin. The cultures were seeded in
animals (i.e., 23 of A. fumigatus and 6 of A. tubingensis); group II com- tubes containing medium Yeast Nitrogen Base (YNB) and incubated
prising 37 isolates from nosocomial environment (i.e., 20 of A. fumiga- at 30°C overnight. The inoculum was standardized in new crops
tus, 10 of A. oryzae, 4 of A. tubingensis and 3 of A. niger); and group III YNB based on the turbidity measured using a spectrophotometer for
comprising 36 isolates from laying hen farms environment (i.e., 20 of a final absorbance of 0.1 (k = 640 nm). The standardized cultures
A. fumigatus, 9 of A. oryzae, 6 of A. tubingensis and 1 of A. niger). The were incubated at 30°C and, after 10 hours anidulafungin was
minimum inhibitory concentrations (MICs) were determined by the added to start the process of induction of resistance. Each isolate was
broth microdilution method according to the CLSI M38-A2 protocol challenged to grow at increasing concentrations of anidulafungin,
and the isolates were considered resistant if they showed a MIC above and the initial concentration of drugs was 0.03 lg/ml and finished
the breakpoints proposed (>2 lg ml
1 for ITZ and VOR; >0.5 lg ml
1 with 8 lg ml
1. These tests were repeated doubling the concentra-
for POS; and >2 lg ml
1 for AMB - Verweij et al., Drug Resist Updat tion of the drug until the final concentration was eight times higher
2009; Arendrup et al., Clin Microbiol Infect 2012). than the initial concentration. From the final culture, the cells were
Results VOR, ITZ and POS showed the highest activity against plated on Sabouraud dextrose agar and a single colony was consid-
Aspergillus spp. strains, whereas AMB the lowest. Two strains of A. ered resistant isolated. The isolates were maintained on distilled
section Nigri (1 of A. tubingensis and 1 of A. niger) isolated from noso- water with 10% glycerol under anidulafungin concentration of resis-
comial environment scored resistant to ITZ (MIC = 64 lg ml
1) and tance (≥8 lg ml
1) at
80°C.
POS (MIC=1 lg ml
1) and A. tubingensis also to AMB Results From 30 isolates of C. parapsilosis which were initially sus-
(MIC = 4 lg ml
1). Resistance to AMB was detected in 19% (12/63) ceptible, after exposition to anidulafungin, resulted at the end of the
of A. fumigatus strains, in 25% (5/20) of A. section Nigri (i.e., 25% A. process analysis two groups: anidulafungin-susceptible (ANF-S) and
tubingensis and 25% A. niger) and in 89% (17/19) of A. oryzae anidulafungin-resistant (ANF-R). After 25 exposures to increasing
regardless of the source of isolation. concentrations of anidulafungin (63 days), we detected the first case
Conclusion Based on these findings, the Aspergillus spp. herein of resistance. It is important to note, that from the concentration of
tested were highly susceptible to azoles and less to AMB. The cross 0.12 lg ml
1 the strains had a delay in their growth requiring more
resistance to azoles registered in 1 A. tubingensis and 1 A. niger sug- time for visualization of growth. After 60 exposures, 265 days after
gests that these strains may circulate in the nosocomial environ- the start of induction of resistance, the susceptibility of anidulafungin
ment, whereas Aspergillus spp. strains, considering their low activity was evaluated resulting in 14 (46.6%) resistant strains. Based on
to AMB, might be widespread. The molecular mechanisms of azole or minimum inhibitory concentration (MIC) range, breakpoints and geo-
AMB resistance, which are poorly investigated in these Aspergillus metric mean of MICs, the resistance was significant,
species, need to be evaluated in future studies. (MG = 2.4 lg ml
1). On the other hand, when considering non-sus-
ceptible strains (intermediated or resistant) the increase was of 18
(59.9%) to ANF-R (Table 1). Significant statistical differences were
observed by comparison between the ANF-S group against ANF-R
P024 (P < 0.05).
Conclusion This study shows that C. paraspilosis acquires resistance
against anidulafungin after in vitro exposure; the impact of resistance
Emergency resistance in vitro to anidulafungin for C.
of C. parapsilosis to anudulafungin requires attention to the suscepti-
parapsilosis strains bility tests with this species.
F. Chassot,1 T. P. Venturini,1 F. B. Piasentin,1 F. K. Tobaldini,2
P. S. Bonfim-Mendonc
a,3 T. I. E. Svidzinski3 and S. H. Alves1
1
Universidade Federal de Santa Maria - UFSM, Santa Maria,
Brazil; 2Universidade Estadual de Maringa/Universidade do
Minho, Maringa, Brazil and 3Universidade Estadual de Maringa,
Maringa, Brazil

Objectives The incidence of C. parapsilosis complex has increased

significantly in the last decades. Some studies have shown that it is

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 61
Poster Presentations

P025 Table. Comparative activity of antifungal agents against Candida spp.

Identification and antifungal susceptibility of Curvularia

strains isolated from human eye infections
C. Vágvölgyi,1 K. Krizsan,1 E. To
 th,1 G. J. Szarkandi,1
P. Manikandan, M. Chandrasekaran,3 S. Kadaikunnan,3
2

N. S. Alharbi3 and T. Papp1

1
University of Szeged, Szeged, Hungary; 2Aravind Eye Hospital
and Postgraduate Institute of Ophthalmology, Coimbatore, India
and 3King Saud University, Riyadh, Saudi Arabia

Objectives Four closely related Curvularia species, C. australiensis, C.

hawaiiensis, C. geniculata and C. spicifera are emerging causative
agents of human phaeohyphomycoses. The classical morphological
examination and the increasingly used ITS-based molecular identifi-
cation proved to be insufficient to discern these species, especially C.
australiensis and C. spicifera. Aims of the study were to develop a reli-
able molecular identification method for these species and to examine Results A total of 421 Candida spp. (374 from blood and 47 from
their antifungal susceptibility. other sterile specimens; 191 from Hem pts and 230 from ICU pts)
Methods Thirty two strains originating from human infections, were included. Species distribution was as follows: 38% (160) C. albi-
plants and soil were involved in the studies. In addition to the ITS cans, 26% (108) C. parapsilosis, 8% (35) C. tropicalis, 8% (34) C. kru-
region, applicability of the calmodulin, b-tubulin and translation sei, 7% (28) C. glabrata, 4.5% (19) C. guilliermondii, 3.5% (15) C.
elongation factor 1-a (tef) genes, as well as the nuclear ribosomal pelliculosa, 3% (12) C. lusitaniae, 2% (10) other species. Prevalence of
intergenic spacer (IGS) were tested to identify the four Curvularia spe- C. albicans was in ICU pts (44% in ICU versus 30% in HEM,
cies. The in vitro effect of 10 antifungal agents against the Curvularia P = 0.003), C.krusei in HEM pts (11.5% versus 5%, P = 0.02).
isolates was also tested and the MIC50 and MIC90 values were deter- Comparative activity of anidulafungin, caspofungin, voriconazole
mined in each case. and fluconazole (MIC50/MIC90/susceptible (S) %) against all Candida
Results The ITS region was suitable only to discern C. hawaiiensis spp.is presented in table.
from the other species. Calmodulin, b-tubulin and tef genes were not Conclusion C. albicans was the predominant Candida spp. especially
enough variable for reliable identification. At the same time, the IGS in ICU pts. Among the predominant isolates, echinocandins had
region proved to be a good choice to distinguish all tested Curvularia reduced activity against C.guilliermondii and some isolates of C.parap-
species. Isolates of C. spicifera proved to be less susceptible to the silosis. Acquired resistance to azoles was for C.parapsilosis and C. albi-
tested antifungals than the other strains. cans. Activity of azoles was less than echinocandins for C.parapsilosis.
Conclusion Among the tested molecular markers, the nuclear ribo- In all cases of invasive cansidiasis the susceptibility to azoles should
somal IGS region contained clearly distinctive species-specific be performed before the beginning of the therapy with these agents.
motives. These discriminating motifs were determined using appropri-
ate reference strains. Majority of the strains isolated from keratomy-
coses belonged to C. spicifera and C. hawaiiensis. Results of the in vitro
antifungal susceptibility tests underline the importance of the correct P027
species identification.

The study was supported by the grants TET_12_DE-1-2013-0009,
OTKA NN 106394 and by the European Union co-financed by the Antifungal susceptibility patterns of cryptic species of

European Social Fund (TAMOP-4.2.2.B-15/1/KONV-2015-0006). Candida glabrata and Candida parapsilosis clades isolated
from patients suffering from oral candidiasis
K. Miranda-Cadena,1 C. Marcos-Arias,2 E. Mateo-Alesanco,2
J. M. Aguirre,1 E. Eraso2 and G. Quindo  s2
P026 1
Universidad del Paıs Vasco/Euskal Herriko Unibertsitatea UPV/
EHU, Leioa, Spain and 2Univ. Paıs Vasco UPV/EHU Facultad de
In vitro activity of echinocandins and azoles against of Medicina y Odontologıa, Bilbao, Spain
invasive isolates of Candida spp: multicenter study in
Russia Objectives To evaluate the antifungal susceptibility patterns of oral
G. Klyasova,1 A. Gracheva,1 M. Maschan,2 O. Kutsevalova,3 isolates of Candida glabrata,Candida bracarensis and Candida nivariensis
I. Molchanova,3 T. Chernenkaya,4 T. Kaporskaya,3 N. Zvezdkina,3 (Candida glabrataclade), and Candida parapsilosis, Candida orthopsilosis
and Candida metapsilosis (Candida parapsilosis clade) from patients suf-
O. Khoreva,3 L. Kraynova,3 S. Shushurina,3 D. Popov,5
fering from oral candidiasis.
R. Khokhlyavina3 and V. Okhmat1 Methods A total of 212 clinical isolates recovered from patients suf-
1
National Research Center for Hematology, Moscow, Russia; fering from oral candidiasis attending the Odontology clinics at UPV/
2
Ctr. of Hematology, Moscow, Russia; 3Hosp., Rostov on Don, EHU from 2003 to 2013 were analyzed. Isolates previously identified
Russia; 4Ctr. of Emerg. Health Care, Moscow, Russia and 5Ctr. by conventional mycological methods as Candida glabrata (114 iso-
for Cardiovascular Surgery, Moscow, Russia lates) and Candida parapsilosis (98 isolates) were re-identified by mul-
tiplex PCR and PCR-RFLP, respectively. Some identifications were
confirmed by the D1/D2 region of 26S rDNA gene sequencing. In
Objectives We investigated the in vitro activity of echinocandins and
vitro antifungal susceptibility testing were performed by disk diffus-
azoles against invasive isolates of Candida spp.
sion method, using the CLSI M44-A protocol, for nystatin, micona-
Methods Susceptibility testing of 421 Candida spp. isolated from
zole, fluconazole and itraconazole against all the isolates.
blood and other sterile specimens to antifungal agent was performed
Microdilution method (CLSI M27-A3 document) was carried out for
by the CLSI the broth microdilution method (CLSI 2012). Isolates
fluconazole.
were collected from 9 hematological (Hem) centers (2003–2014) and
Results The molecular techniques identified the 114 clinical isolates
11 ICU (2009–2014).
of Candida glabrata clade as Candida glabrata sensu stricto, and the 98

ª 2015 The Authors

62 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

isolates ofCandida parapsilosis clade as 95 Candida parapsilosis sensu

stricto, one Candida metapsilosis, one Candida orthopsilosis and other
one as Candida pararugosa. Candida metapsilosis, Candida orthopsilosis
andCandida pararugosa were susceptible to all antifungal agents
tested. None Candida glabrata showed resistance to miconazole or nys-
tatin. However, seven and eight isolates were susceptible-dose-depen-
dent to fluconazol and itraconazol, respectively. Moreover, 14 and
three isolates were resistant to fluconazol and itraconazol, respec-
tively. Only one fluconazole-resistant isolate was confirmed by
microdilution method. There were no resistance to fluconazol, nys-
tatin or itraconazole in Candida parapsilosis, nevertheless, four isolates
presented dose-dependent-susceptibility to itraconazol. Moreover, 46
isolates were intermediate susceptible to miconazole, and one isolate
was resistant to this drug.Six and three isolates with azole cross resis-
tance were detected in Candida glabrata and Candida parapsilosis,
respectively.
Conclusion Cryptic species Candida bracarensis and Candida nivarien-
sis were not isolated from oral candidiasis samples. The presence of
Candida metapsilosis and Candida orthopsilosis was very low. Nystatin
was the most active antifungal agent. Fluconazole was very effective
against Candida parapsilosis but its activity was poor against Candida
glabrata.
Funding: his study was financed in part by Fundaci on Jesus de
Gangoiti Barrera, Fundaci on ONCE ‘Oportunidad al Talento’and pro-
jects GIC12 210-IT-696-13, UFI 11/25 (UPV/EHU).

P028

In vitro activities of six antifungal drugs against Exophiala

spp. isolated from dishwashers and railway sleepers in
Turkey
D. Gulmez,1 O€ Dogan-Ayc
ik,1 B. Boral,1 A. Do
€ gen,2 M.M. Ilkit,3
G.S. de Hoog4 and S. Arikan-Akdagli5
1
Hacettepe University Faculty of Medicine, Ankara, Turkey;
2
Mersin University, Turkey; 3Cukurova Uni. School of Medicine,
Adana, Turkey; 4CBS-KNAW Fungal Biodiversity Centre, Utrecht,
Netherlands and 5Hacettepe University, Ankara, Turkey

Background Exophiala spp. may cause cutaneous, subcutaneous or

systemic infections in immunocompetent as well as immunocompro-
mised individuals. Source of infections is thought to be environmen-
tal in most cases. Data on in vitro antifungal susceptibility profiles of
Exophiala strains are rather limited. This study was undertaken to
investigate the in vitro activities of antifungal drugs against Exophiala
strains isolated from environmental sources in Turkey.
Methods A total of 81 Exophiala strains isolated from dishwashers
and railway sleepers and identified at species level by internal tran-
scribed spacer region (ITS) sequencing were included the study. In vitro
antifungal susceptibility testing for fluconazole (FLU), voriconazole
(VOR), posaconazole (POS), itraconazole (ITR), amphotericin B (AMB)
and terbinafine (TER) was performed according to CLSI M38-A2
microdilution method with modifications. The microdilution plates
were incubated at 25 °C and the incubation period was extended as
needed for sufficient growth. Minimum inhibitory concentration (MIC,
lg ml
1) values were determined visually using MIC-2 endpoint
(~50% inhibition compared to drug-free growth control) for azoles and
terbinafine, and MIC-0 endpoint (complete inhibition of growth) for
AMB. For each individual isolate, MICs were interpreted on two con-
secutive days after sufficient growth was achieved. (3) Among the tested antifungal drugs and in general, TER and
Results No growth was detected for any of the isolates at 24 h in VOR yielded highest in vitro activities, followed by POS, ITR, and
microdilution plates. Sufficient growth was observed at 48 h for all AMB against all Exophiala spp. tested. These five antifungal agents
strains tested and MIC readings were performed at 48 and 72 h, were favorably active in vitro against tested strains.
accordingly. MIC50 and MIC90 values, MIC ranges and geometric mean (4) In contrast, FLU showed highest MICs with no adequate
(GM) MICs at 48 and 72 h are shown in Tables 1 and 2, respectively. in vitro activity in general. Of specific note, FLU GM MICs against E.
Conclusions (1) MIC readings were interpretable at 48 h for all Ex- phaeomuriformis strains were relatively lower as compared to those
ophiala strains tested. against other Exophiala spp.
(2) MIC values at 48 h were similar to those at 72 h for all species (5) Further studies are required to determine correlation of these
and drugs. in vitro results with clinical outcome.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 63
Poster Presentations

P029 P030

Correlation between broth microdilution and disk In vitro activity of amphotericin B by timed-kill curves
diffusion methods for antifungal susceptibility testing of against Cryptococcus neoformans isolated from HIV-
voriconazole and fluconazole against Candida species infected patients with cryptococcal meningitis and
H. Sav,1 A. Baris,1 D. Turan,2 F. Ozakkas,3 S. Sen,1 R. Altinbas1 implications in clinical practice
and N. Kiraz1 O. J. Chagas,1 L. de Oliveira,2 M. Szeszs,2 M. Martins,2 D. Castro
1
Istanbul University, Istanbul, Turkey; 2Istanbul Haydarpasa E Silva,2 R. Buccheri3 and M. Melhem2
Numune Training and Research Hospital, Istanbul, Turkey and 1
Instituto de Infectologia Emılio Ribas/Instituto Adolfo Lutz, Sa~o
3
Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey Paulo, Brazil; 2Instituto Adolfo Lutz, Sa~o Paulo, Brazil and
3
Instituto de Infectologia Emılio Ribas, Sa~o Paulo, Brazil
Objectives Several yeast infections, especially candida infections,
represent a significant health problem in patients at high risk of Introduction Infections due to Cryptococcus neoformans cause severe
infection, leading to increased morbidity and mortality, greater disease, mostly in AIDS patients. Availablesusceptibility tests for C.
healthcare costs and increased duration of hospitalisation.Antifungal neoformans are not useful to detect isolates that are not susceptible to
drugs have been used extensively in prophylaxis, empirical therapy antifungal agents such as amphotericin B. The usefulness of timed-
and treatment of such infections, studies demonstrate that antifungal kill curve (TKC) is to estimate amphotericin B (AMB) fungicidal activ-
resistance is relatively rare. Candida species have varying degrees of ity. Although this method has been explored for Cryptococcus spp. iso-
susceptibility to the frequently used antifungal drugs. For example, lates, few studies have evaluated the correlation between laboratory
while Candida krusei is intrinsically resistant to fluconazole, Candida findings and clinical outcome.
glabrata is less susceptible or has higher MICs than other Candida spe- Objectives The purpose of this study was to correlate AMB TKC for
cies, which makes the correct species identification and susceptibility 27 isolates of C. neoformans with clinical aspects, prognosis and death
tests really necessary. In this study, our aim was dual: (i) to evaluate in 27 HIV-infected patients with cryptococcal meningitis.
the in vitro activities of two drugs with different mechanisms Methods Retrospective study, conducted at the Instituto de Infec-
(voriconazole and fluconazole) of action against clinical Candida iso- tologia Emılio Ribas and Instituto Adolfo Lutz with 27 HIV-infected
lates and (ii) to assess the suitability of results of disk diffusion test patients with cryptococcal meningitis. All of them had cerebrospinal
for use as a screening test in mycology laboratories. fluid (CSF)-positve culture for C. neoformans that was processed
Methods A total of 210 Candida isolates were isolated from 89 through the TKC procedures up to 72 hours of exposition time to
blood, 82 urine, 28 respiratory tract and 11 soft tissue samples of 1 mg of amphotericin B. Strains were divided into two groups
patients in various departments of Istanbul University, Cerrahpasa according to TKC results: Group A (TKC≤24 h) and Group B
Medical Faculty, between November 2011-November 2013 Isolates (TKC>24 h and/or regrowth). The clinical records of all patients
were identified by using conventional methods (germ tube formation, were reviewed and the following variables were analyzed: presence of
microscopic morphology in corn meal-Tween 80 agar) and addition- severe symptoms related to central nervous system (CNS) disease;
ally, through a commercial kit API 20C (Biomeriux, France). Isolates fungal burdens in the first and 2 weeks CSF sample; failure in steril-
are prepared for MALDI-TOF analysis by using a direct on target ization of CSF during induction phase; increased intracranial pressure
extraction method as well. The spectra were analysed by the IVD (ICP); extra-neural Cryptococcosis and outcome. Statistical testing
VITEK MS V.2.0 and SARAMIS 4.12 RUO database Antifungal sus- between groups was assessed by Mann-Whitney U test for continu-
ceptibility testing was performed by reference broth microdilution ous variables after verification of non-normal distribution by Shapiro-
(CLSI M27-A3) and disk diffusion methods (CLSI M44- A2). Wilk test. Fischer’s exact test was used for categorical variables. For
Results Atotal of 210 species of Candida were identified and the dis- survival analysis, a Kaplan-Meier curve with Long-Rank test was
tribution was as follows: 100 C. albicans, 43 C. parapsilosis, 25 C. used to determine the influence of variable. We assumed an IC of
tropicalis, 23 C. glabrata, 7 C. krusei, 6 C kefyr,5 C lusitaniae,1 C. guil- 95% and a < 0.05.
liermondii. Non-Candida albicans species showed higher MICs for the Results Group A comprised 20 (74.1%) patients and Group B the
two antifungal agents when compared with C. albicans isolates.For remaining 7 (25.9%), including 1 patient whose strain was not
fluconazole, the categorical agreement between broth microdilution inhibited by amphotericin B under the test conditions. We observed
and disk diffusion was 91%, with 2 very major error (VMEs), 4 major
error(MEs), and 12 minor errors. In addition, the best agreeement
was determined for voriconazole with five VMEs. For VORI, MEs and
min€ or errors were not observed,
Conclusion Disk diffusion is technically an easier method than
broth microdilution and this method shows very good agreement
with the reference method for fluconazole and voriconazole against
All Candida isolates (91% and 97%, respectively)

Figure 1

ª 2015 The Authors

64 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Table 1

that patients from Group B presented more severe symptoms, ICP,

and failure to sterilized CSF compared to Group A. Moreover, despite
Group B presented a higher median of Cryptococcal yeast counts in
the first CSF and with 2 weeks of induction treatment, there were no
significant clinical finding correlated with TKC in the present study
(Table 1). Despite patients from Group B presented worst clinical find-
ings, they had a lower mortality. Overall, none of these aspects was
significant and TKC >24 h was not associated with increased mortal-
ity in HIV-infected patients with cryptococcal meningitis (Fig. 1).
Conclusion We emphasized the difficulties in interpreting the results
obtained from the susceptibility tests against C. neoformans and their
real clinical meaning when trying to correlate them with the out-
come. The TKC for amphotericin B is a promising tool for the prog-
nosis of HIV-infected patients with cryptococcal meningitis, warrant
future studies encompassing large number of patients. (FLZ), voriconazole (VCZ), Itraconazole (ITZ), miconazole (MCZ), keto-
conazole (KZ), flucytosine (FLU), caspofungin (CAS) and Amfoterisin
B (AMB). C. albicans ATCC 10231, C. parapsilosis ATCC 22019 and
C. krusei ATCC 6258 were included as control reference strains.
P031 Results Species distribution was as follows: 100 C. albicans, 43 C.
parapsilosis, 25 C. tropicalis, 23 C. glabrata, 7 C. krusei, 6 C kefyr, 5C
lusitaniae, 1 C. guilliermondii. The minimum inhibitory concentrations
Species distribution and antifungal resistance profiles (MIC) distribution range, MIC50 and MIC 90 values of isolated Can-
among Candida species isolated from invasive Candida dida species are shown in Table 1. All of C. albicans, C. tropicalis, C.
infections in a Turkish university hospital glabrata, C. krusei, and C. guilliermondii isolates are susceptible to
A. Baris,1 D. Turan,2 H. Sav,3 F. Ozakkas,4 S. Sen,3 R. Oguz5 and caspofungin, whereas all C. albicans isolates were determined as sus-
N. Kiraz3 ceptible to fluconazole. Resistance to fluconazole was determined in 2
1
C. glabrata, 1 C. tropicalis and 2 C. parapsilosis isolates, as resistance
Sisli Hamidiye Etfal Training And Research Hospital, Istanbul, to voriconazole was detected in only 1 C. tropicalis isolate. All C. glab-
Turkey; 2Istanbul Haydarpasa Numune Training and Research rata isolates except two were dose-dependent susceptible to
Hospital, Istanbul, Turkey; 3Istanbul University, Istanbul, Turkey; fluconazole.
4
Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey Conclusion C. parapsilosis was the most isolated species among non-
and 5Istanbul University Cerrahpasa Medical Faculty, Istanbul, albicans Candida species. Non- albicans Candida species showed higher
Turkey MICs for the antifungal agents when compared with C. albicans iso-
lates. Voriconazole and caspofungin were the most active antifungal
in all Candida species.
Objectives Early and accurate diagnosis and appropriate antifungal
therapy are essential for a successful treatment for patients with
invasive Candida infections. Therefore, fast and reliable identification
of pathogenic species is critical, since the sensitivity to antifungal
agents varies depending on the species. In this study, our objective P032
was to evaluate the distribution and the antifungal susceptibility pro-
file of invasive Candida infections agents isolated from various clinical
Antifungal and cytotoxicity activities of Hamamelis
samples.
Methods A total of 210 Candida isolates were isolated from 89
viriginiana glycolic extract for Candida species.
blood, 82 urine, 28 respiratory tract and 11 soft tissue samples of G. N. Back-Brito,1 I. Amendola,1 D. Jesus,1 J. R. Oliveira,1
patients in various departments of Istanbul University, Cerrahpasa F. E. Oliveira,1 A. O. C. Jorge2 and L. D. Oliveira1
Medical Faculty, between November 2011-November 2013. Isolates 1
Institute of Science and Technology - UNESP, Sa~o Jose dos
were identified by using conventional methods (germ tube formation, Campos, Brazil and 2Institute of Science and Technology, UNESP
microscopic morphology in corn meal-Tween 80 agar) and addition- - Univ Estadual Paulista, Sa~o Jose dos Campos, Brazil
ally, through a commercial kit API 20C (Biomeriux, France) and
matrix-assisted laser desorption ionization-time of flight mass spec-
trometry (MALDI-TOF) Vitek MS system (bioMerieux, France). Iso- Objectives The aim of this study was to evaluate the antifungal
lates are prepared for MALDI-TOF MS system (bioMerieux, France) activity and cytotoxicity of Hamamelis virginiana glycolic extract for
analysis by using a direct on target extraction method. The spectra Candida spp. standard strains in planktonic growth and monotypic
were analyzed by the IVD VITEK MS V.2.0 database. In vitro antifun- biofilm.
gal susceptibility testing was determined using the CLSI microbroth Methods The species tested were: C. albicans (ATCC 36801), C.
dilution method M27 A3-S4. The tested drugs included; fluconazole dubliniensis (ATCC MYA 646), C. glabrata (ATCC 9030), C.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 65
Poster Presentations

P033

Aspergillus fumigatus susceptibility testing by CLSI and

EUCAST Guidelines
E. Pinto,1 A. Lopes,2 C. Monteiro,1 S. Suasa-Ard1 and
M. D. Pinheiro3
1
Faculty of Pharmacy-Porto University, Porto, Portugal; 2Porto
University, Porto, Portugal and 3Centro Hospitalar S. Joao EPE,
Porto, Portugal

Introduction Fungal infections caused by filamentous fungi, espe-

cially Aspergillus fumigatus, are rising due to the increased number of
patients with immunosuppression or affected by disorders like AIDS,
diabetes, immunological diseases and COPD. It can cause invasive,
chronic and saprophytic forms of disease. While invasive aspergillosis
(IA), the most common and feared clinical presentation, affects
immunosuppressed patients, chronic and saprophytic aspergillosis
complicates those with pre-existing pulmonary or sinus disease. For
their treatment, azoles are used and voriconazole is the first-line
drug. However, in recent years, Aspergillus fumigatus acquired azoles
resistance has been increasingly reported, especially in some Euro-
pean countries, Asia and Middle East. In contrast the resistance
seems not to affect the USA.
On both sides of the Atlantic Ocean, two antifungal susceptibility
testing guidelines for molds, using broth methodology, are used. In
USA the M38-A2 protocol, proposed by the Clinical and Laboratory
Standard Institute (CLSI) and in Europe the Antifungal Subcommittee
of the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) protocol. The objective of our work was to test Aspergillus
fumigatus strains isolated in patients from Centro Hospitalar S. Jo~ ao,
Porto, Portugal employing both methods and verify their
susceptibilities.
Material and methods A total 49 Aspergillus fumigatus strains, iso-
lated mostly from respiratory samples, between 2010 and 2014,
belonging to 47 patients (21 men and 26 women) with a mean age
guilliermondii (ATCC 6260), C. krusei (ATCC 6258) and C. tropicalis of 55.7 years old (6 months-83 years-old), were tested using the
(ATCC 13803). Antimicrobial activity of Hamamelis virginiana glycolic M38-A2 and EUCAST protocols. The azoles itraconozale, voriconazole
extract was evaluated by microdilution method based on Clinical and and posaconazole, the echinocandins, caspofungin and anidulafungin
Laboratory Standards Institute (M7-A6 and M27-A2) in order to and the polyenes amphotericin B and liposomal amphotericin B were
obtain the minimum inhibitory concentration (MIC) and minimum the antifungal drugs used. We strictly followed the methodological
fungicidal concentration (MFC). Biofilms were formed in microtiter recommendations: for the azoles and amphotericin we read the mini-
plates with suspensions of 107 cells ml
1 and incubation at 37°C mal inhibitory concentration (MIC) and for the echinocandins the
under stirring for 48 hours. Then the biofilms were treated with the minimum effective concentration (MEC). Also to establish the pres-
extract for 5 min with different concentrations for each species. Sal- ence or absence of non-wild-type strains among our isolates we con-
ine was used as control. Biofilms were disaggregated and the decimal sidered the proposed epidemiological cutoff values (ECV).
dilutions were plated on Sabouraud Dextrose agar by drop-plate tech- Results MICs and MECs ranges for each drug/protocol combination,
nique, and incubated at 37 °C for 48 hours. Next, the counting of the range of the essential agreement (EA) between the methods and
colony forming units (CFU ml
1) was performed. Data were com- the EA, considering one/two/three dilutions, are depicted in the table
pared by ANOVA and Tukey test (p ≤ 0.05). Cytotoxicity analyses 1. While for most antifungals the EAs were high, for posaconazole
was performed using the MTT colorimetric method after contact and caspofungin they were quite lower.
between the extract and mouse macrophages cultures (RAW 264.7) Considering the ECV, only one strain of Aspergillus fumigatus exhib-
for 5 min. The absorbance of the wells was read on microplate ited a MIC of 4 mg l
1 for both amphotericin formulations using
reader with wavelength of 570 nm. The optical densities (OD) EUCAST protocol. However, for the same strain, the MIC was
obtained were converted into percentage of cell viability. 2 mg l
1 employing CLSI guidelines. Another strain revealed high
Results MIC and MFC values demonstrated greater resistance for C. MICs to the azoles by both methods (itra/CLSI>16 mg l
1; itra/
albicans and C. glabrata to the concentrations tested in planktonic EUCAST>8 mg l
1; vori/CLSI = 4 mg/L; vori/EUCAST = 4 mg l
1;
growth, MIC = 1.5 mg ml
1 and CFM = 6.2 mg ml
1 for both spe- posa/CLSI = 2 mg l
1; posa/EUCAST = 1 mg l
1).
cies (Table 1). For biofilm growth, the extract was required in higher Conclusions Both methods exhibited good performance, except for
concentrations to obtain a statistically significant reduction (Table 2). posaconazole and caspofungin EAs assessment. More studies are
The extract was not cytotoxic until the concentration of 5%, when likely to provide insights on this point. Furthermore, the difference of
the cell viability was 50.58%. MIC value for amphotericin between EUCAST and CLSI protocols
Conclusions It was concluded that H. virginiana glycolic extract should be investigated. For the azoles resistant strain, both methods
showed antifungal activity against the strains tested, however higher
concentrations were required against biofilm growth. The extract
was not cytotoxic until the highest concentrations, that showed
fungicidal activity against biofilm growth.

ª 2015 The Authors

66 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

performed well. The mechanism of resistance involved in this strain surgery debridement is the most used. However, the high rate of re-oc-
will be investigated. currence is seen. Methylene blue is an aromatic chemical compound.
This work was supported by Grant IN-PT-131-1755 from Gilead Because of low toxicity toward human, it has been used for various
Sciences. clinical purposes including methemoglobinemia, malaria treatment as
well as inactivation of some microorganisms. This study was initiated
to assess the in vitro inhibitory effect of methylene blue on the hyphae
growth of 10 clinically isolated P. insidiosum comparing with growth
P034 rate on media without methylene blue. Various concentrations of
methylene blue ranging from 0.1 to 20 lg ml
1 were tested. The
result revealed that inhibition rate was dose-dependent. Significant
In vitro susceptibility profiles of eight antifungal drugs inhibition rates (85.6  3.9 and 76.5  8.9%) were observed in TSA
against clinical and environmental species of media supplemented with methylene blue at concentration of 20 and
Phaeoacremonium 10 lg ml
1, respectively. In contrast, 0.1 lg ml
1 of methylene blue
H. Badali,1 S. Khodavaisy,2 H. Fakhim,1 G. S. de Hoog,3 concentrations showed minimal effect on the growth rate while
A. Chowdhary4 and J. F. Meis5 approximately 30% inhibition was observed in 1 and 2 lg ml
1 of
1 methylene blue. In addition, slightly decrease of inhibition effect was
Mazandaran University of Medical Sciences, Sari, Iran; 2Tehran observed when tested on blood agar. The results demonstrated that
University of Medical Sciences, Tehran, Iran; 3CBS-KNAW Fungal methylene blue had promising inhibitory effect to the growth of P.
Biodiversity Centre, Utrecht, the Netherlands; 4University of Delhi insidiosum. This agent may be a good candidate for treatment either
- Vallabhbai Patel Chest Institute, Delhi, India and 5Canisius alone or in combination with surgical therapy.
Wilhelmina Hospital and Radboud University Hospital, Nijmegen,
the Netherlands

Background Laboratory diagnosis and treatment of Phaeoacremo- P036

nium infection is problematic and not well defined. The purpose of
this study was in vitro testing of a large collection of Phaeoacremo-
nium strains to eight antifungal drugs, including the novel triazole
Environmental isolates of Hortaea werneckii: enzymatic
isavuconazole. activity and antifungal susceptibility profile
Methods Phaeoacremonium strains (n = 43) from different geographi-  n,1 C. R. Felix,2
D. Heidrich,1 A. Formoso,1 M. Ramırez-Castrillo
cal regions were obtained from the collection of the CBS-KNAW Fungal 
A. C. Tenorio, B. R. Leite, D. M. Pagani, M. F. Landell,2
2 1 1

Biodiversity Centre, Utrecht, the Netherlands, comprising P. para- K. O. Alves,1 Z. M. M. Andrade,1 P. T. Dalbem,1
siticum (n = 16), P. krajdenii (n = 11), P. venezuelense (n = 6), P. infla- M. L. Scroferneker1 and P. Valente1
tipes (n = 3), P. griseorubrum (n = 3), P. rubrigenum (n = 2), and P. 1
alvesii (n = 2). The fungi were identified to the species level by sequenc- Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
ing of the internal transcribed spacer regions of the rDNA region and and 2Universidade Federal de Alagoas, Alagoas, Brazil
partial b-tubulin gene (19, 30–32). In vitro susceptibility was deter-
mined as described in CLSI document M38-A2 for eight drugs. Objectives To evaluate the antifungal activity and virulence factors
Results All strains had low MICs of amphotericin B, voriconazole, through the enzymatic activity of four environmental isolates of Hor-
posaconazole and isavuconazole. Less active drugs were fluconazole, taea werneckii that were isolated from different substrates in Brazil
itraconazole, anidulafungin, and caspofungin. The widest ranges and due to the pathogenic nature of the fungus. Hortaea werneckii is the
the highest MICs were seen for fluconazole and itraconazole etiologic agent of Tinea nigra, an uncommon superficial
(range16- > 64 mg l
1 and 4–16 mg l, respectively). The echinocan- dermatomycosis.
dins had poor activity against both clinical and environmental Methods Two strains were isolated from the salt marsh macrophyte
strains. The geometric mean MICs were as follow in increasing order; Spartina alterniflora in Southern Brazil and two were isolated in
voriconazole (0.35 mg l
1), posaconazole (0.37 mg l
1), ampho- Northern Brazil, one from the bromeliad Portea leptantha and another
tericin B (0.4 mg l
1), isavuconazole (1.1 mg l
1, anidulafungin from the marine zoanthid Palythoa caribaeorum. The identification of
(4.8 mg l
1), caspofungin (6.9 mg l
1), itraconazole (14.8 mg l
1), the strains was confirmed by sequencing of the ITS region or D1/D2
and fluconazole (43.5 mg l
1). domains of the LSU rRNA gene using the universal primers ITS1/
Conclusions Amphotericin B, voriconazole, posaconazole and isavu- ITS4 and NL1/NL4, respectively. The activities of the following
conazole were active against Phaeoacremonium. In contrast all isolates enzymes were tested: proteinase (gelatinase, albuminase and kerati-
were resistant to itraconazole and fluconazole. This susceptibility pro- nase), esterase, lipase, phospholipase, DNase and urease. The samples
file is similar to other melanized fungi. were inoculated into tubes (for evaluation of keratinase and urease)
or at the center of a Petri dish (for the other enzymes). Tests were
performed in triplicate and incubated at 30°C for 14 days. The enzy-
matic activity (Pz) in the plates was determined by the evaluation of
P035 the degradation halo, and the strains were classified as: non-produc-
ers (Pz = 1.0) and producers (PZ<1).The minimal inhibitory concen-
tration (MIC) of 8 antifungal agents was evaluated in microdilution
Inhibition of Pythium insidiosum growth by methylene methods in 98 well plates according to the CLSI protocol M27-A3
blue adapted for H. werneckii (30°C/6 days) and the minimal fungicidal
P. On-Paew,1 T. Chadlane1 and P. Santanirand2 concentration (MFC) were evaluated in Sabouraud dextrose broth
1
Microbiology Laboratory, Department of Pathology, medium in tubes from the wells with no growth.
Results Lipase (Pz medium = 0.365  0.070), esterase (Pz
Ramathibodi Hospital, Bangkok, Thailand and 2Ramathibodi
medium = 0.695  0.074) and urease were positive for all isolates,
Hospital, Bangkok, Thailand
while one strain was positive for gelatinase (Pz = 0.63). All isolates
were negative for albuminase, keratinase, phospholipase and DNase.
Pythium insidiosum is an oomycete pathogenic which causes pythiosis, The MIC/MFC medium obtained were (lg ml
1): tioconazole (0.073/
a life-threatening disease. The infection usually occurs via direct con- 0.210), posaconazole (0.088/0.105), ketoconazole (0.148/0.210),
tact with contaminated fresh water. Although the animal cases have voriconazole (0.176/0.297), terbinafine (0.177/0.177), itraconazole
been reported in various countries, the vast majority of human cases (0.25/1.0), amphotericin B (2.0/4.0) and fluconazole (>64/>64). All
were described only in Thailand. Treatment using antimycotic agents isolates were considered resistant to fluconazole.
is ineffective due to the fact that P. insidiosum is not true fungi. Radical

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 67
Poster Presentations

Conclusion The environmental isolates of Hortaea werneckii pro- vulvovaginal candidiasis (RVVC) which is defined as >3 episodes per
duced some enzymes important as potential virulence factors. The year. Several risk factors are known, such as pregnancy, immunos-
isolates were resistant to fluconazole, and presented low sensitivity to suppression, antibiotic use, AIDS, Innate immunodeficiency and dia-
amphotericin B, showing that resistance to these antifungal agents betes may increase the susceptibility to VVC.
can be found in environmental isolates of H. werneckii, therefore clin- Objective The study undertaken to evaluate clinical, epidemiological
ical administration of fluconazole should be avoided, and the use of and laboratory findings among patients with RVVC, and to analyze the
amphotericin B in severe cases of infection by this fungus should be frequency of different Candida species and their susceptibility profile.
viewed with care. Methods Fifty five women with RVVC from the infectious diseases
service from Hospital Universit ario do Oeste do Paran a (Cascavel/PR/
Brazil) were studied from January 2012- December 2013. Vaginal
swabs were collected and cultured on Sabouraud’s dextrose agar at
P037 35°C and isolates were identified by morphological and biochemical
tests. Antifungal susceptibility of the Candida spp. isolates were deter-
mined by E-test against amphotericin B (AMB), itraconazole (TTZ),
Antifungal susceptibility testing by micro-broth dilution of fluconazole (FCZ), and voriconazole (VCZ), according to CLSI M27
rare Candida species isolated from blood.-a study from a A2 and M27 A3 breakpoints. The following risk factors were studied:
tertiary care center in South India pregnancy, hormonal contraception, corticosteroids, sexually trans-
A. J. Kindo, A. Sivaranjini, V. Rajyoganandh and R. Vijayakumar mitted diseases (STD), HIV infection, diabetes and the previous use of
antibiotics and antifungal agents. Probable external risk factors such
Sri Ramachandra University, Chennai, India
as vaginal douches, use of female intimate soap and use of synthetic
clothes were also investigated. Statistical analysis was performed
Objective Candida species that are closely related to C. haemulonii using the x2 test and p < 0.05 was considered statically significant.
are emerging sources of infection in India. These species show vari- Results Fifty five women were selected with possibility to collect
able patterns of susceptibility to amphotericin B,echinocandins and vaginal specimens although only 24 had positive cultures. The mean
azole antifungal agents. We performed the antifungal susceptibility age was 32 years (range 15–51 year) and 64, 8% were married and
testing to see the resistance pattern of these unusual candida species 90, 7% live in urban area. Only 29% of the women in this study
and to compare the pattern among the common species. gain >3 minimum wage (U$738). The following risk factors were
Methods The microbroth dilution testing was done using CLSI statistic significant by x2 test: vaginal douche (P < 0.0001); preg-
guidelines M27-A3. The list of drugs were amphotericin B, caspo- nancy (P < 0.0001), DST (P < 0.0001), corticosteroids (P < 0.0001)
fungin, micafungin, anidulafungin, fluconazole, voriconazole, and recent antibiotic use (P = 0.0029). The most common species
posaconazole and,itraconazole. isolated in this cohort was C. albicans (67, 85%), C. tropicalis (10,
Results We got 8 rare blood Candida isolates (apart from the com- 71%), C. parapsilosis (7, 14%), C. guillermondi (7, 14%) and C. glab-
mon isolates C.albicans 9, C.tropicalis 20, and C.parapsilosis 9) of rata (7, 14%). All the strains were susceptible to AMB, 92, 5% sus-
which C.haemulonii was 3 C. famata was 3 which were identified by ceptible to FCZ, 76, 9% susceptible to ITZ and 87, 5% to VCZ.
Bio-merieux VITEK -2 compaq and 2 isolates were confirmed by gene
sequencing as C. auris. All cases of fungemia occurred in patients
with severe underlying diseases who had central venous catheters.
Four patients had undergone surgery during the hospital stay. One P039
patient was on steroids, one was on chemotherapy for laryngeal car-
cinoma. Other patients were on long term broad spectrum antibiotics
for various bacterial infections. Invasive candidiasis in Pakistan: Predominant Species and
Among the eight patients from whom rare Candida was isolated Antifungal Resistance (2010–2014)
four patients improved on treatment while four succumbed to the J. Q. Farooqi,1 A. Zafar,1 K. Jabeen,1 R. Mahboob,1 M. Rawala,1
infection. A. Longi,1 A. Deedarali,1 F. Malik,1 S. R. Lockhart2 and
The MIC ranges for the rare candida isolates were 32–64 lg ml
1
M. Brandt2
for fluconazole,0.06–0.5 lg ml
1 for voriconazole, 0.125– 1
1.0 lg ml
1 for Itraconazole, .06–0.25 lg ml
1 for posaconazole, Aga Khan University, Karachi, Pakistan and 2Centers for Disease

1
1
0.5–4 lg ml for AmphotericinB, 0.125–0.25 lg ml for Caspo- Control and Prevention, Atlanta, Georgia, USA
fungin, 0.03–0.125 lg ml
1 for Micafungin and 0.06–

1
0.125 lg ml for anidulafungin Objectives This study reports descriptive epidemiologic data of 753
Conclusion It is very important to speciate the Candida isolated Candida spp isolated from blood and other sterile specimens in Pak-
from blood due to the emergence of newer Candia species which istan (2010–2014), including species identification and antifungal
have high MIC’s to fluconazole and amphotericin B. susceptibility against fluconazole, itraconazole, voriconazole, caspo-
The treatment should be started with one of the echinocandins in fungin, micafungin, anidulafungin and amphotericin. Antifungal
order to prevent mortality from fungemia in the ICU setting. resistance data was compared with a previous invasive candidiasis
survey (2006–2009) published from same center.
Methods The study was conducted at the Aga Khan Hospital Clinical
Laboratories from Jan 2010 to Oct 2014. The isolates were identified
P038 by biochemical characteristics (API 20C AUX BioMerieux, France)
and microscopic morphology. Antifungal susceptibilities were deter-
mined by disc diffusion and MICs on broth microdilution (BMD) as rec-
Clinical, Epidemiological and laboratory findings amoung ommended by CLSI M44-A2 (2009) and M27-A4 (2012), respectively.
women with recurrent vulvovaginal candidiasis (RVVC) in Results Candida tropicalis remained the most common species (36%),
an university hospital, Brazil followed by C. albicans (25%) and C. parapsilosis (22%) respectively.
C. S. Oliveira,1 R. Gandra,1 E. A. Loth1 and Q. F. Telles-Filho2 11% of the infections were due to uncommon Candida species. Isola-
1
Hospital Universitario do Oeste do Parana, Cascavel, Brazil and tion rate of C. glabrata and C. krusei increased from 12.7% (2006–
2
Universidade Federal do Parana, Curitiba, Brazil 2009) to 15.2%. Resistance to fluconazole emerged in C. albicans
(8.0%), C. tropicalis (3.1%)and C. parapsilosis (4.6%). When compared
to previous survey (2006–2009), overall fluconazole resistance
Introduction Vulvovaginal candidiasis (VVC) is a frequent disease increased from 2% to 7.7%.
affecting more than 75% of all women at least once in their lifetime. Conclusion Increasing rates of fluconazole resistant species as well
Approximately 50% of these women will also suffer a single recur- as emergence of fluconazole resistance in previously sensitive Candida
rence. A minority of women, 5–8% experience recurrent

ª 2015 The Authors

68 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

species is concerning. There is an urgent need to develop expertise in P041

both identification and susceptibility testing in laboratories in Pak-
istan. Ongoing surveillance is essential to monitor resistance and any
emerging species. Susceptibility testing of Malassezia furfur, Malassezia
globosa and Malassezia sympodialis. Comparison of disk
diffusion and broth microdilution methods
F. Rojas,1 L. C. Zalazar,2 M. S. Fernandez,1 M. A. Sosa,1
P040 M. E. Cattana,3 L. R. Alegre,1 A. J. Carillo Mun~oz4 and
1
G. E. Giusiano
1
Species distribution and In Vitro and Antifungal Instituto de Medicina Regional- Universidad Nacional del
Susceptibility in Clinically Important Candida Isolated from Nordeste, Resistencia, Argentina; 2Facultad de Humanidades -
Oral Cavity Universidad Nacional del Nordeste, Resistencia, Argentina;
3
P. Andreola,1 J.F. Cano,2 R. Elsemann,3 A. Demathe,3 Instituto de Medicina Regional-UNNE, Resistencia, Argentina
D. Galafassi,1 E. Elsemann1 and A. Gazzoni1 and 4ACIAM, Barcelona, Spain
1
Serra Gaucha Faculty, Caxias do Sul, Brazil; 2Medical School and
IISPV, Reus, Spain and 3School of Dentistry, Caxias do Sul, Brazil Objectives The aims of this study were to evaluate the antifungal
susceptibility of three Malassezia species against fluconazole,
voriconazole, ketoconazole, itraconazole, miconazole, amphotericin B
Objectives Aims of this study were: (a) To evaluated species distri- and terbinafine using both broth microdilution and agar diffusion
bution of isolates of Candida sampled from the oral cavity; (b) To methods, and to compare both methodologies using a model of linear
determined the in vitro susceptibilities to four antifungal agents (nys- regression analysis and categorical agreement.
tatin, fluconazole, itraconazole, voriconazole) in Brazilian strains of Methods A total of 50 Malassezia isolates, including 30 M. furfur,
Candida spp. isolated from oral cavity. 10 M. globosa and 10 M. sympodialis, were studied. Yeasts were iden-
Methods The species-level identification was performed according to tified by PCR-RFLP. The broth microdilution method was performed
standard procedures, i.e., germ tube test, colony morphology on as described in CLSI M27-A3 reference document and disk diffusion
Sabouraud Dextrose Agar (Difco, France) and Chromogenic test was performed on Mueller-Hinton agar using tablets and disks.
CHROMagar Candida medium (Difco, USA). The in vitro activities of To support optimal growth of these yeasts, culture media were sup-
nystatin, fluconazol, itraconazol, voriconazol against 28 oral Candida plemented. In order to correlate both methods, linear regression anal-
were tested by disk diffusion. Reference antifungal susceptibility test- ysis was performed and categorical agreement was determined.
ing was performed exactly as described in Clinical and Laboratory Results Both supplemented media allowed optimal growth of Malas-
Standards Institute Guidelines (CLSI), document M44-A2. Two qual- sezia yeasts and the results were read at 72 h of incubation at 32°C.
ity control (QC) strains, C. albicans (ATTCC 90028) and C. parapsilosis Results of broth microdilution and agar diffusion testsobteined for the
(ATCC 22019) were tested each time a set of clinical isolates was 50 isolates studied are summarized in Table 1.
evaluated. Isolates were classified by zone interpretative criteria Malassezia yeasts were highly susceptible to itraconazole, ketocona-
according to current CLSI Guidelines, document M44-S2. Collected zole and voriconazole. In contrast, a significant dispersion of values
data were analyzed using GraphPad 5.0 for Windows. Candida species was observed for fluconazole, miconazol and terbinafine.
identification and in vitro susceptibilities were done by the Oral The strongest linear association was observed for FLC. The rela-
Microbiology and Pathology Testing Service Laboratory at the School tionship between variables for KTZ and ITZ was not linear. For the
of Dentistry of the Serra Ga ucha Faculty. This study was conducted other drugs, coefficients obtained were not enough to conclude that
according to the principles expressed in the Declaration of Helsinki. It a linear relationship exists between MIC and inhibition zone diame-
was approved by CONEP (National Committee for Research Ethics). ters variables.
Results In this group study, C. albicans was isolated with the highest The highest categorical agreement between both methods was
prevalence followed by C. tropicalis and C. krusei with isolation rates of observed for itraconazole and voriconazole and the lowest for ampho-
72%, 25% and 3% respectively. All clinical isolates Candida albicans tericin and fluconazole.
were fully susceptible for all antifungals. Of the non-albicans Candida Table 2 indicates results of linear correlation analysis and categori-
species isolates, all C. tropicalis strains showed an intermediate suscep- cal agreements when comparing both methodologies.
tibility to fluconazole. This isolates were recovered from patients with Conclusions Susceptibility profiles of Malassezia yeasts against FLC,
prior antifungal exposure to nystatin and triazole-derive agent (flu- AMB and TRB were variable and this is very important considering
conazole). As expected, resistance to fluconazole was displayed in C. that are commonly used drugs. On the other hand, isolates were
krusei isolate. For nystatin, eight clinical isolates were resistant, includ- inhibited at low concentrations of ITZ, KTZ and VCZ, showing to be
ing one C. krusei and seven C. tropicalis. None of the Candida species in highly effective drugs.
our study showed cross-resistance to itraconazole and voriconazole. Although modifications made to both methodologies allowed
Conclusion As expected, we found that the resistance rates for Can- obtaining susceptibility data for Malassezia yeasts, both methods can-
dida non-albicans were higher than those for C. albicans, in particular not be associated through linear correlation model. When analysis
C. tropicalis and C. krusei, which displayed resistance rates of 25%
and 3%, respectively. Our study indicates that despite the widespread
use of fluconazole in Brazil, resistance to this antifungal drug among Table 1. Susceptibility results obtained by broth microdilution and
yeasts isolates from oral cavity is still a fairly restricted phenomenon disk diffusion methods for the 50 isolates tested.
(3%) with 97% of all yeasts isolated being susceptible or susceptible
dose-dependent.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 69
Poster Presentations

Table 2. Linear correlation coefficients and categorical agreements (4.8%) minor errors on disk diffusion and 4 (13.3%) minor errors on
obtained from comparing broth microdilution and disk diffusion E-test.
methods Conclusion Thus, antifungal disk diffusion directly from blood cul-
ture bottles is a rapid and easy method for fluconazole and voricona-
zole susceptibility testing for timely tailoring of candidemia therapy.

P044

Absence of FKS1 and FKS2 mutations in Caspofungin

resistant C. glabrata isolates from candidemia patients in
Delhi, India
A. Prakash,1 A. Singh2 and A. Chowdhary3
1
Vallabhbhai Patel Chest Institute, Univ.of Delhi, Delhi, India;
2
Vallabhbhai Patel Chest Institute, Delhi, India and 3University of
Delhi - Vallabhbai Patel Chest Institute, Delhi, India

Objectives Candida glabrata is the second leading cause of can-

was applied, the determination coefficient values obtained were very didemia in the India. Due to the decreased susceptibility of C. glabrata
low for all drugs test, indicating that based on the diameter of the to fluconazole and cross-resistance to other azoles, echinocandins are
inhibition zone there is a low probability of minimal concentration introduced for therapy in patients with C. glabrata candidemia.
inhibitory prediction using a linear model. Recently, many reports have emerged with increasing number of
The agar diffusion method is a low-cost, fast and simple technique echinocandin resistant C. glabrata isolates. Resistance to echinocan-
that is easily applicable in routine clinical laboratories. The diameters dins is attributed to alteration in the subunits of 1,3-b-D glucan syn-
of the inhibition zones were clearly defined when modifications were thase enzyme encoded by three genes (FKS1, FKS2 and FKS3). The
made in agar diffusion method for Malassezia susceptibility study. present study aimed to screen the FKS1 and FKS2 hotspot region for
However, not a good correlation between this technique and the mutation in the caspofungin resistant (MIC ≥0.5 lg ml
1) Indian C.
CLSI reference method was obtained for evaluating antifungal sus- glabrata isolates.
ceptibility of Malassezia yeasts. This highlights the need for to develop Methods A total of 36 Candida glabrata isolates originating mainly
a reference protocol for Malassezia susceptibility testing in order to from candidemia (n = 33, 92%) and bronchoalveolar lavage (n = 3,
obtain comparable results by reproducible methods. 8%) of individual patients in Delhi, India were analyzed. All isolates
were preliminarily identified based on colony colour on CHROMa-
garTM Candida medium and morphology on rice Tween 80 agar. Fur-
ther all of the isolates were molecularly characterized by
amplification and sequencing of ITS and D1/D2 regions and by
P042 Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spec-
trometry (MALDI-TOF MS). Antifungal susceptibility testing (AFST)
Agreement of the direct antifungal susceptibility testing was performed for azoles, amphotericin B and echinocandins with
from positive blood culture bottles with conventional broth microdilution method (CLSI M27-A3). Isolates with elevated
method for Candida species caspofungin MICs ≥ 0.5 lg ml
1 were subjected to sequencing of
FKS1 and FKS2 hotspot regions for mutation analysis.
J. Q. Farooqi,1 K. Jabeen,1 H. Kumar,1 R. Mahboob,1 M. Brandt2
Results All isolates developed a pink color on CHROMagar[TRADE-
and A. Zafar1 MARK] Candida medium and showed ovoid to elongated budding
1
Aga Khan University, Karachi, Pakistan and 2Centers for Disease yeast cells occurring singly or in pairs. Further ITS and D1/D2
Control and Prevention, Atlanta, Georgia, USA sequences of all the isolates showed ≥99% homology with C. glabrata
in GenBank. The MALDI-TOF yielded a score values of >2 for all C.
glabrata. AFST showed that among azoles, posaconazole had excellent
Objective Early availability of antifungal susceptibilities can ensure
activity (MIC90, 0.25 lg ml
1) followed by itraconazole and
early institution of targeted therapy in candidemia which can
voriconazole (MIC90, 0.5 lg ml
1). No resistance to AMB (MIC
improve patient outcomes. This study prospectively determines agree-
range, 0.25–1 lg ml
1) was observed. Among echinocandins, 24
ment between direct testing of antifungal susceptibilities from blood
(66.6%) blood isolates were found to be resistant to caspofungin
culture bottles by disk diffusion and E-test strips compared to stan-
(MIC, ≥0.5 lg ml
1), whereas 3 fell in susceptible dose dependent
dardized susceptibility testing methods allowing susceptibility results
(MIC, 0.25 lg ml
1). The remaing 9 isolates had caspofungin MIC
to be available 1–2 days earlier.
range 0.06–0.125 lg ml
1. However, all isolates were highly suscep-
Methods A total of 104 blood cultures with different Candida species
tible to other two echinocandins, micafungin (MIC90, 0.015 lg ml
1)
(28% C. albicans, 27% C. parapsilosis, 26% C. tropicalis, etc.) between
and anidulafungin (MIC90, 0.125 lg ml
1). No mutation was seen
Jan 2012 and May 2013 were evaluated for agreement of flucona-
in both FKS1 and FKS2 hotspot regions in any of the resistant
zole, voriconazole and amphotericin susceptibility results by disk dif-
(≥0.5 lg ml
1) C. glabrata isolates.
fusion. Agreement in MICs by E-test was determined for fluconazole
Conclusions The study is first to report the screening of FKS1 and
(21 isolates), voriconazole (28 isolates), amphotericin (29 isolates)
FKS2 hotspot regions in Indian C. glabrata isolates. No mutation was
and caspofungin (29 isolates).
observed in any of the hotspot region in caspofungin resistant C.
Results Kappa scores for categorical agreement were highest for flu-
glabrata isolates (0.5–1 lg ml
1) warranting studies on larger num-
conazole by disk (0.902, SE = 0.076) and E-test (1.00, SE = 0.218)
ber of C. glabrata isolates to determine true prevalence of caspofungin
and amphotericin by disk (1.00, SE = 0.098). Pearson correlation (r)
resistant isolates harboring FKS1 and FKS2 mutations.
of zone diameters was strongest for fluconazole (0.69) and ampho-
tericin (0.70), moderate for voriconazole (0.60); and strongest for flu-
conazole (0.94) and caspofungin (0.88) MICs. However moderate
correlation of amphotericin MICs with zone diameters (-0.42) pre-
cludes the use of amphotericin disk for susceptibility testing. There
were no very major errors; however there was 1 (1%) major and 5

ª 2015 The Authors

70 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P045 have encouraged the search for new compounds with activity
against H. capsulatum. Aldimines, also known as Schiff bases, are
compounds containing an azomethine group (-CH=N-) formed by the
Aspergillus fumigatus resistance surveillance in a tertiary condensation of a primary amine with a carbonyl compound. These
teaching Hospital compounds have been shown to exhibit a broad range of biological
F. Reichert-Lima, L. Lyra, M. L. Moretti and A. Z. Schreiber activities, including antifungal, antibacterial, antimalarial, antiprolif-
State University of Campinas, Campinas, Brazil erative, anti-inflammatory, antiviral, and antipyretic properties. This
research aimed to determine the minimal inhibitory concentration of
aldiminines against Histoplasma capsulatum, the causative agent of
Aspergillus spp. are opportunistic fungal pathogens responsible for histoplasmosis.
high mortality rates, especially in immunocompromised patients, Methods It was determined the minimum inhibitory concentration
mainly neutropenic hosts who become infected after inhaling conidia (MIC) of twenty-four aldimines against five strains of H. capsulatum
dispersed in the air. Primary therapy for invasive aspergillosis has var. capsulatum. The tests were performed according modifications of
been largely limited to amphotericin B (AMB) and the triazole com- the M 38-A2 protocol drawn up by the Clinical and Laboratory Stan-
pounds itraconazole (ITZ) and voriconazole (VRZ). As the use of dards Institute (CLSI). Results: There was wide variation in the anti-
AMB has been limited by its toxic side effects, the azole derivate VRZ fungal activity of the tested aldimines. The geometric means of the
is been considered the first line therapy on aspergillosis treatment. A.- MIC varied from 15.06 lg ml
1 (3976 strain) to 8.23 lg ml
1
fumigatus, the most common specie causing aspergillosis, has been (3962 strain). In general, the aldimines 9, 13, 15 and 20 were the
investigated in several countries because of the emergence of azole most activite against the tested strains, with MIC values that ranged
resistant strains. The University Hospital of Campinas (HC-UNICAMP) from 2 and 8 lg ml
1.
is a tertiary care teaching institution where many high-risk patients Conclusion Some of the tested aldimines have exhibited excellent
are susceptible to opportunistic infectious agents such as the Aspergil- in vitro activity against H. capsulatum, making them potential candi-
lus genus. In our institution, until now, there are no reports of A.fu- dates to future studies on their in vivo activity.
migatus multi azole resistance. However, the monitoring of the Financial support: CAPES, CNPq, FAPEMIG
possible emergence of resistant strains is needed. In this context, this
study aimed to assess the profile of susceptibility to antifungal agents
for clinical A.fumigatus isolated from patients with aspergillosis
assisted at the HC-UNICAMP. Hundred and seventy-two clinical iso-
lates, from 75 patients (2 to 6 from each patient) were evaluated
P047
searching for resistant isolates. Minimal Inhibitory Concentration
(MIC) for amphotericin B (AMB); itraconazole (ITZ); voriconazole Antifungal activity of allylimines alone and in combination
(VRZ); 5-flucytosine (5FC); miconazole (MCZ), fluconazole (FCZ) and with amphotericin B against Cryptococcus gattii strains
the Minimal Effective Concentration (MEC) for micafungin (MCF); M. Resende-Stoianoff,1 T. F. F. Magalhaes,1 C. Silva,1 D. Silva,1
and caspofungin (CPF) were determined by microdilution method as
S. Q. Almeida,1 G. Freitas,1 R. R. Silveira,1 C. Martins,2
recommended by the Clinical and Laboratory Standards Institute (CLSI,
2008) document M38-A2, with minor changes. Molecular analyses A. Fatima1 and D. Santos1
1
allowed to confirm the identification of all A.fumigatus isolates. The Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
MIC ranges (in lg ml
1) were: AMB: 0.25 to 8; 5FC:0.25 to 64; and 2Universidade Estadual Oeste do Parana, Toledo Pr, Brazil
FCZ: 2 to >64; ITZ: 0.25 to 4; VRZ: 0.25 to 8 and MCZ: 0.06 to 4.
The MEC ranges (in lg ml
1) were ≤ 0.015 to 0.06 for MCF and
Objectives Cryptococcosis is an invasive mycosis of global occur-
0.06 to 0.5 for CPF. Ten isolates from 9 patients showed high MICs
rence, mainly caused by Cryptococcus neoformans and C. gattii and
for VRZ and; or ITZ (1 only for ITZ, 7 only for VRZ and 2 for ITZ
affects patients both immunocompromised as immunocompetent. Its
and VRZ). Now, additional studies involving the search of mutations
treatment may be complicated by the toxicity of the drugs used and
are being performed using the cyp51A gene sequencing method.
the emergence of resistant strains. The discovery of new antifungal
Accurate and reliable species identification are important for appro-
compounds and the combination therapy are important tools for the
priate patient treatment, management, and development of hospital
effective treatment of fungal infections. The allylimines exhibit similar
infection control policies. Besides that, monitoring of the possible
structures to the antifungal group of allylamines, containing one
emergence of resistant strains is extremely important due to high
double bond next to the nitrogen atom present in the molecule.
mortality of azole-resistant aspergillosis that can approach 90%.
Methods In the present work we evaluated the in vitro antifungal
activity of three allylimines (A1, A2, A3) alone and in combination
with amphotericin B (AMB) against 12 Cryptococcus gattii strains.
The compounds were evaluated for minimum inhibitory concentra-
P046 tion (MIC), minimal fungicidal concentration (MFC) and the checker-
board assay was used to determinate the fractional inhibitory
concentration index (FIC).
Antifungal activity of aldimines (Schiff bases) against
Results All strains were susceptible to the tested compounds with
Histoplasma capsulatum
MIC values between 2.3 and 7.5 lg ml
1 and MFC values between
M. Resende-Stoianoff,1 D. Silva,1 T. F. F. Magalhaes,1 4.8 and 65.8 lg ml
1. For most of the tested strains the interaction
R. R. Silveira,1 S. Q. Almeida,1 A. Fatima,1 C. Silva1 and of A1 and A2 with AMB was indifferent (0.5 > FIC≤4). The com-
R. C. Hahn2 pound A3, however, was able to interact synergistically with AMB
1
Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (FIC<≤0.5) for one-third of the C. gattii strains tested. Although the
and 2UFMT, Cuiaba, Brazil allylimines A1 and A2 had the best MIC and MFC values, A3
showed better interaction with AMB against the strains tested.
Conclusions Considering the results obtained in this study these
Objectives Histoplasma capsulatum var. capsulatum is the causative allylimines may be considered promising antifungal agents and fur-
agent of histoplasmosis, a systemic mycosis endemic in extensive ther studies will be done in our research group.
areas of the Americas. Clinical manifestations of histoplasmosis are Financial support: CAPES, CNPq, FAPEMIG.
influenced on both by the intensity of exposure to fungal microconi-
dia, as well by the host immunity, and range from asymptomatic to
disseminated infections. Treatment depends on clinical manifesta-
tions, and intolerance to treatment and drug-drug interactions may
occur and lead to failure in antifungal therapy. The restricted alter-
natives and the therapeutic complications for treating histoplasmosis

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 71
Poster Presentations

P048 P049

Comparative genomic and transcriptomic analysis unveil Identification and determination of Drug Resistant
novel features of antifungal resistance in the human Candida species isolated from hospital acquired infections
pathogen Candida glabrata K. Diba, A. Chawshin and N. H. Jazani
N. P. Mira,1 S. Salazar,1 T. Pedreira,1 C. A. N. Wang,2 S. Alves,3 Urmia University of Medical Sciences, Urmia, Iran
R. Henriques,4 M. Sousa,3 M. Lopes,5 I. Sa-Correia,1
S. C. Madeira4 and G. Butler2 Objectives Nowadays use of antifungal drugs group Azoles is cres-
1 cent as well as the number of drug resistant yeast and also frequency
Institute for Bioengineering and Biosciences, Lisbon, Portugal;
2 of Candida infections. The aim of this study is isolation and identifi-
School of Biomolecular and Biomedical Sciences, Conway
Institute, Dublin, Ireland; 3Centre of Molecular and cation of Candida species causing hospital acquired infections and
Environmental Biology, Braga, Portugal; 4INESC-ID, Lisbon, study of antifungal resistance.
Portugal and 5Faculdade de Farmacia da Universidade de Lisboa, Methods Specimens were collected from patients with approved
nosocomial infections in the Urmia educational hospital, Urmia, Iran.
Lisbon, Portugal
Primary tests including direct examination and culture were per-
formed. Differential cultures and molecular test: A molecular diag-
Background and objectives Fungal infections caused by Candida nostic method was used for differentiation and identification.
glabrata are associated to high rates of morbidity and mortality. An Antifungal sensitivities were studied by using the Disk diffusion and
alarming increase in the incidence of infections caused by C.glabrata Micro dilution methods. For the analysis of Candida drug resistance
has been reported in the last years, in part, due to the emergence of we focused on erg 3 gene.
strains resistant to azoles and echinocandins. In this work the gen- Results Total of sixty isolates (23.4%) were obtained including Can-
ome sequences of a C.glabrata clinical isolate (named FFUL887) resis- dida albicans 37 (61.6%), C. krusei and C. glabrata 7 (11.6%) each, C.
tant to voriconazole and fluconazole (MICS of 4 and >64 mg l
1, dubliniensis 5 (8.3%) and C. tropicalis 4 (6.6%). Antifungal sensitivity
respectively) and tolerant to caspofungin (MIC of 0.5 mg l
1) and of analysis resulted C. albicans was not considerably resistant to ampho-
the reference strain CBS182, more susceptible to the above-referred tericin B comparing other Candida species in Disk diffusion method.
drugs (MICs of 1, 16 and 0.06 mg l
1, respectively), were compared. Our findings of Micro dilution method confirmed the above results.
Although much work has been gathered on the elucidation of the Conclusion In spite of our findings that show no considerable drug
molecular mechanisms of resistance to antifungals in C.glabrata, little resistance in Candida isolates, monitoring of antifungal resistant Can-
is still known on the genetic adaptive responses that occur at the didaspecies causing hospital acquired infections can be important.
genomic level, a knowledge which is crucial to fully understand how
this pathogenic yeast develops acquired antifungal resistance. The
results herein obtained also contributed to further elucidate mecha-
nisms of adaptation of C. glabrata to the human host advancing cur-
P050
rent understanding of the pathophysiology of this yeast.
Results and Conclusions The genomic sequence determined for
the FFUL887 isolate includes 12.29 Mb, corresponding to 99.1% of Modified response to antifungals in chromoblastomycosis
the total genome size estimated by flow cytometry. Around 80 000 agents with the inhibition of melanin biosynthesis
SNPs were identified, 10 000 of them corresponding to missense D. Heidrich, P. T. Dalbem, K. O. Alves, Z. M. M. Andrade,
mutations occurring in the coding sequence of 3200 genes, corre- C. Silva and M. L. Scroferneker
sponding to roughly 60% of the predicted C.glabrata ORFeome. No
mutations had been found in the sequence of FFUL887 ERG11 gene Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
indicating that the mechanism by which this isolate acquired resis-
tance to azoles is, apparently, not related with target modification. Objectives To evaluate the correlation between the presence of mel-
Around 115 proteins previously associated with drug resistance in anin in chromoblastomycosis agents and their in vitro response to
C.glabrata were found to harbour mutations in the FFUL887 genome. antifungals.
Among these was the CgPDR1 gene, encoding the most important Methods Five isolates (3 Fonsecaea sp., 1 Phialophora verrucosa and 1
transcriptional regulator involved in the control of drug resistance in Cladophialophora carrionii) were inoculated in potato dextrose medium
C. glabrata, which harboured a mutation not previously described. with and without the addition of 16 lg ml
1 of tricyclazole (melanin
Using a transcriptomic analysis it was possible to confirm that the biosynthesis inhibitor). The tubes were incubated at 30°C for 14 days,
FFUL887 isolate over-expresses several described targets of the CgP- and the conidia suspension were counted in a Neubauer chamber, and
dr1 transcription factor including the drug efflux pumps CgCdr1, the final inoculums were standardized to 3–8 x 104 conidia ml
1. The
CgPhd1, CgQdr2 and CgTpo3, all previously demonstrated to con- minimal inhibitory concentration (MIC) of six antifungal agents (itra-
tribute for azole resistance in C. glabrata. These observations, conazole, ketoconazole, posaconazole, voriconazole, terbinafine and
together with phenotypic data, suggest that the herein uncovered amphotericin B) were evaluated by the microdilution method in 98-
mutation is a new gain-of-function mutation of CgPdr1. The influ- well plates according to the CLSI protocol M38-A2, and the minimal
ence exerted by this GOF mutation in the overall C. glabrata tran- fungicidal concentration (MFC) were evaluated in Sabouraud dextrose
scriptional regulatory network of controlled by CgPdr1 will be broth medium in tubes from the wells with no growth. The MIC and
discussed, combining the information gathered in the genomic and MFC tests were also done in media with or without tricyclazole. The
in the transcriptomic analyses performed. The evolution of the non- MIC and MFC of isolates were analyzed in relation to the biological dif-
coding genome of C. glabrata that was observed to occur between ference of their values between tests with or without the addition of tri-
the CBS182 and FFUL887 isolates will also be discussed. Results cyclazole, considering that there was a biological difference whenever
from genomics and transcriptomics show extensive alteration of the the difference was at least two wells up or down.
expression and coding sequence of genes related with adhesion, Results The geometric mean of MIC (with - without tricyclazole)/
metabolism of alternative carbon sources and stress response, con- MFC (with - without tricyclazole) for each species was (lg ml
1): F.
firming that these functions, essential for maximal adaptation to the pedrosoi (0.25–0.25/2.24 to >16); P. verrucosa (0.097–0.28/0.70–
human host, are under a strong selective pressure. These results are 1.59); C. carrionii (0.17–0.25/4–0.39). The MFCs for F. pedrosoi
consistent with the increased adhesion, stress resilience and diversi- strains were higher in media without tricyclazole. For C. carrionii, the
fied metabolic repertoire of the FFUL887 strain. azolic antifungals obtained higher MFCs for the strain cultivated in
medium with tricyclazole. P. verrucosa had higher MFCs of voricona-
zole and terbinafine in media without tricyclazole, but MFCs of itra-
conazole and ketoconazole were higher in media with tricyclazole.

ª 2015 The Authors

72 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Conclusion There was no difference between MICs of strains with Results A total of 33 Aspergillus isolates were identified as 16 A.
their natural melanin and the same strains with melanin biosynthe- fumigatus, 11 A. flavus, 3 A. niger, 1 A. terreus, 1 A.candidus,1 A sydo-
sis inhibited by the addition of tricyclazole in the media. On the other wii. We determined that voriconazole was the most effective antifun-
hand, MFCs showed differences. The presence of melanin in F. pedro- gal agent againist all isolates. While 33 Aspergillus isolates (100%)
soi reduced the efficacy of the antifungal agents, with the melanized were sensitive to posaconazole, itraconazole and voriconazole with
strains less sensitive. The non-melanized C. carrionii was less sensi- the microdilution method, one A.fumigatus was resistant to Ampho-
tive, while results for P. verrucosa depended on the antifungal agent. tericin B. In vitroantifungal susceptibility results of voriconazole,itra-
These results suggest that the correlation between melanin produc- conazole, amphotericin B, and posaconazole were shown Table 1.
tion and the in vitro response to antifungal agents depends on the Conclusion Among four antifungals tested, the MIC levels obtained
chromoblastomycosis agent. More strains have to be tested to con- for voriconazole were lower compared to posaconazole, itraconazole,
firm this hypothesis. amphotericin B. In conclusion, given the toxic effects of amphotericin
B, voriconazole may be alternative antifungals that can be used by
the clinicians. Well-designed clinical studies are necessary to assist
clinicians in choosing the best antifungal agents.
P051

Antifungal susceptibility of the Aspergillus species by CLSI P052

reference methods
R. Altinbas,1 F. Ozakkas,2 H. Sav,1 E. Akkoyun,1 D. Turan,3
A. Baris1 and N. Kiraz1 Genotypic variability and antifungal susceptibility of
1 Candida spp. isolated from hospital surfaces and hands of
Istanbul University, Istanbul, Turkey; 2Istanbul University, healthcare professionals
Cerrahpasa Medical Faculty, Istanbul, Turkey and 3Istanbul
K. M. Sakita,1 D. R. Faria,1 F. K. Tobaldini,2 E. M. S. Bettega,1
Haydarpasa Numune Training and Research Hospital, Istanbul,
Turkey M. Negri,1 E. S. Kioshima,1 T. I. E. Svidzinski1 and P. S. Bonfim-
Mendonc
a1
Objectives Aspergillus species cause many infections especially in
1
Universidade Estadual de Maringa, Maringa, Brazil and
immunocompromised patients. Generally, Aspergillus infections can-
2
Universidade Estadual de Maringa/Universidade do Minho,
not be diagnosed early due to the lack of reliable and easy diagnostic Maringa, Brazil
tests. However, early diagnosis and initiation of an effective treat-
ment is required in order to get good clinical outcomes. Since resis- Objectives Candida spp. are responsible for 90–95% of hematogenous
tance to these drugs has been seen in patients, susceptibility testing fungal infections. In Brazil and Latin America, C. albicans is the most
may assist with determining the appropriate drug for treatment and common specie, followed by C. parapsilosis and C. tropicalis. Infections
respective epidemiology. Antifungal susceptibility tests should be per- caused by Candida spp. may have their origin in exogenous sources,
formed and interpreted for an effective treatment. The present study transmitted to patients via contaminated infusions, biomedical
evaluated species distribution of Aspergillus isolates which were iso- devices or even by the hands of the hospital staff members. Molecular
lated in our hospital and their antifungal susceptibility results biology techniques such as Randomly Amplified Polymorphic DNA
obtained by microdilution method. (RAPD) can show that the strains found in anatomical sites or abi-
Methods A total of 33 Aspergillus isolates were recovered from clini- otic surfaces have the same pattern genome.Moreover, in the last
cal specimens received at Cerrahpasa medical faculty. Identification decades it has been observed increasing the number of yeasts isolated
of each strain was performed using conventional mycological tech- from hospital environment resistant to antifungals. Thus, the aim of
niques. Minimum inhibitory concentration (MIC) of various antifun- this study was to determine the susceptibility to antifungals and
gal agents (amphotericin B, posaconazole, itraconazole and intraspecies similarity among isolates of different hospital surfaces
voriconazole) were determined by CLSI M38-A2 document. In brief and hands of healthcare professionals.
to induce conidium the isolates were grown on potato dextrose agar Methods The study was conducted with 25 isolates of Candida spp.:
(PDA) slants at 35 °C for 2–7 days before testing. The suspensions of 5 strains of C. albicans and 5 strains of C. parapsilosis isolated from
conidial inocula were prepared from 2 to 7 days old cultures grown hospital surfaces. 5 strains of C. albicans, 5 strains of C. parapsilosis
on PDA slants. The conidia were mixed in sterile saline with 0.05% and 5 strains of C. tropicalis isolated from hands of healthcare profes-
tween 20. The turbidity of the cell suspension was adjusted by spec- sionals. Professionals and surfaces belonged to intensive care units.
trophotometry to an optical density of 0.09 to 0.13 for Aspergillus The minimal inhibitory concentration (MIC) was determined to
spp. Microplates were prepared as described in document and they voriconazole (VOR), fluconazole (FLZ), amphotericin B (AMB) and
were incubated at 35 °C for 48 h. Two CLSI quality control strains, micafungin (MFG) according to M27-A3 of the Clinical and Labora-
C. parapsilosis ATCC 22019 and C. krusei ATCC 6258, were tested in tory Standards Institute (CLSI). To determine the intra-species simi-
the same manner. larity, 3 primers were used: P4 (50 -AAGAGCCCGT-30 ), OPA-18

Table 1. Antifungal susceptibility of Candida spp. isolated from hos-

pital surface and hands of healthcare professionals by the CLSI
method

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 73
Poster Presentations

(50 AGCTGACCGT30 ) and OPE-18 (50 GGACTGCAGA 30 ’). RAPD pro- Conclusion Azole resistance has been described worldwise. Itracona-
files were analyzed using BioNumerics software version 4.6. The zole resistance is common in developed countries (Canada, India,
study was approved by the Ethics in research involving human sub- China and the United States are considered to be the leading coun-
jects, CAAE 0448.0.093.000-11 protocol. tries). However, currently used antifungal susceptibility tests are
Results In relation to susceptibility testing (Table 1), it is important laborious and not sensitive enough. Therefore, implementation of
to highlight that C. parapsilosis showed 80% of MFG resistance. C. modern molecular biology techniques may allow identification of
albicans and C. tropicalis showed reduced susceptibility to VOR, and antimycotics-resistant Aspergillus spp. directly from the sample. The
resistence of the AMB was observed for C. albicans (20%). All amplifi- study of the mutations in cyp51Awill help to determine molecular
cations revealed distinct polymorphic bands. Genetic distances genetic characteristics of azole resistant Aspergillusisolates that may
between each of the isolates were calculated and cluster analysis was lead to development of original system for testing antimycotics-resis-
used to generate a dendrogram showing relationships between them. tant Aspergillus spp.
The analysis of all primers showed similarity greater than 80%
between strains of hands and hospital surfaces for intraspecies.
Conclusion Our work shows that, healthy people and hospital sur-
faces may be colonized by different species yeast. Furthermore, the P054
strains studied had relative resistance to antifungal drugs most fre-
quently used in clinical practice. Finally, there was a high similarity
between samples from hands (hospital staff members) and surfaces, Virulence factors and antifungal susceptibilities of Candida
providing an infection risk to susceptible individuals. Healthy people species isolated from urinary system
working in hospitals can carry yeasts on their hands with the same D. Turan,1 A. Baris,2 H. Sav,3 F. Ozakkas,4 S. Sen,3 R. Oguz5 and
potential virulence, and which therefore offer the same risk of infec- N. Kiraz3
tion. This information should be considered when preventive mea- 1
Istanbul Haydarpasa Numune Training and Research Hospital,
sures are established. Attention to the colonization of hands and
Istanbul, Turkey; 2Sisli Hamidiye Etfal Training And Research
surfaces should not be restricted to high-risk units such as NICUs,
Hospital, Istanbul, Turkey; 3Istanbul University, Istanbul, Turkey;
but should also include other sections of hospitals. 4
Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey
and 5Istanbul University Cerrahpasa Medical Faculty, Istanbul,
Turkey
P053
Objectives Although improvements have been made in many dis-
eases in paralel with medical field in recent years, many fungi kinds
Molecular genetic analysis of the cyp51a gene in azole particularly Candida type yeasts have become opportunist pathogens
resistant Aspergillus spp that could be encountered frequently as an infection agent; they
E. M. Latypova,1 A. E. Taraskina2 and N. V. Vasileva1 have become most frequently isolated fungal pathogens in particu-
1
NWSMU n.a. I.I. Mechnikov Kashkin Research Institute of larly nosocomial urinary tract infections (UTI). Moreover, the impor-
Medical Mycology, Saint Petersburg, Russia and 2Medical tance of virulence factors concerning Candida spp. as well as defence
systems of host is reported in studies to clarify the pathogenesis of
mycology institute named after Kashkin, Saint Petersburg, Russia
Candida infections and develop new drugs against Candida spp. In this
study, it was aimed to research the susceptibilities of antifungal and
Azole drugs are the first line of therapy against Aspergillus spp. Resis- virulence factors of Candida spp. isolated from UTI.
tance to azole antifungal drugs has been associated with treatment Methods A total of 83 Candida isolates were isolated from urine
failure and deaths in patients with aspergillosis. Multi-azole resis- samples of patients in various departments of Istanbul University,
tance increases mortality rate of patients with invasive aspergillosis Cerrahpasa Medical Faculty, between November 2011-November
to 88% in comparison with 30–50% mortality of patients infected by 2013. Isolates were identified by using conventional methods and
susceptible strains. Reduction of the susceptibility to azole is mainly additionally, through a commercial kit API 20C (Biomeriux, France)
triggered by point mutation in the 14a-sterol demethylase (cyp51A) and MALDI-TOF [Vitek MS system (bioMerieux, France)] was utilised
gene, which is the target enzyme for the drug. Nowadays, about 40 for the non-albicans Candida spp.While agar plage technique with
point mutations in cyp51A are identified. However, not all of them egg yolk was used to determine phospholipase activity, medium con-
are accounted for the drug-resistant phenotype. taining 1%bovine serum albumin was employed to determine acid
The aim of the study is a molecular genetic analysis of the proteinase activity from virulence factors and biofilm presence was
cyp51A nucleotide sequence for azole-resistant pathogenic strains analyzed with modified microplate method. In vitro antifungal suscep-
Aspergillus spp. tibility testing was determined using the CLSI broth microdilution
Materials and methods The nucleotide sequences of pathogenic method M27 A3-S4.
Aspergillus spp.cyp51A were aligned with reference sequence from Results The origins were described as following: 50 C. albicans, 10 C.
GenBank database (http://www.ncbi.nlm.nih.gov/genbank/). Optimal glabrata, 10 C. tropicalis, 5 C. parapsilosis, 5 C. lusitaniae, 2 C. kefyr, 1 C.
oligonucleotides sequences were selected for amplification and krusei. The number of strains with positive proteolytic activity was 51,
sequencing of Aspergillus spp.cyp51A. Specificity of the oligonu- C. albicans was determined in 41(50%), C. tropicalis in 5 (6%), C. parap-
cleotides was verified by BLAST algorithm (http:// silosis in 4 (5%) and C. kefyr in 1. Phospholipase activity was deter-
www.ncbi.nlm.nih.gov/blast/). mined as negative in all strains except for C. albicans whereas it was
All Aspergillus isolates were identified by sequencing of internal identified as positive in 34 (41%) C. albicans. Biofilm formations were
transcribed spacer region (ITS) and fraction of the b-tubulin gene. established in 11 strains in total as 8 (9.6%) C. tropicalis, 2 (2.5%) C.
Antifungal susceptibility testing was carried out according to the glabrata and 1 C. lusitaniae. When examined in terms of antifungal sus-
EUCAST microdilution method. ceptibility, it was found that all C. albicans and C. parapsilosis strains
Results To date, we obtained 6 resistant clinical Aspergillus spp iso- were susceptible to fluconazole, voriconazole and caspofungin. One of
lates. The molecular idenification of the isolates by sequencing of ITS the strain of C. tropicalis was resistance to fluconazole and voricona-
and b-tubulin gene revealed that isolates belong to the following spe- zole, on the other hand, other all C. tropicalis were determined to be
cies A.terreus, A.flavus, A.niger andA.calidoustus. According to the susceptible to fluconazole, voriconazole and caspofungin. All C. glabrata
EUCAST, A.terreus and A.flavus were resistant to voriconazole, A.niger were susceptible to caspofungins and their MIK interval was 0.5–
and A.flavus were resistant to ketoconazole, A.flavus and A.calidoustus 32 lg ml
1 and MIK50 ve MIK90 values were identified as 4.
were resistant to voriconazole and itraconazole. The nucleotide Conclusion The presence of virulence factors has gained importance
sequence analysis of the cyp51A gene showed some point mutations in studies conducted to clarify pathogenesis of infections developed
in azole-resistant strains. by Candida and develop new drugs. In the study, higher proteinase
and phospholipase enzyme determination compared to other species

ª 2015 The Authors

74 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

in C. albicans made us think that these two hydrolytic enzymes play of beta-1,3-glucan synthase they have distinct mechanisms of action
an important role in the pathogenesis of C. albicans and they could and target the fungal cell wall and thus present an alternative for
be virulence factors. In addition, the necessity has been understood the tratment of candidiasis. A commercially prepared dried colorimet-
that antifungal susceptibility tests should be performed to define the ric microdilution panel (Sensititre Yeast One, TREK Diagnostic Sys-
type for proper treatment of UTIs caused by Candida types and resis- tems, Cleveland, OH, USA) is able to determine the susceptibility of
tance ratios should be determined. echinocandins and demonstrated excellent corelation with the refer-
ence method M27-A3 from the Clinical Laboratory Standards Insti-
tute (CLSI). Resistance rates were determined by recently revised
CLSI antifungal breakpoints. The aim of this study is to evaluate the
P055 in vitro activity of ANF, CSF and MCF against 162 clinically signifi-
cant Candida strains isolated between June 2012 and December 2014
by Yeast One colorimetric microdilution panel.
Azole susceptibility and resistance in Fusarium spp Methods A total of 162 Candida strains were isolated from bloodcul-
A. D. van Diepeningen, A. al-Hatmi, B. Dalyan Cilo, D. Giosa, tures (n = 69), cultures of urine samples of symptomatic patients
W. J. Bartstra and G. S. de Hoog (n = 55), cerebrospinal fluid (n = 1), corneal abscess (n = 1), bron-
choalveolar lavage (n = 2) and sputum from pediatric patients with
CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands cystic fibrosis (n = 24), abscess (n = 8) and eusophageal biopsy
(n = 2) samples. Isolated strains were checked of purity and were
Objectives Fusarium species are among the emerging agents of identified by classical morphological tests including germ tube forma-
superficial, locally invasive and systemic infections in humans.. The tion in human serum at 37 °C for 3 h, blastoconidia, pseudohyphae,
large genus Fusarium contains many species with either saprophytic true hyphae and chlamydoconidia formation on corn meal agar-
or plant pathogenic life styles and is capable of forming a plethora of Tween 80, integrating with the results of chromogenic agar
mycotoxins. An important problem in dealing with Fusarium is its (CHROMagar, HiMedia, India) and of API 20C AUX kit (Bio Merieux,
high intrinsic resistance to most antifungal compounds applied either France). Identification kit procedure and colorimetric microdilution
in clinical or agricultural settings. In this paper we address the anti- tests were performed according to the manufacturer’s instructions.
fungal susceptibility of clinically relevant Fusarium species to current Quality control was ensured by testing CLSI-recommended strains C.
antifungal drugs and examine the role of the target genes, cyto- parapsilosis ATCC 22019 and C. krusei ATCC 6258. Recently
chrome P450 sterol 14a-demethylase (CYP51) paralogs, in azole described species-specific clinical breakpoint (CBP) values were used
resistance. to categorize the minimum inhibitory concentrations (MICs) of
Methods Many species within the genus Fusarium that were recog- echinocandins as susceptible (S), intermediate (I) and resistant (R)
nized based on morphological characters, have proved to be species against the strains of Candida (Pfaller Diekema 2012).
complexes, with little to no morphological differences, rather than Results Among 162 Candida isolates, 83 were C. albicans, 7 were C.
single species. Most of the identified opportunistic Fusarium patho- glabrata, 45 were C. parapsilosis, 13 were C. tropicalis, three of each C.
gens belong to the F. solani species complex (FSSC), the F. oxysporum kefyr and C. lusitaniae, two of each C. guilliermondii and Rhodotorula
species complex (FOSC), or the F. fujikuroi species complex (FFSC). sp, and one of each was C. kefyr, C. krusei, C. pelluculosa and Tri-
Less frequently encountered are members of the F. incarnatum-equiseti chosporon sp. Regarding to the MIC values obtained, resistance to ANF
(FIESC), F. dimerum (FDSC), or F. chlamydosporum species complexes was observed in one C. albicans, two C. tropicalis, five C. parapsilosis
(FCSC) or species like F. sporotrichioides. Based on multi-locus strains; resistance to CSF was observed in one C. albicans and one C.
sequencing, isolates can be identified to the species level. We used parapsilosis strains; and resistance to MCF was observed in two C. albi-
published antifungal susceptibility (AFST) as well as AFST data from can, one C. tropicalis and five C. parapsilosis strains. Resistance to all
strains identified within our laboratory to create an overview of sus- three echinocandins were observed in two Rhodotorula sp, one Tri-
ceptibility and resistance. chosporon sp and only in one C. albicans strain which showed mul-
Polyene antifungals like amphotericin B and azoles like voriconazole tidrug resistance including to azoles and amphotericin B. The
act by targeting the ergosterol pathway. Whole genome sequencing remaining strains were in vitro susceptible to all echinocandins tested.
data and specific sequencing of the CYP51 paralogs and promoter Conclusion Based on newly established breakpoints for species-
regions in a set of F. proliferatum and F. verticillioides strains and several specific interpretive criteria, the results of this study affirmed that
relatives with known AFST profiles are compared. Experimental evolu- echinocandins exhibit excellent activity against the Candida species
tion under low and high levels of the agricultural azole Carbendazim most frequently involved in human infections.
yields additional mutant strains with increased resistance.
Results and Conclusions The AFST data confirm the high level of
antifungal resistance in Fusarium species to most available antifungal
drugs, but also visualizes that for many species we lack solid data. P057
Fusarium species prove to exceptionally contain three CYP51 paralogs
and these genes have a high level of variation between and within
species, suggesting selection for variation and high levels of resis- Candida haemulonii complex: the true scenario by
tance. Differences in the AFST profiles emphasize the need for at least sequencing and MALDI-TOF among clinical isolates in India
species level identification for adequate treatment. C. Sharma,1 A. Masih,1 P. K. Singh,1 J. F. Meis2 and
A. Chowdhary3
1
Vallabhbhai Patel Chest Institute, Delhi, India; 2Canisius
Wilhelmina Hospital and Radboud University Hospital, Nijmegen,
P056 the Netherlands and 3University of Delhi - Vallabhbai Patel Chest
Institute, Delhi, India
In vitro antifungal activity of echinocandins against 162
invasive isolates of Candida spp Objectives Candida haemulonii complex is unique in being inherently
A. S. Kantarcioglu1 and N. Kiraz2 resistant to the two most commonly used antifungals, namely,
1
Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey amphotericin B and fluconazole. Due to increasing reports of C.
haemulonii and inconsistencies of automated yeast identification sys-
and 2Istanbul University, Istanbul, Turkey
tems, a study was undertaken to molecularly characterize these iso-
lates from cases of candidemia and deep seated infections. We aimed
Objectives The echinocandins anidulafungin (ANF), caspofungin to report the first molecularly confirmed case series due to C. haemu-
(CSF), micafungin (MCF) represent a new antifungal group with lonii complex from India and also evaluate their antifungal suscepti-
potent activity against Candida species. As non-competitive inhibitors bility profiles.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 75
Poster Presentations

Methods A total of 103 clinical isolates initially identified as C. study was to identify fungi isolates from otomycosis and evaluate the
haemulonii by VITEK2 system were subjected for their correct identifi- antifungal susceptibility in vitro. In addition we also evaluated the
cation by ITS sequencing and matrix-assisted laser desorption ioniza- fungicide activity of a derivative of propolis as a possible alternative for
tion-time of flight mass spectrometry (MALDI-TOF). Antifungal otomycosis treatment. Propolis is a natural product collected from
susceptibility testing (AFST) was performed using Clinical and Labo- hives of Apis mellifera L.. Although the antifungal activity of propolis is
ratory Standards Institute (CLSI) brothmicrodilution method (M27- well known, the new derivative product of propolis is still under study.
A3/M27-S3) and VITEK2 AST card. Methods Samples were collected from patients who attended the
Results Of the 104 C. haemulonii, only 15 (13.6%) isolates were con- Teaching and Research Laboratory of Clinical Analysis (LEPAC),
firmed as belonging to Candida haemulonii complex whereas the Division of Mycology, Universidade Estadual de Maring a (UEM), Bra-
remaining 89 were C. auris. The Candida haemulonii complex included zil, from January 2014 to May 2015. This study was approved by
isolates viz., C. duobushaemulonii (n = 8), C. haemulonii (n = 6) and C. the Research Ethics Committee of UEM (approval no. 615.643/
haemulonii var. vulnera (n = 1) identified by both sequencing and 2014). The species isolated from ear secretion were identified by the
MALDI-TOF. Phylogenetic analysis of ITS sequence and spectra by classical method. In vitro antifungal susceptibility testing was per-
MALDI-TOF clearly found different clades for respective species. Of formed using a microdilution method according to the Clinical and
the 15 isolates, 9 (60%) originated from patients with diabetic foot Laboratory Standards Institute (CLSI; protocol no. M38-A2). The
lesions, 5 (33%) fungemia and a solitary isolate from bronchoalveo- agents tested included voriconazole (0.03–16 lg ml
1), amphotericin
lar lavage. All of the 15 isolates showed resistance to amphotericin B B (0.03–16 lg ml
1) and subproduct of propolis extractive solution
(MIC90 16 lg ml
1) by both the methods. Fluconazole had variable (SPES; 11.14 to 5707.4 lg ml
1 of total phenol content expressed in
MIC range (1 to >64 lg ml
1) by both CLSI and VITEK methods. gallic acid). The minimum inhibitory concentrations (MICs) were
Low geometric mean MICs of posaconazole (0.05 lg ml
1) and itra- visually determined after 48 h incubation at 35 °C. Then, the mini-
conazole (0.31 lg ml
1) were observed by CLSI. Barring a solitary mum fungicide concentration (MFC) of SPES was also determined by
isolate of C. haemulonii, which had high voriconazole MIC inoculating each concentration from the MIC test into plates contain-
(4 lg ml
1), all were highly susceptible (MIC range, 0.03– ing Sabouraud Dextrose Agar, that were then incubated at 35 °C for
0.5 lg ml
1) by CLSI. All isolates barring two exhibited low MICs 24 and 48 h.
(range, 0.06–0.5 lg ml
1) against echinocandins by CLSI. Further, a Results In the period of this study, two samples of ear secretion con-
solitary isolate of C. haemulonii var. vulnera exhibited high MIC tained species of Aspergillus, one was identified as A. fumigatus and
(1 lg ml
1) against all echinocandins and one isolate of C. the other as A. flavus. A. flavus presented high MIC value against
duobushaemulonii had high MIC (1 lg ml
1) for anidulafungin and amphotericin B (4 lg ml
1) and it was highly resistant to voricona-
micafungin. All patients that harbored C. haemulonii complex in the zole (≥16 lg ml
1). SPES showed fungicide activity against both spe-
tissue underwent foot amputation, which was the definitive treat- cies, mainly against A. flavus that had higher MIC values for
ment for the gangrenous lesions. Among 5 candidemia patients three common antifungal agents (Table 1).
were treated with voriconazole and remaining two expired before Conclusion Since SPES showedfungicide activity, it has potential to
institution of antifungal therapy. be an alternative topical treatment in cases of otomycosis due to
Conclusions The present study reports the large case series of can- Aspergillus spp. More studies are necessary to investigate the activity
didiasis due to molecularly characterized C. haemulonii complex in of this derivative product and validate its use in vivo.
India. The study reports the isolation of C. haemulonii complex for the
first time from tissue of patients with diabetic foot lesions. It high-
lights the usefulness of molecular characterization to determine the
true prevalence of this rare multi-drug resistant species. P059

Molecular identification and antifungal susceptibility

P058 profile of clinically significant Aspergillus species in a
referral Chest hospital, Delhi, India
C. Sharma,1 A. Masih,1 P. K. Singh,1 J. F. Meis2 and
Fungicide activity of a derivative of propolis against A. Chowdhary3
Aspergillus spp. isolated from otomycosis 1
J. Galletti,1 E. Guilhermetti,1 K. F. Dalben Dota,1 P. S. Vieira,1 Vallabhbhai Patel Chest Institute, Delhi, India; 2Canisius
Wilhelmina Hospital and Radboud University Hospital, Nijmegen,
E. S. Kioshima,2 M. Negri2 and T. I. E. Svidzinski2
the Netherlands and 3University of Delhi - Vallabhbai Patel Chest
1
Universidade Estadual de Maringa, Maringa, Brazil and Institute, Delhi, India
2
Universidade Estadual de Maringa, Maringa, Brazil
Objectives The major Aspergillus species known to cause disease in
Objective Otomycosis is a kind of external otitis that can be caused by humans are included in five Aspergillus sections: Fumigati, Flavi, Nidu-
various species of fungi, amongst which species of Aspergillus are the lantes, Nigri and Terrei. The most common species causing invasive
most common. To use the appropriate treatment, it is necessary to disease is A. fumigatus in section Fumigati. Molecular diagnostic
identify the causal agent of otomycosis. Treatment recommendations approaches have revealed that there are at least 11 species that could
have included local debridement, local and systemic antifungal agents easily be misdiagnosed as A. fumigatus based on their conidial mor-
and discontinuation of topical antibiotics. Sometimes otomycosis pre- phology. Despite being clinically relevant, the identification and thera-
sents as a challenging disease for its long term treatment and follow peutic approaches of rarely reported or cryptic Aspergillus species
up, yet its recurrence rate remains high. Thus, the purpose of this remains ill defined. The accurate identification of species is critical
given that different species may present peculiarities in terms of reser-
voir, virulence factors, natural history of infection and in vitro suscep-
tibility to antifungal drugs. The aim of this study was to molecularly
characterize and to determine in vitro antifungal susceptibility profile
of rare cryptic species in various Aspergillus sections originating from
clinical samples in a referral chest hospital, Delhi, India.
Methods A total of 25 Aspergillus species cultured from respiratory
and CNS specimens of patients with suspected mycotic infections
during 2011–14 were preliminarily identified based on colony col-
our and morphology of the isolates on Czapek dox agar plates incu-
bated at 28 °C for 7 days. All of the Aspergillus species were

ª 2015 The Authors

76 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

identified by amplification and sequencing of b-tubulin and calmod- Methods Leftover BAL samples from hematology patients with sus-
ulin gene. Antifungal susceptibility testing was performed for azoles, pected IA from 5 Dutch and Belgian centers were used. BAL
amphotericin B and echinocandins with broth microdilution method galactomannan ≥1.0 or a positive culture was considered the gold
(CLSI M38-A2). standard for the presence of Aspergillus. Supernatant and pellet
Results During a 4-year survey period, 15.5% Aspergillus species were tested separately and the lowest cyclic threshold (Ct) value
originated from 2117 clinical samples processed. Of these clinically was used. Patients without the EORTC/MSG clinical criteria for pos-
important 25 isolates were investigated. b-tubulin and Calmodulin sible IA but with galactomannan ≥ 1.0 were defined as non-classi-
sequencing identified 3 (12%) isolates each of Eurotium amstelodami fiable. Azole treatment failure and mortality in patients with
and A. sydowii, followed by two each of A. fijiensis, A. ochraceus, A. A.fumigatus containing a PCR-detected RAM was compared with
niveus var. indicus, A. hortai, A. aculeatus and A. terreus. Further, soli- those without RAM. Given the rarity of azole resistance, the 2
tary isolates of A. clavatus, A. wentii, A. chevalieri, A. melleus and A. cases of PCR-detected azole resistance from the previous study
tritici were identified. Also, the teleomorphic stage of A. nidulans, i.e., were pooled with those detected in the current study in the azole
Emericella nidulans and its variety, E. dentata originated from bron- treatment failure analysis. Patients were excluded from this analy-
choalveolar lavage of individual patients of aspergilloma. All the sis if they received < 5 day of azole therapy or had a mixed fun-
Aspergillus species excepting Aspergillus aculeatus exhibited elevated gal infection.
MICs of amphotericin B (range 2–16 lg ml
1). Also, 24% (n = 6) Results 228 BAL samples from 201 patients were available. 9
isolates had high voriconazole MICs of 2 lg ml
1. The species repre- patients had proven, 43 probable, 32 possible and 43 non-classifiable
sented were A. fijiensis, A. niveus var. indicus and A. ochraceus. Excel- IA. 91 of the 228 BAL samples were gold-standard positive. The opti-
lent antifungal activity against all the species tested was exhibited by mal Ct cut-off for the Aspergillus spp PCR was <36. With this cut-off,
posaconazole (range 0.03–0.25 lg ml
1) followed by isavuconazole the PCR was positive in 64 of 91 BAL samples (Fig. 1). 34 of these
(range 0.06–1 lg ml
1) and itraconazole (range 0.03–0.5 lg ml
1). 64 were culture negative. The sensitivity, specificity, PPV and NPV
All the echinocandins revealed good activity against all the species for the detection of Aspergillus spp was 70%, 94%, 89% and 83%. 4
tested. The clinical profile of the patients yielding Aspergillus species of 72 BAL samples with Ct<36 were only positive in the pellet.
ranged from allergic bronchopulmonary aspergillosis, chronic pul- Therefore, PCR testing on pellet had little additional value. The resis-
monary aspergillosis, invasive pulmonary and a solitary case of brain tance PCR demonstrated that 65 patients were infected with a wild-
abscess. Further, in 16% cases these species were isolated from type A.fumigatus and 13 were infected with A. fumigatus with a RAM
patients with pulmonary tuberculosis with damaged lungs. (8 TR34/L98H, 3 TR34/L98H + wildtype, 2 TR46/T298A/Y121F).
Conclusions The present study reports the extension of spectrum of 27 patients were excluded from the azole treatment failure analysis.
Aspergillus species involved in aspergillosis. Accurate molecular iden- Azole treatment failure was observed in 13/43 patients with wildtype
tification is not only important taxonomically, but clinically relevant compared to 6/8 patients with RAM (P=0.04). Mortality at 6 weeks
as many of these newly characterized species exhibit resistance to was 29% higher in patients with detected RAM (21% without versus
antifungal agents thus posing serious implications for the successful 50%, P=0.18).
therapy of aspergillosis. Conclusion This multicenter study confirms that the AsperGe-
niusPCR has a good diagnostic performance on BAL. The assay dif-
ferentiated wildtype from A. fumigatus with RAM and is particularly
of added value because most BAL samples of patients with IA are
P060 culture negative. Most importantly, 75% of the patients with RAM
failed azole therapy. Therefore, PCR-based RAM detection can predict
azole treatment failure as soon as IA is diagnosed.
PCR-based detection of A. fumigatus cyp51A mutations on
bronchoalveolar lavage can readily predict azole treatment
failure. A multi-center validation study in 201 hematology
patients with suspected invasive aspergillosis P061
G.M. Chong,1 M.T. Van der Beek,2 P.A. Von dem Borne,2
J. Boelens,3 E. Steel,3 G. Kampinga,4 L.F.R. Span,5 K. Lagrou,6
Combination antifungal therapy for patients with
J.A. Maertens,7 G. Dingemans,8 G. Gaajetaan,8 J.J. Cornelissen,1 galactomannan (GM) positive probable invasive
A. Vonk9 and B.J.A. Rijnders1 aspergillosis (IA)
1
Erasmus University Medical Center, Rotterdam, Netherlands; J.A. Maertens,1 K. Marr,2 P.G. Pappas,3 J. Perdomo,4 J. Yan5 and
2
Leiden University Medical Center, LEIDEN, Netherlands; 3Ghent J. Aram6
University Hospital, GHENT, Belgium; 4University Medical Center 1
University Hospitals Leuven, Leuven, Belgium; 2Johns Hopkins
Groningen, Groningen, Netherlands; 5University of Groningen,
University, Baltimore, USA; 3University of Alabama, ALABAMA,
University Medical Center Groningen, GRONINGEN, Netherlands;
6 USA; 4Pfizer inc., PARIS, France; 5Pfizer Inc, New York, USA and
UZ Leuven, Leuven, Belgium; 7University Hospitals Leuven, 6
Ho^pital de Hautepierre, STRASSBOURG, France
Leuven, Belgium; 8PathoNostics B.V., Maastricht, Netherlands
and 9Erasmus Medical Center, Rotterdam, Netherlands
Objectives To assess the efficacy of voriconazole (vori) and anidula-
fungin (anid) in combination (combo) versus vori monotherapy in
Objectives Azole resistance in Aspergillus fumigatus is increasingly treating patients with GM-positive IA.
reported. However, the majority of cultures from patients with inva- Methods In a randomized double-blind placebo-controlled trial,
sive aspergillosis (IA) remain negative and hence azole resistance patients were assigned in a 1:1 ratio to receive vori with either anid
remains undetected. The AsperGeniusPCR is a multiplex real-time or placebo for a minimum of 2 weeks; vori monotherapy was contin-
PCR consisting of 2 PCRs: one identifies the clinically relevant Asper- ued to complete 6 weeks of treatment. Patients were stratified at
gillus species, the other detects 4 resistance associated mutations entry according to variables known to have an independent effect on
(RAM:TR34/L98H/ T289A/Y121F) in cyp51A associated with azole mortality: allogeneic (allo-) Stem Cell Transplant (SCT) versus none
resistance. In a recent single-center study the diagnostic performance and pulmonary versus disseminated IA. This analysis is based on a
of AsperGeniusPCR on bronchoalveolar lavage (BAL) was excellent modified intention-to-treat (mITT) population, including all patients
with sensitivity, specificity, positive and negative predictive values with confirmed (by an independent DRC) diagnosis of GM-positive
(PPV, NPV) of 89%, 89%, 73% and 96% (Chong et al. 2015). The probable IA and who received therapy. GM-positivity was defined by
purpose of this multicenter study was to (1)confirm these findings in two serum samples or a single bronchoalveolar lavage (BAL) sample
large patient population, and (2) evaluate if the molecular detection positive for GM, using the U.S. Food and Drug Administration-ap-
of the above-mentioned RAM correlates with treatment failure. proved index cutoff. The primary analysis was a comparison of all-

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 77
Poster Presentations

cause mortality at 6 weeks. Mortality based on enrollment stratifica- P071

tion was also analyzed.
Results 108 patients in the combo and 110 in the vori monother-
apy were identified. Patients had comparable baseline characteristics Comparison of biofilm production, hydrophobicity and
including age (mean 51.8 years, range 18-79 vs 50.5 years, 18–83), proteomic analysis in clinical isolated strains of Candida
gender (males 56% vs 56%) and race (white 71% vs 67%, Asian albicans
25% vs 28%, and black 2% vs 1%) in the combo vs monotherapy E. Valentin-Gomez, L. Cabello-Murgui, A. Valentin-Verdeguer,
arms, respectively. P. Kant, V. Perez-Don~ ate, A. Murgui-Faubel and L. del Castillo-
All-cause mortality at Week 6 was 15.9% (17/108) in the combo
Agudo
and 27.5% (30/110) in vori alone arm [estimated treatment differ-
ence of
11.5% (95% confidence interval [CI],
22.41 to
0.65%), University of Valencia, Burjassot Valencia, Spain
P-value 0.0189]. Interestingly mortality at Week 6 in allo-SCT
patients was 22.2% (8/36) in the combo and 22.6% (7/31) in vori Objectives Study biofilm production and hydrophobicity in 448
alone [difference of
0.4% (95% CI,
20.4 to 19.7%)]; in the non- strains of Candida albicans from patients with fungemia from Hospital
allo-SCT patients, mortality at Week 6 was 12.7% (9/72) in the Universitario la Fe (Valencia, Spain). Classify the C. albicans strains
combo and 29.4% (23/79) in vori alone [difference of -16.7% (95% according to the degree of biofilm production comparing with the
CI,
29.5 to
3.9%)]. Baseline characteristics including host factors, strain C. albicans SC5314 [1] using the Crystal Violet (CV) procedure.
site of infection and underlying conditions did not identify specific dif- Proteomic analysis of the extracellular matrix (EM) from biofilms of
ferences in non-allo-HSCT patients that may explain the mortality different categories of C. albicans.
rate finding. Methods Biofilm formation. Biofilm was formed as described [2] with
Conclusions In patients with probable GM-positive IA, combination some modifications. Cells were counted using a Bio Rad T20 Auto-
antifungal therapy with voriconazole and anidulafungin is associated mated Cell Counter. 105 cells suspended in 100 ll of RPMI medium
with improved mortality rate compared to voriconazole monother- were plated in wells of 96-well microtiter plates and then incubated
apy, especially in non-allo-SCT patients. 24 h at 37 °C.
Biofilm quantification by the CV method. Biofilms were quantified
as described [3] but 0.05% crystal violet was used instead 0.1%. The
OD580 nm values were referred at the OD580 nm of C. albicans
P070 SC5314.
Cell surface hydrophobicity. Cell surface hidrophobicity (CSH) was
measured by the biphasic separation method [4].
Fluconazole versus Voriconazole: Candida glabrata Analysis of tryptic peptides. The tryptic peptide mixtures from EM
biofilms response to different azoles of biofilms were subjected to LC-MS/MS. Database search was per-
C. F. Rodrigues, B. Gonc
alves, M. E. Rodrigues, S. Silva, formed of CAlbicans database.
J. Azeredo and M. Henriques Results We have used 448 strains of C. albicans. The distribution of
University of Minho, Braga, Portugal strains based on CV results was into quartiles to stablish the cut-offs,
comparing with the CV values of the strain C. albicans SC5314, as
follows: Non Adherent (NA) = 0.0–0.2; Weakly Adherent (WA)
Candida glabrata is the second most prevalent yeast in fungal infec- >0.2–0.5; Moderately Adherent (MA) >0.5–0.7 and Strongly Adher-
tions, especially in immunocompromised and/or hospitalized patients. ent (SA) >0.7–1.0. The 448 strains were distributed in this way:
The azole resistance within this species is very well-known and 15.6% SA; 14.5% MA; 30.6% WA and 39.3% NA. The CSH was
results in a low therapeutic response of C. glabrata infections, particu- also analyzed. A relative CSH of around 20% was presented by 402
larly when associated with biofilms. So, the main goal of this work strains (53 SA; 54 MA; 126 WA and 169 NA). Amongst strains
was to understand the different efficacies of two azoles against C. with a CSH >20% the highest percentage was found in the SA
glabrata biofilms: fluconazole (Flu), a long time used drug, and strains. The proteomic profiles of the EM from three strains of each
voriconazole (Vcz), a recent drug used only in hospitals. category, WA, MA and SA were obtained. We identified 1790 pro-
Antifungal (Flu and Vcz) susceptibilities were determined in pre- teins in SA strains, 600 proteins in MA strains and 220 proteins in
formed 24-h-biofilms of different strains of C. glabrata (clinical isolates WA. Of all the proteins identified only 74 were presents in the nine
and a reference strain). The ERG genes expression profiles of C. glab- strains analyzed.
rata biofilms cells were determined. Additionally biofilms’ matrices Conclusions We can conclude that there is not a direct relationship
composition and the retention of the two azoles within the biofilm between the capacity of biofilm formation and the pathogenicity-viru-
matrix were evaluated. lence of C. albicans clinical isolate and that there is not a positive cor-
The results showed that C. glabrata biofilms are more susceptible to relation between the cell surface hydrophobicity and the capacity to
Vcz than Flu. Quantitative Real-Time-PCR results revealed an overex- form biofilm structures. From the proteomic results we can deduce
pression of the three ERG genes in the presence of both azoles. How- that the SA strains secrete larger amount of proteins than the other
ever, the ERG expression was more dependent on the strain than on CV groups. Those proteins could help to maintain the cellular com-
the agent. The matrix content was analyzed following biofilm expo- ponents of the biofilm strongly fixed to each other, and to the surface
sure to antifungal agents and it was noticed a decrease in proteins, where they grow, in the SA strains. Finally, we can conclude that at
an increase in carbohydrates (also b 1,3-glucans) and ergosterol was least 70 proteins could be needed to form a biofilm so they are com-
also found. A further evaluation was made with the determination of mon to all strains analyzed.
the concentration of the agents diffusing through the biofilm, which References
showed a remarkable difference between the two drugs, with Vcz [1] Gillum et al. (1984). Mol Gen Genet 198: 179–182.
reaching more the cells than Flu, which could explain the differences [2] Pierce et al. (2008). Nat Protocol 3: 1494–1500.
in biofilms susceptibilities. [3] Marcos-Zambrano et al. (2014). Int J Med Microbiol 304:
To conclude, this study showed that the better performance of Vcz 1192–8
in C. glabrata biofilms maybe due to its smaller molecule and there- [4] Gelis et al. (2012). FGB 49: 322–331.
fore to its better absorption within the biofilm.

ª 2015 The Authors

78 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P072 although the differences did not reach statistical significance. Non-
Candida biofilms were highly resistant to the three echinocandins.
Conclusions Yeast strains isolated from patients with fungemia and
Antifungal activity of caspofungin, anidulafungin, and showing acquired or intrinsic echinocandin resistance were able to
micafungin against biofilms formed by yeast isolates form biofilms with moderate or low metabolic activity. The activity of
showing intrinsic or acquired echinocandin resistance the three echinocandins against Candida fks-mutant biofilms was
L. J. Marcos-Zambrano,1 M. Go mez-Perosanz,1 P. Escribano,2 higher than the activity against intrinsically-resistant isolates; mica-
O. Zaragoza,3 E. Bouza1 and J. Guinea1 fungin showed the greatest activity against wild-type or fks-mutant
Candida biofilms.
1
Gregorio Maran~on Hospital, Madrid, Spain; 2Hospital General This work was supported by grant numbers PI11/00167 and
Universitario Gregorio Maran~on, Madrid, Spain and 3Instituto de PI14/00740 from Fondo de Investigaci on Sanitaria (FIS).P. Escribano
Salud Carlos III, Madrid, Spain is contracted by Instituto de Investigaci on Sanitaria Gregorio
Mara~ n (grantnumberPI14/00740). J. Guinea is supported by a
no
Objectives Biofilm formation promotes catheter-related fungemia Miguel Servet contract (CP09/00055) from FIS. LJ Marcos-Zambrano
and other forms of fungal disease. Echinocandins have shown potent is supported by a predoctoral PFIS grant (FI12/00265) from FIS.
anti-biofilm activity, although resistance in yeasts causing fungemia
is being increasingly reported. Acquired echinocandin Candida spp.
resistance is a result of mutations in FKS genes whereas non-Can-
dida species commonly show intrinsic resistance. Data regarding bio- P073
film production of isolates showing echinocandin resistance and their
antifungal susceptibility are very scarce. We studied the biofilm pro-
duction of fks-mutant Candida and non-Candidaisolates showing Effect of caspofungin and micafungin in combination with
intrinsic echinocandin resistance. Furthermore, the antifungal sus- farnesol against Candida parapsilosis biofilms
ceptibility of biofilms to caspofungin, micafungin, and anidulafungin , M. Doman, F. Nagy, Z. To
R. Kovács, A. Bozo  th and L. Majoros
was studied. University of Debrecen, Debrecen, Hungary
Methods We studied the biofilm production of the following isolates
from patients with fungemia: Candida spp. strains with fks genes
mutations [C. albicans, n = 1; C. glabrata, n = 1; and C. tropicalis, Objectives The aim of our study was to compare the in vitro anti-
n = 3; Table 1]; intrinsically echinocandin-resistant non-Candidas biofilm activity of caspofungin (CAS) and micafungin (MICA) com-
trains [Rhodotorula mucilaginosa, n = 7; Trichosporon asahii, n = 2; Tri- bined with farnesol in RPMI-1640 against five Candida parapsilosis
chosporon japonicum, n = 1; Trichosporon dermatis, n = 1; and Arxula clinical isolates. Farnesol is a quorum-sensing molecule, which inhi-
adeninivorans, n = 1]; and wild-type Candida isolates [C. tropicalis, bits the yeast-hyphae transition.
n = 6; C. albicans, n = 2; and C. glabrata, n = 2]. Biofilm was mea- Methods Drug interactions were examined by broth microdilution
sured to classify strains as low (LBF), moderate (MBF) and high bio- checkerboard method and an XTT-based colorimetric assay to mea-
film-forming (HBF) (cristal violet assay), or low (LMA), moderate sure metabolic activity of cells. The tested concentrations were 300–
(MMA), and high metabolic activity (HMA) (XTT reduction assay). 1.17 lM, 256–4 mg l
1 and 512–2 mg l
1 for farnesol, CAS and
Preformed biofilm were treated with concentrations of caspofungin, MICA, respectively. Fractional inhibitory concentration index (FICI)
micafungin, and anidulafungin ranging from 0.015 mg l
1 to was used to assess the drug interactions: ΣFIC = FICA+
16 mg l
1 and incubated at 37 °C for 24 h; the metabolic activity FICB = CAcomb/MICAalone + CBcomb/MICBalone, where MICAalone and
was measured by the XTT reduction assay and the sessile MIC MICBalone are the MIC values of agents A and B using alone and
(SMIC50) was defined as the antifungal concentration yielding a 50% CAcomb and CBcomb are the MICs of agents A and B when acting in
reduction in the metabolic activity of the treated biofilm compared to combination, respectively. FICI was defined as the lowest ΣFIC. Syn-
the growth control. The SMIC50 for the three groups of isolates was ergy was specified if the FICI was ≤0.5, between >0.5 and 4 was
studied and compared (Kruskall-Wallis test). indifferent and as antagonistic if the FICI was >4. MICs alone and at
Results The Candida resistant isolates formed biofilms (Table 1) and all of the isoeffective combinations were determined as at least 50%
most of them were MMA. The three drugs showed the highest activ- reduction of metabolic activity compared with control.
ity against biofilms formed by wild-type Candida isolates followed by The results observed in the checkerboard 96-well plate were con-
Candida fks mutant isolates and by non-Candida isolates (P < 0.001) firmed by time-kill experiments. Three concentrations (4, 8,
(Table 2). Micafungin was the drug showing the highest activity 16 mg l
1) were chosen and examined their anti-biofilm effect alone
against biofilms formed by fks-mutant or wild-type Candida isolates and in combination with 75 lM farnesol. Metabolic activity of bio-
films was determined at 0, 3, 6, 9, 12 and 24 h. The XTT reaction
was measured spectrophotometrically at 492/620 nm.
Table 1

Table 1. In vitro interactions of caspofungin and micafungin in com-

bination with farnesol

Table 2

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 79
Poster Presentations

positive blood cultures less than 5 days and matched for age, gender,
intensive care unit stay at onset, and month with 1:1 ratio. The min-
imal inhibitory concentrations (MICs) of antifungals were determined
using the YeastOne microdilution test (Trek Diagnostic Systems;
Thermo Scientific, USA) and interpreted based on the Clinical Labora-
tory Standard Institute (CLSI) species-specific clinical break points.
We determined dynamic biofilm forming ability using a 96-well
plate-based, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(pheny-
lamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay,
and high biofilm forming ability was categorized as the value above
the median one.
Results A total of 106 and 42 isolates collected from 25 case
Figure 1. Time-kill curves of caspofungin (A) and micafungin (B) patients and 25 matched control patients, respectively, were evalu-
alone and in combination with farnesol against five Candida parap- ated. For case patients, a median of 4 isolates per patient (range 2–8
silosis biofilms. isolates) were evaluated with a median 15 days of persistence (range
8–60 days). The demographics, underlying diseases/conditions,
healthcare factors within 30 days prior to onset, infection focus, and
fluconazole non-susceptible rate were not significantly different
One-way ANOVA with Dunnett’s post-testing was used to analyze between case and control patients. Compared with control patients,
the metabolic activity reduction exerted by drugs alone and in com- case patients were less likely to remove central lines within 48 h
bination compared with control. after candidemia onset (28.0% vs. 56.0%, P = 0.085), more likely to
Results The median MIC values of five sessile C. parapsilosis clinical be infected by high biofilm forming isolates (64.0% vs. 36.0%,
isolates were 32–256 mg l
1, 16–512 mg l
1 and ≥300 lM for P = 0.089) and more likely to receive highly active anti-biofilm
CAS, MICA and farnesol, respectively. However, in combination with agents as the definitive therapy (60% vs. 20%, P = 0.009). Multivari-
farnesol these values reduced to 4 mg l
1 and 4–8 mg l
1 for CAS ate analysis showed that removal of central lines within 48 h (ad-
and MICA, respectively. Lower median MICs were detected for ATCC justed odds ratio [aOR], 0.21; 95% confidence interval [CI], 0.06–
22019 alone and in combination (CAS: 2 and 1 mg l
1; MICA: 1 0.81; P = 0.023) and high biofilm forming isolates (adjusted odds
and 0.5 mg l
1). ratio [aOR], 4.55; 95% confidence interval [CI], 1.22–16.95;
Synergistic interactions were observed both for CAS and MICA P = 0.024) were independently associated with persistent can-
combine with farnesol for all of tested clinical isolates. Indifferent didemia. The 28-day mortality in case patients was 40.0% and
interaction was experienced for ATCC 22019 both in case of CAS 52.0% in control patients (P = 0.571). Length of stay after can-
and MICA. Antagonism was never observed. FICIs were shown in didemia onset was longer for case patients (37 days vs. 12 days,
Table 1. P = 0.002).
Our results were confirmed by time-kill investigations (Figure 1). Conclusions Not only prompt removal of central lines but also Can-
Both tested echinocandins without farnesol demonstrated typical con- dida with high biofilm formation contributed to persistent C. tropicalis
centration-dependent activity against biofilms. The metabolic activity fungemia.
of fungal cells significantly decreased for CAS at all of three combina-
tions (4 mg l
1 + 75 lM, 8 mg l
1 + 75 lM, 16 mg l
1 + 75 lM)
between 3 and 24 h compared with control (P < 0.05–0.001). Sig-
nificantly inhibition was observed for MICA with farnesol between 3
and 12 h (P < 0.001) but not at 24 h (P > 0.05). P075
Conclusion Farnesol exerted synergistic interaction in combination
with CAS and MICA against biofilms of C. parapsilosis clinical isolates. Kinetic modeling of Aspergillus fumigatus biofilm
Based on time-kill investigations CAS was effective longer time both developed in vitro using digital image analysis
alone and in combination with farnesol than MICA. Based on our
M. G. Pekmezovic,1 K. M. Rajkovic,2 A. M. Barac,1 M. Z. Kostic3
results farnesol may be a potential adjuvant against biofilms for the
treatment of catheter-associated infections caused by C. parapsilosis. and V. S. Arsic Arsenijevic4
1
Institute of Microbiology and Immunology, Faculty of Medicine
Uni. of Belgrade, Belgrade, Serbia; 2High Chemical and
Technological School for Professional Studies, Krusevac, Serbia;
3
P074 Institute of Microbiology and Immunology, Faculty of Medicine,
Uni. of Belgrade, Belgrade, Serbia and 4University of Belgrade,
Belgrade, Serbia
Impact of biofilm forming ability on persistent Candida
tropicalis fungemia
Y. C. Chen,1 P. Y. Chen,2 Y. C. Chuang,1 U. I. Wu,1 Objectives Detection of biofilm forming fungi is clinically very
important but simple and effective methods for its analysis are lack-
W. H. Sheng1 and S. C. Chang1 ing, so the aim of this study was to perform quantitative analysis of
1
National Taiwan University Hospital, Taipei, Taiwan and Aspergillus fumigatus biofilm in vitro by digital analysis of biofilm pho-
2
National Taiwan University Hospital Jin-Shan Branch, New Taipei tomicrographs. Biofilm size and complexity during time were quanti-
City, Taiwan fied and obtained data were used for development of kinetic model
for description and prediction of the biofilm dynamics.
Objectives From clinical studies, proportions of persistent can-
didemia varied from 11% to 45% with different definitions. Candida
tropicalis is the most common non-albicans Candida species causing
candiemia in Asia and Latin America. Little is known about biofilm
forming ability of C. tropicalis and its impact on clinical outcomes.
This case-control study aims to explore the association of biofilm
forming ability and persistent C. tropicalis fungemia.
Methods Case patients were adult (≥18 years) patients with persis-
tent candidemia (blood cultures positive of C. tropicalis for 5 days or
longer) hospitalized at a 2200-bed teaching hospital in Taiwan dur- Figure 1. Image processing procedure: the grayscale image (A), the
ing July 2011 and June 2013. Control patients were those with binary image (B), the outline image (C).

ª 2015 The Authors

80 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Methods A. fumigatus biofilm was developed in vitro on plastic cover 37 °C for 48 h to produce mature BF. PMN were isolated from
slips (CS). CS were placed in /55 Petri dishes and statically incu- healthy donors by dextran sedimentation/ficoll centrifugation. After a
bated with A. fumigatus spore suspension (106 colony forming units brief washing step of C. albicans mature biofilms, they were incubated
per ml) in Sabouraud dextrose broth with 8% of glucose (Sigma- with PMN for an additional 24 h at effector to target ratio (E:T) of
Aldrich, St Louis, MO, USA) at 28 °C. In each of the six time points 5:1. In another set of experiments, intact 48 h mature BF were
(after 19, 20, 21, 22, 23 and 24 h of incubation), three CS were exposed to the simultaneous action of PMN (5:1) and MFG (0.007–
removed from incubator, washed two times with sterile distilled 4 mg l
1) for 24 h. The PMN-induced BF damage measured as %
water and stained with lactophenol cotton blue (Sigma-Aldrich, St reduction of metabolic activity was assessed by the XTT assay. The
Louis, MO) for 30 min at 37 °C. After washing and air-drying, CS MIC of MFG for BF was determined as the minimum concentration
were mounted on a microscopic slide and the biofilms were pho- that caused ≥50% fungal damage compared to that for the untreated
tographed using a Nikon Camera (total magnification of 400X). Total controls. Statistical analysis was performed by ANOVA with Dun-
of 60 digital images were acquired and stored in JPEG format. Experi- nett’s post-test. Four independent experiments were performed.
ment was performed two times in triplicate. Results The MIC of MFG for mature BF of the three C. albicans iso-
The image analysis was done in software ‘Image J’ in three steps: lates was 0.25 mg l
1. MFG used at 0.5xMIC, 0.25xMIC and
(i) conversion of RGB images into grayscale images (ii) obtaining bin- 0.125xMIC concentrations caused 18%3.7%  2.9 and
ary images by adaptive threshold selection and (iii) procession by 9.8%  2.6 BF damage, respectively. When PMN were added to
image outline (Figure 1). Two biofilm parameters were quantitatively drug-pretreated mature BF there was a significant augmentation of
evaluated: the size (A) and the complexity of biofilm (D). Size was PMN-induced % damage against C. albicans exposed to 0.5xMIC com-
evaluated by measuring the area, while complexity of biofilm was pared to drug-untreated isolates (81%  1.8 vs. 60%  3.5;
done by fractal analysis of image using the box-counting method to P < 0.01). PMN exhibited similar % damage against C. albicans pre-
obtain fractal dimension of contour image which estimated the level treated with 0.25 9 MIC or 0.125 9 MIC compared to drug-un-
of two-dimensional (2D) geometrical complexity of the biofilm. treated isolates, respectively (64%  2 and 67%  2.2 vs.
Finally, A and D values obtained during time were used to develop a 60%  3.5; p = ns). The BF damage induced by the concurrent com-
kinetic model of A. fumigatus biofilm growth. bined treatment of PMN with MFG (0.007 mg l
1-4 mg l
1; 54–
Results Logistic kinetic model was used to describe the increase of 65% BF damage) against intact BF was not significantly different
the two parameters of 2D biofilm image during its growth. The from that of PMN alone (48%5.6).
experimental data successfully fitted exponential equation. The agree- Conclusion Pre-exposure of C. albicans to micafungin at sub-MIC
ment between the predicted and experimental values was observed concentrations increases biofilm susceptibility to the antifungal activ-
which proved that the kinetic model was appropriate for the descrip- ity of PMN. However, the concurrent combined treatment of mature
tion and prediction of A and D parameters. Kinetic model provided biofilms by MFG and PMN does not exert additive biofilm damage.
rate constants for both parameters (asize and acomp for A and D, These findings may be clinically important in the Candida biofilm-as-
respectively) where asize had a higher value than acomp sociated infections and need further study in animal models.
(5.968  0.062 and 0.409  0.024, respectively). A positive linear
correlation was observed between A and D (r = 0.9, P < 0.05).
Conclusion Logistic kinetic model was successfully applied for
description of A. fumigatus biofilm growth by increase of the biofilm P077
size and complexity. Biofilm images used for modeling were obtained
and analyzed using novel, cost-effective and simple approach which
can provide detection and prediction of the biofilm dynamics. This Development of P-113-derived peptides as novel inhibitors
can be useful for further studies on kinetics of fungal biofilm since for drug-resistant Candida spp. and biofilm formation
the prediction of biofilm dynamics can contribute to better clinical W. C. Cheng,1 G. Lin,2 H. Chen,2 M. Liu1 and C. Lan2
management of invasive fungal infections. 1
General Biologicals Corporation, Hsinchu, Taiwan and 2National
Tsing Hua University, Hsinchu, Taiwan

P076 Objectives Candida spp., as major opportunistic pathogens, infects

immunocompromised people and is responsible for a number of life-
threatening infections. The individuals commonly affected include
Pre-exposure of C. albicans biofilms to sub-MIC those with oral candidiasis of human immunodeficiency virus (HIV)-
concentrations of micafungin augments efficacy of human positive patients, of cancer patients undergoing chemotherapy and
neutrophils, but concurrent combined treatment exerts no radiotherapy, of diabetes and xerostoma patients. Moreover, Candida
additive effect against biofilms spp. can develop resistance to multiple antifungal drugs by over-ex-
M. Simitsopoulou,1 D. Kyrpitzi,1 T. J. Walsh2 and E. Roilides3 pression of drug resistance-related targets and other mechanisms.
1 Candida spp. can also form biofilms that are highly resistant to antibi-
Aristotle University of Thessaloniki, Thessaloniki, Greece; 2Weill otics treatment on host tissues or indwelling medical devices. In this
Cornell Medical Center and New York Presbyterian Hospital, New study, we developed a patented anti-fungal peptide P-113 and P-
York, USA and 3Aristotle University, Thessaloniki, Greece 113-derived peptides to overcome the emerging issue of drug-resis-
tance in Candida infections and reduce the resistance of biofilms.
Objectives Candida albicans (CA) has been associated with catheter- Methods The P-113, derived from human histidine-rich histatin 5
related fungemia leading to high mortality. CA forms biofilms (BF) protein, retains a full antibacterial activity and exhibits a wide spec-
on the surface of intravenous central catheters and indwelling medi- trum of activity in vitro against both bacteria and fungi. Several drug-
cal devices. Human neutrophils (PMN) are critical component of (azoles-) resistant clinical strains including C. albicans, C. krusei, C.
innate immune response against fungi. The objective of this study glabrata, and C. tropicalis were isolated from oral candidiasis patients.
was: 1) to investigate whether pre-exposure of C. albicans to sub-MIC We modified P-113 by tandemly repeating sequences (P-113-dimer
concentrations of micafungin (MFG) could subsequently modify the and P-113-trimer) to improve the candidacidal efficiency. Further-
activity of PMN against C. albicans mature BF and 2) to assess the more, the candidacidal activity of P-113 and its derivatives against
effect of the simultaneous combination of PMN with MFG against biofilm cells of C. albicans was also performed. Biofilm was formed in a
intact mature BF. 96-well microplate in YPD medium for 24 h. The biofilm cells were
Methods 106 blastoconidia/mL CA-M61, a well-documented BF-pro- then treated with or without various concentrations of P-113, P113-
ducing CA strain and two BF-forming C. albicans bloodstream iso- dimer, and P113-trimer at 37 °C for 1 h followed by XTT assay.
lates, were grown in RPMI at 37 °C for 24 h in the presence of MFG Results The results showed that P-113 was able to kill these drug-
at 0.5xMIC, 0.25xMIC and 0.125xMIC concentrations. Pretreated resistant strains, suggesting a promising candidacidal activity of P-
and untreated C. albicans isolates were then grown in fresh RPMI at 113 toward drug-resistant Candida spp. To improve the candidacidal

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 81
Poster Presentations

in these infections being the second most prevalent species involved

in human fungemia. This work aims to understand the effect of anti-
fungal agents in C. glabrata’s biofilm formation, specially their role on
matrix composition.
Methods Three antifungal agents, belonging to different classes,
azoles, polienes and echinocandins, were selected for this work,
namely fluconazole (Flu), Amphotericin B (AmB) and Caspofungin
(Csf), respectively. Three strains of C. glabrata were used along this
study. The effect of the agents on C. glabrata biofilm formation was
assessed by Colony Forming Units (CFU) and Crystal Violet (CV)
assays. Matrices’ composition evaluation included the determination
of polysaccharides (phenol-sulfuric acid method), proteins (BCA-Kit)
and b-1,3-glucans (Glucatell Kit) concentrations.
Results Observing the effect of the three drugs on the biofilms, it
was noticed that AmB and Csf showed the best performance in the
reduction of biofilms formed by the three Candida glabrata strains, in
Figure 1. The efficiency of P113-dimer and P113-trimer against C.
opposition to Flu. However, the effect of Csf was the most notorious,
albicans biofilm cells
achieving a biofilm reduction around 85%.
Analyzing the biofilm matrices, it was possible to observe a signi-
ficative change in their composition, when biofilms were formed in
the presence of the three antifungal agents, both in terms of polysac-
charides and proteins. In fact, in the presence of the different antifun-
gal agents two opposite effects were noticed, the amount of
polysaccharides increased, in opposition to the profound reduction in
terms of proteins. For AmB and Csf this decrease was very sgnifica-
tive, being below the minimum detected value range of the BCA
proteins Kit.
Interestingly, the amount of b-glucans on the matrices did not
show important differences in the presence of the drugs, with the
exception of Csf, which induced an increase of 20% of these
compounds.
Conclusion As expected, the three agents had different effects on C.
glabrata’s biofilm formation.
Moreover, matrices0 composition display dissimilarities when
exposed to different antifungal agents, and these differences depend
Figure 2. The candidacidal activity of P-113 is time- & dose- on each drug is used.
dependent. Therefore, AmB, and especially Csf, were confirmed, in this study,
to be the most effective pharmacoterapies for eradication of C. glab-
rata infections associated to biofilms.
activity, we designed and synthesized various P-113-derived dimeric
and trimeric peptides which contain 2- and 3-time tandem repeats.
Among them, the two tandem repeats improved the candidacidal
potency of the peptides, and the dimer and trimer have IC50 values
P079
against C. albicans of 0.5 and 0.4 lM, respectively, compared with
the monomer P-113 (IC50 = 2.3 lM). Moreover, we also found that
both dimer and trimer form of P113 can efficiently inhibit the viabil- The farnesol influence on characteristics of Candida
ity of C. albicans biofilm cells, showing the 50% RMA (reduction in albicans biofilm formation in vitro
the metabolic activity) of dimer and trimer are 20 lM and 8.3 lM, M. Rusakova
respectively.
Conclusion In this study, we showed that P-113 can kill drug-resis-
Odessa Mechnikov National University, Odessa, Ukraine
tant clinical isolates efficiently. Studies with P-113 derivatives further
revealed that P113-trimer can be folded as an a-helix structure (us- Candida albicans is a dimorphic fungus that exists in a number of
ing by Circular dichroism), suggesting the reason that P113-trimer morphological forms under different environmental conditions bud-
can effective against both planktonic and biofilm cells of C. albicans. ding yeast cells, pseudohyphae, true hyphae and chlamydospores.
P-113 and the derivatives show potential for the development of During human candidiasis, especially superficial, this microorganism
novel therapeutics against infections with drug-resistant Candida spp. forms biofilms. C. albicans biofilms usually consist both yeast cells
and inhibit the biofilm formation. and hyphae covered with a thick exopolysaccharide layer. The bio-
film itself is generally resistant to a variety of antifungal agents,
including amphotericin B and fluconazole, and will frequently persist
as a reservoir of infection. The C. albicans biofilm structure, in partic-
P078 ular the component ratio, can be different depending on the substrate
type: artificial medical devices, skin or mucous membranes. But any
environmental factor cannot act alone and under all conditions to
How do Candida glabrata’s biofilms respond to antifungal provoke the C. albicans growth in only one morphological form.
drugs? While some quorum-sensing molecules, for example farnesol (3,7,11-
C. F. Rodrigues, M. E. Rodrigues, S. Silva, J. Azeredo and trimethyl-2,6,10-dodecatriene-1-ol), block the morphological shift
M. Henriques from yeasts to mycelia and suppress filamentation.
The aim of this work was to characterize the C. albicans biofilm for-
University of Minho, Braga, Portugal mation in the farnesol presence under different conditions in vitro.
The work was carried out at Biotechnological Research and Educa-
Objectives Candida species are responsible for recurrent human tional Center using Candida albicans ATCC 18804. It was cultivated
infections, mostly in immunocompromised patients, due to their high in two liquid medium variants: Sabouraud (standard nutrient med-
vulnerability. Candida glabrata has been shown to have a major role ium for yeast like fungus cultivation) and Spider (rich medium for

ª 2015 The Authors

82 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

phenotype growth and colony wrinkling in C. albicans observation).

The farnesol concentration range was 0–500 lM.
In our study it was found that the formation of mature C. albicans
biofilm accompanied by an increase of the cell as well as exopolysac-
charide number. At low temperature biofilm maturation was less
intensive compared with 37 °C. But in both cases, only yeast cells
were observed as part of the biofilm. Such structure of C. albicans bio-
films was noted at urological catheter surface.
The intensity of C. albicans biofilm formation in Spider medium
was slightly lower than in Sabouraud one but only during the last
phase. However at the process beginning the significant number of
hyphae that gradually associated into a continuous system was
noted only in the second nutrient medium. Given the fact that
hyphae production is considered as C. albicans pathogenicity increas-
ing this biofilm structure was a model for the skin candidiasis – the
most aggressive and common form in humans.
It was found in our study that farnesol addition in the cultivation
media did not result in significant changes in the biofilm formation
rate, but considerable modifications of its structure. In the case of
Spider medium cultivation substantially filamentous free biofilm was
detected. The most active farnesol influence was in low concentra-
tions – up to 150 lM.
So, the nutrient medium composition and conditions effect on the
Candida albicans biofilm formation. These factors cause manifestation Table 1. Range of MIC and MIC50, MIC90 of 30 clinical isolates of
of the ability to switch between different forms of microorganism patients with VVC front of the antifungal fluconazole and nystatin.
morphology and overall speed of group production.

P080

Assessment of in vitro biofilm formation and antifungal

susceptibility of Candida albicans isolates from
vulvovaginal candidiasis
D. R. Faria,1 K. M. Sakita,1 F. K. Tobaldini,2 L. Akimoto-
Gunther,1 M. Negri,1 E. S. Kioshima,1 T. I. E. Svidzinski1 and although differences occurred depending on the isolated and conse-
P. S. Bonfim-Mendonc
a1 quently the group. Importantly it was noted that, in general, VVC
and RVVC groups had similar capacity biofilm formation. On the
1
Universidade Estadual de Maringa, Maringa, Brazil and other hand, these groups had less total biomass (average
2
Universidade Estadual de Maringa/Universidade do Minho, Abs = 1,091  0.88) compared with AS group (average Abs =
Maringa, Brazil 1,521  1.32).
Conclusion Although all the samples analyzed are sensitive to anti-
Objectives Vulvovaginal candidiasis (VVC) is an inflammation of the fungals tested research of resistant strains is relevant, since recur-
genital mucosa, which mainly affects the vulva and vagina. Candida rences are related to cases of VVC. Nystatin and fluconazole were
spp. are considered commensal fungus, however, when there is effective in small concentrations for the isolates analysed. All samples
imbalance in the microbiota or the host immune system is compro- were able to form biofilm and the average of the group of asymp-
mised, these can become pathogenic. C. albicans is responsible for tomatic patients greater than the others. Thus, the capacity to form-
most cases of VVC and is able of expressing mechanisms which allow ing biofilm is an important virulence factor in the persistence of
the colonization or infection in the host. These factors related yeasts, microorganisms in infectious processes and represent an increase in
including the growth of strains resistant to antifungal agents and vir- resistance to antifungal and host defense.
ulence attributes (such as biofilm formation) are important in the
development of VVC. In this sense, the objective of this study was to
evaluate the in vitro biofilm formation and susceptibility to antifungal
of C. albicans isolates from patients with vulvovaginal candidiasis. P081
Methods For the study were analyzed 30 clinical isolates of Candida
albicans. The clinical isolates were separated in groups of 10 samples
of the according to symptoms presented by the patients: asymp- Candida albicans biofilm development in various
tomatic (AS), vulvovaginal candidiasis (VVC) and recurrent vulvo- biomaterials using the CDC Biofilm Reactor.
vaginal candidiasis (RVVC). For all isolates were analyzed biofilm 
I. de la Pinta,1 E. Montoya,2 A. M. Zaldua,2 F. J. Alvarez,3

formation and minimal inhibitory concentration (MIC) for flucona- 4 5 6

J. Ibarretxe, T. Guraya, E. Eraso and G. Quindos  6

zole and nystatin. The MIC was performed according to M27-A3 pro- 1
Univ. Paıs Vasco UPV-EHU, Facultad de Medicina y Odontologıa,
tocol of the Clinical Laboratory Standards Institute. Biofilm forming
Bilbao, Spain; 2Leartiker, Markina-Xemein, Spain; 3Cruces
ability was assessed through quantification of total biomass by crys-
tal violet (CV) staining, performed on 96-well microplates containing
University Hospital, Bilbao, Spain; 4University College of Technical
a cellular suspension of 1 9 107 cells ml
1 and incubated for 24 h Mining and Civil Engineering, Bilbao, Spain; 5Industrial
at 37°C. Engineering Technical School of Bilbao, Bilbao, Spain and 6Univ.
Results Antifungal susceptibility testing is showed in table 1. The Paıs Vasco UPV/EHU Facultad de Medicina y Odontologıa, Bilbao,
isolates were tested to the two antifungals. The MIC raging from Spain
0.125 to 2 lg ml
1 for fluconazole and 1 to 4 lg ml
1 to nystatin.
The figure 1 show the quantification of the total biomass. It was evi- Candida biofilms are important and growing problems for the treat-
dent that all the C. albicans isolates were able to form biofilm, ment and outcome of catheter-associated candidiasis. There are not

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 83
Poster Presentations

P082

Bioluminescence Imaging of Candida glabrata adhesion

and biofilm formation
S. Kucharikova,1 G. Vande Velde,2 I. L. S. E. Jacobsen,3
U. Himmelreich,4 M. Brock5 and P. van Dijck1
1
KU Leuven, VIB, Heverlee-Leuven, Belgium; 2Biomedical MRI
unit/MoSAIC, Leuven, Belgium; 3Hans Knoll Institute, Jena,
Germany; 4KU Leuven, Leuven, Belgium and 5University of
Nottingham, Nottingham, United Kingdom

Introduction Candida glabrata is an emerging human fungal patho-

gen causing mucosal and deep tissue infections. Majority of these
Figure 1. Biofilm mean log density formed by C. albicans NCPF infections are associated with biofilm formation on medical implants.
3153 on polyurethane, silicone, teflon, titanium, polycarbonate and Biofilms are difficult to treat as they are tolerant to most antifungals.
borosilicate coupons. Fungal load in biofilms is traditionally analyzed post mortem, requir-
ing host sacrifice and enumeration of microorganisms from individ-
ual biofilms to evaluate the efficacy of antifungal treatment. In this
standardized methods for the study of Candida biofilms and the effect study, we aimed to use bioluminescence imaging (BLI) to detect C.
of antifungal drugs against them. glabrata adhesion and biofilm development in vitro and in vivo.
Objective To evaluate a model of Candida biofilm in coupons of dif- Methods Wild-type bioluminescent strain based on a C. glabrata-
ferent biomaterials used in catheters and other medical devices by codon optimized firefly luciferase was engineered. In vitro C. glabrata
means of the CDC Biofilm Reactor. adhesion and mature biofilms were developed inside serum-coated
Methods Candida albicans NCPF 3153 was cultured overnight at polyurethane catheters and in 96-well polystyrene plates. In vivo bio-
35 °C and adjusted to a concentration of 3 9 106 cells ml
1. One films were studied in catheters implanted on the back of Sprague
millilitre of the inoculum was transferred to the CDC Biofilm Reactor Dawley rats and biofilm formation was imaged with BLI at time of
(BioSurfaces Technologies, USA) that incorporated the removable implantation and followed up after 2 and 6 days. Bioluminescent sig-
materials tested (polyurethane, silicone, teflon, titanium, polycarbon- nal was measured and correlated with colony forming units (CFUs).
ate and borosilicate). The reactor was filled with 500 ml of yeast Results We report significant bioluminescent signal from Candida
extract peptone dextrose (YEPD) and Candida albicans was cultured cells in vitro, which were in agreement with CFU counts at different
onto the coupons for 48 h at 35 °C with an agitation of 120 rpm. stages of biofilm maturation. Data from in vivo BLI signal formed in
Each coupon was rinsed in PBS to remove planktonic cells. Finally, live animals at different time points after catheter implantations will
biofilms were detached from the coupons by sonication (50% ampli- be presented at the conference.
tude, 3 cycles of 20 s) and colony forming units (CFUs) counted in Conclusions We demonstrate the feasibility to non-invasively image
Sabouraud glucose agar plates. All test were done in quadruplicate and quantify C. glabrata adhesion and biofilms making dynamic mon-
and repeated in three different days. Analysis of variance (ANOVA) itoring of biofilm development possible.
and Games-Howell post hoc test was performed in order to find differ-
ences between biofilms developed in the different materials after
48 h.
Results Candida albicans developed sound biofilms on the surface of P083
all coupon tested. Results were repeatable and reproducible. Biofilm
on teflon coupons were significantly higher (5.45  0.09) compared
with the rest of coupons (P < 0.001, Figure). Conversely, titanium
Efficacy of Anidulafungin plus clarithromycin against
coupons (3.85  0.22) showed the lowest density (P < 0.01). Candida tropicalis biofilms growing on teflon and titanium
Borosilicate coupons (4.24  0.19) featured less biofilm density than M. E. Fernandez,1 J. L. del Pozo,2 J. L. Lopez Hontangas,1
teflon, polyurethane and polycarbonate coupons (P < 0.0001). There J. Peman1 and E. Canton1
were nostatistically significantdifferences between biofilm formed on poly- 1
University and Polytechnic Hospital La Fe, Valencia, Spain and
urethane coupons (4.65  0.05) and biofilm formed on polycarbonate 2
University of Navarra Clinic, Pamplona, Spain
coupons (4.74  0.07) (P = 0.724). Likewise, there were not signifi-
cant differences between biofilms developed onto silicone
(4.28  0.16) and borosilicate coupons (P = 0.990). Objectives Up to 60% of all human infections are caused by bio-
Conclusions This model on different biomaterial coupons using the films, causing a significant clinical problem. Candida biofilm-associ-
CDC Biofilm Reactor is valid for the study of Candida biofilm develop- ated infections are difficult to eradicate with antifungal treatment as
ment as it is reproducible and repeatable. Greater biofilms were biofilms cells are more resistant than planktonic cells because the
formed onto Teflon coupons while the biofilm on titanium were the exopolysaccharide matrix reduces the diffusion of antifungals inside
less dense. the biofilm. Previous studies have shown that C. tropicalis biofilms
Funding This study was financed in part by ZabaldUz Program and are resistant to echinocandins. Here we studied the kinetics of biofilm
projects GIC12 210-IT-696-13 and S-PE13AP004 (Gobierno Vasco- formation on teflon and titanium of C. tropicalis and evaluated the
Eusko Jaurlaritza) and UFI 11/25 (UPV/EHU). ability of the combination of anidulafungin (AND) plus clar-
ithromycin (CLA) to eradicate biofilm formed on these biomaterials.
Methods The selected strain of C. tropicalis was isolated in blood
culture and has been demonstrated to be strongly adherent. The
kinetics of biofilm formation on teflon and titanium was performed
using 1.27 cm discs in the CDC Reactor, using an initial inoculum of
107 cfu ml
1 in YNB-100 mM glucose medium. The number of
attached cells was determined at 24, 48, 72 and 96 h. The results
are expressed as Log cfu/cm2. The study of biofilm eradication was
also performed using the CDC Reactor. AND (16 mg l
1) and
AND+CLA (16 + 20 mg l
1) combination was added to the biofilm
of 24 h of maturation formed on titanium or teflon in the CDC

ª 2015 The Authors

84 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

reactor. At 24, 48 and 72 h, the number of viable cells were deter- P085
mined using the same experimental protocol as in the kinetics studies
of growth.
Results A significant difference (P = 0.03) was observed in the The ability of oral Candida albicans and Candida
amount of C. tropicalis biofilm formed on teflon and titanium. The dubliniensis isolates to form biofilm in vitro
average count on teflon was 4.60 log cfu/cm2 and 2.17 log cfu/cm2 A. Zalupska and U. Nawrot
on titanium. The biofilm exposed to AND had an initial adhesion of Wroclaw Medical University, Wroclaw, Poland
4.79 log cfu/cm2 on teflon and 4.23 log CFU/cm2 on titanium. At
72 h, adhered cells were reduced 48.49% (2.47 log cfu/cm2) on
teflon and 41.06% (2.50 log cfu/cm2) on titanium. The biofilm Objectives The aim of this study was to determine the frequency of
exposed to AND+CLA had an initial adhesion of 4.86 log cfu/cm2 on oral candidal colonisation in healthy adults and to evaluate the abil-
teflon and 4.13 log cfu/cm2 on titanium. At 72 h, adhered cells ity of isolated Candida albicans and Candida dubliniensisstrains to pro-
were reduced 61.92% (1.85 log cfu/cm2) on teflon and 54.14% duce biofilm iv vitro.
(1.89 log cfu/cm2) on titanium. Methods Oral swab samples were taken from 120 healthy volun-
Conclusion C. tropicalis formed stable biofilms after 24 h of contact taries, 79 women and 41 man, aged 19–56 years (mean, 24). Mate-
with the biomaterial and persistently adhered to the surface during rial were cultured on chromID Candida and Sabouraud Gentamicin
the 96 h of the experiment. Teflon was the biomaterial on which the Chloramphenicol 2 agars (bioMerieux) and incubated 7 days, first in
greatest amount of biofilm formed. AND alone was capable of eradi- 37 °C (24 h) and then in 25 °C. Cultured strains were identified on
cating 48% of biofilm formed on teflon and 41% of that formed on the base of morphological and metabolic characteristics with the use
titanium. The combination with clarithromycin was capable of eradi- of ID32C panels (bioMerieux). Biofilm production was tested on 96-
cating 62% of biofilm formed on teflon and 54% of the biofilm wells microplates using YNB medium and crystal violet staining
formed on titanium. A combination of AND plus CLA could be an method.
effective strategy for the eradication of C. tropicalis biofilm. Results Positive cultures were obtained from oral samples from 85/
120 (71%) voluntaries, including 27/120 (22.5%) with heavy (>10
colonies) and 58/120 (48%) with scant (<10 colonies) fungal
growth. The most prevalent species was C. albicans (81 isolates), fol-
lowed by C. dubliniensis (3), C. krusei (1) and C. kefyr (1). Biofilm pro-
P084 duced 23/81 (26%) of C. albicans and 3/3 C. dubliniensis isolates. No
relationship between biofilm production and intensity of candidal
Standardization of a method to evaluate biofilm formation growth in sample cultures was found.
by Candida species Conclusion Candida albicans represent a frequent element of oral
A. L. Qasem Morero,1 J. M. Sanchez-Calvo,2 M. D. Lopez Prieto2 microbiota in healthy adults. A quarter part of oral Candida albicans
isolates produced biofilm in vitro. The link between biofilm production
and M. A. Rodriguez-Iglesias1
1
and the ability to induce infection should be elucidated in future
Hospital Puerta del Mar, Cadiz, Spain and 2Hospital Jerez de la studies.
Frontera, Jerez de la Frontera, Spain

Background Candida cells can form biofilms that frequently are

sources of infections and are less susceptible to antifungal drugs. For P086
this reason, is very important to have a method to evaluate biofilm
formation by Candida spp. isolated of clinical samples. The main aim
of this study was standardize a method to evaluate biofilm formation Anti-Candida effect of Matricaria recutita essential oil
in yeast. So, we studied what Candida spp. were more biofilm C. Y. Koga-Ito,1 G. J. Santos,1 A. C. Borges,2 M. A. C. Oliveira,1
producers. N. P. Lopes3 and I. V. Rodrigues3
Material and method We isolated 48 Candida spp.: Candida albicans 1
Universidade Estadual Paulista UNESP, Sa~o Jose dos Campos,
(N = 24) and Candida glabrata (N = 24) from different human reser- Brazil; 2ICT-UNESP, Sa~o Jose dos Campos, Brazil and
voirs (bloodstream, respiratory and vaginal tract). C. albicans was 3
Universidade de Sa~o Paulo, Ribera~o Preto, Brazil
used to standardize this method. Quantitative measurement of biofilm
formation was assessed by XTT reduction assay. We used different
incubation time (24 vs. 48 h) and glucose concentration (2%, 5% Objectives The aim of this study was to evaluate the anti-Candida
and 10%). Finally, we compared the biofilm formation between C. effect of Matricaria recutita essential oil.
albicans versus C. glabrata. We studied if the biofilm formation could Methods Matricaria recutita essential oil was chemically character-
be affected by the site of isolation of Candida spp. All experiments ized by gas chromatography mass spectrometry (GC/MS). The effects
were repeated three times and the results were expressed as average of Matricaria recutita essential oil on Candida albicans (ATCC 18804),
values of absorbance. C. glabrata (ATCC 90030), C. parapsilosis (ATCC 22019), C. krusei
Results The best conditions for biofilm formation by C. albicans were (ATCC 6258), C. tropicalis (ATCC 13803) and C. dubliniensis (NCPF
48 h of time of incubation and 2% glucose concentration. C. albicans 3108) were assessed. Antifungal activity was screened by agar diffu-
strains were more biofilm producer than C. glabrata strains (0.519 sion test. After, the minimal inhibitory concentration values (MIC)
vs. 0.214) (P = 0.007). In both, biofilm formation was higher in were determined by microdilution tests in RPMI broth buffered with
Candida spp. isolated from bloodstream than respiratory and vaginal MOPS. Values of minimal fungicide concentration (MFC) were
tract, respectively, although this was not statistical significance differ- obtained by subculture in Sabouraud dextrose agar. The activity on
ence (P > 0.05). 24 h pre-formed biofilms was also evaluated. Biofilms were formed in
Conclusions This method has demonstrated to be repeatable and RPMI + 2% glucose at 37°C for 24 hours. Then, they were exposed
reproducible in all strains studied. C. albicans strains are strong bio- for 5 min to concentrations 2 and 4 times MIC. The cell viability
film producers and they could be used as model of biofilm formation after exposition was determined by serial dilutions and plating. All
in yeast to study the antibiofilm activity of new agents. No associa- the experiments were performed in duplicate in three different occa-
tion was found between strain’s site of isolation and biofilm sions. Nystatin was included as positive control in all experiments.
formation. The results were compared by ANOVA and post hot Tukey’s test, at
level of significance of 5%. Results: A total of 33 compounds were
detected by GC/MS. Bisabolol oxide was the majority compound, fol-
lowed by b-farnesene. Agar diffusion tests revealed inhibitory activity
against all the tested species. MIC values varied largely among the
tested species (0.1–12.5% v/v). The most susceptible species was C.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 85
Poster Presentations

glabrata and the less susceptible was C. krusei. Fungicide effect was P091
also observed for all the species, with CFM values ranging from 0.30
to 25% v/v. Exposition to 2 and 4 times MIC significantly reduced
cell viability of C. tropicalis, C. parapsilosis and C. krusei pre-formed Experimental study to assess the virulence of clinical
biofilms when compared to non-exposed control (P < 0.01). For C. strains of Candida-psilosis complex, through the use of
tropicalis and C. parapsilosis, final cell counts were similar to those ‘eggs model’ as an animal model
observed after treatment with nystatin (P > 0.05). Conclusion: Matri- V. Prete,1 R. Torelli,2 B. Posteraro3 and M. Sanguinetti3
caria recutita essential oil showed promising anti-Candida activity, in 1
Institute of Microbiology, Universita Cattolica del Sacro Cuore,
particular against C. tropicalis and C. parapsilosis.
Rome, Italy; 2UCSC, Roma, Italy and 3Universita Cattolica del S.
Cuore, Roma, Italy

P090 Objectives Candida spp is a commensal yeast in the healthy human

being but could be pathogen in human being with a reduced or mod-
ified level defenses disorder. In the study of these pathogens, the ani-
Echinocandin resistant Candida parapsilosis bioprosthetic mal models play an important role, embryonated eggs have been
valve endocarditis successfully treated with combined used to study features virulence various bacteria and viruses, instead
medical and surgical treatment of in this study, we have investigated the pathogenesis of Candida-
D. Kofteridis, A. Andrianaki, S. Maraki, A. Christidou, psilosis complex infections in chicken embryos infected via the
G. A. Stamatiades and G. Samonis chorio-allontoic membrane (CAM) and we have analyzed the viru-
lence and pathogenicity.
University of Crete, Heraklion, Greece Methods The embryoneted eggs of chicken are an animal model
used also for the growth of other pathogens. In this study we realize
Objectives Candida parapsilosis is a rare cause of infectious endo- an inoculum Candida parapsilosis ATCC, Candida orthopsilosis ATCC,
carditis, associated with high mortality rate, occurring usually in Candica metapsilosis ATCC in order to provoke an infection in the
prosthetic valve recipients and intravenous-drug abusers. A rare case CAM. The CAM is composed with allantoide and corion, envelops
of echinocandin resistant Candida parapsilosis aortic valve endocardi- completely the embryo.
tis, successfully treated with valve replacement in combination with For each experiment the eggs have been placed in incubator at
liposomal amphotericin B (L-AMB) and micafungin regimen is 37 °C and 60% of humidity, daily have been supervised the quality
described. and vitality of the embryos. Considering the life cycle of embryo, lasts
Case presentation A 54-year-old man with bioprosthetic aortic 21 days before arriving to the inception, in the 8th has been carried
valve presented with fever and pleuritic chest pain. Three months out the inoculum. Candida-psilosis complex has been placed in YPD at
earlier he suffered acute gallstone pancreatitis and underwent chole- 37 °C over night. The day after has been done the inoculum with
cystectomy, complicated by cholorrhea and fever. Stents were placed 108 and 107 CFU of each strains. In order to realize the inoculum,
at the pancreatic and the common bile duct, while two sets of blood two holes have been made in the shell, one in the air chamber and
cultures grew Candida parapsilosis. He was treated with fluconazole the other at 90 grade compared to the first. In the second hole the
until 2 weeks after fever’s resolution. A transthoracic echocardio- eggs have been infected, these eggs have been placed in incubator
gram at that time did not reveal signs of infectious endocarditis (IE). monitoring the temperature, the humidity and the vitality. From the
The patient had history of systemic lupus erythematosus treated with eggs no longer vital have been removed the CAM, and have been
hydroxychloroquine, hypertension treated with metoprolol and qui- done culture on agar plates and histologic examination.
napril and epilepsy treated with oxcarbazepine. Upon the present Results From the data obtained it is evident that some factors nega-
admission he was febrile (38 °C) and examination revealed a systolic tively affect embryo viability and the survival rate drops consider-
murmur of 3/6. An echinocandin resistant Candida parapsilosis has ably. Infection on the developmental day 8 with 108 cfu/egg leds to
been isolated from 4 sets of blood cultures and he was commenced a rapid higher mortality. This depends on the development of the
on L-AMB and fluconazole. A 9 mm aortic valve abscess was embryo and the infectious dose. Infection does of 108 resulted high
detected at the transesophageal echocardiogram, causing severe mortality rate. While the percentage of survival increases consider-
valve dysfunction, while a computed tomography of the abdomen ably with a concentration of 107. The less virulent strain are Candida
revealed multiple splenic emboli. Thus, by Duke’s criteria, the patient orthopisilosis.
was suffering candidal endocarditis of the aortic valve. Due to the Conclusions Through the use of the eggs model, in every experi-
severity of the disease and the persistence of fungemia, he underwent ments that we have been made, all the pathogens analyzed have
aortic valve replacement, and was commenced on L-AMB been noticed by culture and histologic assay. our results suggest that
(5 mg kg
1 od) and micafungin (150 mg od) for a total duration of embryonated eggs can be a very useful alternative infection model to
3 months. Furthermore, biliary and pancreatic stents were removed study Candida spp virulence and pathogenicity.
and a biliary sphincterotomy was performed. He was discharged on a
continuing regimen of oral fluconazole (400 mg d
1). During a
24 months follow-up the infection did not recur.
Conclusions The present case emphasizes the importance of Candida P092
parapsilosis as nosocomial pathogen for endocarditis in prosthetic
valve recipients. Since optimal therapy has not been established yet,
a combination of surgical and prolonged antifungal treatment is Fluconazole treatment response in a Galleria melonella
suggested. infection model using wild-type and trailing C. tropicalis
isolates- a cause for concern?
K. M. T. Astvad and M. C. Arendrup
Statens Serum Institut, Copenhagen, Denmark

Objective Despite being generally fluconazole susceptible some C.

tropicalis isolates display ‘trailing’ growth. Trailing growth was origi-
nally defined for isolates classified as susceptible after 24 h of incuba-
tion but resistant after 48 h using the 80% growth inhibition
endpoint recommended for the CLSI M27A methodology. Such iso-
lates were found susceptible in vitro if using a 50% growth inhibition
endpoint and in vivo in a mouse model of disseminated candidiasis.

ª 2015 The Authors

86 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

On this background the CLSI methodology was subsequently revised Results A dose dependent response was observed for all isolates
in 2011 with recommendation of an endpoint reading after 24 h (table). Only the highest dose of 20 mg kg
1 larvae was able to pro-
using a 50% inhibition. tect >50% of the larvae (table). For this dose mortality was signifi-
Using the EUCAST EDef 7.2 susceptibility testing a noticeable pro- cantly different comparing larvae infected with the five strains
portion of C. tropicalis isolates display only approx. 50% growth inhi- [P = 0.002 log-rank (Mantel Cox)]. Mortality among larvae chal-
bition over a broad concentration range already at 24 h, lenged with the trailing isolates (20 mg kg
1 group) was similar to
complicating MIC determination. How these isolates respond to stan- that for larvae challenged with the resistant isolate and statistically
dard doses of fluconazole is unclear, and recommendations for treat- higher than for larvae challenged with the susceptible (P ≤ 0.0001)
ment is lacking. We therefore tested C. tropicalis isolates with various [log rank (Mantel-Cox)] for T1 whereas only numerically for T2
fluconazole susceptibility patterns in an in vivo larvae model in order (P = 0.18) (figure).
to ascertain whether trailing and wild-type (wt) isolates respond Conclusion The two trailing C. tropicalis isolates were both less sus-
equally well to fluconazole treatment. ceptible to fluconazole in vivo when compared with the wt isolate.
Method Four clinical isolates of C. tropicalis (one wt (MIC: Mortality rates for the trailing isolates were similar to those for the
0.5 mg l
1), two trailing (T1 and T2), one resistant MIC: resistant isolate. For T1 this difference reached statistical significance.
>16 mg l
1) (R)) and one type strain (ATCC750, MIC 1 mg l
1) This observation questions if fluconazole is an appropriate choice for
were selected. Last instar Galleria mellonella larvae weighing approxi- infections due to such isolates and strongly suggests further studies
mately 278 mg (range: 250–325) were chosen. Groups of 15 larvae on this issue are warranted.
were injected with 10 lL aliquots of each Candida suspension
(~ 5*107 CFU ml
1) at the last left proleg (giving a 100% mortality
for all isolates), followed after 30 min with 10 lL of PBS (control) or
fluconazole (adjusted to 0.25, 1, 5, and 20 mg kg
1 larvae) injected P093
in the last right proleg. 20 mg kg
1 provides an AUC of approxi-
mately 408 mg*h l
1 corresponding to that achieved in humans on
standard dosing. Uninfected larvae receiving only PBS or fluconazole Efficacy of caspofungin, fluconazole and micafungin for
were included as control (100% survival). Larvae were kept in the the treatment of the infection caused by Candida glabrata
dark at 37 °C. Survival was monitored daily for 5 days. Target complex in the non-conventional model Caenorhabditis
inoculum concentration was confirmed by CFU determination using elegans
the spot technique (20 ll spots of 10-fold dilutions). A. Hernando, M. Ortega-Riveros, I. de la Pinta, E. Eraso and
G. Quindo  s1
Univ. Paıs Vasco UPV/EHU Facultad de Medicina y Odontologıa,

Bilbao, Spain

Objectives To study the virulence of Candida glabrata, Candida

bracarensis and Candida nivariensis in a Caenorhabditis elegans model of
invasive candidiasis and to evaluate the efficacy of caspofungin, flu-
conazole and micafungin for the treatment of these infections.
Methods Ten thousand nematodes (AU37 mutant strain with
increased susceptibility to various pathogens) were distributed in
groups of 1700 individuals and infected with each of the following
reference strains: Candida glabrata NCPF 3203, Candida glabrata ATCC
90030, Candida nivariensis CBS 9984, Candida nivariensis CECT
11998, Candida bracarensis NCYC 3397 and Candida bracarensis NCYC
3133. The nematodes were distributed onto agar plates with the dif-
ferent species of Candida and allowed to feed for 2 h at 25 °C. After-
Figure 1 wards, 20 individuals were dispensed into each well of a microtitre
plate that contained M9 buffer, 10 lg ml
1 of cholesterol in ethanol,
and 90 lg ml
1 of kanamycin. For the evaluation of treatment effi-
Table 1 cacy, the nematodes were treated with 4 and 8 lg ml
1 of caspo-
fungin and micafungin, and 32, 64 and 128 lg ml
1 of fluconazole.
The plate was incubated at 25 °C overnight and a visually scoring of
live and dead worms was done at 24 h for 6 days. The survival
obtained with each treatment was analyzed with Kaplan-Meier sur-
vival curves and the differences between them were estimated by the
log-rank test with SPSS 15.0 software.
Results The infection by Candida glabrata and Candida nivariensis
strains reduced significantly the survival of Caenorhabditis elegans in
comparison to non-infected nematodes. However, no differences in
survival were obtained among nematodes infected with Candida
bracarensis and uninfected nematodes. Candida nivariensis was less vir-
ulent than Candida glabrata (P < 0.05). Survival of the nematodes
infected with Candida glabrata and Candida nivariensis was significantly
higher after treatment with candins at 120 h (P < 0.05). Whereas
the higher survival of the nematodes infected with Candida glabrata
and Candida nivariensis CBS 9984 was after treatment with
8 lg ml
1 of micafungin, the highest survival of the nematodes
infected with Candida nivariensis CECT 11998 was reached with
8 lg ml
1 of caspofungin.
Conclusions Candida glabrata was the most virulent species. This
Caenorhabditis elegans model was not useful for assessing Candida
bracarensis infection. Candins were effective for the treatment of can-
didiasis caused by Candida glabrata and Candida nivariensis.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 87
Poster Presentations

Funding This study was financed in part by projects PI11/00203

(FIS, Gobierno de Espa~na), GIC12 210-IT-696-13 (Gobierno Vasco-
Eusko Jaurlaritza) and UFI 11/25 (UPV/EHU).

P094

Clinical significance and results of fungal cultures in

surgical patients treated with antifungals due to intra-
abdominal infections
V. Avkan-Oguz,1 M. Abdullayeva,1 O. Eren-Kutsoylu,1
M. Ozbilgin,1 E. Firuzan,2 S. Sokmen,1 M. Doluca-Dereli1 and
N. Yapar3
1
Dokuz Eylul University Faculty of Medicine, Izmir, Turkey;
2
Dokuz Eylul University Faculty of Science, Izmir, Turkey and
3
Dokuz Eylul University, Izmir, Turkey

Objectives Definitions of candidemia, invasive candidiasis and can-

dida peritonitis confuse the minds of physicians in Surgical Patients
with health-care associated infections. This confusion is caused by patients used beta lactam and beta-lactamase inhibitory antibiotics
overuse of antifungal agents. We aimed to determine the rate of anti- was 26 (44%) in culture positive group and 10 (18.1%) in culture
fungal use and to evaluate the results of fungal cultures and clinical negative group (P = 0.002). The most common antifungal used was
management together about fungal intra-abdominal infections in fluconazole (94 patients 82.5%) followed by anidulafungin (5 patient
general surgery patients treated with various antifungals. 4.4%) and caspofungin (4 patient 3.5%), also in 11 (9.7%) patients
Methods The list of patients to whom antifungals were given was antifungal drugs had been changed.
obtained from the hospital pharmacy between January 1, 2012 and Conclusion Indications for use of antifungal drugs are not clear in
December 31, 2014. Mycology laboratory results and patients’ intra-abdominal infections, In our study, especially in immunocom-
records were examined for mycological and clinical data of adult promised patients regardless of culture positivity use of antifungal
(>18 years) patients, retrospectively. Patients were seperated into agents was more common. Significant risk factors detected in the cul-
two groups according to the fungal culture positivity. The data was ture positive group were not different from those for all other candida
analysed statistically. Multivariate, logistic regression analysis was infections. In addition, beta lactam and beta-lactamases inhibitory
performed for the determination of independent risk factors for cul- antibiotic usage in early periods of hospitalization increased fungal
ture positivity. culture positivity. Antimicrobial stewardship strategies will reduce
Results During the study period, 114 patients had used systemic fungal infection.
antifungal treatment. None of the patients received antifungal pro-
phylaxis. Of the 114 patients, 59 (51.7%) had positive fungal cul-
tures, 55 (48.3%) had negative fungal cultures. The demographic
characteristics of the patients and risk factors are presented in the
table 1 and table 2. There were more than one culture positivity in P100
different clinical specimen of 12 (20.4%) patients. Of culture positive
patients, 31 (52.6%) had C. albicans. Laparoscopic procedures were Fungal Rhinossinusitis: a 8 year review study
performed in only two patients in culture positivite group. In multi-
R. Melo Cabral, J. Pimentel, V. Oliveira, M. F. Cruz, C. Toscano,
variate logistic regression analysis, the significant risk factors for fun-
gal culture positivity were determined to be parenteral nutrition J. Batista, D. Silva and P. Escada
(P = 0.04), previous beta lactam and beta lactamase inhibitory Hospital de Egas Moniz, Lisbon, Portugal
antibiotic usage (P = 0.005), postoperative stay in ICU (P = 0.05)
and the initiation of antibiotics in the early period of hospitalization Objectives Fungi have a controversial role in rhinossinusitis and
(P = 0.05). In the first week of hospitalization, the number of there are still some difficulties in the categorization of fungal rhinoss-
inusitis, according to its clinical presentation, and to associated
immune and pathological responses. We present a review of patients
with fungal rhinossinusitis diagnosed and treated in our institution
in the past 8 years.
Methods Case review of patients diagnosed with fungal rhinossinusi-
tis. Clinical charts were reviewed, and data collected concerning clin-
ical presentation, surgical and medical treatment, results of
histophatologic examination, direct examination and culture of
samples.
Results 9 patients were enrolled in this study, including two cases
of invasive disease with accurate diagnosis of the involved fungus,
and 7 cases of non-invasive disease. The later included 4 cases of
fungus ball, in which the agent could be detected from histopatho-
logic examination, but there was no isolation from samples. In the
other 3 cases of non-invasive disease, there was a high index of sus-
picion of a fungal rhinossinusitis from the imaging exams, surgery
findings and the presence of an eosinophilic mucin at histopathologic
examination, but no fungi was found on culture, and they were sub-
sequently classified as eosinophilic mucin rhinossinusitis.
Conclusion Fungi are a relatively rare etiology of rhinossinusitis
and a controversial topic that includes the unclear role of fungi in

ª 2015 The Authors

88 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

diseases such as eosinophilic mucin rhinossinusitis. Culture and

direct examination are sometimes difficult to obtain, however they
are essential for a correct classification of fungal rhinossinusitis. Ulti-
mately it allows the choice of targeted therapy.

P101

Rare cause of the diabetic foot: invasive fungal infections

€ rk, B. Arda, D. Metin and
T. Meltem, S. Uysal, A. Oztu
H. Pullukcu
Ege University Medical School, Izmir, Turkey

Objectives Diabetic foot infections compromise an important part of

complicated skin and soft tissue infections and are expected to
increase even more in the future. Diabetic foot infection complication
rate in diabetes is increasing variety of factors; inefficient treatment
of osteomyelitis and its chronic trend, resistant organisms commonly
Figure 2. Patients demographic data and wound characteristics-2
seen in diabetic foot infection. Most of the infections in diabetic foot
are of aerobic and anaerobic bacterial origin, and in most cases
polymicrobial, which has been characterized in detail although a few
and mycology of the ulcers were summarized in table 1. Four
studies have reported some filamentous fungi and yeasts as etiologi-
patients have history of prior diabetic foot infection and arterial nar-
cal agents of diabetic foot infections. Fungal infections can appear as
rowing causing decreased blood flow on their lower extremities. In
superficial or invasive tissue infections. Here, it was aimed to evalu-
four of the cases antifungal therapy was also given besides the surgi-
ate fungal etiology of diabetic foot infections between June 2012–
cal treatment. Antifungal therapy was not given to two patients
April 2015 prospectively.
because they died before the culture results. Five patients were found
Methods Diabetic foot infection cases that were admitted between
to be positive for fungal culture after the amputation procedure.
June 2012 to April 2015 and followed up by Ege University Faculty
Since surgical wound boundaries were clean, antifungal theraphy
of Medicine, Department of Infectious Diseases and Clinical Microbiol-
was not given to these cases.
ogy were recorded. Demographic data of patients, diabetes character-
Conclusion Although rare, invasive fungal infections can cause dia-
istics, wound characteristics, laboratory findings, physical
betic foot infection. Even the the superficial fungal infections are
examination findings, presence of osteomyelitis, presence of signifi-
more frequent in patients with diabetes we should keep an open
cant vascular stenosis, culture results, PEDIS infection classification
mind and to consider the possibility of invasive funcal infections are
scores, operation types if necessary were recorded. Bone or deep tis-
also the causative pathogen in deep tissue infections. Because of that
sue samples were cultured on two SDA medium which were incu-
we should think fungal pathogens in cases these do not respond the
bated 26 and 35 °C at 10 days. The yeast colonies were identified by
long-term antibiotic therapy. Treatment must include surgical
conventional methods (germ tube and their micromorphological
debridement and antifungal therapy. Fluconazole may be a reason-
appearance on corn meal agar) and ID32C (bioMerieux-France) car-
able alternative to amphotericin B in fungal osteomyelitis. Also
bohydrate assimilation features. Filamentous fungi were evaluated
osteomyelitis caused by fungal pathogens in literature are very few.
depending on their appearance on media and microscopy using lac-
This study is one of the large series in the literature regarding
tophenol cotton blue.
invasive fungal infections in patients with diabetic foot infection.
Results Bone or deep tissue cultures were positive in eleven of 531
cases who were followed with the diagnosis of diabetic foot infection
(8 female, 3 male, mean age 59.7  9 years). Age, gender, diabetes
type, wound characteristics, treatment types and results, bacteriology
P102/M6.1

An uncommon cause of primary adrenal insufficiency in

Central Europe
G. Wagner,1 B. Willinger,2 A. Eckhardt,1 U. Sagel,3 F. Wrba4 and
K. Dam1
1
University Hospital St. Poelten, Clinical Department of Internal
Medicine II, St. Poelten, Austria; 2Medical University of Vienna,
Division of Clinical Microbiology, Vienna, Austria; 3University
Hospital St. Poelten, Institute for Hygiene and Microbiology, St.
Poelten, Austria and 4Medical University of Vienna, Department
of Clinical Pathology, Vienna, Austria

Objectives In Western countries autoimmune processes are the most

common reason for primary adrenal insufficiency (Addison’s disease),
however in countries of high prevalence disseminated tuberculosis or
systemic mycosis are major causes. Aim of our case presentation is
to bear in mind the similarity of tuberculosis and systemic fungal
infections in a patient presenting with bilateral adrenal enlargement
and primary adrenal insufficiency.
Methods We report a single case of a systemic fungal infection asso-
ciated with primary adrenal insufficiency caused by the dimorphic
Figure 1. Patients demographic data and wound characteristics-1 fungus Paracoccidioides brasiliensis.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 89
Poster Presentations

revealed a cortical temporo-parietal located lesion, suspicious for

infection or metastasis. Liquor fluid of lumbar puncture was unre-
markable. Serum levels of basal ACTH and cortisol confirmed pri-
mary adrenal insufficiency, considering bilateral adrenal gland
enlargement and medical history highly consistent with reactivation
of tuberculosis. However, all available reports of prior hospitalizations
did not reveal any microbiological confirmation of mycobacterial
infection. Subsequently CT guided biopsy of the left adrenal gland
and extirpation of a right cervical lymph node were performed, in
neither tissue nor fluid Mycobacterium tuberculosis was detectable.
Fludrocortisone and hydrocortisone were commenced for adrenal
insufficiency. Histology of left adrenal gland biopsy showed granulo-
mas with central necrosis. PAS, McManus and Grocott stain revealed
evidence of fungal pathogens. Paracoccidioides brasiliensis was iden-
tified by microscopy (Giemsa stain), culture (Sabouraud agar) and
PCR. Due to evidence of cerebral involvement the patient was com-
menced on intravenous liposomal amphotericin B followed by oral
itraconazol.
Conclusion Paracoccidiomycosis (South American blastomycosis), a
systemic fungal infection, should be considered for differential diag-
nosis in patients travelled to or lived in South or Central American
countries. Furthermore we want to emphasize the clinical and
histopathological similarities with tuberculosis to avoid misdiagnosis.

P103
Figure 1. Abdominal CT scan
Virulence of Trichoderma longibrachiatum and
experimental treatment of invasive infection
K. V. Paredes,1 J. Capilla,1 D. A. Sutton2 and J. Guarro1
1
Universitat Rovira i Virgili, Reus, Spain and 2The University of
Texas Health Center, San Antonio, USA

Objective To assess the virulence of Trichoderma longibrachiatum, and

to evaluate the efficacy of Amphotericin B deoxycolate (AMB), Lipo-
somal Amphotericin B (LAMB), Voriconazole (VRC) and Micafungin
(MFC) in an immunosuppressed murine model of invasive infection.
Methods Cyclophosphamide-immunosuppressed male OF-1 mice
with a mean weight of 30 g were used. Virulence was evaluated
through survival (8 animals/group) and fungal burden reduction in
kidney, spleen, liver, lung and brain (8 animals/group), after i.v.
infection with 2 strains of T. longibrachiatum (UTHSC 07-3636, 09-
2900) at 1 9 104, 1 9 105, 1 9 106 or 19 107 CFU/animal. For
treatment, mice challenged with 1x107 CFU/animal of T. longi-
brachiatum received AMB at 0.8 mg kg
1 i.v., once a day (QD);
LAMB at 20 mg kg
1 i.v., QD; VRC at 25 mg kg
1 p.o, by gavage
Figure 2. Giemsa stain QD; or MCF at 10 mg kg
1 i.p., QD. Efficacy was evaluated through
survival rates and of fungal load reduction in liver and spleen.
Results The virulence study showed an inoculum size-dependent
Results A 62-year-old man was admitted to our hospital with gener- response with 25%, 25% and 60% mortality 15 days after infection
alized weakness, left sided chest and abdominal pain, weight loss and with 1 9 104, 1 9 105 and 1 9 106 CFUs, respectively, while the
night sweats. Past medical history revealed several episodes of pul- highest inoculum showed a mortality rate of 100%.
monary tuberculosis, initially diagnosed at the age of 7, followed by On day 6 post infection, fungal cells were recovered from all the
several reactivations, recurrently treated with tuberculostatic ther- organs in mice infected with higher inocula. The number of CFUs
apy. Five years prior to admission tuberculosis of adrenal glands was recovered correlated with the inoculum size. Significantly higher
diagnosed by CT guided biopsy at another hospital based on counts were found in animals receiving 1 9 106 or 1 9 107 CFU/
histopathological finding of epitheloid cell granuloma with central animal compared with those challenged with lower inocula. Spleen
necrosis. The patient is currently retired. More than 10 years ago he and liver were the most affected organs regardless of the strain and
lived for several years in a rural area of Peru located next to the the inoculum size assayed.
Amazonas to build a hospital. He had a 45 pack-year history of AMB, LAMB, VRC and MCF were unable to prolong survival of
smoking. CT scan showed an increasing inhomogeneous enlargement mice compared with the controls, irrespective of the infective strain.
of both adrenal glands and alteration of the upper lobe of the left VRC and MCF were not able to reduce fungal loads in spleen or liver
lung, both suspicious for reactivation of tuberculosis. Afterwards per- of mice challenged with any of the strains; however, AMB reduced
formed PET scan revealed an increased metabolic activity of the the burden in liver of mice infected with the strain UTHSC 07-3636,
enlarged adrenal glands, hypermetabolism in cervical lymph nodes (AMB MIC of 0.13 lg ml
1) and LAMB showed a significant reduc-
but not the lung. Ziehl-Neelsen smear of the sputum showed no acid- tion over controls and other therapies in both organs of animals
fast bacteria and PCR as well as culture were negative for Mycobac- infected with this strain (P ≤ 0.0002).
terium tuberculosis. Serology for HIV, lues and adrenal autoantibod- Conclusion This is the first study to develop an immunosuppressed
ies were negative. Cerebral imaging due to neurologic symptoms murine model of disseminated infection by T. longibrachiatum. Only

ª 2015 The Authors

90 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

higher inocula were able to invade and proliferate in all organs

tested.
No therapies improved survival and, with the exception of LAMB,
no therapy was effective in reducing fungal loads. LAMB was effec-
tive against the strain with the lowest MIC for AMB (0.13 lg ml
1),
while no reduction was observed in mice infected with the strain
UTHSC 09-2900 (MIC 2 lg ml
1).
The high level of resistance shown in our study is of concern. Fur-
ther studies should be carried out with more isolates that represent a
wider range of AMB MICs in order to assess whether there is any
relationship between MIC values and efficacy of LAMB.

P104

A cerebral aspergillosis case whom treated with

intrathecal amphotericin B
B. Kurtaran, A. S. Inal, F. Kuscu, A. Ulu, S. Komur, A. Yildirim,
E. Gurkan, H. S. Z. Aksu and Y. Tasova
Cukurova University, Adana, Turkey

Objective Early diagnosis of aspergillosis of central neural system

with non-typically clinical and cerebrospinal fluid findings is so
important. The disease is dramatically progressive, its treatment is
quite difficult, and it has high mortality rates. We represented our Figure 2. Fungal hypha’s which detected with PAS (periodic acid
experience with cerebral aspergillosis patient who treated with non- shift) dye
clasical.
Method CASE: 47 years old, male patient who had chronic lympho-
cytic leukaemia (CLL) since 2011 and treated with 11-dose and confusion were seen in his follow-up but anti-epileptic treatment
chemotherapy and additionally he had been followed for type 2 dia- was started for local convulsions, which were seen on his left arm.
betes mellitus for 10 years. In October 2014 ibrutinib, a tyrosine New lesions were seen in control MRI examination after 4 weeks
kinase inhibitor, treatment were given to him. Weakness in left lower voriconasol treatment and upon this control cerebrospinal fluid cul-
and upper extremities and speech disorder were developed at the ture were done. Voriconasol treatment was prolonged due to nega-
tenth day of his treatment and he was hospitalized with early diag- tive cerebrospinal fluid culture. Occasionally diazepam infusion was
nosis of cerebral mass or bleeding. done because of the on going local convulsions and a generalized
Multiple lesions with peripheric contrast enhancement were seen convulsion. Regression on the contrast and the shapes of the lesions
in fronto-parietal cortex and sub-cortex in his magnetic resonance and a new lesion on right frontal lob were found in control MRI of
imaging (MRI) examination (image 1). Upon this lomber punction the voriconasol treatment on the 52nd day. Due to lack of
were done and non-specific meningitis treatment and trimetoprim- histopathologic diagnosis and new lesions that occurred despite treat-
sulfamethoxasol (TMP-SMZ) medication were started for cerebral tox- ment brain biopsy were planned to the patient. ‘Abscess, necrosis
oplasmosis and cerebral abscess. However on the fourth day of the and fungal hypha’s in necrotic areas’were found as the result of the
treatment, Aspergillus niger were detected in his cerebrospinal fluid biopsy, which were done from frontal lob (image 2). Due to new les-
culture. Upon this, non-specific meningitis treatment and TMP-SMZ sions and confirmation of histopathologic diagnosis, 0.1 mg intrathe-
treatment were terminated and voriconasol treatment was started as cal conventional amphotericin B treatment were added two times in
4 mg kg
1 in every 12 h after 6 mg kg
1 bolus infusion. No fever a week to treatment. Regression with no new lessions was found in
the cerebral MRI with contrast after 8 dose of intrathecal ampho-
tericin B. After additionaly 2 dose intrathecal amphotericin B, patient
was discharged with oral voriconasol treatment.
Conclusion Our experience with this case showed us intrathecal
amphotericin B could use for treatment of the patients who had no
response to voriconasol. This case could be the first case in literature
that shows the usage of intrathecal amphotericin B for this
indication.

P105

A randomised study on the safety of daily, intermittent, or

weekly administration of 1, 3 or 10 mg kg
1 of AmBisome
as prophylaxis in acute leukaemics
C. O. Morrissey,1 A. P. Schwarer,2 S. Patil,1 A. Kalff,1 A. Wei,1
P. Walker,1 S. Avery,1 A. Spencer1 and H. de Silva3
1
Alfred Health, Melbourne, Australia; 2Box Hill Hospital, Box Hill,
Australia and 3Burnet Institute, Melbourne, Australia
Figure 1. Multiple lesions with peripheral contrast enhancement in
fronto-parietal cortex and sub-cortex in cerebral magnetic resonance Background Antifungal prophylaxis is effective in reducing invasive
imaging with contrast fungal disease (IFD) incidence and mortality in patients undergoing

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 91
Poster Presentations

induction-consolidation (IC) chemotherapy for acute leukaemia. Spec- Methods TheMICs of voriconazole (VRC) against 40 strains of S.
trum of activity, bioavailability, drug-drug interactions, and toxicity apiospermum was determined using a microdilution broth method
mean that no one antifungal agent is universally applicable. High (CLSI). Fifteen S. apiospermum strains showing different MICs ranging
dose liposomal amphotericin B (L-AmB) administered intermittently from 0.12 to 8 lg ml
1 were selected for efficacy studies. Groups of
can achieve adequate tissue levels for effective prophylaxis, is safe 16 OF-1 male mice (8 for survival studies and 8 for fungal load
and well tolerated. To date, the safety and efficacy of different dosing determination) were immunosuppressed with 200 mg kg
1 of
regimens of L-AmB as antifungal prophylaxis has not been directly cyclophosphamide every 5 days, starting 2 days before infection.
compared in the acute leukaemic setting. Animals were challenged intravenously with 5x103 CFU via the lat-
Methods In this open-label, parallel-group randomised controlled eral tail vein and received VRC at 40 mg kg
1, starting 1 day after
trial patients ≥18 years receiving IC chemotherapy for acute myeloid infection and for 7 days, or no treatment (control group). When con-
(AML) or acute lymphoblastic leukaemia (ALL), with no history of trol animals began to die, animals were euthanased and kidneys and
IFD were randomised (1:1:1:1) to either 1 mg kg
1 daily, 3 mg kg
1 brain were aseptically removed to determine CFU g
1. Efficacy was
twice-weekly, 3 mg kg
1 three times-weekly or 10 mg kg
1 once- evaluated by fungal burden in brain and kidney and by survival.
weekly of L-AmB. Patients commenced their respective dosing regi- Results In those animals infected with the strains showing the high-
men of L-AmB within 96 h of commencement of each course of est MIC values (4–8 lg ml
1), VRC was not able to increase survival
chemotherapy and were followed up for 52 weeks or until death, if or to decrease fungal burden while for those with MICs of 0.12–
earlier. The primary endpoint was the rate of all study drug-related 0.5 lg ml
1 the drug showed significant efficacy. In those mice chal-
adverse events (AE) by completion of all L-AmB prophylaxis. Second- lenged with strains showing MICs of 1–2 lg ml
1, we observed no
ary end-points included incidence of proven or probable IFD accord- correlation between survival and fungal burden and efficacy seems to
ing to the EORTC/MSG (European Organization for the Research and be intermediate.
Treatment of Cancer/Mycoses Study Group) criteria and overall and Conclusion Our results reveal a correlation between in vitro data
IFD-related mortality. Analysis was by intention-to-treat and included and in vivo outcome. Although more strains will be tested to deter-
all enrolled patients who received at least one dose of L-AmB. This mine more accurately the relationship between MIC values and out-
trial is registered with ClinicalTrial.gov as NCT00451711. come, our preliminary results seem to indicate that VRC MICs ≥ 4
Results 53 eligible patients were recruited from two Australian cen- are indicative of resistance while ≤ 0.5 are indicative of susceptibility.
tres between Aug 2008 and Mar 2014. Seventeen, 3, 18 and 15 were
randomly assigned to 1 mg kg
1 daily, 3 mg kg
1 twice-weekly,
3 mg kg
1 three times-weekly or 10 mg kg
1 once-weekly of L-AmB,
respectively. One patient randomised to 1 mg kg
1 daily arm did not P107
receive a single dose of L-AmB. Fifty-one patients had AML and 2 had
ALL. Of the 52 patients who received at least one dose of L-AmB 6/16
(37.5%), 2/3 (66.7%), 10/18 (55.6%) and 9/15 (60%) of patient Successful treatment of a proven Aspergillus flavus
receiving 1 mg kg
1 daily, 3 mg kg
1 twice-weekly, 3 mg kg
1 three rhinosinusitis in a leukemia patient - Feasibility of
times-weekly or 10 mg kg
1 once-weekly of L-AmB, respectively had intravenous 24 h continuous infusion of Amphotericin B
a study drug-related AE (P = 0.67) with a serious AE recorded in 1/ deoxycholate
16 (6.3%), 0/3 (0%), 2/18 (11.1%) and 2/15 (13.3%), respectively M. Schneemann, Y. Kreis, B. Gerber, E. Marques Maggio and
(P = 0.85). Proven and probable IFD were diagnosed in 1/16 (6.3%), A. Imhof
0/3 (0%), 2/18 (11.1%) and 1/15 (6.7%) of those assigned to
1 mg kg
1 daily, 3 mg kg
1 twice-weekly, 3 mg kg
1 three times- University Hospital, Zurich, Switzerland
weekly or 10 mg kg
1 once-weekly of L-AmB, respectively
(P = 0.89). Overall mortality was similar between the groups: 5/16 Objectives 24 h continuous infusion of Amphotericin B Deoxy-
(31.3%), 1/3 (33.3%), 1/18 (5.6%) and 4/15 (26.7%), respectively cholate is a way of minimizing side effects of amphotericin B while
(P = 0.25). There were 2 IFD-related deaths in the1 mg kg
1 daily maintaining efficacy in invasive aspergillosis as shown previously
arm but no IFD-related deaths occurred in those assigned to either of (BMJ 2001;322:579). The number of proven aspergillus infection,
the other 3 arms (i.e. 3 mg kg
1 twice-weekly, 3 mg kg
1 three though, is low. Therefore it is of interest if a proven infection can
times-weekly or 10 mg kg
1 once-weekly of L-AmB) (P = 0.19). successfully be treated by this approach.
Conclusions Intermittent high-dose L-AmB is safe, feasible and effec- Methods Intravenous 24 h continuous infusion of amphotericin B
tive as antifungal prophylaxis in high-risk patients receiving deoxycholate (Fungizone) in a dose of 1 mg kg
1 body weight was
chemotherapy for acute leukaemia. Such a strategy is likely to have administered as follows: Administration via a dedicated lumen of a
utility in the ambulatory setting, particularly for those intolerant of quadruple central venous catheter. Coadministration of >1000 mL
other antifungal agents. However, further studies are required for normal saline (NaCl 0.9%)/day plus intravenous KCl up to
confirmation, including a comparison to broad-spectrum mould-ac- 160 mmol day
1 and sodium bicarbonate via separate lumina. Daily
tive azoles. control of urine output (>2000 ml day
1) or body weight, serum
potassium levels (>4.0 mmol l
1), serum bicarbonate
(> 20 mmol l
1) and changes of serum creatinine (preferably <100
umol l
1).
P106 Results A 50-year-old female with no relevant comorbidities was
diagnosed with an acute myeloid leukemia, NOS (WHO 2008) in
2010 with a WHO (ECOG) performance status of 1.
Evaluation of the in vitro activity of voriconazole as She was treated with araC 200 mg/m2 (d1-7) and Idarubicin
predictor of the in vivo outcome in a murine model of 12 mg/m2 d1-3. On day 16 the bone marrow was free of leukemia
scedosporiosis and the patient reached complete remission on day 30.
A. Martin,1 J. Capilla,1 M. Lackner,2 G. M. Gonzalez3 and She initially presented with neutropenic fever. She was treated
J. Guarro1 empirically with cefepime and acyclovir. On day 4 a CT scan of the
1 head showed pansinusitis. Amphotericin B deoxycholate was started
Universitat Rovira i Virgili, Reus, Spain; 2Medical University of as continuous infusion and the dose escalated when creatinine was
Innsbruck, Innsbruck, Austria and 3Universidad de Nuevo Leon, stable, up to 1.7 mg kg
1 of body weight (see Methods). The neu-
Monterrey, Mexico trophil count recovered on day 25 (>0.5 G/) but fever and signs of
sinusitis continued. On day 29 the sinuses were operated.
Objectives To determine the possible correlation between in vitro Histopathology showed invasive aspergillosis, cultures grew Aspergil-
activity and in vivo efficacy of voriconazole against Scedosporium lus flavus.
apiospermum in a murine model of disseminated infection. On day 45 the second chemotherapy cycle was started.

ª 2015 The Authors

92 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

On day 59 Amphotericin B dose was tapered down and stopped on C.glabrata was resistant to voriconazole and intermediate resistant to
day 64 because the fever had resolved. The patient successfully fluconazole. C.krusei, which yielded in three patients, was known to
entered allogeneic bone marrow transplantation on day 100 with be, intrinsically resistant to fluconazole and also intermediate resis-
secondary antifungal prophyaxis with voriconazole and caspofungin. tant to flucytosine. Mortality even in treated patients, were found
She is free of leukemia and fungal infection 5 years after treatment. very high. Mortality was found higher in patients with infections of
Conclusion This patient successfully recovered from a proven inva- non-albicans Candida (4/6) than C.albicans (3/7).
sive Aspergillus flavus rhinosinusitis. Amphotericin B deoxycholate Conclusion Candida peritonitis is still associated with poor prognosis.
was administered as a 24 h continuous infusion over >50 days at a Antifungal therapy can be suggested in critically ill patients with
dose of 1–1.7 mg kg
1 body weight. Serum creatinine never raised nosocomial peritonitis where Candida is diagnosed based on perioper-
to more than 29 the baseline levels. Potassium was checked daily ative sampled peritoneal fluid. Rapid detection of Candida might be
and replaced rigorously. There were no acute side effects as chills. beneficial in this regard.
Efficacy in this patient was demonstrated and depended on several
positive co-factors such as type of infection (rhinosinusitis), fungal
agent (Aspergillus flavus), successful surgery, and most importantly
cure of acute leukemia. P109/M11.1

Cerebral Aspergillosis: Report of 2 cases with favorable

P108 response to voriconazole and neurosurgery
F. Queiroz-Telles,1 R. Mialski,2 F. B. Magalhaes,2 F. Queiroz-
Telles,1 H. M. Morales,3 C. S. Oliveira,4 S. G. Gonsalves5 and
Candida peritonitis unrelated with continuous ambulatory
A. L. Colombo6
peritonea dialysis (CAPD) in adult patients in a Turkey
hospital
1
Hosp Clınicas, Univ Fed Parana, Curitiba, Brazil; 2Federal
B. Kurtaran, F. Kibar, A. Ulu, A. S. Inal, S. Komur, G. Sakman, University of Parana, Curitiba, Brazil; 3Hopital do Cajuru, Curitiba,
H. S. Z. Aksu and Y. Tasova Brazil; 4Hospital Universitario do Oeste do Parana, Cascavel,
Brazil; 5University of Sao Paulo, Sa~o Paulo, Brazil and 6Federal
Cukurova University, Adana, Turkey University of Sao Paulo, Sa~o Paulo, Brazil

Objective We aimed to display that the epidemiology, prognosis and Introduction and Objectives Central nervous system aspergillosis
therapy of Candida peritonitis unrelated with CAPD in our hospital. (CNSA) is an often fatal disease that usually affects immunosup-
Methods A retrospective analysis of adult patients with Candida peri- pressed patients. We report two cases of proved CNSA in immuno-
tonitis between April 2011 and February 2014 was done. All of the compromised and immunocompetent patients with favorable
peritoneal fluid samples, which yielded Candida species in microbial response to voriconazole (VCZ).
culture, were included to the study. Patient’s characteristics, microbi- Case Report Case 1. Caucasian 26 years old female, recently diag-
ological feature, prognosis and treatment were reviewed. nosed with autoimmune hepatitis, taking azathioprine
Results Thirteen patients with Candida spp. isolated from peritoneal (50 mg day
1) and prednisone (60 mg day
1) with drowsiness,
fluid were examined. None of the patients were on peritoneal dialysis. mental confusion, hemiplegia, upper right limb spasticity and positive
All of them were considered to have Candida peritonitis. The data on Babinski reflex on right. Neuroimaging were compatible with expan-
these 13 patients were reviewed in the table. Seven of them were sive hypodense lesions with perilesional edema requiring decompres-
died in average 5 days. Most of the cultured Candida species in peri- sive craniotomy. The freezing biopsy was compatible with
toneal samples were C.albicans but C.krusei yielded in three samples hyalohyphomycosis and culture revealed Aspergillus flavus. The
and C.famata in two. And also Candida spp. yielded with bacterial patient received VCZ (400 mg day
1) for 60 days and underwent
pathogens together such as Enterobacteriacea and Enterococcus species abscess drainage with good clinical evolution but with physical
in seven patients. Surgical interventions seemed to be an important sequel. Case 2. A Caucasian 67 years old male, farmer, previously
predisposing factor. Only in one patient, Candida yielded also in blood healthy, presented with daily excruciating headache evolving with
culture. No antifungal resistance was found in C.albicans, but palpebral ptosis and mydriasis on right. Neuroimaging were consis-
tent with expansive lesion on the right sphenoid region. Micro-
surgery was performed with partial resection of the lesion and
revealed an invasion of the cavernous sinus and right orbit, encom-
Table 1 passing carotid with mass effect. Histopathology was consistent with
cerebral hyalohyphomycosis but culture depicted A. fumigatus. Chest
and face sinus images did not show abnormalities. The Galactoman-
nan antigen detection (GM) in serum and CSF was 0.246, 0.544
respectively. He was treated with VCZ (400 mg day
1 for 62 days
with good clinical and neuroimaging responses. The GM index
decreased in the CSF (0.201 and 0.173) at 40 and 60 days,
respectively.
Conclusions Although CNSA is disease for most of the affected
patients, our patients survived after neurosurgery procedures associ-
ated to VCZ therapy. Because VCZ is the smallest molecule (349 Da)
with activity against Aspergillus spp, this compound presents sufficient
penetration across the blood-brain-barrier to attain fungicidal activity
in the CNS. The determination of seriated GM in the CNF was helpful
to evaluate therapy in our second patient.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 93
Poster Presentations

P120 respiratory specimens (N = 36), swabs (N = 10), and miscellaneous

(N = 3).
Results Testing the reference strain collection, the following species
Rapid identification of filamentous fungi by direct MALDI- and species groups gave confident and comparable identification
TOF MS scores using either protocol Aor B, and were correctly identified (des-
A. Riat, J. Schrenzel, H. Hinrikson and V. Barras ignation according BRUKER): Aspergillusflavus (N = 8) A. fumigatus
University Hospital Geneva, Geneva, Switzerland (N = 37), A. nidulans (N = 4), A. niger (N = 13), A. ochraceus
(N = 1), A. terreus(N = 5), A. ustus(N = 2) A. versicolor(N = 1), Cla-
dosporiumherbarum(N = 1), Exophiladermatidis(N = 1), Fusari-
Objectives Identification of filamentous fungi using classical means umdimerum(N = 2), F. oxysporum(N = 1), F. solani(N = 4),
is time-consuming and requires highly skilled technicians. MALDI Lichtheimiacorymbifera (N = 4),Mucorcircinelloides(N = 2)Pae-
TOF MS has the potential to enhance the accuracy and reduce the cilomyceslilacinus(N = 1), P. variotii(N = 1), Penicilliumchryso-
identification time, however, it is not routinely applied for the diag- genum(N = 4), P. citrinum(N = 1), Rhizomucorpusillus(N = 1),
nosis of molds. In order to implement MALDI TOF MS in our routine Rhizopusmicrosporus(N = 2) R. oryzae(N = 1), Scedosporiumapioser-
workflow in the mycology section, we evaluated the BRUKER MALDI mum(N = 6), S. prolificans(N = 1), S. brevicaulis (N = 1).
TOF MS platform testing our mold reference strain collection as well The prospective study on clinical specimens showed that 44 in 49
as human clinical samples in a prospective manner. clinical samples exhibited a score >1.7 and were correctly identified
Methods 111 reference strains of filamentous fungi representing 30 by MALDI TOF MS. For four out of the five remaining specimens
clinically relevant species were analyzed using the toothpick method without reliable score the identification suggested by MALDI TOF MS
(protocol A) and the Sabouraud broth subculture approach (B). In (A. niger, A. unguis, S. apiospermum and R. oryzae) was confirmed
protocol A, primary growth on Sabouraud agar after 24 to 72 h of by internal transcribed spacer sequence analysis. Only one specimen
incubation was collected by means of a wooden toothpick. The bio- without reliable score could not be identified by MALDI TOF because
mass was deposited on an unpolished target plate, air dried, and of the lack of reference data for Uncinocarpusqueenslandicus, the
overlaid with 1 ll alpha-cyano-4-hydroxycinnamic acid. In protocol agent present.
B, sediments of overnight subcultures in Sabouraud broth were thor- Conclusions In conclusion, confident identification of filamentous
oughly washed, extracted with acetonitrile-formic acid, and further fungi by MALDI-TOF MS is feasible without subculturing of primary
processed as above. MALDI-TOF MS was performed using a BRUKER growth. Our findings support the implementation of MALDI-TOF MS
Microflex LT instrument and the BRUKER Biotyper software in con- in the clinical setting in order to promote rapid and reliable diagnosis
junction with the Filamentous Fungi library version 1.0. Identifica- of fungal agents.
tion scores were interpreted as follows: Score ≥ 1.7, confident
identification at species or species group level; score <1.7, no reliable
identification (1, 2). We then prospectively applied protocol A on pri-
mary growth derived from 49 clinical samples which included
P121

Table 1 Induction of sporulation in dematiaceous mould by

Burmese sal bark
T. Kanpanleuk and P. Santanirand
Ramathibodi Hospital, Bangkok, Thailand

Conventional slide culture technique using various artificial culture

media has long been a main method for fungal identification based
on conidia structure of the organisms. However, many strains of
mould may not produce conidia on regular culture media which lead
to delay or unable to identify. Therefore, the aim of this study was to
establish alternative method which mimicked natural habitat by
using bark as a culture based. Seven dematiaceous moulds including
Curvularia spp., Bipolaris spp., Alternaria spp., Nigrospora spp., Chaeto-
mium spp., Lasiodiplodia theobromae and Pyremochaeta remoroi were
used. The organisms grew on Sabouraud dextrose agar (SDA) for 5–
7 days and small pieces of fungal colony were placed onto sterile
bark of Shorea siamensis (Burmese sal). The fungal inoculated barks
were then incubated in moisture chamber at 25 °C. The observation
of conidia production was performed on 3-days interval using tease
mount technique. Conidia production was observed in all tested iso-
lates. Most of the mould produced conidia within 3–6 days. In con-
trast, Pyremochaeta remoroi and Lasiodiplodia theobromae required 15
and 30 days of incubation. The conventional slide culture method of
these two organisms using SDA and potato dextrose agar (PDA)
failed to induce sporulation after 30 days of incubation. This simple
approach may become an alternative method for induction of sporu-
lation and could be applied to other groups of mould.

ª 2015 The Authors

94 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P122/M6.3 P123

Oral paracoccidiodomycosis mimicking lip carcinoma Comparison of the pre-commercial Invasive candidiasis
M. L. Scroferneker1 and F. M. Girardi2 (CAGTA) VirClia IgG Monotest with the CAGTA C. albicans
1
Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil IFA for candidaemia detection in patients admitted to the
and 2Hospital Ana Nery, Santa Cruz do Sul, Brazil ICU.
M. Parra-Sanchez,1 S. Garcıa-Rey,1 C. Castro,1 I. Zakariya,1
A. Rezusta,2 F. Go mez,3 T. Marrodan,4 A. Esteban,4
Introduction Paracoccidioidomycosis (PCM) is a systemic mycosis
characterized by acute or chronic tissue inflammation caused by A. I. Suarez, J. Ayats,6 D. Navarro,7 M. Fajardo,8 A. Bordes,9
5

Paracoccidioides brasiliensis (Pb), a pathogenic thermally dimorphic pez,10 M. Ruiz11 and E. Martin-Mazuelos1
L. Lo
1
fungus that is endemic to Latin America. Patients with chronic PCM Hospital Universitario de Valme, Seville, Spain; 2Hospital
are mostly middle-aged men with smoking and/or drinking habits Universitario Miguel Servet, Zaragoza, Spain; 3Joan XXIII
and who are actively engaged in farming activities. The mucocuta- University Hospital, Tarragona, Spain; 4Complejo Asistencial de
neous lesions caused by Pb are easily mistaken for carcinomatous Leon, Leon, Spain; 5Virgen Macarena University Hospital, Seville,
lesions of the head and neck owing to similar risk factors and clinical
Spain; 6Hospital Universitario de Bellvitge, Barcelona, Spain;
manifestations. In this study, we report a case of PCM with lip mani- 7
festation, referred to our department because of suspected neoplasia.
Hospital Clinico de Valencia, Valencia, Spain; 8Hospital
Case Report A 65-year-old man, actively engaged in farming activi- Universitario Infanta Cristina, Badajoz, Spain; 9Hospital de Gran
ties, with a 90 pack-year smoking history, presented with a painless Canaria-Negrın, Gran Canaria, Spain; 10Hospital de Cruces,
ulcerated lesion of the left labial comissure. He was referred to our Barakaldo, Spain and 11Hospital Universitario Virgen de
department because of suspected neoplasia. The lesion progressed Macarena, Sevilla, Spain
slowly through a 2-year period. Oral examination revealed an exten-
sive ulcer, although flat and with no signs of orbicularis oris muscle Objectives Evaluation of the pre-commercial ‘Invasive candidiasis
infiltration, or involvement of mandibular division of facial nerve, or (CAGTA) VirClia IgG Monotest’ in comparison with the standardized
suspicious neck lymph nodes metastasis. A representative incisional CAGTA C. albicansIFA in a selected non-neutropenic critically ill
biopy was performed. Histopathological examination exhibited adult patients colonized and infected by Candidaspp., with an
chronic granulomatous inflammation on conventional hematoxylin- expected ICU stay of at least 7 days.
eosin staining. Grocott-Gomori staining showed fungal structures. Methods A total of 180 serum samples from 72 non-neutropenic
Clinical and pathological evidences supported the diagnosis of para- patients, admitted to 12 ICUs Spanish tertiary hospitals from October
coccidioidomycosis. Itraconazole was maintained for 6 months with 2012 to February 2014, were analyzed in a prospective cohort,
satisfactory response to proposed therapy. observational, multicenter study. Demographic data, type of patients,
Discussion Active agricultural laborers or those who have a history APACHE II and SOFA scores (on ICU admission), underlying disease,
of agricultural labor are at greater risk for paracoccidiodomycosis. comorbidities, risk factors, antifungal treatment and outcomes were
Masons and civil construction workers rank second in terms of risk. recorded. Clinical assessment, APACHE II, SOFA and Candida score,
Men, on average, have a ninefold increased risk of being affected Candida colonization cultures (rectal swabs, tracheal aspirates, gas-
than do women. The cutaneous and mucous lesions result from tric or pharyngeal aspirates and urine) were performed twice a week
hematogenous dissemination, either by contiguity or, very rarely, by (other samples were taken as clinically indicated). All samples were
direct inoculation under the tissue. Signs and symptoms are related processed by conventional methods. Patients were classified into pro-
to the affected site, and usually appear as visible lesions or non-speci- ven invasive candidiasis (IC): 10 patients (25 serum samples) with
fic symptoms. In cases of mucocutaneous disease, the oral and nasal candidaemia and 17 patients (48 samples) with intra-abdominal
cavities are the most commonly affected sites. infection, Candida colonization (CC): 31 patients (80 samples) and
Similarly to PCM, head and neck carcinomas usually affect middle- neither colonized nor infected (NCNI) 14 patients with 27 serum
aged and elderly white men, mostly those with smoking and/or samples analyzed.
drinking habits, and are characterized by exophytic, infiltrative or These samples were analyzed retrospective with the VirClia assay
ulcerative lesions. As risk factors and clinical manifestations are simi- (Vircell Spain, SL) following manufacturer0 s instructions (positive val-
lar to those of head and neck carcinomas, a differential diagnosis is ues ≥1.1). Results have been compared with a standardized CAGTA
necessary. C. albicansIFA  (Vircell Spain, SL) (positive values ≥1/160).
Results For IC patients with candidaemia, sensitivity, specificity, pos-
itive and negative predictive values and kappa value for kit compar-
ison were: 87.5, 81.3, 70.0, 92.9, and 0.65, respectively.
For IC patients with intra-abdominal infection were: 88.9, 77.3,
61.5, 94.4, and 0.58, respectively. For CC group were: 89.5, 85.2,
81.0, 92.0 and 0.74, respectively. For global comparison were: 88.9,
83.3, 84.4, 88.2 and 0.72, respectively. Overall agreement for both
techniques was 86.1%.
Conclusions VirClia assay (CAGTA enzyme immunoassay) can be
used in the diagnosis of invasive candidiasis with similar results than
CAGTA.
This assay, a semiautomatic method, is easier to perform than
CAGTA IFA. Furthermore, results are semicuantitative and objec-
tively interpretable.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 95
Poster Presentations

P124 Serum GM levels were measured using the Platelia Aspergillus

enzyme immunoassay kit (BioRad, France). BmGT and voriconazole
were quantified by High Performance Thin Layer Chromatography
Bis(methyl)gliotoxin as diagnostic and management tool (HPTLC).
of Invasive Aspergillosis: report of a proven case with Results Serum GM detection was negative in all the four sera col-
negative galactomannan in an immunocompetent patient lected. BmGT was detected in the two samples obtained prior to anti-
with mycotic aneurism fungal treatment (i and ii). The bmGT concentration during ED
M. Vidal-Garcı́a,1 P. Sanchez-Chueca,1 M. P. Domingo,2 admission was 0.19 lg ml
1 and increased to 0.22 lg ml
1 on the
M. P. Soria-Lozano,1 C. Ballester,1 L. Roc,1 I. Ferrer,1 day of surgery. The two samples obtained after 14 (iii) and 22 days
(iv) of correct antifungal treatment did not show detectable bmGT.
M. J. Revillo,1 J. Pardo,3 E. M. Galvez2 and A. Rezusta1 Voriconazole trough levels in serum iii and iv were 2.2 lg ml
1 and
1
Hospital Universitario Miguel Servet, Zaragoza, Spain; 2Instituto 2.8 lg ml
1, respectively.
de Carboquımica ICB-CSIC, Zaragoza, Spain and 3Centro de Histopathological examination of the aorta demonstrated fungal
Investigacion Biomedica de Aragon CIBA, Universidad de structures. A. fumigatus grew in both vitreous and aorta cultures.
Zaragoza, Zaragoza, Spain Conclusion BmGT followed an increasing kinetic until antifungal
treatment and it became undetectable after antifungal initiation. This
findings support the utility of bmGT detection as a diagnostic and
Objectives Bis(methyl)gliotoxin (bmGT) is a derivative of gliotoxin
management tool of IA. GM was falsely negative, showing a real
produced by the mould Aspergillus spp that can be detected in serum
need of reliable biomarkers for IA diagnosis.
from patients at risk of invasive aspergillosis (IA). The aim of this
study was to assess its utility for IA diagnosis in a patient with pro-
ven IA and negative galactomannan (GM). The effect of antifungal
treatment on bmGT kinetics was also evaluated.
Methods A 70-years-old patient presented to the emergency depart- P125
ment (ED) with blurred vision. Endogen endophtalmitis was diag-
nosed and vitrectomy was planned. In the intervention, vitreous was
Abdominal actinomycosis in Saint-Petersburg, Russia
sent for microbiological examination. During his admission for the
surgery, he suffered an ischemia of the right low member. Computed O. P. Kozlova, A. K. Mirzabalaeva and N. Klimko
tomography (CT) angiography of peripheral arteries and thorax North-Western State Medical University named after I.I.
objectified right pedis artery occlusion and a thrombus in aortic arch. Metchnikov, Saint Petersburg, Russia
He received intraarterial fibrinolytic treatment, and aortic lesion
study continued. Infectious endocarditis was then discharged by Objectives To study risk factors, clinical features, and treatment effi-
transesophageal echocardiography. A thoracic CT revealed mycotic ciency of abdominal actinomycosis in Saint Petersburg, Russia.
aneurysm of the ascending thoracic aorta. Graft replacement was Materials and methods Prospective, single-center study of 157
carried out and samples were sent for histological and microbiologi- patients with different clinical forms of actinomycosis (2005–
cal examination. Microbiological report revealed hyphal vision on 2013 yy.). Abdominal actinomycosis was in 44% cases. Women
Gram stain and caspofungin (70 mg d
1 on day 1, followed by
88%, age 27–81 year (median - 47  3). The diagnosis was based
50 mg d
1) was initiated. The next day, A. fumigatus was isolated on histology of postoperative material. The control group was made
and voriconazole (6 mg kg
1 every 12 h on the first day, followed by patients with nonspecific inflammatory process of abdominal
by 4 mg kg
1 every 12 h) was added to the treatment. After sur- organs on 20 people.
gery, patient was stable and extubation was possible. Later he devel- Results Predisposing factor for abdominal actinomycosis were acute
oped severe respiratory failure and intubation was needed again. He appendicitis, particularly with perforation (OR = 5.04 [1.07–23.72],
was gradually getting worse, with severe renal impairment requiring P < 0.05). Pelvic actinomycosis is predominantly associated with
continuous venovenous hemodiafiltration and vasoactive drugs. IUCDS (OR = 7.7[2.5–23.8], P < 0.05). Patients usually present with
Finally, he developed a septic shock with respiratory, renal and hep- a history of prolonged use IUCDS (median 7.6 + /-2.4 years).
atic failure and died. At 67% patients abdominal and pelvic organs were involved in
We detected bmGT, galactomannan (GM) and voriconazole in four inflammatory process. Ovaries (43%), appendix (42%), colon (25%),
sera:(i) a serum obtained during endophtalmitis study, (ii) a serum cecum (22%), uterus (16%) and fallopian tubes (12%) are the most
obtained prior to graft replacement, (iii) a serum obtained after common abdominal sites of actinomycosis. Peritonitis was in 33%
14 days of correct antifungal treatment and (iv) a serum obtained patients.
after 22 days of treatment. Patients may present with non-specific symptoms such as abdomi-
nal pain (94%), fever (80%), and weight loss (39%). In all cases the
diagnosis was based on histology of operational material. The culture
of Actinomyces spp. from ‘sterile’site confirms the diagnosis (A. israelii
- 75%, A. naeslundii - 25%).
Surgery was used in all patients, as well as treatment with ben-
zylpenicillin (12–24 million units a day) for 2–4 weeks, then amoxi-
cillin 1.5–2 g day
1 for 6–12 months. The median duration of
antibiotic treatment was 9  2 months. Efficiency of treatment was
92%.
Conclusion Abdominal involvement was in 44% cases of actinomy-
cosis. Surgical resection of infected tissue and long-term antibiotic
therapy was effective in the treatment of abdominal actinomycosis.

Figure 1. Serum bmGT, GM and voriconazole concentration during

time

ª 2015 The Authors

96 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P126 Methods A reference database was created using high quality spec-
tra from six strains of H. capsulatum previously identified by molecu-
lar methods. Three strains were included in both morphological
Rapid identification of the main Candida Species isolated states (mycelia and yeast) and also the three varieties recognized for
from blood and other clinically significant cultures by the fungus (var. capsulatum, var. duboisii and var. farciminosum) and
Multiplex Real-time PCR the type strain were represented. To construct the database, the fun-
H. Schirmer and V.V. Cantarelli gal isolates were grown for 3 weeks and subjected to protein extrac-
tion following the method recommended by the manufacturer
Universidade Feevale/UFCSPA - Depto. Cie^ncias Basicas da Saude,
(Bruker Daltonics, Germany). Each sample was assessed eight times
Novo Hamburgo, Brazil
and MS-measurement was performed in triplicate. For validating the
new database, 89 fungal isolates were selected from the Collection of
The frequency of systemic infections caused by Candida has increased the Spanish National Centre for Microbiology: 35 strains belonging
considerably in the last few years, especially among hospitalized to different fungal species and a total of 54 strains of H. capsulatum.
patients. It has being considered the fourth agent isolated of blood- Protein extraction was performed after 7 days of culture and all pro-
stream and is associated with significant morbidity and mortality. tein extracts were analysed in duplicate against the MS new refer-
Although Candida albicans still remains as the most common fungal ence database created plus other commercial and in-house MALDI
isolate from clinical specimens, longitudinal studies have detected a databases.
shift towards non-albicans Candida (NAC) species. The non-albicans Results Globally, 81 strains were correctly identified with variable
Candida species are associated with the increase of antifungal drug scores ranging from 1.42 to 2.4 and 66 of them (81.5%) had a score
resistance, and hence, species differentiation became very important. above manufacturer cut-off for reliability at genus level (score ≥ 1.7).
The aim of this study was to standardize a molecular technique to All H. capsulatum isolates were properly identified with scores ranging
rapidly differentiate the most important Candida species isolated from from 1.46 to 2.2. The reliability score ≥ 1.7 was reached by 41 of
the blood cultures. In order to differentiate the Candida species, a set them (76%), 30 in mycelial state (86% of the total of H. capsulatum
of specific primers were designed based on variability in the internal strains in mycelial state) and 11 in yeast form (58% of the total of
transcribed spacer (ITS) region of ribosomal DNA, whose sequences H. capsulatum strains in yeast state). However, when this cut-off was
were aligned using ClustalW. After sequence alignment, a common lowered to score ≥ 1.6, the number of strains properly identified rose
sense primer was selected together with specific anti-sense primer for to 49 (91%), including 33 mycelial forms (94% of mycelial-form iso-
each Candida species detected by this system. This molecular tech- lates) and 16 yeast forms (84% of yeast-form isolates).
nique was initially tested against several know Candida isolates and Conclusion i) The database created provided a suitable identifica-
the reactions were confirmed to be specific for each species being tion for 76% of H. capsulatum isolates when the cut-off score was set
detected. The reaction consisted of a PCR mix containing SYBR green at 1.7 and 91% when this cut-off was 1.6. ii) The database created
as a fluorescent agent, a mixture of primers and fungal DNA in a was able to identify both morphological phases of the fungus and the
total volume of 20 microliters. Reactions were run on the LightCy- three varieties. iii) MALDI-ToF technology yields a prompt and simple
cler instrument (Roche) and Candida identification could be obtained identification from H. capsulatum yeast forms or early mycelial cul-
by analyzing the different melting points of the different species. Two tures. It allows for reducing the time of response and decreasing the
separated reactions were used to identify: C. albicans, C. dubliniensis, risk in manipulating the fungus.
C. tropicalis, C. parapsilosis, C. krusei, C. guillermondii and C. glabra-
ta.The advantage of this methodology is, determine different Candida
species directly from the blood.With molecular detection directly from
the blood, the culture is avoided reducing the releasing time of result. P128
Also, sometimes with the traditional methods used in yeast identifica-
tion is not possible define property the species. So, it methodology
helps to improving the better choice for treatment. Still, this specific Combination of galactomannan detection and computed
method showed good sensibility and specifity in identify the Candida tomographic findings for diagnosis of invasive pulmonary
species. In conclusion, this is a rapid and possible method to apply in aspergillosis
laboratory routine especially that attend hospitalized patients improv- D. Findik, S. Ozbek, G. Demirel, H. Turk Dagi, Z. Koc and F. Ates
ing the epidemiology characterization and treatment of Candida.
Selcuk University Faculty of Medicine, Konya, Turkey

Objectives To evaluate the benefit of the combination of galac-

P127 tomannan (GM) assay and thoracic computed tomography (CT)
images for the diagnosis of invasive pulmonary aspergillosis (IPA).
Methods This retrospective study was performed at the 900-bed ter-
Development of a reference database for the identification tiary-care teaching hospital in Konya/Turkey using the medical
of Histoplasma capsulatum by MALDI-ToF Mass records of the 400 patients between the years 2011 and 2015. The
Spectrometry Platelia Aspergillus Ag EIA assay (Bio-Rad) was used to detect GM
C. Valero,1 M. JBuitrago,1 S. Gago,1 I. Quiles-Melero2 and antigen in serum and bronchoalveolar lavage (BAL) fluid samples.
J. Garcia-Rodriguez2 CT evaluation was performed by radiologists who were unaware of
1 patients’ GM results.
Instituto de Salud Carlos III, Majadahonda, Spain and 2Hospital Results Thorasic CT images of 400 patients with probable or possi-
Universitario La Paz, Madrid, Spain ble IPA were evaluated with respect to the presence of typical radio-
logical findings defined for IPA.The imaging findings were compared
Objectives The diagnosis of histoplasmosis is based on the isolation with the serum or BAL fluids samples’ GM test results of the patients.
of the fungus in cultures. However, it is time-consuming and biosaf- Ground glass parenchymal opacities, cavitations, fungus balls and
ety level 3 containment is needed. MALDI-ToF technology has been patchy consolidations with predominantly subpleural and/or peri-
established as a suitable tool for microbial identification although bronchial distribution were significantly more frequent in GM positive
there are no entries of H. capsulatum spectra in most used commer- group (P < 0.05). On the other hand presence of consolidation with
cial MALDI databases. The main objectives of this work are: i) Con- or without air bronchogram sign, nodule with or without tree-in-bud
struction of a reference database for the identification of Histoplasma appearance, unilateral or bilateral pleural effusion were seen with
capsulatum by MALDI-ToF technology. ii) Validation of this database similar frequency in both GM positive and negative groups.
with isolates belonging to the Collection of the Spanish National Cen- Conclusion It is important to diagnose and differentiate the most
tre for Microbiology. frequent CT findings of IPA that is associated with positive serum or

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 97
Poster Presentations

BAL fluid positive GM tests. Together, these findings may help physi- Table 1
cians to initiate Aspergillus-active antifungal treatment.

P129

Occurrence of fungal DNA contamination in PCR reagents:

Approaches to control and decontamination
S. Czurda, S. Smelik, S. Preuner-Stix, K. Kosulin and T. Lion
CCRI, Vienna, Austria

Objectives Contamination with fungal DNA is a serious threat for and treatment options, infections caused by Candida spp. and Aspergil-
the validity of fungal detection assays based on nucleic acid amplifi- lus spp. are decreasing, while other rare fungal pathogens like Muco-
cation. Here we report the presence of fungal DNA contaminants in rales are on the rise. Mucormycosis is a very severe fungal infection
critical reagents for real-time PCR obtained from commercial provi- and difficult to diagnose. Standard culture is insensitive and micro-
ders. Traces of fungal DNA were detected in lyophilized primers, Taq- scopy as well as radiological imaging unspecific. Most diagnostic pro-
Man probes, and master mix solutions, and resulted in a cedures are invasive and thus cannot be used for screening. DNA
considerable rate of false-positive test results in panfungal real-time detection from serum as described for Aspergillus or Mucorales (Ya-
PCR assays. Controlling this problem is therefore of paramount makami et al., 1996; Millon et al., 2013) by polymerase chain reac-
importance for molecular diagnosis of fungal infections. tion (PCR) is a rapid and very sensitive tool to identify pathogens
Methods Identification of PCR reagent contamination was accom- and can improve diagnosis and treatment.
plished by panfungal real-time PCR screening. A decontamination Methods Forty-six serum specimens from 5 hematological patients
method for PCR components based on the activity of a double-strand were collected from 2008 to 2012 at Medical University Hospitals of
specific DNase was established. DNA-spiking experiments using quan- Innsbruck and Wuerzburg. Sera were drawn twice weekly. According
tified Candida albicans DNA were performed to test the capability of to EORTC/MSG criteria (DePauw et al., 2008), 3 patients were classi-
the DNase to digest typical quantities of contaminating fungal DNA fied having proven/probable IFI either by histology or positive culture
found in PCR reagents. Individual decontamination protocols were from bronchoalveolar lavage (BAL). Sera were available up to
established for each PCR reagent and reduction of false-positive test 127 days before EORTC classification date.
results after decontamination was evaluated. Sequencing of a PCR DNA from 1 ml of serum was manually extracted by using a com-
product obtained from a false-positive reaction was performed to mercially available kit (UltraSens Virus Kit, Qiagen). Extracts were
identify the biological source of contaminating DNA. analysed by real-time PCR assay (qPCR) amplifying the 18S rDNA
Results We established rigorous handling conditions to avoid false- region. This assay was modified using the primers published by Bia-
positive test results from exogenous sources, and found that fungal lek et al. (2005) comprising in addition a Mucorales specific probe
DNA contamination was limited to PCR reagents. We showed that and degenerated nucleotides. A single round PCR cycling was used.
DNase mediated decontamination was capable of digesting a much Amplicons of positive PCR reactions were sequenced and aligned
higher DNA amount than typically found as contaminants in PCR with reference sequences using BLAST analysis search.
reagents. DNase treatment of contaminated oligonucleotides led to Results DNA extracts of serum samples were analysed by a Muco-
more than 10-fold reduction in the frequency of false-positive test rales-specific qPCR assay within 4 h. Four samples of the 3 probable/
results without compromising the overall test performance. The proven Mucorales IFI cases were positive. Sequencing revealed 2 dif-
enzyme was also effective in decontaminating the master mix solu- ferent Mucorales strains (Lichtheimia sp., Rhizopus sp.)confirming
tion used in our assay, albeit with deterioration of the assay perfor- pathogen identification by conventional methods. PCR from serum
mance. Sequencing of the contaminated PCR products revealed detected Mucorales DNA up to 3 days earlier than BAL or biopsy.
sequences showing 100% identity with a conserved region of 28S Day ‘zero’ was defined as time point of EORTC classification (positive
rRNA gene found in representatives of different yeast genera indicat- BAL culture, biopsy). None of the unclassified control samples were
ing that contaminations observed originated from traces of yeast- positive.
derived DNA. Conclusion Using qPCR allowed Mucorales specific detection of
Conclusion Based on our findings, we strongly recommend routine DNA in serum. This probe-based assay detecting a broad spectrum of
monitoring of all reagents used in fungal PCR assays for the presence Mucorales was used as an add-on tool to confirm mucormycosis in
of relevant contaminants. As long as fungal-grade reagents are not serum. Due to easy availability of serum even in severely ill patients,
readily available, pretreatment methods facilitating elimination of qPCR can be used as a screening tool in high-risk patients with sus-
fungal DNA are instrumental for reducing the risk of false-positive pected mould infection. The method used is faster and more sensitive
results in highly sensitive molecular fungal detection assays. than standard methods. It can help to distinguish between infections
with Aspergillus and Mucorales, which is relevant for antimycotic
regimens and patients’ outcome.

P130

Non-invasive detection of mucorales DNA in serum by

mucorales specific real-time PCR assay
J. U. Springer,1 M. Lackner,2 W. J. Heinz,3 B. Risslegger,2
€rl2 and J. Loeffler5
C. Ensinger,4 H. Einsele,3 C. Lass-Flo
1
University of Wu€rzburg, Wuerzburg, Germany; 2Medical
University of Innsbruck, Innsbruck, Austria; 3Med. Clinic 2,
Wuerzburg, Germany; 4Institute of Pathology, Innsbruck, Austria
and 5University Hospital of Wuerzburg, Wuerzburg, Germany

Purpose Invasive fungal infections (IFI) are life-threatening compli-

cations in immunocompromised patients. Due to improved diagnosis

ª 2015 The Authors

98 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P131 Objectives The general objective was to assess the prevalence of

these mycosis in Madagascar. The specific objectives were to charac-
terize the causative fungal species and their habitat in order to pre-
Chromoblastomycosis and sporotrichosis in Madagascar: vent contamination, and; to set up a sustainable clinical and
epidemiology, molecular diagnostic and perspectives laboratory network to allow proper case management and to provide
T. Rasamoelina,1 N. Rakotozandrindrainy,2 M. Raberahona,3 a molecular-based species identification.
F. Rapelanoro Rabenja,4 M. Rakoto Andrianarivelo,1 Methods The study comprised a prospective clinical study that
M. Andrianarison,4 I. Ranaivo,4 B. Contamin,1 started in March 2013. Patients were recruited during field investiga-
tions and consultations provided in a dermatology department. Pus,
L. S. Ramarozatovo4 and M. Cornet5 biopsy and squamous were sampled from the lesions. Informed con-
1
Centre d’Infectiology Charles Merieux, Antananarivo, sent from the patients and ethical approval from the Ministry of
Madagascar; 2UPFR Parasitologie-Mycologie, Antananarivo, Health were obtained. Histopathological and mycological analysis (di-
Madagascar; 3Service des Maladies Infectieuses, Antananarivo, rect examination and culture on Sabouraud-Cycloheximide) were
Madagascar; 4USFR Dermatologie-Rhumatologie, Antananarivo, performed. We have developed a PCR-based strategy that was vali-
Madagascar and 5University Hospital Grenoble, Grenoble, France dated on reference strains provided by BCCM/IHEM (Belgian Coordi-
nated Collections of Microorganism). First, two sets of universal
primers (NL-1/NL4 and ITS5/ITS4) were used to confirm the fungal
Chromoblastomycosis (CBM) and sporotrichosis (SP) are endemic origin of the lesions. Then, specific primers (SSHF31/SSHR97, Fon-F/
mycosis in Madagascar that occurred following injury and telluric Fon-R, EdF/EdR) were used for fungal species identification.
contamination. CBM is mostly due to Fonsecaea pedrosoi or Cladophia- Results Fifty-five patients were enrolled. Mean age of the patients
lophora carrionii and affects usually the subcutaneous tissue, whereas was 38.8 years and men were prevalent with 69.1% of cases. Over-
SP is caused by Sporothrix schenckii by invading the lymphatic system all, 41.8% were farmers, 34.5% self-employed, 16.4% students and
of the arms and legs. These fungal infections are considered neglected 7.3% unemployed. Clinically, 36.4% of cases were suspected having
diseases because of poor resources allocated to their diagnosis, moni- CBM and presented with crusted, verrucous and tumoral lesions;
toring or prevention.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 99
Poster Presentations

40.0% of cases were SP characterized mainly by ulcerative and inhibitions would allow the application of these techniques on com-
nodular lesions of the lymphatic system of the lower limbs; and for plex samples.
23.6% the diagnosis was ambiguous. Mycological analysis revealed Financial support Supported by the UPV/EHU grants (PES13/03,
18 cases of SP and 8 cases of CBM from which the PCR identified 17 GIU12/44 and UFI11/25), the Government of the Basque Country
strains of Sporothrix sp., 1 Cladophialophora sp. and 6 Fonsecaea sp. grant (S-PC11UN007), and the Ministry of Economy and Competi-
One mycetoma probably of bacterial origin was also found. CBM tiveness grant (MICINN CSD2009-00006). Monica Sueiro Olivares
cases were predominant in the north-east, east and south part of the and Xabier Guruceaga have been supported by Pre-doctoral Research
island, whereas SP cases were located mainly in the central Grants of the Government of the Basque Country and Jes us Gangoiti
highlands. Barrera Foundation, respectively. Jimena V. Fernandez Molina has
Conclusion These results confirmed that CBM and SP persist at a been supported by Post-doctoral Research Grants of the UPV/EHU.
high frequency in Madagascar. The availability of a reliable PCR tool
in routine and the clinical expertise gained during this study will
help the national authorities to set up a proper control and preven-
tion program. An environmental survey is planned to describe the P133
spread of the causative agents in the environment in an attempt to
prevent the contamination.
Yeasts identification by mass spectrometry: a comparative
study
V. M. Lopes,1 T. M. Silva,1 A. L. Silva,2 P. J. Pinto2 and
P132 M. H. Ramos2
1
Centro Hospitalar do Porto, Porto, Portugal and 2CHP, Porto,
Rapid and specific detection of Aspergillus spp. at section Portugal
or species levels by multiplex q-PCR
A. Abad,1 J. V. Fernandez-Molina,1 M. Sueiro-Olivares,2 Objectives The technology MALDI-TOF MS (matrix-assisted laser
X. Guruceaga,1 A. Ramirez-Garcia,2 J. Garaizar,1 F. L. Hernando1 desorption/ionization-time of flight mass spectrometry) represents a
and A. Rementeria2 significant advantage for the timely identification of microrganisms
1 in clinical laboratories. The objective of the study was to evaluate
University of the Basque Country, Vitoria-Gasteiz, Spain and the performance of two mass spectrometry MALDI-TOF systems,
2
Universidad del Paıs Vasco UPV/EHU, Leioa, Spain namely VITEK MS BioMerieux and Bruker Daltonics MALDI BioTy-
per, in the identification of clinical yeasts isolates.
Invasive aspergillosis (IA) are opportunistic infections caused by Methods 136 yeasts isolated from clinical specimens were tested: 28
Aspergillus spp. PCR has not been yet accepted as a mycological evi- Candida albicans, 5 C. dubliniensis, 29 C. glabrata, 2 C. guilliermondii, 1
dence of infection by the EORTC, but it is a rapid and highly sensitive C. haemulonii, 2 C. krusei, 1 C. lipolytica, 7 C. lusitaniae, 2 C. norvegen-
technique that can be used to detect fungi DNA and help clinicians sis, 15 C.parapsilosis, 14 C. tropicalis, 21 Cryptococcus neoformans, 3
to obtain quicker diagnosis. Saccharomyces cerevisiae, 1 Trichosporon ashaii, 1 T. asteroides, 4 T.
Objective The aim of this study is to develop a variety of probes that mucoides, 1 Rhodotorula spp. Yeast identification on the VITEK MS
could be combined in multiplex real time PCRs (qPCR) to practical, system (BioMerieux) and on the instrument Microflex LT (Bruker
quick and specific detection and identification at different levels Daltonics BioTyper), was performed as according to the manufac-
within Aspergillus (species, section or genus). turer’s protocol using a short on-plate extraction method. The back-
Methods Three multiplex qPCR were developed based on ITS regions up routine identification methods consisted in the germ tube test,
and benA gene (b-tubuline protein). Primers and specific Taqman chromagar medium, API C AUX and the automated Vitek2 system.
probes were designed to detect and discriminate members of section Results Overall, Vitek MS correctly identified, 90.4% of the yeasts to
Fumigati, Flavi and Nigri (Sections-qPCR); A. fumigatus, A. flavus, A. the genus level, with 88% identifications to the species level; no iden-
niger and A. terreus species (4SP-qPCR); or A. fumigatus-section Fumi- tification results were provided in 8.8% of the strains and misidentifi-
gati species (Fum2). Two DNA extractions methods were assayed. We cations in 0.7%. Bruker Biotyper results were: 89% correct
also designed internal controls to detect inhibition of the reactions. identifications to the genus and species level, no identification results
Finally, several PCR facilitators were added in PCR mixtures to avoid in 11% of the samples and there were no misidentifications results.
false negatives due to PCR inhibitions. The sensitivity and efficiency The Candida strains identification analysis revealed improved results:
of the multiplex real time PCR assay was tested with DNA extracted 96.2% for genus in both systems, with species identification level of
from hyphae and conidia of Aspergillus spp. 93.3% in Vitek MS and 96.2% in the Bruker system. No identifica-
Results The Sections-qPCR demonstrated a high sensitivity with lim- tion results in 2.9% and 3.8% respectively and misidentifications 1%
its of detection of 20 fg and a mean efficiency about 93% for the in Vitek MS.
three probes. On the other hand, 4SP-qPCR showed sensitivity and a In both cases no identification results were found in a higher per-
mean efficiency of 50 fg and 100%, respectively in the detection of centage when testing Cryptococcus neoformans strains. (Vitek MS 38%
A. niger and A. terreus, and 100 fg and 105% in the detection of A. and Bruker 43%).
fumigatus and A. flavus. Moreover, the method also permitted the Conclusions We concluded that regardless the system used, the
detection of only 1 germinated conidium, except for A. terreus (10 technique is very easy to perform and very good results can be
conidia), vs. 102–103 non-germinated-conidia. Finally, the Fum2- obtained very rapidly, contributing to the timely management of
qPCR showed sensitivity and a mean efficiency of 20 fg and 82.16%, patients. For both systems Candida strains identification results were
respectively, in the detection of section Fumigati; and 50 fg and very good while overall results were inferior. This inferiority was
101.23% in the specific detection of A. fumigatus. Furthermore, the related to the, already reported, lower efficacy of this technique in
method also allowed the detection of 1 germinated conidium versus identifying mucoid strains. Although not significant we found a bet-
103 non-germinated conidia. ter performance in Bruker Biotyper in the identification of Candida to
Conclusions The ITS region allows detecting not only genus and the species level.
section level, but also at species level in the case of A. terreus and
Neosartorya pseudofischeri. The high inter-specific variability of the
benA gene has allowed the identification of A. fumigatus, A. niger and
A. terreus, but not the discrimination of A. flavus from their cryptic
species. We also confirmed the detection of hyphae and even a single
germinated conidium, but not non-germinated conidia (<102). The
use of internal control together with strategies to avoid PCR

ª 2015 The Authors

100 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P134

Determination of the detection limit of the polymerase

chain reaction in the Cryptococosis
R. B. Caligiorne,1 C. Graciele-Melo,2 F. Rocha-Silva2 and
P. Christo2
1
Santa Casa Hospital, Belo Horizonte, Brazil and 2Nucelo de Pos-
graduac
a~o Santa Casa Hospital, Mg, Brazil, Belo Horizonte, Brazil

Introduction Cryptococcosis is a fungal infection, with a large epi-

demiological distribution worldwide caused by encapsulated yeasts of
the genus Cryptococcus. The disease affects about one-third of carriers
of human immunodeficiency virus (HIV) presenting an opportunistic
infection. Rapid and specific diagnostic tests for fungal infections are
extremely important to the effectiveness in treating infected patients.
With the advancement of Molecular Biology has been possible to
develop new techniques for diagnosis and identification of Cryptococ-
cus sp.
Objectives This study aimed to determine the detection range of the
genome of Cryptococcus sp by PCR (Polymerase Chain Reaction), in
biological samples of patients with suspect of criptococose.
Methods For the PCR reactions, we used specific primers for Crypto-
coccus sp (CN4-CN5) and global primers for fungi rDNA (ribosomal
DNA - ITS1-ITS4). To determine the detection limit in culture of Figure 1. Venn diagrams-number of agreement or disagreement
Cryptococcus neoformans, we used an ATCC sample, and to determine result between pairs composed of one Mycosis flask (MY) and one
detection limit in cerebrospinal fluid, we used a sample from a flask with resins (AE and/or ANA).
patient with criptococose diagnosis confirmed by examining with
India ink, culture and clinical findings.
Results According our results, PCR was capable to detect 30.1 DNA positivity rate during candidemia; 2) time to positivity (TTP), and 3)
femtograms in culture of Cryptococcus sp and 26.5 DNA picograms in agreement between vials. The presence of SAT was recorded. Uni-
cerebrospinal fluid. variate analysis and multivariate hierarchical model for repeated
Conclusion According to the low level of detection presented, we measures were used.
can suggest that PCR may help in diagnosis of cryptococcosis, Results A total of 125 patients with candidemia and a median age
increasing life expectancy of patients and preventing disease recur- of 61.5 years were considered. Overall, positivity rate was similar in
rence, since it can follow the treatment for the doctor can know the vials with resins and in selective vials. In the presence of SAT, the
right to stop the drug administration time. The next steps will be to positivity rate decreased significantly (OR: 0.16 (95% CI: [0.08;
determine the sensitivity and specificity of the technique, using cere- 0.32] P = 0.01), but this decrease was not linked to the presence or
brospinal fluids of patients diagnosed with cryptococcosis and other the absence of resins in the flask. Overall, TTP was longer in vials
diseases. with resins than in selective vials. SAT prolonged TTP by a factor
1.76 ([1.30; 2.40]; P < 0.01). The impact of SAT on the TTP was
more marked in the selective vials than in those with resin.
The agreement study showed that when the sampling was done
P135 the same day, on a same patient, on both resins and selective BC, in
the absence of SAT, 16 (16%) and 20 (21%) vials were positive only
on resins or only on selective vials respectively, and in the presence
Impact of systemic antifungal therapy on the detection of of SAT, 13 (36%) and 8 (22%) vials were positive only on resins or
Candida spp. in blood cultures with resins or selective only on selective vials respectively (fig. 1).
media, in the clinical setting of candidemia Conclusion In this clinical study, we showed that SAT influences
S. Bailly,1 M. Cornet,2 P. Pavese,2 L. Le Pennec,2 L. Foroni,2 significantly the result of the blood culture, whether adsorbing resins
J. F. Timsit3 and D. Maubon2 are present or not in the vials. We also demonstrated that there is a
1 clear gain in sensitivity to collect concomitantly resins and specific
Grenoble Alpes University, Grenoble, France; 2University
vials, especially when SAT is present. Concerning BC sampling, new
Hospital Grenoble, Grenoble, France and 3Universite Paris recommendations on candidemia diagnostic and follow-up should be
Diderot, Paris, France clarified in a near future to help clinicians in their everyday practice.

Objectives Despite a relatively low sensitivity, gold standard for can-

didemia diagnostic and follow-up still relies on blood culture (BC).
The use of BC specific media is not clearly addressed in guidelines. In P136
vitro studies showed that the presence of an antifungal can signifi-
cantly modify the rate and the time to positivity of BC not containing
adsorbing agents. This can have a direct impact on the diagnostic Case of mycetoma by Scedosporium sp.
and on the monitoring of candidemia. Our objective was to study in M. L. Scroferneker,1 D. Heidrich,1 P. T. Dalbem,1 K. O. Alves,1
clinical practice the impact of systemic antifungal therapy (SAT) on Z. M. M. Andrade,1 C. Silva,1 G. Vettorato,2 I. S. Santos,2
the diagnostic performance of each medium and to highlight their T. G. Amaro2 and M. Zampese3
added value in the management of candidemia. 1
Methods All patients experiencing candidemia, with at least one col- Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil;
lected blood sample, in a 3 years period (2010–2013) were included
2
Hospital Santa Casa de Misericordia de Porto Alegre, Porto
in a retrospective study at the Grenoble University Hospital, France. Alegre, Brazil and 3Private Office, Porto Alegre, Brazil
Vials comprising adsorbent resins (Bactec aerobic PLUS and anaero-
bic PLUS, BD Diagnostics, Sparks, USA) and selective vials without Introduction Mycetoma is a chronic progressive granulomatous
resins (Bactec MYCOSIS IC/F) were considered. We analyzed: 1) the infection of the subcutaneous tissue. It may affect muscles, bones,

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 101
Poster Presentations

cartilage and joints, most often affecting the lower extremities, usu-
ally the foot. The disease is caused by either fungi or bacteria, giving
rise to eumycetomas and actinomycetomas, respectively. It has a
classic triad of soft tissue swelling, draining sinus tracts, and extru-
sion of grains.
Case Report A male, 66 years old, farmer, immunocompetent,
searched medical assistance in a private dermatology office of Porto
Alegre city, Brazil, in march 2015, with lesions typical of mycetoma.
According to the patient, the disease has progressed for 25 years. In
direct mycological examination, hyaline septated hyphae were
observed in the grains, representing an eumycetoma. In cultural
mycological examination, there was growth of a colony in Sabour-
aud’s medium showing grayish pigment. In the optical microscopic
observation of colonies, there were hyaline septated hyphae with
ovoid conidia in simple conidiophores, indicating eumycetoma by
Scedosporium sp. The molecular analysis for identification of the spe-
cies is being performed. The antifungal activity was performed and
the minimal inhibitory concentration (MIC) of seven antifungal Figure 1
agents was evaluated by the microdilution method in 98 well plates
according to the CLSI protocol M38-A2 standardized for Scedosporium
spp., and the minimal fungicidal concentration (MFC) were evaluated laser desorption/ionization-time of flight mass spectrometry (MALDI-
in Sabouraud dextrose broth medium in tubes from the wells with TOF MS) is very powerful tool for precise identification of bacteria, as
no growth. The tubes were maintained for 14 days to 30 °C until well as yeasts.
reading. The concentrations tested for fluconazole ranged from 0.125 Methods In this study PNA-FISH YTL (AdvanDX) for rapid identifi-
to 64 lg/mL, and for the other antifungals, the concentrations ran- cation from yeast positive blood culture and culture based MALDI-
ged from 0.0312 to 16 lg ml
1. The MIC/MFC obtained were TOF MS (Bruker) for identification of yeast cells from blood cultures.
(lg ml
1): voriconazole (0.25/8), ketoconazole (0.5/8), posaconazole 47 yeast positive blood cultures from patients hospitalized in Univer-
(1/>16), itraconazole (2/>16), amphotericin B (4/>16) fluconazole sity Hospital Center Zagreb during 1 year were analyzed. PNA-FISH
(16/>64), terbinafine (>16/>16). Therefore, the isolate was consid- YTL identification was routinely performed for every yeast positive
ered sensitive to voriconazol, ketoconazol and posaconazole; interme- blood culture in gram stain. After cultivation conventional methods
diate to itraconazole; resistant to amphotericin B, fluconazole and for identification and culture based MALDI-TOF MS were performed.
terbinafine. The patient is being treated with 400 mg itraconazole by Results from PNA-FISH YTL were compared with results of identifica-
day and is being monitored. tion with MALDI-TOF MS and convential methods.
Discussion Scedosporiosis is reported infrequently. In Brazil, less Results From 27 positive blood cultures with green fluorescence
than 50 cases were reported. The antifungal susceptibility in infec- MALDI-TOF MS identification reveal 9 C. albicans and 18 C. parapsilo-
tions caused by the genus Scedosporium is important because of the sis. One isolate from patient hospitalized in pediatric intensive care
multi-resistance of the isolates. The MFCs obtained from the isolate of unit did not show any fluorescence and MALDI-TOF, as well as with
this patient were very high, indicating that a successful treatment of conventional methods was identified as C. parapsilosis. From 10 posi-
an immunocompromised patient infected by this strain should be dif- tive blood cultures with red fluorescence MALDI-TOF MS identifica-
ficult. In the present case, in which the patient was immunocompe- tion reveal 8 C. glabrata and 2 C. krusei. Isolates from yeast positive
tent, the choice of treatment was based on the outcome of the blood cultures in gram stain that did not show any fluorescence
antifungal susceptibility tests, the adverse effects of the drugs, mainly MALDI-TOF MS identified as 4 isolates of C. guillermondii, 1 isolate of
because the patients is elderly, and the availability of medication Pichia fabianii, 1 isolate of C. peliculosa, and 3 isolates of C. lypolitica.

publicly distributed by the SUS (Sistema Unico de Saude) in Brazil, (Figure 1) Two of above mentioned blood cultures were mixed and
since the patient belongs to a low income class. As there are ineffec- showed red and green fluorescence. From one bottle MALDI-TOF MS
tiveness reports using itraconazole in certain cases in the literature, identified C. parapsilosis and C. glabrata and from other C. albicans and
the monitoring of this patient is crucial to obtain the cure. C. glabrata.
Conclusion PNA-FISH YTL enables clinical microbiologist to report
very accurate results of Candida identification sooner (even few days)
than with conventional methods and help clinicians to select the
P137 appropriate antifungal drug in order to improve the patients out-
come, as well as report mixed cultures of etiologic agents causing
candidaema especially presence of non-albicans species with intrinsic
PNA-FISH YTL and culture-based MALDI-TOF MS resistance or reduce susceptibility to some of the antifungal drugs.
identification of etiologic agents of candidaemia in
University Hospital Center Zagreb
S. Plesko, M. Jandrlic, V. Rezo Vranjes, V. Tripkovic, M. Novak,
I. Marekovic and V. Plecko P138
University Hospital Center Zagreb, Zagreb, Croatia
Towards non-invasive differential diagnosis of
Objectives Candidaemia is very serious illness with high mortality. cryptococcosis: magnetic resonance spectroscopy reveals
The outcome of patients with candidemia highly depends on early marker metabolites of cerebral cryptococcomas and cell
introduction of appropriate antifungal therapy. The choice of appro- viability
priated antifungal therapy is different according the Candida species L. Vanherp,1 G. Vande Velde,2 J. Poelmans,3 A. Hillen,3
causing the candidaemia since non-albicans species still represent a K. Lagrou1 and U. Himmelreich3
significant burden globally. Peptide Nucleic Acid in Situ Hybridisation 1
Yeast Traffic Light system (PNA-FISH YTL) is molecular test designed KU Leuven, Leuven, Belgium; 2Biomedical MRI unit/MoSAIC,
for identification of 5 most common Candida that are the etiologic Leuven, Belgium and 3KU Leuven, Leuven, Belgium
agent of candidiasis. C.albicans/C.parapsilosis shows green fluores-
cence, C.glabrata/C. krusei shows red fluorescence and C. tropicalis Objectives Central nervous system (CNS) cryptococcosis is associ-
show yellow fluorescence. Identification with PNA-FISH YTL is done ated with significant morbidity and mortality, in part due to the often
in 90 min directly from yeast positive blood culture. Matrix-assisted late diagnosis and associated delay in initiation of antifungal

ª 2015 The Authors

102 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

metabolite profiles from cystic glioblastoma or bacterial abscesses

allows non-invasive distinction between those pathologies. Trehalose
concentrations correlated with the number of colony forming units
and a potentially higher trehalose production by C. gattii compared
to C. neoformans was revealed (Figure 2). These results were con-
firmed by ex vivo NMR spectroscopy of lesion material but also by
fibered confocal fluorescence microscopy (FCFM) (data not shown).
The latter showed a higher cell density in C. neoformans lesions com-
pared to C. gattii lesions.
Conclusion For the first time, this study presents a method for non-
invasive follow-up and characterization of cerebral cryptococcomas
in a mouse model. The combination of these imaging modalities will
contribute to increased understanding of the pathogenesis and
dynamics of cryptococcosis including the in vivo assessment of novel
treatment strategies. MRS identified trehalose as a characteristic
metabolite that can be used for diagnostic purposes and as a quanti-
tative biomarker for in vivo assessment of the fungal load. These find-
ings have great potential to assist in the differential diagnosis of
brain lesions in patients, whereby MRS is a safe, non-invasive and
rapid method in comparison to traditional methods such as CSF sam-
pling or biopsies.
Correction added on 19 October 2015, after online publication.
Affiliation of author Liesbeth Vanherp was changed from University
Figure 1. Localized MRS spectra obtained at different time points Hospital Leuven, Leuven, Belgium to KU Leuven, Leuven, Belgium.
post injection identified marker metabolites.

P139

TRIADx: A comprehensive and rapid invasive aspergillosis

diagnostic testing service for real-time patient
management
G. L. Johnson,1 E. Taylor,1 A. Weddell,1 C. Agrawal,2 D. Ready,1
S. Tiberi,3 S. Wyllie,4 S. J. Sarker,2 R. Holliman,1 M. Wilks,5
S. G. Agrawal3 and R. Manuel1
1
Public Health Laboratory London, Public Health England,
London, United Kingdom; 2Barts Cancer Institute, Queen Mary
Figure 2. Linear regression shows relationship between log-trans-
formed CFU counts and trehalose concentrations.
University of London, London, United Kingdom; 3Barts Health
NHS Trust, London, United Kingdom; 4Portsmouth Hospitals NHS
Trust, Portsmouth, United Kingdom and 5Blizard Institute, Queen
Mary University of London, London, United Kingdom
treatment. Cryptococcus neoformans and C. gattii can cause brain
lesions difficult to distinguish from other pathologies, like cystic
glioblastoma or bacterial abscesses, by means of conventional radio- Objective Invasive Aspergillosis (IA) remains a major concern for
logical examinations such as computed tomography (CT) or magnetic clinicians managing immuno-compromised patients. The Public
resonance imaging (MRI). In addition, it is currently not possible to Health Laboratory London, in collaboration with Queen Mary
distinguish between active and sterile microbial lesions in vivo. We University of London and Barts Health NHS Trust, have recently
aimed at the identification of biomarkers present in cryptococcal implemented a ‘triple-test’ approach for IA (TRIADx) comprising a
lesions by use of localized, in vivo magnetic resonance spectroscopy commercially available galactomannan (GM) test, a novel sero-diag-
(MRS) in a preclinical mouse model of CNS cryptococcosis. We have nostic lateral flow device (LFD) test and a novel (in-house) PCR
tested those in vivo biomarkers on their ability to quantify microbial assay. The rapid, next day turn around time (TAT) provides an IA
load non-invasively. diagnostic service for real-time patient management. Our aim was to
Methods Cryptococcus strains (C. neoformans H99 and C. gattii R265, evaluate the performance of the TRIADx service during the set-up
GFP+, 10 000 CFUs, 1 ll) were stereotactically injected into the phase in high-risk haemato-oncology patients.
right striatum of female BALB/c mice (n = 2–4 animals per group). Methods This prospective study comprised 92 clinical samples
Mice were scanned repeatedly over 2 weeks or at a single time point received for TRIADx testing between July 2014 and March 2015.
using a 9.4 T MRI scanner (Bruker Biospin). We acquired anatomical During the evaluation period, requests were received from outside of
(T2-weighted) and diffusion-weighted MRI as well as single-voxel 1H the Haemato-Oncology setting, including patients on monoclonal
MR spectra of the lesions (voxel size 2 9 2 9 2 mm³). MRS results antibody treatment for rheumatoid arthiritis, HIV and chronic respi-
were quantified by use of jMRUI to determine absolute metabolite ratory disease patients. Samples were also received from external
concentrations. Afterwards, brains were isolated for validation with users. Samples were tested using the ‘triple-test’ approach, as previ-
ex vivo NMR spectroscopy, histology and fungal load quantification ously described (1). The correlation between biomarker test results
(CFU counts). was evaluated, as was TRIADx service TAT.
Results Disease progression could be monitored longitudinally with Results Ninety-two samples were received for TRIADx testing (7
MRI whereby increases in lesion size correlated with increased fungal bronchoalveolar lavage fluids (BALF), 76 sera, 5 EDTA, 1 sputum, 3
load (Figure 1). Diffusion-weighted MRI showed increased diffusion plasma). Five samples were excluded, as EDTA samples are not
inside the lesion compared to normal brain tissue (data not shown). accepted for the TRIADx service. Fifty-two samples were successfully
MRS detected the presence of typical fungal metabolites like trehalose TRIADx ‘triple tested’ (56.5% of requests). Due to LFD manufacturing
and mannitol but also lipids inside the cryptococcal lesions (Figure 1), issues, 32 samples were GM/PCR tested only. Due to insufficient sam-
similar to what has been observed in spectra obtained from cell cul- ple volume received, 2 samples were PCR tested only and 1 GM only.
tures or ex vivo lesion material. Comparison with MRS-based All samples were tested and reported within 48 h of receipt. Out of

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 103
Poster Presentations

52 TRIADx sample ‘triple tests’, agreement across all 3 biomarker BDG was determined using the automated Fungitell assay (Asso-
tests was recorded in 45 samples (86.5%) and between 2 of 3 tests ciates of Cape Cod, Falmouth, MA) and interpreted as following:
in 7 samples (13.5%). Five samples (from 3 patients) were positive <60 pg ml
1 negative, 60–80 pg ml
1 intermediate and
across all 3 biomarker tests; a single patient was triple test positive >80 pg ml
1 positive. 15.4 pg ml
1 is the lower limit of BDG detec-
from a BALF only, a second patient from both a BALF and a serum tion in our setting.
sample and a third patient from 2 consecutive serum samples. For all Results All patients without HD/HDF treatment had negative serum
3 cases, TRIADx results influenced real-time patient management. BDG levels. In patients undergoing HD/HDF 1/72 samples (1.4%)
Out of 32 samples that were GM/PCR tested only, agreement yielded a positive result in the arterial control sample at visit2, prior
between the 2 biomarker tests was 100% (2 positive and 30 nega- to the next consecutive HD/HDF session (146 pg ml
1). This particu-
tive). For both positive cases, TRIADx results influenced real-time lar patient (patient 12) did not receive any kind of medication sus-
patient management. All five (100%) IA positive cases were outside pected to cause false positive BDG levels (e.g. beta-lactam antibiotics
the Haemato-Oncology setting. or blood derivates) or developed signs of IFI without antifungal medi-
Conclusion Initial results from use of the TRIADx ‘triple test’ sug- cation. All other samples remained negative throughout HD/HDF
gest that it impacts positively on patient management. The rapid treatment, resulting in a specificity of 0.986 (95% confidence interval
TAT is essential to supporting real-time clinical management. Sam- 0.925–0.998).
ples from both local and distant centres were processed within the All tested dialysis fluid samples yielded negative results (range
self-imposed TAT. This service has improved quality and safety of <15.4–53.7).
patient care whilst reducing antifungal drug spend. We are currently Conclusion Our results suggest that serum BDG testing is a reliable
setting up an online requesting and reporting system, to assist our biomarker for IFIs in patients with HD/HDF using synthetic
external users. All service users are invited to join the Mycology Con- membranes.
sortium for the Development of Diagnostics (MyCo D2) to allow data
collection and prospective evaluation of the service. Appropriate
recognition will be given to all collaborators.
1. Johnson et al. (2015). JCM.00110–15; Epub 2015/04/24 P141

Falsely elevated serum 1,3-Beta-D-Glucan levels in patients

P140 with gram-negative bacteremia
J. Prattes,1 R. B. Raggam,2 J. Rabensteiner,1 F. M. Reischies,3
I. Zollner-Schwetz,1 T. Valentin,1 F. Prueller,1 R. Krause2 and
Serum 1,3-Beta-D-Glucan levels are reliable in patients
M. Hoenigl1
undergoing hemodialysis or hemodiafiltration 1
J. Prattes,1 E. Jaindl,1 G. Schilcher,1 F. Prueller,1 M. Hoenigl,1 Medical University of Graz, Graz, Austria; 2Medical University
D. Schneditz1 and R. Krause2 Hospital of Graz, Graz, Austria and 3Meduni Graz Section of
1 Infectious Diseases and Tropical Medicine, Graz, Austria
Medical University of Graz, Graz, Austria and 2Medical
University Hospital of Graz, Graz, Austria
Background In absence of invasive fungal infections (IFIs) gram-
negative bacteremia was postulated as potential cause of false posi-
Aim 1,3-beta-d-glucan (BDG) is cell wall component of most patho- tive serum 1,3-beta-d-glucan (BDG) levels. However, previous studies
gen fungi (e.g. Candida spp., Aspergillus spp.) and is used in daily were limited due to small sample sizes and contradictory results.
clinical routine as serum marker for diagnosis of invasive fungal Thus, we evaluated serum BDG levels in a cohort of patients with
infections (IFIs). In the absence of IFIs serum BDG may be elevated gram-negative bacteremia.
in patients undergoing hemodialysis (HD) and hemodiafiltration Methods In our NOBIS cohort, over 1000 blood samples were
(HDF). As data about BDG in HD/HDF patients are contradictory we simultaneously obtained with blood cultures from patients presenting
evaluated the influence HD and HDF using synthetic dialyzer mem- to the emergency department at the Medical University Hospital of
branes on serum BDG levels. Graz, Austria. Out of this cohort 103 samples with positive mono-
Materials and Methods During March and April 2015 we prospec- bacterial blood culture with growth of Pseudomonas spp. (n = 35), En-
tively included 22 patients with chronic kidney disease (CKD) with- terococci (n = 38), and E. coli (n = 30) were tested for BDG retrospec-
out signs and symptoms of invasive fungal infections (IFIs). Ten tively. BDG was evaluated using the automated Fungitell assay and
patients did not have HD/HDF and 12 patients had HD/HDF using
synthetic membranes (see Table 1). Serum BDG was measured once
in patients without HD/HDF to exclude CKD causing falsely elevated
serum BDG levels. Serum sampling scheme for BDG testing in
patients undergoing HD/HDF is displayed in Figure 1. Additionally,
we determined BDG in dialysis fluid as potential source of BDG
contamination.

Figure 1. Sampling scheme for patients undergoing hemodialysis or

hemodiafiltration Figure 1. BDG levels in gram-negative bacteremia.

ª 2015 The Authors

104 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

interpreted as following: <60 pg ml
1 as negative, 60-80 pg ml
1 as

intermediate and >80 pg ml
1 as positive. 15.4 pg ml
1 is the lower
limit of detection in our setting.
Results 25.7% of serum samples with Pseudomonas sp. bacteremia
yielded a positive BDG results, 31.6% of samples with Enterococci and
26.7% of samples with E. coli bacteremia. Chi-quadrat test yielded no
difference regarding the amount of BDG positive sera within our
three groups (P = 0.858).
Median BDG levels plus interquartile range (IQR) are displayed in
Figure 1. Kruskal-Wallis analysis yielded no difference of median
BDG levels within the three groups (P = 0.507).
Conclusion Gram-negative bacteremia may cause false positive BDG
levels in up to 31% of cases. Positive BDG levels in these patients
therefore need to be interpreted with caution.
amplification curves or melting peaks were observed for the tandem
repeat of 34 bp (TR34), but identical melting peaks for T289A were
observed. Remarkably, L98H and Y121F resistance markers showed
P142 different melting peaks for both samples. To confirm these findings,
sequencing of the calmodulin, beta tubulin, ITS and CYP51A gene
Differentiation of A. lentulus and A. felis in two clinical was performed. This resulted in identification of A. lentulus and A.
felis (both in section Fumigati) harbouring both specific melting pro-
cases using the AsperGenius assay
files for Y121F and L98H resistance markers. These findings were
G. M. Chong,1 A. Vonk,1 J. F. Meis,2 G. Dingemans,3 confirmed using additional clinical A. lentulus and A. felis strains.
J. Houbraken,4 F. Hagen,5 G. Gaajetaan,3 D. van Tegelen,3 Conclusion The AsperGenius multiplex assay enables detection and
G. Simons3 and B. J. A. Rijnders1 differentiation of Aspergillus species including members of the Fumi-
1
Erasmus University Medical Center, Rotterdam, the Netherlands; gati complex using the azole resistance markers Y121F, TR34 and
2
Canisius Wilhelmina Hospital and Radboud University Hospital, L98H. The ability to identify Aspergillus on species/complex level by
Nijmegen, the Netherlands; 3PathoNostics B.V., Maastricht, the molecular methods (directly on clinical material) and resistance
mutations improvess correct management of these infections.
Netherlands; 4CBS-KNAW Fungal Biodiversity Centre, Utrecht,
the Netherlands and 5Canisius Wilhelmina Hospital, Nijmegen,
the Netherlands
P144
Objectives The most frequent cause of invasive aspergillosis (IA) is
Aspergillus fumigatus, followed by A. flavus, A. terreus, and A. niger,
but many other Aspergillus sibling species have been associated with Diagnostic performance of galactomannan antigen in
this infection. Limited data is available on the prevalence of Aspergil- cerebral spinal fluid in patients with cerebral aspergillosis
lus sibling species in a clinical setting, which is of concern because G. M. Chong,1 J. A. Maertens,2 K. Lagrou2 and B. J. A. Rijnders1
these species show intrinsic resistance to antifungal agents. Culturing 1
Erasmus University Medical Center, Rotterdam, the Netherlands
Aspergillus has a low success rate and no commercial tests are able
and 2University Hospitals Leuven, Leuven, Belgium
to differentiate sibling species within section Fumigati. We describe
two cases with proven IA caused by section Fumigati siblings and the
contribution of the AsperGenius assay in the diagnosis. Objectives Cerebral aspergillosis (CA) is a rare but often fatal infec-
Case 1 A 68-year-old man known with allogeneic stem cell trans- tion. The diagnosis can be difficult as radiological signs are diverse,
plantation for myelodysplastic syndrome, was admitted to the hospi- obtaining a biopsy may not be feasible, and cerebrospinal fluid (CSF)
tal because of possible IA. Because there was no improvement despite culture is only positive for Aspergillus species in about one-third of
treatment with voriconazole and liposomal amphotericin-B (L-AmB), the cases. The use of galactomannan (GM) antigen testing on CSF
a lung biopsy was performed that showed fungal hyphae and A. has not been well validated for the diagnosis of CA. Based on 3 small
fumigatus complex was cultured. The AsperGenius assay performed studies (n = 25 in total) the test seems promising. In this retrospec-
on this culture was compatible with A. lentulus. Therapy was tive study, we evaluated the diagnostic performance of GM antigen
switched to posaconazole for proven IA. Patient died of severe Graft- test in CSF in patients with suspected CA.
versus-Host disease. Post-mortem, sequencing confirmed A. lentulus Methods Patients from 2004 to 2014 with suspected CA in whom
and MIC’s were 2 lg ml
1 for amphotericin-B, 2 lg ml
1 for CSF GM was determined, were selected. Invasive aspergillosis (IA)
voriconazole, and 0.125 lg ml
1 for posaconazole, respectively. was defined according to the revised EORTC/MSG criteria. To avoid
Case 2 A 54-year-old man with progressive chronic lymphocytic inclusion of the test that we wanted to validate (= CSF GM) into the
leukemia was admitted because of probable IA. Under voriconazole gold-standard to which the CSF galactomannan test had to be com-
the lung abnormalities were progressive and a lung biopsy was per- pared, we removed CSF GM from the microbiology criteria in the
formed. The culture combined with sequencing showed A. felis (no EORTC/MSG definition. To avoid overlap between the definition of
MIC’s available). Therapy was switched to L-AmB. However, treat- CA and IA by EORTC/MSG, we excluded central nervous system radi-
ment was discontinued because there were no therapeutic options for ology from the clinical criteria. We defined probable CA when on top
his disease and patient died shortly thereafter. AsperGenius was per- of a probable IA, cerebral radiological signs compatible with invasive
formed on material obtained post-mortem and was compatible with fungal infections were present. Proven CA was diagnosed when on
A. felis. top of these criteria cerebral pathological evidence of IA or a positive
Methods The AsperGenius multiplex assay was used for the diagno- CSF Aspergillus culture was present.
sis of two patients suspected of IA. The assay can detect and differen- A CSF GM ≥1.0 was used as cutoff to calculate the sensitivity,
tiate A. fumigatus and A. terreus directly from bronchoalveolar lavage specificity, positive and negative predictive values (PPV, NPV). CSF
(BAL) fluid and simultaneously identifies the most prevalent CYP51A GM was correlated with serum GM.
mutations using melting curve analysis. Moreover, other clinical rele- Results In total, there were 38 patients with suspected CA. In these
vant Aspergillus species can be detected, including members of Asper- patients, there were 6 with proven IA, 11 with probable IA and 21
gillus section Fumigati. without IA according to the revised EORTC/MSG criteria. In the pro-
Results Analysis of both BAL samples resulted in signals for A. fumi- ven IA group, there were 4 patients with proven CA and 2 with
gatus and Aspergillus species with the species multiplex assay. No probable CA (these two patients had histopathological evidence of IA

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 105
Poster Presentations

outside the cerebrum). Fifteen of the 17 proven or probable CA had Based on the whole dataset, 18/19 (95%) “PCP”samples had a
a positive GM (all of them ≥2.0). Twenty of the 21 without CA had a positive test whereas 182/184, (99%) “no PCP”samples had a nega-
negative GM. Therefore, the sensitivity, specificity, PPV and NPV was tive test. Specificity and LR of this new assay were higher than that
88.24%, 95.24%, 93.75% and 90.91%, respectively. of DNA qPCR (sensitivity, 0.86 vs. 1.00; specificity, 0.99 vs. 0.87;
Serum GM was available in 16 of the 17 patients with probable or LR, 78.9 vs. 8.0)
proven CA. CSF GM was higher than serum GM in 8 of these 16 Conclusion RNA expression analysis allowed a better discrimination
patients and was lower in 1 patient with probable CA. In patients between PCP and carriage based on the detection of different physio-
with CA, the mean GM in CSF was higher than in serum (4.89 vs. logical states using only two mitochondrial genes. This assay paves
3.72), which illustrates that leakage from serum to CSF does not the road for the implementation of new tests to diagnose fungal or
explain the CSF galactomannan levels as this would result in lower parasitic infectious diseases.
CSF galactomannan levels than in serum.
In the 17 patients with CA, there were two patients who had a
positive culture in the CSF and one who had positive culture on cere-
bral biopsy. A. fumigatus was cultured in these three patients. P146
Conclusion GM detection in CSF shows high diagnostic performance
for CA. CA can be diagnosed or ruled out based on this assay with-
out the need for cerebral biopsy. Fungal isolates from diabetic amputations: histopathologic
spectrum and correlation with culture.
R. P. S. Punia, V. Jassal, J. Chander, A. Attri, E. Jain and
H. Mohan
P145 Government Medical College Hospital, Chandigarh, India

Diagnosis of Pneumocystis pneumonia: switching from Objectives Fungal infections are difficult diagnostic and therapeutic
DNA quantification to RNA expression analysis problems, serious cause of morbidity or mortality in diabetes. Litera-
A. Alanio,1 A. Bergeron,1 A. Sturny-Leclere,2 N. de Castro,1 ture references on fungal infections of diabetic foot ulcers are very
B. Hommel,2 F. Dromer2 and S. Bretagne1 scarce and differ significantly. In diabetic patients, mycotic infection
can open the door to secondary bacterial infections promoting foot
1
Ho^pital Saint Louis, Paris, France and 2Institut Pasteur, Paris, ulcers and gangrene. Because of non-specific nature of the clinical
France findings in fungal foot ulcer infection, the diagnosis depends on two
basic laboratory approaches, mycologic and histopathologic
Context Pneumocystis pneumonia (PCP) is one of the most frequent examination.
fungal infections occurring in HIV-positive and HIV-negative Methods Twenty five diabetic patients undergoing limb amputation
immunocompromised patients. PCP diagnosis still relies on micro- due to gangrene were included in study from a tertiary care teaching
scopic visualization of the microorganism using classical or hospital over a period of 2 years. During surgery specimens from
immunofluorescence stains. However, these classical means fail to gangrenous tissue was taken for culture and KOH examination.
detect low fungal burden associated with PCP in non-HIV immuno- Amputation specimens were subjected to histopathology examina-
compromised patients in contrast to quantitative PCR (qPCR) target- tion. Special stains PAS and Grocott were used in all cases.
ing DNA which has become a valuable tool for PCP diagnosis (ECIL- Results Age of the patients varied from 30 to 90 years with 16
5 guidelines, http://www.kobe.fr/ecil/telechargements2013/ECIL5% males 9 females. On culture/KOH fungi were isolated in 11 cases,
20-%20PjP%20guidelines-%20Biology.pdf). Nevertheless, detection of Candida species 8 cases, Trichophyton rubrum 3 cases, Fusarium 1
DNA is possible in patients with PCP carriage rather than active case. On histopathology with special stains fungi were seen in 4
infection. As a consequence, the distinction between these two situa- cases, Candida species 3 cases, Trichosporon 1 case. In one case Can-
tions has relied until now on quantification of the fungal burden in dida was seen on histopathology while culture/KOH was negative.
the respiratory specimens: high fungal burden in active PCP and low 14 cases were negative for fungal culture after 1-month incubation.
burden in carriers (Alanio et al. CMI 2011). However, large overlaps Conclusion Because of their typical form and size, fungal elements
of quantification between carriage and infection were observed. Con- can be visualized in histopathologic preparations of ulcer using differ-
sequently, qPCR is not fully accepted as a reference diagnostic ent staining procedures. The most common pathogens are: yeast and
method despite its ease, high reproducibility and quantitative results. dermatophytes and the most frequent dermatophyte is Trichophyton
To overcome this limit, we developed a new assay based on the rubrum. Candida infections can be first presenting sign of undiag-
detection and quantification of the expression of two fungal genes nosed diabetes mellitus. The knowledge of fungi strain allows using
(RNA targets) with calculation of the expression ratio between those the best agent, dose and length of management and greatly improv-
genes. We then compared the results to those obtained with our ing the success in treatment.
DNA qPCR assay (Alanio et al., CMI 2011).
Methods RNA from 200 consecutive bronchoalveolar lavages
(BALs) was extracted using RNA minikit (Qiagen). The expression of
two genes (GENE1 and GENE2) was quantified on a LightCycler 480
platform and the ratio between genes was calculated as already
described (Pfaffl et al. NAR 2001). Patients with positive DNA and
RNA qPCR results were classified by two skilled clinicians as
“PCP”(n = 19) or “no PCP”(n = 36) based on clinical, radiological
and immunofluorescence findings. Patients with negative DNA and
RNA qPCR results were assigned to the “no PCP”group.
Results From the 200 samples, 34 (17%) were both DNA and RNA
qPCR positive and 148 (74%) were GENE2 RNA and DNA qPCR neg-
ative (Table 2). In 5 (2.5%) samples, GENE2 DNA but not RNA was
detected, whereas in 13 samples, GENE2 RNA was detected but DNA
was not (Table 2). Sixteen samples with positive GENE2 RNA but
negative GENE1 RNA were associated with “no PCP”patients.
The ROC curve analysis of the 30 samples in which GENE1/GENE2
ratio was calculated showed that a threshold between 1.27 and 1.66
had the highest likelihood ratio (LR: 13.13; sensitivity, 0.94; speci-
ficity 0.93) for distinguishing infection from carriage.

ª 2015 The Authors

106 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P147 P148/M11.3

Galactomannan determination in biological samples of Brain Abcess due to Cladophialophora bantiana: first case
non-hematological patients with invasive aspergillosis in Portugal
caused by different Aspergillus spp. H. S. Brizido,1 D. Carvalho,1 L. Nogueira Martins,1 C. Verıssimo,2
S. M. Ignatyeva,1 T. S. Bogomolova,2 V. A. Spiridonova,3 J. Lavrador,1 L. Marques Lito1 and J. Melo-Cristino1
Y. V. Borzova,4 M. A. Atyukov,5 T. A. Stepanenko,5 1
Centro Hospitalar Lisboa Norte, Lisbon, Portugal and 2National
S. M. Nuraliev,6 O. V. Shadrivova,7 E. A. Desyatik,3 N. Vasilyeva3 Institute of Health Dr. Ricardo Jorge, Lisboa, Portugal
and N. Klimko2
1
Medical mycology institute named after Kashkin, Saint Objectives Cladophialophora bantiana is a highly neurotropic dema-
Petersburg, Russia; 2North-Western State Medical University tiaceous mold that is a rare cause of cerebral abscesses, particularly
named after I.I. Metchnikov, Saint Petersburg, Russia; 3I. in immunocompetent patients, and is associated with a high mortal-
Metchnikov North-Western State Medical University, Kashkin ity (up to 70%). The aim of the authors is to present a rare with a
Research Institut, Saint Petersburg, Russia; 4North-Western State successful outcome case of a brain abscess caused by this species.
Medical University named after I.I. Mechnikov, Saint Petersburg, Few cases have been reported in the literature world-wide, being this
Russia; 5City hospital, Saint Petersburg, Russia; 6Research one the first reported in Portugal.
Institute of Phthisiopulmonology, Saint Petersburg, Russia and 7I. Methods and Results A 56 year-old male, with chronic hepatic dis-
Mechnikov North-Western State Medical University, St. ease (HCV and alcohol/drug abuse), HIV negative, referred to the
neurosurgery department of our hospital with multiple neurological
Petersburg, Russia
deficits and a space-occupying lesion in the left frontal lobe. The
patient underwent multiple surgical interventions to remove an
Objectives To evaluate the sensitivity of “Platelia Aspergillus EIA”test abscess and specimens were sent to the microbiology laboratory. The
for galactomannan (GM) determination in non-hematological first abscess material sent for examination was “dura mater”and
patients with invasive aspergillosis caused by different Aspergillus spp.
Methods The investigation included 115 clinical samples collected
during 1999–2014 years. from 60 non-hematological patients with
diagnosis of invasive aspergillosis (IA) confirmed by classical myco-
logical methods according to EORTC/MSG, 2008. The age of patients
varied from 7 months to 83 years (median –36 year). The main
underlying diseases were: malignancies (22.4%), chronic sinusitis
(15.5%), chronic pyelonephritis (12.5%), chronic obstructive pul-
monary disease (8.6%), pneumonia (6.9%), pulmonary tuberculosis
(6.9%), systemic diseases with pulmonary involvement (5.8%),
chronic bronchitis (9.8%), vasculitis (1.7%), osteomyelitis (1.7$); no
underlying disease – 3.5%. Totally 60 serum samples and 55 samples
of broncho-alveolar lavage (BAL) were examined for GM Aspergillus
spp. antigen by means of the “Platelia Aspergillus EIA”test (Bio-Rad
Laboratories, USA) with calculating of the optical density index (ODI)
of the antigen. The serum sample was considered as positive if ODI ≥
0.5 and BAL sample – if ODI ≥ 1.0. All BAL samples were investi-
gated mycologically including direct fluorescent microscopy with cal-
cofluor white and culture on Sabouraud agar.
Results Cultures of 55 BAL samples from 60 patients with IA
revealed various Aspergillus spp.: A. fumigatus (31%), A. niger (31%),
A. flavus (11%), A. ochraceus (1%). From 20 patients (34%) two or
more different species were isolated including rare species A. ustus
(6%) and A. versicolor (3%). Aspergillus spp. often caused lung disease Figure 1
(68%), more rarely – sinusitis (22%), in 10% of patients > 2 sites of
infection. GM test was positive in 45% of serum samples and 60% of
BAL samples from patients with mycologically confirmed IA. In cases
with A. fumigatus as the causative agent of IA sensitivity of GM test
in serum was 47%, in BAL – 66%. In patients with IA caused by A.
niger or A. flavus sensitivity of the tests was lower: 36% (P = 0.02)
and 53% (P = 0.03) for A. niger; 16% (P = 0.001) and 40%
(P = 0.002) for A. flavus. In biological samples of patients with IA
caused by several species of Aspergillus GM test was positive in 42%
(blood serum) and 70% (BAL) of cases. The results show species
specificity of Aspergillus GM metabolism during the course of
infection.
Conclusion The sensitivity of GM test in blood serum and BAL sam-
ples from non-hematological patients with invasive aspergillosis is
lower than in hematological patients and depends on the type of bio-
logical sample and the species of the etiological agent.

Figure 2

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 107
Poster Presentations

cultures revealed Cladophialophora bantiana and Nocardia nova (Maldi-

TOF score 1.946) but in subsequent abscess material examined (pus)
C. bantiana was the one persistently isolated. Samples were cultured
on Sabouraud dextrose agar (SDA), SDA with gentamicin/chloram-
phenicol and SDA with actidione. After incubation velvety dark colo-
nies were obtained with surface and reverse olive-gray to black color.
Microscopic examination of a lactophenol blue mount preparation
showed brown septate hyphae with long, sparsely branched conidio-
phores bearing wavy chains of smooth oval conidia without pig-
mented scars. The isolate was identified as Cladophialophora bantiana Figure 1. NP tissue processing algorithm for fungal detection
based on its morphological features growth at 42°C and cyclohex-
imide resistance. Total genomic DNA was extracted. The internal Results Our analyses found: (1) almost half of CRSwNP patients
transcribed spacer (ITS) region of ribosomal DNA (rDNA) of these iso- (19/43; 44%) underwent FESS more than once (re-FESS group); (2)
lates was amplified using the primer set ITS1 and ITS4. Sequencing fungi were detected (wF) in 10/43 (23.3%) patients (FESSwF group),
of both strands was performed and nucleotide sequences were edited representing 13/55 culture positive NP tissue (23.6%); (3) re-FESS
using the program Chromas2 and aligned using the program CLUS- group is the most often associated with asthma and aspirin intoler-
TAL X2. The obtained sequences were compared with sequences ance (79; 58%, respectively) compared to ft-FESS group (25; 12%,
deposited in the GenBank and CBS-KNAW Fungal Biodiversity Centre respectively) (P = 0.000, P = 0.002; respectively); (4) mycology eval-
databases: the isolate was identified as C. bantiana with 99% homol- uation of single-cell suspension of NP tissue showed significant
ogy. Molecular results confirmed the microbiological diagnosis. Sus- higher percentage of fungal finding in re-FESSwF than in ft-FESSsF
ceptibility testing was not performed. The patient was initially treated group (42; 8%, respectively; P = 0.01); (5) we observed significantly
with vancomicin, meropenem and liposomal amphotericin B for longer duration of CRSwNP in FESSwF than in fungal negative
28 weeks but the poor clinical and imaging response lead to a switch patients (FESSsF group) (17.4  10.9; 10.4  7.0 years, respec-
to amoxicillin/clavulanic acid and voriconazole. After 16 months of tively; P = 0.033) and (6) predominate strain was Aspergillus flavus
the first brain abscess excision and after 5 months under therapy detected in 6/10 patients.
with voriconazol, the patient improved clinical and imagiologicaly, Conclusion This is the first study which analysed association of
maintaining only minimal neurological deficits. fungi in single-cell suspension of NP tissue and recurrent CRSwNP.
Conclusion New and emerging pathogens have renewed concern We demonstrate significantly higher percentage of positive fungal
about the diagnosis and treatment of brain abscess and equally pose finding in re-FESSwF than in ft-FESSwF group. Although the role of
a challenge to clinical microbiologists. Given the fact that early and fungi in CRSwNP is still controversial, we gave new insight in this
accurate diagnosis saves lives, such specimens should promptly be field showing correlation between fungi and prolonged duration of
sent for microbiological analysis. Brain abscess is not only a neuro- CRSwNP and fungi and relapse of NP after FESS. Further studies are
surgical emergency but also a microbiological emergency and a diag- needed to determine if the nasal lavage with commercial or natural
nostic challenge to both disciplines. antifungals may have negative impact on the fungal growth in upper
respiratory tract and benefit for patients with CRSwNP.

P149
P150
Association of recalcitrant chronic rhinosinusitis with nasal
polyposis and fungal finding in polyps single-cell Construction of a new diagnostic tool for the human
suspension pathogenic members of the Fusarium fujikuroi species
A. M. Barac,1 M. G. Pekmezovic,1 M. Z. Kostic1 and V. S. Arsic complex
Arsenijevic2 A. D. van Diepeningen,1 J. Iltes,1 B. Brankovics,1 G. S. de Hoog,1
1
Institute of Microbiology and Immunology, Faculty of Medicine, J. Bergervoet,2 T. A. J. van der Lee2 and C. Waalwijk2
Uni. of Belgrade, Belgrade, Serbia and 2University of Belgrade, 1
CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands
Belgrade, Serbia and 2Plant Research International, Wageningen, the Netherlands

Objectives In recent years fungi are favoured as origin of recalci- Objectives Construct a new fast, accurate and reliable diagnostic
trant chronic rhinosinusitis (CRS), especially with nasal polyps tool for members of the Fusarium fujikuroi species complex: Fusarium
(wNP). The omnipresence of fungal spores makes the role of fungi in species are emerging agents of superficial, locally invasive and sys-
development of CRSwNP important, but difficult to determine. Data temic infections in humans. Especially members of the Fusarium fuji-
proving the hypothesis that fungi are involved in the pathogenesis of kuroi species complex (FFSC) can cause disseminated fusariosis in
NP are limited mostly because of the difficulty to properly determine immunocompromised – particularly leukemic – patients. Such dis-
fungi and/or to interpret their finding from upper respiratory tract. seminated fusarioses result in a high mortality rate of patients but
Sensitive methods for fungal detection are still absent, therefore we patient survival may increase with rapid diagnosis. While most
used NP single-cell suspension for mycology investigations in aim to Fusarium species have high levels of resistance to antifungal com-
find out association of recalcitrant CRSwNP and fungal finding in pounds, they vary in their susceptibility towards commonly used
patients that underwent functional endoscopic sinus surgery (FESS). antifungal drugs, like amphotericin B, voriconazole and
Methods A prospective case-series study and culture-based mycolog- posaconazole.
ical examination was conducted in patients who underwent FESS for Methods The DNA-based Luminex assay we selected as platform is a
the first time (ft-FESS) and those with repeated FESS (re-FESS). The microsphere phase array-based analysis, that combines a multiplex
study was conducted in a tertiary Otorhinolaryngology Unit of Clini- PCR with hybridization to probes linked to fluorescent microspheres,
cal Centre of Serbia and National Laboratory for Medical Mycology, followed by analysis with a flow cytometer (Luminex Molecular Diag-
Faculty of Medicine, University of Belgrade. A total of 43 consecutive nostics, Toronto, Canada). This technique can detect up to 500-
patients with CRSwNP underwent FESS and 55 NP samples were 1000 different microsphere-linked probes simultaneously, making it
analyzed by culture-based mycological examination. Samples were possible to screen for a wide set of pathogens in one reaction, while
prepared as single-cell suspensions of NP tissue in Petri dish using a enabling the build-in of safeguards and redundancy.
special mesh for the treatment of lymphatic tissue, under sterile con- The FFSC harbours notorious plant pathogens of crops, like maize,
ditions (Figure 1). Patient’s co-morbidity data were collected. rice and sugar cane. At least 13 species of the 34 species within the

ª 2015 The Authors

108 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

FFSC where shown to cause also human infections. As barcoding species. A. tubingensis and A. awamoriwere identified by MALDI as A.
region for species identification of members in the FFSC complex we niger, and A. carneus as A. terreus. A. carbonarius cannot be identified
chose one of the known more variable gene regions in the transla- by MALDI as they are not included in the database. Finally, A. ustus
tion elongation factor 1-a gene (tef-1a). Sequence data of all species and A. calidoustus have identical sequence and MALDI identifies as A.
were collected from specialized curated databases like the FusariumID ustus. The MALDI score median for the most prevalent species con-
and FusariumMLST databases or were generated from well described firmed by sequencing was 2281 (A. fumigatus), 2227 (A. flavus),
isolates in this study. The sequence data of 2-14 strains from each 2094 (A. niger/A. tubingensis), and 2068 (A. terreus). We observed a
species were used to construct consensus sequences for each species. geographicaldistributionnot homogeneous.
Species-specific single nucleotide polymorphisms (SNPs) were selected Conclusion A.fumigatus is the most common species of Aspergillus
for the production of species specific probes. isolated. Cryptic species of Aspergillus have significant prevalence in
Results and Conclusions The tef-1a gene is one of the known most clinical specimens, and A. tubingensis (section nigri) was the most
variable regions within the genus Fusarium, and based on SNPs prevalent. MALDI TOF is very useful in routine identification of
between sibling species it is possible to distinguish between all species Aspergillus spp. However, the database would benefit from additional
– clinical and agricultural – of the FFSC, whether known human species entries to elucidate the capacity of MALDI-TOF MS to differen-
pathogen or not. We are currently in the process of testing all con- tiate between phylogenetically closely related species and to decrease
structed probes and testing the sensitivity and reproducibility of the the rate of nonidentified isolates particularly its database should be
system. To enhance the robustness of the technique even further and expanded to include cryptic species that could be increase the identi-
not to rely on a single gene detection we will expand the set with fication accuracy.
probes based on other barcode genes like the RNA polymerase II sec-
ond largest subunit rpb2.

P152
P151
Polymerase chain reaction-sequencing based identification
of fungal pathogens in formalin fixed and paraffin
Proteomic and molecular identification of Aspergillus spp. embedded tissues
in clinical samples isolated from the South of Spain T. J. Jillwin, S. M. Rudramurthy, A. Bal, A. Das, B. D. Radotra,
(FUNGAE-IFI Study) S. C. Varma and A. Chakrabarti
F. Galan,1 C. Castro,2 D. Miralles,1 M. J. Linares,3 M. L. Serrano,4
Postgraduate Institute for Medical Education and Research,
P. Bermudez,5 C. Pazos,6 C. Freyre,7 W. Sanchez-Yebra,8 Chandigarh, India
E. Clavijo,9 A. I. Suarez,10 E. Martin-Mazuelos2 and
M. A. Rodriguez-Iglesias11 Objectives To standardize and evaluate polymerase chain reaction
1
Puerta del Mar Univ Hosp, Cadiz, Spain; 2Hospital Universitario (PCR) followed by sequencing for the detection and identification of
de Valme, Sevilla, Spain; 3Reina Sofia, Cordoba, Spain; 4Virgen fungal pathogens in formalin fixed and paraffin embedded (FFPE) tis-
de las Nieves Univ Hosp, Granada, Spain; 5Carlos Haya Univ sue biopsies.
Hosp, Malaga, Spain; 6San Pedro de Alcantara Hosp, Caceres, Methods Archived FFPE biopsies with proven fungal infections as
Spain; 7Puerto Real Univ Hosp, Puerto Real, Spain; reported by experienced histopathologists were used for the study.
8
Torrecardenas, Almeria, Spain; 9Virgen de la Victoria Univ Hosp, For optimization of DNA extraction from FFPE tissues, we compared
Malaga, Spain; 10Virgen Macarena University Hospital, Seville, the performance of a commercial kit and the conventional phenol
Spain and 11Hospital Puerta del Mar, Cadiz, Spain chloroform isoamyl alcohol (P-C-I) method on 40 lm sections from
29 FFPE biopsies. For amplifying fungal DNA, the multicopy nuclear
ribosomal DNA was selected for amplification using pan fungal pri-
Objectives To analize the distribution of Aspergillus spp. in mers. Whole Internal Transcribed Spacer (ITS) region, ITS 1 region,
Andalucıa and Extremadura (South of Spain), through a multicenter ITS 2 region and divergent DI/D2 domains of large ribosomal subunit
prospective study involving 16 hospitals, and to determine the perfor- (28S rDNA) were compared for their amplification from FFPE DNA.
mance of MALDI-TOF for routine identification ofAspergillus spp. To check the specificity of the ITS1 target gene, we correlated FFPE
using molecular sequencing as gold standard. tissue sequence results with culture results for 32 samples. Tissue
Methods The study was conducted prospectively from July 2014 to samples for less common hyalohyphomycosis agents such as Fusar-
March 2015. All the patients who were culture positive for Aspergil- ium, Scedosporium and mucoralean agents such as Mucor, Lichtheimia,
lus spp. in the basis of respiratory samples, blood cultures or biopsy Cunninghamella were obtained through disseminated murine infec-
specimens were included. The strains were sent to a reference labora- tion. Also a mucorale specific PCR was performed for biopsies with
tory included in the list of the participants that made proteomic and aseptate hyphae.
genetic identification. For proteomic profile the strains were identified Results The yield of total DNA was higher by the P-C-I method
by on-plate extraction method adding 1 ll of formic acid. The acqui- [mean 298.5 ng ll
1] than the commercial kit [mean
sition and analysis of mass spectra were performed by a Microflex LT 121.7 ng ll
1] with the same amount of starting material. The qual-
mass spectrometer (Bruker Daltonics) using the MALDI Biotyper soft- ity of DNA extracted by the two methods showed no significant dif-
ware package (version 3.0) with the Filamentous Fungi Library 1.0 ference as evidenced by 260/280 ratio: commercial kit [mean 1.92,
(Bruker Daltonics) and default parameter settings and a species cutoff SD 0.15] and P-C-I method [mean 1.79, SD 0.12]. Out of the differ-
of 1.7.Genomic DNA was isolated and molecular identification was ent targets tested, ITS1 region showed higher sensitivity (60.9%) in
performed by sequencing a portion of the b-tubulin gene. All of the PCR, followed by ITS 2 region (52.2%). The whole ITS
sequences were compared with reference sequences from the (ITS1 + 5.8S+ITS2) region showed poor amplification results (17.4%)
GenBank.Different results between MALDI-TOF MS and molecular and the amplification in D1/D2 domains was 34.8%. ITS1 region
identification were categorized as erroneous identifications. sequencing showed good concordance with culture results and also
Results A total of 179 strains were included. The most frequent spe- in tissue biopsies obtained from murine infection. Mucorale specific
cieswas Aspergillusfumigatus (55.8%), followed by A. terreus (13.9%), semi nested PCR increased the detection of mucoralean DNA from
A.flavus (11.2%), A.tubingensis (10.0%) and A.niger (3.3%). Other mucormycosis implicated biopsies (90.2%), than the broad range
cryptic-siblingAspergillus species accounted for 2.2%, and their distri- ITS1 PCR (70.7%). More than one PCR amplicon by pan fungal PCR
bution was as follow: A. calidoustus (1 strain), A. awamori (1 strain), was frequently seen in tissue biopsies from nasal polyps, nasal scrap-
A.carbonarius (1 strain) andA.carneus (1 strain). MALDI-TOF identified ings and palatal biopsies indicating the co-amplification of commen-
175 isolates, with concordant results compared to molecular identifi- sal fungal DNA from these sites which included Malassezia clones,
cation in 88.3%. Erroneous identification mainly involved cryptic uncultured ascomycota fungi and Candida spp, whereas biopsies from

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 109
Poster Presentations

deep tissues yielded single amplicons. Extraneous fungal DNA con- median 85.9 pg ml
1; IQR: 15.4-325.4 mg ml
1; P < 0.001). In the
tamination was observed in 11.7% samples. multivariate COX regression model BDG was the most important pre-
Conclusion In our study, conventional P-C-I method proved better dictor of 30-day mortality. The risk of dying within 30 days after
than commercial kit for extracting DNA from FFPE biopsies for fun- bronchoscopy increased by 0.5% with every 10 pg ml
1 higher
gal PCR. Individual ITS regions, but not whole ITS region is a BALF BDG level.
promising target for broad range PCR-sequencing based detection of Conclusions BALF BDG was the most important predictive variable
fungal signatures in FFPE tissue biopsies. Potential environmental in the COX regression analysis and testing of BDG levels in BALF
fungal DNA contamination should be prevented by following strict may allow for cut-off dependent prediction of 30-day mortality.
precautions.

P154
P153
Calculation of the Galactomannan-Creatinine Index
Bronchoalveolar Lavage 1,3-Beta-D-Glucan Testing for improves the Diagnostic Performance of Galactomannan
Prediction of Overall 30-day Mortality testing from Urine Samples
F. M. Reischies,1 J. Prattes,2 S. Eigl,3 A. List,3 R. B. Raggam,3 F. M. Reischies,1 J. Prattes,2 S. Eigl,3 A. List,4 R. B. Raggam,3
I. Zollner-Schwetz,2 T. Valentin,2 H. Flick,3 A. Woelfler,3 I. Zollner-Schwetz,2 T. Valentin,2 H. Flick,3 A. Woelfler,3
F. Prueller,2 R. Krause3 and M. Hoenigl2 F. Prueller,2 R. Krause3 and M. Hoenigl2
1 1
Meduni Graz Section of Infectious Diseases and Tropical Meduni Graz Section of Infectious Diseases and Tropical
Medicine, Graz, Austria; 2Medical University of Graz, Graz, Medicine, Graz, Austria; 2Medical University of Graz, Graz,
Austria and 3Medical University Hospital of Graz, Graz, Austria Austria and 3Medical University Hospital of Graz, Graz, Austria

Background 1,3-beta-d-glucan (BDG) is a cell wall component of Background Recent studies on urine Galactomannan (GM) determi-
most pathogenic fungi. The objective of the study was to investigate nation did not take into account urine dilution. The aim of this study
the prognostic value of 1,3-beta-D-Glucan (BDG) levels in bron- was to evaluate if an index of urine GM divided by urine creatinine
choalveolar lavage fluid (BALF) samples and to correlate BALF BDG could improve the diagnostic performance of urine GM
levels with Candida colonization. determination.
Methods A total of 300 BALF samples were collected in 272 Methods This prospective cohort study was conducted between 09/
patients between 2012 and 2014 at the Medical University Hospital 2014 and 05/2015 at the Medical University of Graz, Austria. GM
of Graz, Austria, and tested for BDG (using the Fungitell assay) and and creatinine levels were determined in 109 urine samples from 18
growth of Candida spp. in fungal culture. Kaplan Meier analysis was hematological patients with possible/probable or proven invasive
performed for three different BDG-level groups: ≤100 pg ml
1 (group aspergillosis (IA) according to EORTC/MSG, criteria and in 49 urine
1), 101-400 pg ml
1 (group 2), >400 pg ml
1 (group 3). Hazard samples from 49 outpatients without evidence for IA (validation
ratios for 30- day mortality were determined by COX regression anal- cohort (VC)). We calculated the GM/creatinine index by dividing the
ysis for BALF BDG levels, age at time of BALF and Candida optical density index (ODI) value of the urine GM ELISA test (multi-
colonization. plied by 100) by the urine creatinine level. We evaluated the new
Results A significant difference regarding 30-day cumulative sur- GM/creatinine index clinically by comparing the index to conven-
vival was observed between group 1 (17/134, 12.7% died) and group tional GM urine measurement.
3 [22/90, 24.4% died; Log Rank analysis: P = 0.049]. Median BDG Results 96 samples from 17 patients with possible IA and 13 sam-
levels were also found to be significantly higher in patients with Can- ples from 3 patients with probable/proven IA were included in the
dida spp. colonization (n = 83; median 289.1 pg ml
1; IQR: 95.1- IA cohort. Conventional urine GM levels were slightly higher in the
921.1 pg ml
1) compared to patients without colonization (n = 215; IA cohort when compared to the VC (median 0.07 ODI vs. median
0.06 ODI, P = 0.024), while the GM/creatinine index was markedly
higher in the IA cohort when compared to the VC (median 0.17,
IQR 0.11-0.31 vs. median 0.07, IQR 0.03-0.14, P < 0.001).A GM/
creatinine index cut-off of 0.12 was determined by Youdens index
and displayed a sensitivity of 69% and a specificity of 73% for IA.
Conclusions Our data indicate that calculation of the GM/creatinine
index may improve diagnostic performance of urine GM
determination.

Figure 1. Kaplan-Meier curves, showing 30-days cumulative survival

depending on BDG cut-off in BALF samples Figure 1

ª 2015 The Authors

110 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P155 achieving epidemic proportions. Sporothrix schenckii and allied species

are the agents of human and animal sporotrichosis. The global bur-
den of sporotrichosis stress Sporothrix spp. as an important threat the
The regional Paracoccidioides spp. exoantigen for Double health of the warm blooded host. Epidemiological surveillances high-
Immunodifusion Radial (IDR) test in Midwest of Brazil. light an overwhelming presence of the highly pathogenic offshoot
R. C. Hahn,1 D. T. Takarara,1 T. Tadano,1 L. B. Dias,1 Sporothrix brasiliensis during feline outbreaks. With a few exceptions,
M. M. Pesenti,1 S. Amorim,1 A. M. Rodrigues2 and contamination of warm-blooded hosts occurs after traumatic inocula-
Z. P. Camargo2 tion of propagules of Sporothrix spp. into subcutaneous tissue, usually
from an environmental source such as soil or plants, but zoonotic
1
UFMT, Cuiaba, Brazil and 2Federal University of Sao Paulo, Sa~o transmission from the scratches or bites of a diseased cat is a highly
Paulo, Brazil efficient route of disease spread. Upon infection, naturally and
acquired immunity triggered by Sporothrix antigens may balance the
Objective Paracoccidioidomycosis (PCM) is one of neglected tropical host-parasite interplay, the development of clinical symptoms and
disease(NTDs), caused by fungal infection, that0 s one most prevalent control fungal invasion.
systemic mycosis in Latin America, specially in Brazil. In the central- Objectives Using an immunoproteomic approach we characterized
west region of Brazil the studies with regional exoantigen show proteins of potential significance in pathogenesis and invasion that
greater serological reactivity than exoantigen B-339. The new biolog- trigger the humoral response during feline sporotrichosis.
ical specie named Paracoccidoides lutzii. (Teixeira et al. 2009), is pre- Methods We explored the presence and diversity of serum-derived
sent in this central region of Brazil. The Paracoccidiodes brasiliensis antibodies (IgG) in naturally infected cats against S. brasiliensis anti-
complex include species criptics groups as S1, PS2, PS3 e PS4. S1 gens and its potential use for serodiagnosis. For antigenic profiling,
group is predominant in Latin American and S1 has an predomi- we included yeast cell proteins extracts from the closest known rela-
nance in the central-west region of Brazil, therefore studies show rel- tives S. brasiliensis and S. schenckii. Our analyses used ELISA assay
evant results about this serology to monitorate the treatment of and Western blotting, probed with sera from sporotrichotic cats
Paracoccidoidomycosis. The objective is to study serological (double (n = 49), healthy feral cats (n = 19), and cats with other diseases
immunodiffusion technique) of paracoccidioidomycosis patients from (n = 20). In addition, IgG-seroreactive spots (pooled sera; n = 10)
Midwest Brazil using regional exoantigen (Exo-MT) that reacts well were investigated by two-dimensional (2D) gel electrophoresis.
with most sera from patients living in the state of Mato Grosso – Bra- Results ELISA-based quantitation of anti-Sporothrix IgG has high
zil. Methods Tests performed using regional exoantigen (Exo-MT) sensitivity and specificity among sporotrichotic cats (AUC: 1.0; 95%
were reactive with sera from patients with suspected of paracocci- CI: 0.94-1000; P < 0.0001) when compared with controls. A
doidomycosis attended by Julio Muller Hospital (HUJM) and MT Labo- remarkable cross-reactivity was found using both Sporothrix antigens,
ratory (Lacen-MT) from 2011 to 2015 (until the month of may). supporting the hypothesis that antigenic epitopes may be conserved
Results The sera of 319 patients were analyzed. This serology was among close related agents. Our findings based on 1D immunoblot
performed by double immunodiffusion technique in agarose gel and support 3-carboxymuconate cyclase (60-kDa protein in S. brasiliensis
data were obtained by lifting medical records and/or patient files. and 70-kDa protein in S. schenckii) as the immunodomintant antigen
288 (90.3%) patients were male, raging in age from 1 to 84 years in feline sporotrichosis. In addition, 2D immunoblot revealed six IgG-
old, with a mean age of 47.4. The results of serological analysis reactive isoforms of gp60 in the S. brasiliensis proteome, similar to
showed reactive in 134 (42.0%) and non reactive in 166 (58.0%). the response in human sporotrichosis. Although a primary environ-
Of the 134 reactive sera, 99 (73.8%) were reactive with a regional mental function related to the benzoate degradation pathway of aro-
exoantigen (Exoantigen-MT), and only 35 (26.1%) sera reacted with matic polymers has been attributed to orthologs of this molecule, our
exoantigen B-339. findings support the hypothesis that gp60-70 are important for
Conclusion False negative results were previously released to pathogenesis and invasion in feline sporotrichosis.
patients with PCM. Currently the use of regional exoantigen enables Conclusion A convergent IgG-response from different hosts (mice,
improved sensitivity and specificity in IDR tests used in monitoring cats and humans) has important implications for our understanding
the treatment of patients with PCM. If we compare the positivity of of the coevolution of the warm blooded host and Sporothrix. More-
Regional Exoantigen against positivity the Exoantigen-MT, we over, we propose 3-carboxymuconate cyclase as the major target for
observed a sensitive improvement (48%) in serological reactions by the serological diagnosis as for vaccine development.
IDR. This results in a thoughtful and careful look at the exoantigens Financial support: S~
ao Paulo Research Foundation (FAPESP 2009/
this region of Brazil. 54024-2 and FAPESP 2011/07350-1), National Council for Scien-
tific and Technological Development (CNPq 472600/2011-7), and
Coordination for the Improvement of Higher Education Personnel
(CAPES).
P156

Profiling the diversity of the humoral immune response in P157

feline sporotrichosis due to Sporothrix brasiliensis toward
discovery of potential diagnostic and vaccine antigens
Development of RFLP-PCR method for the identification of
A. M. Rodrigues,1 G. F. Fernandes,1 L. M. Araujo,1 P. P. Della
medically important Aspergillus species using single
Terra,1 P. O. Dos Santos,1 S. A. Pereira,2 T. M. P. Schubach,3 restriction enzyme MwoI
E. Burger,4 L. M. Lopes-Bezerra5 and Z. P. de Camargo1 K. Diba,1 H. Mirhendi,2 S. Rezaie2 and A. Namaki3
1
Federal University of Sao Paulo, Sa~o Paulo, Brazil; 2Evandro 1
Urmia University of Medical Sciences, Urmia, Iran; 2Tehran
Chagas National Institute of Infectious Diseases, Rio de Janeiro, University of Medical Sciences, Tehran, Iran and 3Arefian General
Brazil; 3Fundac
a~o Oswaldo Cruz, Rio de Janeiro, Brazil; 4Federal Hospital, Urmia, Iran
University of Alfenas, Alfenas, Brazil and 5Universidade do Estado
do Rio de Janeiro, Rio de Janeiro, Brazil
Objectives In this study we attempted to modify the PCR-RFLP
method using restriction enzyme MwoI for the identification of medi-
Sporotrichosis is a neglected infectious disease that is remarkable in cally important Aspergillus species.
the fungal kingdom due to its occurrence as zoonotic outbreaks or Methods Our subjects included 9 standard Aspergillus species and
sapronoses around the temperate warm regions. Over the last few 205 Aspergillus isolates of approved hospital acquired infections and
decades sporotrichosis has changed from a relatively obscure ende- hospital indoor sources. First of all, Aspergillus isolates were identified
mic infection with scattered cases to an emerging health problem, in the level of species by using morphologic method. A twenty-four

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 111
Poster Presentations

hours culture was performed for each isolates to harvest Aspergillus diagnosis of PCP, IA, IC, and histoplasmosis but was insensitive for
mycelia and then genomic DNA was extracted using Phenol-Chloro- the diagnosis of cryptococal infections, and the less frequent infec-
form method. PCR-RFLP using single restriction enzyme MwoI was tions caused like paraccocidiodomycosis and trichosporiasis.
performed in ITS regions of rDNA gene. The electrophoresis data Conclusion Serum BDG is a useful test for the diagnosis of IFIs in
were analyzed and compared with those of morphologic immunosuppressed patients, in agreement with previous reports. The
identifications. BDG test has a good performance for the diagnosis of IC, IA, PCP
Results Total of 205 Aspergillus isolates included 153 (75%) envi- and histoplasmosis in our cohort. The BDG test does not detect infec-
ronmental and 52 (25%) clinical isolates. A. flavus was the most tions caused by criptococcus and other infrequently found fungal
frequently isolate in our study (55%), followed by A. niger 65 infections.
(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% Keywords Invasive Fungal Infections (IFIs), 1-3 b-D-Glucan (BDG),
each). Invasive aspergillosis (IA), Invasive candidiasis (IC), Pneumocystis
Conclusion MwoIenabled us to discriminate eight medically impor- pneumonia (PCP).
tant Aspergillus species including A. fumigatus, A. niger, A. flavus as
the most common isolated species. PCR-RFLP method using the
restriction enzyme MwoI is a rapid and reliable test for identification
of at least the most medically important Aspergillus species. P159

Evaluation of mueller hinton agar for germ tube test

P158 D. Er,1 H. Uzuner,1 S. Genc,1 S. Keceli1 and F. Otag2
1
Kocaeli University Medical Faculty, Kocaeli, Turkey and 2Mersin
University, Medical Faculty, Mersin, Turkey
Presence of (1-3) b-D-Glucan in patients at risk for invasive
fungal infections
M. Gonzalez,1 C. Sanchez,2 V. Posada Velez,3 A. Segura,3 Objectives To aim of this study is to evaluate Mueller Hinton Agar
(MHA) as a simple medium for the determination of germ tube test
J. J. Acevedo,4 A. Tobon5 and K. Arango5
1
(GTT).
Rutgers-NJMS, Newark, NJ, USA; 2Mayo Clinic, Jacksonville, Methods A total of 86 Candida species isolated from different clinical
USA; 3CES University, Medellin, Colombia, Epidemiology, specimens were included. All isolates were cultured onto Sabouraud
Colombia; 4Boston University., Boston, MA, USA and Dextrose Agar (Salubris, Turkey) and incubated at 37 ͦ C for 24–
5
Corporacion para Investigaciones Biologicas (CIB), Medellin, 48 h. They were identified by VITEK MS (Biomerieux, Frence). For
Colombia serum GTT, a single Candida colony was inoculated into 0.5 ml
freshly obtained human serum. For MHA GTT, an inoculum of single
colony was streaked by Dalmau technique onto MHA and covered by
a sterile coverslip. After incubation at 37 ͦ C for 1.5 h and 3 h, all
Introduction Invasive fungal infections (IFIs) occur in up to 10% of
immunosuppressed patients causing high morbidity and mortality, in
part due to the difficult and delayed diagnosis; improved diagnostic sera and MHA plates were examined using a light microscope at
strategies are needed. (1-3) b-D-Glucan (BDG) is a panfungal nonin- 940. Serum GTT was accepted as gold standard method for the
vasive seric marker that has shown promise for the diagnosis of com- determination of sensitivity and specificity values.
monly encountered IFIs such as: invasive aspergillosis (IA), invasive Results The identification results were as follows: 51 Candida albi-
candidiasis (IC), Pneumocystis pneumonia (PCP) and invasive histo- cans, one C. dubliniensis, 12 C. parapsilosis, 12 C. glabrata, 4 C. kefyr,3
plasmosis (IH). The use of BDG has been validated as an adjunct C. tropicalis, 3 C. krusei. Serum GTT was positive in 30 (58.8%) and
diagnostic tool in different high-risk populations. 36 (70.6%) of C. albicans isolates at 1.5 h and 3 h, respectively.
Objectives To evaluate the diagnostic utility of serum BDG for the MHA GTT was positive in 21 (41.2%) and 40 (78.4%) of C. albicans
diagnosis of IFIs in an unselected population of high-risk patients. isolates at 1.5 h and 3 h, respectively. All non-albicans species
Describe the incidence of BDG according to the different microorgan- except C. dubliniensis were found as negative using both tests. Both
isms causing IFIs. GTTs were positive for C. dubliniensis at 1.5 h and 3 h. MHA sensitiv-
Methods We performed an observational, prospective study between ity and specificity was determined as 58% and 92% at 1.5 h; 86%
November 2013 and May 2014, patients were enrolled from 6 medi- and 81% at 3 h, respectively.
cal institutions. All patients were at risk of IFIs according to the Conclusions MHA is safer and easier medium for GTT that may be
European Organization for Research and Treatment of Cancer/Inva- used as alternative to serum GTT. The evaluation of GTT on MHA
sive Fungal Infections Cooperative Group and the National Institute after 3 h incubation is recommended since it has higher sensitivity
of Allergy and Infectious Diseases Mycoses Study Group (EORTC/ compared to 1.5 h.
MSG) criteria, serum BDG was performed to assess its utility for the
diagnosis IFIs.
Results We collected the serum of 40 patients at risk for IFIs with
proven/probable infections according to the EORTC/MSG criteria. 27 P160
of the patients were male. The most common infections were: IC
27.5% (n = 11), invasive histoplasmosis 17.5% (n = 7), IA 15%
Possible impact of beta-glucan assay results on the length
(n = 6), Cryptococcus infection 12.5% (n = 5), PCP 10% (n = 4),
paracoccidioidomycosis 7.5% (n = 3), histoplasmas/criptococcus 5% of antifungal therapy
(n = 2), candida/aspergillus 2.5% (n = 1), and trichosporiasis 2.5% S. Paoletti,1 G. Spinelli,1 R. Murri,1 M. Fantoni,1 B. Posteraro,2
(n = 1). The most common organs affected were lung 52% (n = 21), E. de Carolis,3 G. Scopettuolo,1 G. Ventura,1 R. Torelli,1
central nervous system 15% (n = 6), skin 12.5% (n = 5), and 40% M. Sanguinetti2 and R. Cauda1
(n = 16) of the IFIs were disseminated involving more than one 1
UCSC, Roma, Italy; 2Universita Cattolica del S. Cuore, Roma,
organ. The BDG test was positive in 67.5% (n = 27), negative in
Italy and 3Catholic University of Sacred Heart, Roma, Italy
25% (n = 10), and indeterminate in 7.5% (n = 10). The overall sen-
sitivity was 65.8%, and the specific sensitivity by microorganism
was: PCP: 100% (4/4), IA 83.3% (5/6), IC 81.8% (9/11), IH 71.4% Objectives To evaluate sensitivity, specificity, positive and negative
(5/7), Criptococcus 0% (0/5), Paracoccidiodes 30% (1/3), Tricosporum: predictive value of beta-glucan (BG) assay and to estimate the impact
0% (0/1), IC/IA 100% (1/1), Histo/cripto 50% (1/2). The corrected of this test on the length of antifungal therapy (AF-Tx) in patients
sensitivity excluding 9 patients (5 Cryptococcus, 3 Paracoccidiodes, 1 with proven candidemia or for which an empirical antifungal ther-
Tricosporum) is 84%. Serum BDG showed good sensitivity for the apy was started.

ª 2015 The Authors

112 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Methods Prospective, monocentric, cohort study. An ID consultation observed for ELISA as a screening test, and for immunoelectrophore-
team was implemented in November 2012 in a 1100-bed university sis and western-blot as confirmation test. Hemagglutination was not
hospital in Rome, Italy, to optimize anti-infective treatment. Data for recommended neither for screening nor for confirmation. The use of
every consultation were prospectively collected by the team using a a confirmation technique was strongly recommended when the
standardized database. In the present study, we included patients screening test is positive (median = 9; CV = 14%). This is also rec-
with proven candidemia, patients for which an AF-Tx was started ommended even in the case of a negative screening, depending on
but without candidemia, and patients who never started an AF-Tx the clinical context (median = 8; CV = 31%) or during follow-up of
but for which a BG test was done. A BG result was considered evalu- patients (median = 8; CV = 33%). When the screening and the con-
able when it was performed  48 h from the positive blood culture firmation techniques are discordant, the experts recommended to
or from starting an empiric AF-Tx. Positive BG was defined when interpret the results giving priority to the confirmation technique
>80 pg ml
1. and integrating the clinical data of the patient (median = 8;
Results At April 2014, an evaluable BG result was available for CV = 28%).
1365 patients. Of these patients, 251 (111 BG positive and 140 BG Conclusion This first investigation on serological tests for Aspergillus
negative) started AF-Tx and 1114 (150 BG positive and 964 BG neg- antibody detection points out the diversity of the practices in France.
ative) did not start AF-Tx. Sensitivity, specificity, positive predictive Only, very few items received a clear response allowing to edict rec-
value and negative predictive value were 44.2%, 86.5%, 42.5%, and ommendations among which, Aspergillus antibody detection should
87.3%, respectively. Including only patients with proven candidemia, include an ELISA testing as screening and precipitin detection using
the sensitivity, specificity, positive predictive value and negative pre- immunoelectrophoresis as confirmation. This poll however underlines
dictive value were 86%, 86.5%, 22.7%, and 99.2%, respectively. the lack of standardization of several methods, including the immu-
Overall, there were 538 (124 with proven candidemia and 414 noelectrophoresis still considered as the reference method. These
without proven candidemia) patients, including the 251 with an results warrant further studies to better evaluate the performance of
evaluable BG result, started an AF-Tx. For this group of patients, those techniques for diagnosis and follow-up.
median age was 67 years (IQR 52–77), 52% were males, 53.7% had
a CVC, and 35.1% received an antibiotic therapy within the previous
30 days. Mean length of antifungal therapy was 12.7 days (SD
11.0) in patients with a negative BG result, 20.2 days (SD 17.5) in P162
patients with positive BG and 15.5 days (SD 12.5) in patients with-
out an evaluable BG result (P at ANOVA <0.0001). Similar results
were found excluding patients who died during the study period. Molecular diagnosIs of Pneumocystis jirovecii pneumonia:
Conclusions As expected, BG assay showed a very high negative is the dilution factor of bronchoalveolar lavage a
predictive value. When used in a real-life setting, BG assay allowed confounding factor for the interpretation of real time PCR
to reduce the mean antifungal treatment length of 3 days. results?
L. Goterris, L. Miguel, M.G.E.M.M. Codina,I. Ruiz-Camps,
A. Anton, A. Villatori, P. Alcubilla and M. T. Martin-Gomez
Vall d’Hebron Hospital, Barcelona, Spain
P161
Objectives PCR-based methods can improve the microbiological
Aspergillus antibody detection: results of the French diagnosis of Pneumocystis jirovecii (PJ) pneumonia (PJP) especially in
Society for Medical Mycology (SFMM) poll among French HIV-immunocompromised patients in which low fungal loads are
experts on the use of technical tools found in cases of PJP. Semiquantitative information obtained by real
J. P. Gangneux,1 F. Persat,2 C. Hennequin3 and S. Societ e time PCR (PCRrt) applied to bronchoalveolar lavage (BAL) may allow
Franc
aise de Mycologie4 differentiation between PJP and PJ colonization status but there is
range of positive values that cannot discern between the two entities.
1
Rennes Teaching Hospital – Inserm U1085, Rennes, France; 2Les One possible hypothesis is that the variable dilution factor of BAL
Hospices civils University Hospital, Lyon, France; 3AP-HP, Ho^pital could interfere with the interpretation of PCRrt results.
St Antoine, Paris, France and 4SFMM Study Group, Paris, France The aim of this study is to assess the effect of the dilution factor in
BAL samples on PCRrt values by comparing their values with the
Objectives The detection of Aspergillus antibody is an essential bio- concentration of human DNA present in the sample in order to nor-
logical argument for the diagnosis of allergic and chronic pulmonary malise PCRrt results.
aspergillosis. For this purpose, numerous commercial and in-house Methods Between November 2013 and November 2014, we
techniques are available. The French nomenclature recommends a received 68 BAL from 68 immunocompromised patients (including
two-steps strategy consisting in a screening test followed in case of HIV+ and HIV- patients) for PJ investigation. A PCRrt which targets
positivity by a confirmatory one. This situation leads to a huge num-
ber of serological practices. In order to better appreciate the usage of
and the confidence in these techniques, the SFMM initiated a poll
among a panel of French experts.
Methods In January 2015, forty mycologists were asked to answer
an on-line questionnaire. For each of the 37 questions, an answer
was mandatory, using a score from 1 to 9: from full disagreement
(1) to full agreement (9). Based on information from a nationwide
French EEQ program, questions investigated eight different tech-
niques: indirect hemagglutination, ELISA (without other precision),
electrosyneresis, double immunodiffusion, immunoelectrophoresis, co-
electrosyneresis and western blot. Descriptive statistics was performed
using box plots to determine the median value and the dispersion of
the values. In addition, the coefficient of variation (CV) was calcu-
lated for each item.
Results 36 questionnaires were completed. CV ranged from 10% to
75%, 38% of them being superior to 50%. Differentiation between
screening and confirmatory techniques was considered overall rele-
vant (median = 7). A median ≥ 8 with a CV <30% was only Figure 1

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 113
Poster Presentations

the dihidropteroate synthase gene (DHPS) was performed to detect Results In silico primer-BLAST searches revealed that candidate
and semiquantify PJ. Human DNA present in samples was semiquan- sequences were conserved intraspecifically through a genetically
tified in 63 samples using a PCRrt, which targets human RNAase P. diverse group of Sporothrix. Moreover, primers were highly sensible
For further analysis, only the 63 samples having results for PJ and being able to detect as much as 1 pg of gDNA in a single round
human DNA were taken into account. Results were expressed in PCR. A simple assay using conidia directly in PCR as a source of
cycle threshold (Ct) values. DNA was effective for the rapid and low-cost genotyping of Sporo-
Demographics and clinical variables of patients were recorded. thrix. We explored a murine model of disseminated sporotrichosis
According to clinical, radiological and laboratory findings, patients and successfully detected S. brasiliensis and S. schenckii DNA from a
were classified as PJP, PJ’s colonization and PJ-unrelated clinical variety of fresh-tissue samples including the spleen (100%), liver
process. (100%), lungs (100%), heart (100%), brain (100%), kidneys (100%),
The accuracy of PCRrt and corrected PCRrt for human DNA was tail (100%) and feces (60%) of the diseased animals, but not from
measured by the area under the Receiver Operating Characteristic the control group (healthy animals). Our results suggest that animal
(ROC) curve (AUC). feces may be valuable samples for ecology and epidemiology of
Results 8 out of 63 patients were classified as PJP; additional 26 sporotrichosis. The knowledge that feces from diseased animals may
patients were categorized as colonisations. Mean and 95% confidence infect adjacent soil and increase the focus and risk of outbreaks in
interval (95%CI) of PCRrt Ct values were 27.9 (23.8-32) and 33,9 endemic areas are important to implement measures to contain the
(32.7-35.1) for PJP and PJ’s colonisation respectively. Mean and progress of the epidemic.
95%CI of PCRrt Ct values corrected for human DNA were 27.9 Conclusion The singleplex PCR-based method was successfully used
(23.9-31.2) and 34.0 (32.4-35.6) for PJP and PJ’s colonisation for the detection and identification down to species level from both
respectively. ROC curve analysis showed an AUC = 0.827 for PCRrt culture and clinical samples. Thus, the feasibility of the species-speci-
values and AUC = 0.861 for PCRrt corrected for human DNA values. fic primer lies on its simplicity, high throughput, and accurate diag-
Cutoff Ct values were 28.3 for PCRRrt and 28.7 for corrected PCRrt. nosis of sporotrichosis. Improvements in diagnosis and surveillance
Both PCRrts (corrected and not corrected) showed identical values: systems may help to identify and tackle future outbreaks.
sensitivity = 75%, specificity=96.2%, PPV = 85.7% and Financial support: S~
ao Paulo Research Foundation (FAPESP 2009/
NPV = 92.6%. 54024-2 and FAPESP 2011/07350-1), National Council for Scien-
Conclusion Introducing a correction for the variable dilution factor tific and Technological Development (CNPq 472600/2011-7), and
of BAL. marginally improves the results obtained with the PJ PCRrt Coordination for the Improvement of Higher Education Personnel
studied. Nevertheless, the slight improvement introduced in the AUC (CAPES).
adds robustness to the Ct values. Further studies aimed to increase
the number of cases included in our series are warranted to assess
the usefulness of this approach.
P165

P164 Genus/species-specific versus panfungal PCR assays for

detection and quantification of fungal pathogens in
clinical samples from immunocompromised patients
Molecular diagnosis based on species-specific nucleic acid M. Lengerova, M. Bezdicek, D. Ricna, I. Kocmanova, J. Mayer
detection of the pathogenic agents of sporotrichosis and Z. Racil
A. M. Rodrigues,1 G. S. de Hoog,2 P. P. Della Terra,1
University Hospital Brno, Brno, Czech Republic
G. F. Fernandes1 and Z. P. de Camargo1
1
Federal University of Sao Paulo, Sa~o Paulo, Brazil and 2CBS- Introduction Molecular methods, the most important of which is
KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands PCR, are routinely used in every day clinical microbiology practice.
Concerning fungal pathogens, molecular methods have their advan-
Sporotrichosis is a chronic infection of the cutaneous and subcuta- tages in great sensitivity and possibility to identify fungal species very
neous tissues caused by thermodimorphic fungi embedded in the accurately. The major point of criticism of its general use is lack of
plant-associated order Ophiostomatales. With major differences standardization, since most of the leading laboratories successfully
between routes of transmission and virulence traits, Sporothrix are use in-house PCR assays and widely validated commercial assays are
emerging as new threat to the vertebrate host, particularly in form missing.
of outbreaks. Large zoonoses and sapronoses are ongoing in South Material and methods For PCR detection of fungi in clinical sam-
America and Asia, respectively. Currently diagnosis methods based ples either genus/species-specific or panfungal assays can be used.
only on morphological and biochemical features are inaccurate due Since lungs are the most common organ affected, bronchoalveolar
to the overlap of phenotypes; nevertheless with the introduction of samples are frequently tested for presence of fungal pathogens. In
dissimilar species with differences in epidemiology, host predilection, our institution we routinely screen BAL samples from patients at
virulence and antifungal susceptibilities there is a critical need for high risk of IFD or symptoms suspicious of IFD (CT findings, positive
developing new diagnostic tools that are sensitive and cost effective. serum galactomannan etc.) with two methods – commercial real-
Objectives In the present study, we have successfully applied spe- time PCR assay for detection of 4 most common Aspergillus species
cies-specific primers to: (1) Identify etiological agents of human and and in-house PCR followed by high resolution melt analysis (HRMA)
animal sporotrichosis circumscribed in the S. schenckii complex; (2) for detection of mucormycetes. If results of both assays are negative
perform large scale screening of Sporothrix in epidemiological studies; and there is still clinical, epidemiological and/or histological suspi-
(3) detect Sporothrix spp. DNA in fresh-tissue and biological samples cion of fungal infection, samples are further tested by panfungal PCR
from experimentally infect animals; and (4) we highlight the utility followed by sequencing or HRMA.
of Sporothrix conidia as a simple, fast-processing source material for Results Advantages or disadvantages of both approaches must be
genotyping. considered. Species-specific primers might miss infections caused by
Methods We developed a panel of novel markers based on calmod- untargeted fungi; conversely, panfungal primers can provide false
ulin (CAL) gene sequences with potential applications to perform positive results due to environmental fungal DNA contamination.
large scale diagnosis and epidemiology of members of the Sporothrix- Results of species-specific methods are usually available within 24 h,
Ophiostoma complex including S. brasiliensis, S. schenckii sensu stricto, while when panfungal PCR assays are done verification of the results
S. globosa, S. mexicana, S. pallida and Ophiostoma stenoceras. CAL by sequencing is necessary. Due to above raised issues, use of pan-
sequences, covering a worldwide array of haplotypes were aligned fungal PCR has been so far focused mainly on sterile samples (serum,
and primers were designed to target species-specific regions. blood, cerebrospinal fluid) or for identification of fungal pathogens in

ª 2015 The Authors

114 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

fresh or formalin-fixed paraffin embedded tissue specimens from P170

patients with culture or histologically proven IFDs.
Conclusions The aim of this presentation is to summarize our experi-
ence and published data concerning testing of various clinical samples Saprolegnia parasitica qPCR Quantification in water: since
(BAL, cerebrospinal fluid, serum, tissue) from immunocompromised the areas of high fish mortality in river to tap water
patients using specific and panfungal PCR assays. S. Rocchi,1 M. Tisserand2 and G. Reboux1
1
University hospital Jean Minjooz, Besancon, France and
2
University of Franche-Comte, Besancon, France

P166 Objectives In the river Loue (Franche-Comte region, East of France),

the death of large numbers of fish is observed for several years. An
Investigation of a Semi-automated DNA Extraction aquatic oomycete, Saprolegnia parasitica, could contribute this impor-
Procedure from Stool Specimen with a Quantitative Real- tant mortality cases observed in Salmo trutta fario and Thymallus thy-
time PCR Assay for the Detection and Identification of mallus. S. parasitica is a secondary pathogen and only colonize
Microsporidial Infections unhealthy and morbid individuals, being the cause of skin mycosis,
respiration trouble and death.
M. Gaboyard,1 R. Khoury,2 O. Robert,1 S. Godichaud,1 Besanc
on city is partly supplied by two pumping water station on
S. Vellaissamy,2 J. Guitard2 and C. Hennequin3 the river Loue. The biggest pumping water station deliver drinking
1
Ademtech, Pessac, France; 2Ho^pital St Antoine – APHP, Paris, water in 39 000 inhabitants of the city of Besanc
on. The presence of
France and 3AP-HP, Ho^pital St Antoine, Paris, France S. parasitica in the Loue river therefore raises the question of its pres-
ence in drinking water and this potential risk for the human health,
even if no link to health problems has ever been described. Health
Objectives Microsporidia are fungi-related pathogens mainly respon-
authorities in the region have requested to develop a specific tech-
sible for diarrhoea in immunocompromised patients. However, the
nique to quantify S. parasitica in the Loue river and use this tool to
spectrum of patients at risk has been recently enlarged making the
verify its absence in drinking tap water.
screening for microsporidial infection more frequently indicated. A
Methods A species-specific quantification assay using real time
number of species-specific PCR and probe-based real time PCR have
quantitative PCR (qPCR) was developed to analyze the abundance
been developed but either fail to cover the diversity of species
variation of S. parasitica in Loue river, during spawn period of Salmo
involved or represent a significant financial effort if used in a screen-
trutta fario and Thymallus thymallus, and in the drinking water.
ing strategy. In addition, the isolation and amplification of DNA from
Water samples (10L) and baiting samples (with hemp seeds) were
stool samples is challenging, due technical barriers and the frequency
performed in the Loue river, once a week during 12 weeks, between
of polymerase inhibitors in stool. In this study, we investigated (i) a
late January and early April 2015. Six stations were defined along
simple semi-automated extraction procedure involving a simple
the river, from the river spring to the interest area (water pumping
chemical lysis of pathogens, and (ii) a novel quantitative PCR based
station temporally closed for renovation). Devices with hemp seeds,
on a fluorescent dye and including a melting-curve analysis for spe-
which are baits for the pathogen, were used to verify its presence.
cies identification.
Tap water samples (10L) were performed in two dwellings too: one
Methods Ten stool specimens previously found positive by micro-
connected to the little pumping station in the headwater of the river,
scopic examination were processed in parallel using ParaGENIE
and another pumping station.
DNA Stool Extraction kit (Ademtech) and QIAamp DNA Stool kit (Qi-
Results S. parasitica DNA was detected in 64% of river samples
agen). In addition, quantified DNA extracted from culture spores of
(n = 72), with quantity between 0.03 to 68 equivalent spores per
different species were tested. qPCR was set up to specifically amplify
liter. Time and space related variations were observed in the river.
rDNA SSU of all microsporidia species. A touch-down amplification of
Hydrologic data have been considered. A dilution effect seems to
40 cycles was performed using an EvaGreen mix on a Stratagen
occur during high flow periods not allowing detecting the pathogen
MX3005 thermal cycler, followed by a final denaturation step for
with qPCR, whereas baiting samplings prove its presence. Greater
melting temperature determination. An internal positive control
quantities seem to be observed at the sampling station near the big
(IPC) was also added to the assay. A prospective study was then initi-
pumping station. No S. parasitica DNA was detected in the tap water.
ated on clinical samples collected from diarrheic patients over a
Conclusion This research work led us to suggest a standardized
4 months period.
detection tool to quantify S. parasitica in the water. Larger quantities
Results qPCR assay produced amplification with all the microscopy-
observed near to the big pumping station showed that at the opening
positive samples with a 100% (range 95-105%) efficiency and a
of the station after renovation, the absence of S. parasitica should be
dynamic range of at least six orders of magnitude. The specificity of
tested in tap water from this station.
the assay was validated against various microorganisms, especially
protozoan. Three different melting temperatures, all different from
that of IPC, allowed the differentiation between Enterocytozoon bien-
eusi/Encephalytozon intestinalis, Encephalitozoon cuniculi and Encephali-
tozoon hellem. Comparative results between the two extraction P171
methods showed a benefit of a mean of 5 cycles threshold using the
ParaGENIE DNA Stool Extraction kit. Besides, this new extraction Evaluation of the susceptibility of phlebotomine sand flies
procedure allowed to process twelve specimens in parallel and took
to Beauveria bassiana and to the essential oil of
half the time compared to the QIAamp DNA miniStool kit. During
the prospective study, among fifty-two tested patients, two cases of
Eucalyptus globulus
microsporidial infections were diagnosed. One of them was found in L. A. Figueredo,1 R. L. Luna,1 D. Miranda,1 K. T. Silva,1
an immunocompetent patient who had not been initially identified C. Cafarchia,2 D. Otranto,2 F. Dantas-Torres,1 S. P. Brandao-
by microscopic examination. Filho1 and L. A. Figueredo1
Conclusion The method set up in this study allows a simple, rapid 1
Centro de Pesquisas Aggeu Magalhaes, Recife, Brazil and
and sensitive screening for microsporidial infection in diarrheic 2
Universita degli Studi di Bari, Valenzano (Bari), Italy
patients. Differentiation of species is partially possible and can be con-
firmed with an additional molecular method. ParaGENIE DNA Stool
Extraction kit, which has been shown to efficiently extract DNA from Leishmaniases are diseases which affects several animal species
Giardia intestinalis, Amoebae, and Cryptosporidium spp, leads to higher including humans, caused by protozoa of the genus Leishmania and
microsporidial DNA recovery, increasing thus the sensitivity of transmitted through the bite of infected female phlebotomine sand
detection. flies. Beauveria bassiana is an entomopathogenic fungus, which can
act as a biological control agent against important vectors of

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 115
Poster Presentations

pathogens. A few studies have demonstrated that Phlebotomus pap- Illumia MiSeq. The data analysis was done using the QIIME pipeline
atasi, the vector of Leishmania major in the Old World, and Lutzomyia and the R package.
longipalpis, the main vector of Leishmania infantum in the New World, For fungal culture and isolation, swab samples from the oral cavity
are susceptible to B. bassiana. Besides that, the essential oils of Euca- and blowhole were collected. The culture was performed using PDA
lyptus trees are natural insecticides for controlling Lu. longipalpis. The and Sabouraud-Dextrose agar and the incubation was performed at
biological control of these vectors would reduce the use of chemical 30°C and 18°C. Fungal identification was performed by macro and
insecticides, resulting in benefits for the human beings and for the microscopic observation, and by sequencing the ITS regions. Fungal
environment. ITS sequences obtained were compared against NCBI nucleotide data-
Objective Therefore, the aim of the present study was to evaluate base for fungal identification.
the effect of the association of B. bassiana with the essential oil of E. Results The metagenomic analysis demonstrated the presence of 29
globulus for the biological control of Lu. longipalpis. fungal genera across all samples studied. Among the genera, 6 were
Methods Phlebotomine sand flies of the species Lu. longipalpis were present only in oral cavity and 12 genera were present only in vagi-
collected using CDC light traps in the municipality of Passira, Per- nal tissue. Malassezia species were the most frequent, followed by
nambuco State, Brazil and separated into five groups comprising 10 Candida, Debaryomyces, Cladosporium, Aspergillus, Penicillium and Rho-
specimens per group. Treatments were carried out using B. bassiana dotorula; and all these genera were found both in oral and vaginal
(107 spores ml
1), E. globulus (0.1 mg ml
1), E. globulus plus B. tissues.
bassiana, distilled water as negative control, and cypermethrin The fungal culture and isolation using different temperatures and
(0.1 mg ml
1) as positive control. The formulations were sprayed media resulted in a total of 92 isolates, including 68 yeasts (74%)
onto a filter paper. Each experiment was performed twice, and Lu. and 24 filamentous fungi (26%). The oral cavity presented a higher
longipalpis survival rates were recorded. number of isolates (45 yeast isolates and 9 filamentous fungi), when
Results The groups treated with B. bassiana, alone or in association compared with the blowhole (23 yeast isolates and 15 filamentous
with E. globulus essential oil presented a reduced survival time. In fungi). No significant differences between the use of different media
particular, the negative control group survived for 13 days approxi- and temperatures were observed. Candida species were the most fre-
mately, while the treated groups presented an average survival time quently found, followed by Rhodotorula sp., Aureobasidium sp., Debary-
of about 6 days (i.e., half of its lifespan). Positive control group sur- omyces sp., Penicillium sp. and Cladosporium sp.
vived for around 24 h. No significant difference was observed Conclusion This study is the first systematic work for evaluation of
between the groups treated or not with E. globulus essential oil. dolphin fungal microbiome in different body sites. The preliminary
Conclusion These results show that the entomopathogenic fungus results demonstrated a high fungal diversity in all body locations
is a good tool to be used in the biological control of Lu. longipalpis. studied and highlight variation in the their distribution according to
On the other hand, the use of E. globulus essential oil did not potenti- organ type. Furthermore, a high variety of known fungal pathogens
ate its pathogenic effect on Lu. longipalpis. Further studies are needed were detected, such as Candida tropicalis, C. guilliermondii, Fusarium
to test a larger number of phlebotomine sand flies and to assess the oxysporum, Scedosporium apiospermum, Cladosporium cladosporioides
efficacy of this biological control agent under environmental condi- and different Malassezia species, confirming these marine mammals
tions. The development of low-cost, biologic control tools against vec- as potential reservoirs.
tors should be encouraged for controlling vector-borne tropical
diseases such as leishmaniasis.

P175
P172
Rhodotorula glutinis catheter-related fungemia in
University of Santo Tomas Hospital: A Case Report
Characterization of the fungal Microbiome in dolphins of R. J. R. Javier, M. L. C. Osabel, M. R. G. Bergantin and
the coast of Portugal P. B. Caguioa
J. I. Carvalho Pereira,1 M. Ferreira,2 J. Vingada,1 C. Eira,3
University of Santo Tomas Hospital, Manila, Philippines
P. Sampaio1 and C. S. Pais1
1
University of Minho, Braga, Portugal; 2Portuguese Wildlife Objective To describe a case of Rhodotorula glutinis catheter-related
Society (SPVS), Quiaios Field Station, Figueira da Foz, Portugal fungemia in an immunocompromised patient with sigmoid adenocar-
and 3CESAM and Department of Biology, University of Aveiro, cinoma who was successfully treated with caspofungin and removal
Aveiro, Portugal of central venous access.
Method We describe the risk factors, course and treatment of a
Objectives Marine mammals are excellent sentinels reflecting the patient who acquired Rhodoturolosis after undergoing abdominal
effects of natural and anthropogenic threats on marine ecosystem. surgery and receiving multiple antibiotics and total parenteral nutri-
However, data on changes in marine mammal health are quite lim- tion through her central vascular access.
ited in animals inhabiting Portuguese and northern Spanish waters. Results The use of a central venous catheter, exposure to broad-
The information available regarding microbiological aspects of mar- spectrum antibiotics and total parenteral nutrition, recent abdominal
ine mammals describes mainly pathogenic bacteria and, despite surgery, and the presence of malignancy are the known risk factors
reports of new or re-emerging fungal infections with epizootic and found in the patient to develop Rhodotorula fungemia. Although
potential zoonotic consequences, studies on fungal species are lack- echinocandins are shown to have high minimum inhibitory concen-
ing. The new emerging diseases elucidate the need for detailed epi- trations against Rhodotorula spp., it was succesfully used in eradicat-
demiological studies to predict outbreaks and facilitate their control. ing this pathogen with concomittant removal of the central vascular
The main objective of the present study was to investigate whether device.
dolphins might serve as potential reservoirs for known and emerging Conclusion Rhodotorula is an emerging opportunistic fungus
human and marine mammals fungal pathogens. reported to cause infection in immunocompromised patients. Most
Methods A total of eight stranded dolphins were studied. Samples common risk factors for developing infection with this organism are
were collected during necropsies by authorized personal of Sociedade malignancy, use of cytotoxic drugs, and central venous catheters. It
Portuguesa de Vida Selvagem and Coordinadora para o Estudio dos is most effectively treated with amphotericin B or flucytosine. This
Mamiferos Mari~ nos, using sampling protocols already established. case is the first reported documentation of Rhodotorula glutinis cathe-
For the mycobiome analysis, total DNA of tissue samples of oral ter-related fungemia with clinical and microbiologic improvement
cavity and vagina was extracted using JetQuick DNA purification Kit after treatment with echinocandin and catheter removal.
and the Internal Transcribed Spacer (ITS) region was sequenced with

ª 2015 The Authors

116 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P176 drugs failed to prompt a response compelling an exploratory laparo-

tomy with right hemicolectomy. Histopathology revealed a granulo-
matous inflammation with Splendore-Hoeppls Phenomenon. Patient
Gastrointestinal Basidiobolomycosis; a new common entity improved clinically and endoscopically with itraconazole.
in the southern region of Saudi Arabia Conclusion Gastrointestinal basidiobolomycosis appears to be ende-
H. A. Alhamzah,1 A. O. Almasri,2 T. G. Semaan2 and mic in the Southern region of Saudi Arabia. It should be considered
A. J. Shehadah2 as differential diagnosis in patient resining in this part of the country
1 with suspicion of intestinal TB or Inflammatory Bowel Disease.
Al-Imam University, Riyadh, Saudi Arabia and 2King Saud
Medical City, Riyadh, Saudi Arabia

Background Basidiobolous ranrum is a fungus that is known to P177

cause subcutaneous infection. Invasive form is a rare entity and
poorly recognized. Clinical presentation is wide, non-specific symp- Phylogenetic diversity of human pathogenic Fusarium and
toms masquerading inflammatory bowel disease, Tuberculosis or
occurrence of uncommon virulent species in Qatar
Malignancy.
Case Report We report a 33-year-old Saudi male suffered from gas- J. Taj-Aldeen,1 H. Salah,1 A. al-Hatmi,2 B. Theelen,2
trointestinal basidiobolomycosis. He was misdiagnosed as malignancy M. Abukamar,1 S. Hashim,1 A. D. van Diepeningen,2
and then as intestinal Tuberculosis. He presented with 4 month-his- €rl,3 T. Boekhout2 and M. Almaslamani1
C. Lass-Flo
tory of abdominal pain, vomiting, diarrhea, recurrent melena, weight 1
Hamad Medical corporation, Doha, Qatar; 2CBS-KNAW Fungal
loss and shortness of breath. Clinical examination and laboratory Biodiversity Centre, Utrecht, the Netherlands and 3Medical
investigations showed unilateral pleural effusion, minimal ascites, ane- University of Innsbruck, Innsbruck, Austria
mia and peripheral eosinophilia. Endoscopic and radiological examina-
tions showed diffuse thickening of the whole gastrointestinal wall from
the upper esophageal sphincter up to the right colon with multiple Objectives Fusarium species cause a broad spectrum of life-threaten-
intramural lesions and stenosis. Treatment with Antituberculosis ing invasive infections especially in immune-compromised patients.
Cutaneous infections, such as keratitis, endophtalmitis or onychomy-
cosis are frequently manifested in immunocompetent hosts and may
be associated with trauma. A phylogenetic study was carried out to
characterize the most common invasive and cutaneous infecting
Fusarium species observed in Qatar.
Methods A set of 44 Fusarium isolates from human clinical speci-
mens diagnosed during the period 2003 to 2014 at Hamad General
Hospital, Qatar were collected. The isolates were molecularly verified
using two molecular markers, the second largest subunit of the RNA
polymerase gene (RPB2) and the translation elongation factor 1
alpha (TEF-1a) that were amplified directly from the genomic DNA
for multi-locus sequence typing.
Results Phylogenetic analysis revealed that the etiological agents
belonged to four speciescomplexes (sc); F. solani sc (FFSC) (75%), F.
oxysporum sc (4.5%), F. fujikuroi sc (13.6%), and F. dimerum sc
(6.8%). The identified species were Fusarium keratoplasticum (n = 14,
31.8%), F. falciforme (n = 9, 20.5%), F. solani (n = 5, 11.4%), F. acuta-
tum (n = 4, 9%), F. petroliphilum (n = 3, 6.8%), F. lichenicola (n = 2,
4.5%), F. proliferatum (n = 2, 4.5%),F. oxysporum (n = 2, 4.5%), F.
dimerum (n = 2, 4.5%) and F. delphinoides (n = 1, 2.27%). Sites of infec-
tion were nails (n = 19, 43.2%), skin (n = 7, 15.9%), cornea (n = 6,
13.6%),blood (n = 3, 9%), wound (n = 4, 6.8%), burn (n = 2, 4.5%),
Figure 1. A scarcely septated broad hyphae surrounded by inflam- and urine (n = 1, 2.27%). Comorbidities associated with invasive infec-
matory cell infiltrates (Grocott’s methenamine Silver GMS stain). tions were hematological malignancy and autoimmune disorders. Mor-
tality was 75% for the blood infections irrespective of antifungal
chemotherapy and was associated with F. petroliphilum and F. solani.
Conclusion The study indicates that Fusarium is an uncommon
agent of invasive fungal infections in Qatar, recalcitrant to antifungal
treatment, and a potentially lethal opportunistic pathogen particu-
larly in immunocompromised patients. Phylogenetic diverse members
of the FSSC, predominatly cause cornea, nail and bloodstream infec-
tions. Fast and more accurate molecular diagnostic tests to species
level may contribute to an earlier and more precise diagnosis and
subsequently optimized treatment.

P178

A case of subcutaneous and gastrointestinal

basidiobolomycosis in Singapore
Y. E. Teh and L. Wijaya
Singapore General Hospital, Singapore, Singapore

Figure 2. Markedly thickened gastric wall more in the antrum with Objectives Entomophthorale is ubiquitous in nature. Entomoph-
multiple ring enhancing lesions. Suggestive of intramural abcesses. thoromycosis due to Basidiobolus is a known rare cause of chronic

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 117
Poster Presentations

subcutaneous zygomycosis affecting the limbs and trunk. It occasion- who had systemic arterial hypertension, ankylosing spondylitis, ter-
ally causes disseminated or invasive disease involving the gastroin- minal chronic renal failure secondary to hypertensive nephropathy,
testinal tract and lungs. Although widely prevalent in tropical and undergoing treatment of automated dialysis peritoneal was admitted
subtropical regions, it is infrequently seen in a developed country like to our hospital, with abdominal
Singapore. However, as residents travel and volunteer abroad more pain and fever. Was performed the culture of peritoneal dialysis fluid
frequently, infections not seen locally are increasingly observed. and after the peritoneal dialysis catheter tip with revealed the fungal
We describe the first case of entomopthoromycosis in Singapore to growth. The yeast was identified as K. ohmeri by Vitek 2 system
increase awareness of this entity. This will in turn facilitate early (Vitek ID-YST, bioMerieux). The patient was started on Micafungin.
diagnosis and allow prompt institution of treatment. Because of fungical peritonitis, peritoneal dialysis was stopped and
Methods We discuss the case of a 67 year old Singapore Chinese submitted the patient to long catheter permanence in the femoral
male with proven subcutaneous entomophthoromycosis at initial pre- vein to do hemodialysis. However, despite the proposing treatment
sentation, who subsequently developed gastrointestinal symptoms, the patient died. K. ohmeri, can be identified by morphology of colo-
due to presumptive gastrointestinal basidiobolomycosis. nies in CHROMagar Candida medium by absorption of carbon com-
Results Our patient presented with a 3 week history of left lower pounds (Vitek API ID32C and 2) or by molecular biology with the
limb swelling in July 2012. He had travelled frequently to rural areas rDNA sequencing. The treatment is not well established in the litera-
of Thailand and Indonesia where he was involved in voluntary con- ture due to shortage of cases, however, some reports obtained a good
struction work. There were no other signs and symptoms. Blood response with the use of Amphotericin B and Micafungin.
investigations were unremarkable. Of note, he did not have
eosinophilia. Imaging confirmed a soft tissue mass in the left inguinal
region. At that time, workup for malignancy, bacteria, fungal, para-
sitic and tuberculous infections was negative. In view of the lack of a P180
definitive diagnosis, he sought alternative treatment. He was readmit-
ted to our institute in May 2013 with a progressively enlarging left
inguinal mass with intra-abdominal extension, causing compression A case of Endophthalmitis and corneal abcess caused by
of the ureters with resulting hydronephroses. He underwent multiple Bipolaris spp.
investigations to ascertain the cause of the mass and to rule out €ksel
S. Keceli, I. Tas, S. Genc and N. Yu
malignancy. These were unyielding. His clinical condition continued
Kocaeli University Medical Faculty, Kocaeli, Turkey
to worsen and he developed gastrointestinal distension and persistent
ileus in July 2013. Repeat imaging showed dilated large and small
bowel loops with no intra or extraluminal obstructing mass. There A built worker’s left eye injured by a piece of iron was applied to
was also nonspecific mucosal thickening extending from the descend- local hospital. He was diagnosed as corneal and scleral penetration
ing colon to the rectum. He underwent a colonoscopy where corre- injury (Zone1-2) and operated. Two weeks later, he was transferred
sponding nodular mucosal thickening was seen. Histology revealed to our University hospital for follow-up and treatment. The patient
tubular adenoma with low grade dysplasia. Due to the absence of was diagnosed as corneal ulcer and endophthalmitis and hospitalized.
malignancy and based on his clinical presentation, he was treated as Then, intravenous (iv) vancomycin, iv ceftazidim (CTZ), iv voricona-
for entomopthoromycosis. Unfortunately, he responded poorly to zole (VRZ), topical vancomycin, CTZ and amphotericin B (AMB)
treatment and passed away 4 months after initiation of therapy. treatment was started. Treatment was continued for 12 days. One
With the help of our counterparts in India, we eventually proved our day after hospitalization, left anterior camera lavage, vitreous tap,
clinical suspicion by polymerase chain reaction on the tissue intravitreal vancomycin plus AMB injections were done. Two days
specimen. later, intravitreal vancomycin, CTZ plus AMB treatment was
Conclusion Basidiobolomycosis is an emerging disease. This is espe- repeated. Ten days later, in direct microscobic investigation of vitreal
cially so with increased international travel which has progressively fluid by KOH, clusters of hyphae were observed. Bacterial culture
led to sporadic cases of mycotic infections outside their previously was negative and there was no bacteria and leucocyte detected in
restricted areas of endemicity. Diagnosis of basidiobolomycosis is gram staining of vitreal fluid. Fungal culture of vitreal fluid on
often delayed due to its nonspecific presentation and myriad of differ- Sabouraud dextrose agar showed a mycelial growth. According to
entials. It is rarely encountered in Singapore, hence collaborative investigation of mold colonies with lactophenol cotton blue under
effort is necessary to establish the diagnosis via histopathology or light microscope, mold culture was identified as Bipolaris spp. After
molecular techniques. This case highlights the importance of the culture result, topical VRZ was given instead of topical AMB and
increased awareness, early diagnosis and prompt treatment of the oral itraconazole was given instead of iv VRZ. Three days later, iv
disease, particularly in travellers returning from regions of the world vancomycin and CTZ treatment was stopped. This treatment was
where these infections are endemic. This will then translate to less continued for twenty days. After the treatment corneal oedema,
morbidity and mortality. inflammation, conjunctival hyperemia, corneal ulcer and endoph-
thalmitis regressed.

P179
P181
Kodamaea ohmeri: case report
Peritonitis caused by Gymnascella hyalinospora in a
B. C. D. A. Zoppas, G. Bortolini, L. W. Y. Yum, C. B. Trevisol and
patient receiving peritoneal dialysis
M. G. Bombel T. S. Bogomolova,1 Y. V. Borzova,1 R. P. Gerasimchuk,2
University of Caxias do Sul, Caxias do Sul, Brazil I. M. Pchelin,1 I. A. Ryabinin,1 Y. L. Avdeenko,1 E. Shagdileeva,1
N. V. Vasilyeva1 and N. Klimko1
We report the first case of Kodamaea ohmeri fungemia in a patient 1
North-Western State Medical University named after I.I.
undergoing treatment of peritoneal dialysis, in a northeast hospital of
Metchnikov, Saint Petersburg, Russia and 2Mariinskaya City
Rio Grande do Sul, Brasil. K. Ohmeri is a yeast and a teleomorph
Hospital, Saint Petersburg, Russia
form of Candida Guillermondii. This is commonly used in the food
industry because of its fermentation properties. Kodamaea ohmeri is a
rare clinical isolate that has recently become known to cause various Introduction Peritoneal dialysis (PD) is a risk factor for fungal peri-
human infections as fungaemia, cellulitis, peritonitis, endocarditis tonitis. Candida spp. are the most common etiologic agents of PD-re-
and wound infection bringing high mortality in both immunocom- lated fungal peritonitis. More rarely the disease may be caused by a
promised and in imunodependentes patients. A 57-year-old woman number of opportunistic molds.

ª 2015 The Authors

118 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Objectives To present a first clinical case of peritonitis due to the although the diagnosis was difficult to make as only a small area
rare ascomycetous mold Gymnascella hyalinospora in a patient with was affected. The infection was detected by histopathology, fluores-
prolonged continuous ambulatory PD. cence microscopy and culture. The isolate was identified as Lichthei-
Case report A 45-year-old female with chronic renal disease had a mia corymbifera by sequencing of the ITS region of its rDNA. The
history of PD since 23.04.2007. On 10.08.2014 she presented with infection was successfully treated by surgical debridement followed
fever, nausea, vomiting, abdominal pain and clouding of dialyzing solu- by administration of liposomal amphotericin B.
tion and was admitted to the Dialysis Department of Mariinskaya City This work was supported by the Palacky University Olomouc Inter-
Hospital. At admission empirical antibiotic therapy (cefazolin nal Grant Agency (project no. IGA_LF_2015_021).
2 g day
1; amikacin 100 mg day
1) was started and cultures of blood,
dialysate and feces were performed. Klebsiella pneumonia was recovered
from feces; repeated blood cultures were negative. Antibiotic therapy
was changed to tienam 1 g day
1 with no clinical improvement. P183
Culture of dialysate from 12.08.2014 grew mycelial fungus which
was identified by DNA-sequencing (ITS region) as Gymnascella hyali-
nospora. The same mold was cultured repeatedly from dialysate sam- Maxillary sinus infection due to Schizophyllum commune
ple taken on 22.08.2014. On 27.08.2014 peritoneal catheter was R. Dobias,1 P. Schwarz,2 T. Ryskova,3 J. Mrazek,3 M. Kantorova1
removed and visible biofilm was observed on the surface of its distal and P. Hamal4
fragment. Direct microscopy of the biofilm revealed narrow hyaline 1
septate hyphae and G. hyalinospora was isolated by culture. Institute of Health in Ostrava, Ostrava, Czech Republic;
2
On 30.08.2014 signs of pneumonia were found at X-ray of lungs University Hospital in Ostrava, Ostrava, Czech Republic;
3
and antifungal therapy with voriconazole was started (6 mg kg
1 on Institute of Health in Ostrava, Dept. of Bacteriology and
day 1; then 4 mg kg day
2), but the patient worsened and on Mycology, Ostrava, Czech Republic and 4Faculty of Medicine and
07.09.2014 she died because of acute intestinal bleeding. Dentistry, Palacky University, Olomouc, Czech Republic
At post-mortem histopathological investigation narrow septate
hyphae were revealed in lung tissue and peritoneum along with toxic Schizophyllum commune is a thermotolerant basidiomycetous wood-de-
damadge. caying fungus which occurs on dead branches and fallen trunks of
Mycology Cultures of G. hyalinospora grew moderately rapidly on deciduous trees. Fungal infections of humans caused by this fungus
Sabouraud dextrose agar at 37◦C. Young colonies were thin, fluffy, are extremely rare. However, In the Czech Republic, however, maxil-
pinkish, without any sporulation. After 3 weeks of growth abundant lary sinusitis due to the fungal species was already found in 2005
white aerial mycelium developed and the surface of colonies became [1].
pale green. Numerous asci aggregated in dense groups, hyaline to Presented is maxillary sinus infection caused by Schizophyllum com-
pale green ascospores were found at microscopic examination of mune in a 49-year-old female patient. She suffered from recurrent
cultures. sinusitis over a period of 5 years. During the last episode, the infec-
Conclusion We report the first case of peritonitis in a patient with tion was suspected to be of bacterial etiology and treated with clin-
chronic renal disease and prolonged peritoneal dialysis, caused damycin only. As the treatment failed and a CT scan revealed almost
by Gymnascella hyalinospora. This mold formed a biofilm on the sur- complete opacification of the right maxillary sinus, the patient was
face of the peritoneal catheter. The patient died in spite of antifungal indicated for endoscopic supraturbinal antrostomy.
treatment and catheter removal. To our knowledge, this is the sec- In the course of the surgery, a polyp, hypertrophic mucosa, and
ond reported case of invasive mycosis due to this fungal species slimy brown mass were found in the maxillary sinus. Fungal infec-
(Iwen P.C. et al., 2000). tion was detected in the surgically obtained sample by a microscopic
finding of hyphae and by culture. The fungal colonies were finally
identified as Schizophyllum commune based on sequencing of the ITS
regions of the rDNA.
P182 The infection was successfully treated by surgical debridement only
and antifungal therapy was not initiated. Currently, the patient is
without any signs of infection.
A case of cutaneous mucormycosis caused by Lichtheimia Reference Dobiasov a S., Dobias R., Klecka P., Kubatova A., Kolarik
corymbifera M. [Filamentous micromycetes as agent of chronic sinusitis.] Abstract
P. Lyskova,1 T. Tyll,2 V. Hubka,3 M. Muller,2 L. Zelenka,2 Book of the 5th Czech and Slovak Interdisciplinary Conference of Medical
M. Curdova,4 M. Kolarik,5 L. Svobodova6 and P. Hamal7 Mycology, Pardubice, Czech Republic, 2007, p. 62.
1
Institute of Health in Usti nad Labem, Prague, Czech Republic; This work was supported by the Palacky University Olomouc Inter-
2 nal Grant Agency (project no. IGA_LF_2015_021).
First Faculty of Medicine, Charles University and Military
University Hospital, Prague, Czech Republic; 3Charles University in
Prague, Prague, Czech Republic; 4Military University Hospital,
Prague, Czech Republic; 5Institute of Microbiology, Czech
Academy of Science, Prague, Czech Republic; 6Palacky University P184
Olomouc, Olomouc, Czech Republic and 7Faculty of Medicine
and Dentistry, Palacky University, Olomouc, Czech Republic Genotyping of multidrug resistant Indian Candida auris
isolates by Multi Locus Sequence Typing, Amplified
Mucormycosis is the second most frequent invasive mould infection. Fragment Length Polymorphism and MALDI-TOF-MS and
It is usually very aggressive due to the angioinvasive nature of the their antifungal susceptibility profile
etiological agents. Cutaneous mucormycosis is the third most com- C. Sharma,1 A. Singh,1 P. K. Singh,1 A. Prakash,2 J. F. Meis3 and
mon clinical manifestation. If surgical debridement is done promptly, A. Chowdhary2
solitary cutaneous infection has a favorable prognosis and a low 1
Vallabhbhai Patel Chest Institute, Delhi, India; 2Vallabhbhai Patel
mortality rate. Early diagnosis of the disease is crucial for successful
management. Chest Institute, Univ.of Delhi, Delhi, India and 3Canisius
Presented is a case report of cutaneous mucormycosis in a 38- Wilhelmina Hospital and Radboud University Hospital, Nijmegen,
year-old healthy male. He was involved in a motorcycle-car accident the Netherlands
in June 2014 and both his lower limbs were amputated during sub-
sequent surgery. Later, he developed fungal wound infection of the Objectives Candida auris is multidrug-resistant yeast that causes a
left stump. Fortunately, the infection was detected very early wide range of infections, especially in intensive care settings. Recent

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Poster Presentations

reports from Asia and South Africa have highlighted the increasing
incidence of Candida auris as a nosocomial bloodstream pathogen
affecting persons of all age groups. The emergence of this pathogen
as an agent of fungemia highlights the concerns of elevated MICs for
azoles and caspofungin in C. auris. Herein, we characterized a large
number of C. auris isolates prevalent in tertiary care hospitals of
India using multilocus sequence typing (MLST), amplified fragment
length polymorphism (AFLP) and matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF) to evaluate
the genetic relatedness among the Indian C. auris isolates.
Methods A total of 120 C. auris isolates originating mostly from fun-
gemia and deep seated infections from various hospitals of India were
analysed. All the isolates were subjected to MLST using 3 loci,
namely, ITS, RPB1 and RPB2. Also, the population structure of C.
auris isolates was determined by AFLP and principal component
analysis (PCA) with MALDI-TOF BioTyper 3.0. Antifungal susceptibil-
ity testing was performed for azoles, amphotericin B and echinocan-
dins with broth microdilution method.
Results MLST demonstrated homogenous population of Indian C.
auris isolates, which were highly related to each other. The AFLP
and PCA analysis of C. auris spectra were in concordance with MLST
demonstrating close relationship among the Indian C. auris isolates.
The large number of invariable fragments observed in AFLP sug-
gested their monophyletic origin. High MIC90 were observed for flu-
conazole (64 lg ml
1), flucytosine (32 lg ml
1), voriconazole
(8 lg ml
1), amphotericin B (4 lg ml
1) and isavuconazole
(2 lg ml
1).
Conclusions The clonality of Indian C. auris isolates by all three Figure 2. Gram stain of Trichosporon asahii showing budding yeasts
techniques suggests their nosocomial transmission. Considering the and barrel shaped arthroconidia
frequent prevalence of multi-drug resistant strains of C. auris in the
intensive care units in the present series, the accurate identification
and antifungal susceptibility testing of this yeast is pertinent for guid-
ing therapy and determining the prognosis in such settings. infections, however, systemic infections are known in immunocom-
promised, cancer, burns, transplant patients as well as patients on
steroids, peritoneal dialysis, prolonged mechanical ventilation and
those undergoing prosthetic valve surgeries. Alimentary tract, respi-
P185 ratory tract, broken skin and mucosa, are possible portals of entry.
Persistent and disseminated infections have poor prognosis. Fatal out-
break in neonates and breakthrough trichosporonosis under antifun-
Trichosporon Asahii urinary tract infections in gal therapy has been reported. Urinary tract infections (UTI) by
immunocompromised hosts Trichosporon are rare and remain scantily reported. This study was
I. D. Khan aimed at studying the characteristics of Trichosporon UTI at an Indian
CHEC Kolkata, Kolkata, India tertiary care hospital.
Methods An active surveillance for Trichosporon UTI was undertaken
after the occurrence of three such cases from various centres in a ter-
Objectives Trichosporonosis is an emerging infection predominantly
tiary care apex teaching and referral hospital. Trichosporon was iden-
caused by Trichosporon asahii. Trichosporon (Beigel, 1985)speciesare
tified phenotypically by a combination of manual and automated
ubiquitous, exclusively anamorphic, yeast like fungi belonging to Tri-
systems. Identification through morphology on cornmeal agar and
chosporonaceae. Trichosporon is implicated in superficial and mucosal
Gram stain, hydrolysis of urea, assimilation of carbon/nitrogen com-
pounds was confirmed VITEK 2 compact automated system
(bioMerieux, France). Antifungal susceptibility was tested by E-test
(AB BIODISK, Sweden) for fluconazole, itraconazole, voriconazole,
Amphotericin B and anidulafungin (Fig 1 and 2). Non repeat positive
cultures were interpreted in conjunction with colony characteristics,
cellular morphology, E-test antifungal susceptibility patterns, clinical
correlates and environmental surveillance.
Results T. asahii and T. inkin were isolated from urine of 20 and two
immunocompromised patients respectively who were receiving in-pa-
tient treatment for multiple comorbidities. Ten patients were con-
firmed HIV positive while the rest were iatrogenically
immunocompromised after solid organ or bone marrow transplanta-
tion. Antifungal susceptibility done by E-test revealed multresistance
with preserved susceptibility to voriconazole (MIC 3 lg ml
1).
Discussion Trichosporon infections present diagnostic and therapeu-
tic challenges. They are likely to surpass routine laboratory identifi-
cation. The ubiquity and biofilm formationposes difficulty in
establishing pathogenicity and delineating community-acquired or
hospital-acquired infections. Risk factors such as prolonged multiple
antimicrobials, indwelling catheter and comorbidities may be contrib-
utory to the establishment of a nosocomial opportunistic Trichosporon
infections. Dedicated efforts targeted at infection control are needed
Figure 1. Antifungal susceptibility of Trichosporon asahii through E- to optimize management and control of Trichosporon infections.
test showing an MIC of 3 lg ml
1 for Voriconazole

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120 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P186 P187

Emerging yeasts and algae in a tertiary healthcare set up Tinea capitis with cicatricial alopecia caused by
I. D. Khan Trichophyton schoenleini
CHEC Kolkata, Kolkata, India S. Carvalho,1 F. J. R. Mota,1 V. M. Lopes,1 G. C. Velho,1
S. Machado,1 H. Ramos2 and M. Selores1
1
Objectives Emerging organisms are organisms that have newly Centro Hospitalar do Porto, Porto, Portugal and 2Microbiology
appeared in a cohort/population or have existed but are rapidly Department, Centro Hospitalar do Porto, Porto, Portugal
increasing in incidence, geographic or host range. One-tenth of all
infectious diseases are attributable to emerging organisms. Opera- Objective Dermatophytes represent the prevailing type of fungi that
tionally defining an organism as emerging is a subjective endeavour. cause infection of the skin and nails. Although dermatophyte infec-
As emerging organisms sporadically affect a relatively small percent- tions are common disorders worldwide, some agents, like Trichophy-
age of population, they are not studied at large. This study was ton schoenleini are rarely seen in the United States and Europe and
aimed at studying the characteristics of emerging yeasts and algae at have been considered eradicated in these areas. Here we report a
an Indian tertiary care hospital. case of tinea capitis with cicatricial alopecia, caused by Trichophyton
Methods 33836 positive isolates obtained from 132646 processed schoenleini that had been previously diagnosed and treated as psoria-
samples during 2011-14 were included. Identification percentage sis for nearly 2 years.
>85% along with inbuilt standards for identification comparison were Methods A 14-year-old caucasian girl was referred to our consulta-
considered for final validation through automated systems.Non tion due to a long standing alopecia of the scalp with associated scal-
repeat positive cultures were interpreted in conjunction with colony ing. She was applying a topical salicylic acid cream since she had
characteristics, cellular morphology, disc-diffusion antifungal suscep- been diagnosed as having psoriasis. Clinical examination revealed
tibility patterns, clinical correlates and environmental surveillance. areas of perifollicular erythema on the scalp with adherent scaling,
The frequency of isolation, sources, referring centres, susceptibility positive traction test of the hair and frontoparietal cicatricial alope-
profiles and phenotypic characteristics. A literature search was done cia. A skin biopsy was performed and scales and hair were collected
to identify reports on human pathogenicity and yeasts and algae for mycological examination. The patient was started on oral itra-
reported fewer than 100 times on PubMed were defined as emerging. conazol and topical ketoconazol shampoo.
Results 332 (0.98%) yeasts/algae were isolated from 33836 isolates, Results Direct microscopy of the hair with potassium hydroxide 20%
of which 174 isolates including 14 yeast species and one algae were revealed hyphae within the hair showing characteristic channeling of
found emerging. Non-albicans Candidemia was caused by 84 emerg- Trichophyton schoenleini, hair infection (favic ringworm). Trichophyton
ing non-albicans Candida isolates comprising ten species in multidis- schoenleini was isolated in culture. The patient completed treatment
ciplinary ICU, NICU and bone marrow transplant centre. Non- with oral itraconazol for 6 weeks and topical minoxidil with clinical
albicans Candida species such as haemulonii, famata, rugosa, guillier- resolution of the infection and partial recovery in hair count.
mondii, lusitaniae, utilis, zeylanoides, sphaerica, krusei and intermedia Conclusions Without adequate diagnosis and treatment, favic hair
were isolated along with Trichosporon asahii, Trichosporon inkin, Malas- infection evolves to cicatricial alopecia as we have observed in this
sezia furfur and non-noeformans Cryptococcus. Twelve Prototheca wick- misdiagnosed case. Although scalp infections due to Trichophyton
erhamii were isolated from blood samples from multidisciplinary schoenleini are uncommon this was not our first favus case, as we
Oncology centre. All non-albicans Candida species were multidrug had already detected this agent in other patients’ scalp infection.
resistant and led to frank sepsis in 24 patients. Environmental Clinicians must be aware and should always workout to exclude a
surveillance was not corroborative. fungal etiology in the differential diagnosis of psoriasis and tinea ami-
Conclusion Emerging yeasts and algae may infect compromised antacea or other situations with scaling and alopecia of the scalp.
hosts and pose difficulty in management due to inadequate identifica-
tion and multidrug resistance. Astute efforts directed at identification
of emerging organisms and containment of infection are required.
P188

Schizophyllum colonization in an orally open antral cyst

(case report)
M. Hell, D. Reisner, L. Baskova, D. Achleitner and A. Gaggl
Paracelsus Medical University, Salzburg, Austria

In a 43-year old otherwise healthy man the abscessed tooth 16 (up-

per right molar) was extracted by a dentist. Four days later the
patient was referred to our clinic because of an apparently open
oroantral fistula. On radiographic examination, a residual root of
tooth 14 was still in place and an antral cyst of appr. 2.5 cm diame-
ter was found (tooth 15 had been extracted many years earlier.).
There was no radiodense object detectable. 3 weeks later the root 14
was extracted, the fistula closed and the antral cyst removed. At fol-
low-up 1 year later the area was clinically and radiographically
without evidence of disease.
The histopathological report of the dark and slimy content of the
Figure 1. Distribution of emerging yeasts and algae
cyst was identified morphologically as a “fungus ball”, suspicous for
aspergillus. Twice performed cultures were negative for fungi. PCR
amplification and sequencing of the ITS2 region revealed Schizophyl-
lum commune and excluded any of Aspergillus spp. as a causing
pathogen. To our knowledge this is the first report of a Schizophyllum
commune colonization in an antral cyst. The duration of the lesion,
however, remains unknown: It is possible that already at the time
when tooth 15 was extracted, the cyst had been opened from the

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Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 121
Poster Presentations

mouth. In this case the fungus colonization could have started many Methods The prospective multicenter study in Saint-Petersburg,
years ago. It is also possible that during the 3 weeks between the Russia, 2012-15 yy. Diagnosis of IM was made according to EORTC/
opening of the cyst now and the final surgery some necrotic debri MSG criteria, 2008.
(e.g. food) could have been colonized by Schizophyllum. As no more Results We observed 1 pediatric and 3 adult patients with IM
details of the patient0 s history were available we cannot arrive at a caused by Trichosporon spp. The median age of patients was
final conclusion in regard to the duration. 34.5 years (range 12–56), 3 males. Underlying conditions were:
acute renal failure, chronic renal disease with peritoneal dialysis,
bacterial meningitis and allogeneic haematopoietic stem cell trans-
P200/M6.2 plantation in a pediatric patient with acute lymphoblastic leukemia.
Diagnosis of invasive infection caused by Trichosporon spp. was
confirmed by culture of cerebrospinal fluid - 2, blood - 1, peritoneal
Paracoccidioidomycosis: a case report mimicking adrenal fluid - 1, and renal biopsy - 1. The etiological agents were T. asahii -
neoplasia 2, T. asahii + Candida albicans - 1 (meningitis), and T. inkin - 1. The
B. C. D. A. Zoppas, M. Sartori, I. F. Guerra, L. W. Y. Yum, clinical variants were meningitis - 1, fungemia + meningitis - 1,
P. R. Zimello and D. Nodari nephritis -1, and peritonitis - 1 (T. inkin).
University of Caxias do Sul, Caxias do Sul, Brazil Antifungal therapy was used in all patients: fluconazole - 3
(adults), and voriconazole - 1 (pediatric). Duration of antifungal ther-
apy was 10-44 days (median - 40.5). Overall survival rate in
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin 30 days was 50%.
America with the highest number of cases arising from Brazil. It is Conclusion Invasive mycoses caused by Trichosporon spp. are rare
characterized by a polymorphism of lesions and can affect any organ, diseases with high mortality. Early identification of yeast isolates
in particular, the skin, the lymph nodes, the lungs, the oral, nasal, and should be done to choose appropriate therapy.
gastrointestinal mucous membranes; caused by dimorphic fungi Para-
coccidioides brasiliensis and Paracoccidioides lutzii. A case of systemic
Paracoccidioidomycosis, in a private hospital in Caxias do Sul, Rio P202
Grande do Sul, Brazil is presented. Male patient, 66 years old, previ-
ously healthy was admitted to the hospital presenting sweat, chills,
fatigue, weight loss (25 kg) and daily fever episodes. The signs and The ROCANET study: results from a prospective
symptoms have started 2 months earlier. He was in hematological fol- observational survey of candidaemia in the Rome city
low-up because of alteration in transaminases and in serum ferritin. V. di Florio,1 B. Posteraro,2 M. Sanguinetti,2 M. Tumbarello,1
Abdominal CT scan showed a bulky lesions affecting both adrenal A. Vella,2 E. de Carolis,3 M. Venditti4 and N. Petrosillo5
glands, measuring 8.8 9 4 9 3.7 cm at right and 11 9 5 9 4.8 cm
at left. The right lesion determined invasion of the inferior vena cava,
1
UCSC, Roma, Italy; 2Universita Cattolica del S. Cuore, Roma,
where was observed 2.3 cm tumor thrombus. Initially, it was sus- Italy; 3Catholic University of Sacred Heart, Roma, Italy; 4Sapienza
pected of neoplastic disease, however, the pathological study of the left University of Rome, Roma, Italy and 5National Institute for
lesion aspiration revealed a high number of leukocytes, a presence of Infectious Diseases ‘L. Spallanzani’, Roma, Italy
necrotic material and spores suggesting Paracoccidioides spp to Grocott
method. The diagnosis was confirmed by culture the fungus. The treat- Objectives Candida species are leading causes of nosocomial blood-
ment was started with Itraconazole 200 mg day
1 with gradual stream infections and are associated with substantial morbidity, mor-
improvement in symptoms and weight regain. The patient still remains tality and costs. While the antifungal susceptibility pattern is closely
with antifungal therapy associated with the use of prednisone linked to the species, it is yet greatly important to understand and
7.5 mg day
1 due to the partial adrenal glands destruction. After monitor the local species epidemiology as well as the antifungal resis-
6 months of treatment, the CT still reveals the presence of adrenal tance rate. The ROCANET (Rome Candida Network) was established in
wounds, however, with a reduction in volume (7.7 at right and 9.3 at December 2012 in order to perform prospective surveillance of all can-
left) compared with the previously CT. Paracoccidioidomycosis can didaemias among patients hospitalized at selected medical centres in
affect locations such as pancreas, adrenal, CNS, and mimic other dis- the Rome city area. Its aim is to improve the knowledge of the burden
eases, delaying diagnosis and leading to severe and fatal prognosis. of candidaemias in different groups of patients, better define patients at
Although the patient in question resides in urban center, he used to risk and understand the epidemiology, species distribution, antifungal
prune vines, a very common activity in the northeast of Rio Grande do susceptibility and outcomes of candidaemias from the study sites.
Sul, therefore establishing connections with rural environment. As so, Methods All the patients admitted to 10 large hospital medical cen-
the main risk factors for acquiring the disease are activities involving tres of Rome (Italy) from January 2013 to December 2014 and diag-
soil management and plantations. nosed with candidaemia will be studied. All the Candida isolates from
the study patients will be collected and maintained at the Clinical
Microbiology Laboratory of the Universit a Cattolica del Sacro Cuore
P201 of Rome. The isolates will be re-identified by the MALDI-TOF MS
method and sequence identified using the ITS gene region, whereas
susceptibility testing will be performed against 7 antifungal agents
Invasive mycoses caused by Trichosporon spp. in Saint-
(anidulafungin, caspofungin, micafungin, fluconazole, itraconazole,
Petersburg, Russia
posaconazole, voriconazole) using CLSI and EUCAST methods. In the
E. Shagdileeva,1 T. S. Bogomolova,1 A. Saturnov,2 O. Pinegina,3 case of antifungal drug-resistant isolates, underlying molecular resis-
A. Volkova,3 K. Ekushov,3 Y. Vasilieva,4 V. Makarov,4 tance mechanisms will be assessed, as well all the isolates will be
A. Kuzmin,4 A. Annanieva,5 N. Vasilyeva1 and N. Klimko1 genotyped using multi-locus sequence typing, as appropriate.
1
North-Western State Medical University named after I.I. Results From January 2013 to October 2013, a total of 668 isolates
Metchnikov, Saint Petersburg, Russia; 2Leningrad Regional of Candida species were studied, including 319 C. albicans, 197 C. parap-
silosis complex (including 4 C. orthopsilosis and 2 C. metapsilosis), 65 C.
Clinical Hospital, Saint Petersburg, Russia; 3Institute Of Children’s
glabrata complex (including 1 C. nivariensis),41 C. tropicalis, 2 C. guil-
Hematology And Transplantology Named R.M. Gorbachova,
liermondii, 12 C. krusei, 7 C. lusitaniae, 5 C. guilliermondii, 5 Rhodotorula
Saint Petersburg, Russia; 4Botkin Clinical Infectious Hospital, Saint mucilaginosa, and other 17 belonging to other 9 species. Overall, resis-
Petersburg, Russia and 5City Mariinskaya Hospital, Saint tance to the echinocandins was very low, with C. albicans (1 isolate)
Petersburg, Russia and C. glabrata (1 isolate) being resistant to anidulafungin, caspofungin
or micafungin and shown to have fks mutations. Resistance to flucona-
Objective Trichosporon spp. are rare etiologic agents of invasive zole was low among isolates of C. albicans (1 isolate) and C. tropicalis (1
mycoses (IM) and publications about these diseases are limited. isolate), whereas 12 isolates of C. parapsilosis complex (11 C.

ª 2015 The Authors

122 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

parapsilosis and 1 C. metapsilosis)were found to be resistant to flucona- P204

zole. Surprisingly only 4 isolates were resistant to azoles. Voriconazole
and posaconazole were active against all Candida species except C. glab-
rata and 2 isolates of C. parapsilosis. Is Aspergillus Fumigatus tr34/l58 h prevalent among
Conclusion Overall, echinocandin and triazole resistance were colonised cystic fibrosis patients? Experience of an Spanish
uncommon. Although no fluconazole and echinocandin co-resistance cystic fibrosis reference center
among C. glabrata isolates was observed, continued close surveillance A. Rando, M. Martin Gomez, L. Goterris, D. Romero, S. Gartner,
is locally warranted. A. Alvarez, S. Rubio, M. Guerrero, F. Barroso and M. A. Cano
Vall d’Hebron Hospital, Barcelona, Spain

P203 Objectives A growing number of clinical A. fumigatus strains carry-

ing the TR34/L58H mutation are reported in Northern Europe (par-
ticularly The Netherlands, Germany and Northern France) and seem
An estimation of burden of serious fungal infections in to be spreading to the South. However, only a few of these strains
France have been isolated in Spain. Moreover, its prevalence in clinical sam-
J. P. Gangneux,1 M. E. Bougnoux,2 C. Godet,3 D. Denning4 and ples in our country is unknown, although it is supected to be very
B. Dupont2 low. Our aim was to study the rate of A fumigatus TR34/L58H
1 among strains recovered from patients diagnosed of Cystic Fibrosis
Rennes Teaching Hospital - Inserm U1085, Rennes, France; (CF) and colonised with A. fumigatus as they may represent a sentinel
2
Necker Enfants Malades University Hospital, Paris, France; population for the emergence of the TR34/L58H mutation in our
3
Poitiers University Hospital, Poitiers, France and 4The University area.
of Manchester and National Aspergillosis Centre, Manchester, Methods 35 consecutive A. fumigatus strains isolated between Jan-
United Kingdom uary 2014 and April 2015 from sputum samples of 22 CF patients
(11 pediatric and 11 adult patients, mean age 20.7 years, range 8–
Introduction Our objective was to estimate the burden of serious 36 years) classified as being A. fumigatus-colonised attending our CF
fungal disease in France, based on epidemiological data. Epidemiol- Unit were studied. The presence of the TR34/L58H mutation was
ogy of several of them remains unknown, whereas precise data are determined using a commercial Real Time PCR kit with specific
available for invasive fungal infections such as invasive aspergillosis, probes for both mutations. As positive external control, we used an
candidemia, cryptococcosis and pneumocystosis. Herein, we report A fumigatus strain isolated in our center from an Invasive Aspergillo-
an estimation of the incidence and prevalence of invasive but also sis case; this strain had been previously characterised as positive for
chronic and immunoallergic fungal diseases in France in order to the TR34/L58H mutation. Genetic material was obtained from pure
provide a basis of reflection in terms of Public Health. cultures of the strains after beadbeating and proteinase K pretreat-
Methods Published epidemiology papers reporting fungal infection ment using an EasyMag apparatus. Real Time PCR was carried out
rates from France were identified. Where no data existed, we used in an SmartCycler termocycler.
specific populations at risk and fungal infection frequencies in those Results None of the 35 CF strains tested positive for the TR34 or
populations to estimate national incidence or prevalence, depending the L58H mutation, whereas the previously known positive strain
on the condition. Population statistics were derived from the Institut isolated from a case of invasive aspergillosis gave a positive result for
National de la Statistique et des Etudes Economiques (INSEE-2014). both probes.
Incidences of invasive fungal infections were derived from Institut Conclusions A fumigatus strains carrying the TR34/L58H mutation
National de Veille Sanitaire (InVS) and the National Center of Refer- conferring pan-azole resistance seem to be absent or, at least, to be
ence for invasive fungal diseases (CNR-MA, Institut Pasteur)(2010). of very low prevalence among CF patients chronically colonised with
Results 82% of the 65.8 M population (2014) are adults. The A. fumigatus in our area, although larger and prospective studies
asthma and COPD prevalences in adults are 6.7% and 7.5%, respec- should be carried out to confirm the observation made by our group.
tively. In this context, the model predicts 124 678 SAFS episodes Nevertheless, it is in the line of previous works published by Spanish
(189 cases/100 000 adults per year), 95 361 ABPA episodes (145/ groups in non-CF related selected populations or samples. Real-time
100 000) and 3450 chronic pulmonary aspergillosis cases (109/ PCR seems to be a fast and useful alternative for screening/confirm-
100 000)(Table 1). These high estimated burdens indicate that such ing the presence of the TR34/L58H mutation in A. fumigatus strains.
patients should be better identified and characterized in order to
improve their management. Good estimates of incidence rates of doc-
umented invasive fungal diseases are available: 3.6/100 000 for can-
didemia, 1.8/100 000 for invasive aspergillosis (IA), 1/100 000 for P205
pneumocystosis, 0.2/100 000 for cryptococcosis and 0.12 for
mucormycosis. However our estimate of IA complicating COPD is
1300 cases against a smaller documented number whatever the
Ethiological profil and epidemiology of tinea capitis in the
underlying condition, suggesting a major diagnostic gap., which may region of Mitidja (BLIDA) in Algeria
be also be true of pneumocystis. These severe infections have persis- M. Chekiri-Talbi,1 K. Ouldrouis-Saoudi,1 L. Rezekallah,1
tently high mortality rates and represent high costs due to hospital- W. Ammour1 and D. Denning2
1
ization, diagnostic management and antifungal drugs use. Finally, Blida 1 University, Blida, Algeria and 2The University of
estimates of Candida peritonitis, oesophageal candidiasis and recur- Manchester and National Aspergillosis Centre, Manchester,
rent vaginal candidiasis rates are O.74/100 000, 13.8/100 000 and United Kingdom
2220/100 000 (rate for adult females only), underline the need for
adjusting such potential high burdens with additional studies.
Conclusion Using local and literature data of the incidence or Introduction Tinea capitis is a mycological affection observed most
prevalence of fungal infections (with the exclusion of tinea capitis), of the time in children. It is due to dermatophytes:Trichophyton and
approximately 1 000 000 (1.47%) people in France are estimated to Microsporum. In Algeria numerous studies in scattered regions of the
suffer from fungal infections each year. Beside a good evaluation of country discussed the mycological and epidemiological character of
5000 cases/year of invasive fungal diseases, recurrent vaginitis and this pathology. We present for the first time the results of a retrospec-
chronic and immunoallergic pulmonary diseases represent a serious tive survey from August, 1999 till August, 2014 in the Mitidja
impact on human health. Prospective or comprehensive registry sur- region of Blida situated in 50 km in the southwest of Algiers.
veys are required to accurately determine their epidemiology and Patients and methods It is a retrospective descriptive study based
improve the management of these disorders. on the data of the registers of the laboratory of parasitology-mycol-
ogy of the teaching hospital of Blida The majority of the patients are

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Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 123
Poster Presentations

from the city of Blida and from the bordering localities. The patients population. PCR-based determination of mating idiomorphs was also
whose personal or clinical information was incomplete were not performed in all strains.
included.our study. Results The sequence analysis based on two genes revealed presence
Results We took all in all 4140 patients among whom 2750 are of five genotypes. Most common genotype (n = 195, 74.4%) was
positive 66.42% is the prevalence the mean age is of 24 years, the characterized by yellow colony reverse on MEA agar with the excep-
extremity of age is between 02 months is 83 years old, the median is tion of isolates from dogs in North America which showed different
of 25 years the mode is of 25 years and the standard deviation is of phenotype. Only MAT1-1 idiomorph was amplified in isolates of this
17 years. The average, the mode and the median being practically genotype from Europe and low variability in microsatellites data was
equal, ca evokes a normal distribution (casting), 2/3 of the patients recorded. In contrast, the isolates of this genotype from North Amer-
got their age fluctuates enter standard deviation between 7 years ica were highly variable by microsatellite data and isolates of both
and 41 years. mating type genes were revealed. The second most common geno-
The samples concerned hair the alopecic region by scratching of type (n = 47, 17.9%) was characterized by dominant red (mating
the lesions with a sterile scraper to collect hairs and then a culture type MAT2) or brown (MAT1) colony reverse on MEA agar. The
on Sabouraud media with antibiotics at 30c is systematic. From third genotype (n = 16), closely related to second genotype, included
4140 patients, only 2750 patients are considered to have Tinea capi- all strains from Japan and some from the CZ and Belgium and was
tis. The direct examination had returned positive for 2420 sick while characterized by both red and yellow colony reverse and MAT1-1
the culture was negative that is 58.45%. Microsporum canis and Tri- idiomorph. The remaining two genotypes were each represented by
chophyton mentagrophytes are the most identified. only two isolates from the CZ.
Conclusion This is the first study done in this region of Algeria, we Conclusion A. benhamiae is a new emerging pathogen in the CZ and
noticed that these pathogenics fungies have zoophile and telluric ori- some other countries in Central and Western Europe region. Accord-
gin although that the lifestyle of the patients concerned by the study ing to recent studies this species is the most important zoophilic der-
is urban.in fact most Algerian living in the cities doesn’t have pets. matophyte in the CZ causing almost 23% of tinea corporis (median
These results could be explicated by the Lack of hygiene which age of patients – 10 years, females - 70%) and 29% of tinea capitis
remains a public health problem in our country. infections. The majority of infections are transmitted from guinea
pigs and other rodents. It is probable that the most common geno-
type of A. benhamiae responsible for the outbreak of infections in the
Central Europe (agent of 79.7% of human infections in the CZ)
P206 spread clonally. This hypothesis is also supported by uniform pheno-
type and mating type in European strains. The american strains of
the same genotype (can be separated by microsatellite data) however
Molecular typization of Arthroderma benhamiae, a showed different phenotype and presence of both mating type genes
zoonotic agent of epidemic dermatophytosis in Central indicating sexual reproduction. Other genotypes contribute only mar-
Europe ginally on the outbreak.
A. Cmokova,1 M. Kolarik,2 T. Vetrovsky,3 S. Dobiasova,4
R. Dobias,5 D. Stubbe,6 M. Skorepova,7 P. Lyskova,8 L. Hoyer,9
N. Mallatova,10 R. Kano,11 P. Nenoff,12 S. Uhrlab,12 A. Peano,13
J. Koubkova,14 K. Mencl,15 H. Janouskovcova15 and V. Hubka1 P207
1
Charles University in Prague, Prague, Czech Republic; 2Institute
of Microbiology, Czech Academy of Science, Prague, Czech Re-emerging paracoccidioidomycosis in Argentina with
Republic; 3Institute of Microbiology of the AS CR, Prague, Czech peculiarities in the diagnosis and clinical manifestations
Republic; 4Institute of Public Health, Ostrava, Czech Republic; G. E. Giusiano,1 M. E. Cattana,2 M. F. Tracogna,3 M. A. Sosa,1
5
Institute of Health in Ostrava, Ostrava, Czech Republic; F. D. Rojas,2 M. S. Fernandez,1 N. Cech4 and A. Arechavala5
6
Scientific Institute of Public Health, Brussels, Belgium; 7Charles 1
Instituto de Medicina Regional- Universidad Nacional del
University in Prague, First Faculty of Medicine, Prague, Czech Nordeste, Resistencia, Argentina; 2Instituto de Medicina
Republic; 8Institute of Health in Usti nad Labem, Prague, Czech Regional-UNNE, Resistencia, Argentina; 3Hospital J.C. Perrando,
Republic; 9University of Illinois at Urbana-Champaign, College of Resistencia, Argentina; 4Hospital 4 de junio, Saenz Pen~a, chaco,
Veterinary Medicine, Prague, Czech Republic; 10Hospital Ceske Argentina and 5Hospital F.J. Mun~iz, Buenos Aires, Argentina
Budejovice, Ceske Budejovice, Czech Republic; 11Nihon University
School of Veterinary Medicine, Fujisawa, Japan; 12Laboratory of
Paracoccidioidomycosis (PCM) is a systemic endemic mycotic disease
medical microbiology, Mo€lbis, Germany; 13Universita degli Studi
caused by the thermally dimorphic fungus Paracoccidioides. The fun-
di Torino, Facolta di Medicina Veterinaria, Turin, Italy; 14Clinical gus has a geographic distribution limited to tropical and subtropical
Veterinary Laboratory Labvet, Prague, Czech Republic and areas from rural areas of Central and South America. PCM is the
15
Pardubice Regional Hospital, Inc., Pardubice, Czech Republic commonest systemic mycosis in Latin America without mandatory
reporting and considered as a Neglected Disease.
Objectives The aim of this study was to find informative molecular Argentina has two endemic areas of PCM; the more extensive is
markers to evaluate population structure of dermatophyte species located in the northeast near Paraguay and Brasil, where the chronic
Arthroderma benhamiae in the studied area. One of the main issues PCM is the historically observed clinical form.
was to determine whether the current outbreak of dermatophytosis In the last years, an increase of the PCM incidence associated with
caused by A. benhamiae in Central Europe is caused by a new more urban cases, patients with rare clinical manifestations or infrequent
virulent genotype which is with the high success rate transmitted to locations and also, cases of juvenile PCM never described before in
humans or whether other causes must be considered (e.g. high that area was observed. In addition, a high percentage of negative
prevalence of the pathogen in animal husbandry). serological diagnosis was detected both in chronic and juvenile clini-
Methods A total number of 262 A. benhamiae strains associated cal forms.
with cases of human and animal dermatophytosis from the Czech In order to know the status of the current clinical and epidemio-
Republic (CZ), Belgium, UK, Germany, Italy, Japan and USA were logical characteristics of PCM in Argentina, a multicentric study of
used for analysis. Ten microsatellite markers were developed and PCM coordinated by Departamento Micologıa of Instituto de Medicina
used for typization of A. benhamiae strains together with sequence Regional of Universidad Nacional del Nordeste (Argentina) was
analysis of ITS region rDNA and glyceraldehyde-phosphate dehydro- started.
genase gene. Bayesian inference analysis and distance methods were We present a retrospective descriptive analysis of PCM cases from
applied on the datasets to reveal the genetic variation of A. benhamiae 2013 to 2015.

ª 2015 The Authors

124 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Table 1 P208

Trichophyton mentagrophytes – a rare zoophilic

dermatophyte isolated in Germany – morphological and
molecular biological characterization of four wild strains
S. Uhrlab,1 C. Mehlhorn,1 V. Hubka,2 J. Brasch,3 T. Maier,4
€ ger1 and P. Nenoff1
C. Kru
1
Laboratory of medical microbiology, Mo€lbis, Germany; 2Charles
University in Prague, Prague, Czech Republic;
3
Universita€tsklinikum Schleswig-Holstein, Kiel, Germany and
4
Bruker Daltonik GmbH, Bremen, Germany

Objective Based on what is known today from molecular biological

analysis, T. mentagrophytesis now only used to refer to the formerly
T. mentagrophytes var. quinckeanum (the pathogen in mouse favus),a
highly uncommon pathogen in WesternEurope. T. mentagrophytes is
predominantly isolated in the Near East,and Asia, as a zoophilic der-
matophyte which has its source in camels and rodents, like mice. In
humans,T. mentagrophytes causes tinea capitis and tinea corporis.
Patients and methods Four wild strains of T. mentagrophytes were
isolated from skin scrapings of patientssuffering from dermatophytosis
or onychomycosis. All strains were characterized based on morpho-
logical andmetabolic features. For molecular biological detection of
the dermatophytes in skin scrapings a uniplex PCR-EIAwas used.
Species identification was confirmed by sequencing of the internal
transcribed spacer region (ITS)of the ribosomal DNA. Further, the
strains were typed by MALDI TOF MS.
Results The four strains of T. mentagrophytes were isolated from 2
children and 2 adults. Patients sufferedfrom tinea corporis or tinea
faciei. In case of a 44 year old woman, T. mentagrophytes was iso-
lated from nailscrapings of onychomycosis or tinea unguium, respec-
tively. The patient had barefooted contact to soil due togardening. A
A total of 67patients with PCM, aged 13–89 years (median guinea pig was source of infection in one out of the four patients.
49 years) were recorded. In 2013 were recorded 23 cases, 32 in The other patients had contactto cats or dogs, however, these ani-
2014 and 12 cases only from January to April 2015, pointing the mals were not considered as source of infection. All here described-
annual increasing. dermatophytoses probably were acquired in Germany. All isolates
A lower male/female relationship (12 : 1) than reported by others had typical white to yellowish stained powderyflat colonies (Fig. 1).
countries was observed. The strains showed small spherical microconidia arranged alongside
Chronic form in 63 patients and juvenile PCM in 4 patients were the thin hyphae,spiral hyphae were seen. Big chlamydospores and
recorded. Without classical manifestations that suggest PCM, about thin-walled macroconidia could be detected. All strainswere con-
15% of patients, only showed rare manifestations or infrequent loca- firmed as T. mentagrophytes by PCR-EIA, sequencing of the ITS rDNA,
tions such as osteomyelitis, cerebral and cerebellar abscesses, cuta- GPD gene and microsatellite typing.In MALDI TOF MS the four
neous lesions, that delayed the diagnosis. Clinical manifestations strains had identical spectra.
observed in all patients are presented in Table 1.
Serological and microbiological diagnosis was achieved in 46
patients, in 6 patients only serological and only microbiological in
15 cases. Negative serological results with microbiological diagnosis
were obtainedin 9 cases (13%).
The knowledge that Paracoccidioides includes P. lutzii and P.
brasiliensis with cryptic species can be an explanation for this find-
ing. Although the impact of this genotypic diversity is not completely
understood, data suggest that false negative results could be obtained
with the standard immunodiffusion test performed using the Gp43
antigen of P. brasiliensis B 399 strain in patients infected with a
strain that has different antigenic profile.
Several unexplained peculiarities have been noted in the diagnosis
and clinical manifestations. The recognition of the re-emergence of
PCM in Argentina with particular characteristics requires urgent
research to explore specifically the circulating Paracoccidioides species
in order to improve the diagnosis and therapeutic tools.

Figure 1. T. mentagrophytes var. quinckeanum

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 125
Poster Presentations

Conclusion The isolation of T.mentagrophytes (which is not T.inter- Methods We searched for existing data and estimated the incidence
digitale incurrentsense) in Germany has tobe considered as a very and prevalence of fungal diseases based on the population at risk
rare finding. In Germany, zoophilic strains of T. interdigitale are and available epidemiological data. Demographic data were derived
standing for the majorityof zoophilic dermatophytes isolated from from the Service (Office) of the Statistics (ONES), World Health Orga-
rodents and affected patients. T. mentagrophytes sensu strictu is still nization (WHO), The Joint Nations Programme on HIV/AIDS
quitecommon in some areas in Asia (in particular in China), how- (UNAIDS) and national published reports. When no data existed, risk
ever, not in Europe. populations were used to estimate frequencies of fungal infections,
using described methodology by LIFE.
Results Algeria had 39.5 million inhabitants in January, 2015 and
a forecast growth of >2% based on population since 2013. Males rep-
P209 resent 50.62% of the population, 28% were under 15 years, and
16.4% were over 50 years. There are about 50 000 new cases of
cancer every year among including 1500 in children, and about 200
Equine dermatophytosis due to Trichophyton bullosum in new cases of leukaemia annually. For HIV 6472 patients are seropos-
the Czech Republic itive and 1422 patients have AIDS. In 2012, there were 8753 sur-
P. Lyskova,1 V. Hubka,2 A. Petricakova,3 R. Dobias,4 M. Kolarik,5 vivors of pulmonary TB, an estimated 317 762 patients with COPD
L. Svobodova6 and P. Hamal7 of whom 20.3% were estimated to be admitted to hospital each year.
1 Using data from Morocco for asthma, 2.84% of the adult population
Institute of Health in Usti nad Labem, Prague, Czech Republic; have asthma, a total of 807 700 people.
2
Charles University in Prague, Prague, Czech Republic; The incidence of invasive aspergillosis in the neutropenic patient
3
Dermatological Outpatient Department, Neratovice, Czech in the center of the country is about 8%, while candidaemia occurs
Republic; 4Institute of Health in Ostrava, Ostrava, Czech in 5% dominated by Candida albicans, Candida parapsilosis and Candida
Republic; 5Institute of Microbiology, Czech Academy of Science, glabrata. Cryptococcosis is less frequent – we count 50 cases/year
Prague, Czech Republic; 6Palacky University Olomouc, Olomouc, and pneumocystosis 30 cases annually.
Czech Republic and 7Faculty of Medicine and Dentistry, Palacky Concerning the superficial mycoses, tinea capitis is by far the most
University, Olomouc, Czech Republic infection which is culture positive, with the pathogenic agents Mi-
crosporum canis and Trichophyton mentagrophytes being common;
favus has almost disappeared. We noticed that Trichophyton rubrum,
Trichophyton bullosum is a zoophilic dermatophyte from Arthroderma
Trichophyton interdigitale and finally Trichophyton violaceum are the
benhamiae complex with poorly known distribution.
most commonly incriminated species in tinea pedis and epidermato-
Presented is a case of dermatophytosis caused by T. bullosum in a
phyties. About ten cases of sporotrichosis have been identified nation-
6-year-old male horse. In October 2013, he developed a weeping skin
ally since 1999.
lesion approximately 10 cm in diameter in a saddle area with subse-
Conclusion Probably at least 1.34% of Algerians have a serious fun-
quent loss of coat. The infection spread rapidly to the upper chest
gal infection each year. This is dominated by recurrent vaginal can-
and to both sides of the trunk. Fungal hyphae were revealed micro-
didiasis and allergic fungal disease complication asthma. Not counted
scopically and, subsequently, fungal colonies grew during culture.
The horse was initially treated with enilconazole solution. After-
wards, the lesions were treated with flutrimazole spray for 3 weeks.
In the course of this antifungal treatment, the skin started to be cov-
ered with soft hairs and then healed completely.
The fungal colonies were suspected as dermatophyte based on their
morphology. Interestingly, the isolate grew better at 35°C than at
25°C and was first identified as Trichophyton verrucosum in slide cul-
ture. However, sequence analysis of the ITS region of its rDNA
revealed T. bullosum.
An epidemiological investigation showed that the horse had been
placed in a racing stable with other horses at the time of infection
and had never been abroad. Subsequent swabs taken from the other
horses stabled in the same building yielded negative results. Before
the infection occurred, the horse had not shown any signs of injury
or visible grazes and had been used for racing. It had always been
stabled with horses only and had never been in direct contact with
cows, sheep or other livestock. Therefore, the source of the infection
remains unclear.
This work was supported by the Palacky University Olomouc Inter-
nal Grant Agency (project no. IGA_LF_2015_021).

P210

The burden of fungal diseases in Algeria

M. Chekiri-Talbi1 and D. Denning2
1
Blida 1 University, Blida, Algeria and 2The University of
Manchester and National Aspergillosis Centre, Manchester,
United Kingdom

Objectives In Algeria superficial mycoses are very commonly diag-

nosed. Deep fungal infections are less often observed. Few data from
Algeria are found in the literature. We report for the first time in this
paper the main causes of these diseases in our country.

ª 2015 The Authors

126 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

here are the most frequent mycoses – superficial. Invasive mycoses P212
are dominated by aspergillosis and candidosis.

The burden of serious fungal infections in Venezuela

M. Dolande,1 M. Panizo,1 A. Alastruey-Izquierdo2 and
P211 D. Denning3
1
Instituto Nacional de Higiene Rafael Rangel, Caracas, Venezuela;
2
The burden of serious fungal infections in Portugal Spanish National Centre for Microbiology, Instituto de Salud
R. F. P. Sabino,1 C. Verıssimo,1 C. S. Pais2 and D. Denning3 Carlos III, Majadahonda, Spain and 3The University of
1 Manchester and National Aspergillosis Centre, Manchester,
National Institute of Health Dr. Ricardo Jorge, Lisboa, Portugal; United Kingdom
2
University of Minho, Braga, Portugal and 3The University of
Manchester and National Aspergillosis Centre, Manchester,
United Kingdom Introduction Venezuela is just north of the equator and has high
rates of endemic fungal infections, HIV and hospital infections. The
burden of fungal infection in Venezuela is not known, despite some
Objectives We here attempt the first estimate of the burden of seri- epidemiological studies. This is the first estimate of the burden of seri-
ous fungal disease in Portugal using deterministic scenario modelling ous fungal disease in Venezuela.
and published incidence and prevalence data. Methods We have used deterministic scenario modelling and pub-
Methods Published epidemiology papers reporting fungal infection lished incidence and prevalence data on both underlying diseases
rates from Portugal were identified. Where no data existed, we used and fungal infection rates from Venezuela to estimate burden. Where
specific populations at risk and fungal infection frequencies in those no data existed, we used specific populations at risk and fungal infec-
populations to estimate national incidence or prevalence, depending tion frequencies in those populations to estimate burden. Population
on the condition. Population statistics were derived from the Statis- statistics were derived from the Instituto Nacional de Estadıstica
tics Portugal and the population census (2011). The incidence and (http://www.ine.gov.ve). Data on respiratory infections were obtained
prevalence of fungal infections affecting HIV patients were obtained and inferred from the WHO data on TB.
from the report on HIV/AIDS 2013 published by the National Health Results In 2011, the population of Venezuela was 29.44M, with
Institute Doutor Ricardo Jorge, I.P. The total number of transplants 71% >15 years of age, and about 9% >60 years of age. An estimated
was obtained from the Authority for Blood Services and Transplanta- 474 720 women (15-50) suffer from recurrent vulvovaginal candidi-
tion (data from 2011). Data on respiratory infections were recovered asis each year. Of the 110 000 HIV positive patients, an estimated
and inferred from the WHO data on TB. 59 000 have CD4 counts <350, and 14 700 are at high risk of
Results The population of Portugal in 2011 was 10.56M, with 75% infection. We estimate that 2699 develop PCP Pneumocystis pneumo-
>15 years of age. An estimated 150 000 women (15–50) suffer from nia in AIDS, 693 cryptococcosis and 1746 disseminated histoplasmo-
recurrent vulvovaginal candidiasis each year. We have not estimated sis, in AIDS. There are an estimated 3800 deaths from AIDS
oral or oesohphageal candidiasis rates. Candidaemia affects 0.88/ annually. Candidaemia is relatively common with affects 1.72/1000
1000 hospital admissions or approximately 8.1/100 000 patients, a hospital admissions or approximately 16/100 000 patients, a total of
total of 856 cases nationally. An estimated 150 patient develop 4798 cases nationally. An estimated 824 patient develop intra-ab-
intra-abdominal candidiasis, post-operatively. Invasive aspergillosis is dominal candidiasis post-operatively. Invasive aspergillosis is thought
less common than other countries as COPD is uncommon in Portu- to affect 1143 people each year. An estimated 226 patients develop
gal, a total of 180 cases annually. An estimated 69 patients develop chronic pulmonary aspergillosis after TB each year, a prevalence of
chronic pulmonary aspergillosis after TB each yearSo, assuming a 711 cases and overall 1422 patients. Assuming ABPA affects 2.5%
15% annual mortality and surgical resection rate, the prevalence is of adult asthmatics, 33 440 cases are likely and 44 141 SAFS cases.
218 cases following TB, whereas the prevalence of chronic pul- Data on dermatophytosis were not included in this study and there
monary aspergillosis for all underlying pulmonary conditions was are few data on fungal keratitis, mucormycosis, coccidioidomycosis
654 patients. In contrast, asthma rates are high and so an estimated and paracoccidioidomycosis.
23 198 SAFS episodes (220 cases/100 000), 17 586 ABPA episodes Conclusion Over 565 685 Venezuleans are affected by serious fun-
(167/100 000). 81 patients developed pneumocystis pneumonia in gal disease annually, with many deaths. Further studies based on
AIDS and 16 cryptococcosis. Data on dermatophytosis and fungal local surveys are required to obtain more precise and complete data.
keratitis were not included in this study.
Conclusion Using published data, we were able to estimate the inci-
dence or prevalence of the above referred fungal infections and
~194 293 (1.8%) people in Portugal suffer from those fungal infec- Table 1
tions each year. Vaginal candidiasis was the most frequent fungal
infection detected in this study. Further inclusion of dermatomycosis
data will largely increase the incidence and prevalence of fungal
infections. This is a preliminary study but constitutes the first report
on the global burden of fungal infections in Portugal. Further studies
based on local surveys are required to obtain more precise and com-
plete data.

Table 1

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 127
Poster Presentations

P213 P214

Clinical, epidemiological and geographic characteristics of Burden of serious fungal infections in Serbia
patients with paracoccidioidomycosis in Southern Brazil: A V. S. Arsic Arsenijevic,1 S. B. Sipetic Grujicic,2 M. G. Pekmezovic3
cross-sectional analysis and D. Denning4
A. Gazzoni,1 L. Deon1 and R. Elsemann2 1
University of Belgrade, Belgrade, Serbia; 2Institute of
1
Serra Gaucha Faculty, Caxias do Sul, Brazil and 2School of Epidemiology, Faculty of Medicine, University of Belgrade,
Dentistry, Caxias do Sul, Brazil Belgrade, Serbia; 3Institute of Microbiology and Immunology,
Faculty of Medicine Uni. of Belgrade, Belgrade, Serbia and 4The
Objectives Aims of this study were: (a) (b) To describe the key clini- University of Manchester and National Aspergillosis Centre,
cal-epidemiological features on the paracoccidioidomicose (PCM) Manchester, United Kingdom
cases in north-eastern part of the Rio Grande do Sul, (Southern Bra-
zil); (c) To investigate the cities of origin for the patients with para- Objective To estimate the annual burden of serious fungal infections
coccidioidomycosis, including its distribution in time and space. (SFIs) in Serbia based on a size of population at risk and limited
Methods We conducted a cross-sectional analysis from PCM cases of available epidemiological databases.
a pathology laboratory in the city of Caxias do Sul, RS, Brazil. This Methods National population data were obtained from Statistical
city is located in north-eastern part of the Rio Grande do Sul, (South- Office of the Republic of Serbia, Institute of Public Health of Serbia,
ern Brazil). Descriptive analysis of PCM cases covering the period Jan- National Reference Medical Mycology Laboratory database as well as
uary 2012 to March 2015. The study was carried out retrospectively from National register for cystic fibrosis. Number of serious fungal
by evaluating the databases of this laboratory. These cases were con- infections among all underlying disease groups was estimated based
firmed by the detection of multiple budding yeast cells typical of P. on the number of risk patients using previously described methodol-
brasiliensis in tissue fragments. All of the cases confirmed by ogy by Leading International Fungal Education (LIFE) and Global
histopathology were included in this study. The following informa- Action Fund for Fungal Infections (GAFFI).
tion was collected for this study using the biopsy data sheets and lab- Results Of the 7.2M population (48.7% male and 51.3% female),
oratory evaluations: age, sex, city of origin, professional activity, 14.3% are children (0–14 years), 67.8% are adults (15–65 years)
course of the disease and location of lesions. The data were compiled and 17.8% of population are >65 years old. The current annual bur-
and descriptively analyzed using the Graph Pad Prism 5.0 (GraphPad den of SFIs in Serbia was estimated to be 109 283 (1517.8/100 000
Software INc. La Jola, CA, USA). This study was conducted according population, Table 1).
to the principles expressed in the Declaration of Helsinki. The study The most common was recurrent vulvovaginal candidiasis (RVVC)
was approved by CONEP (National Committee for Research Ethics). with 95 907 cases per year assuming a literature rate 6% in women
Results A total of 50 patients diagnosed with PCM by histopatholog- between 15 and 50 years old (1.6 M population). This is followed by
ical examination were registered between January 2012 and March severe asthma with fungal sensitisation (SAFS) and allergic bron-
2015. Ages ranged from 15 to 99 years (average = 57 years). chopulmonary aspergillosis (ABPA) that together comprised 10.6% of
Patients ≥ 40 years represented 92% of all diagnostics and 96% were SFIs in Serbia. Chronic obstructive pulmonary disease (COPD) was
men. Information on the area of origin (rural or urban) and occupa- common with 320 000 cases, contributing to the total 236 cases of
tion/profession was obtained for 45 (90%) patients. Of these patients, invasive aspergillosis (IA; 0.2% of all SFIs) together with patients
30 (89%) of them resided in rural areas, and their occupation with cancer, transplantation (Tx) and stay in intensive care unit
involved land-related activities, especially agriculture. The affected (ICU). Using low average literature data for candidemia rate (5/
anatomical regions were the lower respiratory tract, lymph nodes, 100 000) we estimated 360 candidemia cases annually in Serbia.
oropharyngeal mucosa and skin. All cases diagnosed from oropha- Based on 1124 cases of pulmonary tuberculosis, the annual inci-
ryngeal mucosa lesions had associated pulmonary involvement.This dence of new chronic pulmonary aspergillosis (CPA) cases was esti-
type of information, however, was not available for the remaining mated at 371 cases, and prevalence 856 cases. Out of 1692 HIV
13 patients. The city of origin was available for 39 patients. Approxi- patients, Pneumocystis pneumonia was estimated at 26, while oeso-
mately 60% of these patients were from Caxias do Sul. The remain- phageal candidiasis and cryptococcal meningitis were estimated at
ing patients were from Vacaria, Campestre da Serra, Bento 212 and 2 cases, respectively. Data on fungal keratitis and tinea
Gonc
alves, Pinto Bandeira, Nova Prata, Veran opolis, Antonio Prado, capitis was not available.
Vila Ip^e, Flores da Cunha, Nova Petr opolis, Gramado, Carlos Barbosa Conclusion Based on these estimates, approximately 1.52%
and Garibaldi. (n = 109 283) of the population suffer from a SFIs every year in Ser-
Conclusion We found that the Southern of Brazil is particularly bia. Crude rate for SFIs in Serbia is 1517.8 new cases per 100 000
affected by tropical neglected infectious diseases, and PCM is part of population per year. This study serves as a basis but further epidemi-
this list. Paracoccidioidomycosis is a mycosis with an important ological studies are necessary to better categorize, validate and
number of reported incidences in cities of the north-eastern part of extend this estimation.
the Rio Grande do Sul. This study highlights the need to include
PCM as a differential diagnosis of respiratory infection, especially in
patients with oropharyngeal lesions and in rural males.
Table 1. The annual burden of serious fungal infections (SFIs) in
Serbia

ª 2015 The Authors

128 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P215 P216

Molecular epidemiological investigation of clustered Tinea capitis epidemiology in Mali

Pneumocystis jirovecii pneumonias in four recently-liver S. Ranque,1 O. Coulibally,1 A. K. Kone,2 S. Niare-Doumbo,3
transplant patients S. Goita,3 J. Gaudart,1 A. A. Djimde,4 R. Piarroux,1
E. Bailly,1 M. Dominique,1 R. A. Thepault,2 C. Franck-Martel,1 O. K. Doumbo3 and M. A. Thera3
F. Morio,3 J. Chandenier1 and G. Desoubeaux1 1
Aix Marseille University, Marseille, France; 2University of
1
C.H.R.U. Tours, Tours, France; 2Universite Franc
ois Rabelais, Bamako, Bamako, Mali; 3University des Sciences, des Techniques
Tours, France and 3CHU de Nantes, Nantes, France et Technologies de Bamako, Bamako, Mali and 4Universite des
Sciences, des Techniques et Technologies de Bamako, Bamako,
Objectives The main purpose of this study was to look for epidemio- Mali
logical links between four cases of Pneumocystis jirovecii pneumonia
(PCP) in four recently-liver transplant patients, which occurred over Objectives Dermatophytosis, and particularly the subtype tinea capi-
a 5-month period at Tours University Hospital, France. tis, is common among African children; however, the risk factors
Method Bronchial-alveolar lavage fluids (BALFs), which had previ- associated with this condition are poorly understood. This study
ously been found positive for P. jirovecii were processed for Multi aimed at testing whether climate influences the epidemiology of
Loci Sequence Typing (MLST) analysis. DNA was extracted from each dermatophytosis.
sample using the Qiagen DNA mini kit (Qiagen). Molecular typing Methods Cross-sectional surveys were conducted in public primary
was performed at three loci (SOD, CYB and mt26s) as previously schools located in three distinct eco-climatic zones, namely the Sahe-
described (Maitte et al., 2013). Sequences were analyzed using the lian, Sudanian and Sudano-Guinean eco-climatic zones in Mali.
Codon Code Alignersoftware. Nine P. jirovecii positive BALFs, col- Results Among 590 children (mean age 9.7 years) the overall clini-
lected from unrelated patients, five from HIV-positive individuals, and cal prevalence of tinea capitis was 39.3%. Tinea capitis prevalence
four from other immunocompromised patients, were investigated by was 59.5% in the Sudano-Guinean zone, 41.6% in the Sudanian
the same method. Phylogenetic relationships between the isolates zone and 17% in the Sahelian eco-climatic zone. Two anthropophilic
were assessed using the MEGA 6.06 software (Center for Evolution- dermatophytes species, Trichophyton soudanense and Microsporum
ary Medicine and Informatics). audouinii, were the most frequent species associated with tinea capitis
Results Our MLST approach resulted in allowing a reliable discrimi- among primary schoolchildren in Mali. As expected, Microsporum
nation of all the P. jirovecii strains found in unrelated immunocom- audouinii was isolated primarily from large and/or microsporic
promised patients (one single genotype per patient). The allelic lesions, while Trichophyton soudanense was primarily isolated from tri-
profiles were shown to be the same as those previously reported, chophytic lesions. Multivariate logistic regression analysis showed
except for two strains that, to our knowledge, exhibited new Multi- that male gender (OR = 2.51; 95%CI [1.74–3.61], P<10-4) and resid-
Nucleotide Polymorphism (MNP) resulting from previously unknown ing in the Sudano-Guinean eco-climatic zone (OR = 7.45; 95%CI
combinations of mutations in position 279-516-838 of the CYB [4.63–11.99], P<10-4) were independently associated with an
gene. Three of the four P. jirovecii strains hosted in the liver trans- increased tinea capitis risk.
plant patients (14116074, 14113359 and 14111271) were success- Conclusion In this homogenous schoolchild population, our findings
fully sequenced and found to be closely related (bootstrap-score: highlight an increased tinea capitis risk with increasing climate
100%, sequence similitude: 100%) (Figure 1). humidity. Further studies should aim at investigating the role of cli-
Conclusion Our results highlighted the importance of genotyping matic factors in the geographic distribution of dermatophyte species.
during investigation of PCP outbreaks. They confirmed high phyloge-
netic diversity in P. jirovecii strains amongst the unrelated immuno-
compromised patients in the cohort. Besides, we have described for
the first time a very rare example of clustered PCP in liver transplant
P217
patients. Epidemiological investigation showed that concerned
patients were found to be hospitalized at concomitant days, with sev-
eral overlapping dates of outpatient consultations. These findings Outcomes from In Utero, Neonatal and Pediatric
support the evidence of nosocomial PCP outbreak. Environmental Mold Exposure
I. H. Grant1 and J. D. Thrasher2
1
New York Medical College, Tarrytown, USA and 2NA, Citrus
Heights, CA, USA

Objectives Both visible mold growth and malodorous vapors and

are recognized health hazard markers for indoor contamination with
toxin-producing, infectious fungi. Molds such as Aspergillus/Penicil-
lium, Chaetomium and Stachybotrys produce myriad harmful prod-
ucts including carcinogenic, neurotoxic, immunosuppressant
mycotoxins (MCTs), toxic debris, particulates, & volatile compounds.
Despite disinfection, environmental products may remain hazardous.
Both cell-mediated immunity and metabolic detoxification are
poorly developed in early life when hazardous microbial and toxin
exposures are expected to cause more harm.
Methods Environmental spore air trap, microscopy, fungal-specific
PCR, ERMI analysis, MCT measurement, medical examination, analy-
sis: exposure timing/severity, mold spp, obstetrical/pediatric symptom
chronology outcomes, fungal quantitative IGG titers, MCT excretion.
Figure 1. Phylogenetic dendrogram Results Of 66 children (25M, 38 F, 3 embryos. exposed 1st trimester
- 18 yrs.), disease severity was inversely related to age exposed: 3/9
(30%) 1st-trimester died, 3 life-threatening, 17 severe, 28 moderate,
14 mild outcomes with later exposure-ages.
Visible and malodorous microbial exposures contributed the most:
50% total to visible mold and 38% to odors, paralleling disease

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 129
Poster Presentations

severity (44% for mild, 46% moderately ill, 38% severe life-threaten- Conclusions Outcome severity correlated with early hazardous
ing). Chemical and pesticide exposures appeared to contribute to life- exposure.
threatening outcomes. Visible mold and odorous exposures predominated in all disease
Of 18 Inutero exposures (9 1st-trimester, 1 2nd-trimester, 2 3 d, 6 severity levels.
indeterminate), 3 miscarried, 9 were complicated by prematurity or Chemicals and pesticides appear to contribute to severe/life-threat-
organ damage (2 hydronephrosis, 2 congenital heart abnormalities, ening outcomes.
1 microcephalus), 3 had abnormal placentas, 10 birth complications Environmental in utero exposure to toxin-producing, infectious
and 5 neonatal complications. All 6 (100%) breast-fed developed molds and/or hazardous products appears to cause in-utero/congeni-
problems: 4 projectile vomiting, 4 unexplained body dermatitis, 1 tal defects, placental abnormalities.
intussusception with refractory painful anorectal rash, 2 choking Exposure timing, severity correlated with pregnancy complications,
(one apneic requiring ICU treatment for respiratory arrest/pulmonary congenital/placental defects, postnatal & lactation outcomes.
hemorrhage), 3 black oro-nasal drainage, 1 neurologically-impaired Epidemiological studies are urgently needed.
oropharyngeal function from ingesting Ochratoxin(Ochra)/Tri-
chothecene (Trichos) contaminated breastmilk.
The most affected neonates were from 2 mothers in different
homes with severe 1st-trimester exposures: both developed congenital P218
heart disease (PDO, VSD), cognitive/neurological impairment, projec-
tile vomiting and choking breastfeeding. One was hospitalized within
2 weeks with respiratory arrest & pulmonary hemorrhage. The other Candida spp. causing vulvovaginal candidiasis (VVC) in an
developed black oronasal discharge and lost oropharyngeal motor outpatient clinic in Bilbao, Spain
function lactating Ochra/Trichos contaminated milk. Vomiting and I. Arrieta-Aguirre,1 A. Diez,1 I. Fern~andez de Larrinoa,1
discharge resolved when lactation was stopped. M. J. Zabala,2 A. del Val2 and M. D. Moragues1
All breastfed by mother #1 from the Trichos-contaminated home 1
developed rashes. Of 2 not breastfed, one developed chronic fatigue, University of the Basque Country, Bilbao, Spain and
2
speech delay, and cognitive, matching fungal IGGs, urinary Ochra/ Osakidetza, Bilbao, Spain
Trichos excretion impairment after playing extensively in the base-
ment. The other, minimally-exposed to that home was normal. Objective To identify the species of Candida isolated in a population
Overall, skin rashes 42(67%), throat inflammation 42 (67%), nasal of women suffering VVC and (2) to evaluate their susceptibility to
inflammation 40 (63%) (30 PND, 17 nosebleeds), pulmonary 35 Fluconazole (FLC) and Clotrimazole (CLT).
(56%), cough 31 (49%), 25 neurologic & GI (nausea/vomiting 19 Materials And Methods A total of 121 vaginal swabs were ana-
(30%)) cognitive impairment (memory, ADD, Apraxia, learning dis- lyzed from women with symptoms resembling Candida vulvovaginitis.
ability) 22 (33%), developmental delay 9 (14%). Specimens were inoculated onto Candida CHROMogenic Agar
(Conda) to ensure detection of mixed infections. Yeasts were identi-
fied based on carbohydrate assimilation (ID32C-Biomerieux) and/or
by PCR. Susceptibility tests to CLT and FCL, used normally to treat
VVC in our clinic, were performed using the broth microdilution
method according to CLSI guidelines. Since there are no MIC limit
ranges established for CLT microdilution test, we followed the recom-
mendations of other authors [1, 2].
Results Seventy-five (62%) patients were diagnosed with VVC: acute
VVC (17/75; 22.7%) and Recurrent VVC (RVVC) (58/75–77.3%).
Among the VVC patients 42 were pregnant (56%), and 11 (14.66%)
of them had previous treatment episodes with antibacterials, 7
(9.33%) had allergic rhinitis, 6 (8%) were taking hormonal contra-
ceptives and 1 was participating in an experimental vaccination trial
against VVC. There were 46 (38%) negative cases for VVC, and
among them 19 (41.3%) were diagnosed with bacterial vaginosis:
Gardnerella vaginallis (13/19–68.42%), Streptococcus agalactiae (3/19–
Figure 1. Average Age exposed versus Disease Severity. 15.78%) and enterobacteria (3/19–15.78%) were the aetiological
agents. One additional patient was diagnosed with Trichomonas
vaginalis.
With respect to species distribution Candida albicans was the most
frequently isolated yeast (94.66%), followed by Candida parapsilosis
(2.66%), Candida tropicalis (1.33%) and Candida glabrata (1.33%).
Only one patient with VVC showed a mixed fungal infection.
Both azoles tested showed good in vitro activity, only 7 strains
showed MIC values for CLT ≥ 0.5 lg ml
1 (resistance breakpoint
value according to Pelletier et al. [2]), and 3 of them also showed
high FLC MIC (>64 lg ml
1).
Conclusions Although many authors claim that there is a trend
towards the emergence of non-Candida albicans Candida spp., in our
group of study we still found that C. albicans was the most commonly
isolated yeast, followed by far by C. parapsilosis. Regarding antifungal
susceptibility, CLT remains a good topical therapy option.
References Gamarra S., Morano S., Dudiuk C., Mancilla E., Nardın

M.E., de los Angeles E. et al. Epidemiology and antifungal susceptibili-
ties of yeast causing vulvovaginitis in a teaching hospital. Myco-
pathologia (2014) 178:251–258.
Pelletier R., Peter J., Antin C., Gonzalez C., Wood L., and Walsh
T.J. Emergence of resistance of Candida albicans to Clotrimazole in
Human Immunodeficiency Virus-Infected children: In vitro and clini-
cal correlations. J. Clin. Microbiol. (2000) 38(4):1563–1568.
Figure 2. Exposure Type versus Disease Severity.

ª 2015 The Authors

130 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P219 corridors and rooms when ICW occurred. For G2, the RR of A. fumi-
gatus inside was of 2.50 (N = 39, P = 0.26). When windows and
door of wards were opened, A. fumigatus was more present in G2
Evaluation of A. fumigatus aerocontamination dispersion with a RR of 1.86 (N = 51, P = 0.30). As openings were caulked, no
indoor and outdoor hospital blocks during demolition RR could be calculated for A. fumigatus colonisation in G1.
works at hospital Conclusion Although G1 was located just in front of ICW, it was
S. L. Loeffert,1 M. P. Gustin,2 A. Reville,2 P. Cassier,1 the less contaminated by A. fumigatus. On contrary, G2 unit which
C. Dananche,1 T. Benet,1 M. Perraud3 and P. Vanhems1 was further of the ICW has highest A. fumigation colonization. These
1 preliminary findings have already permitted to applied corrective
Edouard Herriot Hospital, Lyon, France; 2Claude Bernard I actions to improve professional practices and develop relevant meth-
University, Lyon, France and 3Edouard Herriot Hospital, Lyon, ods to control and evaluate the microbiological risk during construc-
france, Lyon, France tion works in health institutions.

Objectives A large University Hospital, Edouard Herriot composed of

32 blocks (France) is involved in a large modernization program
which consisted in the demolition of an entire central medical block. P220
Spores may be suspended specially during demolition near immuno-
compromised patients who are at risk to develop severe invasive
aspergillosis (IA). The aim of the study was to evaluate the variability Prevalence and population structure of buccal and
of Airborne A. fumigatus conidia contamination indoor and outdoor cutaneous Candida spp. in a pediatric population
hospital ward during demolition work and to better understand coni- O. Petrini,1 C. Fragoso-Corti,2 A. Crenna,2 F. Schoenherr2 and
dia spread. V. Pezzoli3
Methods Since the beginning of the demolition site in February 1
POLE Pharma consulting, Breganzona, Switzerland; 2Applied
2015, we started a daily environmental survey of fungal loads in 8
Microbiology Lab, Belinzona, Switzerland and 3Ente Ospedaliero
wards of the hospital located all around the demolition site: 4 inten-
sive care units (ICU), 1 unit of kidney and liver transplantation and Cantonale, Lugano, Switzerland
3 other medical wards. We choose to present data from one ICU (G1)
located very closely at south of demolition work site and the unit of Objectives Candida spp are the most frequent fungal commensals on
kidney and liver transplantation (G2) located further at north of the the buccal mucosa and according to some authors their overall
site (Figure 1). The environmental survey consisted in sampling air prevalence in healthy carriers may be as high as 50%. Recent inves-
twice a day, indoor and outdoor, by impaction onto Sabouraud Chlo- tigations in our labs have suggested that this may be an overestimate
ramphenicol agar supplemented with antibiotic chloramphenicol with large geographic variations. This study was thus initiated to
plates (Biomerieux) using a an agar impact sampler (Air-Ideal investigate the prevalence, population structure and antifungal resis-
90 mm). Plates were incubated 48 h at 37°C. After incubation A. tance profile of Candida spp. on the buccal mucosae and on the skin
fumigatus were identified. Data were entered into an Excel database. of healthy children.
Pairwise associations between presence of A. fumigatus aerocontami- Methods 100 healthy children who attended the paediatric depart-
nation and binary variables were evaluated by Fisher exact test. ment of the local hospital for routine checks were included in this
P < 0.05 was considered statistically significant. investigation. Samples from the oral cavity and from skin were col-
Results Between February and May 2015, a mean of 112 air sam- lected using sterile swabs and incubated at 30°C during 48 h on
ples were realised weekly. A. fumigatus was detected outside and chromID Candida Agar medium. Candida species were identified by
inside of the 8 wards with a mean load of respectively 9.24 CFU/m3 MALDI-TOF MS and when needed the identification was confirmed
(n = 278, SD: 23.27) and of 3.94 CFU/m3 (n = 365, SD: 27.99). by sequencing of the ITS region. Population structure analysis was
Outside each monitored wards, A. fumigatus was more frequent out- carried out by microsatellite-based typing using sets of genetic mark-
side when major intensive construction work (ICW) were taking ers with various discriminatory powers. The in vitro susceptibility
place with a relative risk (RR) of 1.22 (n = 206, P = 0.29). Inside profile to azoles, polyenes, and echinocandins was done by
sampled wards same trends was observed, A. fumigatus was more microdilution.
present inside during ICW with a RR of 1.29 (n = 269, P = 0.24). Results and Conclusion C. albicans was the most common species
For G1, the RR of A. fumigatus was of 0.37 (n = 40, P = 0.04) into isolated. C. parapsilosis was also present at lower frequency. The pop-
ulations were predominantly clonal with few or no genetic variation
within each idividual and we could not detect any single widespread
genotype.

P221

Estimated burden of serious fungal infections in Pakistan

J. Q. Farooqi,1 K. Jabeen,1 D. Denning,2 M. Sajjad3 and A. Zafar1
1
Aga Khan University, Karachi, Pakistan; 2The University of
Manchester and National Aspergillosis Centre, Manchester,
United Kingdom and 3University Hospital of South Manchester
Wythenshawe Hospital, Manchester, United Kingdom

Introduction Due to absence of population based surveillance, true-

burden of fungal infection in Pakistan is unknown. Laboratory and
institutional based reports from the country highlight the existence
of these infections in many patient settings. Similarly high risk popu-
lations for fungal infections (TB, diabetes, chronic respiratory dis-
eases, asthma, cancer, transplant, HIV) in the country are also
prevalent. The situation is more complicated due to poor diagnostic
Figure 1. Map of Edouard Herriot Hospital, France capabilities of most laboratories in Pakistan, lack of antimicrobial

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 131
Poster Presentations

stewardship and poor infection control practices. So we estimate here P222

the burden of fungal infections in Pakistan to highlight the public
health significance of these infections.
Methods Population estimates was referred from WHO population The burden of serious fungal diseases in Madagascar
statistics 2013 data. Morbidity data of conditions at risk were deter- D. Denning,1 V. Razanamparany2 and R. A. Rakotoarivelo3
mined by reviewing national and global data. TB rates were taken 1
The University of Manchester and National Aspergillosis Centre,
from WHO TB global report 2014. Asthma prevalence data was Manchester, United Kingdom; 2Pasteur Institute, Antananarivo,
taken from global asthma prevalence rates. COPD rates were taken Madagascar and 3CHU Tambohobe Fianarantsoa, Fianarantsoa,
from BREATHE study. HIV rates were taken from UNAIDS 2013 Madagascar
report. Cancer rates were determined from GLOBOCAN 2012. Dia-
betes rates were taken from national data on NCD by Heartfile. Pub-
lished data from Pakistan reporting fungal infections rates in general Background In Africa, few data on the burden of fungal disease are
and specific populations was reviewed and used when applicable. published, but Madagascar is an exception. We have estimated the
Burden of cryptococcal and Pneumocystis infection in HIV was deter- burden of serious fungal infections in Madagscar.
mined using data from Baqi S et al. and Luxmi S et al. Estimations Methods We searched for existing data and estimated the incidence
for allergic bronchopulmonay aspergillosis (ABPA), severa asthma and prevalence of fungal diseases based on the populations at risk
with fungal sensitization (SAFS) and chronic pulmonary aspergillosis and available epidemiological data. Data were derived from the
(CPA) in asthma, sarcoidosis and TB patients were done using World Health Organization (WHO), The Joint United Nations Pro-
methodologies by Denning DW et al. and Agarwal R et al. Mucormy- gramme on HIV/AIDS (UNAIDS) and national and regional published
cosis and candidaemia rates were calculated using estimated burden reports. When no data existed, risk populations were used to estimate
in India by Chakrabarti A et al. Mycetoma burden was computed frequencies of fungal infections, using previously described methodol-
using Indian data from global burden of mycetoma by van de Sande ogy by LIFE.
et al. Recurrent vulvovaginitis burden was estimated using assump- Results The population of Madagascar is around to 19 625 000;
tion that it affects 6% of women between 15 and 55 years. Invasive 43% are children, and 5% are women >60. Recurrent vulvovaginal
pulmonary aspergillosis (IPA) rates were determined in COPD candidiasis (>4 episodes/year) is estimated to occur in 2 703/
patients assuming it affects 3.9% of admitted COPD patients, as in 100 000 females. Oesophageal candidiasis is estimated to affect
China (Xu et al.). 6.700 HIV patients, given the ~25 000 with <350 CD4 cells not on
Results In the 182 million people in Pakistan, 34% are children ART. An estimated 875 and 1250 patients develop cryptococcosis
(<15 years) and 6% are over the age of 60 years. An estimated and PCP each year, with an overall HIV mortality of 3900 people.
3 216 331 (1.77%) people are affected by a serious fungal infection, An estimated 2.625 have chronic pulmonary aspergillosis after pul-
omitting all cutaneous infection, oral candidiasis and allergic fungal monary tuberculosis 67% of the total burden. ABPA and SAFS were
sinusitis, which we could not estimate. Compared with other coun- estimated in 66.6/100 000 and 87.8/100 000 respectively, in
tries the rates of candidaemia (21/100 000) and mucormycosis (14/ ~522 000 adult asthmatics. A population estimate of chromoblasto-
100 000) are estimated to be very high, and based on data from mycosis was completed 1996 and an rate of 14/100 000 was found
India. Chronic pulmonary aspergillosis rates are estimated to be high (2745 cases). Tinea capitis is estimated to affect a minimum of 10%
(25.6/100 000) because of the high TB burden, with only 20% of of children, over 840 000 cases.
cases not related to TB (i.e. 500 due to sarcoidosis). Fungal keratitis Conclusions The present study indicates that around to 6%
is also problematic in Pakistan with an estimated 24 260 cases (1 155 796) of the population is affected by a serious fungal infec-
(13.3/100 000). Large numbers of COPD patients probably develop tion, predominently recurrent VVC in women and tinea capitis in
invasive aspergillosis (using the 3.9% rate in hospitalizedpatient from children. Further epidemiological studies are needed to validate and
China) (9846 annually). extend these estimates.
Conclusions Pakistan probably has a high rate of certain life or
sight-threatening fungal infections. The unavailability of lipid prepa-
ration of amphotericin, flucytosine and posacozole and limited avail- Table 1
ability of voriconazole prevents good outcomes from many of these
infections.

Table 1. Burden of serious fungal infections in Pakistan

ª 2015 The Authors

132 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P223 Table 1

Epidemiological profile of candidemias in a university

hospital in the Midwest Region of Brazil: 2006–2014
R. C. Hahn
UFMT, Cuiaba, Brazil

Objectives This study aimed to determine the epidemiology and risk

factors associated bloodstream infection caused by Candida spp. in a
university hospital in the city of Cuiaba.
Methods For this, we used a cross-sectional study with laboratorial
exams and secondary data analysis between 2006 and 2014. Eighty-
seven episodes of candidemia were identified. adults) (Yo et al., 2012). It was assumed that no transplantation
Results The prevalence of candidemia among patients hospitalized was done and COPD data was not available.
in the period was 2.5 per 1000 admissions (87/33975) and the Results 56% of the 22.98 million population (2011) are adults, of
attributed mortality rate was 1.3 per 1000 admissions (44/33975). whom only 5% are over 60 years. The estimated HIV prevalence is
The prevalence of candidemia among patients with hospital infection estimated at 1.6 million, with 590 000 with CD4 counts <350.
was 30.5 per 1000 admissions (87/2850) and the attributed mortal- Cryptococcal meningitis incidence was estimated to be estimated
ity rate was 15.4 per 1000 admissions (44/2850). The coefficient of 14 815 cases, Pneumocystis pneumonia 22 125 cases and oesopha-
lethality was 50.6% (44/87). Most patients were females (n = 51; geal candidiasis cases 84 250 annually. Following the approximately
58.6%). The average age was 30 years (SD = 29; 95%CI = 24–37) 40 000 pulmonary TB survivors annually, the annual incidence of
and the mean hospital stay was 53 days (SD = 45; 95%CI = 43–62). chronic pulmonary aspergillosis was estimated at 2596 and the
Overall, 83.9% of patients were prescribed antifungal drugs: ampho- prevalence at 8181, assuming a 15% annual mortality. ABPA com-
tericin B (53.0%) and fluconazole (38.5%) were the most common. plicating asthma is estimated at 14 967 (2.5% rate) and severe
Candida parapsilosis was the most common etiological agent (n = 36; asthma with fungal sensitisation at 19 756, both in adults only. In
41.4%), followed by C. albicans (n = 34; 39.1%); C. tropicalis (n = 13; 2006/7 an estimated 10% of children had tinea capiitis - an esti-
14.9%), C. glabrata (n = 2; 2.3%) and C. guilliermondii (n = 2; 2.3%). mated 1 007 250 children affected. Among fertile women (15–
The distribution of cases by hospital sector showed higher concentra- 50 years), an estimated 303 976 suffer from recurrent vulvovaginal
tions in adult and neonatal intensive care units (75.9%). The follow- candidiasis. No data on fungal keratitis or histoplasmosis were
ing risk factors were identified: central venous catheter (n = 73; available.
83.9%); ventilatory support (n = 64; 73.6%); parenteral nutrition Conclusion Approximately 1 744 000 (7.6%) people in Mozam-
(n = 62; 71.3%); H2 blockers (n = 47; 54.0%); corticosteroids bique are estimated to suffer from a serious fungal infections in any
(n = 41; 47.1%); previous surgery (n = 36; 41.4%) and neutropenia year. Tinea capitis in children recurrent Candida vulvovaginitis are
(n = 24; 27.6%). In newborns, low birth weight was detected in the most common, with HIV fungal complications being the next
51.5% (17/33) and prematurity in 48.5% (16/33). The following more common. Prospective epidemiology studies are required to vali-
risk factors significantly associated with the incidence of death in the date these estimates.
univariate analysis: ventilatory support (OR = 5.6; 95% CI=1.8–
17.0; P = 0.001); parenteral nutrition (OR = 6.8; 95% CI = 2.2–
20.5; P = 0.0003); central venous catheter (OR = 4.7; 95%
CI = 1.2–18.2; P = 0.02). P225
Conclusion Candida non-albicans accounted for 60.9% of the cau-
sative agents of candidemia in this population, this fact reinforces the
importance of considering local mycological profile of bloodstream Burden of serious fungal infections in Greece
infections for the therapeutic management. M. N. Gamaletsou,1 D. Denning2 and N. Sipsas3
1
The National Aspergillosis Centre, University Hospital of South
Manchester, Manchester, United Kingdom; 2The University of
Manchester and National Aspergillosis Centre, Manchester,
P224 United Kingdom and 3University of Athens, Athens, Greece

An estimation of the burden of serious fungal infections Introduction Data on the epidemiology of serious fungal infection
in Mozambique in Greece are scarce. The aim of this work is to calculate the burden
D. Denning1 and J. Sacarial2 of serious fungal infections in Greece.
1 Methods A thorough literature search for papers reporting epidemi-
The University of Manchester and National Aspergillosis Centre, ological data on serious fungal infections in Greece was performed.
Manchester, United Kingdom and 2Faculdade de Medicina, For fungal infections where no Greek data existed, we used a struc-
Maputo, Mozambique tured set of assumptions to estimate their burden, based on specific
population with risk factors for fungal infection, such as immunosup-
Introduction Mozambique is a Portuguese-speaking, sub-Saharan pression, chronic disease, and surgical procedures. Population statis-
African country with a high HIV and high TB burden. Few studies tics were derived from the Greek Statistics Authority and the latest
have reported on the burden of fungal disease in Mozambique and (2011) population census. Data on HIV/AIDS (2014) were obtained
we have attempted to summarise these and estimate the total burden from the Hellenic Centre for Disease Control and Prevention; data for
of the serious fungal diseases there, to assist determine public health transplantations (2012) from the National Organization for Trans-
and research priorities. plantation; data for tuberculosis from the World Health Organization
Methods Published epidemiology papers reporting fungal infection (2012); data on COPD, cystic fibrosis, asthma, abdominal surgeries
rates from Mozambique were identified. Where no data existed, we form the relevant scientific Greek societies; data on the number of
used specific populations at risk and fungal infection frequencies in critical care beds and hospital admissions from the Greek Ministry of
those populations to estimate national incidence or prevalence, Health.
depending on the condition. Population statistics (2011), pulmonary Results 85.5% of the 10.8 M population are adults, 53% are
TB rates (2013) and HIV data were derived from WHO and UNAIDS. women, 27.4% women are over 60 years and 40.3% over 50; 27%
We assumed the rate of asthma was the same as Malawi (4.67% in of population are ≥60 years old. Estimates are: 243 567 Greek

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 133
Poster Presentations

Etest, interpreted according to CLSI breakpoints. Samples from a

single patient, positive for the same fungi, in an interval of up to
30 days, were considered as belonging to the same fungemia
episode.
Results Our lab has performed 206 007 blood cultures in 2014,
20 751 (10.07%) of which were positive. A total of 867 vials were
positive for fungi, which corresponded to 357 fungemia episodes.
More than 56% (490) of these samples were collected from periph-
eral sites, while the others were collected from different types of vas-
cular catheters and devices. Regarding the identified fungi, 97%
(347) of them were yeasts. Most episodes were related to Candida spe-
cies: 104 C. albicans (29.1%), 87 C. parapsilosis complex (24.3%), 61
Figure 1. Fungal infections in Greece C. tropicalis (17.1%), 42 C. glabrata (11.8%); 25 were due to other
Candida species. Of notice, there were 9 episodes of Saccharomyces
cerevisiae and 8 of Trichosporon sp fungemia, all of them in critically
ill and/or immunosuppressed patients. Mean time of detection was
women get recurrent vaginal thrush (≥4x/year). Of 14 434 HIV posi- 38.5 h, ranging from 25 (C. tropicalis) to 55 (C. glabrata) among
tive patients, 1732 are not receiving ARVs. Oral candidiasis is esti- most frequent species. Thirty-two episodes were diagnosed only by
mated to occur at least once in 90% of patients with CD4 cell counts means of specific fungi blood culture (BACTEC Myco/F lytic vial),
<200 lL and oesophageal candidiasis in 20%. We estimated that and they corresponded to 25% of episodes related to Trichosporon sp,
there are 1.296 cases of oral candidiasis and 353 cases of esophageal 16.6% of C. krusei, 10.5% of C. albicans, 8.1% of C. tropicalis, 7.1% of
candidiasis. 2 (2%) of 107 new AIDS cases each year develop crypto- C. glabrata and 6.9% of C. parapsilosis complex episodes. Regarding
coccal meningitis. Annual incidence of Pneumocystis pneumonia is susceptibility, C. parapsilosis and C. tropicalis exhibited the largest pro-
0.26 cases/100 000 in HIV+ patients, 28 cases. Of the 500 cases of portion of Fluconazole resistant isolates (7 each, 8.0% and 11.4%,
TB in 2012, 45 with AIDS, it is estimated that 22 new cases of respectively).
chronic pulmonary aspergillosis (CPA) occurred and that the 5-year Conclusion Fungi are important agents of blood stream infection,
period prevalence is 347 cases (assuming 15% annual mortality). As especially in critically ill and immunosuppressed patients, a large pro-
CPA occurs in multiple other conditions including COPD a preva- portion of which using invasive vascular devices. Although Candida
lence of 1 388 cases is estimated. Estimates of asthma prevalence in albicans remains the most frequent yeast when species are considered
adults are about 9% and assuming 2.5% of asthmatics have ABPA, separetly (29.1%), altogether, non-albicans species were the most fre-
20 805 patients with ABPA are likely and 26 500 with SAFS. quent cause of fungemia (60.2%). Aerobic blood culture vials have
Assuming the rate of candidemia in Greece is 5.0/100 000 popula- good sensitivity for most yeasts, but associated use of fungi specific
tion there are 541 cases. We have estimated 81 cases of post-surgical vials may improve positivity for some species, mainly C. krusei and
candida peritonitis (~60 000 abdominal surgeries/year). Invasive non-Candida yeasts. Resistance to azoles requires attention in non-al-
aspergillosis in immunocompromised patients is estimated at 1125 bicans Candida.
patients annually including 85 cases in intensive care. For mucormy-
cosis, there were 12 cases annually, and 56 cases of tinea capitis.
Conclusion According to our calculations ~ 300 000 (2.72%) peo-
ple in Greece suffer from fungal infections each year. This is the first
attempt to determine the burden of fungal disease in Greece and pro- P227
vides a crude estimate on its impact on public health.
Changing epidemiology of the Non-albicans Candida-
group in clinical specimen in a 10 year- single centre
survey in Austria 2005 to 2014
P226 M. Hell, C. Bernhofer, L. Baskova, D. Achleitner and M. Bender
Paracelsus Medical University, Salzburg, Austria
Epidemiology of fungi isolated in blood cultures after
identification by MALDI-ToF implementation: Brazilian Objectives and background Non-albicans species are reported to
experience in 2014 being emerging yeasts in clincal relevant specimen within the Can-
C. Lázari,1 A. J. Silva,2 D. F. Zamarrenho2 and J. L. M. Sampaio3 dida-group over the last years. Therefore we set up a 10-survey with
1
Sa~o Paulo University, Sa~o Paulo, Brazil; 2Grupo Fleury, Sa~o the aim to identify the most emerging Non-albicans- species.
Paulo, Brazil and 3Sao Paulo University, Sa~o Paulo, Brazil Methods We did a retrospective data analysis of our surveillance
data comparing two time intervalls: 2005–2009 versus 2010–2014.
We screened all relevant clinical isolates at species - level according
Objective Matrix-assisted laser desorption/ionization - Time of Flight to routine diagnostic procedures of our clinical microbiology labora-
(MALDI-Tof) technique has changed microbiology laboratory’s rou- tory. We ranked the top 10 species of each time period and com-
tine, due to its improved accuracy and faster performance to identify pared the proven species-frequency.
microorganisms. Many authors have validated its usefulness to fungi Results 5685 single patient isolates from the first period were com-
identification. In order to provide additional epidemiologic data about pared to 6977 isolates of the second period both including C. albi-
fungemia in Brazil, once no data about private hospitals is currently cans and Non-albicans species.
available, we analyzed the results of all blood cultures performed in a Top 5 non- albicans species were Candida glabrata, C. tropicalis, C.
large private laboratory of S~ ao Paulo after MALDI-Tof full routine parapsilosis, C. krusei and C. lusitaniae in both groups and time peri-
implementation. ods. But there was a move of Candida parapsilosis from the third to
Methods We retrospectively analyzed the results data bank of a the second most frequent position (197/3.5%–238/3.4%) with a
Brazilian private laboratory located in the Southeastern city of S~ ao stable overall percentage. A significant rise was found in Candida
Paulo, the largest and most populated city of Latin America. This lab glabrata (485/8.5% to 777/11.2%) and a decrease in Candida tropi-
analyzes samples from more than 10 hospitals in the main capitals calis (278/4.5% to 236/3.4%).
of Brazil (about 2000 beds). All blood cultures performed during Conclusion The emergence of Candida glabrata and furthermore of
2014 were included, and their results were evaluated regarding posi- Candida parapsilosis which became the second frequent species
tivity, specie identification, site of sample collection, type of positive within the non-albicans-group needs further analysis regarding risk-
vial and, when available, isolate susceptibility. All isolates were iden- factors of patients and antifungal therapeutic consequences.
tified using MALDI-Tof, and the method for susceptibility tests was

ª 2015 The Authors

134 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P228 P229

A 3-year survey of dermatophytosis in Belgium Fungal contamination in one hotel room: Does carpet
R. Sacheli,1 R. Darfouf,1 S. Pateet,2 H. Graide,1 C. Adjetey,1 coating in the floor enhance fungal contamination?
K. Lagrou3 and M. P. Hayette1 C. Viegas,1 T. Faria,1 E. Carolino,1 R. F. P. Sabino2 and S. Viegas1
1
1
CHU Liege, Liege, Belgium; 2University Hospitals Leuven, Environment & Health RG - Lisbon School of Health Technology
Leuven, Belgium and 3UZ Leuven, Leuven, Belgium - Polytechnic Insti, Lisbon, Portugal and 2National Institute of
Health Dr. Ricardo Jorge, Lisboa, Portugal
Objectives Dermatophytosis refers to superficial fungal infections of
keratinized tissues caused by keratinophilic dermatophytes. They are Objectives Several materials used indoors can contribute to enhance
the most common cause of superficial fungal infections worldwide. fungal contamination inside the building and taking this in consider-
Epidemiological studies regarding dermatophyte infections have been ation was developed a study intending to know the carpet influence
conducted in several countries and differences in the incidence and when used in the floor of a hotel room.
in etiological agents have been reported for different geographical Methods Twelve air samples of 250L (six in a room with carpet and
areas. That is why national surveillance of circulating strains caus- six more in a room with wood floor) were collected through an
ing dermatophytosis is crucial. The Belgian National Reference Cen- impaction method with a flow rate of 140 L/min onto malt extract
ter (NRC) for Mycoses conducted a survey on dermatophytes strains agar (MEA) supplemented with chloramphenicol (0.05%), using the
circulating from 2012 to 2014. The present study was performed to Millipore air Tester (Millipore), during cleaning activities. Outdoor
assess the profile of dermatophytosis and to identify the species sample was also performed to be used as a reference. Surface samples
involved. from floor and desks, taken at the same time, were collected by the
Methods The Belgian NRC for Mycosis collected 9138 strains swabbing method. All the collected samples were incubated at 27°C
between January 2012 and December 2014. The isolates were cul- for 5 to 7 days. After laboratory processing and incubation of the
tured from patients clinically suspected for fungal infections of skin, collected samples, quantitative (colony-forming units - CFU/m3)
hair and nails. Isolates were sent by Belgian laboratories to the two results were obtained. Besides fungal contamination, we also assessed
labs of the Belgian NRC (UZ Leuven and CHU of Liege) in order to particulate matter contamination in both rooms during the same
identify the fungus or to confirm the identification. All isolates cul- cleaning tasks. Two metrics were considered: particle mass concen-
tured from patients of UZ Leuven and CHU of Liege were also tration (PMC) - measured in 5 different sizes (PM0.5; PM1; PM2.5;
included. Fungal identification was performed by microscopy after PM5; PM10) - and particle number concentration (PNC) based on
subculture and in case of doubtful identifications, by ITS sequencing. results given in six different diameters sizes, namely; 0.3 lm,
Results Among the 9138 samples, 3587 were identified as dermato- 0.5 lm, 1 lm, 2.5 lm, 5 lm and 10 lm.
phytes. Trichophyton. rubrum (T. rubrum) was the most prevalent spe- Results The air fungal load in the room with carpet ranged from
cies accounting for 56 17% (n = 2015) of the infections from all 1 CFU.m-3 to 68 CFU.m-3 and in the room without carpet from
sources, followed by T. mentagrophytes complex (2183%, n = 783). 1 CFU.m-3 to 112 CFU.m-3. From both rooms, only one air sample
The other main etiological agents of dermatophytosis recorded in this of the one without carpet presented higher counts than the outdoors.
study in descending order of prevalence were M. audouinii (n = 303), Regarding surfaces, the room with carpet presented contamination in
M. canis (n = 120), T. violaceum (n = 112), T. tonsurans (n = 95), T. only one sample (1x104 CFU.m-2) and the room without carpet pre-
soudanense (n = 66), M. praecox (n = 59) and E. floccosum (n = 14) sented statistically significant differences from the carpeted room,
Our data also reveal the predominance of anthropophilic species with the first one having higher counts that ranged from
causing tinea capitis especially M. audouinii responsible for 36 49% 10x104 CFU.m-2 to 115x104 CFU.m-2. The most prevalent fungal
(n = 163/448) of hair/scalp infection. Trichophyton violaceum, rarely genera were the same in the air of both rooms (Penicillium sp. 40.7%
observed in our country, is increasing in frequency these last years - 12.3% and Cladosporium sp. 43.5% - 55.4%). In the analyzed sur-
as 12.8% (n = 57) of the reported cases of tinea capitis are due to faces, isolates belonging to Aspergillus fumigatus complex were the
this species. The retrospective evaluation of data collected also shows only fungi found in the carpeted room, whereas in the other room
that zoophilic strains as M. canis well represented in the past epi- we found Penicllium sp. (63.6%) and Aspergillus sp. (13.6%) as the
demiology of tinea capitis, is decreasing in frequency accounting for most frequent genera. In the case of particles the room with carpet
only 7.2% (n = 32) of clinical cases. Finally, our data confirm the obtained significant higher values for both metrics (PMC and PNC),
high prevalence of T. rubrum commonly observed in Europe as causal showing that carpet may has influence on particles’ contamination
agent of onychomycosis (70.9%, n = 1603) followed by T. mentagro- of the room.
phytes complex (20.9%, n = 455). T. rubrum and T. mentagrophytes Conclusion Taking in account the obtained results, and contrarily
complex are also responsible for the majority of skin infections as they to what was initially expected, carpeted floor does not seem to harbor
represent respectively 40% (n = 386) and 24.75% (n = 239) of skin higher fungal contamination. Nevertheless, when different particles
dermatophytosis during the study period. parameters are analyzed, an increase in PMC and PNC was observed
Conclusions The present work has provided recent data on the in room with carpet, compared to the room without carpet. More
prevalence of several dermatophytes species circulating in Belgium. research had to be made to describe cleaning measures and charac-
Such data is critical for the establishment of therapeutic strategies terize cleaning products since it seems that can have an influence in
and measures for prevention and control of dermatophytes infections. fungal contamination, besides carpet presence.
Our study confirms the predominance of T. rubrum followed by T.
mentagrophytes in the Belgian population but also highlights the
emergence of new anthropophilic species such as M. audouinii and T.
violaceum as causative agents of tinea capitis in children, in relation
with African immigration.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 135
Poster Presentations

P230 P231

Identification of Aspergillus cryptic species in hospital Epidemiology of Candida pelliculosa, Candida utilis and
environment Candida fabianii in the Czech Republic
~es,1
R. F. P. Sabino,1 C. Viegas,2 C. Verıssimo,1 H. Simo L. Svobodova,1 P. Lyskova2 and P. Hamal3
J. C. Brand~ao, C. Martins, K. V. Clemons and D. A. Stevens4
1 3 4 1
Palacky University Olomouc, Olomouc, Czech Republic;
1 2
National Institute of Health Dr. Ricardo Jorge, Lisboa, Portugal; Institute of Health in Usti nad Labem, Prague, Czech Republic
2
Environment & Health RG - Lisbon School of Health Technology and 3Faculty of Medicine and Dentistry, Palacky University,
- Polytechnic Insti, Lisbon, Portugal; 3North Lisbon Hospital Olomouc, Czech Republic
Centre, EPE, Lisbon, Portugal and 4California Institute for Medical
Research & Stanford University, San Jose, Stanford, USA Objectives Clinical yeast isolates belonging to Candida pelliculosa,
Candida utilis and Candida fabianii are difficult to differentiate in a rou-
Objectives Invasive aspergillosis is a fungal infection caused by tine mycology laboratory using standard commercial biochemical
Aspergillus spp. affecting mainly immunocompromised patients. The kits. During the past decade, the use of invasive procedures and
mortality rate can reach 85%. Aspergillus identification should be administration of antimicrobial agents and new technologies such as
based on molecular methods as there are species morphologically bone marrow transplants or chemotherapy have resulted in an
similar but distinct at the molecular level (cryptic species), with vari- increase in the incidence of non-albicans infections such as these
able antifungal susceptibility profiles. Recent studies have shown that three species. The aims of this study were (1) to determine the preva-
cryptic Aspergillus species can cause approximately 10% of the cases lence of C. pelliculosa, C. utilis and C. fabianii in clinical samples col-
of invasive aspergillosis. Since Aspergillus infections in immunocom- lected from 10 Czech hospitals using the biochemical kit ID 32C
promised patients are mainly nosocomial, knowledge of the fungal (bioMerieux) and MALDI-TOF mass spectrometry (Bruker Daltonics)
epidemiology found in hospital environments would have an impor- and (2) to compare their minimum inhibitory concentrations (MICs)
tant role in controlling the development of aspergillosis. Therefore, for 9 antifungals from various aspects.
selected hospital wards, housing patients at higher risk to develop Methods Two hundred and fifty-seven clinical yeast isolates were
invasive fungal infections, were screened in order to understand the included in this study. Type strains of C. pelliculosa (CBS 605), C. uti-
epidemiology and distribution of Aspergillus, especially regarding the lis (CBS 841) and C. fabianii (CBS 5481) were added as controls. The
presence of cryptic species. whole group was first identified using ID 32C and then by the
Methods During a 1-year period, four seasonal samplings, i.e., air MALDI-TOF MS system. Identification of each strain was repeated in
and hard surface, were performed. A total of 101 air samples and 99 triplicate by both methods. In case of questionable identification, a
surface samples were collected from the Hematology, Oncology, and sequencing analysis was performed. MICs of the systemic antifungals
Intensive Care Unit (ICU) wards of a Portuguese Central Hospital. amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole,
Aspergillus isolates were plated for growth as single colonies on malt anidulafungin, micafungin, caspofungin and flucytosine were deter-
extract agar with chloramphenicol to check the colony purity. These mined using the commercially available colorimetric broth dilution
isolates were identified on the basis of microscopic morphology and panels Sensititre YeastOne (TREK Diagnostic Systems). The results
through the use of molecular tools. Genomic DNA was prepared from were compared with respect to patients’ age, gender and site of infec-
each isolate and the sequencing of the Internal Transcribed Spacers tion and location of the hospital.
(ITS) regions, specifically the ITS1 and ITS2 non-coding regions Results From a total number of 257 clinical isolates, 179 were bio-
flanking the 5.8S rDNA was used to determine the species complex, chemically identified as C. pelliculosa, 77 as C. utilisand 1 as Williopsis
whereas b-tubulin and calmodulin sequencing was done to achieve saturnus. The type strain of C. fabianii was determined as C. pellicu-
the correct species identification. losa. Using MALDI-TOF MS confirmed with sequencing, 228 isolates
Results 548 environmental fungal isolates were obtained. Of these, were identified as C. fabianii (88.7%), 21 as C. pelliculosa (8.2%), 6 as
Aspergillus was the most frequently isolated genus (19.7%) and from C. utilis (2.3%) and 2 as Ogataea polymorpha (0.8%). The mean MICs
the total of Aspergillus isolates, 75 were screened for cryptic species (lg ml
1) after 48 h were as follows: amphotericin B 0.77 (range,
detection. The remaining Aspergillus isolates were not speciated either 0.12–2.0), anidulafungin 0.14 (0.015–2.0), micafungin 0.08
because viability was lost, contaminants were impossible to eliminate (0.008–1.0),caspofungin 1.17 (0.03–8.0), 5-flucytosine 7.61 (0.06–
or amplification remained unsuccessful. Six misidentifications at the 64.0),posaconazole 1.20 (0.03–8.0),voriconazole 0.21 (0.008–8.0),
species-complex level (based on morphology) were resolved by ITS itraconazole 1.0 (0.03–16.0)and fluconazole 8.57 (0.5–256.0).The
sequencing. This methodology allowed the identification of ten differ- highest mean MICs were found in yeasts isolated from blood cultures
ent sections within the Aspergillus genus: Versicolores (N = 20), Nigri and central venous catheters. No significant differences in MICs
(N = 11), Flavi (N = 10), Circumdati (N = 10), Fumigati (N = 8), Usti between genders were found.
(N = 4), Terrei (N = 4), Nidulantes (N = 4), Aspergilli (N = 3) and Cre- Conclusion This study showed that, unlike routine biochemical
mei (N = 1). From those, 25 different Aspergillus species were identi- identification, MALDI-TOF MS found C. fabianii to be most prevalent
fied by b-tubulin and calmodulin sequencing, and a high percentage in clinical samples as compared with the other studied species. The
of cryptic species (i.e., not sensu stricto) was found (59%). Sections absence of C. fabianii in databases of commonly used commercial bio-
Usti, Versicolores and Circumdati harbored the highest proportion of chemical kits for yeasts including ID 32C leads to misidentification of
cryptic species [100% (4/4), 95% (19/20) and 90% (9/10), this species. In addition, some strains resistant to two or more anti-
respectively]. fungals were detected.
Conclusion The high number of cryptic species found raises con- Acknowledgement This work was supported by the Palacky
cerns about the possible reduced susceptibility to antifungals of hospi- University Olomouc grant project no. IGA_LF_2015_021.
tal environmental Aspergillus isolates. These data reinforce the
importance of hospital air and surface monitoring, mainly in
immunocompromised patients’ wards. The knowledge of the Aspergil-
lus epidemiology in hospital settings and the use of routine suscepti-
bility testing will allow the monitoring of the rate of resistance in
environmental strains and its potential impact on initial antifungal
choices and therapeutic outcome.

ª 2015 The Authors

136 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P232 Table 1

Occupational exposure to fungi and particles in poultry

and bovine slaughterhouses
C. Viegas, T. Faria, A. Camb~ao, A. Catarino, A. Moreno and
S. Viegas
Environment & Health RG - Lisbon School of Health Technology -
Polytechnic Insti, Lisbon, Portugal

Objectives Dust released due to slaughterhouses activities is largely

organic and can be contaminated with bacteria, fungi and endotoxin
which may represent a health hazard upon inhalation. In addition
several respiratory symptoms have been reported among the exposed
workers. Taking this information in consideration a study was per-
formed intending to assess the fungal contamination and particles
exposure in poultry and bovine slaughterhouses.
Methods Air samples between 100 and 250 L were collected
through an impaction method, while surface samples, taken at the
same time, were collected by the swabbing method and subject to identification and antifungal susceptibility testing with the inclusion
further macro and microscopic observations. Besides fungal contami- of Ambisome in the panel of antifungal agents to study its activity
nation, we also assessed to exposure particles. Two metrics were con- against Mucorales for the first time.
sidered: particle mass concentration (PMC) - measured in 5 different Methods From January 2007 to May 2015, 17 patients had proven
sizes (PM0.5; PM1; PM2.5; PM5; PM10) - and particle number con- (n = 10) or probable (n = 7) mucormycosis according to the EORTC/
centration (PNC) based on results given in six different diameters MSG revised definitions. The available colonies morphologically iden-
sizes, namely; 0.3 lm, 0.5 lm, 1 lm, 2.5 lm, 5 lm and 10 lm. tified as Mucorales were stored and revived to perform molecular
Results Fungal quantification in the poultry slaughterhouse air ran- identification and antifungal susceptibility testing to amphotericin B,
ged from 10 to 970 CFU.m-3 while in bovine slaughterhouse ranged Ambisome, itraconazole, and posaconazole by using CLSI M38-A2
from 10 to 36 CFU.m-3. Surfaces presented results that ranged from methodology. The incidence of the infection during the study period
0 to 10x104 CFU.m-2 in the poultry slaughterhouse and from 0 to was calculated and compared with that found in the same hospital
90x104 CFU.m-2 in the other slaughterhouse. In poultry slaughter- from 1988 to 2006 (M. Torres-Narbona. Med Mycol 2008, 46, 225–
house were detected eight species/genera in the air, being the most 230).
found Scopulariopsis candida (59.5%) and Penicillium sp. (32.8%). Only Results The patients with mucormycosis were mainly male (82%)
Mucor genera was isolated in surface. In the bovine slaughterhouse and mostly admitted to onco/haematology (47%) or ICUs (35%); they
were found eight species/genera in the air being the most isolated had hematological malignancies (59%), or surgical/other invasive
Penicillium sp. (80.8%). Regarding surfaces, five different fungal spe- iatrogenic wounds (41%). In 8 patients the lower respiratory tract
cies/genera were detected and the most prevalent were Scopulariopsis was affected and the remaining 9 patients had skin and soft tissue
brumpti (40.0%) and A. terreus complex (30.0%). infections [3 and 4 patients with surgical or other iatrogenic wound
It was possible to detect the task in each slaughterhouse that was infections, respectively]. Antifungal agents within the month previ-
responsible for higher particles exposure and with priority for preven- ous to diagnosis were administered in 65% of patients, most of them
tive and protective actions: in poultry slaughterhouse (4.669 mg.m- without activity against Mucorales. Patients with proven mucormy-
3) was the handling of the live birds when hanging them in the line cosis had almost exclusively skin and superficial involvement
for slaughtering and in bovine slaughterhouse was the animal cut- whereas patients with probable mucormycosis had lower respiratory
ting without skin (0.456 mg.m-3). Results showed that exposure tract infection (microbiological findings and CT scan). The species
occurs in both units but with significantly higher values in poultry found were Lichtheimia ramosa (n = 4), Lichtheimia corymbifera
slaughterhouse (P < 0.0001). (n = 3), Cunninghamella bertholletiae (n = 3), Rhizomucor pusillus
Conclusion Air fungal load was higher in poultry slaughterhouse (n = 2), Mucor circinelloides (n = 2), Rhizopus arrhizus (n = 2), and
and this can represent higher risk since particle have an important Saksenaea vasiformis (n = 1). In patients in whom several isolates
role as a carrier of fungi and their metabolites for workers respiratory were studied only one species was found. Isolates from patients with
system. Due to species/genera identification we have to consider co- mucormycosis or colonization (n = 39/34, respectively) were studied
exposure not only to fungi and particles, but also to several mycotox- for antifungal susceptibility testing. Ambisome showed the highest
ins, since species with toxigenic potential were isolated. activity followed by posaconazole, amphotericin B, and itraconazole
(Table, P < 0.001). Ambisome was more active than amphotericin B
for all species with the exception of Cunningamella bertholletiae
(P < 0.05). All patients received antifungal treatment, mainly Ambi-
P233 some (88%). In patients in whom surgery was part of the therapy
(59%) mortality was significantly lower (20% vs. 68%, P = 0.015).
The incidence of the infection, in cases per 100 000 hospital admis-
Mucormycosis in a large Spanish hospital from 2007 to sions, increased from 1.2 (1988–2006) to 3.7 (2007–2015)
2015: incidence, epidemiology, and etiological agents (P = 0.004).
P. Escribano,1 T. Pel
aez,1 A. Vena,1 L. Gorostidi,1 L. J. Marcos- Conclusions We detected an increase in the incidence of mucormy-
2
~
Zambrano, P. Munoz,1 E. Bouza2 and J. Guinea2 cosis during the study period compared to that reported from 1988
to 2006. Only half of cased involved the respiratory tract and no
1
Hospital General Universitario Gregorio Maran~on, Madrid, Spain
cases of rhino cerebral mucormycosis were detected. None of the
and 2Gregorio Maran~on Hospital, Madrid, Spain patients had diabetes as underlying condition and although hemato-
logical malignancy was the most common underlying disease, iatro-
Objectives Mucormycosis, although sporadic, is a devastating dis- genic skin trauma was a major predisposing condition. Lichtheimia
ease with a purportedly increasing incidence and changing epidemi- spp. and Cunninghamella bertholletiae were the main species involved.
ology. We reviewed all cases of mucormycosis diagnosed in patients Ambisome was the agent showing the highest antifungal activity
admitted to our hospital during an 8.5 year-period in order to against Mucorales, supporting the need to start early treatment with
describe its incidence and epidemiology. We also obtained an accu- agents showing anti-Mucoralean activity.
rate knowledge of the etiological agents by performing molecular

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 137
Poster Presentations

P234 had the disease confirmed by mycological culture. There were col-
lected 160 samples in the household environments, selecting objects/
sites with the help of owners. Many of these sites were of human-ani-
FungiScope - Global Emerging Fungal Infection Registry mal common use, but they were categorized as follows: 58 samples
D. Seidel, L. Duran Graeff, K. Wahlers, M. J. G. T. Vehreschild, from sites that were of predominant use by the owners (sofas, chairs,
€ hler, F. Mu
P. Ko €ller, J. J. Vehreschild and O. A. Cornely beds, sheets); 48 samples from sites of predominant use by the ani-
mals (animals’ houses, cribs, toys, feeders); 40 samples from general
University Hospital Cologne, Cologne, Germany
flooring (carpet, wood) and 14 samples from other animals that were
in contact with the infected animals.Samples from the animals and
Objectives Number of rare invasive fungal infection (IFI) are rising the environment were seeded on Mycosel agar(BBL-BD) and incu-
worldwide due to increasing patient population at risk. To broaden bated at 25° C for up to 4 weeks. Colonies were submitted to micro-
knowledge on epidemiology of emerging IFD, FungiScope a global culture technique and identified by their macro-and-microscopic
registry has been initiated. Currently, partners from 60 countries characteristics. Dermatophytes were found in 37% (20/54) of the
contribute cases that eventually help determining clinical patterns, samples originated in sick animals. Microsporum canis was the most
improve diagnostic procedures and therapeutic regimens. prevalent dermatophyte (17/20–85%) and it was isolated from dogs
Methods FungiScope uses web-based data capture accessible and cats; Trichophyton quinckeanum (2/20–10%) was isolated from
through www.fungiscope.net. For case enrollment, cultural, histologi- guinea pigs, and Microsporum gypseum from a dog (1/20–5%). Der-
cal, antigen or molecular evidence on the occurrence of infection matophytes were found in 65% (13/20) of the surveyed homes.
with non-endemic fungi is required. Data collected include demo- Among the samples taken from household environments, 29% (47/
graphics, underlying conditions, immunosuppressive medication, 160) were positive, with dermatophytes isolated in 24% (14/58) of
clinical signs and symptoms, sites of infection, diagnostic tests, patho- the sites/objects of predominant use by owners, 37.5% (18/48) from
gen identification, antifungal treatment and outcome. Clinical isolates sites/objects of prevalent animal use, 25% (10/40) from floors and
are collected for centralized identification, molecular analyses and 35%(5/14) from animals that had some contact with infected ani-
exchange between collaborators. mals. The same species of dermatophytes previously detected in ani-
Results To date, 429 cases have been captured. Mucorales mals with lesions were isolated in homes: M. canis, T. quinckeanum
(n = 196; 45.7%), Fusarium spp. (n = 65; 15.2%), yeasts (n = 54; and M. gypseum. Viable fungal spores were found in the home envi-
12.6%), and dematiaceae (n = 48; 11.2%) are the most frequently ronment weeks after sick animals began treatment with antifungal
registered pathogens. Chemotherapy (n = 195; 45.5%) and stem cell drugs and presented healing of the lesions. Sick animals were sources
transplantation for hematological malignancy (n = 102; 23.8%) were of infection for other animals and humans, and it was possible verify
the predominant risk factors, followed by intensive care (n = 95; intra- and inter-species cross-infection in the same residence. Ani-
22.1%), diabetes mellitus (n = 84; 19.6%), and chronic renal disease mals that showed no lesions, but from which dermatophytes had
(n = 35; 8.2%). For 20 cases (4.7%) no risk factor was identified. been isolated, were considered asymptomatic carriers, and they also
Major sites of infection included lung (n = 214; 49.9%), paranasal represent sources of infection, helping to spread the fungus in the
sinuses (n = 79; 18.4%), blood stream (n = 77; 17.9%), and deep soft household environment. Nowadays the contact between humans and
tissue (n = 65; 15.2%). Disseminated infection (n = 72, 16.8%) was pets is becoming more intimate, with greater sharing of the same
mostly associated with lung (n = 51, 70.8%), blood stream (n = 31, space in a home, making dermatophytosis important in medical/vet-
43.1%) and CNS involvement (n = 24, 33.3%). For 212 (52.6%) erinary clinics and in public health as a whole.
patients, complete or partial response to treatment of IFD was docu-
mented. All-cause-mortality and mortality attributable to IFD was
45.2% and 34%, respectively.
Conclusion The clinical relevance of emerging IFD is increasing. P236
FungiScope is a vividly expanding network that attained increasing
interest throughout the years. In a short time period, a wide variety
of cases has been collected that provide a comprehensive view on the A multiple gene genealogical strain typing approach for
epidemiology and clinical presentation of rare IFI. assessing the genetic diversity of a clinical collection of
Aspergillus flavus and its antifungal drug susceptibility
R. Ahmad Raees,1 S. M. Rudramurthy,2 A. K. Ghosh,1 A. Das,2
N. K. Panda,1 A. Gupta,1 S. C. Varma2 and A. Chakrabarti2
P235 1
PGIMER, Chandigarh, India and 2Postgraduate Institute for
Medical Education and Research, Chandigarh, India
Presence of dermatophytes in infected pets and their
household environment Objective To determine the genetic diversity and antifungal drug
J. J. A. Neves,1 A. O. Paulino,2 R. G. Vieira,3 M. C. C. Ramos,4 susceptibility of a clinical collection of Aspergillus flavus.
E. K. Nishida2 and S. D. Coutinho1 Methods Six housekeeping genes (Acetamidase, Polygalacturonase,
1
Paulista University - UNIP, Sa~o Paulo, Brazil; 2Centro Veterinario O-Methyltransferase, b-Tubulin, Acetate regulation and Calmodulin)
of 44 clinical strains of A. flavus were sequenced. Allele types (AT)
Sa~o Jose, Sa~o Paulo, Brazil; 3Africa Pet, Sa~o Paulo, Brazil and
were determined by alignment of individual loci of each strain in
4
Lab&Vet-Diagnostico e Consultoria Veterinaria, Sa~o Paulo, Brazil
Clustal X2. AT was assigned on the basis of single nucleotide poly-
morphism found in each locus. Sequence types (ST) were assigned to
Dermatophytes are important fungi for public health because they each strain based on allele type profile. The multilocus sequence data
are transmitted between animals and humans, causing zoonoses. was analysed in eBURST integrated in PHYLOViZ (Phylogenetic infer-
These fungi have been reported in dogs, cats, guinea pigs and rab- ence and data visualization for sequence based typing methods) and
bits, among other pets. They can be isolated from animals with or minimum spanning tree (MST) was constructed at single locus vari-
without lesions, which represent sources of infection for other ani- ant level (SLV-1). The Discriminatory Power (D) was calculated by
mals and humans. However, researches about their presence in employing Simpson’s Index of Diversity. The antifungal susceptibility
homes where animals live with humans are still scarce. The purpose to polyene amphotericin B, itraconazole, voriconazole, posaconazole,
of this study was to diagnose dermatophytosis in pets with lesions caspofungin, micafungin and anidulafungin was determined by broth
clinically consistent with this infection, and investigate the presence microdilution as per CLSI M38-A2 guideline.
of dermatophytes in their home environment. Samples from haircoat Results All the isolates were from the single centre isolated form
were collected from 54 pets of both genders and varying ages. Of patients with AFRS, fungal ball, invasive FRS, CNS aspergillosis, pul-
these samples, 36 were from dogs, 15 from cats, 2 from guinea pigs monary aspergillosis and keratitis. A total of 24 STs (A-X) were
and 1 from a rabbit. There were visited 20 homes of animals that found in 44 strains included in the study. The discriminatory power

ª 2015 The Authors

138 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

and can be applied for epidemiological investigations of outbreaks

caused due to this agent. The high MIC of amphotericin to A. flavus
should be considered before using this antifungal for treatment of
infections due to A. flavus.

P237

Study of consumption of antifungal drugs in a tertiary

hospital during 7 years
E. Belesiotou, V. Papadopoulou, V. Papandreou, P. Stathopoulou,
M. Vlachou, G. Chatzidimitriou, E. Perivolioti, M. Nepka,
E. Kraniotaki, K. Litsakis, Z. Psaroudaki and A. Argyropoulou
Evagelismos General Hospital, Athens, Greece
Figure 1. On the eBURST network,clinical source of the isolate is
integrated with the genotypes
Objectives Over the past years, the consumption of systemic anti-
fungal agents has been increased worldwide resulting in developing
antifungal drug resistance and causing a significant pharmaco eco-
nomic burden. Antifungal drugs are widely used not only for treat-
ment but for prevention (prophylaxis and empiric therapy) of fungal
infections. The role of appropriate use of antifungal drugs is very
important in this difficult time of economic crisis, in order to achieve
the best cilinical results in conjuction with the best handling of the
economic budget. The purpose of this study is the evaluation of the
total cost of the antifungal treatment and the study of the progressive
role of the usage of the antifungal drugs during the last 7 years in
our tertiary hospital.
Methods Consumption data for systemic antifungal drugs were
obtained through the hospital pharmacy from 2008 to 2014regard-
ing all the medical, surgical wards, intensive care and transplanta-
tion units (931 beds). The consumption of the antifungal agents was
estimated in accordance to the cost and the number of DDDs (Defined
Daily Doses). The consumption of antifungal agents was also esti-
mated during the period of 3 months. As well as the consumption
and the cost in each ward independently (internal medicine, surgical,
ongology, haematology, intensive care units) was calculated. We
used the WHO-ATC/DDD Index 13 for the calculation of DDDs for
antifungal drugs with systemic effect (ATC J02). The quantitation of
results was performed in the level of the active substance of the drug
and the conclusion was expressed in DDD/100 beds-days (DDDs/
100BD).
Figure 2. An eBURST tree drawn at the SLV level with lines con- Results According to the data, the finding results declared that the
necting different MLST genotypes cost of antifungal treatment is almost the half of the total cost of the
antimicrobial therapy and the 1/10 of the total pharmaceutical treat-
ment in our tertiary hospital. The use of antifungal agents was
increased during the last 7 years as these data were calculated with
of this typing method was 0.965. Sequence type I was assigned as DDDs, even the decrease of the cost of the drugs (reduction of the
the founder genotype. With the less stringent definition of 4/6 shared price due to financial crisis). The most often used antifungal drugs
alleles, 3 clonal complexes could be differentiated in the eBURST dia- were triazoles. There was not observed seasonal variation in antifun-
gram (Figure A) The most prevalent ST was the ‘M’ type which was gal drug therapy. The antifungal medication was greater in haemato-
found in 5 strains. Four of these isolates were from AFRS patients logical wards and intensive care units.
and one from pulmonary infection. However, no discernible differ- Conclusion The presented study gives an update status of the anti-
ence between isolates on the basis of clinical manifestations was fungal consumption in a teriary Greek hospital during the past years.
observed (Figure B). ST ‘S’ and ‘Q’ were the unique genotypes found We report an increased utilization of antifungal drugs despite the
only in invasive fungal rhinosinusitis and CNS aspergillosis, reduction in the cost of these drugs. The presented pharmaco-epi-
respectively. demiological data are very important for the initiation and imple-
For amphotericin B, most of the isolates exhibited elevated MICs of mentation of effective antifungal stewardship programmes, despite
8 lg/mL (4.08%) and 4 lg/mL (46.93%) with MIC50 and MIC90 of the variability of the prescribing drugs between the different medical
2.0 lg/ml
1 and 4.0 respectively. All the triazoles and echinocandins wards. They might serve as important initiating information for other
exhibited good in vitro activity against Aspergillus flavus. The MIC50, hospitals with similar structures in order to make practice the appro-
MIC90 andgeometric mean MICs of itraconazole, voriconazole and priate use of antifungal agents.
posaconazole were 0.25, 0.5, 0.22; 0.5, 1.0, 0.31 and 0.12, 0.25,
0.12 lg ml
1 respectively. The MEC50, MEC90 andgeometric mean
MECs of caspofungin, micafungin and anidulafungin were 0.06,
0.12, 0.06; 0.015, 0.02, 0.02 and 0.25, 0.25, 0.22 lg ml
1
respectively.
Conclusion The present study is an attempt to develop multi locus
sequencing approach to type A. flavus isolates. There was consider-
able genetic diversity in the clinical collection of A. flavus isolates.
The technique is highly discriminatory for typing of Aspergillus flavus

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 139
Poster Presentations

P238 38 persons were infected to mycoses of eye site that more frequent
risk are 0 -9 age group, student and most isolated agents were Can-
did and Fusarium.
Aspergillosis of the nose and paranasal sinuses: A review Conclusion Child age groups have most fungal infections of eye and
of 54 cases ear canal sites. That care of eye and ear canal for prevention of ill-
C. B. Severo,1 I. E. Cardoso,2 L. S. Guazzelli,3 F. M. Oliveira3 and ness for this age group strongly recommended.
L. C. Severo3 Key words keratomycosis, otomycosis, Epidemiology, Kermanshah
1
Universidade Federal de Cie^ncias da Saude de Porto Alegre,
Porto Alegre, Brazil; 2Universidade Federal do Rio Grande do Sul,
Porto Alegre, Brazil and 3Irmandade Santa Casa de Misericordia
de Porto Alegre, Porto Alegre, Brazil P240

Introduction Aspergillus species are considered opportunistic fungi Accessing occupational exposure to fungi in a cork
of increasing clinical importance. Information regarding extrapul- industry
monary involvement is scarce. C. Viegas,1 A. Clerigo,2 T. Faria,1 R. F. P. Sabino,3 C. Verıssimo,3
Objective The aim of this study was to isolate the different species of A. Quintal Gomes4 and S. Viegas1
Aspergillus in patients with rhinosinusitis. 1
Environment & Health RG - Lisbon School of Health Technology
Methods A retrospective study was conducted in a university hospi-
tal in Porto Alegre, Brazil (1986–2014). For mycological diagnoses,
- Polytechnic Insti, Lisbon, Portugal; 2Lisbon School of Health
paranasal tissue obtained at surgery was subjected to histopathology Technology - Polytechnic Institute of Lisbon, Lisbon, Portugal;
3
examination and sent for fungal cultures. National Institute of Health Dr. Ricardo Jorge, Lisboa, Portugal
Results Of the 54 samples analyzed, 34 the diagnosis was made by and 4Institute of Molecular Medicine, Faculty of Medicine of
direct examination and culture and in 19 patients, the diagnosis was Lisbon, Lisbon, Portugal
made exclusively by histology with the visualization of the Aspergillus
conidiophore. In one patient, the diagnosis was by direct fluorescent Objectives Different forms of fungal diseases affecting the nose and
antibody staining (Aspergillus and Mucor). The underlying causes of paranasal sinuses are recognized, including invasive and non-inva-
immunodeficiency were: six with transplantation (bone marrow, sive fungal rhinosinusitis. Penicillium glabrum complex is associated
three; lung, two; kidney, one) and two with hematological disease with respiratory diseases such as suberosis, a typical disease of cork
(bone narrow neoplasia, one; leukemia, two). In the present study, industry workers. In addition, Chrysonilia sitophila has been described
the clinical manifestations of rhinosinusitis aspergillosis were: aller- as causing occupational asthma, associated to prolonged exposure to
gic, 20; fungus balls, 20; and acute invasive, 14. The strains isolated high counts of spores. In this study we aimed to access fungal expo-
were: Aspergillus fumigatus, 14; A. flavus, six; A. niger, two; A. terreus, sure in workers from one cork industry through the mycological
one; A. fischeri, one; and Aspergillus sp., three. Two concomitant spe- analysis of their nasal exudate and the environmental fungal con-
cies of Aspergillus were observed in two patients: A. fumigatus and A. tamination of their surroundings as well.
flavus; and A. fumigatus and A. niger. In four patients, Aspergillus was Methods Nasal mucous samples from 127 workers were taken with
associated with other fungi: A. flavus and Fusarium, one; A. fumigatus sterilized cotton swabs. Parallel samples were taken from one nostril.
and Rhyzopus, one; A. flavus and Mucorales, one; and Aspergillus sp. The swabs were rotated against the internal anterior walls of the
and Mucorales, one. The most common strains of Aspergillus that are nostril and then placed in the provided transport tube. The obtained
responsible for paranasal sinus infections are A. fumigatus, A. flavus, swabs were then plated onto malt extract agar (MEA) supplemented
and A. niger. Conclusions Fungal infection of the nose and parana- with chloramphenicol (0.05%), and onto screening media to detect
sal sinuses is rare, although it has been reported more frequently in azole-resistant Aspergillus isolates. Regarding environmental sam-
recent years, it is important to report this vast series on the theme, pling, collections for conventional-based culture studies, were made
highlighting the main clinical, etiological and diagnostics findings, to through the collection of 50–100 L air samples from 5 indoor sam-
alert clinicians as this pathological condition. pling sites by the use of an impaction method. All the collected sam-
ples were incubated at 27 °C for 5 to 7 days and fungal obtained in
positive samples were identified according their morphological char-
acteristics. In addition to cultural methods, four environmental sam-
P239 ples collected from 250L were used to specifically identify the
Penicillium glabrum complex, by Real Time PCR.
Results Eighty workers (63.0%) presented contamination of their
Fungal infections of eye and ear sites in referral patients nose nostrilwith Chrysonilia sitophila, which number of colonies was
to medical mycology lab of special clinic of Kermanshah countless. Talaromyces sp. was another species that also presented a
University of medical sciences countless number of colonies in 3 of the workers. The third most fre-
A. Mikaeili, S. Nemati and F. Shaikhi quently found species/genus with very high colony forming units
School Of Medicine,Kermanshah University Of Medical Sciences, was Penicillium sp. (42.7%). Within the Aspergillus genus, the com-
Kermanshah, Iran plexes Fumigati, Circumdati, Versicolores and Candidi were isolated. No
azole-resistant Aspergillus isolates grew in the selective media used
(screened itraconazole and voriconazole resistance).
Objectives Keratomycoses and otomycoses have different risk fac- Regarding the environmental results obtained by culture-based
tors. This research designed to study of epidemiological parameters of methods, all samples also showed countless Chrysonilia sitophila colo-
fungal infections of eye and ear in referral patients to Kermanshah nies. DNA from the Penicillium glabrum complex was detected in three
medical mycology lab during 1993 - 2011. out of the four samples.
Methods this research is a descriptive study on referral infected Conclusion The fungal species identified in the collected nose swabs
patients to medical mycology lab of special clinic of Kermanshah were shown to be correlated with the results obtained in the environ-
University of medical sciences. In these study epidemiological param- ment. This approach allowed us to estimate the risk associated with
eters such as age, sex, job, infected season, anatomical site of infec- these tasks performance. Moreover, the cork industry is related to
tion, habitant place and diseases in all infected fungal infections of high dust contamination and this can promote exposure to fungi
eye and ear were collected. since dust particles can act as carriers of fungi to the worker’s nose.
Results in all admitted patients, 54 cases have mycoses in ear site, Assessment by molecular tools will ensure the specific targeting of
that more frequent risk are age group 0–9, student. And most iso- DNA from P. glabrum complex in worker0 s nose.
lated agents were Aspergillus and Dermatophytes.

ª 2015 The Authors

140 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P241 P243

Candida lusitaniae fungemia: report of three cases Malassezia japonica isolated from cutaneous microbiome
D. Kofteridis, A. Andrianaki, S. Karageorgos, S. Maraki, of free-living Golden-headed lion tamarins (Leontopithecus
A. Christidou, M. Plataki, N. Spernovasilis and G. Samonis chrysomelas)
University of Crete, Heraklion, Greece S. D. Coutinho,1 J. Marigo,2 M. G. Bueno,3 A. Pissinatti4 and
J. L. Cat~ao-Dias2
Introduction Candida lusitaniae is an uncommon cause of fungemia,
1
Paulista University - UNIP, Sa~o Paulo, Brazil; 2University of Sa~o
frequently resistant to amphotericin B, accounting for about 1% of Paulo, Sa~o Paulo, Brazil; 3Pri-Matas for Biodiversity Conservation
all candidemia cases, with the majority of them being documented in Institute, Rio de Janeiro, Brazil and 4Centro de Primatologia do
patients with hematological malignancies. Three cases of Candida lusi- Rio de Janeiro, Rio de Janeiro, Brazil
taniae fungemia in non hematological patients are described.
Cases presentation Candida lusitaniae fungemia occurred in three Malassezia sp is yeast found in the skin microbiome of domestic ani-
patients (a 22 year-old male, a 57 year-old female and a 14 week- mals and humans. However, when an imbalance with the host
old infant). The male had been operated due to severe traumatic occurs it can cause infections, particularly otitis and dermatitis. Data
brain injury due to traffic accident and was hospitalized for more about the presence of these yeasts in wildlife is still scarce, regarding
than a year, when C.lusitaniae was isolated from the blood. The both its role in the microbiome as well as in infectious processes. The
female had been operated due to uterine sarcoma (not on chemother- golden-headed lion tamarin (GHLT - Leontopithecus chrysomelas) is a
apy) and was admitted to the Intensive Care Unit (ICU) due to hem- species considered EN-Endangered by the Red List of Threatened Species-
orrhagic shock, while C.lusitaniae was isolated from blood 3 days World Conservation Union (IUCN), and appears on the National List of
later. The infant was admitted to the pediatric ICU due to septic Endangered Brazilian Fauna Species. A free-living exotic population
shock. All three had fever, central venous catheters, were intubated, of this species, which was introduced in a State Park in Rio de
receiving total parenteral alimentation and on prolonged antimicro- Janeiro, Brazil, was restrained and translocated to another area, in
bial treatment. After the C.lusitaniae isolation all patients received Bahia, where the species is endemic. The purpose of this study was
micafungin (150 mg od for adults and 10 mg od for the infant) for to investigate Malassezia spp. in the external ear canal and haircoat
14 days the infant, 20 the male and 10 the female. The latter died of GHLT, before the translocation, since there is no research about
due to hemorrhagic shock, while the two others were successfully the presence of these yeasts in such a broad sampling of non-human
treated and the fungus disappeared from their blood and did not primates. There were chemically restrained (ketamine-10 mg kg
1 +
recur during a long follow-up. Catheters were removed. All strains midazolam-0.3 mg kg
1) 199 animals: 102 males (51.3%) and 97
were susceptible to amphotericin B, 5- fluorocytosine, fluconazole, females (48.7%), 77 juveniles (38.7%) and 122 adults (61.3%).
voriconazole and echinocandins. There were collected 597 clinical samples: 398 of cerumen (66.7%)
Conclusion Although rare, C. lusitaniae is an important pathogen, and 199 of haircoat (33.3%). Otic samples were obtained by the
even in non strictly immunocompromised but “susceptible”patients. introduction of sterile swabs in the external ear canals (right and
Surgery, central venous catheters, parenteral alimentation, mechani- left) and from skin surface by rubbing sterile carpets on the haircoat.
cal ventilation, ICU stay and prolonged antimicrobial therapy must Samples were seeded on Sabouraud dextrose and Dixon agar; plates
increase the suspicion index for such a fungemia. were incubated at 32° C for up to 2 weeks. For molecular analysis,
RFLP was carried out after DNA was extracted and 26S rDNA was
amplified through PCR technique. Since all strains presented the
same pattern on RFLP, four strains were randomly selected and their
P242 PCR products were purified and submitted to bidirectional sequenc-
ing. The sequences obtained were compared to mycobank.org. Malas-
sezia sp was isolated from 76 animals (38.2%) and 87 samples
Mycotic Keratitis by Acremonium spp - Case Report (14.6%), with 26 from the cerumen (6.5%) and 61 from the haircoat
B. C. D. A. Zoppas, J. A. M. Vieira, O. E. Souza, C. B. Pissaia, (30.7% - statistically superior). Malassezia sp was isolated from
L. P. Conzatti and E. D. Giustina 43.1% (44/102) and 33.0% (32/97) of males and females, and from
University of Caxias do Sul, Caxias do Sul, Brazil 44.2% (34/77) and 34.4% (42/122) of juvenile and adult animals,
respectively, and there were no statistic differences related to gender
and age. All of the 87 isolates showed lipodependency. In PCR-RFLP
Fungal keratitis is the cornea infection with suppurative and ulcera- techniques all strains showed the same pattern of bands. Four strains
tive aspect. This can be a severe disease and difficult to be treated. submitted to sequencing provided 510pb large subunit (26S) riboso-
More than 60 species of fungi have been recognized like keratitis mal partial sequences that were 99.8% to 100% similar to Malassezia
agents depending on the geographic region. Acremonium is an japonica. Results confirmed that lipodependent Malassezia is part of
uncommon cause of cornea infections. A male patient, agriculturist, the skin microbiome of these animals. However, it is important to
with prior cornea transplantation went to a doctor because of pain notice that M. pachydermatis, which is considered the animals’ species
and redness in the ocular region. He had an ulcerative lesion that and the only non-lipodependent has not been isolated here. Malasse-
improved even after the first medical treatment, thus cornea re-trans- zia japonica has been documented in humans but not in animals,
plantation was necessary. The result of material culture was positive until today. Perhaps because of a closer phylogenetic proximity, the
for Acremonium spp. It chosed to systemic therapy with Ketoconazole fungal skin microbiome of these primates would be more similar to
and maintenance of primary topical therapy with Amphotericin B for that of humans than other domestic or wild mammals. It does not
2 months. Few cases were related on literature and most of them exclude the possibility of being different species.
occurred after procedures like Laser in situ keratomileusis (LASIK).
Fungal keratitis can be caused by filamentous fungi and yeast fungi
and each of these needs a specific treatment based on microbiology
analysis. The disease outcome depends of effective antifungals
medicine.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 141
Poster Presentations

P244 parapsilosis, Candida tropicalis and Trichophyton rubrum. Additionally,

for all antifungals tested in this study showed good activity against
these etiologic agents. Despite the limited number of positive samples
Cases of dermatomycoses in seniors Open University for dermatomycosis, this group of elderly, there was a higher occur-
students of Universidade Estadual de Maringa  rence of yeast than dermatophyte fungi.
J. Galletti, M. C. Gad^elha, F. F. Veiga, E. Guilhermetti,
P. S. Bonfim-Mendonc
a, E. S. Kioshima, T. I. E. Svidzinski and
M. Negri
Universidade Estadual de Maringa, Maringa, Brazil P245

Dermatomycoses are fungal infections which affect the skin, hair, Introduction of an Aspergillus PCR assay to the clinical
and nails. Epidemiological studies have showed that these dermato- mycology service in Iran
mycoses are the most common dermatological infections, which K. Diba,1 A. Namaki,2 H. Mirhendi3 and S. Rezaie3
affect around 20% of the world’s population. Immunosuppression, 1
Urmia University of Medical Sciences, Urmia, Iran; 2Arefian
diabetes and advanced age may increase the risk for developing these General Hospital, Urmia, Iran and 3Tehran University of Medical
infections that usually require extended treatments with high rate of Sciences, Tehran, Iran
relapse and/or reinfection. The Open University ‘Universidade Aberta
para a terceira idade’ (UNATI) is part of The Universidade Estadual de
Maring a (UEM) that provides to senior citizens higher education Objectives Aspergillus species are most abundant and widely dis-
opportunities. Thus, the aim of this study is related with a social pro- tributed in soil, water, air, seed and food. These species are associated
ject that evaluated the occurrence of dermatomycosis in senior citi- with allergic bronchopulmonary disease, mycotic keratitis, otomyco-
zens enrolled at the UNATI/UEM and performed antifungal sis, nasal sinusitis and invasive infection. In this study we developed
susceptibility testing of fungal isolated from these elderly. The stu- a PCR-Single Strand Conformational Polymorphism method to iden-
dents with suspected dermatomycosis were referred to the Teaching tify the most common Aspergillus species.
and Research Laboratory of Clinical Analysis (LEPAC), Division of Methods Our subjects included Aspergillus clinical isolates of an edu-
Mycology, UEM. The inclusion of elderly followed the rules of the cational hospital in Urmia, Iran, also some Aspergillus standard spe-
Research Ethics Committee of UEM and approved (no. 615.643/ cies which obtained from Japanese Collection of Microorganisms. All
2014). Fungi were isolated from skin and nail samples, using the Aspergillus isolates were identified by using the morphological (colo-
method of scraping and skin peeling and nail. The samples were clar- nies and microscopic) features. For the molecular identification, the
ified with 40% potassium hydroxide (KOH) plus Evans blue and were ITS2 region of rDNA gene (approximate length size: 330 bp) was
cultivated in Sabouraud Dextrose Agar dextrose agar and Mycosel. amplified in PCR. The PCR product was incubated at 95°C for 5 min
The fungi identification was performed using classic methods, includ- and then moved quickly into ice bath for an immediately quenching.
ing the macro and micromorphology analysis. The isolates were A vertical electrophoresis with 6%-12% Gradient Poly Acrylamide
maintained in a freeze-dried state in the Mycology Collection of the Gel was used full time cooling at 4°C.
LEPAC/UEM. In vitro antifungal susceptibility testing was performed Results As our result, some of tested Aspergillus species including A.
according to Clinical and Laboratory Standards Institute, document nidulans, A. fisheri, A. fumigatus and A. niger discriminated. SSCP
M27-A3 and M38-A2 to yeast and filamentous fungi, respectively. assay enabled us to identify above Aspergillus species within 8–12 h
The antifungal used were fluconazole, itraconazole, voriconazole, after overnight incubation.
amphotericin B and micafungin. A total of 19 senior citizens’ Conclusion It is concluded that Single Strand Conformational Poly-
UNATI/UEM with suspect of dermatomycosis was observed, 12 were morphism is a simple and rapid method for identification of some
women and seven were men, with mean age of 67 years old. It was medically important Aspergillus but we recommend this as a compli-
collected 28 samples of these elderly (71.43% from skin and 28.57% ment test with other molecular methods such as PCR-restriction frag-
from nail), 53.57% of these were positive for direct mycological ment length polymorphism to cover identification of more Aspergillus
examination and 28.57% for culture. Fungus isolated were Candida species.

P246

Epidemiological characters of Tinea barbae among

admitted patients of medical mycology lab of Kermanshah
University of medical sciences 1994 -2011
A. Mikaeili, H. Hashemi and N. Nazari
School Of Medicine,Kermanshah University Of Medical Sciences,
Kermanshah, Iran

Background and objectives superficial mycoses have different risk

factors. This research designed to study the epidemiological parame-
ters of Tinea barbae among admitted patients of medical mycology
lab of Kermanshah University of medical sciences 1994 -2011
Material and methods This research is a descriptive study on refer-
ral infected patients to medical mycology lab of special clinic of Ker-
manshah University of medical sciences. In this study
epidemiological parameters such as age, sex, job, and infected season,
anatomical site of infection, habitant place and diseases in all
infected fungal infections of face were collected.
Results in 2290 admitted patients, 780(34%) case have dermato-
phytosis, that more frequent age group are 20–29, and student are
most infected group, T. verrucosum more frequent agents of
dermatophytosis.

ª 2015 The Authors

142 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Conclusion In this disease active age group, males, and hot month Methods A retrospective study was conducted on samples examined
has frequent infections. So skin care for prevention of disease most in Microbiology Department of Selcuk University Faculty of Medicine
recommended. in Konya/Turkey in 3 years’ time. Skin scrapings and hair and nail
Key word Tinea barbae, Epidemiology, Kermanshah samples were taken from clinically dermatophytosis suspected
patients. The samples were subjected to potassium hydroxide (KOH)
examination (direct microscopy) and were cultured in Sabouraud’s
dextrose and Mycobiotic agar. The isolates were identified by conven-
P247 tional methods.
Results During 3 years, among 3510 clinically suspected patients
1085 (31.0%) 633 (58.3%) male and 452 female (41.7%) were con-
Dermatophytosis in Iran: clinical, molecular firmed by direct examination and/or fungal culture. The most com-
characterization and in vitro antifungal susceptibility of mon clinical type of infection was tinea pedis (47.6%), followed by
dermatophytes isolates tinea unguium in feet (30.0%) and in hands (4.7%), tinea corporis
M. T. Hedayati,1 S. Ansari,1 K. Zomorodian,2 K. Pakshir,2 (8.4%), tinea inguinalis (4.5%), tinea manum (4.1%), tinea capitis
H. Badali,3 A. Rafiei4 and S. Seyedmousavi5 (0.3%).
1
Invasive Fungi Research Center, Mazandaran University of Fungal elements were examined in 985 (28%) of the 3510 direct
Medical Sciences, Sari, Iran; 2School of Medicine, Shiraz examination-ordered samples. Fungal culture was applied to 2060
samples and the dermatophytes were isolated from 321 (15.5%). Of
University of Medical Sciences, Shiraz, Iran; 3Mazandaran
the 321 culture positive samples fungal elements were examined in
University of Medical Sciences, Sari, Iran; 4Ahvaz Jundishapur 221 (10.7%) by direct examination. On the other hand, in 109
University of Medical Sciences, Ahwaz, Iran and 5Radboud (5.2%) samples fungal elements was observed but culture was nega-
University Medical Centre, Nijmegen, the Netherlands tive. The most common dermatophyte was Trichophyton rubrum
(84.5%), followed by Trichophyton mentagrophytes (12.7%), Trichophy-
Objective Dermatophytosis is a common mycotic infection of skin, ton tonsurans (2.5%), and Trichophyton terrestre (0.3%).
nail and hair caused by dermatophytes belong to three genera, i.e., Conclusion In our area tinea pedis was the predominant clinical
Trichophyton, Microsporum and Epidermophyton, associated with major type and T. rubrum was the most common determined dermatophyte.
public health concern worldwide. Various species of dermatophytes It is important clinically and epidemiologically to determine inci-
show significant differences in susceptibility to antifungals. In the dence, etiological agents and clinical types of dermatophytosis. This
present study we aimed the epidemiology of dermatophytosis and study also shows the need of performing both a direct examination
antifungal susceptibility of the largest series of molecularly identified and culture to improve sensitivity.
dermatophyte isolates from tropical regions of South of Iran.
Methods A total of 9485 clinically suspected patients to dermato-
phytosis from three southern provinces of Iran during October 2012
until April 2014 were involved in the study. The scraped skin sam- P249
ples from all patients were evaluated with potassium hydroxide
examination and culture on Sabouraud’s glucose agar supplemented
with chloramphenicol and cycloheximide. Restriction fragment Candidaemia in a Tertiary Referral London Hospital over
length polymorphism (RFLP) analysis and sequencing of the riboso- an eighteen year period
mal DNA (rDNA) Internal Transcribed Spacer (ITS) regions was per- E. Demertzi,1 N. Fenton,2 R. Lopez,2 B. Azadian2 and M. Petrou1
formed to differentiate dermatophyte isolates. In vitro antifungal 1
Imperial College, London, United Kingdom and 2Chelsea &
susceptibility tests were carried out according to the Clinical and Lab-
Westminster Hospital, London, United Kingdom
oratory Standards Institute (CLSI) M-38A2.
Results The prevalence of dermatophytosis was 11.7%. The most
common dermatophyte infections was tinea corporis (32%) followed Objectives Candidaemia remains the commonest fungal infection
by tinea pedis (21.2%) and tinea cruris (16.1%). Three hundred and worldwide and it is still one of the main causes of mortality and mor-
sixteen dermatophyte isolates originating from patients with der- bidity of all types of patients.We retrospectively reviewed all yeasts
matophytosis. Trichophyton interdigitale was the most prevalent spe- isolated from blood cultures in order to ascertain the prevalence of
cies (n = 156) followed by T. rubrum (n = 60), Epidermophyton species and their susceptibility to antifungals, the choice of antifungal
floccosum (n = 42), Microsporum canis (n = 29), Arthroderma ben- treatment and mortality rates, to compare the distribution in adults
hamiae (n = 17) and T. tonsurans (n = 12). In general, the MIC range and children and identify the medical specialties with the highest
of terbinafine (0.002 to 0.25 lg ml
1) was the most potent antifun- rate.
gal followed by itraconazole (0.004 to 0.5 lg ml
1), griseofulvin Methods The medical notes of patients with candidaemia in our
(0.125 to 8 lg ml
1) and fluconazole (4 to 128 lg ml
1). hospital between 1996 and 2013 were reviewed. We recorded
Conclusion Our results in comparison to previous reports in Iran patients’ demographics and clinical details such as parental nutrition,
have revealed a different epidemiological trends for Arthroderma ben- long line in situ, immune status, treatment and outcome. Candida
hamiae, which showedthe lowest susceptibility toterbinafine. isolates were identified by germ tube test and API or MALTI TOF in
the last 2 years. Susceptibility testing using the CLSI method started
with five and finished with nine antifungals.
Results A total of 234 patients with 240 episodes, 157 (17–
93 years) adults and 77 (1 day to 15 years) children were included.
P248 There was an even distribution for adults whereas there were 12%
more male than female cases among children. The specialties rank
Evaluation of Epidemiological and diagnostic features of order for adults was Intensive Care 35.7%, Surgery 27.4%, Internal
dermatophytosis in Selcuk University Hospital in 3 years’ Medicine 14.6% and HIV Medicine 12.1%, whereas for children
time Neonatal Intensive Care 71.4%, Surgery 11.7% and General Paedi-
D. Findik, Z. Koc, H. Turk Dagi and F. Ates atrics including Oncology 11.7%. In adults Candida albicans
accounted for 60.5%, C. glabrata for 19.1% and C. parapsilosis for
Selcuk University Faculty of Medicine, Konya, Turkey 9.6% whereas for children C. albicans was 62.3% followed by C. para-
psilosis 26% and C glabrata 6.5%. There were no isolates resistant to
Objectives To determine the incidence of dermatophytosis based on amphotericin B or any of the three echinocandins, anidulafungin,
clinical conditions and identification of fungus by direct microscopy caspofungin and micafungin. Only C. glabrata and C. krusei were
and culture methods. resistant to the four triazoles, fluconazole, itraconazole, posaconazole
and voriconazole. Elevated MICs of the three echinocandins against

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 143
Poster Presentations

all isolates of C. parapsilosis, C. lusitaniae and C. guilliermondii were very expensive and local guidelines have been put forward to guide
noticed. Flucytosine was very active against all isolates and in partic- their prescriptions particularly echinocandins in fever-driven, diagno-
ular C. glabrata. Liposomal amphotericin was the most frequent drug sis-driven and targeted treatment approaches. The search for deep
used followed by fluconazole and caspofungin and the overall crude tissue infection and organ involvement is of the upmost importance
mortality in adults was found to be 27.3% and in children 21%. and collaboration between the clinician and the microbiologist is cru-
Discussion The incidence of candidaemia in our hospital was found cial. We have investigated the prescriptions with an antifungal stew-
to be relatively low when compared with similar centres. The species ardship perspective.
and their sensitivity also were found to be similar to previously pub- Methods A retrospective single centre study between 2013 and
lished data with C. albicans being the most frequent isolate followed 2014 was carried out. Each prescription is re assessed as per the
by C glabrata in adults and C. parapsilosis in children. The overall local guidelines and those of IDSA 2009. Inclusion criteria: the
mortality rate is much lower to that published by many institutions positivity of at least one blood culture and/or another presumed ster-
and this might be partly due to the prompt communication of results ile sample culture for Candida spp. Exclusion criteria: negative
by the Medical Microbiologist, as direct germ tube was ready in less peripheral and simultaneous positive deep central catheters’ blood
than 2 h after the blood culture became positive, to the bedside clini- cultures (colonization) and/or positive peritoneal liquid cultures after
cians which ensured prompt and appropriate treatment. The influ- gut perforation.
ence of parameters, such as removal of lines on mortality rates, Results 76 patients were included: 38 patients from ICU, 19 surgi-
predisposing factors and attributable mortality will also be presented cal, 13 medical, 3 oncology and 3 gynaecology. 8 were treated for
in detail. solid cancer, 18 were in septic shock and 19 had deep tissue infec-
tions (endocarditis, pleuropneumonitis, peritonitis, mediastinitis, feb-
rile neutropenia). 59 (78%) presented multiple organ failure (MOF).
Fever-driven, diagnosis-driven and targeted treatment approaches
P251 were done in 12 (16%), 15 (21%), 38 (52%) respectively. Twelve
(16%) had no antifungal chemotherapy. Biological samples included
64 (74%) blood, 3 deep pus (4%), 1 graft conservation medium, 7
The evaluation of the relevance of antifungal (8%) pleural, 5 (6%) peritoneal, 4 (5%) ascites, 1 (1%) amniotic fluid,
chemotherapy prescription in Candida spp bloodstream 1 (1%) native mitral valve, 1 (1%) prosthetic aortic valve and 1 (1%)
infections in a French University Hospital lymphatic node smear cultures. Candida albicans, C. glabrata and C.
C. S. Adjodah, T. Chouaki, H. Dupont and J. L. Schmit
Amiens University Hospital, Amiens, France

Objectives Candida spp bloodstream infections are frequent and

dreaded complications in patients’ care. Anitfungal chemotherapy is

Figure 1 Figure 2

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144 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

tropicalis were found in 62%, 7% and 7% respectively. Caspofungin is P253

used in the front line in 26 cases of MOF of which 10 septic shocks,
2, 3 and 4 cases of endocarditis, pneumonitis and peritonitis repec-
tively. Decrementation to oral fluconazole is observed in 19 (26%) Clinical and epidemiological aspects of the largest
patients where the fungal agent is susceptible (86%) and whose clini- epidemic of sporotrichosis in dogs: 203 cases (2004–2014)
cal status improved (50%). The accordance with the guidelines P. G. Viana,1 I. D. F. Gremi~ao,1 A. B. F. Figueiredo,1
is 19.2%. Transoesophageal ultrasonics, fundus oculi and repeated I. M. S. Antonio,1 L. H. M. Miranda,1 F. B. Figueiredo,1
skin examination are done in only 41 (56%), 13 (18%) and 19 M. Suarez-Mutis2 and S. A. Pereira1
(26%) cases respectively. The global death rate is 38%; it is about 1
55%, 50%, 45% and 17% among patients having bloodstream infec- Evandro Chagas National Institute of Infectious Diseases, Rio de
tions with C. parapsilosis, C. glabrata and C. krusei taken together, Janeiro, Brazil and 2Oswaldo Cruz Institute IOC/Oswaldo Cruz
C.albicans and C. tropicalis respectively. Foundation Fiocruz, Rio de Janeiro, Brazil
Conclusion This study confirms the extremely serious burden of
invasive bloodstream Candida spp infections. The accordance with the Sporotrichosis is a subcutaneous mycosis caused by the Sporothrix
guidelines is weak, not only for the search of deep-tissue involvement complex, which is rarely described in dogs. However, an epidemic of
but also for the decrementation to oral fluconazole. Important task- this mycosis has been occurring since 1998 in the metropolitan
forces have to be implemented on the training and guidelines’ issu- region of Rio de Janeiro, Brazil, affecting humans, dogs and cats. The
ance among clinicians and the distribution of antifungal Evandro Chagas National Institute of Infectious Diseases (INI)/
chemotherapy using a computerized questionnaire, duly filled by the Oswaldo Cruz Foundation (Fiocruz) is a referral center for fungal dis-
clinician and the hospital pharmacist. The right time for oral flu- eases in Rio de Janeiro. In this institution, more than 4000 human
conazole decrementation, the removal of deep central catheters and cases and 4000 feline cases of sporotrichosis were diagnosed between
the search for deep tissue involvement must be done systematically. 1998 and 2012. Usually canine sporotrichosis is described in hunt-
Furthermore, such actions will help to preserve the public hospital’s ing dogs, which acquire the infection through the classic transmis-
fungal ecology, not to mention making substantial savings. sion resulting from the traumatic implantation of the fungus from
soil or contaminated plant material. In the epidemic in Rio de
Janeiro, the transmission to dogs has been occurring through
scratches, bites or contact with exudates from cutaneous lesions of
P252 sick cats, which are the main source of infection of the fungus.
Objective The purpose of this study was to describe the clinical and
epidemiological aspects of the largest series to date of canine
Pulmonary fungal infections among outpatients in medical sporotrichosis.
Mycology Laboratory, of Kermanshah University of Methods The study included dogs with isolation of Sporothrix spp. in
Medical Sciences special clinic 1993- 2012 fungal culture and followed up at the INI/Fiocruz, between 2004
A. Mikaeili, S. Yarali and A. Janbakhsh and 2014. Retrospective data were collected from the medical
School Of Medicine,Kermanshah University Of Medical Sciences, records of the animals (sex, breed, age, contact with cats, origin, clin-
Kermanshah, Iran ical signs, treatment regimens and outcomes).
Results Two hundred and three dogs were diagnosed with sporotri-
chosis in the period of the study. Among the positive dogs, 118 were
Objectives Systemic fungal infections in immunocompromised males (N = 201; 58.7%) and 86 were mongrel dogs (N = 193;
patients have a high prevalence. the goal of this study was to deter- 44.6%). The median age was 48 months (N = 185) and 148
mine the epidemiological characteristics of pulmonary fungal infec- (N = 189; 78.3%) dogs had contact with cats. Regarding the origin,
tions in client’s patients’ of mycology laboratory clinic of 117 dogs (N = 200; 58.5%) were from Rio de Janeiro city. At the
Kermanshah University of Medical Sciences. first clinical examination 165 dogs (N = 194; 85.1%) were in good
Methods In a study using data recorded in the archives of the condition. Respiratory signs were observed in 134 animals (N = 185;
University Mycology Laboratory during 1993 to 2012, the data were 72.4%) and lymphadenopathy in 72 (N = 158; 36.4%).
extracted and analyzed. These dogs have received different treatment regimens using keto-
Results Total admitted patients of Mycology Laboratory; Kerman- conazole, itraconazole or potassium iodide. The antifungal prescrip-
shah University of Medical Sciences with pulmonary clinic during, tion varied according to the clinical picture and the availability of
1993 to 2012 was 297 persons. 145 cases with diagnostic tech- the drug provided free of charge by INI/Fiocruz. To date, the treat-
niques: Direct smear or Culture on selective media has been positive. ment outcome of 97 dogs was described. Seventy-six (78.4%)
Lung and pulmonary Aspergillosis 8 cases, Candidiasis 35 cases and achieved clinical cure, eight (8.3%) died of causes unrelated to
Pnumosistosis have been detected in two cases.Candida albicans and sporotrichosis and one (1.0%) was euthanized due to the severity of
Aspergillus fumigatus more common agent were isolated in this clinical signs.
study. Conclusions To date, the study with the largest number of dogs
Conclusion Prevalence of fungal disease in patient’s mycology labo- with sporotrichosis was performed by Schubach et al. (2006), which
ratories in this study is of high importance. Definitely check fungal evaluated 44 dogs diagnosed at INI/Fiocruz from 1998 to 2003. In
infections in specific groups eligible risk associated with a higher fre- our study, 203 dogs were diagnosed over a period of 10 years,
quency of fungal diseases. The matter is further suspected cases of demonstrating the increasing number of cases due to the epidemic.
high importance. To conclude, sporotrichosis persists as a neglected disease in Rio de
Key words Janeiro, especially in animals.
Candidiasis, Aspergilosis, Pnumosistosis, Epidemiology,
Kermanshah

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Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 145
Poster Presentations

P260 study. Candiduria was defined as isolation of Candida species from

urine cultures of patients. Demographic and clinical data including
age, gender, underlying illnesses, severity of illness and all invasive
Association of lectin pathway proteins with intra- procedures applied were collected from medical reports of all patients.
abdominal Candida infection in high-risk surgical All statistical analyses were performed by using Statistical Package
intensive-care unit patients. A prospective cohort study for the Social Sciences (SPSS, Version 15.0). For the variables that
M. Osthoff,1 A. Wojtowicz,2 F. Tissot,2 U. Flu€ckiger,3 had significance in the chi-square tests, logistic regression analysis
M. Siegemund,1 S. Zimmerli,4 T. Calandra,2 J. Bille,2 was applied. P of <0.05 were considered as statistically significant.
Results In 5 years period, 161 candiduria cases were identified
P. Eggimann,2 O. Marchetti,2 N. Khanna,1 P. Y. Bochud2 and
among 1076 cases (15%). Of the cases 64 (39.8%) were male and
M. Trendelenburg1 97 (60.2%) were female. Mean age was 59.98  22.52. Most com-
1
University Hospital Basel, Basel, Switzerland; 2Lausanne mon Candida species isolated from urinary cultures was Candida albi-
University Hospital, Lausanne, Switzerland; 3Hirslanden Klinik, cans (103 cases-64%) followed by Candida glabrata (24 cases-14.9%),
Aarau, Switzerland and 4University Hospital, Inselspital, Bern, Candida tropicalis (20 cases-12.4%) and Candida parapsilosis (6 cases-
Switzerland 3.7%). Older age, female gender, red blood cell transfusion, presence
of central venous catheter, previous usage of antibiotics and antifun-
gals, duration of central venous and urinary catheters were found as
Objectives Intra-abdominal candidiasis (IAC) is a life-threatening risk factors for candiduria. In 11 patients (6.8%), candidemia was
complication of gastrointestinal (GI) tract perforation or acute necro- also detected with candiduria concurrently.
tizing pancreatitis. Mannose-binding lectin (MBL) has been shown to Conclusion Candiduria is not a rare clinical condition in ICU
bind to Candida albicans leading to enhanced complement activation patients and in some patients candiduria may be an indicator for
and augmented opsonisation in vitro. However, human studies on candidemia. Risk factors associated with candiduria are very similar
the role of MBL in patients with invasive candidiasis have yielded to which are associated with candidemia and those patients should
conflicting results. We investigated the influence of MBL and other be followed up carefully.
lectin pathway proteins on Candida colonization and IAC in a cohort
of high-risk intensive-care unit (ICU) patients.
Methods Prospective observational cohort study (Fungal Infection
Network of Switzerland) of 89 patients with recurrent GI perforation
or acute necrotizing pancreatitis. Levels of lectin pathway proteins at P262
study entry and six MBL2 single-nucleotide polymorphisms (SNP)
were analysed by ELISA and genotyping (Illumina Veracode genotyp- A prospective study of biomarkers to aid diagnosis of
ing platform or KASP[TRADEMARK] system, LGC Genomics), respec- invasive fungal disease in critical care patients
tively, and correlated with 1,3-b-D-glucan levels, development of
heavy Candida colonization (corrected colonization index (CCI) >0.4),
A. F. Talento,1 M. Palmer,2 E. M. Johnson,3 J. U. Springer,4
occurrence of IAC, and mortality during a 4-week period. Patients J. Loeffler,5 P. L. White,6 T. Ryan,7 D. Collins7 and T. R. Rogers1
1
with pre-emptive antifungal therapy were excluded from the analysis Trinity College Dublin, Dublin, Ireland; 2Mycology Reference
of IAC. Laboratory, Bristol, United Kingdom; 3Public Health England,
Results Within 4 weeks after inclusion a CCI>0.4 and IAC was Bristol, United Kingdom; 4University of Wu€rzburg, Wuerzburg,
observed in 42/89 (47%) and 27/69 (39%) patients, respectively, and Germany; 5University Hospital of Wuerzburg, Wuerzburg,
30-day mortality was 8%. Neither serum levels of MBL, ficolin-1,-2,-3 Germany; 6Public Health Wales, Cardiff, United Kingdom and
or MASP2 nor MBL2 genotypes or haplotypes were associated with 7
St. James’s Hospital, Dublin, Ireland
elevated 1,3-b-D-glucan levels, development of heavy Candida coloniza-
tion or 30-day mortality (P > 0.05 for all analyses). Similarly, none of
the analysed proteins was found to be significantly associated with IAC Background and objective Invasive fungal diseases (IFD) are a
with the exception of lower MBL levels (median 0.87 (IQR 0.21–1.93) major cause of morbidity and mortality in critical care patients. The
vs. 1.90 (IQR 0.19–3.44), P = 0.03) at study entry. However, there majority of these are due to Candida spp., however there are reports
was no difference in MBL deficiency (<0.5 lg ml
1), MBL2 haplo- or of a recent increase in the incidence of pulmonary aspergillosis in
genotypes in patients with and without IAC. this cohort. Because the diagnosis of IFD in critical care patients is
Conclusion Lectin pathway protein levels and MBL2 SNPs investi- difficult, non-culture based techniques such as assays for detection of
gated in this study were not associated with heavy Candida colonization fungal antigens and DNA are increasingly being investigated. The
or IAC in a small cohort of high-risk ICU patients. The lectin pathway main objective of this study was to assess the role of biomarkers in
of complement appears to have limited influence on the susceptibility the diagnosis of IFD in our critical care unit.
to invasive Candida infections in high-risk surgical ICU patients. Methods prospective observational study was performed from
December 2012 to January 2014 where patients who had been
receiving critical care for >7 days were recruited. After informed con-
sent, serum samples were taken twice weekly while the patient
remained in the unit. The following assays were performed: detection
P261 of (1–3)- b-d-glucan (BDG), and galactomannan (GAL) using com-
mercial kits, and A. fumigatus DNA by an in house rtPCR. Clinical
Investigation of Candiduria Cases in Intensive Care Unit: a data collected included: age, sex, reason for admission to critical care,
5-year Observational Study underlying diagnosis, host risk factors for IFD, antifungal therapy,
N. Yapar, V. Avkan-Oguz, M. Akan and M. Doluca-Dereli laboratory and radiology results and outcome. The patients were
classified into the following groups: no IFD, colonisation, possible,
Dokuz Eylul University Faculty of Medicine, Izmir, Turkey probable/putative and proven IFD using clinical and laboratory crite-
ria that excluded biomarker results and were adapted from published
Objectives While most of the urinary infections are of bacterial ori- guidelines for invasive candidiasis or a diagnostic clinical algorithm
gin, the incidence of fungal pathogens in urine cultures has been (1) for invasive pulmonary aspergillosis (IPA). Sensitivity, specificity,
reported to be nearly 12%. The most common fungal pathogen iso- negative (NPV) and positive predictive value (PPV) were calculated
lated from urine cultures was Candida spp. In this study, we aimed to to assess the performance of the biomarkers.
investigate the incidence and risk factors of candiduria in intensive Results One hundred patients were recruited into the study. Of
care units (ICU). these, 10 had no IFD, 43 were colonised by Candida or Aspergillus
Methods All patients hospitalized in one of the ICUs of our hospital spp., 14 and 18 had possible and probable invasive candidiasis
for more than 3 days within 5 years period were included in the respectively, 3 had probable/putative invasive aspergillosis and 12

ª 2015 The Authors

146 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

had proven IFD of which 9 were invasive candidiasis, 1 P. jirovecii care units (ICUs). The purpose of this prospective 1-year study was
pneumonia, 1 S. cerevisiae mediastinitis and 1 Candida skin and soft to collect, identify, determine antifungal susceptibility and test biofilm
tissue infection. The sensitivity, specificity, PPV and NPV of a single formation of Candida spp. recovered from blood of ICU patients in 11
positive BDG for the diagnosis of IFD were 63%, 84%, 66% and 83% centers in Serbia.
respectively. With 2 consecutive positive results, specificity increased Methods Study design included: 1) collection of isolates from blood;
slightly to 86%. The area under the receiver operator characteristics 2) identification by culturing, biochemical tests and matrix assisted
curve of the serum BDG was 0.758. A further 8 probable/putative laser desorption ionization time-of-flight (MALDI-TOF); 3) broth-dilu-
IPA cases were identified when results of the BDG, GAL and PCR tion susceptibility testing to amphotericin B, fluconazole, 5-fluorocy-
were incorporated into the diagnostic algorithm (1). tosine, itraconazole, posaconazole, voriconazole, micafungin,
Conclusion The performance of serum BDG detection in our cohort caspofungin and anidulafungin using Micronaut-AM MHK-2 plates
is comparable to previous reports (2). Biomarkers increase the detec- (Merlin Diagnostika, Bornheim, Germany); 4) biofilm formation test
tion of cases of invasive candidiasis and invasive aspergillosis in the using crystal violet assay (Sigma-Aldrich, St Louis, MO) and 5) data
critical care setting and support stewardship programs to optimise analysis using methods of descriptive and analytical statistics.
antifungal therapy. Results A total of 32 Candida isolates were collected from 32
patients: C. albicans (18/32; 56.3%) was the predominant species fol-
lowed by C. parapsilosis (11/32; 34.4%) and C. tropicalis (3/32;
9.3%). No resistance to amphotericin B, 5-fluorocytosine, mica-
P263 fungin, caspofungin and anidulafuingin was observed. Out of 32, 11
isolates were found to be azole-resistant (34.4%). Resistance rates to
fluconazole, voriconazole, posaconazole and itraconazole were 6.25%
Aspergillus Monitoring Project in a large Iranian (232), 15.6% (5/32), 15.6% (5/32) and 31.25% (10/32), respec-
Educational Hospital tively. Azole-resistant strains were predominantly C. albicans (10/11,
K. Diba,1 M. Siedy2 and K. Makhdoomi1 90.9%) and one C. tropicalis (1/11, 9.1%). Among C. parapsilosis
1 strains we detected no resistance, but 45.5% (5/11) of isolates were
Urmia University of Medical Sciences, Urmia, Iran and 2Tabriz
susceptible-dose dependent to itraconazole. When the difference in
University of Medical Sciences, Tabriz, Iran
minimum inhibitory values (MIC) between C. albicans strains and
non-albicans Candida strains (NAC) was tested, C. albicans had signifi-
Objectives The main object was monitoring of Aspergillus infections cantly higher MIC values for itraconazole, voriconazole and
and epidemiological approaches in the greatest university hospital of posaconazole, while NAC had significantly higher MIC values for
Urmia, Iran. micafungin and caspofungin (P < 0.05). The ability of biofilm forma-
Methods The subject of our study included bronchial fluid and spu- tion was present in 84.4% (27/32) of tested strains: 88.8% of tested
tum were collected from the hospitalized patients with acute respira- C. albicans (16/18), 72.7% of C. parapsilosis (8/11) and all C. tropi-
tory symptoms. All clinical specimens were transported to the calis strains (3/3, 100%). No association between planktonic antifun-
medical mycology lab, UMSU, for the diagnostic purposes and fungal gal susceptibility and biofilm formation was observed.
identifications. For environmental detection of Aspergillus isolates, Conclusion C. albicans was the most commonly isolated species
some specimens were collected from air and environment surfaces. A from the blood of Serbian ICU patients (56.3%). Antifungal resis-
morphological study was firstly performed including; growth charac- tance was restricted to azoles (6.25–31.25%). All isolates were sus-
teristics and microscopic features of Aspergillusspecies on mycological ceptible to echinocandins which should be regarded as the
media. For the Confirmation of Aspergillusisolates which similarly empirical treatment of choice for candidaemia in severely ill patients
found in clinical and environmental sources, PCR-RFLP using a or in the presence of risk factors. The majority of strains were able
novel restriction enzyme Mwo I and an additional molecular tech- to form biofilm (84.4%) so the next step in this study is to correlate
nique of RAPD were carried out. biofilm formation with clinical patient data on risk factors, therapy
Results A total of 102 fungal isolates, including Candida species 82 and outcome.
(80%), Aspergillus spp.20 (19.6%) and the other fungi 2 (0.4%).
Among the clinical isolates of Aspergilli; Aspergillus flavus (8.4%),
Aspergillus fumigatus (4.2%) and Aspergillus niger (2.8%) were identi-
fied, as well as environmental Aspergillus isolates Aspergillus flavus P265
(19.4%), Aspergillus niger (6.5%) and Aspergillus fumigatus (6.5%).
Discussions Comparing the clinical and environmental findings, 2
of 11 clinical Aspergillus isolates were matched with environmental Beta-D-glucan and Candida albicans germ tube antibody in
isolates in RAPD method and the other environmental sources were ICU patients with invasive candidiasis
not confirmed. E. Martin-Mazuelos,1 S. Ruiz-Santana,2 A. Loza,1 C. Castro,1
P. Saavedra,3 D. Macıas,1 I. Zakariya1 and C. Leon4
1
Hospital Universitario de Valme, Sevilla, Spain; 2Hospital
Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain;
P264 3
Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran
Canaria, Spain and 4Hospital Universitario de Valme/Hospital
Invasive candidiasis in intensive care units in Serbia: Universitario Dr Negrın, Sevilla, Spain
preliminary results on species distribution, antifungal
susceptibility profile and biofilm formation of Candida Purpose To assess the value of (1?3)-ß-D-glucan (BDG) and Can-
spp. isolates from blood dida albicans germ tube antibody (CAGTA) for the diagnosis of inva-
M. G. Pekmezovic,1 A. M. Barac,1 M. Z. Kostic2 and V. S. Arsic sive candidiasis (IC), their kinetics in the presence of systemic
Arsenijevic3 antifungal treatment, and the influence of main confounding factors
1
Institute of Microbiology and Immunology, Faculty of Medicine on BDG levels in unselected, non-neutropenic ICU patients.
Methods Prospective cohort study of 107 ICU patients admitted ≥
Uni. of Belgrade, Belgrade, Serbia; 2Institute of Microbiology and
7 days. BDG (cutoff positivity ≥ 80 pg ml
1) and CAGTA (cutoff posi-
Immunology, Faculty of Medicine, Uni. of Belgrade, Belgrade,
tivity ≥ 1/160) assays were performed twice a week. Confounding
Serbia and 3University of Belgrade, Belgrade, Serbia factors included amoxicillin-clavulanate and piperacillin-tazobactam
treatments, recent surgery, Gram-positive bloodstream infection,
Objective Candida species are an important cause of morbidity and renal replacement therapy, and enteral nutrition. Patients were clas-
mortality in the critically ill patients, especially those in intensive sified as neither colonized nor infected (n = 29), Candida spp.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 147
Poster Presentations

colonization (n = 63) (low-grade, n = 32; high-grade, n = 31), and Conclusion In the particular ICU setting, C. parapsilosis has emerged
invasive candidiasis (IC) (n = 15). as a significant bloodstream pathogen, steadily outranking C. albi-
Results A total of 465 measurements of biomarkers were performed cans. The emergence of fluconazole resistant C. parapsilosis strains
(4.3 per patient). BDG values were higher in patients with IC and raises questions about the superiority of fluconazole as a better drug
high-grade colonization than in the remaining groups (P = 0.012), against C. parapsilosis. Therefore, it is of great value to monitor the
and two consecutive measurements ≥ 80 pg ml
1 discriminated IC local epidemiology so as to guide the timely and successful empirical
from the remaining groups (sensitivity 80%, specificity 73%). Signifi- anti-Candida treatment.
cant changes of BDG and CAGTA kinetics in IC patients treated with
antifungals were not observed. In patients neither colonized nor
infected or with low-grade Candida spp. colonization, none of the con-
founding factors was associated with a significant increase in BDG P267
positivity.
Conclusions Two consecutive BDG values ≥ 80 pg ml
1 allowed
discrimination among IC and high-grade colonization. Systemic anti- Effectiveness and safety of anidulafungin: A real-life
fungal therapy could not be monitored with biomarker kinetics, and multicenter data in Turkey
BDG levels were not interfered by confounding factors in neither col- T. Meltem,1 O. E. Kutsoylu,2 H. Pullukcu,1 S. Sayin-Kutlu,3
onized nor infected patients or with low-grade colonization. B. Ozturk,4 O. Kaya,5 O. Turhan,6 S. Senol,7 S. Alp-Cavus,2
Supported by a research grant from Instituto Salud Carlos III.
M. Kutlu,3 G. Mermut,8 D. Metin,1 B. Baysan-Ozhak,6 C. Ergin,3
Madrid. Spain (FIS PI 10/02110)
C. Cetin,7 M. B. Ertugrul,4 V. Avkan-Oguz9 and N. Yapar2
1
Ege University Medical School, Izmir, Turkey; 2Dokuz Eylul
University, Izmir, Turkey; 3Pamukkale University, Denizli, Tunisia;
4
P266 Adnan Menderes University, Aydin, Turkey; 5Suleyman Demirel
University, Isparta, Turkey; 6Akdeniz University, Antalya, Turkey;
7
Celal Bayar University, Manisa, Turkey; 8Ege University, Izmir,
Surveillance of Candidaemia in a Greek Intensive Care Unit Turkey and 9Dokuz Eylul University Faculty of Medicine, Izmir,
from 2009 to 2014 Turkey
V. Mamali,1 G. Vrioni,2 O. Zarkotou,1 P. Tselioti,1 A. Prekates,1
A. Themeli-Digalaki1 and A. Tsakris3 Objective Fungal infections are a growing global problem. The most
1
Tzaneio General Hospital, Athens, Greece; 2University of common global mycoses are due to infections by Candidiasis, Crypto-
Athens, Medical School, Athens, Greece and 3Medical school, coccosis, Aspergillosis. More antifungal agents have reached clinical
University of Athens, Athens, Greece use in the past two decades than at any other time. The echinocan-
dins have been a welcome addition to this group, with the latest
Objectives The aim of the present study was to investigate the local being anidulafungin. Anidulafungin is a semisynthetic product of
epidemiological data of candidaemia (species distribution and suscep- echinocandin B and it inhibits enzyme complex 1,3-b-D-glucan syn-
tibility profile), collected from 2009 to 2014, in a 12-bed Intensive thase and thereby inhibits fungal 1,3-b-D-glucan synthesis. Anidula-
Care Unit (ICU) of a large tertiary-care hospital in Greece. fungin is FDA approved for the treatment of invasive candidiasis,
Methods All candidaemia cases were recorded. Non-duplicate Can- candidemia and esophageal candidiasis in 2006. This drug is used
dida isolates were identified to species level by using CHROMagar since 2012 in our country. We aimed to examine the real-life data of
Candida Medium (Becton Dickinson) and Vitek 2 system (bioMerieux), the effectiveness and side effects of anidulafungin in the multicenter
supplemented with examination of the morphology on cornmeal Dal- study,
mau plates. All C. parapsilosis strains were characterized as C. parap- Methods The patients whose had proven fungal infection or based
silosis sensu stricto with MALDI-TOF (Bruker). In vitro antifungal on their candida colonization index/candida score or empirically
susceptibility testing was performed by broth microdilution method, anidulafungin treatment was started by the Infectious Diseases spe-
according to CLSI M27-A3 guidelines. Recently revised CLSI M27-S4 cialists from seven different centers are retrospectively screened
species-specific clinical breakpoints were applied. between the dates of 1st January 2012 and 31st December 2014.
Results A total of 92 episodes of candidaemia from 83 ICU patients Results Total number of 262 patients (115 female, 147 male, mean
were registered during the study period. Ninety-six Candida blood- age 58.65 + 19.51, min:15 max:95) from seven centers were
stream isolates were studied. Of these 96 isolates, 46 (47.9%) were involved in this study. 140 (53.4%) patients were hospitalized at the
C. parapsilosis, 23 (24%) were C. albicans, 17 (17.7%) were C. glabrata internal medicine departments and the other 122 (46.6%) patients
and 8 (8.3%) were C. tropicalis; there was 1(1%) isolate of C. krusei
and 1 (1%) isolate of C. lusitaniae. C. parapsilosis was the predomi-
nant species, always first at the rank order over the study period. Table 1. Risk factors
Decreased fluconazole susceptibility, defined as MIC >4 lg ml
1 was
detected in 9/46 (19.5%) C. parapsilosis isolates (MIC range 8–
32 lg ml
1). Despite the finding that 50% of the C. parapsilosis clini-
cal strains tended to have elevated anidulafungin MICs, clustering at
2 lg ml
1, no resistance to anidulafungin (MIC ≥8 lg ml
1) was
demonstrated. Only 5 out of 46 C. parapsilosis strains were catego-
rized as intermediately susceptible to anidulafungin (MIC of
4 lg ml
1). The MIC values obtained for caspofungin were in gen-
eral 1 to 2 dilution steps lower than those for anidulafungin, among
C. parapsilosis isolates. All 23 C. albicans strains were fully susceptible
to azoles and candins. All C. glabrata isolates were categorized as sus-
ceptible dose-dependent to fluconazole (MIC90 = 8 lg ml
1) and
100% as susceptible to anidulafungin (MIC ≤0.12 lg ml
1); 64.7%,
(11/17) were classified as being intermediately susceptible to caspo-
fungin (MIC of 0.25 lg ml
1). A single C. tropicalis isolate was cate-
gorized as susceptible dose-dependent to fluconazole with a drug MIC
of 4 lg ml
1, while for another C. tropicalis strain, the MIC values
were suggestive of resistance to caspofungin (MIC of 1 lg ml
1) and
susceptibility to anidulafungin (MIC of 0.016 lg ml
1).

ª 2015 The Authors

148 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

were at the surgical departments. 206 patients (78.6%) were fol- P269
lowed at the 3rd level of intensive care units. Treatment was started
for culture positive 163 (62.2%) patients. Blood cultures were posi-
tive for candida spp. (67 C.albicans, 33 C.parapsilosis, 17 C.tropicalis, Additional value of cytochrome B gene sequencing to
13 C.glabrata, 10 others) in 140 patients. 67 patients were treated internal transcriber spacer sequencing for the correct
with the other antifungal agents (48 fluconazole, 12 caspofungin, 5 identification of Trichosporon species
voriconazole, 2 amphotericin B) and their treatments were switched C. Oliviera Dos Santos,1 G. Kampinga,1 A. D. van Diepeningen2
to anidulafungin afterwards. and E. Bathoorn1
In 121 (%86.4) of 140 blood culture positive patients, source was 1
central venous catheter and 59 (48.8%) of them were removed after University Medical Center Groningen, Groningen, the
the culture results. There was significant difference comparing the Netherlands and 2CBS-KNAW Fungal Biodiversity Centre, Utrecht,
catheter removed group and unremoved group in terms of mortality the Netherlands
rates (P = 0.046; Fisher’s E. Test).
In blood culture positive group mortality rate was 52.9% (79 of Objectives Trichosporon species are emerging fungal pathogens in
140 patients). These high mortality rates due to not only candidemia immunocompromised patients. Trichosporon species are generally sus-
but also other co-morbidities. During the course of anidulafungin ceptible to triazoles and amfotericine B. Correct species identification
treatment, 1 anaphylaxis, 1 skin eruption, 1 trombocytopenia were of Trichosporon species is challenging. Here we show the additional
seen as side effects. value of sequencing the cytochrome B gene for identification of Tri-
Conclusion In this study which has large number of patients, chosporon species of a high-level triazole resistant Trichosporon isolate.
anidulafungin treatment was seen as effective in candidemias and This strain was isolated from a recipient of a liver re-transplant
low side effects. Considering invasive candida infections in clinically admitted to the surgical intensive care unit.
severe patients, we believe that anidulafungin can be preffered as ini- Methods Species identification was based on matrix associated time
tially and spectrum of treatment can be adjusted after the culture of flight mass spectrometry (Maldi-TOF) and sequencing of the inter-
results. nal transcriber spacer (ITS) of the 18S ribosomal subunit. The addi-
tion of sequencing the cytochrome B gene lead to definitive
identification. Susceptibility of the isolate was tested using a commer-
cial microdilution test (Yeast Sensititre OneTM) by the national refer-
P268 ence center.
Results Trichosporon spp was isolated from abdominal fluid sample of
recipient of a liver re-transplant receiving fluconazole and ampho-
Species distribution and susceptibility testing of Candida tericin B. Provisional susceptibility testing by e-test indicated resis-
spp. isolated from patients with invasive candidosis in tance to triazoles. Microdilution testing showed MICs to
Russia amphotericine B 0.25 mg l
1, fluconazole >256 mg l
1, itraconazol
N. Vasilyeva,1 E. Raush,2 T.S. Bogomolova,2 E. Shagdileeva2 and >16 mg l
1, voriconazol >16 mg l
1, posaconazol >8 mg l
1 caspo-
N. Klimko2 fungin >16 mg l
1 and 5-flucytosine >64 mg l
1. The isolate was
1 identified as Trichoporon mucoides with a score of 2.17 by Maldi-TOF.
North-Western State Medical University named after I.I.
Mechnikov, Saint Petersburg, Russia and 2North-Western State
Medical University named after I.I. Metchnikov, Saint Petersburg,
Russia

Objectives To study etiology of invasive candidosis and susceptibility

of Candida spp. isolates to antifungal agents: fluconazole, voricona-
zole, posaconazole and caspofungin.
Methods Species identification was performed by MALDI-TOF-mass-
spectrometry and DNA-sequencing. Susceptibility testing was con-
ducted according to CLSI M27-A3 (S4, 2012) microdilution method.
Results A total of 322 Candida spp. isolates from normally sterile
body sites of patients with invasive candidosis hospitalized in 37
medical centers of 6 Federal regions of Russia have been studied
prospectively (2011–2014 yy.). Species distribution was as follows:
C. albicans
43.2%, C. parapsilosis - 20.2%, C. glabrata - 11.5%, C.
tropicalis -9.6%, C. krusei - 6.2%, C. guilliermondii -5.3%, C. lusitaniae
- 1.3%, C. pararugosa - 0.6%, C. dubliniensis - 0.6%, C. inconspicua -
0.3%, C. kefyr - 0.3%, C. utilis - 0.3%, C. bracarensis - 0.3%, C. lipolyt-
ica - 0.3%. Prevalence of C. albicans decreased from 54% (2012 y.) to
32.8% (2014 y.), P = 0.015. Prevalence of C. parapsilosis increased
from 16.4% (2012 year.) to 27.9% (2014 year), P = 0.012. Differ-
ences in etiology of invasive candidosis were revealed in various geo-
graphic regions. Proportions of C. albicans were significantly more in
Siberian Federal District (75%) and Ural Federal District (57.8%),
than in Northwestern FD - 37% and Southern FD - 35% (P < 0.05).
Susceptibility patterns to antifungals: fluconazole - S - 75%, SDD-
13%, R- 12%; voriconazole -S - 97.5%, SDD - 0.6%, R - 1.6%; caspo-
fungin - S - 100%. MIC100 of posaconazole - 0.25 mg l
1.
Conclusion Among etiologic agents of invasive candidosis in Russia
the proportions of C. albicans is decreasing and C. parapsilosis -
increasing.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 149
Poster Presentations

Identification by ITS sequencing could not differentiate between T. P271

mucoides and T. dermatis subspecies (figure 1). Combining the data
from ITS sequencing and the sequence data from the cytochrome B
gene lead to the correct identification T. dermatis (figure 2). Invasive candidiasis in ICU patients. Diagnostic usefulness
Conclusion Here we present a highly triazole resistant T. dermatis of anti-mycelium antibodies, 1-3-Beta-D-Glucan, mannan/
isolate. The subspecies was misidentified as T. mucoides using Maldi- antimannan antibodies, and detection of Candida DNA
TOF, and by ITS PCR, now differentiation was possbible between T. (PCR). Cava Trem Project
dermatis and T. mucoides. The addition of cytB was needed for correct S. Ruiz-Santana,1 E. Martin-Mazuelos,2 A. Loza,2 C. Castro,2
species identification of T. dermatis. Correct species identification is D. Macıas,2 I. Zakariya,2 M. Parra,2 A. Ubeda,3 P. Saavedra,4
essential for epidemiology of resistance of Trichosporon species.
A. Rezusta,5 A. Rodriguez,6 I. Fernandez,7 C. Leo  n8 and
C. T. Cava Trem Study Group2
1
Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria,
P270 Spain; 2Hospital Universitario de Valme, Sevilla, Spain; 3Hospiten
Estepona, Estepona, Spain; 4Universidad de Las Palmas de Gran
Canaria, Las Palmas de Gran Canaria, Spain; 5Hospital
The impact of a diagnostics-driven antifungal stewardship Universitario Miguel Servet, Zaragoza, Spain; 6Hospital Joan XXIII,
programme in a UK tertiary referral teaching hospital Tarragona, Spain; 7Complejo Asistencial de Leon, Leon, Spain
R. Richardson, E. Muldoon and M. Richardson and 8Hospital Universitario de Valme/Hospital Universitario Dr
University Hospital of South Manchester, Manchester, United Negrın, Sevilla, Spain
Kingdom
Objectives To assess the usefulness of biomarkers (BM) (Candida
Background Candidaemia is a serious complication in critically ill albicans germ tube antibodies [CAGTA], (1,3)-b-D-glucan [BDG],
patients, and associated with high mortality. The University Hospital mannan antigen [MN], anti-mannan antibodies [AMN] and Candida
of South Manchester, UK (UHSM) candidaemia guidelines for the DNA PCR) in the diagnosis of invasive candidiasis (IC) in critically ill
ICUs recommend early initiation of antifungal therapy based on adult patients with primary acute gastrointestinal injury* and ICU
strong clinical suspicion and the presence of specific risk factors. stay ≥7 days.
Echinocandins are the first line therapy, and micafungin is the Methods Prospective, cohort, observational and multicentre study
echinocandin on formulary. Serum b-1-3-D-glucan (BG) should be (12 ICUs/18 months). Twice a week: BM assays and Candida PCR.
used to guide discontinuation of therapy in the absence of other Techniques included CAGTA (Vircell kit), BDG (Fungitell), MN and
microbiological evidence of candidaemia as it has a very high nega- AMN (Platelia TM Candida Ag Plus) and detection of DNA/Candida
tive predictive value. The aim of this study was to evaluate the com- by PCR-TR multiplex. Cutoff positivities: CAGTA: ≥ 1/160, BDG ≥
pliance of the 3 ICUs (including cardio-thoracic & transplant, burns, 80 pg ml
1, MN: ≥ 60 pg ml
1; AMN: ≥ 10 UA ml
1, and PCR (+)
acute medical) in UHSM (total of 71 beds) with the current guideline, or (-). BM were considered positive in the presence of two consecu-
the impact of the UHSM antifungal stewardship programme on anti- tive results of CAGTA, MN and AMN, maximal geometrical mean of
fungal consumption. two consecutive observations for BDG and at least one positive isolate
Methods The usage of micafungin at UHSM between April and July for PCR. For each patient, these assessments were performed at or
2014 was reviewed using pharmacy databases, and by reviewing before the episode of IC. When an episode of IC did not develop, the
patient records. All prescriptions were reviewed by the audit team highest value of all recorded values was used. Patients were classified
and a decision made as to whether or not the use of micafungin was into: neither colonized/nor infected (NCI), Candida spp. colonization of
appropriate in each individual case. Antifungal consumption data low grade (LGCC) and high grade (HGCC), intra-abdominal candidia-
was analysed for the general adult ICU (19 beds) for the past sis (IAC) and candidemia (C). Categorical variables were expressed as
24 months. frequencies and percentages, continuous variables as mean and stan-
Results A total of 72 patients admitted to ICU were started on mica- dard deviation (SD) or median and interquartile range (IQR, 25th-
fungin during the 4-month study period. Of these, 45 were treated 75th percentile). Data were analyzed with the chi-square test, the
for suspected or proven candidaemia, and 25 for suspected or proven Fisher’s exact test and the Kruskal-Wallis test as appropriate. ROC
aspergillosis or scedosporiosis. Of those treated for candidaemia,
seven (16%) had Candida spp. isolated from blood cultures. Two of
these were ECMO patients. In 14 cases (31%) the local guideline was
not fully followed. In eight cases the documented reason for starting
micafungin was isolation of Candida spp. in a single superficial sample
such as sputum or urine. In four cases microbiological tests had not
been taken as per guideline, and in two cases negative BG result was
not acted upon. Of the 38 cases without evidence of invasive candi-
dosis when the patients were reviewed by a member of the Infectious
Diseases team, micafungin was discontinued in 19 cases (50%). In
two further cases, discontinuation of micafungin was recommended,
however the primary team (both surgical) chose not to follow the
advice given. Two patients had been prescribed higher (150 mg od)
dose in the absence of clinical suspicion of aspergillosis or oesopha-
geal candidosis. There were no deaths due to candidaemia during the
study period. The monthly micafungin expenditure on the AICU was
some 13 000€ per month before the audit and was reduced to
£9200€ during the audit and has stayed on that level until now.
Conclusion True candidaemia is a medical emergency and mica-
fungin a well-tolerated drug. Therefore, it would be inappropriate to
restrict the initiation of first-line therapy in cases of suspected candi-
daemia. Our study shows that with the active presence of Infectious
Diseases Consultants on ICU, and with the appropriate use of diag-
nostics, it is possible to safely discontinue echinocandin therapy
when it is not indicated. This has important implications for antifun-
gal stewardship and cost saving in intensive care.

ª 2015 The Authors

150 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

curve analysis for BDG to discriminate between IC and the remaining mechanical ventilation (84%), parenteral nutrition (82%), steroids
groups (80% sensitivity of the cutoff selected). In both cases (BDG (37%) and renal replacement therapy (25%). There were 48 patients
geometric mean and maximum values), the cutoff was 80 pg mL
1. in the NCI group, 130 in the LGCC, 24 in the HGCC and 31 (13.3%)
Statistical significance was set at P < 0.05. in the IC. Positive samples for Candida spp. accounted for 34.7%
Results 233 patients: NCI 48, LGCC 130, HGCC 24 and IC 31 (IAC (n = 885) of all 2549 cultures, with multifocal colonization in 120
20 and C 11). Surgical 187 (80%), medical 39, T, 7)], age: 66.7 (49%) patients. Pharyngeal exudates were the most frequent colo-
(13.2) years and M/F: 67/33%. Hospital/ICU stay: 15 (10;25)/37 nized samples (59.6%) followed by rectal and respiratory samples
(21;57) days. Antifungal treatment: 119 (50.8%). Overall mortality: (51% each). The most frequently isolated Candida spp. were C. albi-
68 (29.4%). Samples: BM: 860 (median 3.69 [1–10] per patient) and cans (60.1%) and C. glabrata (11.7%). There were 31 cases of IC (20
PCR 213 measurements. Table 1 shows BM and PCR in relation to intra-abdominal infection, 11 candidemia), with C. albicans, C. glab-
the groups of patients, and Table 2 their values in the diagnosis of rata and C. parapsilosis isolated in 16, 7 and 3 cases, respectively. C.
IC, with BDG (cutoff >80 pg/mL) showed a 77% sensitivity and 42% tropicalis, C. krusei, C. famata, C. dubliniensis and C. albicans + C. glab-
specificity (AUC ROC 0.67). For the diagnosis of candidemia, BDG rata, in 1 case each. Two cases were catheter-associated candidemias.
with a cutoff of 235 pg ml
1 showed 82% and 74% sensitivity and Twenty-three (74%) patients showed multifocal colonization. The
specificity, respectively (AUC ROC 0.79). The remaining BM and PCR mean time to develop IC from hospital and ICU admission was 17.4
did not show relevant values. The association of BDG with other BM (15.7) and 7.1 (6.0) days, respectively. A total of 119 (51%) patients
and PCR did not improve the diagnostic capacity of BDG for IC and received antifungal therapy, 29 and 90 patients from the IC and CC/
specifically for candidemia. NCI group, respectively. The overall mortality rate was 68 (29.4%).
Conclusion (1,3)-b-D-glucan was the only biomarker allowing dis- Conclusions In this cohort, a high rate of multifocal multicoloniza-
criminating candidemia from the remaining groups analyzed. cion was observed. C. albicans followed by C. glabrata were the most
* Blaser AR et al. ICM 2012;38:384–94 frequently isolated species. A high percentage of patients (38.6%)
Supported by a research grant from Astellas S.A and Instituto Salud without documented Candida infection were treated with antifungal
Carlos III. Madrid. Spain (FIS PI 13/01168) agents.
* Blaser AR et al. ICM 2012;38:384–94
Supported by a research grant from Astellas Pharma and Instituto
Salud Carlos III. Madrid. Spain (FIS PI 13/01168)
P272

Invasive Candidiasis in ICU patients. Epidemiological P273

Study. Cava Trem Project
E. Martin-Mazuelos,1 S. Ruiz-Santana,2 A. Loza,1 C. Castro,1
D. Macıas,1 I. Zakariya,1 A. Ubeda,3 P. Saavedra,4 A. I. Suarez,5 Directed/empirical antifungal treatment in non-
 pez,9 C. Leo
 n10 and neutropenic critically ill patients with abdominal
J. Ayats,6 D. Navarro,7 A. Bordes,8 G. Lo
conditions. Differential characteristics. Cava Trem Project
C. T. Cava Trem Study Group1
1
S. Ruiz-Santana,1 E. Martin-Mazuelos,2 A. Loza,2 A. Ubeda,3
Hospital Universitario de Valme, Sevilla, Spain; 2Hospital D. Macıas,2 M. Gurpegui,4 A. Rodriguez,5 R. Gonzalez,6
Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain;  n10 and C. T. Cava
3 M. A. Gonzalez,7 J. Ballus,8 J. Puig,9 C. Leo
Hospiten Estepona, Estepona, Spain; 4Universidad de Las Palmas 2
de Gran Canaria, Las Palmas de Gran Canaria, Spain; 5Virgen Trem Study Group
1
Macarena University Hospital, Seville, Spain; 6Hospital Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria,
Universitario de Bellvitge, Barcelona, Spain; 7Hospital Clinico de Spain; 2Hospital Universitario de Valme, Sevilla, Spain; 3Hospiten
Valencia, Valencia, Spain; 8Hospital de Gran Canaria-Negrın, Estepona, Estepona, Spain; 4Hospital Universitario M. Servet,
Gran Canaria, Spain; 9Hospital para el Nin~o Poblano, Puebla, Zaragoza, Spain; 5Hospital Joan XXIII, Tarragona, Spain;
6
Mexico and 10Hospital Universitario de Valme/Hospital Complejo Asistencial de Leon, Leon, Spain; 7Hospital
Universitario Dr Negrın, Sevilla, Spain Universitario V. Macarena, Sevilla, Spain; 8Hospital Universitario
de Bellvitge, Barcelona, Spain; 9Hospital Clinico Universitario de
Objective Epidemiological study of selected non-neutropenic (pri-
Valencia, Valencia, Spain and 10Hospital Universitario de Valme/
mary acute gastrointestinal injury*) critically ill adult patients colo- Hospital Universitario Dr Negrın, Sevilla, Spain
nized and infected by Candida spp., with an expected ICU stay of at
least 7 days. Objectives To assess the characteristics of antifungal therapy (AFT)
Methods Prospective, cohort, observational, multicentre study car- in non-neutropenic critically ill patients with surgical abdominal con-
ried out during 18 consecutive months in 12 Spanish ICUs from ter- ditions or non-operated pancreatitis with an ICU stay of ≥ 7 days.
tiary hospitals. Demographic data, type of patients, APACHE II and Methods Prospective, cohort, observational and multicentre study
SOFA scores (on ICU admission), underlying disease, comorbidities, carried out during 18 consecutive months in 12 ICUs. Demographic
risk factors, antifungal treatment and outcomes were recorded. Clini- data, type of patient, APACHE II and SOFA scores, comorbid condi-
cal assessment, APACHE II, SOFA and Candida score, Candida colo- tions, risk factors, AFT and clinical outcome were recorded. Type,
nization cultures (rectal swabs, tracheal aspirates, gastric or doses and duration of AFT, given in monotherapy, combined or fol-
pharyngeal aspirates and urine) were performed twice a week (other lowing sequential regimens were registered. Patients were classified
samples were taken as clinically indicated). All samples were pro- into the groups of neither colonized/nor infected (NCI) and Candida
cessed by conventional methods. Patients were classified into: neither spp. colonization of low grade (LGCC) and high grade (HGCC), intra-
colonized/nor infected (NCI), Candida spp. colonization of low grade abdominal candidiasis (IAC) and candidemia (C). Categorical vari-
(LGCC) and high grade (HGCC), intra-abdominal candidiasis (IAC) ables were expressed as frequencies and percentages, continuous
and candidemia (C). Categorical variables are expressed as frequen- variables as mean and standard deviation (SD) or median and
cies and percentages, and continuous variables as mean and stan- interquartile range (IQR, 25th-75th percentile). The Student’s t test
dard deviation (SD). (means) and the Kruskal-Wallis test (medians) were used for the
Results A total of 233 patients (67% men, mean [SD] age 66 [13.0] analysis of continuous data. Statistical significance was set at
years) were included. The mean APACHE II and SOFA scores on ICU P < 0.05.
admission were 18.4 (6.3) and 7.2 (3.3), respectively; 211 (90.6%) Results Directed treatment: 31 patients (13.2%) presented invasive
patients underwent abdominal operations (344 surgical procedures). candidiasis (IC), 20 IAC and 11 C (catheter-related in two cases).
Risk factors included central venous catheter (99%), urinary catheter AFT was administered to 29 patients (93.5%). The median time
(98%), broad-spectrum antibiotics (97%), arterial catheter (88%), elapsed from hospital and ICU admission to diagnosis was 16 (6;19)

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 151
Poster Presentations

and 7 (3;13) days, respectively. AFT was started on the day of IC P281
diagnosis (21/29), before (6/29) and 1 day later (2/29), and was
maintained for a mean of 14 (8.0) days. Initial AFT included azoles
(15) and candins (14). Sequential AFT with a second (4) and third Gastrointestinal mucormycosis in a patient with acute
(1) antifungal agents was also recorded. Empirical treatment: 90 myeloblastic leukemia
patients (38.4%) without IC received AFT, 81 (90%) undergoing G. G. Solopova, N. G. Uskova, D. M. Konovalov, A. A. Maschan
abdominal surgery, overall mortality 31.1% (28 cases). The indica- and G. A. Novichkova
tion of AFT was associated with severe sepsis (66 [73.3%]) and/or
multifocal colonization (39 [43.3%]). The mean time of starting AFT Federal Research Centre of Pediatric Hematology, Oncology and
from hospital and ICU admission was 14.8 (15.2) and 7.08 (6.0) Immunology, Moscow, Russia
days, respectively. Initial AFT included candins (56) and azoles (34)
with a mean duration of treatment of 12.1 (6.9) days. Eleven Objectives In patients with hematological malignancy mucormyco-
patients received a second course of treatment: de-escalation (5), sis is the third most common invasive mycosis after aspergillosis and
poor clinical course (3) and other causes (3). All antifungals were candidiasis. The most expected localisation is paranasal sinuses, orbit
administered in monotherapy or sequentially. The mean APACHE II, and brain or lungs. We report a rare case of gastrointestinal
SOFA and Candida score at the beginning of AFT, directed and empir- mucormycosis. Most cases of gastrointestinal mucormycosis are asso-
ical, were 16.8 (4.9) and 17.7 (6.2); 6.1 (3.5) and 6.8); and 3.9 ciated with diagnostic delay and high mortality.
(1.0) and 3.0 (0.9), respectively. The comparative analysis of these Methods Patient 6 month-old boy diagnosed with AML and treated
variables did not reveal statistically significant differences. with stadart chemotherapy in a regional hospital. Remission was
Conclusion In this type of patients, neither clinical features nor achieved after first course of induction therapy. After consolidation
severity-related scores were different between both groups (directed course (Cytosine arabinoside 3000 mg/m2 every 12 h day 1–4, Idaru-
and empirical treatment) at the time of starting AFT. In both groups, bicin 10 mg/m2 day 2–4) the patient developed sepsis with pneumo-
AFT was initiated around 7 days of ICU stay. The number of empiri- nia, enterocolitis. Candida krusei was recovered from blood. One week
cal AFT was very high. Types of antifungals and duration of treat- later massive GI bleeding started and gastroscopy revealed extensive
ment seems adequate. duodenum ulcer with thrombotic and necrotic mass. GI bleeding was
Supported by a research grant from Astellas Pharma and Instituto treated conservatively. Febrile neutropenia developed after second
Salud Carlos III. Madrid. Spain (FIS PI 13/01168) consolidation (Cytosine arabinoside 1000 mg/m2 every 12 h day 1–3
and 8–9, mitoxantrone 10 mg/m2 day 2–3, L-asparaginase 6000 ME/
m2 day 4 and 10) and was complicated by recurrent GI bleeding.
P280 During gastroscopy perforation of duodenum ulcer was find out.
In very poor condition he was admitted to our hospital. After
examination, wich revealed leakage of radiopaque substance from
Imaging findings in patients after allo-HSCT with
duodenum to the right part of abdominal cavity and necrosis of the
pulmonary mucormycosis
right kidney, child was immideately taken to the operating room.
I. Nikolaev,1 A. Volkova,2 M. Popova1 and N. Klimko1 Abdominal cavity revision showed perforation of duodenal ulcer,
1
First I. Pavlov State Medical University, Saint Petersburg, Russia total rupture of vertical part of duodenum, perforation of cecum and
and 2Raisa Gorbacheva Memorial Institute, First Pavlov State total necrosis of the right kidney and paranephral tissues. Surgery
Medical University, Saint Petersburg, Russia included resection of horizontal part of duodenum and duodeno-je-
junoanastomosis, resection of ileocecal angle and ileo-ascendoanasta-
mosis, right nephroectomy, gastrostomy.
Objectives to describe computer tomography (CT) features of pul-
Results In histopathological specimen of resected duodenum, cecum
monary mucormycosis in patients after allogeneic hematopoietic
and kidney the picture of invasive mycosis was seen: wide, ribbon-
stem cell transplantation (allo-HSCT).
like mycelium with no regular septation and different angles of
Materials and methods A prospective single center study in 2010–
branching such as 90 °. All these characteristics confirm gastro-in-
2014 yy. We include 15 patients (age from 4 to 59 year) after allo-
testinal form of mucormycosis. Most probably, that kidney necrosis
HSCT with probable and proven (n = 4) pulmonary mucormycosis
was secondary. Considering the complicated situation: septic condi-
according to EORTC/MCG 2008 criteria. All the patients underwent
tion of patient, nephroectomy in one hand and age of 9 month, gas-
CT guided bronchoscopy with comprehensive study of broncho-alveo-
trostasis, bad intestinal absorbtion in other hand, we decided to start
lar lavage fluid (BAL). The samples were sent to the laboratory for
with combined antifungal therapy. One month he recieved lipid for-
microscopy, culture and galactomannan test. If the result of BAL
mulation amphoB 5 mg/kg/every day with posaconasol
fluid was negative, we performed further diagnostic procedures. Two
15 mg kg day
1 followed by 4 months posaconasol monotherapy. In
patients underwent forceps biopsy and one percutaneous biopsy
a good condition with normal nutritional status in complete hemato-
under ultrasonography navigation control.
logical remission he was discharged without any sign of infection.
Results The majority of the patients (60%) had unilateral lesion on
Conclusion Despite gastro-intestinal form remain to be very rare,
CT scan. Main lesions were pleural effusion (47%), solitary mass with
we should keep it in mind, that in children especially before one year
the halo sign (46%), later with the appearance of reversed the halo
old it happens 3 times more frequent than in general population.
sign (31%), small ill-defined infiltrates (27%), and multilobar consoli-
Prompt diagnosis is very important, which means minimal invasive
dation (27%).
technics with histological examination, not cultures only. Therapy
Conclusions Pulmonary mucormycosis usually appears as a pleural
should be multimodal and includes agressive surgery combined with
effusion and unilateral mass with the halo sign followed by transfor-
liposomal/lipid formulations of ampho B. Posaconasol can be a com-
mation in mass with the reversed halo sign. Multilobar consolidation
plimentary/step-down therapy.
and small ill-defined infiltrates are less common.

ª 2015 The Authors

152 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P282 P283

Delphi-based study and selection of key risk factors for High frequency of HIV-seronegative patients colonized by
invasive fungal infection (IFI) in hematologic patients Pneumocystis jirovecii in a Community Central Hospital in
L. Vázquez,1 I. Ruiz,2 M. Lizasoaın,3 J. Gayoso,4 N. di Benedetto5 Lisbon, Portugal
and M. Salavert5 O. M. G. Matos,1 M. C. Carvalho,2 F. Esteves3 and
1
Hospital Universitario de Salamanca, Salamanca, Spain; A. Galzerano2
2
Hospital Vall d0 Hebron, Barcelona, Spain; 3Hospital Universitario 1
Instituto de Higiene e Medicina Tropical, Lisboa, Portugal;
12 de Octubre, Madrid, Spain; 4Hospital Universitario Gregorio 2
Hospital de Egas Moniz, Centro Hospitalar de Lisboa Ocidental,
Maran~on, Madrid, Spain and 5Hospital Universitario La Fe, Lisboa, Portugal and 3Faculdade de Cie^ncias Medicas,
Valencia, Spain Universidade Nova de Lisboa, Lisboa, Portugal

Introduction New risk factors for IFI have been identified in recent Pneumocystis jirovecii is an important opportunistic agent that causes
years and should be classified, in order to improve patient manage- one of the most severe respiratory infections in immunocompromised
ment and prognosis. patients, known as Pneumocystis pneumonia (PcP). Although PcP is
Objective: The purpose of this study was to identify key risk factors commonly associated with HIV infection, the numbers of PcP cases
for the development of IFI caused by filamentous fungi (particularly and P. jirovecii asymptomatic carriers are increasing among HIV-
of the Aspergillus genus) in oncohematologic patients in various clini- seronegative patients with other predisposing immunodeficiencies. In
cal settings, excluding allogeneic transplants. the last decades, molecular techniques have allowed the detection of
Methods A national Delphi-based prospective study was conducted very low burdens of P. jirovecii, usually not detectable by the classic
during November 2014, in order to reach a consensus. All risk fac- microscopic methods.
tors were identified following a literature review and then assigned to The aim of this study was to determine the frequency of P. jirovecii
the following groups: allogeneic hematopoietic stem-cell transplanta- infection in HIV-seronegative Portuguese patients from a community
tion (HSCT), acute leukemia/myelodysplastic syndrome, and lym- central hospital in Lisbon, using molecular approaches.
phoma/multiple myeloma. The study was performed anonymously A total of 274 respiratory specimens were obtained from HIV-
by e-mail contact with hematologists. A risk factor was considered seronegative patients, underlying medical conditions included: 139
key for a specific group when at least 70% of experts rated the risk malignancies, 45 chronic obstructive pulmonary diseases (COPD), 37
as ‘maximum’or ‘high’in their response. pneumonias (not PcP), 17 tuberculosis (TB), 16 autoimmune dis-
Results The panel was composed of 42 hematologists, 28 (66.7%) of eases, nine solid-organ transplantation and 11 other causes.
whom completed the questionnaire. Risk factors associated to Allo- All samples were analyzed by real-time PCR (RT-qPCR) targeting
HSCT were presented at EBMT 2015 and therefore the following the P. jirovecii nuclear single-copy kexin-like serine protease (KEX1)
table lists the results for acute leukemia/myelodysplastic syndrome, gene and by nested-PCR directed to the P. jirovecii mitochondrial
and lymphoma/multiple myeloma multicopy large-subunit rRNA (mtLSU rRNA) gene.
Conclusions The Delphi method is a useful tool to determine and The analysis of the results, indicate that P. jirovecii DNA was
classify key risk factors for IFI caused by filamentous fungi in patients detected in 51 (36.7%) malignancies, 20 (44.4%) COPD, nine
with hematologic cancer. This identification can aid the management (24.3%) pneumonias (not PcP), one (5.9%) TB, five (31.2%) autoim-
of patients at high or changing risk for IFI, when deciding on pro- mune diseases, eight (88.9%) solid-organ transplantation and four
phylaxis adjustments or initiation of early antifungal treatment. The (36.4%) other causes. There was a statistically significant association
method can be used to construct and validate a score with the risk between solid-organ transplantation HIV-seronegative (P = 0.003)
factors selected, so that the most adequate strategy can be adapted patients and the presence of P. jirovecii DNA.
according to an individual assessment of each case. This study confirms the importance of the HIV-seronegative
patients, with other predisposing immunodeficiencies, colonized by P.
jirovecii in a hospital environment. This population is at risk of devel-
oping PcP or may transmit the pathogen to other susceptible
patients, and may play an important role in the circulation and
transmission of P. jirovecii in the community.

P284

Non-Aspergillus fungal rhinosinusitis at a tertiary-care

hospital and the first report of human infections by
Trichoderma asperellum
L. S. Guazzelli,1 C. B. Severo,2 I. E. Cardoso,3 F. M. Oliveira1 and
L. C. Severo1
1
Irmandade Santa Casa de Misericordia de Porto Alegre, Porto
Alegre, Brazil; 2Universidade Federal de Cie^ncias da Saude de
Figure 1 Porto Alegre, Porto Alegre, Brazil and 3Universidade Federal do
Rio Grande do Sul, Porto Alegre, Brazil

Objective Describe a cohort of 27 cases of fungal rhinosinusitis

caused fungi other than Aspergillus, which have been diagnosed at
university hospital in Porto Alegre, Brazil during a 24-year period,
with a particular emphasis on their etiologic agent and the predispos-
ing factors. In this series we also report the first case of rhinosinusitis
and human infection by T. asperellum.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 153
Poster Presentations

Methods A retrospective study was conducted in our institution rhinosinusitis, highlighting the main clinical, etiological and diagnos-
(1989–2014). For mycological diagnoses, paranasal tissue obtained tics findings, to alert clinicians.
at surgery was subjected to histopathology examination and sent for
fungal cultures, patients must have fulfilled the following criteria:
positive nasal sinus cultures and/or biopsy specimens demonstrating
fungal hyphae. P285
Results Fungal cultures were obtained in 20 cases and showed no
growth in 07 cases. Classification of mycotic disease of the nose and
paranasal sinuses in invasive (9) and non-invasive (18) based on Fusarium infections in pediatric oncology patients: 13
clinical, radiological, and histopathological. A total of 14 of 27 cases years’ experience in a single Brazilian institution
were in women, and 13 cases were in men. The mean age of F. Carlesse,1 A. Seber,2 M. L. Martino Lee,1 S. S. Gonc
alves,3
patients was 45.26 years. The most common pathogens were Histo- V. G. Zecchni,1 H. M. Lederman1 and A. L. Colombo3
plasma capsulatum (n = 4), Scedosporium apiospermum (n = 2), Al-
ternaria alternata (n = 2), Schizophyllum commune (n = 2),
1
Oncology Pediatric Institute; Federal University of Sao Paulo, Sa~o
Pseudallescheria boydii (n = 1), Penicillium sp (n = 1), Absidia corymb- Paulo, Brazil; 2Samaritano Hospital, Sa~o Paulo, Brazil and
ifera (n = 1), Xylaria enteroleuca (n = 1), Trichoderma asperellum
3
Federal University of Sao Paulo, Sa~o Paulo, Brazil
(n = 1), T. harzianum (n = 1), T. viride (n = 1), Fusarium solani
(n = 1), Cladosporium sp (n = 1) and Cryptococcus neoformans (n = 1). Background Fusarium species are ubiquitous moulds that may
From the ones that have non-growth, four was classified as hyalohy- cause severe opportunistic infections in immunosuppressed patients.
phomycosis and three mucormycosis by the histopathological find- In Brazil this pathogen is one of the most common molds causing
ings. In addition, we describe the first well-documented case of invasive fungal disease in patients with hematological malignancies
rhinosinusitis and human infection by T. asperellum, in a 44-year-old and/or undergoing hematopoietic stem cell transplantation (HSCT).
woman, with a history of asthma, chronic rhinosinusitis, and allergic Infections occur by inhalation of airborne conidia or skin breakdown
rhinitis since childhood. due trauma, burns or central lines. There is scarce literature data on
Conclusions Fungal infection of the paranasal sinuses is an uncom- fusariosis in pediatrics.
mon disease. Aspergillus and Mucor are the most commonly impli- Aim To describe clinical and epidemiological aspects of 15 consecu-
cated fungal organisms in invasive rhinosinusitis. Nevertheless, tive pediatric patients with invasive fusariosis in a single institution.
numerous fungi can colonize the paranasal sinuses, and it is not sur- Methods We reviewed all culture-proven Fusarium sp infections
prising that many them can cause symptomatic infections, but rarely diagnosed at the Pediatric Oncology Institute (IOP/GRAACC-UNI-
documented. It is important to report this series of non-aspergillus FESP), Sao Paulo, Brazil. The case definition was based on EORTC/

ª 2015 The Authors

154 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

MSG criteria. Fungal isolates were identified by phenotypic and antigen (Platelia Candida, Bio-Rad, California, USA). All tests were
molecular methods. performed according to the manufacturer’s instructions.
Results From 2002 to May 2015, 15 children were diagnosed with Results All 7 detected IFD cases were IC; 4 were proven and 3 prob-
invasive fusariosis. The mean age was 7 years (9 months–18 years) able IFDs and were observed mainly in leukaemia patients (3F/4M,
and 7 were male. Fusarium oxysporum caused seven catheter-related 1.7–16.3 years, 5 ALL, 1 AML, 1 NHL). The invasive candidiasis
bloodstream infections (BSI) (Group 1 -Table 1). Fusarium solani was was detected as candidaemia (29 C. albicans, 19 C. fabianii), hepa-
isolated in seven out of eight disseminated tissue infections and F tolienal with/without affection of other organs (3x) and pulmonary
oxysporum in one (Group 2- Table 2). Group 1 (N = 7) included (1x). Glucan was positive in all 7 patients with IC, false positivity
patients with mean age of 2.5 years (9 months-8 years), 6/7 had an was observed in 13 patients (test evaluation: sensitivity 100%, speci-
underlying solid tumor and only one patient was neutropenic at the ficity 50%). The onset of glucan positivity correlated with the blood
time of the positive blood culture. Patients had positive cultures culture or PCR positivity or with the development of the hep-
drawn from the central line, catheter tips and swab from the port-a- atosplenic infiltrates after restoration of haematopoiesis in pancy-
cath reservoir. All peripheral blood cultures, chest CT and serum topenic patients. The long-term evaluation of glucan in single patient
galactomannan were negative. All patients had the central line showed persistent positivity in IC cases. Candida PCR was positive in
promptly removed and voriconazole was the therapy of choice in all 4 patients (39 Candida spp., 19 C. glabrata). PCR reaction was inhib-
but one patient who received liposomal amphotericin B. All patients ited in 1 patient in sample correlating with the onset of the disease
survived without any further complication. Group 2 included chil- and there were 2 false positive patients detected (test evaluation:
dren with disseminated tissue infections (N = 8) with mean age of 80%, 92%). In 2 IC cases PCR was the only proof of the pathogen.
11years, 4 were male, 7/8 had underlying acute leukemia: 5 had Mannan was positive in 4 IC (test evaluation: 57%, 88%).
active disease and 2 were in remission undergoing allogeneic HSCT. Conclusion The study confirmed accessory value of new methods in
Most patients (6/8) were receiving prednisone (mean dose of 2.5 IFD diagnostic to the conventional methods. In our cohort, we con-
mg kg day
1) and neutropenia was present in 6, lasting for 5 to 50 firmed all IC cases by glucan; in one case it was the only positive
days before the diagnosis of fusariosis. The infection involved lungs technique. PCR helped identify the etiological agent in 2 additional
(7), skin (6), sinus (1), and liver (1). Five had tissue involvement and cases.
a positive blood culture and 3 only positive tissue biopsies. Galac- Acknowledgement The study was supported by Renishaw Diagnos-
tomannan was tested in 5 patients and positive in two. It was possi- tics Ltd, Glasgow, UK, Associates of Cape Cod, East Falmouth, MA,
ble to identify the portal of entry in 6 patients: airway in 5 and USA and by the project of Ministry of Health of the Czech Republic
central line in one. Only one (non-neutropenic) patient survived, but for conceptual development of research organization 00064203
Fusariosis was the immediate cause of death in just 1/8 cases. (University Hospital Motol, Prague, Czech Republic)
Conclusion This is the largest case-series of invasive Fusarium infec-
tion in a pediatric oncology cohort. Catheter-related blood stream
fusariosis caused by F. oxysporum in non-neutropenic patients has
excellent outcome with the central line immediately removed and P288
systemic therapy. Patients with disseminated Fusarium tissue infec-
tions and underlying hematologic malignancies, on steroids or under-
going HSCT had high mortality. Galactomannan presented low Risk factors and clinical outcome of breakthrough yeasts
sensitivity to diagnose fusariosis in this pediatric setting, especially bloodstream infections in patients with hematological
for F. oxysporum infection. malignancies in the era of new antifungal agents
S. H. Kim,1 J. Choi,1 S. Y. Cho,2 D. Lee,2 S. H. Park,1
S. M. Choi1 and J. Yoo1
1
College of Medicine, The Catholic University of Korea, Seoul,
P287 South Korea and 2College of Medicine, Catholic Univ. of Korea,
Seoul, South Korea
Retrospective evaluation of new diagnostic methods for
the detection of invasive candidiasis in paediatric Objectives In recent years, breakthrough candidemia constitutes a
haematooncology patients substantial proportion of candidemia in patients with hematological
V. Chrenkova,1 P. Keslova,1 P. Hubacek,1 J. Tkadlec,1 malignancies, ranging from 19% and 72%. In the same population,
L. Sramkova,1 P. Sedlacek2 and P. Drevinek1 the rare yeasts bloodstream infections (BSIs) are increasingly seen
1 and associated with high mortality rate. This study was performed to
Charles University and Motol University Hospital, Prague, Czech
identify the risk factors and clinical outcome of breakthrough yeast
Republic and 2Charles University and University Hospital Motol, BSIs in these patients in the era of new antifungal agents.
Prague, Czech Republic Methods We reviewed medical records of all consecutive patients
with hematological malignancies developing yeasts BSIs at a ter-
Objectives Invasive candidiasis (IC) is important cause of morbidity tiary care center from January 2009 to December 2014. Break-
and mortality in immunocompromised host. The detection of invasive through yeasts BSIs were defined as recovery of at least one yeast
fungal diseases (IFD) remains a challenge and there are few data in by blood culture in patients receiving one or more systemic antifun-
paediatric patients published. Therefore we performed retrospective gal agents for ≥3 days before the first positive blood culture. We
study evaluating two methods of detection: (1?3)-b-D-glucan and performed a case-control study comparing breakthrough BSIs with
PCR, as they are rarely published in children. de novo BSIs.
Methods We tested 157 serum samples of 33 patients (20F/13M, Results In this population-based, prospective, observational study,
age 0.7–20.1, median 6.64 years) in high and intermediate risk of 21 (43%) of 49 patients with yeasts BSIs met the definition for
IFD treated in period 07/2012 to 06/2013 in Department of Paedi- breakthrough yeasts BSIs. Overall, the most common yeast was Can-
atric Haematology and Oncology, Motol University Hospital, Prague, dida tropicalis (12 of 51 isolates, 24%). Among 4 non-Candida yeasts
Czech Republic. All cases of IFD were classified according to EORTC/ (2 Saccharomyces cerevisiae, 1 Trichosporon asahii, and 1 Pseudozyma
MSG 2008. Serum samples were retrospectively tested for the pres- aphidis) isolated from 4 patients, 3 cases were breakthrough BSIs.
ence of glucan (Fungitell, Associates of Cape Cod, East Falmouth, Patients with breakthrough BSIs were receiving various kinds of anti-
MA, USA) and Candida spp. DNA (RenDx Fungiplex Assay, Renishaw fungal agents (fluconazole [n = 11], itraconazole [n = 3], posacona-
Diagnostics Ltd., Glasgow, UK). DNA extraction was performed by zole [n = 1], amphotericin B [n = 3], caspofungin [n = 2], or
QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Obtained micafungin [n = 1]). In multivariate analysis, acute leukemia or
results were added to results of the standard methods used in our myelodysplastic syndrome as underlying hematological malignancies
hospital: blood culture (Becton Dickinson, NJ, USA) and mannan (OR 9.735, 95% CI 1.916–49.449, P = 0.006) and neutropenia

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 155
Poster Presentations

within 3 days prior to yeasts BSIs (OR 11.274, 95% CI 2.062– (HSCT) are at risk for the development ofInvasive Aspergillosis (IA).
61.633, P = 0.005) were significantly associated with breakthrough This complication has often been reported to yield a high mortality
BSIs. The proportions of C. krusei and C. tropicalis were significantly rate. However, patients with a generally unfavourable prognosis with
different between breakthrough BSIs and de novo BSIs (32% vs. 3%, regard to their hematological condition have a higher probability to
P = 0.015; 5% vs. 38%, P = 0.007, respectively). As the first-line develop IA as well as a greater risk of death due to other disease
antifungal agents, amphotericin B (57%) was most frequently admin- related causes. To assess the short and long term association of IA
istered, followed by echinocandins (22%) and triazoles (20%). The with mortality in this vulnerable patient population, full considera-
appropriateness of first-line antifungal regimen was not different tion of changing patient characteristics over time is necessary to
between breakthrough BSIs and de novo BSIs (86% vs. 93%, obtain reliable estimates of the correlation of IA with death.
P = 0.639). The 6-week and overall mortality rates of breakthrough Methods Consecutive patients who initiated remission-induction
yeasts BSIs were not different from de novo BSIs (33% vs. 43%, therapy for AML or MDS at the Leiden Academic Medical Centre
P = 0.564; 62% vs. 64%, P = 1.000). Independent predictors for 6- were included. During the period of study an assertive protocol for
week mortality were refractory neutropenia (HR 64.488, 95% CI diagnosis of suspected fungal infection was executed instead of stan-
10.488–368.852, P < 0.001) and Pitt bacteremia score (HR 1.886, dard antifungal prophylaxis. Population characteristics and data
95% CI 1.142–3.114, P = 0.013). acquisition was previously described and published. IA was classified
Conclusion Breakthrough yeasts BSIs were associated with acute according to the 2008 revised European Organization for Research
leukemia or myelodysplastic syndrome and neutropenia. The distri- and Treatment of Cancer criteria. We studied the effect of IA on risk
bution of yeasts species in breakthrough BSIs was significantly differ- of death by means of four different survival analyses, using the Cox
ent from de novo BSIs. However, breakthrough BSIs did not show a proportional hazards model, considering (1) time from diagnosis to
higher mortality rates compared with de novoBSIs. death before HSCT, (2) time from diagnosis to HSCT, (3) time from
HSCT to death after HSCT and (4) time from diagnosis, both before
and after HSCT.
Adjustment for confounding variables was performed (the use of
P290 granulocyte colony stimulating factor (GCSF), age, sex, WHO hema-
tologic risk category, number of chemotherapy courses needed to
accomplish complete remission, previous lung disease, secondary or
A new time dependent analytical approach for assessment relapsed hematologic disease and a day-by-day appraisal of the pres-
of the impact of Invasive Aspergillosis on short and long- ence of neutropenia). The presence of neutropenia was entered as
term survival time dependent covariate. The analyses were performed using STATA
R. J. van de Peppel, P. A. von dem Borne, S. Le Cessie and v 12.0.
M. G. J. de Boer
Leiden University Medical Center, Leiden, the Netherlands

Objectives Patients with hematological malignancies treated with

chemotherapy courses and haematopoietic stem cell transplantation

ª 2015 The Authors

156 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Results In 55 of 167 enrolled patients, at least possible IA was diag-

nosed at some point during treatment. Hazard ratios for death after
diagnosis of IA were calculated by using the above described models,
and adjusting for the confounding variables (table 1). Hazard ratios
(HR) and 95% Confidence Intervals (95%CI) on mortality after the
diagnosis of IA within 30 days, between 30 and 60 days, between
60 and 90 days or more than 90 days were 5.0 (2.7–9.4), 3.1 (1.3–
7.6), 2.4 (0.83–7.0) and 0.97 (0.57–1.6) respectively in model no.4.
The HR to reach the point of stem cell transplantation was not sig-
nificantly altered by the occurrence of IA prior to HSCT (figure 1).
Conclusion The results provide new insight in short and long-term
survival of patients diagnosed with IA. By applying a new analytical
approach, we found that the association of IA with mortality
remained substantial. However, while an increased risk of death was
significantly present in the first and second month after diagnosis of
IA, no unfavourable association with mortality was observed in the
later course of treatment. Other patient characteristics conferred
comparable HRs for death. Furthermore, the occurrence of IA did
not affect the probability of attaining HSCT in our population.

P291

Galactomannan Testing and Aspergillus PCR in same-day

bronchoalveolar lavage and blood samples obtained from
patients with hematological malignancies at risk for
invasive mould infection
S. Eigl,1 J. Prattes,2 F. M. Reischies,3 R. B. Raggam,4 B. Spiess,5
M. Reinwald,5 D. Buchheidt,6 C. R. Thornton,7 R. Krause,1
H. Flick,1 P. Neumeister8 and M. Hoenigl2
1
Medical University Hospital of Graz, Graz, Austria; 2Medical Conclusions Our results indicate that among patients with hemato-
logical malignancies at risk for IPA, PCR and GM testing should
University of Graz, Graz, Austria; 3Meduni Graz Section of
preferably be performed in samples from the site of infection (i.e. BAL
Infectious Diseases and Tropical Medicine, Graz, Austria; or biopsy), as the diagnostic yield when these tests are performed in
4
Medical University Hospital Graz, Graz, Austria; 5Heidelberg blood might be significantly lower.
University Hospital, Mannheim, Germany; 6Mannheim University
Hopsital, Mannheim, Germany; 7University of Exeter, Exeter,
United Kingdom and 8Medical University of Inssbruck, Graz,
Austria
P292
Objectives Invasive pulmonary aspergillosis (IPA) is a live threaten-
ing disease in patients with hematologic malignancies. Early and reli- Cost of antifungal drugs in different haemato-oncology
able diagnosis is needed for rapid initiation of first line antifungal settings
(AF) therapy, which is crucial in order to reduce the mortality rate. S. G. Agrawal and T. L. Dexter
In recent years galactomannan antigen testing (GM) and also Asper- Barts Health NHS Trust, London, United Kingdom
gillus PCR have become increasingly important for IPA diagnosis.
Whether or not these tests need to be performed with bronchoalveo-
lar lavage fluid (BALF; i.e. primary site of infection), or testing of Objectives Invasive fungal disease (IFD) remains a difficult area in
blood samples is sufficient, remains, however, a matter of debate. Haemato-Oncology, with significant clinical and financial impact.
Methods We evaluated the diagnostic performance of GM ELISA, This study calculates the cost of antifungal medications per admis-
Aspergillus PCR and other tests (e.g. Panfungal PCR, Aspergillus speci- sion over a 5 year period, costs per type of leukaemia and transplant,
fic lateral flow device test, culture) for invasive mould infection (IMI) and the costs during induction versus subsequent cycles of
by using bronchoalveolar lavage fluid samples and blood samples chemotherapy. In addition, the EORTC scores for each episode were
obtained at the same day (within 0–4 h). All patients with hematologic correlated with the cost of antifungal drugs.
malignancies, who received bronchoscopy and BALF sampling Methods A retrospective analysis was performed of 528 inpatient
between April 2014 and April 2015 at the Medical University of Graz, episodes (representing 303 patients) in the Division of Haemato-On-
Austria were screened for includion in this prospective study. IPA and cology at St Bartholomews Hospital between 2004 and 2009. EORTC
IMI were defined according to the revised EORTC/MSG criteria. scores (2008) were calculated. Anti fungal prices were extracted
Results 42 bronchoalveolar lavage/biopsy samples from 34 patients from the local formulary (July 2014) as cost per mg of the drug.
were included in the final analyzes. One patient had proven invasive Costs were calculated per episode (admission) and statistical signifi-
mucormycosis (with positive panfungal PCR result from biopsy speci- cance analyses applied to determine the differences between cohorts
mens, but negative PCR result from blood). Of the remaining 41 of patients. The data set includes 118 episodes where voriconazole
cases, 8 (20%) had probable or proven IPA, 9 (22%) possible IPA, was used principally for primary prophylaxis of acute meloid leukae-
while 24 (58%) did not fulfill IPA criteria. Sensitivity, specificity, pos- mia (AML) induction chemotherapy according to local protocol.
itive predictive value, negative predictive value, and diagnostic odds Results Of a total 528 episodes, 408 were described in patients with
ratio of the different tests for diagnosing probable/proven IPA are AML or high-risk myelodysplastic syndrome. Acute lymphoblastic
shown in table 1. The combination of positive GM result and/or leukaemia (ALL) was the primary diagnosis in 37 episodes. Prophy-
Aspergillus PCR was significantly more sensitive for detection of prob- lactic antifungals alone were prescribed in 320 episodes, while in
able and proven IPA when performed in BAL (75% sensitivity), when 157 episodes prophylactic and treatment antifungals were given.
compared to same day blood specimens (sensitivity 13%; P = 0.04). There were 137 episodes of bone marrow transplants including 111

ª 2015 The Authors

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Poster Presentations

reduced-intensity allografts (sibling and unrelated), 12 ablative allo- P293

grafts (unrelated and sibling) and 14 autografts.
In the whole cohort (n = 528), the mean total cost of prophylactic
antifungals was £1150.80, compared to the mean total cost of treat- Fungal infections by filamentous fungi in hematologic
ment, which was £1762.10. The mean total cost of antifungal drugs patients at high risk. It’s useful looking for fungal DNA in
in all 528 episodes was £2912.90. serum?
The total antifungal cost per episode associated with AML inten- V. Favuzzi, R. Koncan, A. Centonze, G. Lo Cascio, C. Tecchio,
sive chemotherapy (n = 408) was higher (mean £3196.60) than I. Ferrarini and A. Sorrentino
those with ALL (n = 37; £2706.70) (P = 0.509). Moreover, antifun-
gal therapy during induction chemotherapy for AML (n = 68) cost Azienda Ospedaliera Universitaria Integrata di Verona, Verona,
more (£3643.60) than during post induction regimes (n = 280; Italy
£2817.70) although this difference is not statistically significant
(P = 0.123). Objectives The aim of this study was to compare three different
The antifungal costs for different transplants were a mean £3262 commercial available RT-PCR assays in hematological patients at
vs £2769.70 vs £1720.20 for ablative allografts (n = 111), reduced high risk of fungal infection for early diagnosis of invasive aspergillo-
intensity allografts (n = 12) and autografts (n = 14), respectively. sis (IA). The objective is to assay the sensitivity of each of the kits in
In 118 episodes where voriconazole was used as prophylaxis, the the three methods of extraction and amplification.
total antifungal costs for these episodes were significantly more Methods From January to March 2014, 86 samples of serum from
expensive than the mean total antifungal cost for the 353 episodes patients admitted to the CTMO (Center Bone Marrow Transplant)
where fluconazole was used as primary prophylaxis (£3861.50 vs and at the Hematology of Verona were analyzed; the patient were
£2330.60) (P ≤ 0.001). Using the 2008 EORTC/MSG scoring system, University of Verona, Italy selected according to seric fungal
total costs for treatment and prophylaxis were: £1 351 302.60 in biomarkers results: 59 negative for galactomannan assay (GM), 27
the ‘none’category (i.e, no evidence of IFD; n = 489; mean of positive; 58 negative for (1-3) Beta-D-Glucan (BG) assay, 28 positive.
£2763.40 per episode); £115 410.40 with a ‘possible’ score (n = 28; The detection of serum DNA of filamentous fungi was per-
mean £4121.80); £35 225.60 for a ‘probable’ score (n = 4; mean formed using three diagnostic kits: Liferiver TM fungus real time
£8806.40); and £36 056.30 for a ‘proven’ IFD (n = 7; mean PCR kit (Shanghai ZJ Bio -Tech Co) which provides extraction and
£5150.90). amplification reagents.
Conclusion These data provide average costs for antifungal drug MycoGENIE Aspergillus fumigatus Real-Time PCR kit. DNA extrac-
use for AML, ALL and different types of transplant. Mould-active pro- tion was made automatically with AutoMag solution (Automated
phylaxis was associated with increased total antifungal drug expendi- DNA Extraction-Ademtech, Pessac, France).
ture. Interestingly, the vast majority of antifungal drug expenditure Myconostica Myc Assay TM Aspergillus Cepheid Smartcycler serum.
occurs in episodes classified as unlkely to have an IFD by the In this case extraction was performed with High PCR template prepa-
EORTC/MSG criteria. ration Kit by Roche Diagnostics (Mannaheim, Germany).
All Real time PCR were run on a Smart Cycler II, software version
3.0 (Cepheid).
We have analyzed the three systems of amplification with the
three extraction kit to evaluate the efficacy
Results To analyze the limit of detection of the three assays we per-
formed the Real-Time PCR on serial dilutions of DNA of Aspergillus

ª 2015 The Authors

158 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

fumigatus in water, starting from a concentration of 20 lg ul
1 to a therapy (ART) was commenced for 5 patients; 2 were already on
final concentration of 2 9 10
7 pg ll
1. ART at screening.
The amplification kit gave the following results: Real-Time PCR Conclusion The NNT to detect a case of new antigenemia among
Myconostica Myc Assay TM were positive up to 2 9 10
4 pg ll
1 of children and adolescents was comparable to the NNT for adults. Chil-
DNA, Real-Time PCR MycoGENIE 2 9 10
6 pg ll
1, Real-Time PCR dren and adolescents with CD4 count <100 cells ll
1 could also be
Liferiver TM 5 pg ll
1. included in a cryptococcal screen-and-treat program.
We analyzed 19 patients. For each patient multiple sera were
analyzed.
The results are summarized in table 1.
Conclusion Latest guidelines for the diagnosis of IA do not recom- P295
mend any molecular test for the diagnosis, neither any preferable
biology sample because lack of standardization. Diagnostic performance of 1,3-beta-d-glucan Serum
We compared three commercial kits using serum from at risk Screening in Patients receiving Hematolopoietic Stem Cell
hematological patients. Our study showed comparable results for Transplantation
Myconostica and MycoGENIE kit, even with different extraction sys-
F. M. Reischies, J. Prattes, S. Eigl, A. List, R. B. Raggam,
tems and even with some reinterpretation of positivity cut off. Lower
sensitivity exhibit the Liferiver TM kit, which showed an inferior per- I. Zollner-Schwetz, T. Valentin, H. Flick, A. Woelfler, F. Prueller,
formance also in the analysis of the limits of detection. This prelimi- R. Krause and M. Hoenigl
nary results confirm the utility of PCR in early diagnosis of IA. Meduni Graz Section of Infectious Diseases and Tropical
Medicine, Graz, Austria

Background 1,3-beta-d-glucan (BDG) is used as a biomarker for

P294 invasive fungal infections (IFI). Patients receiving allogeneic or autol-
ogous hematopoietic stem cell transplantation (HSCT) are considered
Reflex Cryptococcal antigen screening among children and a highly vulnerable group for IFI development, therefore early detec-
adolescents in two districts in Gauteng province, South- tion and treatment of IFIs are crucial to improve survival. We evalu-
Africa ated the diagnostic performance of serum BDG screening in HSCT
recipients.
C. Sriruttan,1 N. Naidoo,1 R. Mpembe,2 R. Smith,3 G. Greene,3
Methods 33 HSCT recipients were prospectively enrolled in this
A. Adelekan,3 V. Deyde,3 S. Oladoyinbo,4 T. Maotoe,4 study, which was performed between September 2014 and May
T. M. Chiller3 and N. P. Govender2 2015 at the Medical University hospital of Graz, Austria. Serum BDG
1 screening was performed twice weekly (total 225 samples) by using
National Institute For Communicable Diseases, Johannesburg,
South Africa; 2National Institute for Communicable Diseases, the Fungitell assay. All samples were classified according to the
Johannesburg, South Africa; 3Centers for Disease Control and 2008 EORTC/MSG criteria (at the time the sample was obtained),
Prevention, Atlanta, USA and 4United States Agency for with serum BDG results being not considered for classification. To
International Development, Pretoria, South Africa evaluate the diagnostic performance of BDG testing for IFI we calcu-
lated sensitivity, specificity, negative predictive value (NPV), positive
predictive value (PPV) and diagnostic odds ratio (DOR) for all
Objective To compare the number needed to test (NNT) to detect a samples.
case of new cryptococcal antigenemia for children, adolescents and Results 27 (71.1%) patients received allogeneic HSCT and 11
adults during implementation of reflex cryptococcal antigen (CrAg) (28.9%) autologous HSCT. 24/38 patients were male and 14/38
laboratory screening in South Africa. Current guidelines recommend female. Acute myeloid leukemia (n = 17) was the most frequently
screening for adults only; however, reflex laboratory testing was per- observed underlying disease, followed by non-hodgkins lymphoma
formed irrespective of age, enabling determination of NNT for chil- (n = 10), acute lymphatic leukemia and myelodisplastic syndrome
dren and adolescents. (each 4) and multiple myeloma (n = 3). 167/225 (74%) serum sam-
Methods All persons with a CD4 count <100 cells ll
1 were reflex- ples were collected from patients under ongoing antifungal prophy-
ively screened for CrAg at 106 healthcare facilities in two Gauteng laxis. Performance of BDG screening is displayed in Table 1.
provincial districts from September 2012 through August 2014. Lab- Conclusions Our data suggest that serum BDG screening in HSCT
oratory/clinical data were collected from patients with a positive patients may be associated with a very high NPV of over 99% and
plasma CrAg test at 45 facilities. Cases of incident cryptococcal therefore a helpful tool to rule out IFI.
meningitis (CM) were reported to national surveillance. CrAg screen-
ing and CM surveillance data were cross-matched to determine if
screened patients had prior CM. Children, adolescents and adults
were defined as persons aged ≤9 years, 10–19 years and ≥20 years,
respectively.
P296/M11.2
Results The overall prevalence of antigenaemia was 4.5% (820/
18357). Among those with recorded age, 3.7% (637/17149) were Paecilomyces lilacinus and Fusarium solani central line
aged ≤19 years, 31 (4.9%) of whom had a positive plasma CrAg test; infection in a paediatric patient with acute lymphoblastic
24/31 (77.4%) were adolescents. The prevalence of antigenemia leukaemia
among adults, adolescents and children was 4.5% (735/16512), E. Demertzi,1 J. Hatcher,2 V. Schwierzeck,3 W. Alsaud3 and
5.0% (24/483) and 4.5% (7/154) respectively. The number needed
M. Petrou1
to test (NNT) to detect a case of new antigenemia among adults, ado- 1
lescents and children was 27 (16512/623), 28 (483/17) and 31 Imperial College, London, United Kingdom; 2Imperial College
(154/5) respectively. Healthcare NHS Trust, London, United Kingdom and 3Chelsea &
For 31 children/adolescents with a positive plasma CrAg test, the Westminster Hospital, London, United Kingdom
median CD4 count was 13 cells ll
1 (IQR, 8–31). Nine of these 31
(29.0%) had prior CM. Seventeen of 22 (77.3%) cases of new antige- Objectives Fungaemia due to moulds is rare and the presence of
naemia occurred among adolescents. Clinical data were available for more that one mould at the same time is exceedingly rare. We
9 of these 22 cases; 2 were symptomatic. All 7 asymptomatic describe a case of central line infection caused by Paecilomyces lilaci-
patients were prescribed fluconazole, CSF specimens were negative in nus and Fusarium solani in a paediatric patient with acute lym-
all 4 where lumbar puncture was performed and no subsequent labo- phoblastic leukaemia treated with voriconazole and central line
ratory-confirmed CM was recorded for these 7 patients. Antiretroviral removal.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 159
Poster Presentations

Methods Paecilomyces spp. and Fusarium spp.both cause hyalohy- mounted on glass slides and examined with a light microscope. Each
phomycosis. Paecilomyces spp. are found ubiquitously within the entire coverslip was scanned at a magnification of 9 1000. Results:
environment. These are uncommon pathogens with P. lilacinus and May-Grunwald Giemsa stain permitted the visualization of para-
P. variotii the two species most frequently associated with human ill- sitophorous vacuoles (PV) containing mature microsporidian spores
ness. Ocular and cutaneous disease are the most common presenta- in the cytoplasm of the infected cells. Conclusion: The stain used in
tions, however fungaemia is reported and associated with this study is commonly used in hematology laboratory routine but
haematological malignancies and indwelling prosthetic material. has not been used to identify microsporidia spores. This result sug-
Fusarium spp. are the most common cause of fungal keratitis gests that May-Grunwald Giemsa is adequate for visualize the PV
worldwide. In patients with immunodeficiency Fusarium spp. can containing spores of microsporidia inside the infected cell cultures to
cause disseminated disease and is second only to Aspergillus spp. study this pathogenic fungi in vitro.
within this population. Cutaneous manifestations of fusariosis can
precede fungaemia and blood cultures may be positive in up to 40%
of cases.
Both fungi were identified from the cultural characteristics and P298
microscopic appearance as well as MALDI-TOF. Susceptibility to nine
antifungals was performed using the Clinical and Laboratory Stan-
dards Institute (CSLI) recommended method. Encephalitozoonosis in B-cell deficient XID mice
Results A seven years old female was treated for relapsed acute lym- A. Pereira,1 L. Costa,2 A. Saraiva,2 E. Hurtado,2 P. R. Rocha,2
phoblastic leukaemia with methotrexate, cytarabine and cyclophos- D. Spadacci-Morena3 and M. Lallo1
phamide. Chemotherapy was given by central venous access via a
Hickmann line. She attended our institution with neutropenic fever
1
Paulista University and Centro Universitario Sao Camilo, Sa~o
with no localising symptoms or signs. She was at the end of a once Paulo, Brazil; 2Paulista University, Sa~o Paulo, Brazil and 3Instituto
daily outpatient teicoplanin course for a recent line infection episode, Butantan, Sa~o Paulo, Brazil
when blood cultures via the Hickmann line had grown Bacillus sp.
and Rhodococcus sp. She was commenced on piperacillin-tazobactam Encephalitozoon cuniculi is an opportunistic pathogenic fungi for peo-
and vancomycin with resolution of the fever. Seventy 2 h after ple with AIDS or other immune deficiencies. The adaptive immune
admission blood cultures flagged positive and filamentous fungi were response is essential for the elimination of E. cuniculi, but evidence
identified on Gram stain. The microscopic appearance of the fila- indicates that the innate immune response is responsible for initiat-
ments were consistent with Paecilomyces spp. however the culture ing and setting up the pathogen or will not survive. B-1 cells act as
yielded Fusarium spp. which produce chlamydospores when they dis- antigen-presenting cells, phagocytes, produce IL-10 and had other
seminate. As Fusarium solani sporulates faster than Paecilomyces lilaci- immune functions. The possible role of these cells in the dynamics of
nus, extended incubation was required to differentiate the two the inflammatory process of various etiologies is unknown. Objective:
moulds. The MICs of P. lilacinus and F. solani to voriconazole were This study aimed to investigate the role of B-1 cells in E. cuniculi
0.25 and 0.125 mg l
1 respectively however the MIC to ampho- infection. Methods: BALB/c and BALB/c XID (deficient B-1 cells) were
tericin B for P. lilacinus was >8 mg l
1. The patient received intra- inoculated with fungal spores. The B-1 and B-2 cells, macrophages
venous voriconazole and the central line was removed 16 days after and CD4+ and CD8+ lymphocytes obtained from the spleen and peri-
admission. No deep seated focus was identified and she remained well toneal washes were quantities by flow cytometry. The Th1 cytokines
and afebrile after 30 days. and Th2 serum were quantified by flow cytometry by CBA assay.
Conclusion This is the first report, to our knowledge, of a mixed Results: We identified significant increase in macrophages population
fungal central line infection with P. lilacinus and F. solani. This case in the peritoneal cavity of infected BALB/c mice, but there was no
highlights the importance of antifungal drugs susceptibility testing difference in BALB/c XID infected or not. Despite the increase in the
for moulds to target treatment. This allowed administration of single number of CD4+ lymphocytes and CD8+ cells in BALB/c mice XID,
agent voriconazole. Removal of prosthetic material is critical in the these animals were still susceptible to infection. Conclusion: We show
successful management of fungal line infection. that BALB/c XID mice were more susceptible to infection experimen-
tally induced E. cuniculi, so these animals can be used as a biological
model for encephalitozoonosis.

P297

Use of May-Grunwald Giemsa stain for microscopic

P299
evaluation of cell culture inoculated with Microsporidia
in vitro The case of succesful treatment of pulmonary mycosis
A. Pereira,1 A. Saraiva2 and M. Lallo1 caused Exophiala Dermatitidis in a patient with acute
1
Paulista University and Centro Universitario Sao Camilo, Sa~o myeloblastic leukemia (m4)
Paulo, Brazil and 2Paulista University, Sa~o Paulo, Brazil E. Shagdileeva,1 S. Khostelidi,1 O. Ruzhinskaya,2 O. Riabykina,2
O. Uspenskaya,2 T. S. Bogomolova,1 M. V. Rudneva,3
Microsporidia are obligatory intracellular spore-forming microorgan- S. M. Ignatyeva4 and N. Klimko1
1
isms that infect a wide range of invertebrate and vertebrate species. North-Western State Medical University named after I.I.
Traditionally, they were considered protozoa but recently have been Metchnikov, Saint Petersburg, Russia; 2Leningrad Regional
reclassified phylogenetically as fungi. They have been recognized as Clinical Hospital, Saint Petersburg, Russia; 3North-Western State
human pathogen particularly in immunodeficient patients and after Medical University named after I.I. Mechnikov, Saint Petersburg,
the advent of HIV and AIDS, interest in the in vitro culture of them Russia and 4Medical mycology institute named after Kashkin,
has been increased. In our laboratory we have been used in vitro cul- Saint Petersburg, Russia
ture to study the genera and species Encephalitozoon cuniculi and E.
intestinalis in monkey and rabbit kidney cell lines (Vero and RK-13
respectively). Objective: This study aimed to investigate the May- Frequency of invasive mycoses due to rare pathogens in hematologi-
Grunwald Giemsa stain for microscopic evaluation of cells inoculated cal patients has increased in last years. The number of publications
with E. cuniculi in vitro. Methods: Cell lines were incubated in sterile about these mycoses is limited.
circular glass coverships in 24-well plates and were inoculated with Methods A clinical case of successful treatment of pulmonary myco-
spores of E. cuniculi. After the inoculation and incubation, the cover- sis caused by Exophiala dermatitidis in a patient with acute myeloblas-
slips were fixed with methanol, stained with May-Grunwald Giemsa, tic leukemia (M4).

ª 2015 The Authors

160 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Results Male, 56 years old, was admitted to the Leningrad regional organs involvement. Overall and IPA-related mortality were not dif-
clinical hospital 31.10.2014 with mucous membranes hemorrhages, ferent between the two groups.
weakness, and fever up to 38.5 °C. The diagnosis was acute myeloid Conclusion Non-fumigatus consists one-third of culture-positive IPA
leukemia (M4 variant). He was treatment with ‘7 + 30 , ‘HAM’ cyto- in this institute. Multiple organ involvement, especially PNS, was fre-
static chemotherapy. The patient’s condition deteriorated on the quent in non-fumigatus IPA compared to A. fumigatus IPA. Differ-
background of the prescribed treatment (short of breath, rare cough, ences between non-fumigatus and fumigatus IPA should be the focus
persisted subfebrile body temperature). Period of severe neutropenia of future studies with in vitro susceptibility test of Aspergillus isolates
(<0.5 9 109) was 49 days. with large number of patients.
CT scan of the chest was made on 16.12.2014: increased lung
pattern due to the interstitial component in S6 and basal segments of
the lungs. Antibacterial therapy was enhanced (imipenem, van-
comycin, linezolid) with partial clinical effect, then empirical antimy- P301
cotic therapy was prescribed - amphotericin B (1 mg kg day
1)
19.12.2014. On control CT scan (26.12.2014), negative dynamics
was identified, signs of lesions of both lungs (on the boundary of S2 Outcomes of Hematologic Patients with Invasive
and S6, S6, left in S6 and S10 - diameter 4.8 mm, 4.6 mm, 12 mm, Aspergillosis before Allogeneic Stem Cell Transplantation
15 mm, respectively), small hydrothorax on the right (up to S. Y. Cho, D. Lee, J. Choi, H. Lee, S. H. Kim, S. H. Park,
7.2 mm). Test «Platelia Aspergillus EIA» (Bio-Rad) was negative in S. M. Choi, J. Choi and J. Yoo
BAL. At microscopy of BAL the fungal elements not detected. Numer-
ous colonies of black fungus were cultined. The isolate was identified College of Medicine, The Catholic University of Korea, Seoul,
as Exophiala dermatitidis morphologically and by ITS-sequensing. South Korea
Amphotericin B were replaced by itraconazole 400 mg day
1. The
patient CSF. Clinical improvement of the patient and the positive Background Previous invasive fungal infection (IFI) is one of the
dynamics of changes in the lung during a CT study (a minimum set- risk factors for recurrence of fungal infection and mortality in allo-
back of infiltrative changes decrease from interstitial 08.01.2015). geneic stem cell transplantation (SCT) recipients. Until now, there is
Invasive fungal infection has stabilized after two months of no report about the outcome of patients with remained invasive
antimycotic therapy. Due to the presence of risk factors the patient aspergillosis (IA) before SCT. The aim of this study was to evaluate
continued antifungal therapy with itraconazole. the clinical characteristics and outcomes of patients continuing treat-
Conclusion Onco-hematological patients with neutropenia need the ment of IA before the allogeneic SCT for the hematologic diseases.
fiber-optic bronchoscopy with mycological examination BAL, and Method All consecutive cases of IA were reviewed retrospectively
mandatory identification of the pathogen. Successful treatment of from January 2011 to March 2015 at the Catholic Blood and Mar-
fungal infections were requires a combination of adequate antifungal row Transplantation Center. Only proven or probable IA cases in
therapy and correction of neutropenia. adult hematologic patients were included in this study.
Results During the study period, there were 365 patients with pro-
ven/probable IAs, of which 96 patients had experienced IA before
allogeneic SCT. Patients who had a history of IA that had been
P300 resolved (n = 17), coinfected with another fungus (n = 13), partici-
pated in clinical trials (n = 1) were excluded from this study. A total
of 65 patients were included in the analysis. The median age was
A Comparison between Invasive Pulmonary Aspergillosis 49 years (interquartile range [IQR], 42 - 57). Sixty percent (39 of
(IPA) caused by Aspergillus fumigatus and non-fumigatus 65) of patients were male. Median time from the diagnosis of IA to
Aspergillus species SCT was 92 days (IQR, 57 - 130). All of the patients kept taking
S. Y. Cho, D. Lee, J. Choi, H. Lee, S. H. Kim, S. H. Park, voriconazole per oral or intravenously according to the tolerability or
S. M. Choi, J. Choi and J. Yoo severity of the mucositis. Relapse or progression of IA were observed
in 8 of 65 patients (12.3%). Median time to relapse of IA was
College of Medicine, The Catholic University of Korea, Seoul, 253 days (IQR, 114 - 454). These 8 patients experienced relapse of
South Korea underlying hematologic diseases or graft-versus-host disease (GVHD)
that needs ≥ 2 immunosuppressant or high-dose steroid therapy. IA-
Background Aspergillus fumigatus is the most common pathogen related mortality and overall mortality was 9.2% (6 of 65) and
that causes invasive pulmonary aspergillosis (IPA) in hematologic 35.4% (23 of 65), respectively. Independent risk factors for death
patients. However, recent studies have reported an emergence of IPA were refractory state of the underlying hematologic diseases (adjusted
caused by non-fumigatus aspergillus species. The aim of this study odds ratio [OR] 12.072, 95% confidence interval [CI] 2.058 -
was to evaluate the epidemiology, characteristics, and outcomes of 70.796, P = 0.006) and GVHD (adjusted OR 0.134, 95% CI 0.036–
culture-positive IPA and to compare the clinical characteristics of A. 0.504, P = 0.003).
fumigatus and those of non-fumigatus IPA. Conclusion Performing SCT for underlying hematologic diseases
Method All consecutive cases of invasive fungal infections (IFIs) might be feasible during the treatment of IA. Further criteria for the
were reviewed retrospectively from January 2011 to February 2015 clinical conditions of IA patients or optimal timing of SCT should be
at the Catholic Blood and Marrow Transplantation Center. Only cul- studies in this group of patients.
ture-positive IPA cases in hematologic patients were included in this
study.
Results During the study period, there were 361 proven/probable
IFIs, of which, proven/probable IPA and culture-positive IPA were P302
323 (89.5%) and 63 (17.5%) cases, respectively. The isolated patho-
gens consisted of 38 (60.3%) A. fumigatus, 20 (31.7%) non-fumigatus,
and 5 (7.9%) of multiple species with A. fumigatus and non-fumigatus Invasive pulmonary aspergillosis in a kidney transplant
Aspergillus species. There were no significant differences in baseline recipient
patient characteristics, including age, sex, underlying hematologic K. Ozden, A. Karaman, G. Ozturk, B. Aydinli, A. Uyanik,
diseases, treatment for hematologic diseases, severity of neutropenia, E. Cankaya, M. Uyanik, F. Alper and M. Akgun
and graft-versus-host disease between A. fumigatus and non-fumigatus
group. However, as an aspect regarding the manifestation of IPA, Ataturk University, Erzurum, Turkey
two or more organ involvement of IFI was more common in non-fu-
migatus group (2.6% vs. 20.0%, P = 0.041). Paranasal sinus (PNS) Objectives Invasive pulmonary aspergillosis is a fungal disease with
involvement was most common (75%) in IPA patients with ≥ 2 high mortality rate that especially encountered in patients with

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 161
Poster Presentations

voriconazole treatment, serum galactomannan antigen level was

negative. While fever decreased, WBS and CRP levels returned to
normal limits. In the fourth week of intravenous voriconazole, it
switched into oral voriconazole. There was a regression on follow up
chest x-ray and HRCT images of the patients and no complication
occurred (Figure 2).
Conclusion In kidney transplant recipients, invasive pulmonary
aspergillosis should be considered in the patients with the clinical
findings of pneumonia and especially in the ones who did not
respond empirical antibacterial treatment. For the diagnosis, obtain-
ing serum galactomannan antigen and HCRT should be kept in
mind.

P303/M6.4

First report of the bloodstream infection associating with

brain lesion by Schizophyllum commune in Brazil.
M. M. E. Oliveira, A. S. Lemos, M. L. C. Gonc
alves, R. Almeida-
Paes, V. R. G. Valviesse, M. A. S. Lima, C. C. Lamas and
Figure 1. Chest radiography after 10 weeks of treatment R. M. Zancope-Oliveira
Fundac
a~o Oswaldo Cruz- Instituto Nacional de Infectologia
Evandro Chagas, Rio de Janeiro, Brazil

Infections due Schizophyllum commune are rare in humans. The first

report was an onychomycosis case occurred in 1950. Recently, a
review of 114 cases revealed three cases presenting as brain abscess,
including the first case of the brain abscess by S.commune from
Brazil.
Objectives The objective of this study is to report the first case of
bloodstream infection associated with brain lesion by S.commune, in a
HIV-infected person, from Instituto Nacional de Infectologia Evandro
Chagas, Brazil.
Methods and Results A 49 years old man, admitted in our insti-
tute with a difficult-to-treat HIV - tuberculosis (TB) coinfection. He
was antiretroviral na€ıve, with baseline CD4 count was 106 cells ll
1
at that time. Aside respiratory symptoms and fever, the patient pre-
Figure 2. Chest radiography before treatment sented headache and neck stiffness. A CT-scan pointed a single round
hypodense lesion in right capsular nucleus, without contrast enhanc-
ing, measuring 2.5 cm, and other smaller hypodense lesions in left
cerebellar hemisphere and right parietal subcortical region. CSF anal-
hematological malignancies but also affect the immunosuppressive yses revealed a typical inflammatory pattern, but cultures were nega-
therapy patient population. We aimed to remind that invasive pul- tive. The clinicians decided to treat as tuberculosis meningitis, adding
monary aspergillosis could be seen in kidney transplant recipients. steroids to therapy and the patient was discharged. Antiretroviral
Case A 50-year-old male, who underwent kidney transplantation a therapy was initiated in ambulatory setting, but after two weeks, the
month ago, admitted to our hospital with complaints of cough, fever patient was admitted again, with new neurological findings. A new
(38.7 °C) and poor general condition was hospitalized. The patient, CT-scan showed worsening of lesions. Two days after admission,
who was considered as pneumonia, underwent chest x-ray and mycology laboratory reported the growing of a rare fungus, and
empirical antibacterial treatment was. Because of persistent fever on amphotericin B deoxicolate (1 mg kg day
1) was started. Culture
the fifth day of the treatment, sputum, urine and blood cultures were was did and 37 °C grew a fast expanding cottony white mycelium.
obtained, serum galactomannan antigen test was performed, and Culture this moth initial in medium PDA (Potato Dextrose Agar)
pulmonary HRCT (high resolution chest tomography) was obtained. after a week produce a distinctive fruity odor, cottony white and
The patient was on treatment of steroid and tacrolimus for solid turned light grey with appears as hyaline, septate nondichotomously
organ transplantation. His laboratory examination was as follows: branching hyphae in direct potassium hydroxide. It was not possible
white blood cell (WBC): 14.400/mm3, CRP: 168 mg l
1 (0–5), crea- to make a diagnosis using only conventional techniques of mycology
tinine: 3.3 mg dl
1 (0.67–1.17), BUN: 43. Serum galactomannan being realized the partial sequencing of the region internal tran-
antigen test was positive (2.44, normal range between 0 and 1). scribed spacer (ITS). The sequences were edited using the Sequencher
HRCT revealed multiple pulmonary nodules bilaterally with the high- 4.9 software and compared by BLAST (Basic Local Alignment Search
est diameter of 7 mm in the left lung, which were considered in Tool) with sequences available from NCBI/GenBank being this 100%
favor of fungal infection (Figure 1). With the diagnosis of invasive concordant with sequences of S. commune deposited in Genbank.
pulmonary aspergillosis, the patient was prescribed intravenous After one month on HAART, antituberculous and antifungal ther-
voriconazole. The patient was closely followed for the possibility of apy, without any other complication, the patient got better, CD4
the accumulation of sulfobutylether-b-cyclodextrin, which is present count of 424 cells ll
1. New CSF analysis showed improvements of
in the intravenous form of voriconazole, in the patients with renal inflammatory pattern and cultures were negatives, and important
insufficiency. Because of a possible interaction with voriconazole, healing noted in CT-scan. Until now, this patient is well. We chose
tacrolimus dose was decreased and its level was monitored. Ampho- doing maintenance treatment with fluconazole, because of its better
tericin B treatment was initially not considered to be appropriate for penetration through hematoencephalic barrier. HIV immunodefi-
this patient due to potential nephrotoxicity of the drug in a kidney ciency can change the medical course of diseases, and lead rare
transplant who had high creatinine clearance. On the ninth day of opportunistic diseases.

ª 2015 The Authors

162 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Conclusion Differently from the other cases reported, we did not iso- P305
late the fungi in the CSF or in the brain tissue, but we had a positive
blood culture, which was the first case described. We also believe this
report is relevant, as it was the first case in the world that the Sphingosine kinase inhibition for the treatment of invasive
patient with probably brain abscess due S.commune who survived. aspergillosis
C. Vinci
Imperial College London, London, United Kingdom

P304 Invasive aspergillosis (IA) is an increasingly common cause of death

in immunocompromised individuals. During IA, the activation of
Rhino-orbital-cerebral Mucormycosis in a known diabetic phagocytes leads to a pro-inflammatory cytokine response with
patient in Nigeria excessive production of TNF-a leading to tissue destruction. The low
R. O. Oladele,1 A. Ettu,2 O. James,2 W. B. Adeyemo,2 efficacy of antifungal drugs currently available and a rising immuno-
compromised population has resulted in an increasing need for more
U. Osigwe2 and D. Denning3
1
effective treatments. Sphingosine kinase 1 (SphK1) is a lipid kinase
University of Manchester, Manchester, United Kingdom; 2Lagos that generates the bioactive sphingolipid sphingosine-1- phosphate
University Teaching Hospital, Lagos, Nigeria and 3The University (S1P) from sphingosine. S1P regulates many cellular functions and is
of Manchester and National Aspergillosis Centre, Manchester, recognized as a critical regulator of inflammation.
United Kingdom Inhibition of fungal-dependent TNF-a in the presence of FTY720
and the sphingosine kinase 1 specific inhibitor SKI-II in murine
Introduction Mucormycosis is a rare, fulminant and potentially macrophages demonstrates that pro-inflammatory cytokines are
lethal invasive opportunistic infection caused by Mucorales. The mor- SphK1 dependent. FTY720 (Gilenya) is currently used as a treatment
tality rate in patients with systemic mucormycosis is as high as 50%, for multiple sclerosis and has been shown to block the S1P G pro-
while in patients with cerebral involvement, it exceeds 80%; 50% of tein-coupled receptor S1P1.
these patients have diabetes mellitus (DM). In a murine study a reduction in lung consolidation and mRNA
Case Report A 60-year-old known type 2 diabetic and hypertensive levels of TNF-a in whole lung homogenate indicate that FTY720
male, who defaulted clinic attendance 5 years ago, presented with a 6- reduces inflammation in response to a fungal challenge. Further
week history of persistent headache. He was rushed to hospital from murine data indicated that FTY720 directly reduces fungal burden
church where he had collapsed. The headache was severe and right as well as having anti-inflammatory properties. Additionally, there is
sided; there was an associated right sided dark pigmented necrotic facial a known homology between mammalian and fungal sphingosine
eschar with nasal congestion and proptosis (Fig 1). He also had progres- kinases, and anti-fungal studies demonstrate FTY720 to have fungi-
sive loss of vision in his right eye and pain in the right ear. The patient static effect at high doses, with SKI-II having significant anti-fungal
also complained of fluids taken orally dripping through the nose. activity at a low dose. These data suggest that the sphingolipid path-
There was necrosis of the palate extending from upper right sec- way is important in fungi for normal growth, and therefore a novel
ond premolar to distal to the last molar. antifungal target.
Differential diagnoses were rhinocerebral mucormycosis, T-cell Overall, my findings indicate that mammalian sphingolipid path-
lymphoma and necrotising fasciitis. Incisional biopsy was done and ways are important mediators of fungal induced inflammation and
specimen sent for histopathological and microbiological examina- that FTY720 has unexpected direct antifungal properties that lead to
tions. Histology revealed broad non-septate hyphae and cultures enhanced outcomes in animal models of aspergillosis.
yielded a heavy growth of Lichtheimia species (Fig 2). CT scan of skull
revealed hypodensity of skin and soft tissue overlying the anterior
wall of the right maxilla. The right orbital content could not be
delineated as the area showed a diffuse hyperdensity. Liposomal P306
amphotericin B was initiated one week later due to the need to pro-
cure it from the US since it is not available in Nigeria. Surgical
Cutaneous infection caused by Scopulariopsis brevicaulis
debridement of the necrotic region and right orbital exenteration
were performed by a combined team of oral and maxillofacial sur- in a renal transplant recipient
geons and ophthalmologists. The extent of the lesion was confirmed E. Pinto,1 A. Lopes,2 C. Monteiro,1 M. Faria3 and M. D. Pinheiro4
1
intra-operatively to have extended into the optic canal and temporal Faculty of Pharmacy-Porto University, Porto, Portugal; 2Porto
and infra-temporal fossae making complete debridement very diffi- University, Porto, Portugal; 3Faculty of Pharmacy, University of
cult. The necrotic palatal tissue was excised. His clinical condition, Porto, Portugal and 4Centro Hospitalar S. Joao EPE, Porto,
however, degenerated on the second postoperative day with eventual Portugal
demise of the patient.
Discussion The prevalence of DM in Nigeria is 0.6–11% and it is esti-
mated to be highest in Africa. Rhinocerebral or sino-orbital mucormy- Introduction Scopulariopsis brevicaulis, a filamentous fungus, is
cosis are common among diabetic patients, especially those who are widely distributed in soil and in environment. It presents, primarily,
poorly controlled, as was the case in this patient. Studies have demon- as a fungal nail infection and, rarely, as cutaneous or disseminated
strated that DM alters the immunological response to resist mucormy- infection. These situations are noticed mainly in immunosuppressed
cosis through a reduction of the phagocytic properties of granulocytes patients where the cutaneous involvement is usually secondary to
in the presence of acidosis and hyperglycemia with an inability of dia- disseminated pulmonary or visceral disease; rarely, it is consequent
betic serum to inhibit Mucorales in vitro. In addition, Mucorales thrive to primary skin inoculation. Here, we present a case of cutaneous
best in an acidic and glucose-rich environment. infection by Scopulariopsis brevicaulis in a renal transplanted patient,
Successful management of rhinocerebral or sino-orbital mucormyco- following skin trauma [1].
sis depends on early diagnosis, early intervention with ablative sur- Case On October 2013, a 72 years-old Caucasian female was
gery, and intravenous Amphotericin B, as standard treatment. referred to our hospital due to a ESBL positive Escherichia coli
However in Nigeria, Amphotericin B is not readily available and pyelonephritis; she began treatment with meropenem that continued
voriconazole is the only licensed available parenteral antifungal avail- for fourteen days. The patient had been kidney transplanted five
able in Nigeria. years before, due to autosomal dominant polycystic kidney disease,
Conclusion There is dire need to increase the awareness of clini- and was medicated with tracolimus and prednisolone since. On the
cians in Nigeria regarding Mucormycosis in diabetic patients and a physical exam a nodular erythematous-exudative lesion, surrounded
critical need to provide essential antifungal agents when indicated. by smaller satellites, were observed on both legs; the patient remem-
bered a skin trauma with a rake about a year before. A swab from

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 163
Poster Presentations

the lesion exudate was sent to the laboratory and Scopulariopsis brevi- Methods We conducted a cross-sectional survey in three centers in
caulis was identified. In the following days, two additional swabs and Nigeria. Adults HIV positive and negative consenting patients who
two cutaneous biopsies from both legs lesions were performed and are at the end or are in their last month of TB treatment (smear
sent to pathological and mycological study, which revealed fungal and/or Genexpert positive = documented TB) or currently being trea-
structures and the same fungus growth. Imagiological exams to ted for ‘smear negative TB’ were recruited from DOTS, chest and
exclude systemic infection were all negative. The patient was medi- ART clinics in Lagos and Ilorin states, Nigeria. All were assessed with
cated with caspofungin and voriconazole. Seven days later, cryosur- clinical assessment, chest X-ray and Aspergillus IgG serology. Asper-
gery was employed to remove the skin lesions. Treatment continued gillus-specific IgG was measured by ELISA using Dynamiker, which
with voriconazole and caspofungin for seven days and then caspo- has a sensitivity of 77% and specificity of 97% for the diagnosis of
fungin was replaced by terbinafine; this dual therapy schedule lasted CPA. Sputum was collected for fungal culture in those producing
for four months. No relapse was observed after one year of follow-up. sputum. Patients’ demographic and clinical data was collected using
Microbiology The three swabs and two biopsies sent to the labora- a semi-structured questionnaire. Healthy blood donors were used as
tory were cultured on Sabouraud with gentamicin and chloram- control. CPA was defined as a positive Aspergillus IgG titre (>xx
phenicol; in all, tan powdery colonies were grown; the microscopic IU ml
1), compatible chest X-ray and pulmonary or systemic symp-
exam evidenced branched annellides and chained round conidia with toms, despite anti-TB therapy.
truncated base, suggestive of Scopulariopsis brevicaulis. The sequence Results 209 patients were recruited between December 2013 and
of the internal transcribed sequence (ITS) region of the ribosomal September 2014. 150 (75%) were HIV positive. Mean age was 39.46
RNA showed similarity with Scopulariopsis brevicaulis (GenBank (95% CI), 59.9% were female and 18.9% were unable to work. Mean
Accession: KP117309; CBS Accession: DTO 001-F7/002-C9/012-E6/ CD4 (meanS.E) was of all HIV patients was 246.20  39 and
012-E7). Susceptibility testing was made following the Clinical Labo- 244.8  45.4 in the HIV infected patients with positive Aspergillus
ratory Standard Institute protocol M38-A2 [2] and the MIC results IgG. One hundred and thirteen (54%) had documented TB and
were amphotericin 2 mg l
1, fluconazole >64 mg l
1, ketoconazole 70.5% had productive cough. Forty-one patients had hemoptysis,
8 mg l
1, itraconazole >16 mg l
1, posaconazole >16 mg l
1, while one 8/41 had frank blood hemoptysis. Only one patient had a
voriconazole >8 mg l
1, anidulafungin 0.25 mg l
1, caspofungin co-existing chronic obstructive airway disease (COPD). The preva-
0.5 mg l
1 and terbinafine 8 mg l
1. These high MIC values are lence of Aspergillus IGg was 30.0%; 48/149 (32.2%) in HIV positive
according to the reported in vitro resistance of Scopulariopsis to anti- and 17/60 (28.3%) in HIV negative. HIV co-infection had no signifi-
fungal agents [3]. cant impact on the frequency of CPA (p-0.645). Patients with weight
Conclusions The case emphasizes the increased frequency of unu- loss, fatigue, ongoing or worsening respiratory symptoms + Aspergil-
sual fungal infections affecting immunocompromised patients. In lus IgG were defined as probable CPA, pending radiological review
contrast with the frequent systemic involvement, in this case, it was (on going). There were 48 probable CPA HIV positive patients, 17
confined to the skin, likely to be port of entry following local trauma. HIV negative CPA patients, 11 documented TB patients with proba-
Scopulariopsis brevicaulis, the identified fungal agent was effectively ble CPA and 44 smear negative/genExpert negative probable CPA
treated with local cryotherapy and commonly used antifungal drugs, patients.
despite the in vitro voriconazole high MIC. Conclusion CPA is a neglected disease and represents a significant
[1] Perusquıa-Ortiz AM, et al.J Dtsch Dermatol Ges. 2012, public health challenge. CPA will fit into the WHO diagnostic criteria
10:611–21; quiz 621–2. doi: 10.1111/j.1610-0387.2012.07994.x. for ‘smear-negative’ tuberculosis. Accurate diagnosis of CPA in this
[2] Clinical and Laboratory Standards Institute. 2008, CLSI docu- group would avoid unnecessary and potentially toxic TB therapy
ment M38-A2. [3] Sk ora M et al. 2014, 52:723–7. doi: 10.1093/
mmy/myu039. Epub 2014 Jul 21.

P308
P307
Seroprevalence of cryptococcal antigenemia in
antiretroviral naive and antiretroviral experienced HIV
Evaluation of chronic pulmonary aspergillosis (CPA) as a infected patients with CD4 + count below 250 cells/mm3
cause of smear negative TB and or anti-TB treatment in Lagos, Nigeria.
failure in HIV infected Nigerians R. O. Oladele
R. O. Oladele,1 A. Akanmu,2 N. Irurhe,3 A. Nwosu,3 University of Manchester, Manchester, United Kingdom
F. Ogunsola,3 M. Richardson4 and D. Denning5
1
University of Manchester, Manchester, United Kingdom; 2Lagos Introduction and objectives Cryptococcal meningitis accounts for
University Teaching Hospital, Lagos, Nigeria; 3College of high mortality in HIV infected persons in Africa. This is a pre-
Medicine, University of Lagos, Lagos, Nigeria; 4University Hospital ventable outcome when early screening and preemptive therapy is
of South Manchester, Manchester, United Kingdom and 5The administered. WHO recommends screening for <100 CD4+ cells/
University of Manchester and National Aspergillosis Centre, mm3, this is presently not done in our setting and the antifungals
Manchester, United Kingdom recommended for treatment of this infections are not readily avail-
able in Nigeria. We evaluated the cryptococcal prevalence (CrAg),
Introduction and Objectives Nigeria has an estimated 3. 2million possible associated factors and outcomes in HIV infected patients
people living with HIV/AIDS. Tuberculosis (TB) is the recognised being managed in a tertiary hospital in Lagos, Nigeria.
leading cause of death in people with HIV and thousands of Nigeri- Methods Sera were collected from 216 consenting HIV infected par-
ans are believed to be living with a multidrug-resistant strain. CPA ticipants attending PEPFAR clinics, with CD4 + counts less than
has been linked to pulmonary tuberculosis. Our primary objective is 250, between November 2014 and May 2015. CrAg LFA (IMMY)
to report the prevalence of Aspergillus antibodies along with the was used for testing. Pertinent clinical data was from patients and
prevalence of CXR changes consistent with CPA in patients with TB. their case notes.
We compared the rate of CPA in the HIV positive and negative Results Of the 214 participants, 96 (44.9%) were male and 118
patients with tuberculosis described to establish whether HIV status (55.1%) were female. Mean age was 41.25 + /= 9.350. Majority
is associated with a different risk of developing the disease. We also (>95%) were ART experienced. The seroprevalence of cryptococcal
compared the rate of CPA in HIV positive patients with high and low antigenemia was 9.7% overall, of which 38.1% were ARV na€ıve and
CD4 counts to determine if advanced immunosuppression is associ- 19.04% were ART defaulters. The rate of CrAg posivity was 6/19
ated with developing CPA. We then assessed any other potential risk (38.1%) in those with CD4 counts <100, with CD4 counts 100–200
factors. 6/19 (28.6.9%) and 7/19 (33.3%) with CD4 count of 200 = 250.
(38.1%) had CD4+cell count below 100 cells/mm3 and 13 (61.9%)

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164 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

above CD4+cell count above 100. 27 (12.1%) had associated oral P310
thrush, comorbidities such as diabetes mellitus (1) and Tuberculosis
(2). Potential meningitic symptoms were common in the studied
group but was not statistically significant when both CrAg positive Clinical significance of immunological parameters in
and CrAg negative were compared. Of the 21 participants with posi- hematological patients with invasive aspergillosis
tive CrAg, 3 died and 10 were lost to follow up. Empirical fluconazole O. V. Shadrivova,1 N. Klimko,2 E. V. Frolova,1 A. E. Uchevatkina,1
200 mg thrice weekly was commonly given to those with low CD4 L. V. Filippova,1 T. S. Bogomolova,2 S. M. Ignatyeva4 and
counts >100 cells/mm3 (WHO guidelines) (p-0.018), unrelated to N. Vasilyeva5
Crag positivity.. 1
Conclusion We have reported a seroprevalence of 9.7% cyprococcal I. Mechnikov North-Western State Medical University, St.
antigenemia in as setting where amphotericin B, flucytosine and Petersburg, Russia; 2North-Western State Medical University
intravenous fluconazole are not readily available. HIV infected named after I.I. Metchnikov, Saint Petersburg, Russia; 4Medical
patients frequently present with comorbidities. Public health officials mycology institute named after Kashkin, Saint Petersburg, Russia
in Nigeria should consider adding CrAg screening to existing and 5I. Metchnikov North-Western State Medical University,
national guidelines for HIV/AIDS care, as recommended in the WHO Kashkin Research Institut, Saint Petersburg, Russia
guidelines.
Objective To study of immunological parameters dynamics and it’s
prognostic value in hematological patients with invasive aspergillosis
(IA).
P309 Materials and methods We observed 83 hematological patients
with IA. Immunological parameters were evaluated within 2–
4 weeks after IA diagnosis (median–14 days), after 2–3 months, and
An estimation of the Pneumocistose, Aspergillose and
before the end of antifungal therapy. For the diagnosis of IA criteria
Histoplasmose diseases in Mozambique
EORTS/MSG, 2008 were used.
J. Sacarial,1 A. Passanduca,1 N. Elisabeth2 and O. M. G. Matos3 Group I included 48 patients after cytotoxic chemotherapy (CT),
1
Faculdade de Medicina, Maputo, Mozambique; 2Hospital age 18 - 78 years, median - 46 (33  58); males - 42%. Group II -
Central de Maputo, Maputo, Mozambique and 3Instituto de 35 recipients of allogeneic hematopoietic stem cell transplant (allo-
Higiene e Medicina Tropical, Lisboa, Portugal HSCT), age - 18 - 59 years, median - 26 (21  44); males - 51%.
Lymphocyte subsets and immunoglobulin levels were examined.
Blood cell supernatants were tested for IFN-c, IL-6, IL-10, IL-17,
Introduction Pneumocystis jirovecii, Aspergillus fumigatus, and
TNF-a and G-CSF. Receiver operating characteristic (ROC) analysis
Histoplasma capsulatum are organisms with global distribution, and
was performed to determine prediction rules for clinical outcome of
important opportunistic agents responsible for a high rate of morbid-
IA.
ity and mortality of pneumonia in HIV positives patients. The wide-
Results IA was diagnosed between 5 and 50 days (median - 30)
spread use of prophylaxis with trimethoprim-Sufametoxazol for
after CT course in group I. IA developed in the early post-transplant
pneumonia by PCP, Amphotericin B and other antifungals, as well
period (100-day) in 64% of patients of group II; 56% of patients had
as the Antiretroviral Treatment, caused a considerable decline in the
unrelated donors, HLA-matched - 31% and HLA-mismatched - 13%.
incidence of three diseases in industrialized countries. In Africa until
In both groups, the prevailing underlying diseases were acute leuke-
the early twenty-first century, the PCP was considered a rare disease,
mias - 56% and 66%, respectively. All patients received antifungal
but recent studies have reported a significant increase in the fre-
therapy after diagnosis of IA, voriconazole predominantly (79%).
quency of this particular AIDS-associated disease, while for Aspergil-
12th weeks overall survival was 90% and 71%, respectively. We
lus and Histoplasmas was still lack studies. In Africa, few studies was
identified significant decrease in the absolute number of lymphocyte
performed on the genetic characterization of identified isolates of P.
subsets in the both groups at the early stage of IA. In allo-HSCT
jirovecii, Aspergillus fumigatus, and Histoplasma capsulatum, to
patients were found significant increase in T-helper cells CD4 +
determine their virulence. In Mozambique, particularly in HIV-in-
number (P = 0.01), natural killer cells CD16 + (P = 0.001) and a
fected patients, suspected cases of pneumocystis pneumonia,
lower production of IgG and IgA (P = 0.007 and P = 0.0003),
aspergillosis and histoplasmosis have been increasing, as well as case
respectively, compared with CT patients. Reduction of proinflamma-
reports alone was confirmed. However, knowledge of the epidemio-
tory cytokines production (IFN-c, TNF-a, IL-17, G-CSF and IL-10)
logical behaviour of the three diseases in the country, it is essential
were detected in all hematological patients with IA, in allo-HSCT
so that you can think about and implement control measures of
group it was the most significant. We found positive dynamics of
these opportunistic diseases.
immunological parameters in patients with a favorable outcome. In
Objectives Estimate incidence of pneumonia by P.jirovecii, A. fumi-
CT patients increase in TNF-a (P = 0.02) and G-CSF (P = 0.001) pro-
gatus and H. capsulatum in adults patients infected with HIV and
duction, in allo-HSCT recipients - increase of absolute number
admitted in Maputo Central Hospital
CD4 + (P = 0.03) and the ability of leukocytes to TNF-a (P = 0.04),
Methods A prospective study in admitted adult’s patients HIV posi-
G-CSF (P = 0.02), IL-10 (P = 0.03) and IL-6 (P = 0.04) production
tives with pneumonia. The patients are admitted into the study after
were detected. Immunological parameters recovery are necessary for
signing inform consent. We collecting 15 ml of pulmonary samples
successful course of IA.
and it is analyzing in Microbiology Laboratory. Vital data, chest X-
We found positive predictors of a 12-week survival in hematologi-
ray and sputum induction are collected from patients. A slide is read
cal patients with IA: the absolute T-helper CD4 + number >
by immunofluorescence to identify the P. Jirovenci with use of com-
0.177*109/L, (P = 0.002), and the TNF-a level > 215 pg ml
1,
mercial kits and posteriorly we will do molecular analysis.
(P = 0.001).
Results The study started in May 2015 and we expected we will
Conclusion The absolute number of T-helper CD4 + > 0.170*109/L
recruit around 100 patient until end of September 2015. We will
and the blood cells ability to produce TNF-a > 215 pg ml
1 are sig-
present the prevalence of this 3 disease in Adults HIV positives
nificance prognostic markers of a favorable outcome of invasive
patients after analyzing data in end of September 2015.
aspergillosis in hematological patients.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 165
Poster Presentations

P311 P312

Initial Use of Combination Treatment Does Not Impact Antifungal prophylaxis in high-risk, adult hematological
Early Survival of 106 Patients with Hematologic patients - results from multicenter, real life MIFI study
Malignancies and Mucormycosis: a Propensity Score L. Drgona,1 M. Mistrik,2 J. Chudej,3 T. Guman,4 E. Kralikova,5
Analysis S. Palasthy,6 L. Demitrovicova,1 J. Gabzdilova,4 D. Horvathova,2
A. Kyvernitakis, H. A. Torres, Y. Jiang, G. Chamilos, R. E. Lewis E. Schweighofer-Bednarikova,1 J. Sokol,7 E. Mikuskova,1
and D. P. Kontoyiannis F. Alsabty,2 J. Holasova5 and K. Kahankova8
University of Texas MD Anderson Cancer Center, Houston, USA 1
Comenius University and National Cancer Institute, Bratislava,
Slovak Republic; 2Comenius University, Bratislava, Slovak
Objectives Mucormycosis is an emerging opportunistic mold infec- Republic; 3Jessenius Faculty of Medicine, Comenius University,
tion in patients with hematologic malignancies (HM) and hematopoi- Martin, Slovak Republic; 4Louis Pasteur University Hospital,
etic cell transplant (HCT) recipients. In view of the poor outcomes Kosice, Slovak Republic; 5FDR Hospital, Banska Bystrica, Slovak
associated with mucormycosis, antifungal combinations are fre- Republic; 6University Hospital, Presov, Slovak Republic; 7Jessenius
quently used, yet the value of such strategy remains unclear. Faculty of Medicine in Martin, Comenius University in Bratislava,
Methods We retrieved the records of patients with HM and probable Martin, Slovak Republic and 8JDSoftware, Bratislava, Slovak
or proven mucormycosis (EORTC/MSG criteria), seen at The Univer- Republic
sity of Texas MD Anderson Cancer Center from 1994 to 2014.
Monotherapy was defined as single agent treatment with ampho-
tericin-B or posaconazole. Combination treatment included any com- Objective To assess the overall efficacy of antifungal prophylaxis
bination of amphotericin-B, posaconazole and echinocandins, given (AFP) in patients with hematological malignancies in real life setting;
as initial treatment. The primary outcome was mortality at 42 days to compare posaconazole and fluconazole in adult patients with acute
after treatment initiation. Univariate and logistic regression analyses myeloblastic leukemia (AML).
were used to determine predictors of mortality. A multivariate analy- Methods Management of invasive fungal infection (MIFI) is ongoing
sis with a propensity score for receiving combination treatment was database project in Slovakia. The aim of this project is to cumulate
performed to adjust for baseline imbalances. and analyze the data about antifungal strategies used in participating
Results Of the 106 patients identified, 44% received monotherapy centres with the focus mainly on prophylaxis of the high-risk hema-
and 56% combination treatment. Patients who received combination tological patients. Data input is performed via web based registry.
therapy were more likely to be on voriconazole prophylaxis (69% Data from six center (study period 2012–2014) were retrospectively
versus 38%; P = 0.001), be HCT recipients (61% versus 38%; analyzed. Withdrawn of antifungal drug regardless of reason was
P = 0.02), have active graft-versus-host disease (36% versus 17%; considered as a failure of prophylaxis. The predefined reasons for fail-
P = 0.03), or develop mucormycosis after 12/2004 (83% versus ure were non-compliance, toxicity and breakthrough invasive fungal
40%; P < 0.01). In contrast, patients who received monotherapy disease (proven, probable, possible) according to EORTC/MSG criteria
were more likely to be on fluconazole prophylaxis (34% versus 14%; 2008. Comparison of primary prophylaxis of patients with AML was
P = 0.01), when compared to those who received combination treat- performed using statistical analysis.
ment. There were no differences in delayed initiation of therapy (≥ Results 664 adult patients, 325 (49%) males and 339 (54%)
6 days, 38% versus 41%; P = 0.8) or 42-day mortality (43% versus females with average age 54.2 years were evaluated. The most com-
41%; P = 0.9) between monotherapy and combination treatment mon hematological diseases were AML (69%), malignant lymphomas
groups. Patients who died within 42 days were more likely to have (13%) and ALL (5%). 181 (27%) patients underwent hematopoietic
disseminated mucormycosis (30% versus 11%; P = 0.02), active HM cell transplantation with 52% of allogeneic transplants. At the start
(80% versus 55%; P < 0.01), higher APACHE II scores (median, 15 of AFP 583 (88%) patiens were neutropenic, 618 (93%) received
versus 13; P < 0.01), neutropenia (≤500 cells ll
1, 48% versus broad-spectrum antibiotics, 504 (73%) had inserted central venous
29%; P = 0.05), lymphopenia (≤500 cells ll
1, 84% versus 50%; catheter and steroids were administered to 159 (24%) patients.
P < 0.01), or be admitted in ICU at diagnosis (39% versus 8%; Posaconazole was used in 353 (53%) and fluconazole in 272 (41%)
P < 0.01). Patients who survived were more likely to have localized patients. Average duration of prophylaxis was 23 days and 26 days
mucormycosis (21% versus 2%; P < 0.01) or receive hyperbaric oxy- in patients with successful AFP. Overall success was documented in
gen therapy (16% versus 2%; P = 0.02). In multivariate analysis, 374 (56%) patients; posaconazole and fluconazole were successful in
lymphopenia (OR = 5.3; 95% CI = 1.9 - 15; P < 0.01) and ICU 220 (62%) and 135 (50%) patients, respectively. The reasons of fail-
admission at diagnosis (OR = 7.7; 95% CI = 2.2 - 27.6; P < 0.01) ure were breakthrough IFD in 122 (17%) patients, but only 10 (2%)
were associated with increased 42-day mortality. Localized mucormy- were proven. Uncompliance was presented in 136 (20%) patients
cosis was associated with better outcome (OR = 0.07; 95% CI = 0.01 and adverse effects in 32 (5%) patients. Survival at day +100 from
- 0.7; P = 0.02). Initial combination treatment had no impact in the start of AFP was 79% in total and 90% in group with successful
mortality, even after propensity score adjustment (OR = 0.8; 95% CI AFP, whereas in group with AFP failure survival was only 50%. We
= 0.3 - 2.3; P = 0.6). performed an analysis comparing patients with posaconazole and flu-
Conclusions With the current status of mucormycosis diagnosis, conazole AFP in adult patients with AML. The number of risk factors
there was no difference in mortality in HM patients, whether they was similar in both groups with some differences in the proportion of
received monotherapy or combination treatment. Earlier diagnosis risk factors. Average duration of AFP in posaconazole group was
and immune reconstitution are unmet needs to affect outcomes. 24.5 days (1–416 d) and in fluconazole group 21 days (1–316 d).
Comparison of posaconazole and fluconazole primary AFP in adult
AML patients showed successful AFP in 59% of 246 patients in
posakonazol group and in 42% of 176 patients in fluconazole group
(P = 0.001). Survival at day + 100 was 81% and 74% in posacona-
zole and fluconazole group, respectively (P = 0.072).
Conclusion Real - life retrospective data on AFP in high-risk hema-
tological patients is the important source of valuable information out-
side the clinical trials. We have confirmed the position of AFP in this
population of patients - patients with successful AFP had better sur-
vival. Our analysis documented superior efficacy of posaconazole in
comparison to fluconazole with trend to better survival at day +100
in adult patients with AML.

ª 2015 The Authors

166 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P313 P314

Investigation of pulmonary fungal infections leading to Epidemiology of candidaemia in burn-injured patients: A

hospitalization in HIV-infected adult patients Croatian nine-year study
A. I. M. Passos,1 R. P. Dertkigil,1 A. Busso,1 E. O. Ribeiro,1 E. Mlinaric-Missoni,1 A. Gveric-Grginic,2 M. Domijan,2
P. Trabasso,1 T. Matsuzawa,2 Y. Mikami,2 I. C. Toro,1 A. Munjiza,2 H. Tomicic,2 Z. Loncar2 and E. Missoni3
A. Z. Schreiber,1 K. Kamei2 and M. L. Moretti1 1
Croatian National Institute of Public Health, Zagreb, Croatia;
2
1
State University of Campinas, Sa~o Paulo, Brazil and 2Chiba Clinical Hospital Centre Sestre milosrdnice, Zagreb, Croatia and
3
University, Chiba, Japan University of Zagreb, Zagreb, Croatia

Objectives To study the occurrence of pulmonary fungal infections Objectives Advancements in burn care therapy have extended sur-
in HIV-infected patients hospitalized with acute respiratory symptoms vival of seriously burned patients, exposing them to increased risk of
Methods Cross-sectional study that included patients HIV-infected ≥ infectious complications. According to several published reports, dif-
18 years, hospitalized for community-acquired pneumonia (CAP) ferent Candida species have become increasingly common causes of
from 2012 to 2015. The diagnosis criteria of the pulmonary infec- bloodstream infections in these high risk patients within hospital set-
tions was based on the review of the cases by specialists and the ting. However, published data regarding these infections in Croatian
results of following exams: Culture and direct microscopic examina- burn-injured patients are scarce. Therefore the main objective of this
tion of sputum and bronchoalveolar lavage (BAL), serum LDH, blood study was to determine the annual and the overall incidence of can-
cultures, chest radiography and tomography, serum (1,3) b-D-glucan didaemia in adult patients hospitalized in the Reference Centre for
and galactomananan, LAMP-PCR for P. jirovecii in sputum, BAL and Burns, Zagreb Clinic of Traumatology, Croatia, during a 9-year per-
oral lavage. Risk of mortality was evaluated by the CURB-65 score. iod. In addition, we have provided species identification of Candida
Results Fifty-three patients were included with mean age bloodstream isolates together with their susceptibility profiles to
43.64  9.99 years; 34 (65%) patients were male; median time of amphotericin B, flucytosine, fluconazole, itraconazole, and
the diagnosis of HIV infection was 12.14  7.62 years and 35(65%) voriconazole.
patients had an intermediate mortality risk. Abdormalities in chest Methods Prospective single-centre cohort study had commenced in
X-rays and in chest tomography were found in 95% of 53 patients January 2006 and lasted until January 2015. Blood samples were
and in 96% of the 29 patients respectively. The median CD4+ T lym- taken in accordance with clinical indication in patients with superfi-
phocytes was 87 cel/mm3 and median LDH serum level was 443. cial, partial-thickness or full-thickness burn injuries of ≥ 10% total
Twelve patients (23%) had undetectable viral load and 33 (62%) had body surface area. Yeast isolates from Sabouraud glucose agar were
CD4 + T lymphocytes ≤ 200 cel/mm3. Two patients had positive spu- identified using an API ID 32C kit (bioMerieux, France) and on the
tum culture for Mycobacterium tuberculosis and two for Mycobac- basis of their cornmeal agar morphology. In vitro antifungal suscepti-
terium avium. Blood cultures were positive in 6 patients bility testing was performed using the ATB FUNGUS 3 (bioMerieux,
(4 Streptococcus pneumoniae; 1 micrococcus; 1 Acinetobacter baumanii); France) microdilution method, and the results were interpreted in
(1,3) b-D-glucan values were > 80 pg (reference value) in 22 (44%) compliance with the European Committee on Antimicrobial Suscepti-
patients and for galactomannan, 3 (6%) patients had values >0.5. bility Testing (EUCAST) recommendations.
LAMP-PCR was positive for P. jirovecii in 1of 2 BALs, 4 (21%) of 19 Results Candidaemia was determined in 16 out of 355 patients
samples of sputum and negative in 13 samples of oral lavage. The (4.5%). The lowest incidence was established in 2008 (0%), and the
probable diagnoses were: pneumocystis pneumonia (PCP): 14; CAP: highest in 2014 (7.7%). During the studied period, the average inci-
15; PCP + CAP: 3; Tuberculosis: 3; Mycobacteriosis + PCP: 2; Nocar- dence of candidaemia amounted to 1.09 per 100 admissions and
diosis: 1; Histoplasmosis: 1; Cryptococcosis: 1; Lower respiratory 0.61 per 1000 patient days. It annually ranged between 0 and 4.1
infection: 8; Pulmonary embolism: 1; Unconclusive: 3; Strongyloidia- per 100 admissions and between 0 and 1.77 per 1000 patient days.
sis: 1. Overall mortality was 15% (8 deaths) and 21% (3 deaths) for Species identification revealed that C. albicans was the most predomi-
the 14 patients with PCP. The diagnosis of PCP had a significant nant (50% of isolates), followed by C. parapsilosis (25%), C. glabrata
relationship with: late diagnosis of HIV infection (P = 0.008), higher (18.8%), and C. lusitaniae (6.2%). No in vitro resistance to ampho-
values of LDH (P = 0.002), lower CD4 + T lymphocytes count tericin B, flucytosine, fluconazole, itraconazole, and voriconazole was
(P = 0.001) and values > 80 pg of (1,3) b-D-glucan (P = 0.0002). observed in C. albicans isolates. However, non-albicans Candida spe-
Patients with undetectable viral load had significantly lower values cies isolates displayed a decreased susceptibility to fluconazole. The
of (1,3) b-D-glucan (P = 0.04). There was a significant relationship highest value of the frequency of C. albicans bloodstream infection
between higher values of (1,3) b-D-Glucan and the presence of dif- never exceeded 0.75 per 1000 patient days. Opposed to that, the
fuse infiltrate in chest radiography (P = 0.02). The sensitivity and non-albicans Candida species candidaemia frequency proved to be sig-
specificity of (1,3) b-D-glucan in PCP cases were 82% and 80%, nificantly higher, reaching as much as 1.42 per 1000 patient days
respectively. precisely in the final year of our study.
Conclusion Unlike the present data from developed countries, PCP Conclusions Our findings demonstrated a low incidence of candi-
was the leading cause of lung disease in patients with AIDS, followed daemia in patients hospitalized at the Zagreb Reference Centre for
by CAP. Pulmonary fungal infection represented 40% of the leading Burns in the period from 2006 until 2015, despite the fact that this
causes of hospitalization in HIV/AIDS patients. The combination of incidence was becoming more frequent toward the end of the con-
chest radiography and tomography with serum biomarkers was use- ducted study. We furthermore established that the presence of more
ful in the etiological characterization of lung infections in HIV adult resistant non-albicans Candida species isolates from blood cultures
patients. has been on the rise, which emphasizes the need for further investi-
gation of local candidaemia epidemiology in order to be able to prop-
erly implement systemic antifungal therapy.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 167
Poster Presentations

P315 Thus, we evaluated the relationship between VVC and RVVC with
diabetes as a potential risk factor for vulvovaginal candidiasis
Methods This prospective study included 254 women sexually
Virulence potential of Candida albicans isolated from oral active, aged between 15 and 50 years old (33.5  8.95), with or
cavity of patients with chronic renal failure on without signs and symptoms of VVC and RVVC. Vaginal cultures
hemodialysis were performed for yeasts isolation and identification by classical
J. S. R. Godoy,1 B. A. Ratti,1 P. S. Bonfim-Mendonc
a,1 methods. Fasting blood was collected for plasmatic glycemia and
F. K. Tobaldini,2 S. O. S. Lautenschlager1 and T. I. E. Svidzinski1 insulin measurements. Insulin levels were determined employing
chemiluminescent microparticle immunoassay using the Architect-
1
Universidade Estadual de Maringa, Maringa, Brazil and Abbott analyzer. The insulin sensitivity was derived from fasting glu-
2
Universidade Estadual de Maringa/Universidade do Minho, cose and insulin data, using the homeostasis model assessment
Maringa, Brazil (HOMA) mathematical model. Measurement of plasma glucose was
performed by enzymatic colorimetric GOD-PAP method (Diasys) using
Objective In patients with chronic renal failure (PCRF), the fre- the equipment Vitalab Selectra2. Data distributions were expressed as
quency of colonization of the oral cavity by yeasts of genus Candida mean  standard deviation. Significant differences among means
spp. is high compared with healthy individuals. These yeasts have were identified using GraphPad Prism 5.0 software. Bonferroni test
virulence factors that may contribute to the persistence of coloniza- was used to calculate the multiple comparisions and Chi-square (v 2)
tion and the development of these infections. The aim of this study test using the STATA for Statistics and Data Analysis 9.1 software
was evaluate aspects of virulence from Candida albicans isolated from was used to investigate variables between control group and positive
oral cavity of PCRF on dialysis. culture group. All variables were expressed as absolute and relative
Methods This study was inittialy conducted with 49 clinical samples frequencies, p values < .05 were considered significant.
of C. albicans. The virulence factors assayed were produce of biofilm, Results Yeasts were isolated from 48 (19%) women: 22 (46%) in
germ tube, determination of adherence in oral epithelial cells and acute episode (VVC) and 26 (54%) with RVVC. Control group (nega-
evaluation of resistance to the antimicrobial action of neutrophils tive culture) comprised 206 (81%) women. The average values found
and mononuclear cells. in fasting blood glucose was very similar in all groups. Abnormal
Results All isolates were highly efficient in forming biofilms on poly- glycemia (≥100 mg dl
1) was detected in 16 (7.8%) control women
styrene microplates, where 94% of the samples formed 4 + biofilm. and in 5(20.5%) of the positive culture group (VVC+ RVVC). The
Used as a screening test, of which three isolates were selected with HOMA Index (HI) indicating insulin resistance were detected in 42
different degrees of ability to form biofilm to assess other indicators of (20%) women of control group and in 16(64%) of the positive cul-
virulence. Overall, the isolates exhibited different characteristics ture ones.Compared to control group, women with positive culture
regarding the virulence factors analyzed. It was also observed that (VVC + RVVC), were significantly associated with abnormal glucose
the hypophosphorous acid (HOCl), production, one of leading inflam- metabolism (P = .0002; OR= 4.6;CI=2.0–10.9) and insulin resistance
matory mediators with fungicidal action, also varied especially when (P = 0.0317; OR= 2.2;CI= 1.05–4.84).
the neutrophils, and not mononuclear cells, were stimulated with dif- Conclusion Our data suggest a significant association between
ferent samples. (Figure 1). abnormal blood glucose levels and the incidence of vulvovaginal can-
Conclusion Therefore, our results indicate that C. albicans, is not didiasis. Women with blood glucose above the reference values and
only the most common species in the oral cavity of CRFP on dialysis, with insulin resistance (HOMA IR) presented at least twice the risk to
but also it presents the main virulence attributes, which reinforces have positive culture for Candida spp.
the importance of monitoring of these patients towards the preven-
tion of fungal infections.

P317

P316 Impact of Prior Invasive Fungal Disease on the outcome of

Allogeneic Hematopoietic Stem Cells Transplantation
Hyperglycemia and increased risk of vulvovaginal E. S. Soulaiman,1 M. Popova,2 A. Volkova,3 S. Bondarenko,3
candidiasis (VVC) M. Vladovskaya,3 O. Pinegina,3 S. Ignatyeva,4 T. S. Bogomolova,5
L. Akimoto-Gunther,1 P. S. Bonfim-Mendonc
a,1 J. S. R. Godoy,1 N. Klimko,5 L. Zubarovskaya3 and B. Afanasyev3
G. Takahachi,2 H. K. Anjos,1 M. R. N. Oliveira,1 M. E. Lopes- 1
Tishreen Hospital, Hematology and Bone Marrow
Consolaro1 and T. I. E. Svidzinski1 Transplantation Department, Damascus, Syrian Arab Republic;
1
Universidade Estadual de Maringa, Maringa, Brazil and 2
First I. Pavlov State Medical University, Saint Petersburg, Russia;
2
Universidade Estadual de Maringa - UEM, Maringa, Parana, 3
Raisa Gorbacheva Memorial Institute, First Pavlov State Medical
Brazil University, Saint Petersburg, Russia; 4First Pavlov State Medical
University, Saint Petersburg, Russia and 5North-Western State
Objective The vulvovaginal candidiasis (VVC) is a prevalent oppor- Medical University named after I.I. Metchnikov, Saint Petersburg,
tunistic mucosal infection that affects a significant number of healthy Russia
women, with a peak incidence between 20 and 40 years old. The
recurrent form of the disease (RVVC) is defined usually as 3 or more Objectives The objectives of the study was to estimate impact of
episodes of VVC that occur within a 12-month period. According to prior IFD on overall survival (OS) after alloHSCT, the incidence of
many authors, diabetes mellitus is a predisposing factor for vulvo- IFD event and related risk factors during alloHSCT, and evaluate the
vaginal candidiasis, along with pregnancy, use of broad-spectrum effectiveness of preventing measures against IFD event.
antibiotics, high-estrogen-dose oral contraceptives, obesity and drug Methods In retrospective and prospective observational single-center
addiction. Others studies consider hyperglycemia as the major cause study 256 HSCT recipients (303 alloHSCT, 256 first and 47 second
of increased susceptibility of diabetic patients to vulvovaginal candidi- HSCT) were included from Jan 2012 to Dec 2013 in Raisa Gor-
asis. It is known that increased glucose levels in genital tissues bacheva Memorial Institute of Children Oncology, Hematology and
enhance yeast adhesion and growth and also that hyperglycemia Transplantation, First Pavlov State Medical University of Saint-Peters-
may impair several aspects of humoral host defense, resulting in burg. Most of patients had acute leukemia (71.6%), 162 males, and
decreased random motion of neutrophils, chemotaxis, phagocytosis, median age 44 years (1–67 years). AlloHSCT from HLA-matched
and microbial killing. Despite those findings, there are few and incon- unrelated donor were performed in 73.9%, HLA-matched related
clusive scientific reports concerning diabetes and VVC and RVVC. donor - 18.8%, haploidentical - 7.3%, predominantly with reduce

ª 2015 The Authors

168 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

intensity conditioning regimen (76.2%) with complete remission of dyspnea were observed. At 28.04.2012 a lung CT revealed alveolar
underlining disease in 56.1%. EORTC/MSG 2008 criteria for diagno- and interstitial infiltration with indistinct, rough contours in S1 + 2
sis IFD and response to therapy were used. ‘Active IFD’ is the IFD and S6 of the right lung and an area of consolidation average and
diagnosed immediately prior HSCT. OS and effectiveness of antifun- big intensity with indistinct, rough contours, roundish form, with a
gals (fungal free survival (FFS) - survival without any events: relapse, diameter of 8–13 mm in S3, S6 of the left lung. At 29.04.2012 the
progression, and new episode of IFD) estimated by the Kaplan-Meier normalization of level of leukocytes and body temperature was regis-
analysis at 1 year after alloHSCT. trated. At that time the patient refused from diagnostic bron-
Results The incidence of IFD before alloHSCT was 27% (83/303): choscopy. At 02.05.2012 clinical and hematologic remission of AML
proven/probable (PP)-IFD (65), possible IFD (18). In PP-IFD group was established. Due to the changes in lungs the next course of the
the most frequent IFD was invasive aspergillosis (IA) 85%, invasive chemotherapy was postponed. At 10.05.2012 control lung CT
candidiasis/candidemia (IC) - 4.5%, some of cases had two patho- revealed cavities in S1–2 of left lung and in S3, S6 of right lung, and
gens: IA+IC (3), IA+Fusarium (2). At the moment of HSCT active IFD size of cavity in S6 increased in left lung. At 12.05.2012 bron-
was 30.8%, PR - 43.1%, CR- 26.2%. The main sites of infection were choscopy was done. Test for galactomannan was negative in bron-
lungs 70.1%, other localizations were observed less frequently and choalveolar lavage, Rhizomucor sp. was cultured. Treatment with
mostly in combination with lung involvement - 12%. Most of amphotericin B 1 mg kg d
1, then posaconazole 800 mg d
1 was
patients received voriconazole (88.1%) during HSCT. Possible IFD used. At 10.06.2012 the resection of S1 + 2 and S3 of the left lung
group consist of two types: possible according EORTC/MSG with typi- was performed. The cytostatic chemotherapy was continued success-
cal radiological signs and negative mycological criteria (n = 7) and fully with antifungal secondary prophylaxis with posaconazole.
group of patients with host factors, atypical radiological signs and Conclusions Hematological patients have the high risk of rare fun-
negative mycological criteria but were treated with voriconazole with gal infections. The complex treatment with surgical debridement
effect (n = 11) because of high risk IFD - AML/MDS, Diagnostic pro- allow to treat acute myeloid leukemia in patient with mucormycosis.
cedures included bronchoscopy (50%), culture of blood and respira-
tory substrate (68.2%), GM in BAL (50%), GM in serum (80%).
Voriconazole was used as secondary prophylaxis during alloHSCT in
72% of cases, caspofungine - 17%, other - 11%. One-year FFS for P319
patients who had prior IFD from the day of HSCT was 81.9%. Cumu-
lative one-year incidence of IFD event was 18% (15). There was no
difference in cumulative incidence of IFD event and OS between pos- Cryptococcosis in and around Delhi, India: Clinical,
sible IFD and PP-IFD group (27.8% vs 15.6%- P = 0.436, 77.8% vs antifungal and molecular pattern
56.3% P = 0.068, respectively). Twelve patients had progression of M. R. Capoor, T. Shende, S. Raghvan, B. K. Tripathi and
prior IFD after HSCT. Three patients developed the new IFD due to P. Varshney
Paecilomyces variottii; Candida krusei andTrichosporon. ‘Active’ (re-
lapse/progression) underlying disease in early (before Day+100) post Vardhman Mahaveer Medical College and Safdarjung Hospital,
transplant period was the only risk factor for any IFD event after Delhi, India
alloHSCT (9.1% vs 37%, P = 0.001). The one-year OS after HSCT in
patients with prior IFD was 61% and 63.8% in patients without his- Objective To analyse the clinical, antifungal and molecular pattern
tory of IFD (P = 0.870). of cryptococcosis in and around Delhi, India.
Conclusion Prior IFD did not impair the outcome of alloHSCT with Methods Cryptococcosis was diagnosed by direct microscopy, latex
effective methods of diagnosis and prophylaxis being used. agglutination and culture. Cryptococcal species and variety was con-
firmed by sequencing. Antifungal susceptibility was carried out by
broth microdilution as per CLSI 2012 guidelines.
Results A total of 129 isolates of C. neoformans were isolated from
P318 101 patients, as nineteen cases were of disseminated cryptococcosis
over a period of six years. These were isolated from CSF (99), blood
(11), respiratory sample (sputum/BAL/pleural fluid) (5) and urine
The case of successful treatment of invasive pulmonary (13), soft tissue (1). One hundred one cryptococcosis cases included
mucormycosis (Rhizomucor sp.) in patient with acute 79 HIV positive cases and 22 immunocompetent. Among HIV nega-
myeloid leukemia tive patients the known risk factors were leucopenia (9), neutropenia
L. A. Tarakanova,1 V. N. Semelev,1 A. M. Zhivopistceva,1 (2), Hodgkin’s lymphoma (1), lymphocytosis (2), tuberculosis (1) and
D. A. Gornostaev,1 O. V. Shadrivova,2 V. V. Tyrenko1 and apparently immunocompetent (7). Overall mortality was 29 (28.7%).
Mortality (25), Relapse (4) and dissemination (18) were more in HIV
N. Klimko3
1
positive than HIV negative group (4), (1) and (1), respectively. Co-in-
Kirov Military Medical Academy, Saint Petersburg, Russia; 2I. fection with tuberculosis was seen in HIV (6) AND HIV seronegative
Mechnikov North-Western State Medical University, St. (4) patients. HIV positivity was the most important risk factor of
Petersburg, Russia and 3North-Western State Medical University cryptococcosis. Predictors of mortality in HIV positive patients were
named after I.I. Metchnikov, Saint Petersburg, Russia CD4 + count ≤50 mm3, non-usage of ART, inadequate dose of flu-
conazole. Predictors of mortality in HIV negative patients were con-
Introduction Rare fungal infections in immunocompromised trol of underlying condition. HIV seronegative patients, were
patients became actual in recent years. We report a case of invasive observed to have better survival (77.3%) as compared to HIV positive
pulmonary mucormycosis (Rhizomucor sp.) in patient with acute patients (72.2%). Symptoms of fever, headache, altered sensorium
myeloid leukemia. were observed to have better survival. Patients who exhibited any
Case report In April 2012 27 years old male with acute myeloid degree of neurological deficit at first examination had a higher pro-
leukemia (FAB-M4) was treated with chemotherapy ‘7 + 3’(cytara- portion of deaths (35.5%) due to cryptococcal meningitis as opposed
bine 100 mg/m2, mitoxantrone 12 mg/m2). Prophylactic fluconazole to those without any deficit (22.9%). Patients with normal findings
300 mg/d was used in neutropenia. On the 8th day of the on first CT (head & neck), had a higher proportion of survivors
chemotherapy fever was noted. A chest and paranasal sinuses x-ray (87%) at the end of the study period, as compared to those with any
was unremarkable. Antibacterial therapy (ertapenem 2 g/d, linezolid abnormal finding (69.2%). Patients who were treated with a combi-
1.2 g/d) was used, but fever persisted. Lung CT scan was unremark- nation of antifungals and ATT/ART (as required) had maximum
able, but in paranasal sinuses CT pansinusitis was revealed. Vori- number of survivors (86.1%) as opposed to use of either antifungals
conazole (800 mg d
1 in 1st day, then 400 mg d
1) was added. alone (68.5%) or ART/ATT alone (55.6%). The antifungal suscepti-
Blood test for galactomannan was negative. Despite antibacterial and bility of the isolates to amphotericin B, fluconazole, itraconazole,
antifungal therapy high fever persisted (38–39 °C). Coughing and voriconazole, posaconazole were in the susceptible range. MIC of all
the C. gattii isolates was higher as compared to C. neoformans

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 169
Poster Presentations

serotype A & D isolates. A total of 40 isolates were sequenced, target- P321

ing the 26S ribosomal subunit by using ITS-1, ITS-2 and ITS-4
region. Out of these isolates 32 were C. neoformans var. grubii, 2 were
C. neoformans var. neoformans, 6 were C. gattii. Aspergillossis and Cryptococcal meningitis in HIV-infected
Conclusions Cryptococcosis is fatal despite despite timely diagnosis, patients in C H U Oran
institution of antifungal therapy and the sensitive isolates to antifun- Z.B. Benmansour
gals. It is common in HIV seronegative patients. C. gattii can also University of Oran, Oran, Algeria
infect HIV population. More ecological, phenotypic, host-defense and
molecular studies are warranted to study this mycoses.
Objective The invasive fungal infections in hospital is a subject of
concern both for the healthcare professionals .The infection at the
hospital is a major risk for the patients the invasive acts, in first row
of morbidity, mortality.
P320 Materials and methods A retrospective study was conducted from
september 2011 to Mars 2014, the fungal infections from immuno-
Invasive fungal infectious in HIV positive patients compromised patients and the occurrence of Cryptococcus neofor-
H. Pullukcu,1 P. K. Ekren,2 M. Tasbakan,2 F. Bacakoglu2 and mans admitted in unites of disease infections ,with Aspergillosis
€kengin2
D. Go investigation serology .Cryptococcal meningo-encephalitis was diag-
1
nosed in 23 individuals. The median age of the patients under study
Ege University Medical School, Izmir, Turkey and 2Ege was39.25 years With male preponderance . Typical presentations
University, Izmir, Turkey were persistent headaches (27 cases/36), neck stiffness (16/36),
altered consciousness (14/36), fever (12/36) and convulsions (9/
Objective Individuals with human immunodeficiency virus (HIV)/ 36).Cryptococcal meningitis highly contributes to mortality in HIV-
AIDS are at significant risk of invasive fungal infections (IFIs). Pul- infected patients. A need exists to improve strategies for clinical man-
monary and oesophageal candidiasis, Pneumocystis jiroveci pneumo- agement of AIDS patients and systhematique Aspergillosis serology
nia are AIDS-defining fungal infections. Invasive aspergillosis is all samples sent to Laboratory for mycological analysis. A question-
relatively rare. In this sudy it is amimed to evaluate invasive fungal naire was conducted in which there is information for each sam-
infections in HIV(+)/AIDS patients. pleInclusion criteria: The patients admitted in risk servicesExclusion
Methods The patients records hospitalized because of invasive fungal criteria: - Patients under antifungal treatment430 samples from dif-
infections in infection diasease department and respiratory intensive ferent departments .Cryptococcal meningitis in HIV-infected patients
care unit were evaluated retsospectively between 2006 and 2015. is an important fungal pathogen in immunocompromised patients.
Inclusion criteria for each specific IFI were adapted from the 2002 High mortality was related to delayeddiagnosis. A need exists to
guidelines of the European Organization for Research and Treatment improve strategies for clinical management of AIDS patients.
of Cancer/Invasive Fungal Infections Cooperative Group and the Results prevalence of the meningo-encephalitis cryptococcose at the
National Institute of Allergy and Infectious Diseases Mycoses Study patients infected by the HIV was 0.4 %. frequency practically
Group. Data for each IFI were collected including patient demograph- decreased while it was 2.09 % for the ten years.
ics and clinical characteristics, comorbidities, and concomitant bacte- Discussion the man is preferentially touched. The average time evo-
rial infections. Patient characteristics included HIV-specific lution of the symptomatologie before hospitalization represented 1
information such as CD4 cell counts, and viral load. Detailed infor- week. The mode of installation of the disease is progressive,fever,
mation about the IFI was collected and included the specific fungal headache,immunosuppressed patient, with a rate of CD4 lower than
pathogen and species, infection site. 200/mm3, any suspicion of meningitis has to make look for Crypto-
Results The data of 11 patients (9 male, mean age 49.6  13.7, coccus neoformans . positive in 72.7 % the death arose in 54.5 %
min:30-max:73) had invasive fungal infections were evaluated. The Conclusion The fungal infection relegated to the last rows neu-
data of patients were summarized in table. romeningeal cryptococcose Aspergillosis ,necessarily in systematic
Conclusion Fungal infections remain a clinical challenge in HIV study from subjects HIV + the prescription of effective systematic
patients with severe immunosuppression. Our data also suggest that antifungals is primary prevention with Antiretrovirals in front of a
HIV patients with CD4 cell counts >100 cells ll
1 and other under- therapeutic
lying conditions may be susceptible to invasive fungal infections
including pulmonary aspergillosis.

Table 1. The data of patients were summarized

Figure 1. Parasitology and mycology CHU Oran

ª 2015 The Authors

170 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P322 P323

Antifungal Susceptibility Testing to Predict Appropriate Invasive aspergillosis in patients with hematological
Initial Antifungal Therapy and Outcome in non-Aspergillus malignancies in Czech, Slovak and Croatian
Invasive Mold Infections hematooncological departments: Fungal INfection
F. Lamoth1 and B. D. Alexander2 Database (FIND) analysis (2001–2014) - an update
1
CHUV, Lausanne, Switzerland and 2Duke University Medical B. Weinbergerova,1 Z. Racil,1 I. Kocmanova,1 M. Lengerova,1
Center, Durham, USA E. Janousova,2 L. Drgona,3 M. Kouba,4 M. Hricinova,4
A. Ostojic,5 R. Vrhovac,5 J. Gabzdilova,6 T. Guman,6
Objectives Non-Aspergillus invasive mold infections (NAIMI) are V. Petecukova,7 J. Novak,7 K. Forsterova,8 J. Haber,8 B. Ziakova,9
associated with high mortality. Mucoromycotina, Fusarium spp. and E. Bojtarova,9 A. Zavrelova,10 S. Vokurka,11 V. Chrenkova,12
Scedosporium spp. account for the majority of NAIMI and exhibit dis- P. Sedlacek,13 B. Tkacikova,14 P. Mudry,14 V. Zatezalo,15
tinct susceptibility profiles with resistance to many antifungal classes. N. Gredelj,15 N. Mallatova,16 P. Timr,17 D. Sejnova,18
The lack of established clinical breakpoints precludes the interpreta-
D. Tanuskova,18 A. Chocholova,18 J. Horakova,18 M. Navratil,19
tion of minimal inhibitory concentration (MIC) values for these
molds. The aim of this study was to assess the association between R. Hajek,19 J. Chudej,20 J. Sokol,21 M. Rolencova,1 P. Zak,10
MIC and clinical outcomes and the possible role of antifungal suscep- P. Cetkovsky4 and J. Mayer1
1
tibility testing in guiding antifungal therapy of NAIMI. University Hospital Brno, Brno, Czech Republic; 2Institute of
Methods Medical charts of patients with positive cultures for the Biostatistics and Analyses, Faculty of Medicine, Masaryk
most relevant non-Aspergillus mold species collected at Duke Univer- University, Brno, Czech Republic; 3Comenius University and
sity (Durham, NC, USA) between 2009 and 2013 were retrospec- National Cancer Institute, Bratislava, Slovak Republic; 4Institute of
tively screened. Patients with proven/probable NAIMI (EORTC-MSG Hematology and Blood Transfusion, Prague, Czech Republic;
definitions) were included in the analysis. Antifungal susceptibility 5
University of Zagreb and University Hospital Centre Zagreb,
testing for amphotericin B, voriconazole, posaconazole, micafungin
and caspofungin was retrospectively performed according to CLSI
Zagreb, Croatia; 6Louis Pasteur University Hospital, Kosice, Slovak
procedure. Patient characteristics, pathogen MIC values, antifungal Republic; 7University Hospital Kralovske Vinohrady, Prague,
therapy and NAIMI outcome were analyzed. Czech Republic; 8General Faculty Hospital, Prague, Czech
Results 39 NAIMI episodes were included (19 mucormycosis, 12 Republic; 9St. Cyril and Methodius Hospital, University Hospital
fusariosis, 4 scedosporiosis, 3 Paecilomyces spp. infections and 1 Scop- Bratislava, Bratislava, Slovak Republic; 10University Hospital
ulariopsis spp. infection). Twenty-two (56%) patients had hematologi- Hradec Kralove, Hradec Kralove, Czech Republic; 11University
cal malignancies and 13 (33%) were solid-organ transplant Hospital Pilsen, Pilsen, Czech Republic; 12Charles University and
recipients. Mortality at week 12 was 59% (44% before week 4). Motol University Hospital, Prague, Czech Republic; 13Charles
Amphotericin B had variable in vitro activity against Mucoromy- University and University Hospital Motol, Prague, Czech Republic;
cotina (median MIC 0.5 lg ml
1, range 0.125 - 4) and Fusarium 14
Masaryk University and University Hospital Brno, Brno, Czech
spp. (2 lg ml
1, 1 - 4), but had minimal activity against other mold Republic; 15University of Zagreb and University Hospital Merkur,
species. All Fusarium spp. exhibited high MICs to voriconazole Zagreb, Croatia; 16Hospital Ceske Budejovice, Ceske Budejovice,
(≥16 lg ml
1) and 53% of Mucoromycotina had high MICs to Czech Republic; 17Ceske Budejovice Hospital, Ceske Budejovice,
posaconazole (>16 lg ml
1). Initial antifungal therapy was found to Czech Republic; 18Pediatric University Hospital, Bratislava, Slovak
be crucial for NAIMI outcome. Success rate at week 6 was 75% for Republic; 19University Hospital Ostrava and Medical Faculty of
initial appropriate antifungal therapy defined as use of an antifungal the Ostrava University, Ostrava, Czech Republic and 20Jessenius
drug with a MIC cut-off ≤0.5 lg ml
1 during the first 72 h of treat-
Faculty of Medicine, Comenius University, Martin, Slovak
ment versus only 13% in cases of MIC >0.5 lg ml
1 or absence of
Republic
antifungal therapy (p value = 0.001). There was no other significant
association with better outcome including immunosuppressive status
(neutropenic vs non-neutropenic) or underlying condition (hemato- Objectives ‘Fungal InfectioN Database’(FIND) represents interna-
logic malignancies vs others). tional database of invasive fungal infections in Czech, Slovak and
Conclusion NAIMI remain associated with a very high mortality Croatian hematooncological departments. FIND – aspergillus covers
rate. Because of the unpredictable susceptibility profile of Mucoromy- all case of invasive aspergillosis (IA) in participating centers since
cotina and Fusarium spp. to amphotericin B and azole compounds, 2001.
antifungal susceptibility testing is useful. A MIC cut-off of Methods The goal of our retrospective analysis was to evaluate inci-
≤0.5 lg ml
1 is a good indicator of appropriate initial antifungal dence, early diagnostic procedures and effect of antifungal therapy in
therapy, which is associated with better outcomes. proven and probable IA that occurred in 18 institutions participating
in FIND database between 2001–2014. Till 2009 we followed
EORTC/MSG 2002 and from 2010 EORTC/MSG 2008 criteria in
evaluation of IA diagnosis and therapy response.
Results 444 probable and 94 proven IA (87.6% isolated pulmonary
IA, IPA) have been documented. Prolonged, profound neutropenia
(60.4%) and long-term use of corticosteroids (28.4%) were identified
as the major risk factors of IA. 58.4% pts. had consecutive positivity
of serum-galactomannan (S-GM) (OD index >0.5). 82.0% pts. with
IPA and bronchoalveolar lavage (BAL) had positive GM in BAL fluid
(OD index ≥ 0.5). In pts. with IPA only 12.8% BAL fluids and 11.8%
sputum samples had positive microscopy for filamentous fungi and
19.7% BAL fluids and 49.6% sputum samples had positive culture
for Aspergillus spp. The primary antifungal therapy of IA was used
in 82.2% pts. - 51.6% voriconazole (VORI), 5.9% echinocandins
(ECHINO), 19.0% VORI+ECHINO, 4.5% amphotericine B deoxy-
cholate (C-AMB), 13.1% lipid-based AMB (LBA) and 5.9% other.
Overall RR to primary therapy of IA was 51.6% - VORI 57.9%, VOR-
I+ECHINO 57.1%, C-AMB 35%, LBA 44.8%, ECHINO 23.1%. There
was a statistically significant difference in overall RR to targeted

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 171
Poster Presentations

therapy in pts. with neutrophil count <0.1 and >1.0 9 109/l at the P332
end of therapy (28.3% vs. 53.4%; P = 0.004). The overall mortality
rate was 57.6%, with 30.7% attributable to IA.
Conclusion Based on our analysis we have confirmed typical risk Invasive Fungal Infections after Autologous Hematopoietic
factors for IA and critical role of S-GM and CT for early diagnosis Stem Cell Transplantation in Children and Adolescents: the
and prompt start of antifungal therapy of IA. A reasonable treatment Mu € nster Experience
response was achieved using VORI, VORI+ECHINO or LBA in pri- C. Linke, M. Ahlmann, B. Fro €hlich, M. W€altermann,
mary therapy of IA. We have confirmed neutropenia at the end of B. Burkhardt, C. Ro€ssig and A. H. Groll
antifungal therapy as the major predictive factor for therapeutic
University Children’s Hospital Mu€nster, Mu€nster, Germany
response. On behalf of CELL - The Czech leukemia study group for
life.
Background High dose chemotherapy followed by autologous
hematopoietic stem cell transplantation (HSCT) carries risks of infec-
tious morbidity. Little is known, however, about the occurrence of
invasive fungal diseases (IFDs) in pediatric patients undergoing autol-
P330
ogous HSCT. We therefore analyzed the epidemiology and manage-
ment burden associated with IFDs in a cohort of children and
Invasive Candidiasis in children with organic aciduria adolescents undergoing autologous HSCT for solid tumors or
Z. D. Pana,1 A. Tragiannidis,1 T. Marquardt,2 F. Rutsch,2 lymphoma.
O. Makarova,2 E. Idelevich2 and A. H. Groll2 Patients and methods In a retrospective single center study, epi-
1 demiology and management burden associated with IFDs were ana-
Aristotle University, Thessaloniki, Greece and 2University lyzed in all pediatric cancer patients who underwent autologous
Children’s Hospital Mu€nster, Mu€nster, Germany HSCT for solid tumors or lymphoma between 2005 and 2015. Clini-
cal, radiographic, and microbiological data were assessed until
Objectives Organic acidemia or aciduria (OA) refers to a heterogenic engraftment and up to 100 days post-transplant. The primary end-
group of inherited metabolic disorders, resulting from dysfunction of point was the incidence of proven, probable, and possible IFDs
a specific step in amino-acid catabolism usually due to deficient according to current EORTC/MSG definitions. Further endpoints
enzyme activity. Invasive candidiasis (IC) has not been recognized to included the use of systemic antifungal agents for prevention and
be associated with OA. On the other hand, acidotic metabolic state management of suspected or proven/probable IFDs; non-infectious
(as in diabetic ketoacidosis) has been previously implicated in and infectious co-morbidities; and survival until day +100. Patients
impaired neutrophil function (impaired adherence, chemotaxis, were housed in single non-HEPA-filtered rooms and received non-ab-
phagocytosis, pathogen killing, and respiratory burst) and therefore sorbable polyenes and penicillin plus ciprofloxacin for antimicrobial
in predisposition to invasive Candida infections. Herewith we report a prophylaxis. Piperacillin/tazobactam plus gentamycin was used as
case series of OA pediatric patients with significant metabolic acidosis initial empiric regimen. Interventions for persistent or recurrent fever
and simultaneous presence of Candida bloodstream infections (CBI), consisted of pulmonary computed tomography (CT) imaging and
enhancing the hypothesis that OA may predispose to the occurrence appropriate modification of antibacterial empiric therapy. The use of
of IC. systemic antifungal prophylaxis and of empiric antifungal therapy
Methods Children with OA and occurrence of CBI hospitalized at were at the discretion of the respective attending physician.
the University Hospital of Muenster (for the period 2006–2014) were Results During the ten-year observation period, 95 patients under-
enrolled. Demographic data, clinical features, laboratory exams, went 103 autologous HSCT procedures (solid tumors 92, lymphoma
microbiology data, antifungal treatment and outcome were retrospec- 11; mean age 9.9, range 0.75-20). High-dose chemotherapy
tively collected. Statistical analysis was performed with SPSS statisti- included treosulfan plus melphalan (50), carboplatin based (33), and
cal package12. various other (20) regimens. The mean time to neutrophil engraft-
Results Five patients with OA were enrolled with median age ment and to discharge were 12 (r, 7–29) and 16 (r, 11–72) days,
10.5 yrs (IQR 11) of whom 4 (80%) were males. Three children respectively.
were diagnosed with methylmalonic acidemia, one patient with glu- Systemic antifungal prophylaxis was administered to 49 HSCT pro-
taric aciduria and one with multiple acetyl CoA dehydrogenase defi- cedures (47.5%) and consisted mostly (43) of fluconazole. At least
ciency, respectively. All patients had a permanent central venous one CT scan was performed in 21 procedures. There was no single
catheter and experienced at least one episode of bacteremia while case of invasive yeast infection. Similarly, no single case of proven/
hospitalized for metabolic deterioration. Prior to Candida infection, all probable mold infection was diagnosed. Nine cases (8.7%) fulfilled
five children presented with metabolic deterioration with significant criteria of a possible pulmonary mold infection during neutropenia
acidosis, high ammonia levels and neutropenia with median neu- and received mold active antifungal therapy for a median of 14 days
trophil count 742 ll. The median duration of hospitalization was (r, 7–35). In an additional 12 procedures, empiric antifungal therapy
22 days. Two weeks prior to Candida infection, 2 pts presented infec- with mold active agents was given for a median of 8 days (r, 3–
tion due to St. epidermidis and in one patient a Kocuria rhizophila 105). Considering the total antifungal treatment burden of the
infection was recorded. Blood cultures grew in 3/5 of patients Can- cohort, systemic antifungal treatment was administered in 63 proce-
dida albicans, in 1/5 Candida parapsilosis and in 1/5 Candida lusitaniae, dures (61.2%), either as systemic prophylaxis only (42; 40.8%), pro-
respectively. Initial antifungal treatment consisted of caspofungin phylaxis followed by empirical or directed treatment (7; 6.8%) and
and liposomal amphotericin B (LamB) in two cases each, and of com- empirical or directed treatment only (14; 13.6%).
bined treatment with flucytosine and LamB in one case. Median time Grades III/IV mucositis was recorded in 51 procedures and micro-
for bloodstream sterilization of Candida spp. was 5 days. In all biologically documented non-fungal infections in 17. Five patients
patients CVCs were removed with a median time of removal after were transferred to the ICU. One patient died from biopsy docu-
Candida diagnosis 5 days. None of the patients died due to invasive mented toxic endothelial damage at day 83 post-transplant, account-
Candida infection. ing for an overall mortality rate of <1%.
Conclusion The present case series suggest an increased risk of Conclusions Autologous HSCT for solid tumors or lymphoma was
patients with OA for invasive Candida infections during metabolic associated with low morbidity from IFDs at our institution. However,
decompensation with acidosis, granulocytopenia, high concentration utilization of pulmonary CT imaging and use of systemic antifungal
glucose infusion, antibacterial treatment and presence of central agents for prevention and management of suspected IFDs were
venous catheters as contributing factors. considerable

ª 2015 The Authors

172 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P333 Different methods of anesthesia were performed, depending on the

age and the degree of respiratory failure of patients with mandatory
monitoring of vital functions of the body and oxygen levels. The
Successful treatment of knee Actinomycosis obtained samples were sent immediately to the laboratory for micro-
O. P. Kozlova, A. K. Mirzabalaeva and N. Klimko scopy with calcofluor white, culture, and galactomannan (GM) detec-
North-West State Medical University n.a. I.I. Mechnikov, Saint tion (Platelia Aspergillus, Bio-Rad).
Petersburg, Russia Results No major complications occurred during bronchoscopy.
According to EORTC/MCG 2008 criteria IPA was seen in 30%
patients with different various lesions on computed tomography,
Objectives Actinomycosis of a knee is rare, with few published 16% of them have been proven by biopsy. In multivariate analysis,
cases. We report here a case of successful treatment of knee GM test in BAL fluid showed a high sensitivity compared to micro-
actinomycosis. scopy and culture (83.3% and 46.3%, respectively). When the index
Materials and methods A 8-year-old girl was admitted at March of the optical density was 0.925, diagnostic accuracy shown in the
2014 with painless swelling of the left knee after daily activities. One area under the ROC curve (AUC) analysis was 0.794 (95% CI:
year before trauma of left foot (stab wound with a rusty nail) 0.665–0.923). The most diagnostic efficiency was achieved by using
occured. The patient was diagnosed as ‘infectious-allergic arthritis’. all three methods - microscopy, culture, and GM test in BAL: AUG
She was treated with non-steroid anti-inflammatory drugs. At was 0.914 (95% CI: 0.853–0.975).
August 2014 movements of the affected joint was limited. Lab tests Conclusions - Bronchoscopy with modern video endoscopy equip-
(hemoglobin and total white counts, total protein, ALT, AST, blood ment and adequate anesthesia can safely be used for diagnosis of
sugar, rheumatologic tests) were normal. CT scan revealed inhomo- invasive pulmonary aspergillosis in children after allo-HSCT.
geneous contents in the projection prepatelly bags, extending to the - The most diagnostic efficiency is achieved by using microscopy,
anterolateral surface with clear contours, size 50 * 8 mm. Intraoper- culture, and galactomannan test of bronchoalveolar lavage fluid.
ative inspection showed edema and fibrosis of the tissue surrounding
patella left knee, education dimensions of 8 * 6 * 2 cm, yellowish-
brown color, with areas of necrosis. Histology showed inflammatory
infiltrate comprised of neutrophils, lymphocytes and plenty of foamy
macrophages around a sulphur granules. P335
She was treated for two weeks with IV penicillin 24 000 000
units/d with rapid clinical response and disappearance of all symp- Invasive Fungal Infection (IFI):one center experience with
toms. Then she used oral amoxicillin 750 mg d
1 for 4 months. She rare filamentous fungi
has remained asymptomatic since this time. She has remained
T. J. a Jamal Mohamed,1 K. A. Mohamed Razali1 and E. J. Abdul
asymptomatic since this time. General state of health is satisfactory:
no pain, full motion of the knee joint. CT imaging of left knee are Rahman2
1
normal. Paediatric Infectious Diseases Div,Hospital Kuala Lumpur, Kuala
Conclusion Intravenous and oral penicillin therapy for 4.5 months Lumpur, Malaysia and 2Paediatric Oncology Division, Kuala
to which patient showed excellent response. Antimicrobial therapy Lumpur, Malaysia
with surgery is the treatment for actinomycosis.
Introduction IFI has increased over years as a major cause of mor-
bidity and mortality among hematological malignancy.In paediatric
population not only candida cause IFI but filamentous fungi had
P334 been implicated due to increasing immunosuppresion and invasive
procedures.
The role of bronchoscopy in the diagnosis of invasive Objective We report here our center incidence of IFI and case
reports of rare infection by mucomycosis and one disseminated
pulmonary aspergillosis in children after allogeneic
fusariosis.
hematopoietic stem cells transplantation Methods Retrospective analysis of IFI from January 2014 to Decem-
A. Volkova,1 N. Klimko,2 M. O. Popova,3 O. V. Shadrivova,4 ber 2014 in paediatric oncology ward of Paediatric Institute,Hospital
T. S. Bogomolova,2 S. M. Ignatyeva,5 B. I. Smirnov,6 Kuala Lumpur,Malaysia.
L. Zubarovskaya1 and B. Afanasyev1 Results 10 cases of fungaemia with 7 cases of candidaemia and 3
1
Raisa Gorbacheva Memorial Institute, First Pavlov State Medical cases involving filamentous fungi (2 fusarium spp,1 of rhizomucor
University, Saint Petersburg, Russia; 2North-Western State spp.).
1st case report of 2 year old child with meduloblastoma with per-
Medical University named after I.I. Metchnikov, Saint Petersburg,
sistent fever and respiratory symptoms with rhizomucor spp isolated
Russia; 3Raisa Gorbacheva Memorial Institute of Children from both central and peripheral lines. Despite chemoport removal,
Oncology, Saint Petersburg, Russia; 4I. Mechnikov North-Western fever persists and examination of skin, sinus or lung and CNS were
State Medical University, St. Petersburg, Russia; 5Medical normal. Imagings show pneumonia with no sinusitis or CNS involve-
mycology institute named after Kashkin, Saint Petersburg, Russia ment. Responded to prolong couse of liposomal amphotericin B with
and 6Saint-Petersburg Electrotechnical University „LETI„, Saint posaconazole (off label use).
Petersburg, Russia 2nd case; a 9 year old girl diagnosed with precursor B ALL in
October,2014.Following chemotherapy develop vesicular nodular
Background Early diagnosis of invasive pulmonary aspergillosis lesion with crusting and bleeding from nose. Examination showed a
(IPA) in patients undergoing hematopoietic stem cell transplantation febrile child with multiple skin lesions consisting of papulonodule
(HSCT) is one of the factors positively influencing the survival of over arms, legs with bilateral crusting of both nostrils (EUA: anterior
adult patients. The role of bronchoscopy in diagnosis of IPA in chil- septal perforation 2 9 2 cm with necrosis of cartilage. Lt side: ante-
dren after HSCT is not clear. rior perforation). Other systema;revealed pneumonia with hep-
Materials and methods We evaluated the safety and efficacy of atosplenomegaly and normal CNS examination. Skin biopsy and
bronchoscopy with bronchoalveolar lavage (BAL) fluid (n = 222) nasal tissue during EUA grew Fusarium spp. Imaging show sinusitis;
testing for the diagnosis of IPA in 150 children after allogeneic multiple lung nodules and cavity at rt upper lobe, splenic nodules
hematopoietic stem cells transplantation (allo-HSCT). The median of with no intracranial extension. She had poor response to liposomal
age was 12 years old. We used EVIS EXERA II Olympus endoscopic amphotericin B but infection resolved with combination of triazole
video system with an external diameter of the distal end tubes of the and terbinafine.Environmental screening were negative for both
3.6 mm and 4.9 mm in a specialized endoscopy room or ICU (12%). mould.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 173
Poster Presentations

Conclusion IFI by filamentous fungi is now common in children Dermatophytes are spread by direct contact with other people (an-
and isolation even a rare mould need prompt action from clinician to thropophilic organisms), animals (zoophilic organisms), and soil (geo-
prevent mortality. philic organisms), as well as indirectly from fomites.
Case report Female patient, 11 years old, presenting erythematous
and scaly plaque with vesicles and pustules in lesion on the chest.
P336 The lesion appeared four months before the medical assistance, and
was associated to itching and burning. The adult guardian claims to
have conducted several treatments, including antifungal medications,
Chronic Hepatosplenic Candidiasis with related IRIS in achieving a partial improvement. Transmission of the fungus by
young child with leukaemia Cavia porcellus (guinea pig) was suspected, as the patient said that
T. J. a Jamal Mohamed,1 K. A. Mohamed Razali,1 M. Mohamed2 she frequently took her pet and put near the site of the injury. The
and N. S. Che Hussin3 animal had scaly lesions on the ear, which began before the appear-
1 ance of lesions in the patient. Direct mycological examination of the
Paediatric Infectious Diseases Div,Hospital Kuala Lumpur, Kuala patient’s skin material revealed the presence of arthroconidia charac-
Lumpur, Malaysia; 2Paediatric Oncology Division, Kuala Lumpur, teristic of dermatophytes. In the cultural mycological examination of
Malaysia and 3Hospital Kuala Lumpur, Kuala Lumpur, Malaysia the material from the patient and the guinea pig there was growth
of a filamentous fungus with the same whitish and powdery aspect.
Introduction Invasive candidiasis(IC) is common among children The microscopic cultures showed hyphae, macro and microconidia
undergoing intensive treatment of haematological malignancies. In caracteristic of Trichophyton sp. The urease test was positive, indicat-
our institute, the incidence of candidaemia is 13 cases out of 14 fun- ing T. mentagrophytes or T. interdigitale, which can be identified only
gaemia in year 2012. Chronic form of IC is a rare form of dissemi- by molecular methods that are currently being performed. The anti-
nated fungal infection seen during neutrophil recovery seen in subset fungal susceptibility tests were performed for both isolates, and the
of patients with IC. minimal inhibitory concentration (MIC) of eight antifungal agents
Case report We report here a case of infant who had febrile neutrope- were evaluated by the microdilution method in 98-well plates
nia complicated by Candida tropicalis infection following chemotherapy according to the CLSI protocol M38-A2. The MICs (lg mL
1) for
for acute lymphoblastic leukaemia. Despite clearance of candidaemia patient/guinea pig were: posaconazole (0.03/0.03); terbinafine
and removal of central venous catheter, fever persisted. Search for (0.03/0.125); tioconazole (0.125/0.06); voriconazole (0.125/0.125);
other fastidious infections including mycobacterium and filamentous itraconazole (0.25/0.25); amphotericin B (0.5/0.5); ketoconazole (8/
fungi were negative. Repeated bone marrow showed child to be in 8); fluconazole (32/32). These results indicate that the isolates had
remission.Splenectomy was performed but despite that and almost similar MICs for all antifungals, most of them low, except ketocona-
three months of amphotericin B, there was no resolution of sign and zole and fluconazole. The girl and her pet were treated with terbina-
symptoms hence immune reconstitution was thought of and empiric fine and the lesions were regressing until cure.
steroid started. Symptoms finally abated after 12 weeks of steroids. Discussion The anthropophilic subspecies of T. mentagrophytes, as
Method Retrospective analysis of episodes of candidemia in paedi- well as many of the zoophilic strains, formerly differentiated as var.
atric oncology ward of Paediatric Institute of Hospital Kuala Lumpur mentagrophytes or var. granulosum, are indistinguishable and are now
from January to December 2014 and the presence of chronic form of designated T. interdigitale. The morphological differentiation between
IC in this subgroup. anthropophilic and zoophilic T. interdigitale strains by classical micro-
Results There were seven cases of candidaemia in children admitted scopical and biochemical methods is often problematic. Molecular
to paediatric oncology ward throughout 2014.Only one child had identifications methods are required to differentiate between the zoo-
chronic hepatosplenic candidiasis.Ultrasound abdomen showed sple- philic strains of T. interdigitale and T. mentagrophytes. Zoophilic der-
nic micro abscesses after 2 weeks of neutrophil recovery. Serial ultra- matophytes produce acute severe inflammation with pustules and
sounds later show involvement of liver and kidney. Blood cultures vesicles as described in our case. The household exposure increases
grew candida tropicalis and serum mannan was 560 pg Ml
1. the appearance of dermatophyte infection in pre-puberty population,
Echocardiogram showed pericardial effusion, eye examination was and the extent of inflammation depends on the causal pathogen and
normal and splenic tissue cultures were negative for bacteria, fungi the host immune response.
and mycobacterium. Histopathological examination of spleen showed
necrotizing granuloma with presence of fungal elements suggestive of
candida. Serum galactomanan was repeatedly negative. Ultrasound
after 5 months of antifungal; initial monotherapy of amphotericin B P339
followed by combination showed resolution of micro abscesses with
serum mannan turning negative.
Conclusions When dealing with chronic disseminated candidiasis, need In vitro hemolytic activity and biofilm production by
to think of immune reconstitution inflammatory response (IRIS) and trial isolates of yeast species from immunosuppressed pediatric
of steroid given after exclusion of concomitant bacteria,viral other fungal host
infections if symptoms did not resolve with antifungal alone. V. Lora, M. L. Perez and G. Lo
pez
Hospital para el Nin~o Poblano, Puebla, Mexico

P338 Introduction The ability of pathogenic organisms to acquire ele-

mental iron has been shown to be of pivotal importance in their sur-
vival and ability to establish infection within the mammalian host.
Dermatophytosis transmission from guinea pig to child: a The pathogen is equipped with a mechanism that destroys the heme
case report moiety and enables it to extract the elemental iron. The enzymes
M. L. Scroferneker,1 D. Heidrich,1 K. O. Alves,1 G. Vettorato,2 mediating such activity are broadly classified as hemolysins. Medi-
I. S. Santos,2 T. G. Amaro,2 D. de Villa,2 K. S. Bastos2 and cally important biofilms can be split into two major types: those that
A. Grill2 develop on the surface of implanted devices and those that form
1 directly on the host tissues. Yeast cells in biofilms differ greatly from
Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil their planktonic counterparts in terms of their susceptibility to
and 2Hospital Santa Casa de Misericordia de Porto Alegre, Porto antimicrobials and evasion of host defenses, and there appear to be
Alegre, Brazil multiple mechanisms of antimicrobial resistance.
Objective To study isolates of yeast cells species recovered from pedi-
Introduction Dermatophytosis or Tineas are superficial fungal infec- atrics patients for the capacity to produce biofilm and hemolytic
tions of the hair, skin and nails of humans and animals. activity.

ª 2015 The Authors

174 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Methods Biofilm formation was determinated by inoculating each clinical evidence; 39 (72.2%) of 54 patients were false positives
strain of yeast species into a glass tube containing Sabouraud Dex- results because sepsis evidence and presence of galactofuranose in
trose Broth and other glass tube containing Brain Heart Infusion. cereals foods. Aspergillus fumigatus was in 11 of 15 invasive mycosis
Tubes were incubated at 37ᵒC for 24–48 h without agitation. After with detected galactomannan antigen 0.622-4.101 ng/ml; Aspergillus
incubation, the culture broth in the tube was aspirated and the tubes versicolor with 0.598 ng/ml antigen Galactomannan; Penicillium spp
were washed once with distilled water. The walls of the tubes were with 1.225 ng/ml, Fusarium solannii with 1.071 ng/ml, and Candida
stained with safranin after media and yeast cells were discarded. tropicalis with 0.880 ng/ml as antigen value results. The death rate
Hemolysin production was evaluated using a plate assay. Each strain was 33.3% (5 of 15) and 66.7% (10 of 15) for successful medical
of yeast species was inoculated on a Sabouraud dextrose agar supple- treatment with Voriconazole and Caspofungin acetate. Aspergillus
mented with 5% sheep blood. The plates were incubated at 37ᵒC for fumigatus was etiological agent for invasive mycosis in 4 of 5 dead
48 h. The presence of a distinct translucent halo around the inocu- patients, 2 Fallot’s tetralogy, 1 fibrocystic disease of breast, and 1
lums site, viewed with transmitted light, indicated positive hemolytic hemolytic-uremic syndrome. Fusarium solanii was etiological agent
activity. Results. 228 isolates were obtained from leukemia, cancer, for invasive mycosis related to Fallot’s tetralogy in 1 of 5 dead
tuberculosis, fibrocystic disease of breast, meningocele and Fallot’s patients only.
tetralogy patients. The isolates were cultured from urine (n = 108) Conclusion Utilizing biopsy to diagnose Invasive Mycosis is ham-
and respiratory specimens (n = 120). The yeast strains including pered by the physiologic condition of the patients, as most cannot
132 Candida albicans, 44 C.tropicalis, 21 C.parapsilosis, 11 C.glabrata, tolerate invasive procedures due to thrombocytopenia, coagulation
10 C.lusitaniae, 6 Trichosporon asahii and 4 C.krusei. A total of 132 disorders and/or their critical condition. Positive test results for galac-
(57.9%) of 228 yeast species isolates obtained were biofilm positive. tomannan antigen with no clinical signs have been reported, espe-
Biofilm production was most frequently observed for isolates of C.kru- cially in young children; however, current data support the
sei (100%, 4 of 4); C.tropicalis (93.2%, 41 of 44); T.asahii (83%, 5 of usefulness of the galactomannan antigen detection as a biomarker
6); and C.parapsilosis (71.4%, 15 of 21). At 48 h postinoculation, for Invasive Mycosis to be used in conjunction with other diagnosis
alpha hemolysis and beta hemolysis could be observed circumscrib- test as radiographic procedures and clinical findings.
ing the yeast colony. A total of 153 (67.1%) of 228 yeast species iso-
lates obtained produced hemolytic activity. Hemolysin production
was most frequently observed for isolates of C.krusei, C.parapsilosis, P341
C.glabrata and C.tropicalis.
Conclusions Even though non-albicans species are considered less
Disseminated mucormycosis in a 6 month old infant
invasive and virulent than C.albicans, some species are inherently less
susceptible to common antifungals and some species such as tropi- following congenital heart defect corrective surgery: Case
calis, glabrata and parapsilosis; have the ability to produce biofilm and report
hemolysins as components of fungal virulence and cause disrupting P. Brogueira,1 N. Carvalho,2 J. Batista,1 E. Goncalves,1
host cell membranes. R. F. P. Sabino,3 C. Verıssimo,3 K. Mansinho,1 R. Anjos2 and
C. Toscano1
1
Hospital Egas Moniz, Lisboa, Portugal; 2Hospital de Santa Cruz,
Lisboa, Portugal and 3National Institute of Health Dr. Ricardo
P340 Jorge, Lisboa, Portugal

Galactomannan antigen detection as a biomarker for Objectives (Introduction) Invasive fungal infections in children are
invasive mycosis in immunosuppressed pediatric host becoming more frequent primarily due to an increased survival of
 pez, M. L. Perez, A. Bonifaz and C. Velasco
V. Lora, G. Lo children with primary or secondary immune deficiencies. Although
uncommon, mucormycosis has been increasingly identified among
Hospital para el Nin~o Poblano, Puebla, Mexico
immunocompromised patients and carries a high fatality rate. There
are several risk factors specially related to an immunodeficiency state
The diagnosis of Invasive Mycosis is based on clinical suspicion, such as hematologic malignancy with prolonged neutropenia and
imaging studies, and collection from affected tissue for pathologic bone marrow or solid organ transplant, illustrating the role of phago-
and microbiologic examination. Because the prognosis of the disease cytic capacity as well as cellular immunity in the prevention of
improves with early commencement of specific therapy, diagnosis mucosa and tissue invasion.
should be made as quickly as possible. The detection of galactoman- Methods (Clinical case) A 6 month old infant was admitted to our
nan antigen in pediatric patients immunosuppressed serum samples intensive care unit due to central cyanosis and heart failure. He had
used in conjunction with other diagnostic procedures can support past history of prematurity (35 week and 6 days), neonatal cholesta-
the diagnosis of Invasive Mycosis. sis, atrial dysplasia with multifocal atrial tachycardia, right ventricle
Objective Galactomannan antigen test in serum for neutropenia leu- hypoplasia with ventricular tachycardia and inferior vena cava inter-
kemia and immnunosuppressed pediatric patients to support invasive ruption with continuity to superior vena cava through azygos. He
mycosis diagnosis. was submitted to medical and surgical correction and was on
Method Immunosuppressed pediatric patients galactomannan anti- mechanical ventilation and central venous catheterization. During
gen test was used by immunoenzymatic sandwich microplate assay hospital stay he suffered from several nosocomial infections treated
in serum samples. A first serum sample was tested for galactoman- with large spectrum antibiotics. On day 48 he was diagnosed with
nan antigen as individual reference value for each patient and 72 sepsis. A Rhizomucor pusillus was isolated from urine and 4 days later
hours after a second serum sample tested was compared with the it was also detected from several tracheal aspirates. Due to his clini-
first value due to galactofuranose in cereals foods as interference cal condition, tissue biopsy was not an option. He was treated with
probable. Serum samples subsequent were tested weekly. Invasive liposomal amphothericin B for 40 days. Caspofungin was added for
Mycosis diagnosis established with related host, clinical, microbio- 27 days. Also vesical irrigations with amphotericin B deoxycholate
logic and histopatologic criteria. Results Serum samples were stud- was done as initial therapy. He was on peritoneal dialysis because of
ied for detection of galactomannan antigen in 121 pediatric patients; acute renal injury AKIN (Acute Kidney Injury Network) III.
83 (68.6%) male and 38 (31.4%) female; 98 of 121 (81%) with neu- Immunological study revealed decreased lymphocyte absolute num-
tropenia in leukemia and oncosis patients; 10 of 121 (8.3%) ber, mainly due to CD8 subpopulation. Renal echography showed
immune-deficiency syndromes; 9 of 121 (7.4%) Fallot’s tetralogy; 3 2 mm bilateral nodules suggestive of fungal etiology. He was extu-
of 121 (2.5%) cystic fibrosis; and 1 of 121 (0.8%) hemolytic-uremic bated on day 90 with success. Due to clinical recovery but persis-
syndrome. Galactomannan antigen was detected in 54/121 patients tence of dialysis dependence, he was transferred to a pediatric
(44.6%) with >0.500 ng/ml. Only 15 (27.8%) of 54 patients were nephrology unit after 122 days.
true positives results in conjunction with mycological culture and

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 175
Poster Presentations

Results (Laboratory findings) A classical urine culture on blood P346

agar revealed the presence of a filamentous non-septate fungus after
24 h incubation at 37 °C. Based on its microscopic morphology it
was identified as Rhizomucor pusillus. A direct examination of the Characterization of the in vitro interactions between
centrifuged urine showed broad, irregular, hyaline, non-septate Aspergillus fumigatus and the mouse macrophage cell line
hyphae. Culture of respiratory secretions yielded growth of the same RAW 264.7
fungus and on the wet mount non-septate hyphae were clearly visi- X. Guruceaga,1 A. Balmaseda-Rubina,1 S. Etxenagusia,1
ble. One repeated specimen of urine and two respiratory specimens J. Fernandez-Molina,1 M. Sueiro-Olivares,1 A. Ramirez-Garcia,1
collected in the following days revealed growth of the same fungus.
A. Abad-Diaz-de-Cerio,2 J. Garaizar,1 F. L. Hernando2 and
As morphological identification may be misleading, the isolate was
sent to identification by sequencing of genomic DNA fragments. A. Rementeria1
Conclusion This is a rare case of possible invasive fungal disease in 1
Universidad del Paıs Vasco UPV/EHU, Leioa, Spain and
2
an infant without known inherited or acquired immunodeficiency University of the Basque Country, Vitoria, Spain
with positive cultures for Rhizomucor pusillus both in urine and tra-
cheal aspirate. Cellular immunity defect with a decrease in TCD8
Objectives The study of the virulence of Aspergillus fumigatus and
positive cells may have played a critical role in this rare clinical spec-
their behavior during the first steps of an infection may involve the
trum of mucormycosis.
use of animal models. However, it requires the approval of the Ethics
Committees, which recommend that animal models are replaced or
limited. In this context, the use of cell cultures is increasing in impor-
tance as an alternative method to study the role of the most impor-
P345 tant cells during an infection.
That is why, the aim of this study is to standardize and character-
ize an in vitro cell culture model based on the interaction between
Platelets and complement in the pathogenesis of invasive
macrophages and A. fumigatus.
fungal infections
Methods The in vitro model consisted of co-incubating A. fumigatus
C. Speth,1 H. Deshmukh,1 M. Hagleitner,1 D. Sheppard,2 Af293strain conidia and the murine macrophage RAW 264.7 with a
€ rl1 and G. Rambach3
M. Lee,2 C. Lass-Flo Multiplicity of infection (MOI) of 10. The study was carried out using
1
Medical University of Innsbruck, Innsbruck, Austria; 2McGill RPMI 1640 medium containing 2 mM glutamine and penicillin-
University, Montreal, Canada and 3University of Inssbruck, streptomycin and, in presence and absence of 10% heat-inactivated
Innsbruck, Austria fetal bovine serum (FBS), during 8 h at 37 °C humidified 5% CO2
atmosphere. The parameters studied were: a) Phagocytosis and ger-
mination b) Reactive oxygen species (ROS) and NO2 production, and
Objectives Aspergillus and mucormycetes species are leading causes d) Killing by MTT reduction.
for invasive fungal infections. To better understand the antifungal Results A progressive increase in the conidia germination was
immune reaction and relevant pathomechanisms we studied the shown, being in the co-incubation higher than in the control of the
interaction of the fungi with platelets and the complement system, fungal growth. The maximum phagocytosis rate of 73.04  6.97%
two important elements of the innate immunity. Our recent studies was reached after 4 h of infection with A. fumigatus. The presence of
showed that Aspergillus fumigatus secretes soluble galactosamino- FBS in the culture medium increased the phagocytosis rate but also
galactan (GAG) which induces platelet activation and complement the conidia germination. Moreover, we observed that NO2 production
deposition on their surface. We expanded these studies to other of the macrophages during the infection was a relatively quick pro-
Aspergillus species as well as to different mucormycetes. cess, showing statistical difference regarding to the control condition,
Methods Clinical isolates of different Aspergillus andmucormycete- whereas the production of ROS was more unstable. Finally the killing
species were grown in RPMI medium for 48 h followed by filtration study showed a progressive reduction in the fungus viability during
to obtain the fungal supernatants (SN).Activation and complement infection.
deposition on platelets derived from healthy donors were investigated Conclusions The incubation of A. fumigatus with RAW 264.7 pro-
by FACS analysis using specific antibodies. GAG production was motes the germination of the fungus, which is also increased by FBS
detected on the hyphae of Aspergillus and mucormycetespp. as addition. The presence of FBS also enhances 7 conidia uptake by
stained with FITC-labeled SBA lectin. RAW 264.7 phagocytes. However, at the end of the experiment there
Results The culture supernatants of all tested clinical isolates of were 40% of fungal cells that remained active. On the other hand,
Aspergillus fumigatus and Aspergillus flavus triggered significant plate- NO2 production by macrophages showed to be a very fast process,
let activation as measured by quantification of the activation marker whereas there was not a significantly higher ROS production in con-
CD62P. In parallel, SN-induced deposition of complement factor C3 tact with the fungus than cells alone.
on the platelet surface as well as formation of the terminal comple- Finally, the results of this work analyze the main parameters of
ment complex (TCC) could be demonstrated. However, no or only the fungal-macrophage interaction, which may explain what happen
weak induction of platelet activation or complement deposition on during an infection by A. fumigatus without the use of any animal
their surface could be achieved using the culture supernatants model.
derived from Aspergillus niger, versicolor or terreus as well as from all Financial support This work was partly funded by the UPV/EHU
mucormycetes species except one isolate of Mucor racemosus. These grants (PES13/03, GIU12/44 and UFI11/25), the Government of the
results correlated perfectly with the expression of GAG by the differ- Basque Country grant (S-PC11UN007), and the Ministry of Economy
ent fungi, as demonstrated by immunofluorescence using specific and Competitiveness (MICINN CSD2009-00006). Xabier Guruceaga
staining with SBA lectin. has been supported by Pre-doctoral Research Grants of the Fun-
Conclusion The correlation between GAG expression by the Asper- dacion Jes
us Gangoiti Barrera. Monica Sueiro Olivares has been sup-
gillus and mucormycete species and isolates with the capacity of the ported by Pre-doctoral Research Grants of the Government of the
fungal supernatants to stimulate platelet activation and opsonisation Basque Country. Jimena V. Fernandez Molina has been supported by
underlines our hypothesis that the polysaccharide GAG might be an Post-doctoral Research Grant of the University of Basque Country
important fungal immunomodulatory molecule. Putative conse- (UPV/EHU).
quences of its activity are platelet-mediated antifungal attack but also
the formation of platelet thrombi.

ª 2015 The Authors

176 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P347 RNAs (lincRNAs). Pathway analysis using these eQTL genes revealed
pathways that could be implicated in anti-Candida host immune
defence, including the well-described type I Interferon (IFN) sig-
A systems genetics approach to Candida infection nalling pathway, as well as novel pathways (coagulation and
V. Matzaraki,1 I. Ricano,1 I. Jonkers,1 L. Franke,1 Y. Li,1 lipoprotein metabolism). Furthermore, our SNPs were significantly
C. Wijmenga,1 M. G. Netea2 and V. Kumar Magadi Gopalaiah1 correlated with Candida-induced IFN-gamma, TNFa, IL-8 and IL-6.
1 Lastly, both in vitro and in vivo gene perturbation experiments con-
University Medical Center Groningen, Groningen, the
firmed a significant role for our gene candidates in host immune
Netherlands and 2Radboud University Medical Center, Nijmegen, defence against Candida infection.
the Netherlands Conclusions Our system genetics approach has revealed not only
genetic variants associated with disease susceptibility but, impor-
Objectives Candida albicans is an opportunistic fungal pathogen that tantly, molecular pathways implicated in the anti-Candida host
ranks fourth among pathogenic causes of systemic bloodstream infec- immune defence. 0ur study also suggests a role for lincRNAs in host
tions (candidemia). Immunocompromized patients have an increased immune defence against C. albicans. Ultimately, knowledge derived
risk of Candida infections, making adjunctive immunotherapies an from our approach could be used for identification of new
attractive strategy to improve outcomes for these patients. Not all at- immunotherapeutic targets for susceptible patients.
risk individuals develop Candida infections, implying that host genes
must influence disease susceptibility, and making insight into genetic
susceptibility a first step towards new treatment strategies. Unfortu-
nately, genome wide association studies (GWAS) to identify suscepti- P348
bility genes are challenging due to the necessity for large cohorts. In
addition, GWAS alone cannot implicate functional genetic variants
and pathways. To overcome these challenges, we applied a systems Neutrophils functionality against Aspergillus fumigatus in
genetics approach to candidemia in which multiple layers of molecu- allogenic hematopoietic stem cell transplant recipient
lar data were integrated, and which allowed us not only to identify S. Imbert, L. Gauthier, P. Besler, V. Leblond, D. Mazier,
susceptible variants for candidemia, but also provided deeper biologi- S. N’Guyen and A. Fekkar
cal insights into susceptibility genes and molecular pathways impli-
cated in the host immune defence against C. albicans. Ho^pital Universitaire Pitie Salpe^triere, Paris, France
Methods We performed Immunochip-wide association (IWA) analy-
sis by using the largest candidemia cohort to date to identify genetic Background Aspergillus fumigatus is a major threat for immunocom-
variants associated with candidemia susceptibility. Using RNA promised patients, including allogenic hematopoietic stem cell trans-
sequencing data and genotype data from 629 healthy-donor blood plant (HSCT) recipients. Although polymorphonuclear neutrophils
samples, cis-expression quantitative trait loci (cis-eQTL) mapping was cells are key effectors against the mould, little is known concerning
performed to study the effect of genetic variants identified by Immu- their functionnality in HSCT recipients during the recovery and over
nochip analysis. The functional specificity to Candida infection of the time.
cis-eQTL genes was further validated in peripheral blood mononu- Objectives The aim of this study was to assess the functionality of
clear cells (PBMCs) upon time-dependent Candida stimulation. Can- neutrophils against A. fumigatus in allogenic HSCT recipients at sev-
didemia SNPs were then correlated with cytokine levels from Candida eral points of the graft.
stimulated PBMCs, providing further evidence for their functional Methods: the study is a prospective longitudinal study. Thirty-
role in anti-Candida host immune defence. Pathway enrichment anal- seven patients who benefited from a matched related donor stem cell
ysis was performed on the candidemia genes we identified using the transplantation were included. The functions of their neutrophils
co-expression-based GeneNetwork database. By integrating these were compared to the functions of the neutrophils sampled from the
multiple sources of molecular data (Figure 1), we selected gene can- donors. Recipients were sampled at the recovery time, 2 months,
didates for functional (knock-down) experiments to further investi- 6 months and 10 months after the transplant. At each point, neu-
gate their role in host immune defence against C. albicans. trophils ability to inhibit the fungal growth was assessed. Production
Results The IWA analysis revealed three genome-wide significant of reactive oxygen species (ROS) and expression of neutrophils major
loci (P < 5 9 10
8) and 28 SNPs with suggestive, independent asso- surface molecules (CD11b, CD62L, CD66, TLR-2, TLR-4, Dectin-1)
ciations (P < 9.99 9 10
5) with candidemia. From eQTL mapping, were also evaluated by flow cytometry.
we found 19 SNPs that significantly affect the gene expression levels Results Our results indicate that the ability of the neutrophils to
(P < 0.01). We also found, for the first time, that some of the can- inhibit A. fumigatus hyphal growth is weakened during the recovery.
didemia SNPs affect the expression of long intergenic non-coding We found that the impairment relies on the use of calcineurine inhi-
bitors, such as cyclosporine. Indeed, we found that neutrophils sam-
pled in patients with an insufficient plasma concentration of
calcineurin inhibitor (without other immunosuppressive drugs) at
recovery time were as efficient as neutrophils get from donors. Then
we performed in vitro experiments that confirm this result.
Interestingly, cessation of immunosuppressive drugs 10 months
after the graft leads to a retrieval of the inhibition capacity. No differ-
ence was observed concerning the ROS production and the surface
molecules expression between recipients and donors.
Conclusion Innate immunity against A. fumigatus driven by neu-
trophils is impaired among allogenic HSCT recipients. During the
recovery, the impairement seems to be dependant on the use of cal-
cineurin inhibitor, such as cyclosporin. However, the mechanism
responsible of this impairment is not yet elucidated. ROS production,
one of the major mechanisms used by neutrophils against pathogens
as well as surface molecules expression keep stable over time. There-
fore, others pathways such as the neutrophil extracellular traps
(NET) production have to be investigated.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 177
Poster Presentations

P349 P350

In vivo laser therapy as an adjuvant treatment in Platelets and invasive Candida infections in vitro and
experimental paracoccidioidomycosis: effect on neutrophils in vivo
E. Burger,1 A. Mendes,1 G. M. A. C. Bani,1 P. Pereira,1 G. Rambach,1 K. Pfaller,2 C. Eberl,2 M. Hagleitner,2
V. Gaggino,1 Z. P. Camargo2 and L. M. Verinaud3 M. Hermann,2 R. Bellmann,2 I. Lorenz,2 M. Stro € hle,2 C. Lass-
1
Federal University of Alfenas, Alfenas, Brazil; 2Federal University €rl2 and C. Speth2
Flo
of Sao Paulo, Sa~o Paulo, Brazil and 3State University of 1
University of Innsbruck, Innsbruck, Austria and 2Medical
Campinas, Campinas, Brazil University of Innsbruck, Innsbruck, Austria

Introduction Paracoccidioidomycosis (PCM) is a systemic mycosis Objectives Platelets are nowadays recognized as an important part
caused by the thermodimorphic fungus Paracoccidioides brasiliensis of innate immunity. They can be activated in response to contact
(Pb). The presence of granulomatous inflammatory reactions highly with fungal pathogens, which may lead to multifaceted antimicrobial
permeated by neutrophils is typical in this infection. Neutrophils are effects, but also to thrombosis or excessive inflammation.
crucial in the initial stages of PCM, participating in the innate As common inducers of fungal septicaemia, Candida species come
immunity and directing the acquired immune response towards an in close contact with platelets in the bloodstream. Putative subse-
effective response. PCM patients show decreased neutrophil activity quent processes such as mutual binding, activation and decrease of
compared to healthy individuals, suggesting that Pb has a modulat- viability can profoundly influence the clinical outcome. For that rea-
ing effect on these cells. The treatment of this disease includes the sons, we studied platelet-Candida-interactions in vitro as well as in the
use of antifungals which, in addition to the prolonged administra- blood of patients with Candidaemia.
tion time needed, have many side effects; so research for more effi- Methods Human platelets were isolated from the blood of healthy
cient drugs or adjuvant treatments is needed. Low level laser donors and subsequently incubated with clinical isolates of different
therapy (LLLT) was shown to be an effective activator of immune Candida species. Furthermore, blood samples from patients with pro-
cells. The aim of this study was to develop a treatment capable of ven candidaemia were taken every other day. Interactions of platelets
stimulating neutrophils arriving at the injury site elicited by either with Candida were studied by confocal immunofluorescence micro-
infection with Pb or inoculation with Zymosan (Z) in subcutaneous scopy and scanning electron microscopy. Platelet activation and via-
air pouches. bility were investigated by flow cytometry. Fungal growth and
Materials and Methods We used in vivo application of LLLT at viability were quantified by luminometric assay and plating.
two points on each hind paw of mice on alternate days focusing Results In vitro, the adhesion rate of platelets to yeast cells and
the bone marrow of femur, in animals either infected with the hyphae/pseudohyphae of Candida was rather low. Furthermore, no
highly virulent Pb isolate or inoculated with Zymosan (Z) in subcu- or only marginal activation of platelets could be demonstrated after
taneous air pouches; the control groups were infected with Pb or co-incubation with clinical isolates of different Candida species (albi-
inoculated with Z but did not receive LLLT therapy. The highly cans, glabrata, parapsilosis, tropicalis, dubliniensis, lusitaniae, rugosa).
pure neutrophils population isolated from the air pouches was ana- Similarly, the presence of platelets did not affect growth or viability
lyzed after 10 days of infection or inoculation for the number and of the fungus. To monitor whether Candida-derived secreted com-
viability of neutrophils, protein production, mitochondrial activity, pounds had a stimulatory effect, we incubated platelets with fungal-
reactive oxygen species production (ROS) and fungicidal capacity by culture supernatants, but still could not detect any activation.
quantification of colony forming units. In a second set of experiments we mimicked the situation in vivo
Results LLLT increased the production of ROS, the metabolic activity using a whole-blood-model, where the interaction between Candida
and the fungicidal activity in both Pb and Z groups. Neutrophils were and platelets occurs in the presence of other cellular and soluble ele-
re-exposed to Pb18 (virulent) and Pb265 (avirulent) yeast cells and ments of the immune defence. However, no platelet activation by
ROS and protein production were analyzed, showing a higher deacti- incubation with Candida was visible in this setting.
vating effect of Pb18 than of Pb265. Neutrophils submitted to LLLT All these in vitro results are in contrast to the situation in vivo in
and exposed to Pb18 produced similar amounts of ROS and of total candidaemic patients. Our pilot study showed that platelets derived
proteins than PMNs exposed to Pb265. LLLT rendered PMN more from the blood of these patients were significantly stimulated with
active metabolically, leading to higher fungicidal activity against Pb, enhanced levels of the characteristic activation markers CD62P and
probably due to increased production of ROS and independently of CD63 as well as increased numbers of circulating platelet-derived
primary or secondary exposure to Pb. microparticles. Furthermore, a decrease of platelet membrane integ-
Conclusions Our results suggest that Pb exerts modulating effect on rity and viability could be monitored, which are typical consequences
neutrophils, that this effect depends on the virulence of the fungal of platelet activation. The kinetic of the activation markers and plate-
isolate and that in vivo LLLT treatment reverts this effect, rendering let viability in the course of the disease differs between the patients,
PMN more active metabolically, resulting in higher fungicidal activ- presumably due to various underlying diseases and drug regimens.
ity against Pb, probably due to increased production of ROS, indepen- Conclusions We hypothesize that the survival of Candida in blood
dently of the primary or secondary exposure to the fungus. We and the establishment of sepsis is facilitated by the lack of direct pla-
propose this treatment as a complementary therapy for telet activation, as shown by in vitro experiments. However, stimula-
paracoccidioidomycosis. tion of platelets and subsequent antimicrobial effects may be induced
Supported by grants CNPq 486135/2012-8 and 304827/2012-6 in vivo by indirect activation mechanisms, as found in patients suffer-
and FAPEMIG CBB-PPM-00119-14. ing from candidemia.

ª 2015 The Authors

178 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P351 Results The decrease in local levels of IFN-c and IL-10 were detected
in all hematological patients compared with healthy group. Not also
detected significant changes in the levels IL-17, the main cytokine of
The Candida albicans factor H binding molecule Hgt1p - the early stage of inflammation, which could prevent the fungi inva-
in vivo evidence that it functions as virulence factor sion into the tissues of the oral cavity mucosa. We also determined
U. Binder,1 D. Gr€  ra,2 D. Orth-
assle,1 V. Staudinger,1 M. Sko the increase of IL-6 and IL-8 in all patients with hematological
Ho€ller and R. Würzner
1 1 malignancies compared with healthy people. It was found that the
1 level of hemoattractant MCP-1 in oral fluid in hematological patients
Medical University of Innsbruck, Innsbruck, Austria and in groups I and II was significantly higher compared to healthy indi-
2
Jagiellonian University Medical College, Krakow, Poland viduals (88 (44154) and 156 (62188) vs 44 (3053) pg ml
1,
P < 0.05, respectively), although in patients with OFC increasing of
Objectives The complement system is tightly controlled by several its production was weaker than in patients without infection.
regulators. In particular Factor H (FH) is preferentially acquired by Conclusion Thus, the increase in cytokines production, activating
pathogens conveying resistance to complement attack. neutrophils and other cells of the innate immune system, may indi-
The aim of the study was to determine whether the FH binding cate the increase of antimicrobial protection in the conditions of T-
molecule ‘high affinity glucose transporter 1’(CaHgt1p) of Candida dependent mechanisms deficiency, but can cause inflammatory
albicans, a potentially life-threatening yeast, is a significant virulence mucosal damage, which in turn may facilitate its invasion by fungi.
factor in vivo. The data indicate the need to study other factors of local immunity,
Methods The gene coding for this molecule was initially identified significant for the resistance to C. albicans.
by probing an expression library and homozygous deletion mutants
of the respective gene have been constructed previously. An in vivo
study employing the Galleria mellonella model has now been used to
investigate whether this complement evasion molecule is a virulence P353
factor, i.e., whether Galleria inoculated with the knock-out mutant
(i.e. lacking CaHgt1) are surviving longer than those inoculated with
the wild type. Chronic widespread dermatophytosis due to Trichophyton
Results Especially at 30 °C, but also at 37 °C, Galleria larvae inocu- rubrum: a syndrome associated with a Trichophyton-
lated with 10^5 homozygous hgt1D/D deletion mutant yeast cells per specific functional defect of phagocytes
larva significantly (P < 0.05) lived longer than those inoculated with G. Benard,1 G. B. Santana,2 P. R. Criado3 and M. G. T. Sousa4
the restored strain, in which HGT1 was reintegrated, or inoculated 1
Medical School, University of Sa~o Paulo, Sa~o Paulo, Brazil;
with the wild type strain. 2
Laboratory of Medical Investigation #56, Sa~o Paulo, Brazil;
Conclusions The multifunctional complement evasion molecule
CaHgt1p is not only a complement inhibitor, but also a virulence fac-
3
Division of Clinical Dermatology, Sa~o Paulo, Brazil and
tor, corroborating in vitro data.
4
Laboratory of Medical Investigation #53, Sa~o Paulo, Brazil

Background and objective Dermatophytes are agents of usually

benign superficial infections. However, an increasing number of sev-
P352 ere infections in immunocompromised hosts have been reported. We
aimed to understand the factors underlying the existence of a cohort
of patients presenting chronic widespread dermatophytosis (CWD)
The role of cytokines in the regulation of inflammation in due to Trichophyton rubrum, but no signs of immunodeficiency.
oropharyngeal candidiasis in patients with hematological Methods Fourteen patients meeting the criteria for CWD were stud-
malignancies ied: T. rubrum culture-proven cutaneous lesions with ≥10 cm in at
E. U. Danilova,1 N. V. Shabashova,1 T. S. Bogomolova,2 least one dimension, involving at least 3 non-contiguous localizations
V. Frolova,1 L. V. Filippova,1 A. E. Uchevatkina3 and for >1 year duration, and no predisposing conditions. For compar-
ison, we also studied 13 acute Tinea pedis (Tp) patients. Macrophages
I. V. Vybornova1
1
and neutrophils were isolated and tested for T. rubrum conidia phago-
Kashkin Research Institute of Medical Mycology, Saint cytic and killing activity. H2O2, NO, and pro- and anti-inflammatory
Petersburg, Russia; 2North-Western State Medical University cytokines release were measured. All experiments were run with age
named after I.I. Metchnikov, Saint Petersburg, Russia and and sex-matched healthy donors’ cells in parallel.
3
Metchnikov North-West State Medical University, Saint Results CWD’s macrophages and neutrophils present reduced T.
Petersburg, Russia rubrum-phagocytic and killing abilities, and reduced H2O2 and NO
release compared with healthy donors. CWD’s macrophages secreted
lower levels of the pro-inflammatory cytokines IL-1beta, IL-6, IL-8
Introduction It is clear that patients with hematological malignan-
and TNF-alpha, but enhanced levels of the anti-inflammatory cyto-
cies have systemic defects of immune response, which is initially
kine IL-10. Neutrophils’ secretion followed closely this unbalanced
caused by functional imbalance of the bone marrow and later - by
pattern. In contrast, responses to the positive controls zymosan, LPS
therapeutic interventions. The transition from commensalism to
and PMA were comparable with those of healthy donors. The same
infection in the oral mucosa is caused by changes in the local oral
experiments were performed with Tp’s macrophages and neutrophils
microenvironment, overgrowth off C. albicans and oropharyngeal
and showed no differences when compared with matched healthy
candidiasis (OFC). However, according our data, patients with hema-
donors.
tological malignancies are not highly prone to OFC. It will occur only
Conclusion Patients with CWD and a T. rubrum-related deficiency of
in 9% of all hematological patients. Understanding of immunity to
phagocytesmay represent a new specific syndrome in the complex
Candida may serve the development of new additional immunoregu-
spectrum of the Trichophyton-host interaction.
latory therapy, which lies in their ability to generate and/or enhance
the appropriate host immune response required for the control of
microorganisms.
Objective This study aimed to investigate cytokine levels in oral
fluid of hematological patients with OFC.
Materials and methods We observed three groups. Group I
included 38 hematological patients with OFC, Group II: 85 hemato-
logical patients without OFC, Group III: 19 healthy people

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 179
Poster Presentations

P354 P355

Antifungal activity of anti-Candida albicans germ tube Microglia shows inefficient phagocytic capacity against the
antibodies (CAGTA) from patients with Invasive neurotropic fungus Lomentospora prolificans
Candidiasis A. Pellon,1 A. Ramirez-Garcia,2 I. Buldain,1 A. Antoran,3
G. Carrano,1 L. Lainz,1 I. Olazabal,2 J. C. Garcıa-Ruiz,2 C. Matute,1 A. Rementeria2 and F. L. Hernando1
M. S. Cuetara,3 M. J. Sevilla1 and M. D. Moragues4 1
University of the Basque Country, Leioa, Spain; 2Universidad del
1
University of Basque Country, Leioa, Spain; 2University Hospital Paıs Vasco UPV/EHU, Leioa, Spain and 3University of the Basque
Cruces-Barakaldo, Barakaldo, Spain; 3Hospital Severo Ochoa, Country UPV/EHU, Leioa, Spain
Madrid, Spain and 4University of the Basque Country, Bilbao,
Spain Objectives The filamentous ascomycete Lomentospora prolificans is an
opportunistic pathogen that, when gets disseminated into the blood-
Objective mTo characterize the antifungal activity of antibodies stream, tends to infect the Central Nervous System (CNS), being ter-
raised by patients with Invasive Candidiasis (IC) against Candida albi- med as a neurotropic fungus. In order to deeply understand the
cans. Effect on viability and metabolic activity of C. albicans. underlying mechanisms of this neurotropism, the aim of this study
Materials and methods Immune sera from 29 patients with IC was to analyze the interactions of fungal cells with microglia, the
from different clinical units (Hospital Severo Ochoa-Madrid and innate immune cells in the CNS. Moreover, in order to determine if
University Hospital Cruces-Barakaldo, Spain). Group I: 15 patients microglial cells are inefficient against this fungus, we compared our
infected with C. albicans. Group II: 14 patients infected with non-albi- results with the activity of monocytes.
cans Candida spp. Methods Lomentospora prolificans strain CECT 20842 was subcul-
- Anti-C. albicans blastospores antibodies (CABLA): human sera tured on potato dextrose agar at 37 °C, being conidia obtained by
were adsorbed with heat inactivated C. albicans blastospores and the washing plates with sterile saline. Then, BV2 microglial and J774A.1
reacting IgG antibodies were eluted and purified with the Melon Kit monocyte cell lines, and rat primary microglial cultures were used in
(Pierce, USA). co-incubation experiments at a multiplicity of infection (MOI) of 1 so
- C. albicans germ tube antibodies enriched IgG fraction (CAGTA-fr) as to measure phagocytosis of conidia, conidial germination and
was obtained from human sera that had been previously adsorbed hyphal branching, pro-inflammatory cytokines, and reactive oxygen
with C. albicans blastospores. IgG fraction was purified with the (ROS) and nitrogen species (RNS). Animal experimental procedures
Melon Kit. were approved by the Ethics Committee for Animal Welfare of UPV/
- CAGTA were eluted with sodium iodide from C. albicans germ EHU. All cell cultures and fungus-cell co-cultures were maintained in
tubes that had been incubated with immune sera previously DMEM/10% FBS/2 mM L-Glutamine in a humidified atmosphere at
adsorbed with heat inactivated blastospores [5]. Eluted antibodies 37 °C and 5% CO2.
were dialyzed against PBS. Results Phagocytosis dynamics during co-incubation showed that,
- The effect of antibodies on the metabolic activity of C. albicans while L. prolificans is recognized and engulfed by BV2 microglial cells,
was estimated with a colorimetric XTT assay [2, 4]. phagocytic indexes were low in comparison with previously pub-
- The viability of C. albicans cells was assessed by plating on lished data concerning this fungus. Accordingly, microglia was not
Sabouraud agar plates and colony counting (CFU). Cells treated with able to inhibit conidial germination. In fact, L. prolificans even
different antibody fractions were also stained with vital fluorescent increased the production of branched hyphae in the presence of
dyes CFDA and DiBAC [1, 3]. microglial cells. Moreover, while microglia was able to significantly
Results The CABLA fraction, mainly anti-mannan antibodies, produce ROS and RNS, pro-inflammatory cytokines TNF-a and IL-6
reduced the metabolic activity of C. albicans by 22% at 100 lg ml
1. were poorly released. All these data made us hypothesize that micro-
- The CAGTA-fr of patients infected with C. albicans, at concentra- glial cells may be inefficient when infected by this fungus. Hence, we
tions ≥100 lg ml
1, reduced the metabolic activity of C. albicans cells performed co-cultures with a monocyte-like cell line to compare the
by at least 69%. This inhibitory effect was more effective for CAGTA- response of both cell types, J774A.1 cells showing improved phago-
fr purified from patients with the highest titers of these antibodies cytic and secreting capacities. To verify and validate our results, we
than those with low titers. used primary cultures of microglia that, in accordance with the
- On the contrary, the CAGTA fractions of patients infected with results obtained with the BV2 cell line, showed similar features when
non-albicans Candida strainsshowed a reduced inhibitory effect (range exposed to L. prolificans.
38–43% at concentrations ≥100 lg ml
1) that was not statistically Conclusions The evaluation of L. prolificans-microglia interactions,
significant with reference to the untreated cells control. and their comparison with other phagocytes, has allowed us to deter-
- Concomitantly, at concentrations ≥100 lg ml
1, CAGTA-fr mine microglial inefficiency during these fungal infections. In this
reduced C. albicans CFU by 58%. way, we have identified a decreased phagocytic capacity, both in the
- Staining with CFDA and DiBAC proved that purified CAGTA (40 cell line and primary cultures, and a poor pro-inflammatory cytokine
and 20 lg ml
1) were fungicidal for C. albicans blastospores and fila- production. In consequence, although more in depth studies should
mentous cells. be performed in order to understand the molecular basis of microglial
Conclusions 1. The activity of anti-blastospore antibodies, mainly inefficiency, the results obtained from this work might explain the
anti-mannan antibodies, on the metabolic activity of C. albicans did neurotropism that L. prolificans shows during disseminated infections.
not justify the effect of IgG from whole sera of patients with Invasive Financial support This works was partly funded by the UPV/EHU
Candidiasis. grants (PES13/03, GIU12/44, UFI11/25, and EHUA13/14), the Bas-
2. The CAGTA enriched IgG fraction is the main responsible for que Government grant (S-PC11UN007).
the in vitro antifungal effect of the IC immune sera on viability and AP, and AA and IB, are recipients of FPI grants from the UPV/
metabolic activity of C. albicans. Thus, the antigens recognized by EHU and the Basque Government, respectively.
these antibodies could be the basis for the development of future vac-
cines against Candida invasive infection.
References Bowman JC et al. Antimicrob. Agents Chemother. 2002;
46(9):3001–3012.
Cabezas J. PhD Thesis. 2011. University of the Basque Country
UPV/EHU.
Liao RS et al. Antimicrob. Agents Chemother. 1999; 43:1034–1041.
Peeters E et al. J. Microbiol. Meth. 2008; 72:157–165.
Saez-Roson A et al. Int. Microbiol. 2014; 17:21–29.

ª 2015 The Authors

180 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P356 HES with pulmonary nocardiosis and empyema due to using long
term corticosteroid.
Case Report A 32 year-old male patient was admitted to a local
Enolase, cyclophilines and heat shock protein 70 are the university hospital for dyspnea, chest pain and hemoptysis 8 months
major antigens of Lomentospora prolificans recognized by ago. Laboratory examination showed white blood cell count (WBC)
human salivary IgA 5.71 9 109/L, eosinophil 63.9%. The peripheral blood smear and
I. Buldain,1 A. Ramirez-Garcia,2 A. Pellon,1 A. Rementeria,2 bone marrow biopsy showed eosinophilia. FIP1L1/PDGFRA rear-
F. L. Hernando1 and A. Antoran3 rangement was positive. He was diagnosed HES and the treatment of
1 high dose prednisolone was started. In the following 5 months pred-
University of the Basque Country, Bilbao, Spain; 2Universidad nisolone dose was reduced. Following the reoccurrence of the same
del Paıs Vasco UPV/EHU, Leioa, Spain and 3University of the symptoms, dose was increased again. He was admitted to our hospi-
Basque Country UPV/EHU, Leioa, Spain tal in February 2012 with the same symptoms. Physical examination
revealed both of the lower zones of the lung sounds reduced and the
Objective Lomentospora prolificans is an opportunistic pathogenic liver and the spleen were palpable. Laboratory examination showed
fungus, which mainly infection route is the airways. Once inside the WBC count 7.8 9 10 9/L, eosinophil 0.2%, erythrocyte sedimenta-
host, one of the most important defense mechanisms to protect the tion rate 101 mm h
1. In 5 days he had become febrile. For the
mucosa against microorganisms is salivary immunoglobulin A (IgA). treatment of HES imatinib 400 mg day
1 was started. Pulmonary
These antibodies are able to inhibit pathogen adhesion and tissue computed tomography angiography revealed no pulmonary trom-
invasion, and promote phagocytosis. boemboly. There was a large pleural effusion (3.5 cm of wideness) in
Thereby, the main objective of this project was to identify the most the left hemithorax. There were atelectasis by the pleural effusion.
important conidial antigens of L. prolificans recognized by human There were necrotic consolidation areas at the left lung upper lobe
IgA, and the study of their prevalence and localization. Moreover, 8 9 9 cm and 4 9 5 cm at the right lung upper lobe. The thoracen-
the immunome of this fungus was compared to the phylogenetically tesis was performed to the left pleural effusion. The empyema was
related fungi Scedosporium apiospermum and Scedosporium aurantiacum. diagnosed and thorax tube was placed for the drainage. Nocardia
Methods Conidia from L. prolificans CECT 20842, S. aurantiacum growed in the culture of pleural fluid. For the severity of the disease
CBS 116910 and S. apiospermum CBS 93851 were grown firstly on trimethoprim-sulfamethoxazole 392 gr day
1, imipenem
Potato dextrose agar plates and secondly on Potato dextrose broth, 4 9 500 mg day, and linezolid 2 9 600 mg day were started. Dur-
both at 37 °C for 7 days. Finally, conida were recovered by centrifu- ing the clinical follow-up his fever was controlled and the complaints
gation and washed with sterile saline. Then, in order to obtain total disappeared.
and cell wall protein extracts, conidia were disrupted by glass bead Discussion Pulmonary involvement and hepatosplenomegaly are
beating, or incubated during 10 min at 100 °C on extraction buffer, usual signs of HES. The best characterized and most frequently
respectively. observed chromosomal aberration is an interstitial deletion on chro-
The most prevalent antigens of L. prolificans were detected by 2-DE mosome 4q12 resulting in the fusion of two genes, that for Fip1-
and immunobloting, using saliva from 20 healthy donors, and identi- like1 (FIP1L1) and PDGFRA. For all patients with the FIP1L1/
fied by LC-MS/MS. Afterwards, their presence in the salivary immu- PDGFRA mutation (even if asymptomatic), imatinib mesylate is rec-
nomes of S. aurantiacum and S. apiospermum was also analyzed using ommended for initial therapy, in preference to other available agents
the same methods. (Grade 1B). Nocardia spp have the ability to cause localized or sys-
Results Enolase, cyclophilines and heat shock protein 70 (Hsp70) temic suppurative disease in immunocompromised humans and ani-
were the most prevalent antigens of L. prolificans recognized by mals. Despite the success of TMP-SMX in the treatment of
human salivary IgA, being detected by 90%, 80% and 75% of the nocardiosis, combination therapy with other agents is warranted in
samples, respectively. In addition, they were studied in the cell wall patients with severe infections. In vitro susceptibilities and animal
extract of L. prolificans, being the cyclophilines the most intense. To models of disease have demonstrated activity against Nocardia with
finish, the three antigens were also recognized by IgA on S. apiosper- a variety of antibiotics, including amikacin, imipenem, meropenem,
mum and S. aurantiacum immunomes. and linezolid.
Conclusions Enolase, cyclophilines and Hsp70 are the most preva-
lent antigens of L. prolificans recognized by human salivary IgA.
Their presence on total and cell wall extracts not only from L. prolifi-
cans but also from S. aurantiacum and S. apiospermum has been also P358
proved. Therefore, although it must be researched more in depth,
these results and the fact that these antigens are phylogenetically
very conserved in eukaryotic and prokaryotic organisms, it may be Influence of Laminarin in colonisation process of Candida
concluded that their high prevalence, is likely due to cross-reactivity albicans
with other fungi. P. S. Bonfim-Mendonc
a,1 F. K. Tobaldini,2 I. M. Batalini,1
I. R. G. Capoci,1 J. S. R. Godoy,1 E. S. Kioshima,1 M. Negri1 and
T. I. E. Svidzinski1
1
Universidade Estadual de Maringa, Maringa, Brazil and
P357 2
Universidade Estadual de Maringa/Universidade do Minho,
Maringa, Brazil
Hypereosinophilic syndrome with pulmonary Nocardiosis
M. Gurkan, G. Ozgur, I. Erturk, G. Mert, O. Nevruz, G. Somak, Objective Candida albicans is responsible for the majority of cases of
F. Avcu, M. Guney and T. Cetin vulvovaginal candidiasis (VVC), one of the most important candidal
GATA Military Academy, Ankara, Turkey virulence factors is the ability to adhere to host surfaces. Chemother-
apies that seek to improve the host immune response are an alterna-
tive to control fungal infections. b-glucans are polymeric
Objectives The hypereosinophilic syndromes (HES) are a group of carbohydrates that have been reported to modulate human inflam-
disorders marked by the sustained overproduction of eosinophils, in matory responses in vitro and in vivo. The aim of this study was to
which eosinophilic infiltration and mediator release cause damage to determine the influence of Laminarin (LAM) a b-glucan on C. albicans
multiple organs. Nocardiosis is an uncommon gram-positive bacterial virulence, namely colonisation of HeLa cells.
infection caused by aerobic actinomycetes. Nocardiosis is typically Methods To assess the role of LAM in the cell colonization process,
regarded as an opportunistic infection especially in the patients who HeLa cells were previously treated or not with 3 mg mL
1 of LAM
require prolonged glucocorticoid therapy.We report a rare case of (b-glucan extracted from Laminarina digitata) for 30 min at 37 °C,

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 181
Poster Presentations

and prosthetic material infections are the Staphylococci including

MRSA, however fungi such as C. albicans are of paramount impor-
tance as they form biofilms at the site of infection thus preventing
the activities of the immune system and antifungal drug. Several
studies have shown that when microorganisms are exposed to SW it
damages their cell membranes thereby increasing permeability due to
the disturbance of the osmotic gradient, allowing electrolyte leakage
leading to cell death. The aim of this study was to determine whether
SW at levels used for treating patients can kill C. albicans planctonic
cells and to determine the effect SW exert on tissue.
Methods Four sets of duplicate experiments were performed in
which C. albicans cells were sandwiched in animal muscle in semiso-
lid of similar viscosity to pus, 1 mL cylinder of 0.6% agar, thus mim-
icking the conditions during infection. The Candida cells were treated
with SW at various settings using the Storz Modulith SLX-F2 litho-
tripter. After SW treatmentthe cylinder containing the cells was
transferred into physiological saline, dispersed and viable counts were
estimated using decimal dilutions. The viable counts were compared
to those of untreated controls as well as controls that did not come
into contact with animal muscle. MICs of the SW treated and
untreated cells were tested for Amphotericin B, Flucytosine, Flucona-
zole, Itraconazole, Voriconazole, Posaconazole, Anidulafungin, Caspo-
fungin and Micafungin directly after treatment and after re-culturing
using the CLSI recommended method.
Results Exposure of Candida cells to animal muscle did not affect
their viability. All settings resulted in cell death with 90% killing
5% CO2. Three clinical isolates (5V, 7V and 9V) obtained from when 4000 SW were applied at 3 Hz and energy flux density 6 and
female vaginal secretions and one reference strain (ATCC 90028) large focus. The SW also caused a physical effect on the muscle at
were used in the study. These strains were separated according to the interface of different viscosities but did not cause any changes in
symptoms presented by the patients. Colonization assays were susceptibility.
assessed for 2 h incubation at 37 °C, 5% CO2, with 2x105 HeLa Conclusion This study shows that SW exert a significant killing
cells mL
1 treated or not with LAM and 1x107 yeast mL
1 of differ- effect on C albicans. We believe that the physical effect seen on both
ent clinical isolates of C. albicans. After colonization assays, adherent sites of the muscle at the interface between low and high viscosity, a
C. albicans cells were harvested by detaching the cervical cells mono- phenomenon not normally observed in the clinical situation because
layer to evaluation of viable cells (colony forming units). of the blood flow of the surrounding tissues that can dissipate heat,
Results In this study, LAM significantly decreased the interaction of will play a crucial role in clearing infections by permitting antifungal
VVC clinical isolates with Hela cells (Figure 1). For ASS and VVC iso- drugs and above all the immune system to penetrate the biofilms
lates, there was a similar reduction in the number of viable cells dur- and the surrounding necrotic tissues.
ing colonization process, approximately one log (P < 0.05).
Moreover, RVVC isolate showed a reduction more expressive, approx-
imately two log (P < 0.05).
Conclusion The pathogenesis of VVC involves the initial adherence
of the yeast to the vaginal mucosa, followed by asymptomatic coloni-
P360
sation, ultimately leading to infection (symptomatic vaginitis). This
study was able to show that LAM a b-glucan can negatively modu- The absence of TLR-4 receptor in vivo increased the
late the process of interaction between HeLa cells and Candida albi- severity of Sporothrix brasiliensis infection
cans. These results show that this carbohydrate might be a promising L. Rossato, L. Ferreira and S. Almeida
agent for preventing the first contact between yeast and vaginal
epithelium, and consequently the development of VVC. Universidade de Sa~o Paulo, Sa~o Paulo, Brazil

Introduction Sporotrichosis is a subacute or chronic infection

caused by fungals thermo-dimorphic Sporothrix complex. Recent
P359 molecular studies show that the genus Sporothrix comprises at least
six distinct species. Among them the leading cause of epidemic out-
breaks, proven virulence and lack of evaluation of immune response
The use of Shock waves for the killing of Candida albicans is the Sporothrix brasiliensis species.
M. Petrou, M. A. Hanna, K. Periyasamy, M. I. A. Hafez, Objectives The aim of this study was to elucidate the role of Toll-4
J. W. Ramsay and R. H. Coombs in the immune response against S. brasiliensis 5110.
Material and Methods Bone-marrow derived macrophages (BMMs)
Imperial College, London, United Kingdom from C57BL/6 mice (wild-type and Toll-4-/-) were incubated with S.
brasiliensis yeast for 3, 6, 12 and 24 h. The interaction was evalu-
Introduction Unprotected sailors swimming in the vicinity of depth ated by optical microscopy and IL-6, TNF-a and IL-10 levels were
charges in World War II were killed with deep tissue changes and measured by ELISA. Groups of 4 C57BL/6 mice (wild-type and Toll-
minimal external trauma. This was attributed to the unknown effects 4-/-) were infected with S. brasiliensis yeast intraperitoneally. Animals
of Shockwaves (SW). were maintained for 14 days before being euthanized. Liver and
Early studies on the effects of SW on living tissues such as muscles spleen were collected for determination of fungal burden and cyto-
and lung led to effective use of SW in the 1980s for the treatment of kine measurements for CBA (IL-10, IFN-c, IL-4, TNF-a, IL-6, IL-2).
renal stones. SW therapy has been extended for a variety of ortho- Results The in vitro results showed that in the absence of Toll-4
paedic conditions including chronic non-unions, rotator cuff prob- receptor, there was a minor amount of yeast phagocytosed. In in vivo
lems, tennis elbow, and plantar fasciitis. Infection has been perceived tests, in mice Toll-4-/-we observed that after 14 days of infection, the
as a contraindication to SW treatment in the orthopaedic context liver and spleen showed more fungal burden. In relation the cytoki-
due to fears that it will disseminate an otherwise localised infection. nes, interestingly, we observed absence in the production in wild-
The main group of microorganisms implicated in deep tissue, bone type and Toll-4 -/-, which needs to be further investigated.

ª 2015 The Authors

182 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Conclusions Therefore, it can be suggested that Toll-4 absence P367

interferes with the course of the infection induced by the fungus S.
brasiliensis.
Development and evaluation of a quantitative droplet
digital PCR for the molecular diagnosis of invasive
aspergillosis
P366 I. Montesinos Hernandez,1 H. Pehlivan,1 R. de Mendonc
a,1
M. Delaunoy,1 K. Lagrou,2 S. Pateet,3 M. Dodemont1 and
Detection of Pneumocystis jirovecii DHPS genotypes in M. Hites1
1
bronchoalveolar lavages from immunocompromised Hopital Erasme, Brussels, Belgium; 2UZ Leuven, Leuven, Belgium
patients at a University Hospital in Belgium and 3University Hospitals Leuven, Leuven, Belgium
I. Montesinos Hernandez, F. Brancart, M. Hites, M. Delforge,
F. Jacobs and O. Denis Objectives Several PCR assays have been described as a possible
Hopital Erasme, Brussels, Belgium diagnostic tool for the diagnosis of invasive aspergillosis (IA). Droplet
digital PCR (ddPCR, Biorad) is a method that uses emulsion PCR
technology to divide complex samples into small, manageable and
Objectives Trimethoprim-sulfamethoxazole (TMP-SMX) is the recom- highly diluted DNA containing subunits. It provides absolute and
mended first line treatment and prophylaxis regimen for Pnemocystis accurate target quantification without standard curve. In this study,
jirovecii pneumonia (PCP). This combination inhibits 2 enzymes in we have optimised and evaluated ddPCR for the diagnosis of IA.
folate metabolism: dihydropteroate synthase (DHPS) and dihydrofo- Methods Primers and probes were specifically designed on the basis
late reductase (DHFR) respectively. Mutations in DHPS are associated of the ITS region from A. fumigatus and A. flavus. Limit of quantifica-
with prophylaxis failure of TMP-SMX in PCP due to sulfa drug resis- tion (LOQ) was defined as the amount of DNA (genomic units (GU)/
tance. In this study, we aimed to determine the presence of P. jiro- ll) that generate at least 3 positive droplets. LOQ were determined
vecci harbouring DHPS mutations in our hospital and its impact on on quantified DNA from A. fumigatus and A. flavus. Different strains
treatment efficacy. (13 fungi, 8 yeasts, and 11 bacteria) were used to determine the
Methods Between January 2010 and September 2014, P. jirovecii analytical specificity. Diagnostic performance was evaluated on two
was detected by real-time PCR (RT-PCR) in bronchoalveolar lavages groups of patient’s samples: 1) galactomannan (GM) positive (Plate-
(BALs) from 121 patients hospitalized in Erasme Hospital. Pneumocys- lia-Aspergillus, Bio-Rad), composed of 15 broncoalveolar lavage fluid
tis positive BALs were selected for DHPS analysis: extracted DNA was samples (BALs) (index ≥1) and 8 sera (index ≥0.5) and 2) GM nega-
amplified by nested PCR to amplify a 278-bp region of DHPS gene tive, composed of 15 BALs (index <1) and 5 sera (index <0.5).
encompassing polymorphic nucleotide positions 165A/G and 171C/T. Revised European Organization for Research and Treatment of Can-
The nested PCR products were sequenced using BigDye Terminator cer/Mycoses Study Group (EORTC/MSG) criteria for the diagnosis of
chemistry and the DNA sequences were analysed with Bionumerics IA were applied by an infectious disease expert to classify the patients
6.5. Patients’ characteristics were acquired retrospectively by medical in proven, probable, possible and non IA. DNA extraction was per-
chart review. formed by QIAsymphony DSP virus/pathogen extraction kit (Qiagen).
Results Amplification of the DHPS gene was attempted in 73 of the DdPCR was performed on QX200 Droplet Digital PCR system (Bio-
121 BAL samples and in these cases DHPS locus was successfully Rad). An home-brew pan-Aspergillus real-time PCR (with excellent
genotyped. No amplification was observed in 48 BALs probably due performance in European Aspergillus PCR Initiative (EAPCRI) evalua-
to low fungal burden. Sixty eight patients harboured P. jirovecii wild- tions) was performed to confirm ddPCR results.
type. Two DHPS mutation genotypes were observed in 5 BAL from Results Analytic performance results showed that LOQ of ddPCR
four patients (6.8%): single mutation resulting in Pro57Ser amino was 0.15 GU ll
1 of A. fumigatus and A. flavus DNAbothdiluted in
acid substitution (M2 P. jirovecii) and double mutation resulting in sterile water or spiked in BAL. Among all 32 strains tested for speci-
Thr55Ala and Pro57Ser (M3 P. jirovecii). Patient number 1, a renal ficity, only A. fumigatus complex strains(A. lentulus)and A. ustus were
transplant patient, was treated with TMP-SMX for PCP due to M2 P. amplified. Patient’s demographics, clinical characteristics and labora-
jirovecii, but died 15 days later of respiratory failure and septic shock. tory results are shown in the following tables. Considering proven
Patient number 2 had primary Sj€ ogren’s syndrome and was chroni- and probable IA as true positives, and absence of IA as true nega-
cally treated with corticoids and immunosuppressive drug (azathio- tives, the diagnostic performance results showed 31% of sensitivity
prine). Two populations of P. jirovecii were observed in this patient: and 96% of specificity. Concordance with RT-PCR was good (70%).
M2 P. jirovecii and wild type. This patient was switched successfully
to pentamidine after poor clinical response despite 10 days of TMP-
SMX treatment. Patient number 3 had severe rheumatoid arthritis Table 1
treated with corticoids and methotrexate and was hospitalized in the
intensive care unit for respiratory distress due to influenza A infec-
tion. This patient harboured M3 P. jirovecii, considered to be colo-
nization, and thus was not treated. These 3 patients were not
receiving TMP-SMX prophylaxis before P. jirovecii detection by PCR.
Patient number 4 was a HIV patient with low CD4 count and not on
antiretroviral therapy nor on TMP-SMX prophylaxis. This patient
harboured M2 P. jiroveci during a first episode of PCP treated success-
fully with TMP-SMX. Nine months later, on TMP-SMX prophylaxis,
M3 P. jirovecii was detected during a new PCP episode not observed
in the previous sample. This patient was treated successfully with
pentamidine.
Conclusions As only low mutation rate of P. jirovecii harbouring
DHPS mutations was observed in our hospital, it’s difficult to attri-
bute prophylactic and/or treatment failures. However 2 patients had
failure to TMP-SMX treatment or and 1 to TMP-SMX prophylaxis,
which suggests a possible correlation. Further studies are required to
better understand the clinical impact of these mutations.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 183
Poster Presentations

Conclusions ddPCR is a promising new technology that allows Results Considering reference and clinical strains together, 23 out of
extremely accurate quantification. However, further prospective eval- 27 Candida species could be clearly distinguished by HRMA and 4
uation is necessary to evaluate the real diagnostic performance of species were grouped in 2 pairs. Candida species could be identified
this ddPCR in the diagnosis of IA. by applying the mean Tm  3 SD values of reference strains, the
shape of the derivative melting curve and, in some cases, the normal-
ized and temperature - shifted difference plot against C. krusei.
Conclusion Real-time PCR followed by HRMA is a simple, rapid and
P369 inexpensive tool to identify wide spectrum of Candida species. It seems
to be a suitable complement to current clinical diagnostic approach
based on usage of commercially available biochemical kits.
Caspofungin elicits highly-dynamic and specific The study was supported by the grant CKTCH No. 201
phosphatidylinositol-(4,5)-bisphosphate (PIP2) and cell wall
integrity pathway (CWIP) responses in Candida albicans,
which correlate with killing and paradoxical effects
C. Clancy, H. Badrane and M. H. Nguyen P371
University of Pittsburgh, Pittsburgh, USA
Candida parapsilosis clinical isolates resistant and
Background CSP exposure for as brief as 5 min mislocalizes PIP2 to susceptible to azoles with identical genetic profile
aberrant plasma membrane (PM) sites, and results in sustained kill- determined by PFGE-RFLP
ing of C. albicans (Ca) over 24 hrs. In baker’s yeast, PIP2 activates D. Y. Thomaz,1 M. C. Giudice,2 J. J. Gaudereto,1 R. C. Grenfell,3
the CWIP. Our objective was to study PIP2 dynamics and CWIP acti- G. M. E. Lima,4 M. O. Nunes,4 D. S. Y. Figueiredo1 and
vation upon exposure to CSP and other agents.
Methods We visualized and quantitated PIP2 over 3 hrs by live-cell
G. M. B. del Negro5
imaging of Ca SC5314 expressing CaPH-GFP, measured CWIP activa-
1
School of Medicine - University of Sa~o Paulo, Sa~o Paulo, Brazil;
tion by Mkc1p phosphorylation, and performed 24 hr time-kills.
2
Institute of Tropical Medicine, University of Sa~o Paulo, Sa~o
Results CSP (0.25x-49 MIC) induced PIP2 mislocalization in the Paulo, Brazil; 3School of Medicine, Federal University of Sa~o
PM within 5 mins, which time-lapse imaging revealed to be highly Paulo, Sa~o Paulo, Brazil; 4University Hospital, Federal University
chaotic over 3 hrs. Mislocalization increased in conjunction with of Mato Grosso do Sul, Campo Grande, Brazil and 5Clinics
PIP2 accumulation in a dose-response manner, and culminated in Hospital, School of Medicine, University of Sa~o Paulo, Sa~o Paulo,
arrested, unidirectional septae at aberrant sites. At ≥89 MIC, these Brazil
responses progressively declined. CWIP activation was transient, and
maximal within 10 mins in the same dose-response manner. Like-
wise, CSP exerted dose-depended killing at ≤4x, but paradoxical Objectives to characterize the genetic profiles of C. parapsilosis clini-
growth occured at ≥89 MIC. PIP2 abnormalities were not evident in cal isolates presenting different patterns of susceptibility to azoles.
response to fluconazole (49 MIC), amphotericin B (49 MIC), cal- Methods nine C. parapsilosis sensu stricto isolated from sterile sites
cofluor white (50 lg ml
1) or H2O2 (10 lg ml
1), even though the (blood, bone marrow aspirate and central venous catheter) were ana-
latter 3 agents were fungicidal. H2O2 was the only other agent to lyzed. These isolates were obtained from seven intensive care unit
activate the CWIP. patients, one patient from the emergency unit and one from the
Conclusions CSP exerts rapid and highly-dynamic PIP2 and CWIP nephrology service, at a tertiary care hospital of the Universidade
responsesthat are specific to b-D-glucan inhibition, correlate with the Federal do Mato Grosso do Sul, Brazil from 2012 to 2015. PFGE of
extent of Ca killing and paradoxical effects, and are not induced by the SfiI restriction fragments was carried out to evaluate the genetic
other agents or caused by cell death. Taken with previous studies of profiles of all C. parapsilosis isolates. The dendrogram analysis was
PIP2 and septin mutant strains from our lab, the data demonstrate performed by using Bionumerics software. The results were expressed
that dynamic regulation of a novel PIP2-septin-CWIP network is a employing the position tolerance setted at 1% and optimization at
crucial early regulator of CSP responses. Failure to either activate or 3%. The Dice coefficient was used to analyse the similarities of the
down-regulate the network increases susceptibility to CSP. band patterns and the unweighted pair group method using arith-
metic averages (UPGMA) was used for cluster analysis. Susceptibility
testing was performed by the standardized broth microdilution tech-
nique described by EUCAST with amphotericin B, fluconazole (FCZ)
and voriconazole (VCZ), and results were interpreted according to
P370 EDef 7.2.
Results the antifungal susceptibility tests showed seven isolates with
High resolution melting analysis for rapid identification of azole cross-resistance to FCZ (MIC ranging from 32 to 64 mg l
1)
a wide range of Candida species from culture and to VCZ (MIC ranging from 0.5 to 2 mg l
1). One isolate was
resistant only to FCZ (MIC 8 mg l
1) and the other presented MIC
E. Nemcova,1 M. Cernochova,1 F. Ruzicka,2 B. Malisova,1
4 mg l
1. All isolates were susceptible to amphotericin B. The PFGE-
M. Vanerkova,1 H. Suranska,1 P. Nemec1 and T. Freiberger1 RFLP presented the same pattern of bands ranging from 600 to
1
CKTCH Brno, Brno, Czech Republic and 2Masaryk University and 50 kb for all isolates and the UPGMA dendrogram indicated 100% of
St. Anne’s University Hospital Brno, Brno, Czech Republic similarity.
Conclusion PFGE-SfiI was able to identify closely related clonal iso-
lates from different patients, anatomic sites and nosocomial settings,
Objectives Although Candida albicans remains the most common
indicating a possible common source. However, despite being closely
fungal isolate from blood, many studies have described an increasing
related according to the PFGE-SfiI, the isolates showed distinct pat-
trend in non–albicans infections. Correct identification of Can-
terns of susceptibility. Therefore our results suggest that the PFGE-
dida species is important for targeted antifungal therapy and for epi-
RFLP is not accurate enough to detect possible genetic alterations
demiological purposes. The aim of this study was to develop method
associated with antifungal susceptibility.
for broad range Candida species identification from culture.
Methods We have tested real-time PCR followed by high resolution
melting analysis (HRMA) on 25 reference Candida strains and vali-
dated on additional 143 clinical isolates. HRMA outcomes were com-
pared with biochemical tests results. All reference and clinical strains
revealing discrepancy in species identification between biochemical
tests and HRMA were sequenced using ITS2 region.

ª 2015 The Authors

184 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P372 to estimate the impact of a commercial available multiplex PCR sys-

tem in the diagnosis of Candida spp. among patients with suspected
sepsis in intensive care units (ICUs).
Typing of Scopulariopsis and Microascus fungi by Random Methods Blood samples from patients with presumed sepsis were
Amplified Polymorphic DNA (RAPD) incubated into a Bactec 9240[TRADEMARK] system (Becton Dickin-
M. E. Kordalewska,1 T. Jagielski2 and A. Brillowska-Dabrowska1 son, Heidelberg, Germany) and blood in EDTA from the same
1
Gdansk University of Technology, Gdansk, Poland and patients subjected to analysis with a commercial multiplex real-time
2 PCR (LightCycler SeptiFast assay, Roche Molecular Systems) accord-
University of Warsaw, Warsaw, Poland
ing to the manufacturer’s instructions. LightCycler Septifast (LC-SF)
assay uses a renovated technology that enables simultaneously the
Objectives Scopulariopsis species and their teleomorphs of the direct detection of a wide panel of Gram-negative, Gram-positive and
genus Microascus are commonly isolated from soil, decaying plant fungal microorganisms (5 Candida species and Aspergillus fumiga-
material and indoor environments. Moreover, certain Scopulariop- tus), commonly involved in systemic infections.
sis and Microascus species are recognised as opportunistic human Results From 352 patients examined during 5-years period (from
pathogens. Although most species can be identified by detailed mor- 2010 to 2015), 56 (16%) were detected as positive (31 patients with
phological study, phenotypic characters appear to overlap in several Gram-negative microorganisms, 16 with Gram-positive microorgan-
cases and morphology seems to be insufficient for establishing species isms and 9 with Candida spp.). Especially in patients with candi-
limits in these fungi. Molecular typing has improved understanding daemia: C. albicans was detected in 4 patients, C. parapsilosis in 4
of species concepts in many fungal groups, but such a study has not patients, while mixed C. albicans and C. tropicalis was obtained from
been performed for Microascus and Scopulariopsis genera. The aim of one case. In six of 9 cases, the pathogen was confirmed by a positive
this study was to investigate the relationships between Scopulariop- BC and the rest were characterized as negative by BC. In the case of
sis and Microascus isolates and evaluate the differentiation potential mixed Candida bloodstream infection (BSI), only C. tropicalis was
of six Random Amplified Polymorphic DNA (RAPD) assays towards detected in BC. LC-SF results were available in 7–15 h, in contrast to
fungi of Scopulariopsis and Microascus genera. the 48–72 h required for BC. All patients had risk factors for Candida
Methods The study included DNA samples of 38 Scopulariop- BSI: use of broad-spectrum antibiotics, presence of a central vascular
sis and Microascus isolates representing 24 species, isolated with Bril- catheter and mechanical ventilation. According to the LC-SF results,
lowska-Daz browska method (J Clin Microbiol 2007; 45(4):1200–4). initial therapy was inadequate in 6 patients, and antifungal treat-
The isolates were obtained from international culture collections ment was added promptly in all patients with Candida BSI.
(CBS; BCCM/IHEM; DSMZ) and Molecular Biotechnology and Microbi- Conclusion The rapid multiplex pathogen detection LC-SF system
ology Department collection of fungi (Gda nsk University of Technol- complemented the traditional culture-based methods and offered
ogy, Gda nsk, Poland). Six primers: LIAN2004-4, LIAN2004-7, some added diagnostic value for the timely detection of causative
CgAP50-1, (GACA)4, CgRAPD-EDP4, CgRAPD-EDP8 were evaluated fungal pathogens having a relevant impact on clinical management
in RAPD assays. RAPD mixtures, of 20 l each, consisted of 10 ll of for a subset of patients with clinically suspected sepsis.
29 PCR Master Mix Plus High GC (A&A Biotechnology, Gdynia,
Poland), 0.2 ll of primer at 100 lM, and 2 ll of DNA. RAPD time-
temperature profile included initial denaturation for 3 min at 94 °C
followed by 40 cycles of 45 s at 94 °C, 45 s at 45 °C, and 90 s at
P374
72 °C. The presence of amplicons was examined electrophoretically
on 2% agarose gels, stained with ethidium bromide.
Results Application of RAPD analysis of DNA extracted from Scopu- A new Aspergillus fumigatus typing method based on
lariopsis and Microascus fungi revealed differences between species hypervariable tandem repeats located within exons of
and even single isolates among species. We were able to recognise surface protein coding genes (TRESP)
between 20 and 24 genotypes, depending on the primer used. R. Garcia-Rubio,1 H. Gil,2 M. Monteiro,3 T. Pelaez1 and
Conclusion In this study we found, that RAPD is sufficient for typ-
E. Mellado3
ing of Scopulariopsis and Microascus fungi. Obtained results support
recent redefinition of Scopulariopsis, Microascus and allied genera by
1
Hospital General Universitario Gregorio Maran~on, Madrid,
Sandoval-Denis et al. (Persoonia 2016; 36:1-36). Spain; 2Program for Public Health Microbiology Training EUPHEM
Acknowledgements The study was in part financed by the Polish & ECDC, Stockholm, Sweden and 3Instituto de Salud Carlos III,
Ministry of Science and Higher Education «Iuventus Plus» grant Majadahonda, Spain
(IP12013023672) to TJ.
Objectives Many studies have shown a high genotypic diversity of
A. fumigatus in the air. Most cases of invasive aspergillosis are caused
by A. fumigatus, whose conidia are ubiquitous in outdoor and hospi-
P373 tal air. Aspergillus fumigatus strain typing can fulfill many needs both
in clinical settings and otherwise. Short tandem repeats of A. fumiga-
tus (STRAf) is widely accepted as the first choice for A. fumigatus
Rapid diagnosis of candidaemia using a multiplex PCR test
genotyping. However, difficulties associated with sizing of the
directly to blood from intensive care units patients with obtained PCR, reproducibility results among laboratories and avail-
presumed sepsis ability of the specific technology in some laboratories encourage the
G. Vrioni,1 K. Theodoridou,1 C. Tsiamis,1 V. Pitiriga,1 development of novel typing techniques. Recent research has shown
A. Stavropoulou1 and A. Tsakris2 the presence of hypervariable tandem repeats within exons of surface
1
University of Athens, Medical School, Athens, Greece and protein coding genes (TRESP). The purpose of this study was to cre-
2
Medical school, University of Athens, Athens, Greece ate a panel based on these markers which would make possible to
differentiate epidemiologically and geographically non-related A.
fumigatus strains.
Objectives Sepsis is a serious medical condition that requires rapidly Methods Ninety two A. fumigatus strains (48 azole susceptible and
administered and appropriate antibiotic treatment. The rapid detec- 44 azole resistant), were used in this study. They belong to our own
tion of pathogens in blood is critical for a favorable outcome of fungal collection obtained from clinical setting and from some inter-
patients with suspected sepsis. Especially for Candida bloodstream national collaborators. These strains were characterized by PCR
infections (BSI), although blood cultures (BC) are essential for diag- amplification and sequencing, with a panel of three TRESP:
nosing candidaemia, their sensitivity to detect Candida is 50–75%, Afu3 g08990,(cspA for cell surface protein A), Afu2 g05150 (MP-2
with lower sensitivity rates reported in neutropenic patients and antigenic galactomannan protein), and Afu6 g14090 (hypothetical
those undergoing antifungal treatment. In this study, we attempted

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 185
Poster Presentations

protein with a CFEM domain). For each strain a TRESP type was
assigned according to the composition and number of repeats. The
type of Afu3 g08990 (cspA) was assigned according to the types pre-
viously described. A final genotype for each strain was assigned com-
bining the types obtained from the three TRESP genes.
Results A total of 64 genotypes among the 92 strains of A. fumiga-
tus were identified with our method, showing a high level of discrimi-
nation (Simpson index (D) = 0.968). For Afu3 g08990,we identified
15 types, 25 types for Afu2 g05150 and in the case of
Afu6 g14090, we identified 17 types. Azole susceptible isolates were
distributed across a total of 43 types (D = 0.995). However, resistant
isolates were distributed among only 25 genotypes (D = 0.880).
Conclusions We have developed a novel genotyping method
(TRESP), based on PCR and sequencing, which has showed a high normalization of the microarray data. Despite this appreciation,
discriminatory power. In addition, it is reproducible, easy to perform, quantile method of normalization seems to have less variability and
and could be specially useful for studying outbreaks; (ii) Interpreta- give more consistent results than the ones offered after the normal-
tion of the sequence information does not require sophisticated algo- ization using the scale method.
rithms or software and thus can be easily translated into the clinical Conclusion The normalization process is essential to compare the
microbiology laboratory; (iii) Interestingly, a lower index of diversity varying conditions of microarray experiments. While there are sev-
is obtained when resistant strains are tested compared to the index eral methods, in our experience, quantile method seems to work best
obtained from the analysis of susceptible strains, suggesting a com- in reducing all the differences in expression values for replicates sam-
mon ancestor among resistant strains, as previously reported. ples and gives the most consistent results.
Financial support This work was funded by the UPV/EHU grants
(PES13/03, GIU12/44 and UFI11/25), the Government of the Bas-
que Country grant (S-PC11UN007), and the Ministry of Economy
P375 and Competitiveness (MICINN CSD2009-00006). Research Grants of
the: Fundaci on Jes
us Gangoiti Barrera, Government of the Basque
Country and University of Basque Country (UPV/EHU), have sup-
Effect of microarray expression data normalization for the ported Guruceaga X., Sueiro-Olivares M., and Fernandez-Molina J.
choice of differentially expressed genes of Aspergillus respectively.
fumigatus during an intranasal infection
X. Guruceaga,1 G. Ezpeleta,2 S. Etxenagusia,1 A. Balmaseda-
Rubina,1 J. Fernandez-Molina,1 M. Sueiro-Olivares,1 A. Abad-
Diaz-de-Cerio,3 A. Ramirez-Garcia,1 F. L. Hernando,3 J. Garaizar1 P376
and A. Rementeria1
1
Universidad del Paıs Vasco UPV/EHU, Leioa, Spain; 2Univ. Paıs Trichophyton anamorph of Arthroderma benhamiae - an
Vasco UPV/EHU Facultad de Medicina y Odontologıa, Bilbao, emerging pathogen in dermatomycology - morphological
Spain and 3University of the Basque Country, Vitoria, Spain and molecular biological characterization of six wild
strains
Objective Microarray technologies are widely used for massive S. Uhrlab,1 J. Brasch,2 V. Hubka,3 T. Maier,4 C. Kru€ ger1 and
screening of differentially expressed genes under different experimen- P. Nenoff1
tal situations. However, the analysis of this amount of data remains 1
Laboratory of medical microbiology, Mo€lbis, Germany;
a challenging issue due to changes in experimental and hybridization 2
Universita€tsklinikum Schleswig-Holstein, Kiel, Germany; 3Charles
conditions, but also to the existence of many data preprocessing and University in Prague, Prague, Czech Republic and 4Bruker
normalization methods which attempts to compensate for such effects Daltonik GmbH, Bremen, Germany
through use of internal controls. The aim of this abstract is to com-
pare and assess the differences between combinations of seven meth-
ods of background correction and two methods of array data Objective In the past few years, the zoophilic dermatophyte Tri-
normalization in a microarray dataset obtained during an experimen- chophyton (T.)anamorph ofArthroderma (A.)benhamiae was increas-
tal animal model of Aspergillus fumigatus intranasal infection. ingly isolated as causative agent of often
Methods After intranasal infection during 4 days in a mouse model, inflammatorydermatophytosis predominantly in children and adoles-
and taking daily samples, the transcriptome of A.fumigatus was stud- cents, however, also in adults. Source of infectionof T. anamorph of
ied using the AWAFUGE expression microarray (v.1) designed by our A. benhamiae are small rodents, first of all guinea pigs.
research group. After hybridization of the RNA samples extracted Patients and methods Six wild strains of T. anamorph of A. ben-
from the lungs of mice with the Agilent custom chip and following hamiae were isolated from skin scrapingsof patients suffering from
recommendations about pre-processing array expression dataset pre- dermatophytosis. All strains were characterized based on morphologi-
sent in the literature, combination of different methods of back- cal and metabolicfeatures. For molecular biological detection of the
ground correction and dataset normalization were tested. Regarding dermatophytes in skin scrapings a uniplex PCR-EIA was used.Species
to the background correction, exponential-normal method and con- identification was confirmed by sequencing of the internal tran-
volution model were tested combined with quantiles and scale nor- scribed spacer region (ITS) of the ribosomalDNA. In addition to ITS
malization methods were also studied. All these methods and their rDNA, GPD gene and microsatellite typing scheme were used for
analyses were implemented using the limma package contained in identification of the strains.Further, the strains were typed by Matrix
the Bioconductor project. Assisted Laser Desorption/Ionisation Time-Of-Flight MassSpectrome-
Results After the analysis of differentially expressed target genes try (MALDI TOF MS).
over the different groups only comparisons between groups 3 and 4 Results The six strains of T. anamorph of A. benhamiae were isolated
were statistically significant (P < 0.01). However, differences among from 4 children and 2 adults suffering fromtinea corporis or tinea
the different normalization and background correction methods were faciei. Guinea pigs were the source of infection in 5 out of 6 patients,
recorded, and the results are on the table attached (table 1). the sixth patient haddifferent pets at home. 5 out of the 6 isolates
Based on the data contained on table 1, the normexp background had typical yellow stained colonies (Fig. 1), one isolate was the
correction yields to the largest number of differentially expressed socalled white-colony-type (Fig. 2). Microscopically, the yellow
genes identified regardless the method use to perform the strains showed few microconidia, however ring hyphaewere detected.

ª 2015 The Authors

186 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P378

Sporothrix brasiliensis infection in cats: a mycological and

molecular study of 47 clinical isolates from Rio de Janeiro,
Brazil
S. A. Pereira,1 J. S. Boechat,1 M. M. E. Oliveira,2 R. Almeida-
Paes,2 I. D. F. Gremi~ao,1 A. C. Sa-Machado,1 K. B. Silva,1
R. M. Zancope-Oliveira2 and T. M. P. Schubach1
1
Fundac
a~o Oswaldo Cruz, Rio de Janeiro, Brazil and 2Fundac
a~o
Oswaldo Cruz- Instituto Nacional de Infectologia Evandro
Chagas, Rio de Janeiro, Brazil

Sporotrichosis is a subcutaneous mycosis caused by the Sporothrix

complex. Since 1998, in Rio de Janeiro, Brazil, it has been diagnosed
a wide number of cases in humans and animals, especially cats.
Based on phenotypic and molecular studies, a high intraspecific vari-
ability between morphologically isolates identified as S. schenckii was
demonstrated, which led to proposition of a complex of six species: S.
schenckii, S. brasiliensis, S. globosa, S. mexicana, S. pallida and S. luriei.
S. brasiliensis has been described as an emerging specie, highly patho-
genic for humans and animals, with a regional distribution in Brazil.
Studies involving the characterization of cat isolates are scarce.
Objectives The objective of this study was to characterize 47 Sporo-
Figure 1. T. anamorph of A. benhamiae strains with yellow colonies thrix spp. isolates from cats obtained before start of antifungal treat-
ment and assisted at the Evandro Chagas National Institute of
Infectious Diseases/Fiocruz, Rio de Janeiro, Brazil.
Methods For characterization in species level were used character-
ized phenotypically, according to the identification key, including
analysis of conidial morphology and the auxonogram analysis using
raffinose and sucrose as carbon sources. However, identification
based on this key could be inconclusive due to phenotypic variability
within these species being used the genotypic identification of species
by T3B PCR fingerprinting. The control strains of S. brasiliensis, S.
globosa, S. schenckii, and a S. mexicana were included in this study.
Results In phenotypic identification thirty-five isolates were charac-
terized phenotypically, according to the identification key as S.
brasiliensis. Twelve did not show a typical phenotype with any spe-
cies of the complex, being classified as Sporothrix spp..
Applying the molecular analysis of the T3B PCR fingerprinting all
strains included in this study were identification as S. brasiliensis.
This methodology was also able to distinguish the species of strains
misidentified by the phenotypic analysis.
Conclusion S. brasiliensis is the only species in feline sporotrichosis
in endemic area of Rio de Janeiro until now. However, the correla-
tion between phenotypic and molecular methods are necessary for
species characterization.
Figure 2. T. anamorph of A. benhamiae strains with white colonies

The white strain showed abundant microconidia. Urease test was P379
negative in all yellow strains, thewhite strain showed strong positive
urease reaction. Hair perforation test was negative in all strains. All
strains wereconfirmed as T. anamorph of A. benhamiae by PCR-EIA, Multilocus sequencing of the micromycetes-biodestructors
sequencing of the ITS rDNA, GPD gene and microsatellitetyping. In Stachybotrys spp.
MALDI TOF MS the 5 yellow strains had identical spectra, the white M. V. Rudneva,1 E. V. Dorshakova,2 D. M. Lavnikevich,2
strain showed marked differences.All six strains used in this study S. M. Ignatyeva,2 A. E. Taraskina2 and N. Vasilieva2
have been deposited in the Centraalbureau voor Schimmelcultures 1
North-Western State Medical University named after I.I.
(CBS), Utrecht,The Netherlands: No. CBS 139072, 139073, 139074, Mechnikov, Saint Petersburg, Russia and 2Medical mycology
139075, 139076 and 139077.
institute named after Kashkin, Saint Petersburg, Russia
Conclusion The dermatophyte T. anamorph of A. benhamiae repre-
sents an ‘emerging pathogen’ both in Germany,but also in the Czech
Republic and other European countries. Within the species T. ana- Micromycetes Stachybotrysspp. are commonly found in water-dam-
morph of A. benhamiae strainswith yellow colonies can be distin- aged buildings and often considered to be an indicator organism of
guished from those with white colonies. There are clear indoor air problems. This fungus is known to produce mycotoxins
morphological and moleculardifferences between these two types. that has been associated with a number of human and veterinary
health problems. Many investigators noticevariation inthe levels of
toxin production among Stachybotrys individuals that can be pre-
dicted by nucleotide analysis. The most variable genes of Stachy-
botrys are the trichodiene synthase gene and the chitin synthase
gene.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 187
Poster Presentations

Objective of this study is intraspecies typing of Stachybotrysspp. by germination or biofilm formation. In conclusion, this antibody, alone
multilocus sequencing for discriminating toxic and nontoxic strains. or in combination with commonly used antifungal drugs, may be of
Methods We examined 16 strains of Stachybotrysspp. All isolates interest for the treatment of immunocompromised patients suffering
were tested biochemically, the quantity of mycotoxins were mea- from C. albicans infections.
sured. DNA was extracted from 10-day samples using a modification Financial support This works was partly funded by the UPV/EHU
of a CTAB extraction protocol. PCR was performed by the trichodiene grants (PES13/03, GIU12/44, UFI11/25, and EHUA13/14), the Bas-
synthase loci using primer pairs Tri5 and STOX, and by the chitin que Government grant (S-PC11UN007).
synthase loci with primer pair Chs. Sequencing was performed by AP, and AA and IB, are recipients of FPI grants from the UPV/
Sanger method using the analyzer ABI Prizm 3500. Sequences EHU and the Basque Government, respectively.
obtained from Tri5, STOX, and Chswere aligned in the program
MEGA 5.2. Isolates were analyzed by unweighted pairgroup method
of arithmetic averages (UPGMA) cluster analysis.
Results The amplification program was modificated. Reaction per- P381
formed with 0.5 ng of DNA and annealing temperature 52 °C. There
were 10 high-toxic and 6 low-toxic strains investigated biochemi-
cally. The trichodiene synthase gene fragment presented in all sam- Genetic adaptive mechanisms mediating response and
ples. Phylogenetic trees that represent connections between tolerance to acetic acid stress in the human pathogen
strainswere constructed. We inwestigated several distinct groups of Candida glabrata: role of the CgHaa1-dependent signaling
strains with similar sequences. This strains in each group had also pathway
similar amountof mycotoxins. N. P. Mira,1 R. Bernardo,1 D. Cunha,1 C. A. N. Wang,2
Conclusion The intraspecies typing by multilocus sequencing can be H. I. R. O. JChibana,3 S. Silva,4 I. Sa-Correia,1 J. Azeredo5 and
used for discriminating toxic and nontoxic strains of Stachybotrys
spp. G. Butler2
1
Institute for Bioengineering and Biosciences, Lisbon, Portugal;
2
School of Biomolecular and Biomedical Sciences, Conway
Institute, Dublin, Ireland; 3Medical Mycology Research Center,
P380 Chiba, Japan; 4CEB, Centre of Biological Engineering, Braga,
Portugal and 5University of Minho, Braga, Portugal
Characterization of the effect of anti-KRE9 monoclonal
antibody against Candida albicans Background and objectives C. glabrata is a commensal found in
the human genitourinary tract but under certain conditions this
A. Antoran,1 A. Ramirez-Garcia,2 A. Pellon,3 I. Buldain,3 harmless colonization evolves to a mucosal infection and, in more
A. Rementeria2 and F. L. Hernando3 serious cases, to disseminated mycosis. To thrive in the acidic vaginal
1
University of the Basque Country UPV/EHU, Leioa, Spain; tract C. glabrata has to cope with the presence of a competing com-
2
Universidad del Paıs Vasco UPV/EHU, Leioa, Spain and mensal microbiota that restrains the overgrowth of pathogens by
3 producing acetic and lactic acids, among other interference effects.
University of the Basque Country, Leioa, Spain
The persistent emergence of C. glabrata strains resistant to currently
used antifungals demands the implementation of novel therapeutic
Objectives Candida albicans is an opportunistic fungus, being the strategies based on non-conventional targets. Genes contributing to
main fungal pathogen responsible for the nosocomial infections. increase C. glabrata competitiveness in the vaginal tract by mediating
Among all its proteins, C. albicans KRE9 is an essential and highly tolerance to the organic acids found therein are a cohort of interest-
mannosylated protein which is involved in beta-1,6-glucan biosyn- ing, and yet unexplored, set of therapeutic targets. Thus, the objec-
thesis. Therefore, the aim of this study was to generate an anti-KRE9 tive of this work is the identification of key genes/pathways/
monoclonal antibody (mAb), and to test its specificity towards KRE9 mechanisms underlying survival of C. glabrata to acetic and/or lactic
and its effect on C. albicans growth. acids. In particular, the characterization of the involvement of a new
Methods The anti-KRE9 mAb was produced using the hybridome signalling system, controlled by the putative transcription factor
technique. Among the positive cell lines generated, the most reactive CgHaa1, in C. glabrata tolerance and response to acetic acid is
one was selected to be cultured and to purify the mAb used in this aimed.
work. Results and conclusions Elimination of CgHAA1 gene from C. glab-
The specific recognition of the protein was tested by 1-D and 2-D rata genome dramatically increased susceptibility of this pathogenic
electrophoresis and Western Blot. On the other hand, in order to yeast to concentrations of acetic acid similar to those found in the
localize the protein in the yeast, an indirect immunofluorescence vaginal tract. A transcriptomic analysis revealed that CgHaa1
(IIF) was also performed; using different strains of live and sodium impacted the expression of roughly 70% of the overall set of C. glab-
metaperiodate (50 mM)-treated C. albicans. Finally, the effect of the rata genes that are activated in response to acetic acid stress, con-
mAb over yeast growth and endothelial cell activation was also firming the crucial role of this transcriptional regulator in the control
measured. of genomic expression under these conditions. Functional clustering
Results The anti-KRE9 mAb recognized specifically one band, corre- of the genes activated by CgHaa1 under acetic acid stress shows an
sponding to KRE9 protein, on the extract of C. albicans. However, enrichment of those involved in carbohydrate metabolism, transport,
using IIF it was only detected on the cell surface of the metaperiodate cell wall maintenance, regulation of internal pH and nucleic acid
treated yeast, thus, when mannoses of the cell wall had been processing. The mechanisms by which the CgHaa1 pathway mediate
removed. On the other hand, the mAb was able to reduce C. albicans tolerance to acetic acid in C. glabrata were further dissected, explor-
growth, both of the reference strain and the clinical isolate CECT ing a transcriptomics approach, being of notice the involvement of
13062, at least at a concentration of 10 ng/ml. Endothelial cell acti- this regulatory system in the control of internal pH and in reducing
vation induced by C. albicans was also significantly diminished by the the internal accumulation of the acid. In the presence of acetic acid
antibody at the same concentration. CgHaa1 enhanced adhesion and colonization of reconstituted vaginal
Conclusion The anti-KRE9 mAb specifically binds to C. albicans human epithelium by C. glabrata, an in vitro model of vaginal infec-
KRE9. However, since this protein is highly mannosylated, the detec- tion. Consistently, CgHaa1 expression exerted a positive effect over
tion by IIF was unable unless C. albicans cells were treated to remove the expression of several adhesin-encoding genes and increased C.
the mannoses present in the cell wall. Even so, the mAb was able to glabrara adherence to the extracellular matrix proteins fibronectin
reduce significantly yeast growth and endothelial cell activation by and vitronectin.
C. albicans. Therefore, although promising results have been On the overall the results obtained show similarities, but also
obtained, more studied should be carried out and in the near future remarkable differences, in the way by which the ScHaa1 and
other effects will be studied in depth, such as inhibition of

ª 2015 The Authors

188 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

CgHaa1 pathways mediate tolerance to acetic acid in S. cerevisiae P383

and in C. glabrata, indicating a ‘functional expansion’of the network
in the later species. Further details on the evolution of this network
from S. cerevisae to C. glabrata will be discussed. The role of the Epidemiological aspects and genotypic characterization of
CgHaa1-pathway in the extreme acetic acid-tolerance exhibited by T.violaceum strains collected during a Belgian National
vaginal C. glabrata isolates will also be discussed, along with other survey on anthropophilic tinea
uncovered mechanistic insights that were found to be on the basis of R. Sacheli,1 C. Dekkers,1 B. Geron,1 H. Graide,1 R. Darfouf,1
the acid tolerance phenotype exhibited by these isolates. C. Adjetey,1 C. Meex,1 J. Descy,1 P. Huynen,1 P. Melin,1
J. Andre,2 J. Arrese3 and M. P. Hayette1
1
CHU Liege, Liege, Belgium; 2CHU Brugmann, Bruxelles, Belgium
P382 and 3University Hospital of Liege, Liege, Belgium

Objectives The last 2 years, clinical cases of tinea capitis caused by

Standardization of a two step real-time PCR based method Trichophyton violaceum (T. violaceum), have been identified in Belgium.
for species specific detection of medically important To better understand the emergence of this species in the population,
Aspergillus sp. the Belgian National Reference Center (NRC Liege) launched a 1-year
P. Das, S. Bhattacharya, A. Harishankar and M. Chandy national survey in 2013. Epidemiological aspects and genotypic char-
Tata Medical Center, Kolkata, India acterization of the strains were included.
Methods The study was conducted from March 2013 up to Febru-
ary 2014. All Belgian laboratories were asked to send M. audouinii
Objective To standardize a two step real-time PCR based method and T. violaceum strains isolated from hair to the NRC with a form to
using 28S rDNA and beta-tubulin gene, calmodulin gene specific tar- fill in including epidemiological data. The fungal strains were identi-
get as a tool for rapid detection of aspergillosis from whole-blood fied by microscopy or ITS sequencing in case of doubtful identifica-
samples. tion. The genotypic analysis was performed by the DiversiLab
Method Five medically important Aspergillus sp. i.e. three reference system (bioMerieux) for DNA fingerprinting and analysis. Epidemio-
isolates Asp. fumigatus (ATCC-204305), Asp. niger (ATCC-16404), logical data were analyzed with the help of a biostatistician.
Asp. flavus (ATCC-204304) and two clinical isolates Asp. terreus, Asp. Results Amongst the collected isolates, 23 strains were confirmed as
clavatus, were used for standardization. The inoculum size was T.violaceum (results concerning the 116 M. audouinii strains have
adjusted to a concentration of 5 9 106 to 1 9 108conidia ml
1 by already been reported). Analysis of the epidemiological characteristics
hematocytometer. Total genomic DNA was extracted by bead beating of the infected population shows that the main age category con-
with thermal shock treatment prior to the use of commercial DNA cerns 0–4 year-old children (n = 9, 39.1%) with a sex-ratio M/F of
extraction kit. Extraction yield and purity was checked by NanoDrop 1.875. Data concerning the ethnical origin of the family were pre-
2000 and gel electrophoresis. Efficiency of extraction was checked in sent in 82.6% of the cases and reveal that patients were mainly of
real time PCR. A PAN fungal PCR was performed using custom Ethiopian origin (n = 8, 57.9% of known cases). One patient was
designed primers and probe targeting 28S rDNA for screening (Step- also from Burundi showing that T. violaceum strains probably circu-
I) followed by species specific primer targeting beta-tubulin gene and late mainly in East Africa. The genotypic analysis by DiversiLab led
calmodulin gene as targets (Step-II). All positive PCR products from to the distinction of 2 variants of T. violaceum. The major group was
each of these reactions were then sequenced in duplicate or triplicate composed of 17 strains which were mainly collected in the North of
using standard BIGdye3 chemistry and methods on an ABI3100 (Ap- Belgium and included also the reference strain (18/23, 83.3%). The
plied Biosystems, CA). other group (6 strains) was close to the major group but the analysis
Result DNA extracted was observed to have a concentration of the spectral superposition showed some differences between these
between 40 to100 lg ll
1. In terms of purity the 260/280 ratio two groups, defining two distinct variants of T. violaceum in the Bel-
were between 1.2 to 2.0. Taq Man based real time-PCR showed satis- gian population. This second variant was mainly recovered from
factory sensitivity and specificity for Asp. fumigatus, Asp. flavus, Asp. South Belgium (5/6, 83.3%). No correlation could be made between
niger, Asp. terreus. Analytical sensitivity for PAN Aspergillus 28S the genotypic group and a particular ethnical origin as Ethiopian
rDNA primer and probe was found to be 102 cfu ml
1. Species speci- subjects were found in both groups.
fic detection of Aspergillus was made for four species i.e. Asp. fumiga- Conclusion The DiversiLab system proved to be an efficient
tus, Asp. niger, Asp. flavus, Asp.terreus and specificity was checked method to investigate the molecular epidemiology of dermatophytes
against 37 different microbial species including eukaryotes and infections as reported previously for M. audouinii. These results show
prokaryotes. All the species specific primers BLASTn, result were then that two distinct isolates co-exist in Belgium providing evidence of
carefully analyzed in graphical and statistical form. The graphical genetic heterogeneity and a possible spread of one genotypic variant
form was used to ensure that the all of the four products of the query in a restricted geographic area or the co-existence of two variants
sequence entered into the search was subsequently aligned with the circulating in different African communities. However, no clear cor-
high scoring database sequence returned (99–100% query coverage relation could be established between the appartenance to a group
and 99–100% identity). and epidemiological factors, such as age or ethnical origin.
Conclusion Data from this study indicate the efficacy of the current
DNA extraction method using physical sharing and it is found to be
specific, sensitive, stable and easy to serve as a useful tool for rapid
detection of aspergillosis from whole-blood samples.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 189
Poster Presentations

P384 Agarose Gel Electrophoresis was performed to visualize the

amplicons.
The ITS (Internal Transcribed Spacer) region TEF1a (Translation
Environmental isolation of Cryptococcus gattii molecular Elongation Factor 1a) gene from all the 17 Fusarium isolates were
type VGII and Cryptococcus neoformans molecular type sequenced (SciGenom labs, Cochin, Kerala, India). The sequences
VNI from wooden poles located in the deep Amazon Rio results were then used for nucleotide search using the BLAST algo-
Negro basin rithm at the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/)
F. Brito-Santos,1 L. Trilles,1 B. Wanke,1 D. V. Santos,1 and Fusarium ID BLAST (http://isolate.fusariumdb.org/index.php).
F. A. Carvalho-Costa2 and M. S. Lazera1 Hits more than 99% were considered.
1 Result
National Institute of Infectology/FIOCRUZ, Rio de Janeiro, Brazil BLAST analysis ITS sequences - Out of the 17 isolates, 14 (A47,
and 2Institute Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil A68, A71, A72, A73, A97, A98, A99, A103, A109, A74, A96,
A100 and A108) sequences gave definitive results and 3 out of 17
Background Primary cryptococcosis caused by molecular type VGII (A95, A102 and A110) gave more than one result in the NCBI blast.
(serotype B, MAT alpha) prevails in immunocompetent patients in In Fusarium ID blast, 5 out of 17 (A71, A72, A95, A97 and A 99)
the North and Northeast of Brazil, revealing an endemic regional pat- gave definitive results, 3 out of 17 (A73, A102 and A110) gave two
tern of cryptococcosis by this molecular type. results where both belonged to the same species complex but
Objective This study investigated the presence of cryptococcosis
agents on wooden poles in neighborhoods located in the Upper Rio
Negro, municipality of Santa Isabel do Rio Negro, Amazon, Brazil.
Methods The decaying wood was collected from the poles, and this
material was diluted and plated on agar bird seed. Dark brown colo-
nies were identified phenotypically and genotypically by URA5 poly-
morphism restriction fragment (RFLP) analysis.
Results The samples were positive for C. gattii molecular type VGII
and C. neoformans molecular type VNI by URA5 polymorphism
restriction fragment (RFLP) analysis.
Conclusions For the first time C. gattii molecular type VGII and C.
neoformans molecular type VNI were isolated from wooden poles, lar-
gely used by the rural population of the mainland forest of the Ama-
zonia basin. These findings correlate with the endemic status of
cryptococcosis in Amazonia and complement previous study in the
same area of Santa Isabel do Rio Negro. In this municipality, differ-
ent MLST subtypes of C. gattii VGII were isolated from indoor dust
from typical wooden houses (Brito-Santos et al. 2015). Besides the
dust indoor, the wooden poles may act as potential sources of human
infection by agents of cryptoccocosis, which are associated with a
specialized niche resulting from the natural biodegradation of wood
(Lazera et al. 2000).

P385

Standardization of PCR and molecular speciation of 17 Figure 1. Gel showing amplicon of TEF region
cases of Fusarium keratitis from South India using
sequences of the ITS and TEF 1a genes
A. Tupaki Sreepurna,1 S. Sharma,2 S. Natarajan1 and A. J. Kindo3
1
Sri Ramachandra Medical College and Research Institute,
Chennai, India; 2L.V. Prasad Eye Institute, Hyderabad, India and
3
Sri Ramachandra University, Chennai, India

Objectives To standardize an in-house method of PCR using primers

specific to ITS-1 & 2 and Translation Elongation Factor - a (TEF-1a)
genes to detect and differentiate clinical Fusarium isolates.
Sequencing of PCR amplicons and sequence analysis and molecu-
lar identification using BLAST
To confirm if TEFa is a better target gene than ITS for unambigu-
ous differentiation of Fusarium species.
Methods 17 Fusarium isolates of human keratitis from Tamilnadu
and Andhra Pradesh in south India were studied. Growth in stan-
dard media like Sabouraud’s Dextrose Agar and microscopic features
of slide cultures using Oatmeal Agar were noted. Genus level identifi-
cation was done but speciation wasn’t possible in majority of isolates.
DNA isolation - performed using growth from Sabouraud’s Dextrose
Broth following Phenol-Chloroform method. No commercial kit used.
Primer sequences for ITS and TEF-1 genetic regions were obtained
from Mitchell et al., 1994 & Jackson et al., 2006 respectively. Fusar-
ium solani and Fusarium oxysporum isolates confirmed by gene
sequencing were used as control.
Figure 2. Gel showing amplicon of ITS region

ª 2015 The Authors

190 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

different groups, 9 out of 17 (A47, A68, A98, A103, A109, A74,

A96, A100 and A108) gave more than one result where all
belonged to different species. Furthermore, the results obtained in
NCBI blast and Fusarium ID blast did not correlate with each other.
TEF 1a sequences - Out of the 17 Fusarium isolates, 10 were
Fusarium falciforme, which belonged to Fusarium solani species com-
plex (isolate numbers-A47, A72, A74, A95, A96, A99, A100,
A103, A108and A109. The remaining 7 isolates belonged to Gib-
berella fujikuroi species complex. Thevarious Fusarium species which
were perceived in the Gibberella fujikuroi species complexwere 1
Fusarium proliferatum (A110), 1 Fusarium sacchari (A98), 1 Fusarium
verticilloides (A71), 1 Fusarium thapsinum and 1 Fusarium subqluti-
nans/Fusarium pseudocircinatum. Isolate A102 belonged to Fusarium
oxysporum species complex. Isolate A97 showed BLAST result as
Fusarium solani in NCBI database and as Giberella fujikuroi in Fusar-
ium ID database leaving it ambiguous.
Conclusion ITS region provides adequate genetic information to reli-
ably detect Fusarium upto species level, which serves ideal for rapid
molecular test for diagnosis in a clinical setting, TEF-1a is the most
phylogenetically informative gene and is ideal for molecularspeciation
in epidemiological studies.
Correction added on 19 October 2015, after online publication. 7(3.7%), and 3(1.5%) M. furfur in boys and 6 (3.1%) M. restricta, 3
Presenting Author was changed from S. Sharma to A. Tupaki (1.5%) M.sympodialis, M. globosa 4(2.1%), and 3(1.5%) M. furfur in
Sreepurna. girls. Chi-square analyses.
Conclusion This study revealed that there is no correlation signifi-
cant difference between Malassezia Species distribution, PV and gender
and also shown that oily skin contained more M. restricta than dry
ones and dry and normal ones contained more M. sympodialis, M.
P386 globosa and M. furfur compared to only ones.

Molecular epidemiology of Mallasezia species in students

of Sabzevar universities
H. Moallaei P387
Sabzevar university of medical Sciences, Sabzevar, Iran
Spectral subtraction signal post-processing of Candida
Objectives The genus Malasezia currently includes fourteen species parapsilosis noisy microsatellite fragment data typing
that have been considerd as part of normal flora of skin in healthy profiles
and diseased human and animal skin. However, there were differ- C. Trobajo-Sanmartı́n,1 G. Ezpeleta,1 E. Sarasola,2 C. Marcos-
ences with respect to the species most commonly isolated, not only  s1
Arias,1 E. Eraso1 and G. Quindo
in patients with various skin diseases but also between healthy indi-
viduals. The aim of this study was to analyze the prevalence of
1
Univ. Paıs Vasco UPV/EHU Facultad de Medicina y Odontologıa,
Malassezia species from clinically normal skin of the scalp and trunk Bilbao, Spain and 2Hospital Universitario Basurto, Bilbao, Spain
in students of Sabzevar universities and to examine if the range of
species varies according to body site, gender and age. Objectives Microsatellite markers have demonstrated their suitability
Methods The study was conducted at the Department of Microbiol- for typing clinical isolates from different species of Candida. Despite
ogy, Sabzevar University of Medical Sciences, Iran from September the apparent simplicity of this technique, many problems and sources
2012 to November 2013. One hundred and eighty-nine healthy stu- of error could lead us to confounding conclusions and must be con-
dents of Sabzevar University with no skin diseases and aged from 18 sidered before producing accurate genotypes. The aim of this work
to 28 years were studied. The samples were obtained by scraping the was to evaluate the reliability of a spectral subtraction signal analysis
skin surface from the upper and middle part of trunk and from scalps (SSSA) (a method which removes noise from noisy signals in the fre-
of all subjects and then all samples were prepared with KOH 20% quency domain) in a set of microsatellite data obtained from two
and methylene blue for staining and undergone microscopic exami- Candida parapsilosis sensu stricto reference strains.
nations and also inoculated in plates containing Glucose-Neopepton- Methods Two C. parapsilosis reference strains, ATCC 22019 and
Yeast extract medium (pH 6.2) with additional olive oil (20 mL.L
1), CDC-317, were used for this study. Yeast cells were grown on Sabour-
Tween 80 (2 mL.L
1) and Glycerol monostrated (2.5 mL.L
1) incu- aud glucose liquid medium for 24 h previous to DNA isolation using
bated at 31 °C for 2 weeks and examined at frequent intervals for a UltraClean Microbial DNA Isolation kit, according to the manufac-
developing colonies. turer’s instructions. PCRs for all microsatellite markers were per-
DNA extraction was performed from colonies. The ITS1 region of formed according to the protocols established by Sabino et al. (J. Clin.
rDNA from isolates of Malassezia species were amplified by PCR reac- Microbiol. 2010; 48 (5): 1677–1682). Subsequent fragment size
tion. The PCR were digested by Cfo I enzyme. determination was performed on a 3130 Genetic Analyzer using 1 ll
Quantitative data were analyzed by the group of t-test. The data of of a 1:10 dilution of each PCR product obtained, 13.7 ll of for-
the students were analyzed using chi-square test. Correlation of pre- mamide and 0.3 ll of the internal size standard. After size determina-
disposing factors with Mallasezia speciesas well as the difference tion, data were analyzed using the Peak Scanner software and the
between isolates from students was evaluated by Fisher exact test. A alleles obtained were designated by their sizes (in mer). The noise
q-values of <0.05 were considered as significant. removal algorithm was implemented using Matlab and Sig-View soft-
Results The products of restriction digestion are shown separately in ware. Briefly, the corresponding spectrums of the different signals
Fig. 2; the bands generated were of the predicted sizes. Using CfoI obtained after microsatellite analysis were computed using the FFT,
(Roche Diagnostics,Mannheim, Germany), we could distinguish four removing the noise from the noisy spectra, and reconstructing the sig-
different species, including Malassezia furfur,s, Malassezia globosa,, nal back into the time domain using the inverse Fast Fourier Trans-
Malassezia restricta and Malassezia sympodialis. form (IFFT). The performance of the algorithm was evaluated by
The prevalence of different Mallesezia species among students were calculating the Signal to Noise Ratio (SNR). Frame averaging using
as follows: Ten (5.3%) M. restricta, 5(2.6%) M. sympodialis, M.globosa different windows was an optional technique to improve the SNR.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 191
Poster Presentations

Results Microsatellite analysis lead to four different sized PCR prod- P389
ucts which lengths varied from 150 to 330 bp. However, stutter effect
was remarkable making sometimes the allele designation difficult. On
the contrary, results from the SSSA showed better signals profiles Molecular epidemiology of Candida albicans from
which made the allele designation an easier task. The Signal to Noise vulvovaginal Candidasis
ratio ranged from 8.1 to 12.3 depending on the windows and the F. Shangrong, L. I. Ting, S. Yingying, L. Xiaoping and Z. Yuxia
frame length used. No frames averaging showed the best performance Peking University Shenzhen Hospital, Shenzhen, China
in removing noise from noisy microsatellite fragment data profiles.
Despite these promising results we also observed that SSSA tend to dis-
miss the amplitude and wide the length of the reconstructed peaks Objectives To investigate ABC genotype and mating type of C. albi-
which could lead to inaccurate fragment size determination. cans from vulvovaginal candidiasis (VVC). Analysis of virulence gene
Conclusion Despite microsatellite analysis is a useful technique for expression and antifungal susceptibility of the C. albicans.
C. parapsilosis typing, careful design of primers and PCR conditions Methods C. albicans were collected from the vulvavaginal of patients
should be considered for a reliable allele determination. SSSA could with VVC from January 1, 2003 to September 30, 2014. The ABC
be a novel post-processing technique of such data when adverse type was also determined, based on the presence or absence of an
design conditions are found. However, limitations found make advis- intron in DNA sequences encoding rRNA. The mating type, i.e., a/a,
able to recommend more research in this field to assess the robust- a/a, or a/a, was determined by PCR. HWP1, ALS3, SAP1 gene
ness of this typing technique. expression was also determined by using RT-PCR analysis. Antifun-
Funding This study was financed in part by Fundaci on Jes
us de gal susceptibility testing, performed on 658 of the isolates, performed
Gangoiti Barrera and projects GIC12 210-IT-696-13, UFI 11/25 using the broth microdilution technique. The results based on contin-
(UPV/EHU). uous measurement were analyzed using a one-way analysis of vari-
ance (ANOVA).
Results Among the 1427 strains investigated, 1319 isolates (92.4%)
were found as genotype A, 77(5.39%) were genotype B and 31
(2.17%) were genotype C. There were no genotype D and genotype
P388 E. 1419 isolates were MTL locus heterozygous isolates and 8 were
MTL locus homozygous isolates. Of the 8 homozygous isolates, 7
Standardization of nested-Polimerase chain reaction for were MTLa and one was MTLa. Expression levels of virulence-related
detection of Histoplasma capsulatum genome in batA? ~ A
^ 0s genes Sap1 was a little higher in heterozygous isolate cells than in
guano droppings. homozygous isolate cells (1.02  0.68 V.S 0.83  0.31, F = 3.55,
R. B. Caligiorne,1 T. T. Silveira,1 C. Assuncao,1 J. Ianni,1 P = 0.07). Hwp1 was significantly higher in heterozygous isolate
cells than in homozygous isolate cells (1.71  1.42 V.S1.20  0.62,
R. Acaiah1 and F. Rocha-Silva2 F = 5.14, P = 0.03). There was no statistical difference of the expres-
1
Santa Casa Hospital, Belo Horizonte, Brazil and 2Nucelo de Pos- sion of virulence genes HWP1, ALS3 and SAP1 in the different ABC
graduac
a~o Santa Casa Hospital, Mg, Brazil, Belo Horizonte, Brazil genotypes. The resistant rate of azoles including voriconazole, flu-
conazole, itraconazole, clotrimazole and miconazole in genotype A
Introduction Histoplasmosis is a systemic, opportunistic infectious was more to than those in genotype B and genotype C. Flucytosine
disease caused by inhaling the fungus Histoplasma capsulatum micro- and caspofungin were susceptible to all genotypes,
conıdeos. Histoplasmosis started to gain more prominence in the Conclusion C. albicans genotype A and MTL locus heterozygous iso-
1980s and 1990s, with the advent of acquired immunodeficiency lates was the most prevalent among patients with VVC. Hwp1 was
syndrome (AIDS) and other diseases that affect the immune response, significantly higher in heterozygous isolate cells than in homozygous
as leukemic patients, transplant and taking chemotherapy. Rapid isolate cells. The resistant rate of azoles in genotype A was more to
and specific diagnostic tests for fungal infections are extremely impor- than those in genotype B and genotype C.
tant to the effectiveness of treatment of infected patients. With the
advancement of molecular biology has been possible to develop new
techniques for diagnosis and identification of Histoplasma capsulatum.
Objectives The present study aimed to standardize the technique of P390
nested PCR for detection of H. capsulatum genome in bat guano.
Methods For this purpose, 14 guano0 s samples were isolated from a
cave near the town of Unai, Minas Gerais, Brazil. The primers pairs Features of Aspergillus flavus, A. oryzae and related
Fungus I and II; Histo I and II, described for the amplification of isolates revealed by MALDI-TOF-MS
regions of ribosomal DNA (rDNA) were used, generating a fragment I. A. Riabinin, O. D. Vasilyev, N. V. Vasilyeva, M. V. Rudneva,
of approximately 231 bp. The DNA of H. Capsulatum was detected in T. S. Bogomolova and G. A. Chilina
78.5% of sample analyzed (n = 11). North-Western State Medical University named after I.I.
Conclusion The level of detection of H. capsulatum DNA by nested- Mechnikov, Saint Petersburg, Russia
PCR technique, proposed by the study, was 22.6 9 10–2 ng ul
1.
This result showed that the PCR is capable of detecting very low con-
centrations of DNA in an environmental sample. Therefore, the Objective of this study is to find out the possibility of correct species
nested-PCR technique can facilitate the detection of fungu0 s DNA in identification and strains grouping of Aspergillus spp. from section
guanosine, without the needing of fungus isolation in culture media. Flavi by MALDI-TOF-mass-spectrometry and related software.
Funding/support FAPEMIG - Fundac
~ao de Amparo a Pesquisa do Methods 38 isolates of Aspergillus spp. belonging to section Flavi
Estado de Minas Gerais, Brazil, CNPq - Conselho Nacional de Desen- were collected from sputum, bronchoalveolar lavage, paranasal
volvimento Cientıfico e Tecnologico, Brazil. sinuses washing fluids, biopsy materials, corneal scraping and
Key words histoplasmosis, Histoplasma capsulatum, PCR, DNA, glass another kinds of specimens. Strain were subcultivated in Eppendorf
beads, rDNA tubes with 0.5 ml of Sabouraud broth with 2% dextrose at 37 °C
overnight. For extraction of proteins from cultures we performed
standard method situated for MALDI-TOF-MS with using ethanol,
formic acid and acetonitrile. MALDI-TOF-mass-spectrometry of pro-
tein extracts form cultures was performed in Autoflex speed TOF/TOF
(Bruker Daltonics, Germany). Some strains were underwent described
manipulations 2–3 times to obtain several spectra as a control of sta-
bility of the discrimination of strains in different groups. Totally 48
mass-spectra were collected. Mass-spectra were identified in Biotyper

ª 2015 The Authors

192 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P391

Optimization of the heterologous expression enzyme

Chorismate Synthase from Paracoccidiodies brasilensis -
focus in drug development
F. A. V. Rodrigues, J. S. R. Godoy, F. A. Seixas, P. S. Bonfim-
Mendonc
a, M. Negri, T. I. E. Svidzinski and E. S. Kioshima
Universidade Estadual de Maringa, Maringa, Brazil

The aim of this work is to optimize the main conditions necessary to

express the chorismate synthase from Paracoccidiodies brasilensis with
focus in drug development. For the heterologous expression of CS
from Paracoccidioides brasiliensis (PbCS), the gene was chemically syn-
thesized using preferential codon of Escherichia coli and cloned into
the expression vector pET series (Novagen). For optimization of PbCS
recombinant production many parameters were evaluated: E. coli
host strains (BL21(DE3) and BL21(DE3) pLysS), induction periods (0
to 24 h), and different culture media (Luria Broth - LB, Terrifc Broth
-TB, 4YT and mininal medium - M9). The IPTG concentration
(1 mM) and temperature (37 °C), were maintained constant during
the tests. The PbCS recombinant (PbCSr) expression in different con-
ditions was analyzed by SDS-PAGE and Western blotting. The struc-
tural characterization of enzymes involved in the metabolic processes
of pathogens with selective toxicity may be an alternative to drug
development. The Chorismate Synthase (CS) has been shown to be
an excellent target for antifungal drug, as it is involved in a meta-
Figure 1 bolic pathway that is present only in plants, fungi and bacteria. The
high-level expression of PbCSr was observed both in inclusion bodies
and cell lysate supernatant. The maximum amount of PbCSr in
OC with ‘Fungi Library’database. Although the difference in mass- native form was obtained using the strain E. coli BL21 (kDE3) culti-
spectra profiles (MSP) of A. flavus and A. oryzae and grouping of vated in medium TB to 4 h of induction (Figure). During the PbCSr
strains by construction of the dendrogram of hierarchical principal purification process, the best imidazole concentration in the washing
components analysis (hPCA) were investigated with using of the buffer was 80 mM and in the elution buffer was 250 mM. The con-
same software. Morphological features of selected strains were stud- centration of purified protein was 0.2 mg ml
1 of supernatant. The
ied in Sabouraud dextrose agar (SDA) and Czapek yeast autolysate PbCSr purification was confirmed by Western Blotting using anti-His
agar (CYA). antiserum. In this work, we developed an efficient procedure for the
Results 41 mass-spectra (MS) were identified as A. flavus and 7 - as PbCSr production and optimizing protein purification conditions. Val-
A. oryzae. 5 MS from group of ‘A. flavus’corresponded with strains, idation steps of enzyme activity, characterization of the structure and
that characterized of orange-brown colorings of cultures without any crystallization of the recombinant protein, may be carried out with
yellow or green shades in CYA and SDA witch not typical neither to the aim of increasing the basic knowledge of this enzyme, and, in the
A. flavus, nor to A. oryzae. The result of the strains grouping by future, the development of antifungal drugs or vaccine directed to
hPCA is shown in fig. 1. Strains were subdivided into 5 groups. The this target.
most of A. oryzae spectra are situated in group B, but this group con-
tains some spectra of A. flavus too. 2 spectra of A. oryzae obtained
from one strain were placed in another groups (A and D). The com-
parison of mass-spectra-profiles identified as ‘A. flavus’and ‘A.
oryzae’demonstrated at least 2 ranges of M/Z in which the differences
between this group of species-specific MSP are most pronounced:
4450–5800 Da and 7450–7850 Da. Both ranges include several
small peaks in A. oryzae, witch not situated in A. flavus. The compar-
ative analysis of MSP from strains with orange-brown colonies not
allowed to identify of any regular differences with each other as well
as with ‘A. flavus’and ‘A. oryzae’groups. Matching of positions of
mass-spectra in hPCA-dendrogram allowed to reveal that spectra
from identical strain are situated in different groups.
Conclusion Our study demonstrates that species identification of
aspergilli from section Flavi by standard technique of MALDI-TOF-MS
is very difficult. We consider, that one of the probable reason of this sit-
uation is in obtaining of poor detailed mass-spectra. For solving this
problem an improved protocol of proteins extraction is needed to
P392
obtain more itemized mass-spectra from cultures of filamentous fungi.
Molecular characterization of Aspergillus infections of an
Iranian educational hospital using RAPD-PCR
K. Diba,1 A. Namaki2 and K. Makhdoomi1
1
Urmia University of Medical Sciences, Urmia, Iran and 2Arefian
General Hospital, Urmia, Iran

Objectives The nosocomial infections by Aspergillus species are

associated with constructions and increased dust loads in hospital

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 193
Poster Presentations

indoors. Our main object was to find the environmental sources of contend that there is no single ‘gold standard’in fungal identifica-
Aspergillus species causing hospital acquired infections. tion testing and that a combined approach is the best approach for
Methods The clinical and environmental samplings were performed identifying fungi.
during 18 months from spring 2010 to summer 2011 in Imam edu-
cational hospital, Urmia, Iran. A morphological diagnosis was per-
formed including microscopic characterization of isolated aspergillus
from cultured specimens and Polymerase Chain Reaction - Restric- P400
tion Fragment Length Polymorphism (PCR-RFLP) for the identifica-
tion in the level of species. Random Amplified Polymorphic DNA -
PCR RAPD-PCR using random primers for rDNA gene was performed In vitro activity ASP2397 against Aspergillus isolates with
to compare Aspergillus isolates of clinical cases with the relevant and without acquired resistance mechanisms
environmental sources. M. C. Arendrup1 and M. Cuenca-Estrella2
Results Use of RAPD method resulted various differential patterns, 1
Statens Serum Institut, Copenhagen, Denmark and 2Spanish
so that some Aspergillus isolates from the clinical and hospital indoor
National Center for Microbiology, Madrid, Spain
were completely matched (matched pairs) and some other Aspergillus
isolates were not matched. In the case of matched pairs, A. niger and
A.flavus isolated from broncoalveolar lavage and sinus discharge were Objectives ASP2397 is a new compound with activity against
relevant to those of air conditioner and walls surfaces, respectively. Aspergillus and C. glabrata. It is actively transported into the fungal
Conclusion The hospital sources for the Aspergillus clinical isolates cells via the siderophore transporter Sit1. The mode of action is novel
included air condition and walls. RAPD-PCR analysis can play a triv- and yet not known, but different from that of the azoles and ampho-
ial role to find the hospital sources of Aspergillus clinical isolates. tericin. The purpose of this study was to investigate the in vitro activ-
ity against a well characterised panel of wild type susceptible (wt)
and azole resistant A. fumigatus and A. terreus isolates by the EUCAST
and CLSI methodologies and to compare that with the activity of
P393 amphotericin B (AMB), itraconazole (ITC), posaconazole (PSC) and
voriconazole (VRC).
Methods 34 isolates were included: A. fumigatus: 4 wt, 5 with TR/
Molecular Identification of Yeast: Gold Standard or Fool’s L98H, 9 with M220, 9 with G54 CYP51A alterations and 1 with a
Gold? HapE mutation. A. terreus: 2 wt and 1 with an M217I CYP51A alter-
A. W. Fothergill, D. I. Mccarthy, J. Lindner, H. Fan and ation. EUCAST EDEF9.2 and CLSI M38-A2 MICs were read visually
N. P. Wiederhold day 2 at a 100% inhibition endpoint. Reference strains were: A. fumi-
gatus ATCC 204305, F6919 and ATCC MYA-2636.
UT Health Science Center San Antonio, San Antonio, USA Results MIC50 (MIC range) (mg l
1) determined by EUCAST and
CLSI for ASP2397 were: 0.5 (0.25–1); 0.25 (0.06–0.25) against A.
Objective Traditionally, fungal identification has been considered a fumigatus wt isolates and similar to those against A. fumigatus har-
labor-intensive process with few automated options as compared to bouring azole resistance mutations: 0.5 (0.125 to >4); 0.125 (0.06
bacterial identification. Yeast lend themselves to automated systems to >4). The similar values for the comparator drugs against wt iso-
allowing their identification to become more routine. In the last dec- lates were: ITC 0.125 (0.125–0.25); 0.125 (0.06–0.25), PSC ≤0.03
ade, molecular testing has shortened the time needed for species (≤0.03–0.06); ≤0.03 (≤0.03–0.06), VRC 0.5 (0.5–1); 0.25 (0.25–
identification and is presumed by many to be the ‘gold standard’. We 0.5) and AMB 0.25 (0.125–0.5); 0.25 (0.125–0.25) but higher for
reviewed 130 clinical yeast isolates submitted to the Fungus Testing the azoles against A. fumigatus harbouring azole resistance muta-
Laboratory for identification to determine the efficiency of sequencing tions:: ITC >4 (1 to >4); 4 (0.5 to >4), PSC 0.5 (0.06 to >4); 0.5
data alone to obtain a final identification to the species level. (≤0.06 to 4), VRC 0.5 (0.25 to >4); 0.5 (0.125 to 4) and not for
Methods A total of 130 clinical yeast isolates were received for iden- AMB 0.25 (0.125 to 1); 0.25 (0.125 to 1). For A. terreus the pattern
tification in 2014. All isolates were sequenced, with ITS and D1/D2 was somewhat different as both azole and ASP2397 MICs were ele-
being the primary loci used for this purpose. The resulting sequences vated for the CYP51A mutant isolate: ASP2397 EUCAST MIC: >4 vs.
subsequently underwent BLAST searches to determine the percent 0.5–1 for wt and CLSI MIC: 2 vs. 0.5–1 for wt isolates. Similar MIC
identity with species available in public databases (e.g., GenBank, elevation was also observed for the azole compounds but not for
CBS-KNAW). In addition to sequencing, the isolates underwent AMB (data not shown). Further studies of ASP2397 against more
extensive physiologic and morphologic testing, including carbohy- isolates of A. terreus are needed before the implication of the findings
drate assimilation patterns (API 20C AUX, bioMeriuex, St. Louis, for this species can be interpreted.
MO), cycloheximide susceptibility, temperature tolerance, and micro- Conclusion ASP2397 displayed in vitro activity against A. fumigatus
scopic morphology on Cornmeal Agar. Additional tests that were per- and A. terreus isolates. The activity was independent of the absence
formed when appropriate included urea hydrolysis, nitrate utilization, or presence of azole target gene resistance mutations in A. fumigatus.
and L-Canavanine Glycine Bromothymol Blue Medium (CGB) This finding holds promising implications at a time where azole resis-
reactions. tant is emerging globally in this species.
Results Of the isolates tested, sequencing alone provided a clear and
correct identification in only 30 isolates (23%). For 60 isolates (46%)
additional testing other than solely by molecular sequence analysis
was required to differentiate between two to three separate species. P401
Furthermore, 26 isolates (23%) required additional testing to differ-
entiate between four to five species. Interestingly, additional assays
were required to discriminate between six to up to twelve species for In vitro activity of Isavuconazole and Comparators against
the remaining 13 isolates (10%). Each of these isolates was docu- Clinical Isolates of the Mucorales Order
mented as having a high percent identity among different yeast spe- M. C. Arendrup,1 R. H. Jensen1 and J. Meletiadis2
cies. For 2 of the isolates, identification to the exact species was not 1
Statens Serum Institut, Copenhagen, Denmark and 2Clinical
possible. Microbiology Laboratory, Attikon Hospital, Athens, Greece
Conclusions This data reveals that sequencing alone is not suffi-
cient for yeast identification, and that a combined approach is
required to obtain a correct identification to the species level. Background The in vitro activity of isavuconazole (ISC), a new
Although only yeast were tested, several strains had 100% identity broad spectrum azole, has been studied using the EUCAST methodol-
with moulds such as Aspergillus, Exophiala, and Trichophyton spp. ogy against Candida and Aspergillus, however, data on in vitro activity
One strain surprisingly had 100% identity with a parasite. We against Mucorales isolates is sparse. The purpose of this study was to

ª 2015 The Authors

194 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

investigate the in vitro activity against clinical isolates of the Muco- In summary our approach in finding novel antifungals by screen-
rales order by the EUCAST and CLSI methodologies and to compare ing a library of drug-like molecules has yielded a promising novel
that with the activity of amphotericin B (AMB), posaconazole (PSC) compound, hemofungin, which inhibits the process of fungal heme
and voriconazole (VRC). biosynthesis and is strongly inhibitory towards a panel of fungal
Materials and Methods 72 clinical isolates were included: Lichthei- strains while only weakly affecting the growth of mammalian cell
mia (L) corymbifera 12, L. ramosa 5, Mucor (M) circinelloides (Group I) lines and is active and non-toxic in an insect model of fungal infec-
5, M. circinelloides (Group II) 9, Rhizomucor pusillus 9, Rhizopus (R) tion. Further investigation of this promising compound in additional
microsporus 26 and R. oryzae 6. Species identification was confirmed animal models of fungal infection is strongly warranted.
by ITS DNA sequencing. MICs were read visually day 1 and 2 for
EUCAST EDef 9.2 and day 2 for CLSI M38-A2 (no visible growth day
1).
Results MIC50 (MIC range) (mg l
1) determined by EUCAST (day 1), P403
CLSI (day 2) and EUCAST (day 2) for ISC were: 1 (0.125 to 16); 1
(0.125 to 2); 4 (0.5 to >16) across all isolates with a high agreement
between the EUCAST day 1 and CLSI day 2 MICs. The similar values In vivo Antifungal activity of compound selected against
for the comparator drugs were: PSC 0.25 (≤0.03 to >16); 0.25 (0.06 thioredoxin reductase from Candida albicans
to >16); 1 (0.06 to >16), AMB 0.06 (≤0.03–0.5); 0.06 (≤0.03– J. S. R. Godoy,1 A. K. R. Abadio,2 P. S. Bonfim-Mendonc
a,1
0.25); 0.125 (≤0.03–1) and VRC 16 (2 to >16); 8 (1 to >16); >16 M. S. S. Felipe,3 V. Leroux,4 B. Maigret,5 T. I. E. Svidzinski,1
(8 to >16) in agreement with the lack of clinical efficacy of VRC
F. A. V. Rodrigues1 and E. S. Kioshima1
against Mucorales. Species dependent ISC activity was observed with
the lowest MICs for: L. corymbifera 1 (0.5–2); 1 (1–2); 2 (1–4), L.
1
Universidade Estadual de Maringa, Maringa, Brazil; 2University
ramosa 0.25 (0.125–0.5); 1 (0.5–2); 2 (0.5–4), Rhizomucor pusillus from the State of Mato Grosso, Nova Xavantina, Brazil;
0.5 (0.5–1); 1 (0.125–1); 2 (1–2), R. microsporus 1 (0.5–4); 0.5
3
Universidade de Brasılia - UNB, Brasılia, DF, Brazil; 4LORIA,
(0.125–1); 4 (1–8), and R. oryzae 1 (0.5–4); 1 (0.125–2); 4 (0.5–8). Lorraine University, Nancy, France, Nancy, France and 5University
The highest MICs were found for M. circinelloides Group I 8 (4–8); 4 Henri Poincare-Nancy I, Nancy, France
(2–4) and 16 (2–16) and M. circinelloides Group II 8 (1–16); 8 (1–8);
16 (4 to >16). This pattern was also observed for the in vitro activity
Objective Invasive fungal infections (IFIs) have emerged as a public
of PSC but not for AMB.
health problem worldwide. The therapeutic options currently avail-
Conclusion ISC displayed in vitro activity against Mucorales isolates
able are limited and restricted to four antifungal classes. Further-
with exception of M. circinelloides. The MICs were in general 1–3
more, the resistance and side effects caused by these therapies,
steps higher than those for PSC. However, in the clinical setting this
indicate significant need for the new antifungal development. There-
may be compensated by the higher exposure (AUC ranges: ISC: 100
fore, the aim of this work was to select compounds by virtual screen-
vs PSC 15–35 mg.h ml
1).
ing against thioredoxin reductase from Candida albicans, contributing
to the new antifungal development for the treatment of important
fungal infections worldwide.
Methods There is no crystallographic structure presently available
P402 for pathogenic fungi thioredoxin reductase. Therefore, the 3D struc-
tures of Trrr from Candida albicans, in different conformation, were
constructed by homology modeling based on known structures with
Identification and characterization of Hemofungin, a novel
high percentage of identity in amino acid sequences. The stability of
antifungal compound which inhibits the final step of 3D structures was analyzed by Dynamic Molecular (DM) Simulation.
heme biosynthesis The program NAMD was employed in conjunction with the
D. Ben-Yaacov, A. Rivkin, G. Mircus and N. Osherov CHARMM22 force field to DM simulation. After that, a bank of com-
Tel-Aviv University, Tel-Aviv, Israel mercially available compounds from Life Chemicals database was
docked with the model by virtual screening simulations. The minimal
inhibition concentration (MIC) determination was performed to Can-
The incidence of fungal infections such as invasive pulmonary
dida, Cryptococcus and Paracoccidiodies species, according to M27-A3
aspergillosis (IPA) caused by Aspergillus fumigatus (A. fumigatus), has
guidelines (CLSI) with modifications to new compounds. The cytotox-
risen dramatically throughout recent years, especially among
icity studies was performed in HeLa cells, using the CellTiter 96
immunocompromised patients. Despite this rise in invasive infections,
assay (Promega, Madison, WI, USA), based on the reduction of MTS
there is only a limited number of antifungal drugs active against fun-
(3-[4,5-dimethylthiazol-2-yl]-5-[3- carboxymethoxyphenyl]-2-[4-sul-
gal pathogens and even with treatment, the mortality rate remains
fophenyl]-2H-tetrazolium). The experimental model of candidemia
high. Therefore, there is an urgent need to develop novel effective
was used to evaluation of in vivo antifungal activity.
antifungal drugs to treat fungal infections.
Results Three 3D structures were constructed to Trr1 from C. albi-
The aim of this study was to evaluate CW-208/hemofungin as a
cans, one the oxidized flavin conformation (FO) and two others in the
novel antifungal compound. This molecule was previously identified
flavin-reducing (FR) conformation (Figure A). Since these
in our laboratory by a library screening of synthetic drug-like com-
pounds and shown to cause swelling and lysis of growing fungal cells.
Hemofungin inhibited growth of pathogenic Aspergillus, Candida,
Fusarium and Rhizopus isolates at micromolar concentrations, while
only weakly affecting the growth of mammalian cell lines. Genetic and
biochemical analyses in A. nidulans indicated that hemofungin primar-
ily inhibits ferrochelatase, the last enzyme in the heme biosynthetic
pathway that converts protoporphyrin IX to hemin, suggesting that
its effect on the cell-wall is indirect. Hemofungin significantly reduced
mortality rates of larvae infected with A. fumigatus, in a dose-depen-
dent manner without signs of toxicity. Several additional findings
strengthened our hypothesis that ferrochelatase is the target of hemo-
fungin- (i) addition of the end-product hemin canceled inhibition by
hemofungin in A.fumigatus in a dose-dependent manner. (ii) enzymes
in the hemin biosynthetic pathway were strongly up-regulated in A.
fumigatus treated with hemofungin (iii) HPLC analysis revealed higher
levels of protoporphyrin in A. fumigatus treated with hemofungin.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 195
Poster Presentations

conformations are important in the catalytic mechanism. The DM bound of the 95% CI (-7.8, 5.7) for the adjusted treatment difference
simulations results show that systems apparently remained stable was lower than the NIM of 10%. ACM through day 42 in the mITT
over the course of 10 ns. By docking simulation, the small molecules population was 19.6% (isavuconazole) and 23.3% (voriconazole).
that interact with the models were ranked. Finally, eleven molecules ACM through day 84 in the ITT population was 29.1% (isavucona-
were selected as putative inhibitors of Trr1. Two compounds (LMM5 zole) and 31.0% (voriconazole) and in the mITT population was
and LMM11) showed antifungal activity in vitro against the patho- 30.1% (isavuconazole) and 37.2% (voriconazole). Overall Response
genic fungi. The MIC values of LMM5 ranging from 8 to 32 lg ml
1 rates at EOT in the mITT population were 35.0% (isavuconazole)
against Candida species. For Cryptococcus species the MIC values were and 36.4% (voriconazole). Treatment-emergent adverse events (AE)
32 lg ml
1 and to Paracoccidiodides species was 4 lg ml
1. In other were reported in 96.1% (isavuconazole) and 98.5% (voriconazole) of
hand, the MIC values of LMM11 ranging from 32 to 64 lg ml
1 patients. The most common AEs (i.e. nausea, vomiting, pyrexia, and
against Candida species. For Cryptoccocus sp. and Paracoccidiodes spp. diarrhea) were reported at similar rates between treatment groups.
the MIC values were the same observed to LMM5. These compounds Drug-related AEs were reported in 42.4% (isavuconazole) and 59.8%
not showed in vitro cytotoxicity on HeLa cells. The in vivo results are (voriconazole) of patients. Fewer (P < 0.05) AEs were reported in the
promising for LMM5 compound which showed a significant reduc- isavuconazole treatment group in the System Organ Classes of Skin
tion in CFU in the kidneys of mice treated with this compound, when (33.5% v 43.5%), Eye (15.2% v 26.6%) and Hepatobiliary Disorders
compared to animals that received only PBS control group in experi- (8.9% v 16.2%).
mental candidemia (Figure B). Conclusions Isavuconazole is effective for the primary treatment of
Conclusion One of compounds showed in vitro and in vivo activity invasive fungal disease caused by Aspergillus species or other filamen-
antifungal against important human pathogenic fungi, thereby creat- tous fungi. Isavuconazole was well tolerated relative to voriconazole,
ing additional perspectives for innovation and technological develop- with fewer study drug related AEs and AEs of the Skin, Eye and
ment of antifungal drugs. Hepatobiliary system.
(Encore abstract: Previously presented at the European Congress of
Clinical Microbiology and Infectious Disease [ECCMID], Barcelona,
Spain; May 10–13, 2014; O230a).
P404

Phase 3 Randomized, Double-Blind Trial Evaluating P405

Isavuconazole vs. Voriconazole for Primary Treatment of
Invasive Fungal Disease Caused by Aspergillus Spp. or
Other Filamentous Fungi (SECURE) Outcomes in Patients with Invasive Mold Disease Caused
J. A. Maertens,1 T. Patterson,2 G. Rahav,3 D. P. Kontoyiannis,4 by Fusarium or Scedosporium spp. Treated with
K. Marr,5 R. Maher,6 M. Lee,7 B. Zeiher6 and A. Ullmann8 Isavuconazole: Experience from the VITAL and SECURE
1 Trials
University Hospitals Leuven, Leuven, Belgium; 2The University of
O. A. Cornely,1 L. Ostrosky-Zeichner,2 G. Rahav,3 R. Maher,4
Texas Health Science Center at San Antonior, San Antonio, USA;
3
Sheba Medical Center, Tel Hashomer, Israel; 4The University of B. Zeiher,4 M. Lee5 and J. Perfect6
1
Texas MD Anderson Cancer Center, Houston, TX, USA; 5Johns University Hospital of Cologne, Cologne, Germany; 2University
Hopkins University, Baltimore, USA; 6Astellas Pharma Global of Texas, Houston, USA; 3Sheba Medical Center, Tel Hashomer,
Development, Inc, Northbrook, USA; 7Astellas Global Pharma Israel; 4Astellas Pharma Global Development, Inc, Northbrook,
Development, Inc., Northbrook, USA and 8University Hospital of USA; 5Astellas Global Pharma Development, Inc., Northbrook,
Wuerzburg, Wuerzburg, Germany USA and 6Duke University, Durham, USA

Objectives Isavuconazole is a novel triazole with broad-spectrum Objectives Isavuconazole is a novel, broad-spectrum, triazole anti-
antifungal activity in vitro and is offered in an IV and bioequivalent fungal developed for the treatment of invasive fungal diseases. Fusar-
oral formulation. The objectives of this randomised, double-blind, ium and Scedosporium spp. are associated with high mortality in
multinational study were to assess efficacy and safety of isavucona- immunocompromised patients; however, treatment options are lim-
zole versus voriconazole in patients with invasive fungal disease ited. We report outcomes in a subset of patients enrolled in the
(IFD) caused by Aspergillus spp. or other filamentous fungi. VITAL and SECURE trials with invasive mold disease (IMD) caused
Methods Patients meeting the EORTC criteria for proven/probable/ by these pathogens.
possible disease were randomized 1:1 to isavuconazole or voricona-
zole. Isavuconazole patients received 200 mg IV TID for 2 days, fol-
lowed by 200 mg QD (either IV or oral). Voriconazole patients
received 6 mg kg
1 IV BID on day 1, then 4 mg kg
1 IV BID on day Table 1. Patient demographics and outcomes.
2, then either 4 mg kg
1 IV BID or 200 mg PO BID. Study drug
could be administered for up to 84 days. The primary efficacy end-
point was non-inferiority for day 42 All-Cause Mortality (ACM) in
the ITT population based on a pre-specified non-inferiority margin
(NIM) of 10%. The key secondary efficacy endpoint was Overall
Response at end of treatment (EOT) as determined by an independent
blinded data-review committee (DRC) in the mITT population. The
mITT population included patients with proven or probable disease
as determined by the DRC.
Results 527 patients were randomized, 516 (258 per group)
received at least one dose of study drug and comprised the ITT popu-
lation. The mITT population included 143 isavuconazole and 129
voriconazole patients (85% aspergillosis). Baseline characteristics
were balanced between treatment groups; 92% pulmonary involve-
ment, 84% haematologic malignancies, 65% neutropaenic and 20%
allogeneic haemotopoetic stem-cell transplantation. ACM through
day 42 for the ITT population was 18.6% (isavuconazole) and 20.2%
(voriconazole). The primary objective was achieved since the upper

ª 2015 The Authors

196 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Methods VITAL and SECURE were Phase 3 trials that evaluated effi- Table 1
cacy and safety of isavuconazole treatment in patients with IMD.
Dosages were isavuconazole 200 mg TID for 2 days followed by
200 mg QD (IV or PO). Proven/probable IMD (EORTC/MSG criteria),
and overall response at end of treatment (EOT) were determined by
independent, data-review committees. Mortality rates, safety and tol-
erability were also analyzed.
Results Demographics and outcomes data are shown in Table A1.
Conclusions Isavuconazole may be a promising treatment for
patients with invasive Fusarium or Scedosporium spp. infections. Data
are limited for patients with these infections and require further
investigation.
ClinicalTrials.gov Identifiers VITAL (NCT00634049) and SECURE
(NCT00412893).
(Encore abstract: Previously presented at the 54th Interscience
Conference on Antimicrobial Agents and Chemotherapy [ICAAC];
Washington, DC, USA; September 5–9, 2014; M-1760).

P406

Primary treatment of invasive mucormycosis (IM) with

isavuconazole (VITAL Study) or amphotericin formulations
(FungiScopeTM): case matched analysis
M. J. G. T. Vehreschild,1 J. J. Vehreschild,1 F. M. Marty,2
J. Perfect,3 L. Ostrosky-Zeichner,4 G. Rahav,5 B. Zeiher,6 M. Lee,7
R. Maher,6 C. Lovell,6 M. Engelhardt8 and O. A. Cornely1
1
University Hospital of Cologne, Cologne, Germany; 2Brigham
and Women’s Hospital, Boston, USA; 3Duke University, Durham, on this matched case comparison of mortality rates from the VITAL
USA; 4University of Texas, Houston, USA; 5Sheba Medical study and FungiScope[TRADEMARK].
Center, Tel Hashomer, Israel; 6Astellas Pharma Global (Encore abstract: Previously presented at the American Society of
Development, Inc, Northbrook, USA; 7Astellas Global Pharma Hematology [ASH] annual meeting; San Francisco, CA, USA; Decem-
Development, Inc., Northbrook, USA and 8Basilea Pharmaceutica ber 6–9, 2014; 1151).
International Ltd, Basel, Switzerland

Objectives Invasive fungal diseases (IFD) are on the rise due to the
increasing numbers of immunosuppressed patients including those P407
undergoing high-dose chemotherapy and haematopoietic stem-cell
transplantation. Isavuconazole is a novel, broad-spectrum antifungal
triazole, available as a water-soluble prodrug in IV and oral formula- Successful Outcomes in Patients with Invasive Fungal
tions. IM is a life-threatening IFD with significant mortality and lim- Disease due to C. gattii and C. neoformans Treated with
ited treatment options. Isavuconazole: Experience from the VITAL Trial
Methods The VITAL study was a Phase 3, multicentre, open-label, F. Queiroz-Telles,1 O. A. Cornely,2 J. Perfect,3 L. Kovanda,4
single-arm trial conducted to evaluate efficacy and safety of isavu- B. Zeiher4 and J. Vazquez5
conazole treatment in patients with rare IFD, including IM. Eligibility 1
Hosp Clınicas, Univ Fed Parana, Curitiba, Brazil; 2University
criteria and evaluated outcomes are outlined in clinicaltrials.gov, Hospital of Cologne, Cologne, Germany; 3Duke University,
NCT00634049. Patients received IV or PO isavuconazole 200 mg
Durham, USA; 4Astellas Pharma Global Development, Inc,
TID for 2 days followed by 200 mg day
1. FungiScope[TRADE-
Northbrook, USA and 5Georgia Regents Univ, Augusta, USA
MARK] - Global Emerging Fungal Infection Registry maintains a glo-
bal web-based database on rare IFD, including IM. Entrance criteria
are outlined in clinicaltrials.gov, NCT01731353. Twenty-one Objectives Cryptococcosis is associated with significant morbidity
patients from the VITAL study who received isavuconazole for the and mortality. Isavuconazole is a novel, broad-spectrum, triazole
primary treatment of proven/probable IM were matched (blinded to antifungal agent (IV and PO) developed for treatment of invasive fun-
mortality status) to up-to three proven/probable patients with IM gal diseases (IFD). It displays potent activity in vitro against Crypto-
who received a formulation of amphotericin B entered in FungiScope coccus spp. We report outcomes in a subset of patients enrolled in the
[TRADEMARK] based on three dichotomous risk factors: severe dis- VITAL trial with cryptococcosis.
ease (i.e. CNS/disseminated), surgical debridement, and haematologic Methods VITAL was a Phase 3, open-label, multi-center trial con-
malignancy. All-cause mortality through day 42 was summarised. ducted to evaluate efficacy and safety of isavuconazole in patients
Results Thirty-three FungiScope[TRADEMARK] matched controls with emerging IFD. Patients received isavuconazole 200 mg TID for
were identified; 14 VITAL cases were matched to a single control 2 days followed by 200 mg QD (IV or PO). Proven/probable IFD
each (n = 14), two VITAL cases were matched to two controls each (EORTC/MSG criteria) and overall response at end-of-treatment (EOT)
(n = 4), and five VITAL cases were matched to three controls each were determined by an independent, data-review committee. Mortal-
(n = 15). Demographics, treatments, matching criteria and mortality ity and safety were also assessed.
outcomes are shown in Table 1. The crude mortality rate (33.3%) Results Nine patients were treated with isavuconazole for cryptococ-
through day 42 from the VITAL cases was similar to the mortality cosis. The pathogens isolated were Cryptococcus neoformans (n = 4)
rate (39.4% crude; 41.3% weighted) from the matched controls of and C. gattii (n = 3); one patient had histological evidence only, and
FungiScope[TRADEMARK]. one had positive antigen testing only. Three patients had isolated
Conclusions Isavuconazole was as effective as amphotericin B for- pulmonary disease and two had central nervous system (CNS) dis-
mulations in the primary treatment of invasive mucormycosis based ease. The remaining patients had disseminated disease in the lung,

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 197
Poster Presentations

CNS, and/or blood or other organ. CLSI MICs were available for
seven patients and ranged from 0.008 to 0.12 mg l
1. Mean dura-
tion of therapy was 132 days (range 6–182 days). Six patients had
primary therapy with isavuconazole; two were intolerant to ampho-
tericin B (AmB; alone or with fluconazole), and one was refractory to
AmB + fluconazole. Eight patients were alive through ≥84 days: six
were treatment successes (two complete, four partial) and two were
treatment failures (both stable) at EOT. All patients with C. gattii
infection were treatment successes. One of the stable patients was
switched to amphotericin B + 5-flucytosine at Day 25. One of the
nine patients died on Day 7 and was considered a treatment failure
(progression) at EOT. Adverse events (AEs) and drug-related AEs
were experienced by eight and three patients, respectively.
Conclusions Cryptococcosis, caused by either C. neoformans or C.
gattii, was successfully managed in this small group of patients, sup-
porting further research into this disease.
ClinicalTrials.gov Identifier Figure 1. Comparison of Median LOS days for ITT patients receiving
NCT00634049 (Encore abstract: Previously presented at the Inter- Isavuconazole vs. Voriconazole, by Age, BMI and Renal Impairment
science Conference on Antimicrobial Agents and Chemotherapy categories.
[ICAAC] annual meeting, Washington, DC, USA; September 5–9,
2014; M-1773)
Conclusions Median LOS days was found to be lower for patients
treated with isavuconazole vs voriconazole; this difference was statis-
tically significant in patients with renal impairment.
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and N. Azie2 aspergillosis: disease spectrum, treatment practices, and outcomes. I3
1 aspergillus study group. Medicine (Baltimore) 2000; 79: 250–60.
Basilea Pharmaceutica International Ltd, Basel, Switzerland and
2 (Encore abstract: Previously presented at IDWeek, Philadelphia,
Astellas Pharma Global Development, Inc, Northbrook, USA
PA, USA; October 8–12, 2014; A826).
Objectives Invasive aspergillosis (IA) is an emerging clinical problem
and remains an important complication especially among immuno-
compromised patients. IA is associated with significant increases in
morbidity, mortality, length of hospitalization stay (LOS) and costs1. P409
Case fatality is estimated to be at 60% in immunocompromised popu-
lations2-4. Despite improved treatment options and advances in diag- An open-label phase 3 study of isavuconazole (VITAL):
nostic testing, patient outcomes remain suboptimal. The SECURE focus on mucormycosis
trial was a Phase III, double-blind, randomized, multi-center, non-in-
F. M. Marty,1 J. Perfect,2 O. A. Cornely,3 K. M. Mullane,4
feriority study of isavuconazole versus voriconazole. Patients
>18 years of age, who had proven, probable or possible invasive fun- G. Rahav,5 M. Lee,6 M. Ito,7 R. Maher,7 B. Zeiher7 and
gal disease caused by Aspergillus species or other filamentous fungi L. Ostrosky-Zeichner8
1
were randomized 1:1 to receive isavuconazole or voriconazole. Brigham and Women’s Hospital, Boston, USA; 2Duke University,
Methods Primary objective of this analysis was to compare initial Durham, USA; 3University Hospital of Cologne, Cologne,
LOS during hospitalization and 30-day all-cause hospital readmission Germany; 4University of Chicago Medicine, Chicago, USA;
rates in patients who received isavuconazole or voriconazole. Out- 5
Sheba Medical Center, Tel Hashomer, Israel; 6Astellas Global
comes in subgroups of interest by age, Body Mass Index (BMI) and Pharma Development, Inc., Northbrook, USA; 7Astellas Pharma
renally-impaired patients (eGFR-MDRD category <60 ml/min/
Global Development, Inc., Northbrook, USA and 8University of
1.73 m2) were conducted. Ratio of total days on IV over total num-
ber of days of (IV + oral) therapy for study drugs and total number
Texas, Houston, USA
of additional days on potentially mould-active systemic antifungal
therapy after end of study treatment were also analyzed. Objective Isavuconazole is a novel, broad-spectrum triazole antifun-
Results A total of 516 patients were included in the Intent-to-Treat gal, available as a water-soluble prodrug in IV and oral formulations,
(ITT) group. Median LOS was calculated with descriptive statistics for the treatment of invasive fungal disease (IFD). The objective of
(13.0 vs 15.0 days) and using Kaplan–Meier estimates (15.0 vs 16.0 this analysis is to report the overall response, survival, and safety in
days, P = 0.607) for isavuconazole vs voriconazole, respectively. The a subset of patients with invasive mucormycosis (IM) who were trea-
30-day readmission rate for isavuconazole compared to voriconazole ted with isavuconazole.
patients was 18.3% vs. 24.4% (P = 0.114), respectively. Ratio of Methods VITAL was a Phase III, multicenter, open-label trial con-
days on IV formulation to total days of (IV + oral) therapy were simi- ducted to evaluate safety and efficacy of isavuconazole treatment in
lar (isavuconazole 0.38 [SD 0.39]; voriconazole 0.38 SD [0.38]). patients with rare IFD. Eligibility criteria and evaluated outcomes are
Median additional days on potentially mould-active systemic anti- outlined in clinicaltrials.gov, NCT00634049. Patients received IV or
fungal therapy were comparable isavuconazole vs voriconazole PO isavuconazole 200 mg TID for 2 days followed by 200 mg day
1
(32.0 days vs. 33.0 days). Median LOS days were similar across vari- until day 180, end of treatment (EOT). An independent data review
ous subgroups (Figure 1), except in renal impaired subgroup where committee (DRC) categorized patients as having proven or probable
the difference was statistically significant in favor of isavuconazole IFD by EORTC/MSG criteria. DRC-assessed overall response at EOT,
(isavuconazole 9.0 days; voriconazole 19.0 days, P = 0.0032). survival, and adverse events (AEs) using standard definitions are
reported for patients with proven/probable IM.

ª 2015 The Authors

198 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Table 1. Baseline demographics, study outcomes, and survival. activity was investigated on 13 clinical isolates of Candida spp. C. albi-
cans (n = 5) C. kefyr (n = 2), C. krusei (n = 2), C. glabrata (n = 2), C.
lusitaniae (n = 2) and 4 Trichophyton spp, T. rubrum (n = 2) and T.
mentagrophytes (n = 2) by a disk diffusion assay. In vivo efficacy of
the econazole imprinted textile was evaluated on a murine cutaneous
candidiasis model using econazole cream as reference and the textile
alone as placebo.
Results In vitro antifungal disk diffusion tests showed a comparable
activity to commercial formulation on all Candida species and slightly
lower on dermatophytes species. Nine days post-infection, the retro-
cultures of biopsy samples demonstrated an effective infection in the
control with high density of Candida albicans. In contrast, the treat-
ment with the econazole imprinted textile lead to a high reduction of
the yeast cutaneous burden. This reduction was less important in the
group treated by econazole cream.
Conclusions Overall, the econazole imprinted textile exhibited a sig-
nificant in vitro antifungal activity against Candida species and Tri-
chophyton. These results showed that this technology is promising to
develop pharmaceutical textiles for the treatment of fungal superficial
infections. Furthermore this technology could improve patient com-
pliance while maintaining drug activity.

P411

Propolis potential activity against Candida tropicalis

adhered cells and its biofilms
F. K. Tobaldini,1 P. S. Bonfim-Mendonc
a,2 H. C. Rosseto,2
M. L. Bruschi,2 M. Henriques,3 J. Azeredo,3 M. Negri,2 S. Silva3
and T. I. E. Svidzinski2
1
Universidade Estadual de Maringa/Universidade do Minho,
Maringa, Brazil; 2Universidade Estadual de Maringa, Maringa,
Brazil and 3University of Minho, Braga, Portugal

Objectives Invasive fungal infections, such as candidiasis, represent

a public health problem of major importance and Candida tropicalis
has been highlighted among the main agents of candidiasis. Adhe-
Results Overall 37 patients with IM received isavuconazole for a med- sion, which is the attachment of microbial cells to biological or non-
ian of 84 days (range 2–882). Baseline demographics, study outcomes, biological substrates, has a critical role in the initial stages of biofilm
and survival are shown in Table 1. Survival at 180 days was 53%. formation. Additionally, one of the major contributions to C. tropicalis
Overall 95% of patients experienced an AE, 76% experienced a serious virulence is its versatility in adapting to a variety of different habitats
AE (SAE). Only 3 (8%) SAEs were attributed to isavuconazole. and the formation of microbial communities known as biofilms.
Conclusion Isavuconazole appears to be a safe and promising agent Moreover, from the clinical perspective, the most important feature of
for the treatment of mucormycosis in a particularly high-risk Candida biofilms is its intrinsic tolerance to conventional antifungal
immunocompromised population. therapy. This scenario encourages the search for alternative thera-
(Encore abstract: Previously presented at IDWeek, Philadelphia, pies. Natural matrixes, such as propolis, compromise a multitude of
PA, USA; October 8–12, 2014; A824) bioactive properties, in particular phenolic extracts have evidenced
significant antimicrobial properties against a multiple of opportunist
invaders, including Candida species. Thus, the main objective of the
present work was to evaluate the potential antifungal effect of propo-
P410 lis against Candida tropicalis adhered and biofilm cells.
Methods This study was conducted with four clinical strains of C.
tropicalis and one reference strain, from the American Type Culture
Antifungal activity of a new econazole imprinted textile Collection (ATCC 40042). Adhesion and biofilm formation were car-
P. Le Pape,1 M. A. Hossain,2 F. Pagniez,3 P. Simard4 and J. Chain5 ried out on 96-well microplates containing a cellular suspension of
1 1 9 105 cells ml
1 and incubated for 2 h and 24 h, respectively at
CHU de Nantes, Nantes, France; 2University of Montreal,
37 °C. Adhered cells and pre-formed C. tropicalis biofilms were trea-
Montreal, Canada; 3University, Nantes, France; 4Biomod
ted with propolis (ranging from 0.47 to 1.42 mg ml
1), during 24 h
Concepts Inc, Sainbte-Julie, Canada and 5University of Montreal, at 37 °C and its effect assessed through quantification of the number
Montreal, Canada of colony forming unit (CFU).
Results It was evident that all C. tropicalis strains tested were able to
Objective Fungal skin infections are widespread, potentially conta- adhere and form biofilm and that propolis was able to reduce around
gious and have important economic consequences. Failure in treat- 2.5 logs (P < 0.05) and 3 logs (P < 0.05) on adhered cells and pre-
ment is most often due to low patient compliance, since antifungal formed biofilm, respectively. Moreover, in general the propolis effect
cream should be applied daily for several weeks or months. In this was similar among all the C. tropicalis clinical isolates.
work, the antifungal activity of a textile imprinted with solid lipid Conclusions The results of this study are promising, considering
microparticles of econazole (ECN) was investigated. that biofilms are difficult to control by conventional antibiotherapy,
Materials Solid lipid microparticles containing econazole were propolis can be considered an efficient and safe antifungal alternative
imprinted on textiles using a screen-printing method. Antifungal strategy to combat yeast biofilm associated infections.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 199
Poster Presentations

P412 absence of cytotoxic effect in the MIC concentration (64 lg ml
1) for
24 h. Therefore, the virtual screening of chemicals libraries offered
alternative for new antifungal agents development against human
Antifungal activity of compound selected by virtual pathogens. Then, this study showed that CP1 is capable of killing P.
screening against chorismate synthase from brasiliensis in vitro and lower cytotoxicity to human cells. Thus, our
Paracoccidioides brasiliensis results show great therapeutic potential for compound CP1 owing to
F. A. V. Rodrigues, E. S. Miazima, P. S. Bonfim-Mendonc
a, its important antifungal activity in vitro.
F. A. Seixas, M. Negri, T. I. E. Svidzinski and E. S. Kioshima
Universidade Estadual de Maringa, Maringa, Brazil
P413
The aim of the present work was to evaluate the CP1 antifungal
activity with focus in the development of effective and safe antifun-
gal. Firstly, the minimal inhibition concentration (MIC) determina- In vitro antifungal activity of two perylene bisimide
tion was performed to P. brasiliensis (five isolates) and P. lutzii (Pb01 derivatives and a dehydroabietylamine against Fusarium
isolate), according to M27-A3 guidelines (CLSI) with modifications. and Scytalidium species causing onychomycosis
The CP1 was prepared in a range of concentrations from 64 to A. C. Mesa-Arango,1 Y. Figueroa-Vargas,1 S. Flo rez-Mun
~oz,1
0.5 lg ml
1, diluted in DMSO and Amphotericin B ranging from 16 M. Gallego2 and M. Gonzalez3
to 0.03 lg ml
1. The yeast cells were collected aseptically in sterile 1
saline (0.85%). The inoculum suspensions were diluted in RPMI Group of Investigative Dermatology,School of Medicine,
1640 medium (the final concentration was 0.5 to 1 9 104 University of Antioquia, Medellin, Colombia; 2Medical Mycology
cells ml
1). The plates were incubated 15 days at 35 °C. The MIC Group, School of Medicine, University of Antioquia, Medellin,,
endpoint was defined as the lowest concentration of amphotericin B Medellin, Colombia and 3University of Valencia, Valencia, Spain
and CP1, showing 90% and 100% growth inhibition, repectively.
The in vitro fungicidal and fungistatic activities were determined for Objective To evaluate the antifungal activity of two perylene bisi-
CP1 after 15 days by subculture in brain heart infusion (BHI) agar mide derivatives and a dehydroabietylamine against Fusarium and
plates, at 37 °C. The CP1 cytotoxicity was performed in HeLa, J774 Scytalidium species causing onychomycosis.
macrophages and HUVEC cells, using the CellTiter 96 assay (Pro- Methods In vitro antifungal activity of twoperylene bisimide deriva-
mega, Madison, WI, USA), based on the reduction of MTS (3-[4,5- tives (72M and 73M) and a dehydroabietylamine (81M) was tested.
dimethylthiazol-2-yl]-5-[3- carboxymethoxyphenyl]-2-[4-sul- Molecules were dissolved in DMSO, and five concentrations 25, 12.5,
fophenyl]-2H-tetrazolium). The Chorismate Synthase (CS) has been 6.25, 3.125 and 1.56 lg ml
1 were evaluated. Antifungal activity of
shown to be an excellent target for antifungal drug development, as the compounds was determined following the European Committee on
it is involved in a metabolic pathway that is present only in plants, Antibiotic Susceptibility Testing Subcommittee on Antifungal Suscepti-
fungi and bacteria. By in silico methodologies, a new compound, bility Testing protocols. Fifteen clinical isolates of Fusarium spp, an
which acts as CS inhibitor from Paracoccidioides brasiliensis, was ATCC strain (F. oxysporum ATCC 48112) and six clinical isolates of of
selected by virtual screening. The dynamic molecular simulation Scytalidium spp were evaluated. The minimum inhibitory concentra-
showed a strong interaction with this enzyme from P. brasiliensis. tion (MIC) was defined as the lowest compound concentration that
This thermally dimorphic fungus causes Paracoccidioidomycosis resulted in total inhibition of growth. A molecule was considered
(PCM) is a systemic granulomatous disease. It is a prevalent systemic active with a MIC value of ≤25 lg ml
1. Plates with inocula and com-
mycosis in Latin America which requires prolonged antifungal treat- pounds were incubated at 28 °C for 48 h. The minimum inhibitory
ment. These therapies have several limitations, such as drug interac- concentration tests were performed by duplicate in two different
tions, infusion-related events and nephrotoxicity. Therefore, the assays. As a positive control, itraconazole (Sigma) and terbinafine (Re-
development of drugs that act selectively in target pathogenic fungi calcine Laboratories, Santiago de Chile, Chile), in concentration ranges
without producing collateral damage to mammalian cells is a daunt- from 16 to 0.5 lg ml
1 and from 4 to 0.5 lg ml
1, respectively, eval-
ing pharmacological challenge. All isolates tested were sensible the uated with Fusarium oxysporum ATCC 48112, were included. Also, all
amphotericin B, with MIC values ranging from 0.5 to 1 lg ml
1 and clinical isolates were evaluated with these two antifungals.
for CP1 32 to 64 lg ml
1. Regarding, the fungicidal activities of the Results OverallScytalidium spp isolates were more sensitive to the
CP1 is illustrated in Figure 1. The CP1 demonstrated to have fungici- three molecules (MIC values range 6.25–12.5 lg ml
1) than Fusar-
dal activity against P. brasiliensis. The cytotoxicity assays in J774 ium spp (6.25 to ≥ 25 lg ml
1). Also, MIC values with terbinafine
macrophages, HUVEC and HeLa cells exposed to CP1 revealed were lower for Scytalidium spp (MIC value range 0.5 to ≥4 lg ml
1),
while for Fusarium spp, MIC value was ≥4 lg ml
1 in all cases. The
susceptibility to itraconazole was low in all isolates evaluated (MIC
values ≥16 lg ml
1). Dehydroabietylamine 81M was active against
19/21 isolates (MIC value range 12.5 to 25 lg ml
1) and with F.
oxysporum ATCC 48112 (6.25 lg ml
1). On the other hand, pery-
lene bisimide derivative 73M was more active against Scytalidum spp
(MIC value 6.25 lg ml
1), but Fusarium spp susceptibility was strain
dependent (MIC value range 6.25 to ≥25 lg ml
1). Perylene bisimide
derivative 72M was active against Scytalidum spp (MIC value
12.5 lg ml
1), but it was not active against 11 Fusarium spp isolates
(MIC value ≥25); the other isolates showed an MIC value of
25 lg ml
1.
Conclusions Although onychomycosis is most frequently caused by
dermatophytes and Candida spp., filamentous fungi such as Fusarium
spp and Scytalidium spp are also increasing as a cause of this infec-
tion in tropical countries. Species of these genera are highly resistant
to antifungals used in onychomycosis treatment. In addition, some
Fusarium species are phytopathogenic. Therefore, identifying active
molecules against these fungi could be important for both human
health and treatment of plant infections.
Results indicate that mainly perylene bisimide derivative 73M and
dehydroabietylamine 81M have important antifungal activity in vitro,

ª 2015 The Authors

200 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

suggesting that these molecules are candidates for further studies of bacterial lysate. Trr1 was purified with high purity and presented
toxicity on skin cell lines, determination of efficacy in animal models specific enzymatic activity. Structural changes of the protein were
and mechanisms of action. observed at different pHs and temperatures, showing high thermal
stability at pH’s 7.0 and 8.0. The administration of Trr1 recombinant
increased titers of the Trr1-specific antibody (Figure A) and appeared
to induce protective immunity against C. albicans, since reduced
P414 number of CFUs in kidney as compared with the control (Figure B).
Conclusion Therefore, our results demonstrated the facility to
obtaining with the heterologous expression, high stability and effi-
Thioredoxin reductase from Candida albicans as a cacy in the immunogenic response of thioredoxin reductase from C.
promising immunogen against candidiasis albicans,narrowing our search for a probable and promising antifun-
J. S. R. Godoy,1 A. K. R. Abadio,2 P. S. Bonfim-Mendonc
a,1 gal target, as well as candidate for a vaccine against candidiasis.
M. S. S. Felipe,3 S. M. Freitas,3 T. I. E. Svidzinski1 and
E. S. Kioshima1
1
Universidade Estadual de Maringa, Maringa, Brazil; 2University
from the State of Mato Grosso, Nova Xavantina, Brazil and
P415
3
Universidade de Brasılia - UNB, Brasılia, DF, Brazil
Anti-Candida albicans activity of acetonic phenols-enriched
Objective The aim of this work was the heterologous expression of fraction of Buchenavia tomentosa extract and gallic acid
thioredoxin reductase from Candida albicans in the Escherichia coli sys- G. R. Teodoro,1 A. V. Gontijo,1 A. C. Borges,2 A. C. B. Delbem,1
tem, evaluate its structural stability at different temperatures and pH, F. L. Brighenti,1 M. J. Salvador3 and C. Y. Koga-Ito4
contributing to an potential antigen candidate to vaccine develop- 1
Universidade Estadual Paulista - UNESP, Sa~o Jose dos Campos,
ment against candidiasis. Since the vaccination could be an impor- Brazil; 2ICT-UNESP, Sa~o Jose dos Campos, Brazil; 3Universidade
tant complementary strategy to pharmacotherapy. Estadual de Campinas - UNICAMP, Campinas, Brazil and
Methods The gene coding for the thioredoxin from Candida albi- 4
Universidade Estadual Paulista UNESP, Sa~o Jose dos Campos,
cans was cloned into pET21a expression vector, achieving the best
expression condition. The protein with high purity was subjected to
Brazil
enzymatic assays using 5,50 -dithiobis(2-nitrobenzoate) (DTNB) and
thioredoxin from Candida albicans (Trx1-Ca) and NADPH as a sub- Objectives The aim of this study was to in vitro evaluate the anti-
strate and cofactors, respectively. Then, the secondary structures and Candida albicans activity of acetonic phenols-enriched extract (FE)
protein stability at different pHs and temperatures were evaluated by from B. tomentosa leaves and gallic acid (GA). Their influence on vir-
circular dichroism (CD) and the study the intrinsic fluorescence infor- ulence factors was also assessed.
mation was obtained for the structure of the native protein and its Methods Minimal inhibitory (MIC) and minimal fungicide concen-
possible conformational changes by changes in pH, temperatures and trations (MFC) of FE and GA were determined for C. albicans reference
binders. Trr1 recombinant was administrated to mice by subcuta- strains (ATCC 18804, SC 5314) and 29 clinical isolates by EUCAST
neous injection before challenging them with a lethal dose of C. albi- methodology. Pre-formed 24, 48 and 72 h biofilms of reference
cans. After vaccination of Trr1, antibody responses were observed. To strains and 3 clinical isolates were exposed to concentrations of 2, 4
evaluate the vaccination effect of Trr1, 5 days after the challenge and 10 times MIC to evaluate the eradication potential. The effect of
they were sacrificed and the number of viable C. albicans cells in the the presence of sub-inhibitory concentrations of FE and GA on the
kidney and spleen were determined by number of CFUs. development of biofilms for 24, 48 and 72 h was assessed. The
Results Thioredoxin reductase (Trr1) is a flavoprotein widely dis- effects on the production of hydrolytic enzymes (proteinase and phos-
tributed that catalyzes the NADPH-dependent reduction of thiore- pholipase) and adherence to buccal cells were also studied. All the
doxin, which compose the thioredoxin system. This system plays a experiments were performed in triplicate. The results of effect on bio-
critical role in maintaining the cytoplasm redox state, participating film viability (expressed in number of fungal cells) and adherence to
in important functions to the cellular viability this fungus. Therefore, buccal cells (expressed in number of yeast cells adhered to 25 cells)
this system has proven not only to be a promising target for the were compared by ANOVA and post hoc Tukey’s test at level of sig-
development of new antifungal drugs using rational approaches, nificance of 5%. For production of hydrolytic enzymes, the results
against problems as few therapeutic options and the emergence of were expressed by Pz values ranges.
resistant strains to antifungal agents, as well as candidate for a vac- Results MIC values ranged from 10 to 1.25 mg ml
1 to FE and 10
cine against candidiasis. The heterologous Trr1 was efficiently to 2.5 mg ml
1 to GA. No fungicide activity was detected. Gallic acid
expressed in Escherichia coli, resulting a protein with molecular and FE were able to promote significant reduction in the number of
weight (Mr) of approximately 35 kDa, in the soluble fraction of the viable cells of pre-formed 24 h biofilm (P = 0.021 and P = 0.034,
respectively). The presence of sub-inhibitory concentrations of gallic
acid reduced significantly the development of biofilms of 24
(P = 0.008) and 48 h (P = 0.002). No effect on proteinase (Pz range:
0.22–0.58) and phospholipase (Pz range: 0.36–0.54) was found. Sig-
nificant reduction in the adherence ability was detected after exposi-
tion to FE (P < 0.001) and GA (P < 0.001).
Conclusion Acetonic phenols-enriched fraction of Buchenavia tomen-
tosa extract and gallic acid showed promising anti-Candida albicans
activity, with inhibitory effect on biofilm and cell adherence.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 201
Poster Presentations

P416 method and performing susceptibility tests against PES, SPES and
conventional antifungal agents. The anti-biofilm effect and cytotoxic-
ity tests of the PES and SPES were evaluated.
Evaluation of propolis and its subproduct as an inhibitor Results In 38.48% (112/291) of culture was positive for Candida
of growth and biofilm formation in vaginal yeast from species. There were patients with two different species, being a total
pregnant women of 115 yeasts (82.61% C. albicans; 6.08% C. glabrata; 5.22% C. tropi-
L. B. Moraes,1 T. P. Salci,1 P. S. Bonfim-Mendonc
a,1 calis; 5.22% C. parapsilosis and 0.87% C. krusei). PES and SPES were
F. K. Tobaldini,2 L. A. S. Toledo,1 M. Negri,1 M. L. Bruschi1 and effective, even against isolates resistant to conventional antifungal
(Table 1) and reduced about 25% C. tropicalis biofilm, besides pre-
T. I. E. Svidzinski1
senting its low toxicity in the concentrations of fungicides.
1
Universidade Estadual de Maringa, Maringa, Brazil and Conclusion Thus, in addition to the PES, SPES can also be a promis-
2
Universidade Estadual de Maringa/Universidade do Minho, ing alternative treatment, especially in this population.
Maringa, Brazil

Objectives The treatment of vulvovaginal candidiasis (VVC) is still

unsatisfactory, especially in pregnant women, being promising to the P417
utilization of alternative therapies. Propolis extract solution (PES) has
demonstrated antifungal efficacy and low toxicity. In addition, the
subproduct of propolis extract solution (SPES) is produced during the Antifungal activity and time-kill assay of essential oil from
process of preparing PES and is usually discarded, but can still sub- Cymbopogon nardus against Candida parapsilosis
mit substances responsible for biological effects, such as the polyphe- L. G. Toledo,1 M. A. S. Ramos,2 L. Sposito,2 E. M. Castilho,1
nols, responsible for the therapeutic activity of propolis. SPES have B. G. Almeida,1 A. G. Santos,2 T. M. Bauab2 and
not been investigated or used as an antimicrobial agent. Thus, the M. T. G. Almeida1
objective of the present study was to investigate the effect of PES and 1
Medical School - FAMERP, Sa~o Jose do Rio Preto, Brazil and
SPES on Candida spp. isolated from the vaginal material of pregnant 2
Sa~o Paulo State University - UNESP, Araraquara, Brazil
women.
Methods Vaginal samples from 291 pregnant women were collected
and cultivated for yeasts, which were identified by the classical Objectives Candida parapsilosis has emerged as one of the most
important opportunistic pathogens, and the second most commonly
isolated Candida species from blood cultures in Latin America, Europe,
Canada and Asia. In addition, drug-resistant isolates are emerging
Table 1. Minimum inhibitory concentration (MIC) values (lg ml
1) quickly with the increasing clinical use of conventional drug, con-
to amphotericin B, nystatin, fluconazole, PES and SPES. tributing to the therapeutic failure. Thus, the aim of this study was
to test the antifungal activity of essential oil from Cymbopogon nardus
(EO) against C. parapsilosis ATCC 22019 and clinical isolate.
Methods The minimal inhibitory concentration (MIC) of EO was
determined according to the protocol described by Ara ujo et al., with
modifications. The initial concentration of EO was 2000 lg ml
1.
0.1 mL was placed in a 96-well microtiter plate containing RPMI
1640 medium. Each well was inoculated with 0.1 mL of a suspen-
sion containing 2.5x103 cfu ml
1 of yeast. Amphotericin-B and flu-
conazole were used as controls of the antifungal activity. The plates
were incubated for 48 h at 37 °C. The MIC of sample was detected
following the addition of 0.02 mL 2.0% triphenyltetrazolium chlo-
ride. The time-kill assay was performed according to Zore et al., with
modifications. In brief, Sabouraud broth medium (10 mL) containing
2.5 9 103 cfu ml
1 of C. parapsilosis and 2 9 MIC of EO were

ª 2015 The Authors

202 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Table 1. Inhibitory effects of amphotericin B (AmB) and its salts

(ChAmB and C16PyAmB) and C16PyCl are reported as EC50 values
(lM).

Methods The synthetic route and characterisation of AmB salts will

be reported elsewhere (collaborative work with Prof. Luıs C. Branco).
Aspergillus fumigatus and A. terreus biofilm susceptibility assays (EC50
values determination and checkerboard assay) and cytotoxicity
assays (mIMCD-3) were performed. Expression analysis of AmB-re-
sponsive genes was performed by qRT-PCR.
Results ChAmB and C16PyAmB displayed improved solubility in
incubated and aliquots of 0.4 mL were removed at different time
physiological solution and significantly enhanced antifungal activity
intervals (30 min, and 1, 2, 4, 8, 12, 24 and 36 h), and re-sus-
against A. fumigatus biofilm when compared with AmB (Table 1).
pended in Sabouraud broth medium and 100 lL were inoculated on
Aspergillus terreus showed higher resistance to ChAmB and
Sabouraud agar plates. All plates were incubated at 37 °C for 48 h.
C16PyAmB, which could be seen as a preliminary evidence of the
Amphotericin-B (32 lg ml
1) was used as control. The number of
preserved mode of action. The individual contribution of the ions in
colonies was counted and compared with controls.
C16PyAmB was investigated by combining C16PyCl and AmB in a
Results The results showed EO had effective antifungal activity with
checkerboard assay. Surprisingly, equimolar concentrations of the
MIC of 1000 lg ml
1 (ATCC and clinical isolates), while fluconazole
two compounds caused inhibitory effect similar to AmB alone. There-
and amphotericin-B had MIC >64 lg ml
1 (resistant) and
fore, a possible explanation for the stronger antifungal activity of
8.0 lg ml
1 (resistant), respectively. The time-kill assay curve
C16PyAmB may rely in the fact that AmB lost its zwitterionic nature.
showed that EO killed 100% of the ATCC strain and clinical isolate
We are currently analysing further this hypothesis. In addition, cyto-
within 24 h of exposure (Fig. 1 e Fig. 2).
toxicity of ChAmB and C16PyAmB towards mIMCD-3 cells increased
Conclusion In conclusion, the findings show that EO is a promising
tremendously when compared to AmB and this aspect needs to be
antifungal agent in the treatment of candidiasis caused by C. parap-
carefully considered.
silosis. Furthermore, on time-kill assay, EO exhibit similar or higher
Conclusion Creating new opportunities in antifungal therapy is cru-
antifungal activity than amphotericin-B.
cial for defeating induced resistance occurrence and emergence of
Financial Support Fundac
a ~o de Amparo a  Pesquisa do Estado de
novel fungal pathogens. The concept of converting biologically active
S~
ao Paulo-FAPESP
molecules to a salt form as to improve their physical properties was
explored before. We have herein demonstrated that such alteration
potentiates the antifungal activity of AmB and possibly leads to dual
biological action. A recent mechanistic study which proposed that
P418 large extramembranous AmB aggregates actually extract ergosterol
from lipid bilayer is defying the well-established ion channel model.2
Modulating antifungal activity of amphotericin B through We will attempt molecular dynamics simulation to further elucidate
the possible interaction of AmB salts with ergosterol and we believe
its conversion to a salt form
that our findings can contribute to the better understanding of AmB
M. Petkovic Garcia, D. Hartmann and C. Silva Pereira mode of action, guiding novel synthetical routes for efficient AmB
Instituto de Tecnologia Quımica e Biologica (ITQB) Antonio derivatives.
Xavier, Oeiras, Portugal References 1. Gautam, P. et al., Antimicrob. Agents Chemother. 52,
4220–4227 (2008).
Amphotericin B (AmB) is still the number one choice in invasive 2. Anderson, T. M. et al., Nat. Chem. Biol. 10, 400–6 (2014).
aspergillosis therapy. AmB interacts with ergosterol in the plasma
membrane and its main molecular targets in Aspergillus fumigatus are
ergosterol biosynthesis, oxidative stress response, cell wall mainte-
nance and transport proteins.1 Resistance to AmB has been rarely P419
reported, except for some A. terreus strains.
AmB is insoluble in saline at normal pH and therefore it is usually
administrated with sodium deoxycholate. The major drawback in
Antifugal activity of Brazilian Cerrado Pouteria ramiflora
AmB use is its high toxicity. Three commercialised lipid-AmB formu- plant extract against Candida spp
lations have slower release and therefore reduced toxicity; however, Z. D. Lopes Da Silva, I. M. Teoto ^nio, D. Silveira, Y. Fonseca,
increased cost is also associated. L. Gandolfi, R. Pratesi and Y. K. M. No  brega
Aiming at improved physical and antifungal properties, novel AmB Universidade de Brasılia, Brasılia, Brazil
derivatives are herein proposed, namely salts combining the anionic
form of AmB with several cations, e.g. cholinium (ChAmB) or
cetylpyridinium (C16PyAmB). We focused on A. fumigatus biofilm, as Objectives Invasive candidiasis is recognized as a cause of morbidity
it presents increased resistance to antifungal agents and is the pre- and mortality in tertiary care hospitals worldwide. Although the
vailing growth mode in vivo. The mode of action of these AmB-based majority of candidiasis infection cases are attributed to Candida albi-
salts and their cytotoxicity were also subject of investigation. cans, rates of infection by non-C. albicans species are increasing

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 203
Poster Presentations

worldwide. Additionally, the limited number of available antifungal 0.0070 w/cm2) for MB, green LED light (511 nm, 0.0058 w/cm2)
drugs and the increasing frequency of resistant cases stresses the for RB and blue LED light (460 nm, 0.013 w/cm2) for PpIX and
urgent need for developing new drugs with antifungal activity. In CUR. The resultant suspensions were subcultured onto agar Sabour-
this context compounds originating from medicinal plants become an aud to determine the viable yeasts by colony-forming units counting
important field of research. The genus Pouteria belongs to the family (CFU ml
1). Appropriate control experiments were carried out.
Sapotaceae and shows a widespread distribution around the World, Results APDT with MB reached a reduction of 6 log10 in the num-
being mainly found in tropical and subtropical regions of Asia and ber of CFU/mL of C. albicans using concentrations between 80 and
South America. Species of this genus have shown several biological 160 lg ml
1 with white light and 80 lg ml
1 with red light. In the
activities such as antioxidant, anti-inflammatory, antibacterial and case of C.krusei, the same log reduction was allowed with 320–
antifungal properties. However, the potential of this genus as source 640 lg ml
1 and 320 lg ml
1 of MB with white and red light
of new drugs or phytomedicines remains unknown. Therefore, this respectively. In the case of C.parapsilosis, 160–320 lg ml
1 were
study aim to determine the antifungal activity of Pouteria ramiflora needed either with white and red light.
specie, originated from the Brazilian Cerrado. Conversely, concentrations of RB, CUR or PpIX higher than
Methods Three standard strains from the American Type Culture 2560 lg ml
1 were not enough to show a significant reduction in
Collection (ATCC) of Candida species, Candida albicans (ATCC 40277), any of the experimental conditions.
Candida glabrata (ATCC 40136) and Candida parapsilosis (ATCC Conclusion In vitro PDT-MB has a significant fungicidal effect on
40038) were used in this study. The strains were grown on Sabour- Candida (C. albicans>C. parapsilosis>C. krusei)whereas RB, CUR and
aud dextrose agar at 25 °C for 24 h. The evaluation of the antifun- PpIX were not effective PSs.
gal activity of Pouteria ramiflora extract was performed using the As it was expected, red light is the most efficient, although white
microdilution method M27-A2 broth (CLSI). From pure ethanol light can be also a good option.
extract serial dilutions were realized using a solution containing Acknowledgements This work has been supported by grant
DMSO <1% and Ethanol <10%. The final concentration of the extract CTQ2013-48767-C3-2-R from the Spanish Ministerio de Economıa y
in each well ranged from 50 mg ml
1 to 0.1 mg ml
1. For the Competitividad.
assay, the plant extract was added to the 1 9 103 cells ml
1 yeast
suspension in Sabouraud medium that had been previously seeded in
each well. The cultures were observed at 6, 24 and 48 h to measure
the activity profile of the plant extract on the yeast. After this incu- P421
bation period, the contents of each well was seeded in Sabouraud
dextrose agar medium at 25 °C for 24 h to allow the counting of the
Colony Forming Units (CFU) and determining the Minimum Inhibi- Modulation of morphogenesis in Candida albicans by
tory Concentrations (MICs). All assays were performed in triplicate. atmospheric pressure plasma jet
Results The Pouteria ramiflora extract was able to inhibit all three A. C. Borges,1 T. M. C. Nishime,2 R. Y. Honda,2 K. G. Kostov2
Candida species disclosing MIC values ranging from 6,25 mg ml
1 to and C. Y. Koga-Ito3
12.5 mg ml
1. Candida glabrata showed MIC with a lower range
when compared to Candida albicans and Candida parapsilosis.
1
ICT-UNESP, Sa~o Jose dos Campos, Brazil; 2FEG-UNESP,
Conclusion In vitro results obtained with ethanol extract of the Guaratingueta, Brazil and 3Universidade Estadual Paulista UNESP,
Pouteria ramiflora showed potential antifungal effect against Candida Sa~o Jose dos Campos, Brazil
albicans, Candida glabrata and Candida parapsilosis.
Objectives The aim of this study was to evaluate the effect of atmo-
spheric pressure plasma jet on Candida albicans viability, morphogene-
sis and hydrolytic enzymes (proteinase and phospholipase)
P420 production.
Methods Standardized suspensions of C. albicans SC 5314 and ATCC
18804 (106 cell ml
1; k = 550, OD: 0.380) were exposed to atmo-
In vitro effect photodynamic therapy with methylene blue, spheric pressure plasma jet working with helium (flow: 5.0 L min
1)
rose bengal, protoporphyrin IX and curcumin on Candida at 1.5 cm distance from the device tip to the liquid surface. The
albicans, C. parapsilosis and C. krusei. exposition times were 30, 60, 90 and 120 seconds. To determine the
erez-Laguna,1 L. Perez-Artiaga,1 V. Lampaya,1 Y. Gilaberte,2
V. P minimum exposition time necessary to reduce cell viability, cell sus-
S. Samper,3 M. C. Alejandre,4 M. J. Revillo1 and A. Rezusta1 pensions were ten-fold diluted and cultivated on Sabouraud dextrose
1 agar (37 °C, 24 h) to obtain CFU ml
1 values. The effect of plasma
Hospital Universitario Miguel Servet, Zaragoza, Spain; 2San jet on C. albicans morphogenesis was evaluated by exposing standard-
Jorge Hospital, IIS Aragon, Huesca, Spain; 3Hospital Universitario ized cell suspensions to plasma jet for 30 and 60 seconds (sub-in-
Miguel Servet, IIS Aragon, Zaragoza, Spain and 4Ayuntamiento hibitory exposition times). After exposition to plasma jet, cell
de Zaragoza, Zaragoza, Spain suspensions were supplemented with fetal bovine serum (20%) and
after incubation (4 h, 37 °C) hyphae and yeasts were counted using
Objectives Candida albicans, C. parapsilosis and C. krusei are yeasts a hemocytometer. To evaluate protease and phospholipase produc-
that can be important pathogens commonly involved in infections. tion, the suspensions were exposed to plasma jet also for 30 and
Antimicrobial photodynamic therapy (APDT), based on the applica- 60 seconds. Then, they were inoculated on agar containing bovine
tion of a photosensitizer (Ps) activated by visible light to generate albumin or egg yolk emulsion. After 5 days of incubation (37 °C),
reactive oxygen species that are cytotoxic to microorganisms, could halos diameters were measured and Pz values were determined. All
be an alternative treatment. Methylene blue (MB), rose bengal (RB), the experiments were performed in triplicate in three different situa-
protoporphyrin IX (PpIX) and curcumin (CUR) are promising Pss for tions. Non-exposed control groups were included in all experiments.
APDT. Results were compared statistically by ANOVA and post-hoc Tukey
The aim is to compare the efficacy of APDT on C. albicans, C. para- test. Results: Significant reduction in cell viability was observed from
psilosis and C. krusei using MB, RB, PpIX and CUR. 90 seconds of exposition to plasma jet (P < 0.001). After exposition
Methods C. albicans ATCC10231, C. parapsilosis ATCC 22019 and C. of 30 and 60 seconds, the percentage of filamentation was signifi-
krusei ATCC 6258 suspensions containing >107cells ml
1 were pre- cantly reduced for both ATCC 18804 and SC 5314 (P < 0.001) cells.
pared. Different concentrations two-fold serial dilution of PSs (from No difference in enzymes production was observed between exposed
0.15 lg ml
1 to 2560 lg ml
1) were added. Irradiation at a fluence and non-exposed groups, P > 0.05).
of 36 J/cm2 was carried out using white light-emitting diode (LED) Conclusion Atmospheric helium plasma jet showed promising
(0.024 w/cm2) in all cases and different LEDs lamps depending on effects on Candida albicans, reducing cellular viability and modulating
the most appropriate wavelength for each Ps: red LED light (625 nm, morphogenesis.

ª 2015 The Authors

204 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P422 diseases, other species, such as Candida parapsilosis, Candida glabrata

or Candida krusei, cause an increasing number of candidiasis. These
emergent species frequently show a reduced susceptibility to common
Molecular modeling of a heat shock protein (HSP90) as antifungal drugs. New compounds are needed for the treatment of
strategy for drug development against invasive and recalcitrant superficial candidiasis. Human uterine cer-
paracoccidiodomycosis vical stem cells conditioned medium (HUCSCCM) has antitumour and
A. K. R. Abadio,1 E. S. Kioshima,2 P. A. Silva,3 J. S. R. Godoy,2 antibacterial activities [1,2] and it could be a potent antifungal drug.
C. P. Bravo,3 A. M. Nicola,4 M. S. S. Felipe5 and B. Maigret6 Objectives To evaluate and to compare the antifungal activities of
1 human uterine cervical stem cells conditioned medium (HUCSCCM)
University from the State of Mato Grosso, Nova Xavantina, and adipose tissue stem cells conditioned medium (ATSCCM).
Brazil; 2Universidade Estadual de Maringa, Maringa, Brazil; Methods Antimicrobial in vitro activities of HUCSCCM and ATSCCM
3
University of Brasilia, Brasilia, Brazil; 4Catholic University of against one strain each of Candida albicans, Candida dubliniensis, Can-
Brasilia, Brasilia, Brazil; 5Universidade de Brasılia - UNB, Brasılia, dida glabrata, Candida krusei, Candida lusitaniae and Candida parapsilosis
DF, Brazil and 6University Henri Poincare-Nancy I, Nancy, France were compared with a microdilution method on RPMI 1640 using
the BioScreen C microbiological incubator.
Objectives In order to develop new antifungal treatments against Results Antifungal activity recorded as a reduction of cell growth in
Paracoccidioides lutzii, we proposed to evaluate the heat shock protein respect to the control at 24 h of culture at 37 °C is shown in table
of 90 kDa (HSP90) as drug target. As there is no crystallographic 1. The inhibitory activity of HUCSCCM was excellent against Candida
structure presently available for HSP90 of P. lutzii, we performed the glabrata (91.5%, Figure), Candida dubliniensis (73.4%), Candida albicans
homology modeling of the N-terminus domain of P. lutzii HSP90 pro- (57.5%), and Candida lusitaniae (46.8%). However, there was not
tein and the molecular dynamics simulations to obtain the HSP90 antifungal activity against Candida krusei and this activity was low
3D model with stability. against Candida parapsilosis. ATSCCM showed also a very good activ-
Methods The 3D model of HSP90 P. lutzii was constructed by homol- ity against Candida albicans (96.8%), Candida krusei (83.6%), Candida
ogy modeling based on known structures with high percentage of iden- parapsilosis (74.98%), and Candida dubliniensis (74.7%). Conversely,
tity in amino acid sequences. The known template structures were Candida glabrata was resistant to ATSCCM, with a paradoxical growth
searched in the Protein Data Bank (PDB). The amino acid residue of this species in its presence, and the activity against Candida lusita-
sequences of HSP90 were compared with the primary sequences of the niae was low.
structures deposited in the PDB using the BLAST program. The homolo- Conclusions HUCSCCM exerts an excellent antifungal activity
gous sequences allowed the construction of a 3D model of HSP90 using against the most common species of Candida but Candida krusei.
the homology module of the Insight II software package (Biosym/MSI, ATSCCM shows very good antifungal activity but it is ineffective
San Diego, Accelrys Inc. 2001). This model was further submitted to against Candida glabrata.
molecular dynamics simulations in order to gain a better relaxation and References
a more correct arrangement. The NAMD program version 2.6 was 1. Eir
o N, Sendon-Lago J, Seoane S, Berm udez MA, Lamelas ML,
employed in conjunction with the CHARMM22 force field. Garcia-Caballero T, Schneider J, Perez-Fernandez R, Vizoso FJ. Poten-
Results The 3D model of N-terminus domain of P. lutzii HSP90 pro- tial therapeutic effect of the secretome from human uterine cervical
tein was obtained. To investigate the stability of 3D model, molecular stem cells against both cancer and stromal cells compared with adi-
dynamics simulations were performed and revealed that the evolu- pose tissue stem cells. Oncotarget. 2014;5:10692–708.
tion of the system is very stable. The folding stability of the model 2. Berm udez MA, Sendon-Lago J, Eiro N, Trevi~ no M, Gonz alez F,
during the simulations was checked by monitoring secondary struc- Yebra-Pimentel E, Giraldez MJ, Macia M, Lamelas ML, Saa J, Vizoso
ture conservation over simulation time. With the model stable, vir- F, Perez-Fernandez R. Corneal epithelial wound healing and bacterici-
tual screening for select the main small molecules that interact with dal effect of conditioned medium from human uterine cervical stem
this target will be performed. cells. Invest Ophthalmol Vis Sci. 2015;56:983–92.
Conclusion This work describes the prediction of HSP90 model of P.
lutzii and its stability by molecular dynamics simulations aiming to
identify new antifungal compounds against paracoccidioidomycosis. In
the absence of structures solved experimentally, the available homol-
ogy modeling tools were extremely useful for the structural prediction
of the HSP90 protein of P. lutzii. From these steps we obtained relevant
information for future technological development. Moreover, these
results are being used to virtually screen chemical libraries, which are
under progress, generating new perspectives on technological develop-
ment and innovation of antifungal agents to these human pathogens.

P423

Antifungal activity of the human uterine cervical stem cells

conditioned medium against medically important species
of Candida
C. Marcos-Arias,1 E. Mateo-Alesanco,1 N. Eiro ,2 F. Vizoso,2
R. Perez-Fern 3 1 4
andez, E. Eraso, J. Schneider and G. Quindo  s1
1
Univ. Paıs Vasco UPV/EHU Facultad de Medicina y Odontologıa,
Bilbao, Spain; 2F. para la Investigacion con C. Madre Uterinas
FICEMU/Fundacion Hospital de Jove, Gijon, Spain; 3F. para la
Investigacion con C. Madre Uterinas FICEMU Santiago de
Compostela, Gijon/Santiago de Compostela, Spain and 4F. para
la Investigacion con C. Madre Uterinas FICEM Rey Juan Carlos,
Gijon/Madrid, Spain

Candidiasis are major causes of human morbidity and mortality.

Although Candida albicans is the prevalent aetiology of these fungal

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 205
Poster Presentations

Funding: This study was financed in part by Fundacion Jes

us de diffusion technique using different concentrations of methanolic and
Gangoiti Barrera, Fundaci
on ONCE ‘Oportunidad al Talento’and pro- ethanolic extracts.
jects GIC12 210-IT-696-13, UFI 11/25 (UPV/EHU). Methods Isolates from the scalp, skin, toes and feet of forty individu-
als (mainly children) were obtained from four locations namely Aluu,
Choba, Rumuosi and Emohua areas of Rivers State. The methanolic
and ethanolic herb extracts that were tested are Ficus exasperata
P424 vahl, Ocimum gratissimum, Jatropha curcas leaves, Commelina beng-
halensis, Mitracarpus villosus, Harungana madagascariensis and Chloro-
phora excelsa. The phytochemical contents of the extracts were also
Photoinactivation with red LED lamp of Candida albicans determined.
using Methylene Blue, alone or in combination with Results The qualitative phytochemical analysis showed that the
fluconazole or sertaconazole extracts contained alkaloids, cardiac glycosides, tannins, saponins
erez-Laguna,1 V. Lampaya,1 L. Perez-Artiaga,1 S. Samper,2
V. P and flavonoids. Anthraquinones and phlobatanins were absent. The
Y. Gilaberte,3 M. C. Alejandre,4 M. J. Revillo1 and A. Rezusta1 study showed significant inhibitory effect by O. gratissimum, F. exas-
1 perata vahl and J. curcas on the fungal isolates, at five different con-
Hospital Universitario Miguel Servet, Zaragoza, Spain; 2Hospital centrations (250 mg ml
1, 200 mg ml
1, 150 mg ml
1,
Universitario Miguel Servet, IIS Aragon, Zaragoza, Spain; 3San 100 mg ml
1 and 50 mg ml
1) used. The other plant extracts of C.
Jorge Hospital, IIS Aragon, Huesca, Spain and 4Ayuntamiento de excelsa, M. villosus, C. benghalensis, and H. madagascariensis showed
Zaragoza, Zaragoza, Spain some levels of inhibition at higher concentrations (250 mg ml
1 and
200 mg ml
1).
Objectives Candida albicans is a yeast frequently found in skin and Conclusion Assessment of the various minimum inhibitory concen-
mucous that can be important pathogen commonly involved in trations (MIC) showed that Ocimum gratissimum had the most poten-
infections. tial for use as an antidermatophytic agent. The phytochemical
Antifungals like fluconazole or sertaconazole are used to treat the constituents revealed may also be useful to the pharmaceutical
infection by C. albicans. Antimicrobial photodynamic therapy (APDT), industries and could be used as effective nutraceuticals. However,
based on the application of a photosensitizer like Methylene Blue (MB) further studies are needed to isolate and purify the bioactive com-
activated by visible light to generate reactive oxygen species that are pounds of these useful traditional plants for industrial drug
cytotoxic to microorganisms, could be an alternative treatment. formulation.
The aim of this study was to compare the efficacy of APDT-MB on
C. albicans at different times of preincubation in darkness alone and
joined to antifungals.
Methods C. albicans ATCC 10231 suspensions containing P426
>107cells ml
1 were prepared. Different concentrations two-fold
serial dilution of MB (from 2.5 lg ml
1 to 640 lg ml
1) combined
with 0, 10, or 20 lg ml
1 of fluconazole and sertaconazole were Nepenthes rafflesiana pitcher liquid has antifungal activity
tested using different preincubation times in darkness (0, 0.5, 1, 2 or against Candida spp.
24 h). Irradiation was performed with a red light-emitting diode H. Yolanda, I. M. Makahinda, M. Aprilia, N. Sanjaya,
(LED) lamp (625 nm, 0.007 w/cm2) at a fluence of 18 J/cm2. The H. Gunawan and R. Dewi
resultant suspensions were subcultured onto agar Sabouraud with Atma Jaya Catholic University of Indonesia, Jakarta, Indonesia
added chloramphenicol to determine the viable yeasts by colony-
forming units counting (CFU/mL). Dark and irradiated controls, with
and without MB, fluconazole or sertaconazole were included. Objectives To develop new effective antifungals, it is essential to
Results A reduction of 6 log10 CFU ml
1 were reached without and search for antifungal compounds from plants such as Nepenthes spp.,
with 0.5, 1 and 2 h of preincubation with 80–160 lg ml
1 of MB which have their greatest diversity in Indonesia. Since chitin-induced
diminishing the concentration to 80 lg ml
1 at 24 h. Adding 10 or liquid (CIL) from Nepenthes khasiana pitchers has antifungal activity,
20 lg ml
1 of fluconazole or sertaconazole, the MB concentrations due to their naphthoquinone content, this study aimed to evaluate
required remain the same. antifungal activity of Nepenthes rafflesiana pitcher liquids on Candida
Conclusion In vitro PDT-MB has a significant fungicidal effect on spp.
C.albicans and 0 min of preincubation with the MB is enough to Methods Collected pitcher liquids were of 2 types: non-induced liq-
obtain the maximum effect. This is very convenient to translate this uid (NIL) and chitin-induced liquid (CIL). NIL was collected from
therapy into the clinic. fresh naturally opened pitchers and CIL from closed pitchers after
PDT-MB seems to have not synergic effect in vitro with fluconazole 5 days of chitin solution injection. Chitin solution was prepared from
or sertaconazole against C. albicans. dried prawn exoskeleton. The antifungal activity of the liquids
Acknowledgements This work has been supported by grant against C. albicans, C. glabrata, C. krusei, and C. tropicalis were
CTQ2013-48767-C3-2-R from the Spanish Ministerio de Economıa y detected by disc diffusion. The medium was Mueller Hinton agar add-
Competitividad. ing with glucosa 2% and metilen blue dye 0.5 mg l
1. The 5 mm
sterile discs were made from Whatman filter paper No. 42. The ster-
ile disc soaked in aquabidest and placed on the seeded medium was
used as a control. The test was performed in triplicate. The inhibition
P425 zone diameters were recorded.
Results The colour of NIL was white cloudy. On chitin induction,
the colour of pitcher liquid changed to orange red (figure 1). Acidity
Effects of Local Herbs on Common Dermatophytes and of NIL was 3.1 and CIL was 2.6. All samples were slimy. Inhibition
causative agent of Pityriasis versicolor in Rivers State, zone diameters of NIL and CIL against C. albicans were
Nigeria 36.44  2.49 mm and 30.00  2.00 mm, respectively, while for C.
C. Mbakwem-Aniebo and I. O. Okonko glabrata the zone diameters were 22.55  3.53 mm and
University of Port Harcourt, Port Harcourt, Nigeria 28.89  1.17 mm, respectively (figure 2). There were significant dif-
ferences (P < 0.05) between NIL and CIL to both of fungi. No inhibi-
tion zones were found for NIL and CIL against C. krusei and C.
Background The antifungal activities of seven (7) local herbs used tropicalis.
by traditional medicine practitioners against the Dermatophytes (Tri- Conclusions NIL and CIL of N. rafflesiana have antifungal activity to
chophyton, Microsporum, Epidermophyton) and Malassezia furfur (the C. albicans and C. glabrata, but no antifungal activity to C. krusei and
etiological agent of Pityriasis versicolor), were studied by well-in-agar C. tropicalis. The pitcher liquid of N. rafflesiana has antifungal

ª 2015 The Authors

206 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

importance. Recently, in addition to conventional microwave and

sodium hypochlroide disinfection regimes, strong and weak acidic
electrolyzed waters are introduced. Regarding to this information,
this study evaluated the effect of different disinfection regimes on C.
albicans biofilm which is formed on acrylic denture base resin,
in vitro.
Methods The samples were subjected to the previously mentioned
disinfection process. At this stage, samples were transferred into
200 ml of sterile distilled water for the microwave disinfection at
650 W for 3 min. The chemical disinfection method was carried out
transferring each sample into 1 ml disinfection solution. The disinfec-
tion process was carried out with weak and strong electrolyzed acid
water and sodium hypochlorite for 1, 1 and 10 min, respectively.
The disinfection was not adjusted on the control group.
The C. albicans ATCC (90128) suspensions were subcultured into
fresh medium and incubated at 37 °C for 24 h. After 24 h of incuba-
tion, liquid cultures were washed three times. The optical density of
the yeast stock solution was adjusted to a 0.5 MacFarland solution.
1 ml yeast suspension was incubated with the sample for 48 h at
37 °C in a horizontal shaker. To remove non-adhering fungi, all
samples were carefully washed three times in PBS. Samples taken
from the experimental group and control group were transferred into
1 ml of PBS and vortexed for 60 sec. to resuspend any microorgan-
isms present. 0.1 ml of this resuspensions was plated on Sabouraud
dextrose agar and incubated aerobically for 48 h at 37 °C. After
incubation, the microbial colony count of each plated denture was
quantified by the eye counting method and the logarithm of colony
forming units (CFU) per ml was then calculated.
Results All four types of disinfection methods are completely elimi-
nated C. albicans biofilm on acrylic resin specimens.
Conclusion This results revealed that electrolyzed acid waters are
effective against C. albicans biofilm and they can be used to disinfect
dentures for the patients with denture stomatitis.

P428

Antifungal Activity of Tesso Nilo Wild Honey Against

Candida albicans in Vitro
Y. Putra,1 I. Gracia1 and H. Yolanda2
1
Fakultas Kedokteran UNIKA Atma Jaya, Jakarta, Indonesia and
2
Atma Jaya Catholic University of Indonesia, Jakarta, Indonesia
properties, presumably due to the presence of many potentially active
substances, such as naphthoquinones, as has been proven in other
studies. Objectives Candida sp. is a normal flora inhabiting the human skin
Key words antifungal, chitin-induced, disc diffusion, Nepenthes, and mucosa. Candida sp. may cause a disease called candidiasis, espe-
Candida cially in immunocompromised patients. Candida albicans is the main
pathogen in candidiasis cases. Candidiasis among immunocompro-
mised patient often requires a long term usage of antifungal agents
which leads to the emergence of the side effects. Candida sp. resis-
tance against antifungal agents is also often reported. This condition
P427 has been an important issue in medical scope, so the various studies
had been performed to find a better alternative antifungal agents.
The effectiveness of different denture disinfection Honey is used as an alternative antifungal agent that had been pro-
methods on Candida albicans biofilm activity ven by researchers. Tesso Nilo Wild Honey (TNWH) which originated
€r1
N. Karacan,1 T. Didinen,2 Y. Keskin2 and M. Izgu from Tesso Nilo National Park - the largest rain forest in Sumatera,
1 Indonesia - account as one of the Green and Fair Product by World
Faculty of Veterinary Medicine, Ankara University, Ankara, Wildlife Fund Indonesia, so the quality of this honey can be guaran-
Turkey and 2Faculty of Dentistry, Ankara University, Ankara, teed. This study is performed with the aim of investigating the anti-
Turkey fungal activity of TNWH against C. albicans in vitro.
Methods This is an experimental in vitro study using the macrodilu-
Objective Denture stomatitis is a widespread infection of complete tion method. C. albicans which has been incubated on 37°°C for
denture users. The condition is related to multiple factors such as 24 h is dissolved by normal saline with optical density equal to 0.5
using old dentures, denture trauma and insufficient oral hygiene. McFarland in 530 nm wavelength. Furthermore, the inoculum is
Among all of these factors, the candidal biofilm formed on tissue diluted by 1:2000 in Sabouraud Dextrose Broth (SDB). In this study
surfaces of dentures are believed to be the primary etiological factor there are 4 test groups with concentration of TNWH of
of denture stomatitis. Various types of candida species are con- 250 mg ml
1, 500 mg ml
1, 750 mg ml
1, and 1000 mg ml
1.
tributed in this process, while Candida albicans is believed to be the The positive control used in this study is C. albicans in SDB and also
most important one. Because of this, to prevent and to treat den- 3 negative control consist of TNWH, SDB and mixture of both. All
ture stomatitis, it is believed that elimination of C. albicans biofilm test groups are inoculated on Sabouraud Dextrose Agar and incu-
with various types of disinfection procedures is of paramount bated on 37 °C for 24 h. The main idea of this study is to compare

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 207
Poster Presentations

Figure 1. Colony Distribution.

Figure 1. The colonies of C. albicans in SDA for counting

the quantity of C. albicans numbers of colony in each test group to

the positive control.
Results The numbers of colony for test group with concentration of Table 1. Statistical analysis of total colonies among groups
TNWH of 250 mg ml
1, 500 mg ml
1, and 750 mg ml
1 are 34
(33–49), 10 (9–10), and 0 (0–1), consecutively, whereas the positive
control is 2760 (2520–4400). The test group with concentration of
TNWH of 1000 mg ml
1 and all negative control have no growth of
C. albicans. Results are processed with SPSS using the Kruskal-Wallis
statistic test and continued with the Post Hoc Mann-Whitney statistic
analysis. The test group with the concentration of 500 mg ml
1 and
750 mg ml
1 had proven to had significant difference if compared to
the positive control with p equals to 0.043 and 0.046 respectively.
Conclusion TNWH has an antifungal activity against C. albicans
in vitro according to the macrodilution method. The test group with
the concentration of 500 mg ml
1 shows an optimal result with the
significant difference in numbers of colony.
Key words antifungal, Candida albicans, macrodilution, Tesso Nilo
Wild Honey.

concentrations of SLI were less than negative control. Antifungal

activity of SLI 100% w/v has no significantly difference with positive
control (P = 0.114).
P429 Conclusions The SLI has antifungal activity against C. albicans.

Antifungal activity of soursop leaf infusion against

Candida albicans
H. Yolanda, M. Florensia and D. A. Soeselo P430
Atma Jaya Catholic University of Indonesia, Jakarta, Indonesia
Anti fungal activity of three Terpyridine derivates on
Objectives Candidiasis is a disease caused by Candida sp, mainly pathogenic Candida species
Candida albicans. Antifungals used to treat candidiasis have side K. Diba,1 P. Asgari2 and A. Namaki3
effects such as disruption in liver and kidney functions, also produce 1
Urmia University of Medical Sciences, Urmia, Iran; 2Hamadan
resistency. Annona muricata, known as soursop, have been used as
University of Medical Sciences, Hamadan, Iran and 3Arefian
folk medicine as antimicrobe and anticancer. In Indonesia, traditional
remedies are often processed by boiling, such as infusion and decoc-
General Hospital, Urmia, Iran
tion. The aim of this study is to detect antifungal activity of sourso-
pleaf infusion (SLI) against C. albicans in vitro. Objectives Recently, some Terpyridine based chemical compounds
Methods This was a laboratory experimental study. The SLI was have been used as antibacterial agents. In this study, three Ter-
prepared in various concentrations: 20% w/v, 40% w/v, 60% w/v, pyridine based compounds including [Cd(Clphtpy)(NO3)2H2O], [Cd
80% w/v, and 100% w/v. Antifungal activity of SLI was assessed by (Clphtpy)(N3]2 and [Cd (Clphtpy)(NCS)(NO3]n with similar structural
macrodilution methods and continued with colony counting. Keto- and antibacterial activities were studied. This is a experimental study
conazole 0.5 mg ml
1 was used as positive control. Negative control to develop using Terpyridine derivates on human pathogenic Candida
was fungal suspension in Sabouroud dextrose broth. Statistical analy- albicans.
sis was performed using Kruskal-Wallis test and post hoc Mann- Methods A hundred Candida isolates of human infections and a
Whitney from SPSS 17. standard Candida strain were profiled to the agar diffusion method
Results Total colony counting from SLI 20% w/v, 40% w/v, 60% by using a pour plate culture of Candida isolates which punched and
w/v, 80% w/v, and 100% w/v were 206 (185–262), 162 (161– loaded by 100 ll of each three drugs. The cultures incubated at
176), 14 (9–25), 8 (6–12), 3 (0–3), consecutively. Whereas, total 30 °C for 48 h. Fungal activity was determined by measuring inhib-
colony counting from negative control was 451 (371–561), and no ited zone of the drug wells. The antifungal amphotericine B were
colonies was found in positive control. Total colonies from all used as positive control of inhibitory activity.

ª 2015 The Authors

208 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Results One hundred clinical isolates of pathogenic Candida albicans P436

were used in this study. The clinical sources included sputum, bron-
cho alveolar lavage and vaginal discharge of suspected patients to
fungal infections. The average inhibitory zone of Terpyridine based Predisposing factors to Vulvovaginal Candidiasis (VVC) and
compounds on studied Candida isolates included 18.2 mm, 29.6 mm Recurrent Vulvovaginal Candidiasis (RVVC)
and 20.8 mm for drugs 1,2 and 3 respectively. L. Akimoto-Gunther,1 P. S. Bonfim-Mendonc
a,1 G. Takahachi,2
Conclusion Comparing data of Terpyridine derivates with that of H. K. Anjos,1 F. K. Tobaldini,3 M. R. Oliveira,1
amphotericine B showed a low level of antifungal activity for men- M. E. L. Consolaro1 and T. I. E. Svidzinski1
tioned drugs. Perhaps these compounds are useful to develop some
forms of antifungal drugs.
1
Universidade Estadual de Maringa, Maringa, Brazil;
2
Universidade Estadual de Maringa - UEM, Maringa, Parana,
Brazil and 3Universidade Estadual de Maringa/Universidade do
Minho, Maringa, Brazil
P435
Objectives Vulvovaginal candidiasis (VVC) and recurrent vulvovagi-
nal candidiasis (RVVC) are diseases commonly diagnosed in women
Insights into the synthesis of b-1,3-glucan in Candida in reproductive age and is mainly caused by yeasts belonging to the
albicans: relevance for echinocandins inhibition genus Candida. It has been demonstrated that cellular immune
R. Rocha,1 J. H. Morais-Cabral2 and C. Pina-Vaz1 response and defective neutrophil function may increase susceptibility
1
FMUP, Porto, Portugal and 2IBMC, Porto, Portugal to infections by C. albicans. The cell mediated immunity appears to be
important in limiting the proliferation of Candida spp., suggesting that
clinical conditions like exposure to chronic stress or deficient antioxi-
Fungal b-1,3-glucan synthase is an essential enzyme with no human
dant micronutrients that alters the proper functioning of the immune
homologue responsible for the biosynthesis of b-1,3-glucan. This
system, could facilitate the primary VVC and RVVC occurrence. A
polysaccharide is the main structural component of fungal cell wall
low overall antioxidant capacity has been associated with immune
providing structural stability and rigidity through covalent cross-link-
deficiency, and some authors suggest that the antioxidant mecha-
ing with chitin and mannoproteins. The catalytic subunit of b-1,3-
nism action is the modulation of signal transduction factors involv-
glucan synthase (known by FKS) is inhibited by the echinocandins,
ing transcribing cells and immune-mediated cytokine production. On
the newest antifungal class.
the other hand, attenuated levels of morning salivary cortisol and
Objectives In order to get insights into the synthesis of b-1,3-glu-
hyporesponsiveness of the hypothalamus-pituitary-adrenal axis have
can, a biochemical approach was established for heterologous expres-
been shown previously to be indicative of chronic stress. In addition,
sion and purification of FKS cytoplasmic domains. The Candida
authors suggest that stress may increase the risk for impaired func-
albicans FKS has two cytoplasmic domains: a N-terminal domain and
tion of the immune processes. Examine chronic stress and low levels
the catalytic domain surrounded by two sets of transmembrane
of overall antioxidants capacity as a host intrinsic factors that could
helices. The functional role of N-terminal and transmembrane
predispose women to developing VVC and RVVC.
regions is currently unknown.
Methods In a population of 254 women with or without signs and
Methods C. albicans Fksp cytosolic domains were cloned into differ-
symptoms of VVC and RVVC, vaginal cultures were performed for
ent expression vectors. The approximate boundaries of each cytoplas-
yeasts isolation and identification by classical methods. Cervical-vagi-
mic-facing region were predicted using multiple protein sequence
nal cytology and vaginal bacterioscopy were performed to analyze
alignments and predictions of secondary structure or unfolded/disor-
inflammatory process and microbiota. Fasting blood was collected for
dered regions.
plasmatic cortisol and overall antioxidant capacity analyses. Cortisol
Results The N-terminal cytoplasmic domain was successfully
levels were determined employing chemiluminescent microparticle
expressed in bacteria and purified at large-scale using an affinity tag.
immunoassay in the equipment Architect (Abbott, IL, USA), and the
Recombinant purified protein was subsequently analyzed by analyti-
overall antioxidant capacity was determined by Trolox equivalent
cal gel filtration chromatography to assess protein stability and
antioxidant activity (TEAC), which was performed as described by
homogeneity. TheN-terminal FKS domain is stable and elutes as a
Miller et al. and Re et al. to evaluate plasma non-enzymatic antioxi-
monomer although this domain shows tendency to oligomerize when
dants.Data distributions were expressed as mean  standard devia-
protein concentration increases. Domain oligomerization could be rel-
tion (SD). Significant differences among means were identified using
evant for the assembly of b-1,3-glucan synthase in the plasma mem-
GraphPad Prism 5.0 software. Bonferroni test was used to calculate
brane since b(1,3)-glucan exists as a triple-helical structure in the
the multiple comparisions and Chi-square (v2) test using the STATA
cell wall.
for Statistics and Data Analysis 9.1 software was used to investigate
Similarly to N-terminal domain, expression and purification of C.
variables between control group and positive culture group. All vari-
albicans catalytic domain was also attempted. However, the catalytic
ables were expressed as absolute and relative frequencies. P < 0.05
domain is unstable and its expression as soluble protein is very low.
were considered significant.
Different approaches were therefore employed to increase domain
Results Yeasts were isolated from 48 (19%) women: 22 (46%) in
expression including co-expression of both catalytic and N-terminal
acute episode (VVC) and 26 (54%) with RVVC, and mainly C albicans
domains. All these strategies failed suggesting that Fksp catalytic
was isolated in all clinical profiles. Control group (negative culture)
domain is unstable in the absence of the Fksp transmembrane region.
comprised 206 (81%) women. VVC and RVVC groups showed lower
In order to understand FKS, a theoretical three-dimensional model
means level of cortisol than control groups. RVVC group showed
was generated by comparison with the crystal structure of bacterial
lower mean levels of antioxidant capacity than all other groups.
cellulose synthase.
RVVC showed a similar vaginal inflammatory process than control
Conclusion C. albicans FKS catalytic domain is not stable alone in
and lower than VVC groups. Only CVV group showed a reduction in
the absence of the transmembrane region, in contrast with the N-ter-
vaginal lactobacilar microbiota.
minal domain.
Conclusion Our data suggest that both, chronic stress (decreased
early morning cortisol levels) and low overall antioxidant capacity
can be considered specific host predisposing factors to VVC and
RVVC.

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Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 209
Poster Presentations

P437 P438

Aspergillus fumigatus transcriptome during a disseminated Effect of antimycotic drugs on human platelets and their
murine infection reveals the metabolic adjustments once fungal killing capacity
the infection has established €rl,1
C. Speth,1 B. Salzgeber,1 M. Hagleitner,1 C. Lass-Flo
M. Sueiro,1 J. V. Fernandez-Molina,1 A. Abad-Diaz-de-Cerio,1 1
M. Hermann and G. Rambach 2

X. Guruceaga,1 A. Ramirez-Garcia,2 J. Garaizar,1 1

Medical University of Innsbruck, Innsbruck, Austria and
F. L. Hernando,1 J. Margareto3 and A. Rementeria2 2
University of Inssbruck, Innsbruck, Austria
1
University of the Basque Country, Leioa, Spain; 2Universidad del
Paıs Vasco UPV/EHU, Leioa, Spain and 3Tecnalia Research & Objectives Platelets become more and more established as crucial
Innovation, Leioa, Spain players of the innate immunity and thus influence the course of
invasive fungal infections. Recent own studies showed that some
Introduction Aspergillus fumigatus is a saprophytic mold that can antimycotic drugs modulate the activity of platelets. This effect might
act as an opportunistic pathogen. In order to succeed in the develop- influence the capacity of platelets to mediate antifungal killing and to
ment of the infection, not only does this pathogen have to evade and discharge their role in the immune network, but also impact pro-
confront the immune system of the host, but it also has to deal with cesses such as excessive inflammation and thrombosis. For that rea-
numerous microenvironments. On the other hand, murine models son we performed a broad screening of antimycotic drug effects on
have been used to study the pathogenesis of this fungus, which platelets.
enables to understand what happens during a real infection and Methods Platelets were either mock-treated or incubated with
whose results might be extrapolated to human cases. increasing concentrations of various antimycotics. Putative activation
Objective The aim of this study is to analyze the transcriptome of A. of the platelets was tested by FACS. Fungal killing by platelets in
fumigatus throughout an experimental disseminated infection in order presence or absence of the antimcyotics was investigated by cal-
to determine the metabolic changes that take place once the infection cofluor quantification of Aspergillus fumigatus growth and life-time
has established. microscopy. Interaction of platelets with fungal hyphae was studied
Methods Three independent murine infections were performed, by confocal microscopy.
being one infected kidney from each day used for RNA isolation. Results The antimycotics amphotericin B and caspofungin were
RNA samples were hybridized with a customized A. fumigatus expres- both capable to stimulate platelets in a time- and dose-dependent
sion microarray. Transcriptomic data were compared with a previous manner, as shown by increase of the activation marker CD62P on
study of the germination of this pathogen at 37 °C (Sueiro-Olivares the platelet surface. In contrast, the azoles fluconazole and voricona-
et al., 2015) and results were validated by RT-qPCR. Moreover, a zole as well as the echinocandins micafungin and anidulafungin did
clustering process was performed to determinate which genes had an not affect platelet activity.
increasing expression. Finally, these genes were classified according Furthermore, we studied a putative synergistic effect between
to their functional classification and the enrichment p-value of the antimycotics and platelets on Aspergillus killing. For that purpose, the
functional groups was determined. fungus was incubated with increasing numbers of platelets in the
Results Statistical analysis of each day of infection using the A. presence of partially inhibitory concentrations of the respective drugs.
fumigatus germination at 37 °C as the control condition showed that It could be demonstrated that platelets alone induced already fungal
4,080, 377, 3,604 and 1,645 genes were differentially expressed on killing in a ratio-dependent manner. Interestingly, the platelet effect
day 1, 2, 3 and 4 of infection, respectively. Genes involved in iron, on fungi in the presence of caspofungin, micafungin and posacona-
zinc and nitrogen metabolism appeared down-regulated during the zole is ambivalent: whereas low (1:100) and high (1:4000) ratios of
infection. However, genes related to gliotoxin biosynthesis as well as platelets have an additive effect on drug-induced fungal killing, the
others encoding proteases and phospholipases described as virulence drugs even appeared to protect the fungus against intermediate plate-
factors were up-regulated along the infection. Focusing on the clus- let ratios (1:1000).
tering process, 5 clusters showed an increasing expression pattern We also studied the capacity of the antimycotics to alter the platelet
relative to the germination at 37 °C or throughout the infection. binding rate to the fungal surface. For that purpose, Aspergillus fumiga-
According to the enrichment p-values of the functional categories, tus was grown in the presence of the drugs before adding the platelets
secondary metabolism and polysaccharide metabolism were signifi- and studying the interaction microscopically. It could be demonstrated
cantly represented. Nevertheless, among the 257 genes included that pretreatment of the fungus with partially inhibitory posaconazole
within these clusters, 79 and 36 coded for hypothetical or unclassi- concentrations resulted in enhanced binding of the platelets.
fied proteins. Conclusions Various antimycotics can affect the interaction
Conclusion It seems that once A. fumigatus infection has established between platelets and Aspergillus, either by altering the fungal surface
in the host, a reduction of nitrogen, iron and zinc requirements takes in a manner that platelets show enhanced binding, or by modulating
place and/or this pathogen obtains these nutrients through different the activity of platelets and their fungal killing. These processes
mechanisms. Secondary and polysaccharide metabolisms are the might crucially influence the course of invasive aspergillosis as well
functions significantly expressed during the infection, standing out as as other of fungal diseases and will be further studied in the future.
indispensable metabolisms to spread the infection. However, the large
number of hypothetical and unclassified proteins stresses the impor-
tance to focus researches on them, as they could shed light on novel
virulence factors, and even on possible diagnostic targets. P439
Supported by: the UPV/EHU (grants PES13/03, GIU12/44 and
UFI11/25), the Government of the Basque Country (grants S-
PC11UN007), and the Ministry of Economy and Competitiveness Profiling Candida albicans gene expression during human
(MICINN CSD2009-00006). Monica Sueiro Olivares has been sup- intra-abdominal candidiasis by RNA-Seq affords insights
ported by Pre-doctoral Research Grants of the Government of the into pathogenesis and antifungal responses
Basque Country. Jimena V. Fernandez Molina has been supported by C. Clancy,1 C. Meslin,1 N. Clark,1 W. Nierman2 and M. H. Nguyen1
1
Post-doctoral Research Grants of the UPV/EHU. Xabier Guruceaga University of Pittsburgh, Pittsburgh, USA and 2JCVI, Rockville,
has been supported by a Jes us Gangoiti Barrera Foundation grant. USA

Background Profiling C. albicans gene expression during human

infections may provide unique insights into pathogenesis and
responses to antifungal treatment. Intra-abdominal candidiasis (IAC)

ª 2015 The Authors

210 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

is suited to Candida transcriptional profiling because yeast concentra- slightly more resistant to Congo red, was not able to grow in the
tions in infected fluid are relatively high, host cells are less abundant presence of SDS, and presented sensitivity to caffeine, in comparison
than in tissue or bloodstream infections, and drainage is part of with glucose-grown cells.
treatment. The increased transcription of genes involved in cell adhesion cor-
Methods We used an Illumina platform to perform RNA-Seq on bil- related well with adhesion and biofilm assays, in which Drlm1/
iary fluid recovered from a 61 year old man with C. albicans cholan- Drlm1 mutant presented greater biofilm formation than WT in cells
gitis. Day 1 (D1) samples were collected from two distinct sites at grown in both carbon sources. However, in cells adapted to lactate
time of drainage (RUQ-D1, LUQ-D1); a follow-up sample was col- biofilm formation was more pronounced. In general, lactate-grown
lected from one site 24 h later, after fluconazole treatment was insti- cells were less efficiently killed in comparison to glucose-grown cells,
tuted (RUQ-D2). Differentially expressed genes were identified using while Drlm1/Drlm1 mutant cells were more resistant when adapted
EdgeR Bioconductor, and defined by ≥4-fold change and false discov- to glucose than to lactate. The production of TNF-a and IL-10 by
ery rate <0.001. macrophages was lower in response to Drlm1/Drlm1 mutant and the
Results Fluid pH was 8.0. Cultures revealed C. albicans as sole cellular toxicity, measured as extracellular lactate dehydrogenase
pathogen; all cells were yeasts on Gram stain. 1.8 to 3.8 million activity, was significantly lower in comparison with the WT and
reads mapped to C. albicans coding sequences; ≤380 000 reads complemented strains in glucose-grown cells. However, when C. albi-
mapped to human sequences. Reads mapped to ≥93% of C. albicans cans cells were grown on lactate the Drlm1/Drlm1 mutant was
open reading frames. Only 2% of genes were differentially expressed slightly less resistant to macrophage killing but enhancing the
between D1 samples. Adhesion and oxidation-reduction were pro- expression of anti-inflammatory IL-10 cytokine.
cesses over-represented among upregulated genes at RUQ (cholecys- Conclusion Candida albicans regularly colonizes niches that are poor
tostomy tube) vs. LUQ (drain). 23% of genes were differentially in glucose, depending thus upon alternative carbon sources for
expressed in D2 vs. D1 samples. Adhesion, filmentation, pathogenesis growth. C. albicans cells adapted to different carbon sources behave
and ergosterol synthesis were over-represented, upregulated processes differently, affecting important virulence parameters, such as stress
on D2. D1 (non-fluconazole) results were compared to RNA-Seq data resistance, adherence, biofilm formation, and infection outcome. This
from in vitro, ex vivo, and mouse kidney and tongue datasets. By study reinforces evidence that assimilation of alternative carbon
principal component analysis, samples from given conditions clus- sources increases the yeast fitness, impacting Candida-host
tered most tightly. Similarity to human expression was: mouse kid- interactions.
ney, tongue>ex vivo>in vitro. Oxidation-reduction, glyoxylate
pathway, fatty acid oxidation, host entry and carbohydrate transport
were over-represented, upregulated processes in human IAC vs.
in vitro. Oxidation-reduction, glyoxylate pathway, fatty acid oxida- P441
tion, host entry and carbohydrate transport were over-represented,
upregulated processes in human IAC vs. ex vivo. Adhesion, pathogen-
esis, filamentation and biofilm were over-represented, upregulated Immunological features in patients with asthma and
processes in ex vivo vs. human IAC. Fatty acid oxidation, osmotic fungal sensitization
stress and TCA cycle were over-represented, upregulated processes in Y. I. Kozlova,1 E. V. Frolova,2 O. V. Aak,1 A. E. Uchevatkina,2
human IAC vs. mouse kidney. Adhesion, pathogenesis, filamentation L. V. Filippova,2 E. V. Burygina1 and N. Klimko2
and biofilm were over-represented, upregulated processes in mouse 1
kidney vs. human IAC. North-Western State Medical University named after
Conclusions C. albicans gene expression in vitro or under ex vivo I.I.Mechnikov, Saint Petersburg, Russia and 2I. Mechnikov North-
conditions is poorly representative of gene expression during human Western State Medical University, St. Petersburg, Russia
IAC. RNA-Seq is a powerful tool for transcriptional profiling of C.
albicans in vivo, offering a snap shot of genes involved in different Objectives To study immunological parameters in patients with
stages of pathogenesis and in responses to fluconazole treatment. asthma and fungal allergy.
Materials and methods We observed 32 patients with asthma.
Main group included 16 patients with asthma and fungal allergy (8
men and 8 women), aged 18–71 years (median–49 years). The com-
P440 paring group consisted of 16 patients with asthma without fungal
allergy (9 men and 7 women), aged 21–69 (median - 34 years). The
control group included 12 healthy people (median - 29 years).
Role of Candida albicans RLM1 in the interaction with Allergological examination included skin prick tests with six fungal
macrophages: the impact of the carbon source allergens, determination of total IgE level (by ELISA) and specific fun-
C. S. Pais, J. P. Pacheco, C. Carneiro and P. Sampaio gal, epidermal, household allergens (MAST-panel, Hitachi Chemicals
University of Minho, Braga, Portugal Diagnostic). Positive skin prick-test and/or detection of specific IgE to
fungal allergen in serum (level of IgE antibodies to mold allergens in
serum - class ≥1) were considered as the criteria of fungal
Objectives Previously we showed that Candida albicans RLM1 partic- sensitization.
ipates in the cell wall biogenesis, with the mutant rearranging its Production of interleukines (IL-2, IL-4, IL-10, IL-8, TNF-a, GM-
metabolic pathways to allow the use of alternative carbon sources. CSF, IL-6) and interferon-c (IFN-c) was determined in peripheral
In this study we aimed to evaluate the effect of different carbon blood cell supernatants after 24 h stimulation with phytohemagglu-
sources in response to cell wall damaging stress agents and in the tinin using Bio-Plex Pro Human Cytokine 8-Plex Panel (Bio Rad).
interaction with macrophages. The obtained results were statistically processed using the STATIS-
Methods Candida albicans a ^ˆ†rlm1/^aˆ†rlm1 mutant, WT and comple- TICA for Windows (version 6.0).
mented strains were adapted to lactic acid or glucose, and the hyper- Results In the main group the highest frequency of fungal sensitiza-
sensitivity of these cells to Congo Red, Calcofluor White and tion was connected with Aspergillus spp. (64%), Penicillium spp. -
caspofungin was evaluated. Expression levels of GPD1, AGP2, GCV2, 52%, Alternaria spp. - 36%, Mucor spp. - 30%, Cladosporium spp. -
SOU1, PUT2 and CIT1 were also evaluated by RT-PCR in cells 27%, Rhizopus spp. - 12%. The total IgE level ranged from 3 to
adapted to the different carbon sources. The cell viability and pro-in- 2100 U ml
1 (a median – 591) in this group. In the comparing
flammatory capacity was evaluated after incubation with the J774 group total IgE level was significantly lower – 1–693 U ml
1 (a med-
murine macrophages cell line. ian – 181), P < 0.05.
Results Candida albicans a ^ˆ†rlm1/^
aˆ†rlm1 mutant displayed pheno- Production of GM-CSF was increased in all patients with asthma
types associated to cell wall deficiency, such as hypersensitivity to as compared with the control group. Strong positive correlation were
Congo Red and caspofungin, both in glucose- and in lactate- grown found between the IL-4 levels and total IgE, GM-CSF and IFN-c
cells. However, the Drlm1/Drlm1 mutant grown in lactic acid was (r = 0.59, r = 0.90, r = 0.79 P < 0.05, respectively). However,

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 211
Poster Presentations

significantly higher production of IFN-c (median 1275 (6342163) P444

vs 474 (321527) pg ml
1; P = 0.001) and IL-2 (median 1416
(9261893) vs 573 (235970) pg ml
1; P = 0.001) was detected
in patients with asthma and fungal allergy as against the comparing The value of a multimodal imaging approach for the non-
group. The levels of IL-10, TNF-a, IL-6 and IL-8 did not differ from invasive assessment of invasive pulmonary aspergillosis in
the control values in patients of both groups. a mouse model
Conclusion Immunological features in patients with asthma and J. Poelmans,1 G. Vande Velde,2 L. Vanherp,3 A. Hillen,1
fungal sensitization were increased levels of total IgE, high frequency J. A. Maertens,3 T. Dresselaers,1 U. Himmelreich1 and K. Lagrou4
of sensitization to Aspergillus spp. and increased concentrations of IL- 1
2 and IFN-c, which suggested the mixed type of inflammation due to KU Leuven, Leuven, Belgium; 2Biomedical MRI unit/ MoSAIC,
fungal sensitization. Leuven, Belgium; 3KU Leuven, Leuven, Belgium and 4UZ Leuven,
Leuven, Belgium

Objectives Preclinical research plays an important role in gaining a

P443 better understanding of the pathogenesis of Aspergillus infections.
Standard techniques to evaluate experimental infections in small ani-
mals are histology and fungal load quantification in tissue samples.
Sph3: a glycosyl hydrolase required for biofilm formation Due to their invasive character, these techniques cannot provide us
by Aspergillus fumigatus and potential therapeutic agent. with any insight in dynamic disease processes as the available infor-
D. Sheppard,1 N. Bamford,2 D. Little,2 P. Baker,2 B. Snarr1 and mation is based on snapshot data of individual animals in a highly
L. Howell2 variable disease model. Thereby, disease severity may be underesti-
1 mated due to a low sensitivity of culture techniques (1,2).
McGill University, Montreal, Canada and 2The Hospital for Sick
We aimed at developing a multimodal imaging approach to non-
Children, Toronto, Canada
invasively investigate the pathogenesis of invasive pulmonary
aspergillosis in a mouse model. The combination of non-invasive
Objectives Aspergillus fumigatus is the second most common cause of imaging techniques, such as computed tomography (CT), magnetic
hospital acquired fungal infections. In the infected lungs, A. fumigatus resonance imaging (MRI) and fibered confocal fluorescence micro-
lives in multicellular communities encapsulated in self-produced scopy (FCFM), were evaluated to obtain dynamic information on dis-
matrices, termed biofilms. The galactosaminogalactan (GAG) polysac- ease progression in individual animals.
charide is an important adhesin in A. fumigatus biofilm formation and Methods BALB/c mice were immune suppressed by intraperitoneal
has immunosuppressive effects in vivo. A cluster of genes in the A. injections of cyclophosphamide 4 and 1 day prior to intranasal instil-
fumigatus genome has been linked to GAG biosynthesis and biofilm for- lation of 20 lL GFP-expressing A. fumigatus FGSC A1258 (5.105
mation. Bioinformatic analysis suggests that one of these genes, sph3, spores, n = 5–8) or saline (n = 5). The animals were scanned at
is a membrane bound protein that may adopt a fold similar to known baseline and daily after infection using CT (SkyScan 1076, Bruker
glycosyl hydrolases (GHs). We hypothesized that Sph3 may play a role microCT), MRI (9.4 T, Bruker Biospin) and FCFM (Cellvizio, Mauna
in the synthesis of GAG and in GAG-dependent biofilm formation. Kea Technologies). On day 4 after infection, the lungs were isolated
Methods Recombinant Sph3 from A. fumigatus and Aspergillus clava- for validation of the imaging results by CFU counts and histology.
tus were expressed and purified from E. coli. Structural and func- Results Within the lungs of infected mice, an increased attenuation
tional studies were carried out using the resulting proteins. A was observed on the CT images and hyperintense signals were
deletion mutant of Sph3 was constructed using the split marker tech- detected on the MR images (Fig 1A-B). The relative signal intensity,
nique. Biofilm assays were conducted in 96 well plates and the quan- lesion volume and total lung volume calculated from the CT and MR
tified with crystal violet staining. GAG quantification on hyphae was images gradually increased over time, which corresponded to lung
performed using fluorescent lectin staining and confocal microscopy. disease progression. In addition, hyphae and sporulating structures
Results Deletion of sph3 resulted in a complete loss of GAG produc- could successfully be visualized at a microscopic scale within the
tion and biofilm formation by A. fumigatus. Complementing this lungs of living animals by using in vivo FCFM (Fig 1C).
mutant with a wild-type allele of sph3 restored GAG production and Conclusion We successfully visualized the development of fungal
biofilm formation. Recombinant Sph3 from A. fumigatus (AfSph3) lesions within the lungs of mice infected with A. fumigatus. Lung CT
was recalcitrant to crystallization, however, an Sph3 ortholog from and MR imaging techniques provided valuable insight in the global
A. clavatus (AcSph3) formed crystals that could be optimized using extent and 3D distribution of developing lesions, while FCFM allowed
streak seeding. The structure was solved using selenium incorpora- for the real-time visualization of fungal hyphae independent of sur-
tion and the single- wavelength anomalous dispersion technique. rounding inflammation and necrosis. In addition, disease onset and
The structure reveals a (b/a)8 barrel with similarity to GHs from progression was successfully quantified in individual animals by cal-
families 18 and 27. Reducing sugar assays using crude GAG isola- culating signal intensities and fungal lesion volume from the CT and
tions show glycoyl hydrolase activity. Low concentrations of the MR images. By combining these complementary imaging techniques,
recombinant hydrolase domain of Sph3 hydrolase decreased cell-as- an overall longitudinal picture can be provided about the dynamics
sociated GAG, disrupted preformed A. fumigatus biofilms. Introduction of fungal lung infections on both a macroscopic and microscopic
of a point mutation into the predicted catalytic domain of Sph3 level in a completely non-invasive manner in individual animals.
reduced the anti-GAG and anti-biofilm activity of recombinant Sph3.
Conclusion We propose that Sph3 is a GAG membrane bound gly-
cosyl hydrolase responsible for processing GAG during biosynthesis,
and that recombinant Sph3 hydrolase domains could be developed as
a novel therapeutic enzyme against biofilm- dependent A. fumigatus
infections.

Figure 1. In vivo CT, MR and FCFM images of the lungs 4 days after
intranasal instillation with A. fumigatus (right) or saline (left).
References
1.Latge JP. Clin microbiol rev. 1999;12(2):310–50.
2.Hope WW. Lancet Infect Dis. 2005;5(10):609–622.
Correction added on 19 October 2015, after online publication.
Affiliation of author Liesbeth Vanherp was changed from University
Hospital Leuven, Leuven, Belgium to KU Leuven, Leuven, Belgium.

ª 2015 The Authors

212 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P445 10 min with 30 second on ice. RNeasy Mini kit (Qiagen, Germany)
was used for purification. The concentration and purity of RNA was
evaluated with NanoDrop (Thermo Scientific 200c). The cDNA was
Adhesion of s and m strains of Candida albicans to hacat obtained using Superscript III RT Supermix kit (Invitrogen), accord-
cell line ing to the kit manufacturer. For detection of ALS family transcripts,
M. Z. Mandelblat,1 M. Frenkel2 and E. Segal2 specific pairs primers were designed. The complete gene sequences of
1
Maccabi Health Services, Rehovot, Israel and 2Tel Aviv ALS genes for C. albicans were obtained from the GenBank database.
University, Tel-Aviv, Israel Real-time PCR (CF 9 48 Real-Time PCR System; Applied Biossys-
tems) was used to determine the relative levels of ALS family mRNA
transcripts, with ACT1 (actin)and CEF3 (translation elongation factor
Objectives In the frameofour study which focuses on a comparison 3)as reference housekeeping genes for normalization. The relative
of phenotypic and genotyping characteristics of Candida albicans iso- quantification of ALS family expression was performed by the DDCt
lates from patients with blood stream Candida infection (S strains) method. Each reaction was performed in triplicate and mean values
with isolates from patients with vaginal infection (M strains), we of relative expression were determined for each gene.
compared the adhesion to a human keratinocyte cell line - HACAT. Results The results of the adhesion assay are shown in Fig. 1. All
Methods Cell Line - The HACAT cell line was maintained in Dul- ALS genes were expressed, however, the amount of ALS genes
becco’s Modified Eagle Medium (DMEM) at 37 °C under 5% CO2. For expressed were strain- dependent. The ASS isolate expressed the
the adhesion assay the cells were seeded in 96 well microtiter plates highest number of ALS genes and the expression was homogeneous
at a concentration of 2x105 ml and grown for 48 hrs. to complete among 2 and 6 h of adherence to cervical cells. For this strain, only
confluence. ALS2 and ALS3 expression differed significantly between 2 and 6 h,
Strains’ maintenance, growth and FITC labelling - The C.albicans with a decrease of 90 and 60%, respectively (P < 0.05). VVC isolate
strains were maintained on Sabouraud’s dextrose agar (SDA) at was also able to express a large number of adhesins, however, most
4 °C. For the adhesion assay fresh 18 hrs. cultures grown in yeast of the genes were expressed in low amounts. Among these genes,
extract broth under shaking were adjusted to a concentration of ALS3 was significantly expressed compared to ALS6 (six-fold,
1 9 108 ml and labelled with Fluorescein isothiocynate (FITC) at a P < 0.05). In general, RVVC isolate expressed a lowest number of
concentration of 2.5 mM/0.5% DMSO in carbonate buffer. adhesins. Although, the expression levels of ALS2 and ALS3 at 6 h
Adhesion- The adhesion assay was carried out in the 96 well were significantly higher to RVVC than ASS and VVC isolates,
microtiter plates containing 2 9 104 HACATcells/well to which P < 0.05. Furthermore, it was possible to observe that ALS3 increase
1x107 FITC labelled C.albicans yeasts were added for 2 h. at 37 °C. expression over time (2 h to 6 h), significantly.
Assessment was done by TECAN Infinite 2000 microtiter plates Conclusions Were observed that despite the ASS isolate, have
reader measuring the fluorescence by an excitation and emission at a greater adhesion capacity, showed down-expression of pathogenicity-
wave lengths of 480 and 530 nm, respectively. related genes. And yet, that despite VVC and RVVC having less
Results We assessed 20 S and 24 M strains. As control we used the potential adhesion, which were able to markedly up- regulation
C.albicans strain CBS 562. Each strain was tested at least 3 times. genes involved in the pathogenicity as ALS1 and ALS3.
The M strains were compared to the S strains as a group using the
Student T test. The evaluation showed that M strains demonstrated
higher adhesion values than the S strains, with a statistically signifi-
cant difference. This observation is in contrast to our concurrent
in vivo experiments in mice, where no such differences between viru-
lence of the S and M strains, as expressed in survival rates and mean
survival time, were noted.
Conclusions These data indicate that apparently the M strains rep-
resenting mucosal infection exhibit as a major virulence attribute,
higher tendency to adhere than strains from blood stream infection.

P446

Quantitative expression of ALS family genes of Candida

albicans in the experimental cervical epithelial infections
P. S. Bonfim-Mendonc
a,1 F. K. Tobaldini,2 I. R. G. Capoci,1
M. Negri,1 E. S. Kioshima1 and T. I. E. Svidzinski1
1
Universidade Estadual de Maringa, Maringa, Brazil and
2
Universidade Estadual de Maringa/Universidade do Minho,
Maringa, Brazil

Objectives Investigate the potential interaction of vulvovaginal can-

didiasis isolates asymptomatic (ASS), with one episode (VVC) and
recurrent VVC (RVVC), with a human vaginal epithelium and the
relative expression of ALS genes family in same condition.
Methods Four strains of Candida albicans were used, one reference
strain (ATCC 90028) and three clinical isolates (5V, 7V and 9V)
obtained from female vaginal secretions. The clinical isolates were
separated according to symptoms presented by the patients. To assess
the adhesion ability of C. albicans to human epithelial cells by 2 and
6 h of incubation, was used as model HeLa cells. After adhesion
assays, adherent Candida albicans cells were harvested by detaching
the cervical cells monolayer to perform transcriptional analyses. The
total RNA was extracted using Trizol reagent (Invitrogen), adding
to solution acid-washed glass beads (Sigma) and vortexing for

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 213
Poster Presentations

P447 lipase, 0.5% carboxymethyl cellulose and 1% tributyrin oil is added

as an enzyme substrate. All the plates are incubated at 30 °C for 6
to 7 days and observe for zone of lysis around the fungal colony.
Could the determination of Aspergillus fumigatus mating Enzyme activity index was calculated for each isolate by taking ratio
type have prognostic value in invasive aspergillosis? of lysis diameter to colony diameter as described by Blanco et al.
M. Monteiro, R. Garcia-Rubio, L. Alcazar-Fuoli, T. Pelaez and (2002).
E. Mellado Results 51 Penicillium sps, comprising 23 clinical strains (including
P. marneffei, n = 13) and 28 strains of environmental Penicillium spp.
Instituto de Salud Carlos III, Majadahonda, Spain
was evaluated for extra cellular enzymes activity. It was noted that
no single strain of P. marneffei showed activity in protease and few
Objectives A clear link between mating type and virulence has been strains of the isolates have mild activity in lipase (Table 1). However,
demonstrated for some fungal pathogens, but has not been estab- cellulase (n = 7) and ∞ amylase (n = 12) activity were detected in
lished yet for Aspergillus fumigatus. However, some research in the significant number of P. marneffei. Although, the study detected non-
field has pointed out genes associated with or linked to mating type marneffei Penicillium spp. with activity of cellulase and ∞ amylase,
idiomorphs could play a role in pathogenicity. In addition, a statisti- 43% of non marneffei Penicillium spp. exhibited to have hydrolytic
cally significant association between mating type and invasiveness activity in protease. Additionally, clinical isolates produced 8 different
has recently been established. The main aim of this work was to spectrum of enzymes hydrolysis of which ∞ amylase (30.4%) was
search for an association between mating type and virulence in an commonly hydrolyzed followed by combined cellulase and ∞ amy-
alternative host model of infection, and also in the clinical setting. lase (21.7%). Similarly, environmental isolates could produce 12 dif-
Methods We determined mating type proportion (Mat1.1:Mat1.2) of ferent spectrums of extra cellular enzymes. Further, the enzyme
a collection of 200 A. fumigatus, (150 azole susceptible and 50 azole activity index to determine the invasiveness and aggressive nature of
resistant strains). In order to investigate the pathogenicity of Mat1.1 the fungal strains showed higher index from clinical source with a
and Mat1.2 from both, azole susceptible and resistant strains, we value as much as 1.8 (cellulase) and 2.2 (∞ amylase) compared to
tested 4–5 strains of each group in the Galleria mellonella model of environmental Penicillium spp. (1.3; cellulase and 2.1; ∞ amylase).
infection. Moreover, we retrospectively analyzed the outcome of Conclusion Although P. marneffei showed no activity in protease, its
patients that were infected with A. fumigatus of each mating type. potential in causing infection cannot be underestimated. Further,
Results The results obtained showed 48.5% of the analyzed strains their ability to hydrolyze cellulase and ∞ amylase could be a sugges-
were Mat1.1 and 51.5% were Mat1.2, which is in agreement with tion of environmental origin.
previous studies. However, we found that these percentages changed
when the strain collection was divided in azole susceptible and resis-
tant strains. The 150 azole susceptible strains kept these proportions
with 44.2% Mat1.1 and 55.7% Mat1.2, but among the 50 azole P449
resistant strains we found 60.8% Mat 1.1 and 39.2% Mat1.2.
In the infection model, strains with Mat1.1 genotype were consis-
tently more pathogenic than strains Mat1.2, independently of their Lactobacillus spp. directed inhibition of Candida albicans
phenotype (azole susceptible or resistant). There was a significant dif- in a Galleria mellonella infection model
ference (P < 0.0009) in median survival time between larvae R. D. Rossoni,1 B. B. Fuchs,2 M. S. Velloso,1 A. O. C. Jorge,1
infected withmating type Mat1.1 strains (3.1 days) and those ones J. C. Junqueira1 and E. Mylonakis2
infected with the Mat1.2 (5.1 days). In addition, when we looked at 1
the clinical outcome associated to 25 patients infected with A. fumi- Institute of Science and Technology, UNESP - Univ Estadual
gatus strains, we found that Mat 1.1 was linked to a mortality rate of Paulista, Sa~o Jose dos Campos, Brazil and 2Rhode Island Hospital,
64% while the ones associated to Mat1.2 genotype were 15%. Alpert Medical School and Brown University, Providence, USA
Conclusions (i) Our collection presents an equal strain distribution
of Mat1.1/Mat1.2 as previously reported. (ii) Strains with Mat1.1 Although a naturally colonizing microbe, the fungus Candida albicans
genotype were consistently more pathogenic than strains Mat1.2, can be responsible for disease and health problems during weakened
indicating that differences in pathogenicity are dependent on the health or changes to dental structures. A potential means of control-
mating type genotype but are not related to the strain phenotypic ling this infective microbe is through interactions with a probiotic
drug susceptibility. (iii) In the clinical setting, Mat1.1 was linked to a bacteria.
higher mortality than Mat1.2 genotype. Altogether, it would suggest Objectives Our study evaluates the inhibitory effect of Lactobacillus
that the mating type determination at the time of the diagnosis species against C. albicans using the greater wax moth Galleria mel-
might have a prognostic value of disease development. However, fur- lonella as an in vivo infection model .
ther studies would be necessary to a better understanding of the rela- Methods We used nine clinical strains of Lactobacillus recovered
tionship between differences in pathogenicity observed in the G. from the oral cavity to investigate the influence to C. albicans. The
mellonella model and the clinical outcome.

Table 1. Spectrum of extra cellular enzymes activity of Penicillium

spp.
P448

Extra cellular enzymes activity of Penicillium spp

R. Khuraijam and R. B. Yendrembam
Regional Institute of Medical Sciences, Imphal, India

Objective To screen for the production of important fungal enzymes

(Protease, Lipase, Cellulase and ∞ - amylase) which could act as
putative virulence factors among the Penicillium isolates and to eluci-
date if there is any difference in the spectrum of enzymes secreted by
Penicillium marneffei and other non marneffei strains.
Methods For analysis of protease, cellulase, lipase and a-amylase,
the isolates are inoculated onto skimmed milk agar, potato dextrose
agar, tributyrin agar and starch agar respectively. For cellulase and

ª 2015 The Authors

214 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

oral Lactobacillus strains were isolated from the saliva of 41 healthy

patients including the following species: L. paracasei (n = 5), L. rham-
nosus (n = 2) and L. fermentum (n = 2). In vitro analysis interrogated
the effects of Lactobacillus on the biofilm formation. Since C. albicans
uses biofilm formation during a lethal infection in G. mellonella, the
changes to larvae survival were monitored during a dual infection
process.
Results The in vitro results showed that Lactobacillus spp. were able
to inhibit C. albicans biofilm formation. In the in vivo study, injection
of Lactobacillus into G. mellonella larvae infected with C. albicans pro-
longed survival of these animals. The number of C. albicans CFU/mL
recovered from the larval hemolymph was lower in the group inocu-
lated with Lactobacillus compared to the control group. Furthermore,
some groups with strains of Lactobacillus were able to increase the
hemocyte count compared to the control group with C. albicans.
Conclusion Lactobacillus spp. inhibited in vitro biofilm formation by
C. albicans and protected G. mellonella against experimental candidia-
sis in vivo suggesting prokaryotic-eukaryotic interactions.
Figure 2. Phagocytosis of C. tropicalis in vitro by peritoneal
macrophages.

P450
macrophages. Phagocytic indices were determined by counting with
Candida tropicalis isolated from oral colonization of microscope analysis and the phagocytic index (PI). All experiments
patients with head and neck cancer present changes when were done in duplicate. It was to observe that all colonies before irra-
exposed to gamma radiation diation exhibited macro morphology of annular spicules (Figure 1a
F. A. V. Rodrigues, E. M. S. Bettega, B. Kischkel, E. S. Miazima, and 1b) and after irradiation the colonies missed these characteristics
completely, becoming smooth (Figure 1c and 1d). The PI of irradi-
F. T. Lucio, M. Negri and T. I. E. Svidzinski ated yeast was significantly smaller than non-irradiated yeast (Fig-
Universidade Estadual de Maringa, Maringa, Brazil ure 2). These results suggest that gamma radiation may influence
the virulence factors of yeast, making them more aggressive. Thus, it
Candida tropicalis isolated from the oral cavity of a patient with head is interesting to evaluate whether the impact on increased yeast viru-
and neck cancer submitted to in vitro radiation therapy in order to lence in vitro is the same in vivo, since invasive fungal infections
simulate a procedure used in cancer patients. A culture containing caused by C. tropicalis are emerging significantly, especially in tropi-
1x105 yeast ml
1 on Sabouraud Dextrose Broth tubes were exposed cal countries such as Brazil, affecting frequently in patients with neo-
to gamma radiation. The following conditions were calculated and plasia. The authors thank Sant’ana Radiation Oncology Center of
employed: irradiated (area 9 9 26 cm) to the right and left sides Maring a for radiation procedure and Fundac
a ~o Araucaria and CNPq
with spine angle 270 and 90 respectively. The source skin distance for financial support of this study. The authors declare that they
equipment was 80 cm; target depth 6.5 cm 72.8% depth dose. The have no conflict of interest.
irradiation rate was 50.93 cGy/min lasting 2m25s in 40 applications
of 180 cGy/day for each side, totaling 7,200 cGy. Thus, cultures
were irradiate once a day, five days a week using a diary fraction
dose of 180 cGy for 8 weeks. After irradiation of yeast, it was evalu- P451
ated the colony morphology and phagocytosis in vitro using mouse
peritoneal macrophages added at a ratio of 5:1 yeast to
Incubation period and early events of the natural history
of the acute form paracocciodioidomycosis: lessons from
patients with a single Paracoccidioides spp. exposure
R. Buccheri,1 Z. Khoury,1 L. Barata2 and G. Benard3
1
Instituto de Infectologia Emılio Ribas, Sa~o Paulo, Brazil; 2Hospital
e Maternidade Santa Catarina, Sa~o Paulo, Brazil and 3Medical
School, University of Sa~o Paulo, Sa~o Paulo, Brazil

Introduction At difference from the most prevalent, chronic form of

Paracoccidioidomycosis, the acute form represents a continuum from
the initial respiratory infection to full-blown disease. However, as this
form of the disease generally develops in individuals living in ende-
mic areas and thus repeatedly exposed to the fungus, it has been dif-
ficult to determine when the initial infection with Paracoccidioides
spp. has occurred. This missing information has hindered the under-
standing of the first steps of natural history of this disease and the
determination of its period of incubation.
Objectives and Methods Herein we describe for the first time two
cases of AF PCM in patients with a single time point exposure to
Paracoccidioides spp. in whom we were able to determine precisely the
time lapsed from exposure to the fungus to the onset of signs and
symptoms.
Results The two patients always lived downtown S~ ao Paulo city.
The first patient went to a one-day ecological trekking and the other
Figure 1. A e B) C. tropicalis without radiation exposure; C e D) C. traveled for a weekend in a farm in S~ ao Jose dos Campos, both cases
tropicalis after 7,200 cGy radiation exposure. at the Paraiba do Sul river valley. In case 1 the initial pulmonary

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 215
Poster Presentations

Figure 1

infection was unapparent, and after forty-one days laboratory data of

an ongoing but subclinical infectious process such as marked eosino-
philia and radiological chest X-ray showing normal pulmonary par-
enchyma but an enlargement of left peri-hilar node, compatible with
deep lymphatic involvement (Fig. 1A) wasdemonstrated. The first
manifestations of the AF disease appeared 4 months after Paracoccid-
ioides spp. exposure, such as fever, weight loss, papular erythematous
cutaneous lesion (Fig. 1B), lymph node enlargement (cervical, supra-
clavicular, pre-auricular, axilar) and tender hepatosplenomegaly. A
new chest X-ray showed normal pulmonary parenchyma and no
peri-hilar enlargements. After 1 month a biopsy of a dorsal cuta- Figure 1. Serum beta-d-glucan levels according to citrulline levels
neous lesion provided the diagnosis of PCM (Fig. 1C). Serum anti-P.
brasiliensis antibodies titer was 1:16. Treatment with itraconazole
was started and improvement was seen after 1 week. He was dis- the Fungitell assay (Associates of Cape Cod, Falmouth, MA). BDG
charged and continued his treatment with sulfadiazine. The patient levels were interpreted as follows: <60 pg ml
1 negative, 60–
has been cured of PCM. In case 2, the patient presented fever, non- 80 pg ml
1 intermediate, >80 pg ml
1 positive.
productive cough and night sweats 45 days after infection. Fifteen Citrulline serum levels were used as a surrogate marker for mucosi-
days later, she referred diffuse abdominal pain, erythematous patches tis. Citrulline levels were determined at the University Hospitals Leu-
on the face, trunk, and upper limbs and palpable bilateral cervical ven, Belgium, using liquid chromatograph-tandem mass spectrometry,
and axillar lymph nodes. The persistent cough was investigated ini- and interpreted as follows: <10 lm diffuse destructive mucositis,
tially with a chest X-ray, which showed a mild left lung basal infil- >10 lm mild mucositis or no evidence for intestinal mucositis.
trate (Fig. 1D). The cough subsided but the other signs and Results Primary underlying disease was acute myeloid leukemia in
symptoms progressed. Laboratorial results revealed eosinophilia and 17 patients (73.9%), acute lymphoid leukemia in 4 patients (17.4%)
anemia. A cervical lymph node biopsy diagnosed PCM and serum and myelodysplastic syndrome in 2 patients (8.7%).
anti-P. brasiliensis antibodies titer was 1:256. The patient was treated 17 cases (42.5%) had citrulline levels <10 lm and 23 cases
with Amphotericin B followed by itraconazole and has been cured of (57.5%) >10 lm.
PCM. Median BDG levels according to citrulline levels are displayed in
Conclusion Our patients confirm that, few weeks after infection, Figure 1.
Paracoccidioides spp. affect the pulmonary lymphatic system,causing Spearman-Rho analysis did not show a correlation between serum
no or mild to moderate self-limited symptoms, and eventually abnor- BDG and citrulline levels (r =
0.034, P = 0.833).
malities on chest X-ray, all of which spontaneously subside. These Conclusion Our results suggest that intestinal mucositis is not a
cases provide some insights on the natural history of this mycosis, cause of elevated serum BDG levels in hematological malignancy
the extent of the host exposure to the fungus, and the determination patients.
of its incubation period.

P453
P452
A morphological study on evolution of cutaneous
Intestinal mucositis in hematological malignancy patients chromoblastomicotic infection before and after therapies
is not a cause of elevated serum 1,3-beta-d-glucan levels R. Minotto,1 M.L. Scroferneker2 and M.I. Edelweiss2
1
J. Prattes,1 K. Vanstraelen,2 F. M. Reischies,3 F. Prueller,1 ISCMPA, Porto Alegre, Brazil and 2Universidade Federal do Rio
T. Valentin,1 I. Zollner-Schwetz,1 R. Krause,4 R. B. Raggam,4 Grande do Sul, Porto Alegre, Brazil
I. Spriet2 and M. Hoenigl1
1
Medical University of Graz, Graz, Austria; 2University Hospitals Objectives This study compared patients with elevated lesions of
Leuven, Leuven, Belgium; 3Meduni Graz Section of Infectious chromoblastomycosis and patients with flat lesions evaluating the
Diseases and Tropical Medicine, Graz, Austria and 4Medical morphological components of biopsies in cases with regular follow-up
before and after treatments.
University Hospital of Graz, Graz, Austria
Methods: Historical cohort study with before and after type of
analysis without control group.
Background In the absence of an active invasive fungal infection Results The study included 67 patients who were divided into two
(IFI) serum 1,3-beta-d-glucan (BDG) may be a reasonable indicator groups, according to the clinical course of disease and to the type of
of gut mucosal barrier impairment and microbial translocation. The dermatological lesion: 24 of them had flat and 43 had elevated
aim of this study was to investigate whether serum BDG levels are lesions, biopsied before and after therapies. “Bad responders”were
elevated in hematological malignancy patients without IFI but with found in 95% of cases with elevated lesions. “Good responders”were
intestinal mucositis. found in 92% of cases with flat lesions. Patients with elevated lesions
Methods We determined same day serum BDG and citrulline levels had persistence of high intensity of the histopathological findings
in 40 samples obtained from 23 patients with underlying hematolog- associated with a unsatisfactory response to therapy and those with
ical malignancies and without IFI/signs of pneumonia. BDG evalua- flat lesions showed more decreasing of the intensity of this findings
tion was performed at the Medical University of Graz, Austria, using (with significant statistical analisys to all elements through odds ratio

ª 2015 The Authors

216 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

and P = 0.01) with a satisfactory response to therapies. After treat- [2] Catal
an Gonzalez M, Montejo Gonzalez JC. Anidulafungin: A
ments the relative risk of high intensity of these components in the new therapeutic approach in antifungal therapy. Pharmacology of
elevated lesions were higher than those of flat lesions (P < 0.001). anidulafungin. Rev Iberoam Micol 2008;25:92–100
Conclusion The high and low intensity of the histopathological ele- Funding This work has been funded by UFI11/25 (University of the
ments in association with the type of lesion denoted the presence of Basque Country), GIC07 123-IT-222 and by SAI13-112, Saiotek
polarity of the granulomas in this disease either before or after thera- 2013 program (Basque Goverment).
pies. The pathogenic events may be better understood and its
histopathology findings better associated with clinical aspects. Little
is known regarding the pattern of histopathological aspects in flat
and elevated lesions before and after therapies. The cutaneous lesion P461
presenting as a verrucous plaque had a granulomatous reaction with
a suppurative granuloma (with high intensity of the elements) with
several fungi cells. It was suggested that patients with this type of Posaconazole Pharmacokinetics among Lung Transplant
lesion have a type Th2 immunological response (“bad response”to Recipients
therapy), while in erythematous atrophic plaque had a granuloma- C. Clancy, M. H. Nguyen, E. Press and R. K. Shields
tous reaction less supurative resembling a tuberculoid granuloma
University of Pittsburgh, Pittsburgh, USA
(low intensity of the elements) with few fungi cells and a type Th1
response (“good response”to therapy). These findings confirm the
hypotheses that is a polar disease relating either to polar granuloma Background Posaconazole (POS) is a promising agent for prevention
or polar lesions respectively. and treatment of invasive fungal infections among lung transplant
recipients (LTR), but there are very limited pharmacokinetic (PK)
data in this population. We previously showed that POS trough
P460 levels >0.5 are associated with successful outcomes following LT. We
hypothesized that LTR, in particular those with cystic fibrosis (CF),
would have difficulty achieving PK targets.
Pharmacokinetic/pharmacodynamic simulation of Methods We conducted a prospective study of POS oral suspension
anidulafungin in an in vitro model against Candida PK among LTR with or without CF. Steady-state (SS; after a mini-
parapsilosis clade mum of 5 days of therapy) blood samples were obtained at 0, 2, 4,
S. Gil-Alonso,1 N. Jauregizar,2 I. Ortega,3 E. Eraso,1 E. Su
arez2 6, 8, and 12 h after oral administration. POS levels were determined
and G. Quindos  1 by a validated HPLC with fluorescence detection assay; calibration
curves were linear over 0.02–3 lg ml
1. Maximum, minimum, and
1
Univ. Paıs Vasco UPV/EHU Facultad de Medicina y Odontologıa, average plasma concentrations were calculated (Cmax, Cmin, Cavg).
Bilbao, Spain; 2Faculty of Medicine, University of the Basque Area under the curve (AUC) was determined from time 0 to 12 h.
Country, Bilbao, Spain and 3FAES FARMA, Leioa, Spain Results 20 LTR were enrolled (7 with CF). 60% and 85% were men
and white, respectively. POS doses ranged from 600 -
Objectives To simulate the expected time-kill curves of anidulafungin 1200 mg day
1. There was no correlation between dose and level.
for Candida parapsilosis, Candida metapsilosis and Candida orthopsilosis Median age and weight were lower for CF pts (36 yrs and 57 kg)
using a previously developed mathematical Emax sigmoid model and compared to non-CF pts (61 yrs and 78 kg). Mean time to Cmax
human pharmacokinetic data for a typical dosing regimen. was 4.7 h. Only 20% of pts had Cavg levels >0.5 lg mL
1. Mean (s-
Methods Three reference strains were included in this study, C. parap- tandard deviation) Cmax (0.4 [0.3]), Cmin (0.3[0.2]), Cavg (0.3
silosis ATCC 22019, C. metapsilosis ATCC 96143 and C. orthopsilosis [0.3], and AUC (3.7 [3.2]) were lower among CF pts than non-
ATCC 96139. Time-kill curve analysis and mathematical modelling of CF pts (1 [0.8], 0.6 [0.5], 0.7 [0.6], and 8.6 [7.4], respec-
the time-kill curve data were performed using a nonlinear mixed-effect tively) (P = 0.04, 0.05, 0.03, and 0.03, respectively). Mean oral
approach as appropriate with NONMEM 7 (ICON Development solu- clearance (L h kg
1) was 189 higher among CF pts (10.6  18)
tions, USA). A previously described adapted Emax model [1] was tried versus non-CF pts (0.6  0.4; P = 0.03). After dose normalization,
to fit to the log-transformed data of the static time-kill curve experi- mean POS AUC for CF pts was decreased by 57% compared to non-
ments of anidulafungin. Simulations of the expected time-kill curves CF pts (P = 0.06). Trough levels were well correlated with AUC
for each isolate were made using the estimated pharmacodynamic (R2=0.95, P < 0.0001); however, significant inter-patient variability
parameters, from the Emax model, and human pharmacokinetic data was observed for both CF and non-CF pts.
obtained from in vivo pharmacokinetic parameters of anidulafungin Conclusions POS levels are suboptimal among LTR, particularly
(t1/2 = 25.6 h; Vd = 33.4 L) [2]. Phoenix WinNonlin (Certara, USA) those with CF. Trough levels are representative of SS AUC and ideal
software program was used to simulate plasma concentration-time for therapeutic drug monitoring. Given the challenging PK of POS
profiles for recommended multiple intravenous dosing regimen of oral suspension in this population, there is a pressing need to study
anidulafungin (loading dose/maintenance dose, 200/100 mg with a PK of new formulations.
dosing interval of 24 h up to 6 days) and then applied to the adapted
Emax model to simulate in vivo time-kill curves.
Results The results of the simulations showed that anidulafungin at
the selected dosing schedule would be effective in a species-dependent P462
way at easily achievable serum levels. A rapid fungicidal activity
would be exerted against C. metapsilosis ATCC 96143. Similarly,
against C. orthopsilosis ATCC 96139, anidulafungin would be fungici- Voriconazole blood levels and its major metabolite,
dal. A prolonged fungistatic activity against C. parapsilosis ATCC voriconazole-N-oxide: Utility on Therapeutic Drug
22019 was observed, with reductions from starting inocula that Monitoring
were predicted to exceed 1 log. A. Gomez-Lopez,1 L. Bernal-Martınez1 and A. Alastruey-
Conclusion The mathematical Emax sigmoid model can be used to Izquierdo2
simulate expected time-kill data for typical anidulafungin dosing regi- 1
CNM-ISCIII, Majadahonda, Spain and 2Spanish National Centre
men. Our approach of combining in vitro time-kill data with existing
for Microbiology, Instituto de Salud Carlos III, Majadahonda, Spain
in vivo pharmacokinetic data, might serve in the future to define opti-
mal antifungal regimens against candidiasis.
References [1] Li Y, Nguyen MH, Cheng S, Schmidt S, Zhong L, Background Voriconazole is considered the first-line agent in the
Derendorf H, et al. A pharmacokinetic/pharmacodynamic mathemati- treatment of invasive aspergillosis and one of the best positioned
cal model accurately describes the activity of voriconazole against Can- alternatives to handle emerging fungal infection with high resistance
dida spp. in vitro. Int J Antimicrob Agents 2008;31:369–74 profiles (Scedosporium, Fusarium . . .). However, certain peculiarities

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 217
Poster Presentations

such as nonlinear pharmacokinetic profile are reflected in large vari- influence on the hepatic and renal function due to the use of the
ability in exposure and therefore in difficulties in adjusting the effec- drug and if there are differences in patients undergoing bone marrow
tive dose. One of the mechanisms responsible for such variability is transplantation or in patients taking cyclosporine.
related to their hepatic metabolism conditioned by the presence of Methods Thirty haematology patients under prophylactic treatment
concomitant therapy and individual genetic factors. Primary route of with posaconazole as mono-therapy (600 mg d
1 in three divided
metabolism involves oxidation of N-fluoropyrimidine ring leading to doses) were enrolled after written consent and record of all their rele-
the formation of major metabolite, N-oxide VRC (VRC-NO) which has vant medical details. Serum specimens were collected at the end of
lower activity. The evaluation of the relationship [metabolite/active the first and the second week of treatment, just before the morning
drug] while monitoring blood concentration is proposed as a useful dose, 2 h and 6 to 8 h afterwards. Levels were measured with a vali-
strategy to predict metabolic profile and individually estimate the dated bioassay in 157 specimens and with HPLC in 51 of them.
drug exposure. This strategy might help to maximize the effectiveness ALT, AST, ALP, bilirubin, c-GT, urea and creatinine were measured
of this antifungal treatment. on each occasion. Analysis was done with non-parametric
Objective The objective of this study is firstly, to develop and validate methodology.
a chromatographic method to detect and quantify VRC and its major Results All inter and intra daily posaconazole levels were correlated
metabolite in serum samples, valid for estimating individual metabolic (r > 0.8, P < 0.05), and there was not a statistical difference
rate. Secondly, to describe the metabolic rate from serum samples between them irrespective of the timing (P > 0.05). Interestingly,
received in our department to monitor voriconazole concentrations. there was a slight decrease at the 2 h post dose specimens, although
Method A new high-pressure liquid chromatography assay was not statistically significant. The same results were found with HPLC
developed using a stepwise gradient elution profile. The proposed measurements. There was not a statistical difference (P = 0.056)
method enables the simultaneous quantification of VRC and its between bioassay (1.59  1.03 mg l
1) and HPLC levels
metabolite in 150lL serum aliquots, after a first step of protein pre- (1.24  0.80 mg l
1), while they were significantly correlated
cipitation and direct injection of resulting supernatant. The analytical (r = 0.86, P = 0.000). There was no inter-day variation of the hep-
run lasting <16 min per sample. For the validation procedure linear- atic or renal function markers which were within the reference
ity, accuracy and precision parameters were determined. ranges, nor a correlation with posaconazole levels, while the levels of
Results The validation procedure establishes that the method was the drug were similar, independently of the cyclosporine or other
linear between concentrations ranging from 0.125 and 8 mg l
1 for immunosuppressive regimen and previous bone marrow transplanta-
both components (analytical range). The correlation coefficient was tion or not.
higher than 0.9. The retention time for VRC-NO and VRC was 7.3  Conclusions Thus, it appears that in clinically stabilized patients a
0.06 and 9.3  0.14 min, respectively. In 42% of the samples evalu- random specimen on any day after steady state serum concentrations
ated voriconazole concentration remained at therapeutic levels (1– have been achieved is adequate in order to monitor posaconazole
5.5 mg l
1, average 2.6 mg l
1). The metabolic rate in these sam- levels. Moreover, when the drug is used as monotherapy a bioassay
ples remained at values close to 1 (average 1.29). 50% of the sam- is an acceptable, alternative to the HPLC. The drug was well and
ples showed subtherapeutic levels (<1 mg l
1, mean 0.31 mg l
1). safely tolerated in all thirty patients in this study, there was no
For these samples the average metabolic rate exceeded the value of 1 breakthrough fungal infection during treatment and the levels were
(mean 6.65). The remaining samples showed voriconazole levels not affected by the use of cyclosporine or other immunosuppressive
above the therapeutic value (> 5.5 mg l
1), and significantly lower drugs or previous bone marrow transplantation.
metabolic rate (mean 0.43).
Conclusions 1) The method proposed allows the simultaneous
quantification of VRC and its major metabolite VRC-NO in serum
samples. 2) The estimation of the metabolite/active drug relationship P464
could quickly detect patients with impaired voriconazole metabolism.

Drug interaction profiles of isavuconazole, voriconazole

and posaconazole with immunosuppressants metabolized
P463 by CYP4503A4 (CYP3A4)
R. Townsend,1 A. Desai,1 N. Azie,1 M. Jones,2 M. Engelhardt2
and A. H. Schmitt-Hoffmann2
Evaluation of the methodological approach for therapeutic 1
drug monitoring of prophylactic posaconazole in Astellas Pharma Global Development, Inc, Northbrook, USA and
2
haematology patients Basilea Pharmaceutica International Ltd, Basel, Switzerland
T. A. Vyzantiadis,1 E. Yannaki,2 D. A. Oliver,3 E. M. Johnson,4
A. M. Markantonatou,1 Z. Bousiou,2 A. Louka,1 E. Zachrou,1 Objectives Isavuconazole is the active moiety of isavuconazonium
A. Anagnostopoulos2 and N. Malissiovas1 sulfate, a water-soluble prodrug for oral and intravenous administra-
1 tion. Isavuconazole is a broad-spectrum triazole with in vitro activity
1st Dep. of Microbiology, Medical School, Aristotle University of against clinically relevant pathogens including Aspergillus spp., Can-
Thessaloniki, Thessaloniki, Greece; 2General Hospital G. dida spp., Cryptococcus spp., and Mucorales.
Papanicolaou, Thessaloniki, Greece; 3Public Health England (PHE), This makes isavuconazole potentially useful to treat patients with
Mycology Reference Laboratory, Bristol, United Kingdom and invasive mould disease (IMD) including those receiving immunosup-
4
Public Health England, Bristol, United Kingdom pressants (bone marrow/solid organ transplant recipients), at risk of
renal/hepatic impairment and with an increased potential for drug-
Objectives Although tablet and IV formulations of posaconazole are drug interactions (DDIs) due to multiple co-medications.
now available, the oral suspension is still widely used and the sys- Isavuconazole shows predictable pharmacokineticsi, high oral
temic exposure of this formulation is significantly affected by acid, fat bioavailabilityi and no food effectii. Isavuconazole is a moderate
and food. It has a long half-life and levels reach steady state towards CYP3A4 inhibitor. Unlike voriconazole, isavuconazole does not inhi-
the end of the first week of use. A recommended target for prophy- bit CYP2C9 or CYP2C19. A review of clinical pharmacology informa-
laxis is a trough level of 0.7 mg l
1. It is metabolized mainly in the tion was therefore performed to compare the potential for DDIs
liver, interacting with several drugs that use similar pathways. between immunosuppressants metabolized by CYP3A4 and the tria-
The aims of the study were: to analyse intra and inter-day stability zoles isavuconazole, voriconazole, or posaconazole.
and assess whether a unique daily specimen could be used for TDM Methods A review of US and EU prescribing information was con-
thus facilitating and lowering the cost of the procedure; to determine ducted to identify clinical pharmacology information related to multi-
if a bioassay and an HPLC procedure produce similar results to allow ple doses posaconazole or voriconazole on co-administration with
use of whichever method is available; to reveal if there is an midazolam (typical CYP3A4 probe substrate for clinical DDI studies)
and immunosuppressants metabolized by CYP3A4. A literature

ª 2015 The Authors

218 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Table 1 resistance mechanism to antifungals. Because of the high resistance

of C. albicans biofilms to antifungals, new pharmacological strategies
to treat these infections are needed. Non-steroidal anti-inflammatory
drugs (NSAIDs) have antibiofilm activity on C. albicans. On the other
hand delivery of exogenous NO through a NO donor molecule, has
shown to inhibit C. albicans biofilms formation and to potentiate the
effect of conventional antifungals. NO releasing aspirin (NO-ASA),
NCX-4040 has been proposed for treatment of different cardiovascu-
lar affections and inflammatory conditions, since maintain the anti-
inflammatory effects of aspirin, besides the beneficial effects of NO,
such as endothelial protection. According to this information, in this
work we evaluated the antifungal/antibiofilm effect of NO-ASA on
Candida albicans clinical isolates obtained from denture stomatitis
patients.
Objectives 1) characterization clinical isolates of Candida spp.
obtained from oral mucosa in denture stomatitis patients; 2) evaluate
susceptibility of clinical Candida spp. strains to fluconazole, NO-ASA
and their combination.
Methods Candida spp. obtained from oral mucosa of denture stomati-
tis patients (N = 60), were identified through CHROMagar Candida
search was also performed to obtain missing information. Clinical Medium BDTM and by sequencing the ITS1-5.8S rDNA-ITS2 region
pharmacology information for isavuconazole was derived from pub- using the primers ITS1 and ITS4. To evaluate antifungal susceptibil-
lished abstracts. The effects of isavuconazole, voriconazole or ity, isolated strains were standardized to 0.5 McFarland and then
posaconazole on the exposure of the respective CYP3A4 substrates - grown on sabouraud agar plates and disk diffusion tests were per-
expressed as area under the curve (AUC) - were compared. formed in presence or absence of fluconazol, NO-ASA and their
Results The AUC of oral midazolam (probe substrate for CYP3A4) combination.
was increased 2-fold with isavuconazole, 5-fold with posaconazole Results from the 60 strains, 55% (N = 33) were identified as C. albi-
and 10-fold with voriconazole. Results with immunosuppressants cans while 27% corresponded to C. tropicalis (n = 16) and remaining
metabolized by CYP3A4 are presented in the table below. 18% was identified as C. glabrata (n = 18). According to susceptibility
Conclusion Considering midazolam probe substrate, isavuconazole assays 6 strains of C. albicans were classified as resistant to flucona-
exhibits moderate inhibition of CYP3A4 (≥2 and <5 -fold), whereas zole (10%) and the rest as susceptible. NO-ASA had no antifungal
posaconazole and voriconazole are strong CYP3A4 inhibitors (≥5- effect on any of the strains assayed. Addition of 25 ug of NO-ASA to
fold). Clinical pharmacology studies demonstrate that the effect of fluconazole standard disks (25 ug) resulted in marginal increase in
isavuconazole on CYP3A4 substrates is substantially less pronounced susceptibility, ranging from 6 to 10% increase. In resistant strains
than posaconazole or voriconazole. The clinical pharmacology profile however, NO-ASA was able to increase the effect of fluconazole in
indicates potential advantages of isavuconazole in treating patients 20–30% (P < 0.05, ANOVA), reversing the resistant behavior of
receiving immunosuppressants metabolized by CYP3A4. those strains.
References (i) Schmitt-Hoffmann, Antimicrob Agents Chemother Conclusions NO-ASA was able to potentiate antifungal effect of Flu-
2006;50:279–285; (ii) Schmitt-Hoffmann, ICAAC 2008:A008; (iii) conazole in resistant strains obtained from denture stomatitis
Desai, ICAAC 2012:A1936; iv Saari, Clin Pharmacol Ther patients. Since NO-ASA has no antifungal effect, we hypothesize that
2006;79:362–70; (v) Yamazaki, ASCPT 2013:PIII-76; (vi) Yamazaki, prostaglandins synthesis inhibition or NO releasing effect alters drug
ASCPT 2013:PIII-75; (vii) Desai, ASCPT 2013:PIII-71; (viii) Saad, resistance mechanisms. We are currently working to elucidate that
Pharmacotherapy 2006;26:1730–44. postulate.
ASCPT: American Society for Clinical Pharmacology & Therapeu-
tics; ICAAC: Interscience Conference on Antimicrobial Agents &
Chemotherapy.
(Encore abstract: Previously presented at the European Congress of P470
Clinical Microbiology and Infectious Disease [ECCMID], Copenhagen,
Denmark; April 25–28, 2015; P0216)
Immunoreactive proteins of Lichtheimia corymbifera for
farmer’s lung serodiagnosis.
L. Millon,1 B. Rognon,1 C. Barrera,1 B. Valot,2 A. Bellanger,1
P465 M. Quadroni,3 J. Dalphin,1 G. Reboux4 and M. Monod3
1
University Hospital, Besancon, France; 2UMR/CNRS Chrono
Effect of NO-aspirin on susceptibility to fluconazole in Environnement, Besanc
on, France; 3University of Lausanne,
Candida albicans clinical isolates obtained from denture Lausanne, Switzerland and 4University Hospital J. Minjoz,
stomatitis patients Besanc
on, France
A. E. Molina, F. Madariaga, B. Urzua Orellana, R. Ramires
Fernandez, J. A. Jara-Sandoval, R. Fernandez-Soto, C. Zarate and Objective Lichthteimia corymbifera has been described as one of the
E. Herreros main causative agents of farmer’s lung disease (FLD). Serodiagnosis
is currently routinely performed by immunoassays with crude anti-
Institute for research in dental sciences, University of Chile, gens. Serodiagnosis is based on immunoprecipitation techniques with
Santiago, Chile homemade crude antigens and is not standardized. The objectives of
this study were to identify immunogenic proteins from a strain of L.
Candida albicans form biofilms on diverse biomaterials and is fre- corymbifera isolated from hay, and to synthesize the corresponding
quently present in denture prosthesis participating on developing of recombinant antigens for the development of a standardized ELISA
denture stomatitis, one of the most common oral pathology in elderly test.
people. In a large percentage of denture stomatitis patients, C. albi- Methods An immunoproteomic approach based on comparative
cans biofilms function as an infection reservoir that in susceptible western blot analysis with serum samples from FLD patients and
individuals could lead to invasive candidiasis. Biofilms presence is healthy exposed controls (HEC) was used to identify FLD specific
crucial for their persistence in the host, acting as a multidrug proteins.

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 219
Poster Presentations

We recruited 41 FLD patients and 43 HEC from five university 20 days. To reduce the size of the capsules, the yeast cells were then
hospital pneumology departments in France and Switzerland. L. seeded in acid Sabouraud agar plus 2.9% NaCl, and incubated at
corymbifera proteins were extracted from the reference strain BBCM/ 30 °C for 48 h. For each strain, the capsule size was measured in
IHEM 3809 isolated from FLD-linked hay. Proteins were separated by light microscope using the software Axiovision. At least ten different
two-dimensional electrophoresis and subjected to western blotting, fields of the slides were randomly chosen and 40 to 50 capsule cells
with sera from FLD patients (n = 7) or controls (n = 9). FLD-specific were measured. Subsequently, the strains were analyzed by MALDI-
proteins were identified by mass spectrometry (LC-MS/MS) and were TOF mass spectrometry after standard extraction protocol with etha-
produced as recombinant antigens as previously described1. The diag- nol 70% and formic acid 70%. Then, the supernatants were placed
nostic performance of ELISA tests using the recombinant antigens in MALDI target plate wells, dried at room temperature and overlaid
was assessed with all the sera from FLD patients and controls. with the matrix containing a saturated solution of HCCA. Mass spec-
Result When compared western blot membranes revealed by FLD tra (MS) were generated by the Autoflex MALDI-TOF mass spectrom-
and HEC serum, 25 spots were considered as FLD specific. The 25 eter and compared to main spectra of both C. neoformans and C.
FLD specific spots were cut from the gel and analyzed by LC-MS/MS. gattii from the database Biotyper 3.1. To ensure the reproducibility of
Sixty-nine different proteins were identified from the 25 spots. Six spectra each strain was tested in quadruplicate in eight different
proteins were selected to be produced as recombinant antigens: experiments. Results were expressed as log score of 1.700–1.999
acylCoAdehydrogenase, proteasome alpha, pyruvate kinase, malate indicating a probable genus identification and log score above 2.000
dehydrogenase, ATP synthase alpha, dihydrolipoyl dehydrogenase. indicating secure genus and species identification.
ELISA tests were performed using each recombinant antigen, with Results the strains of C. gattii developed larger capsule sizes in com-
all the sera from FLD patients (n = 41) and controls (n = 43). Dihy- parison with C. neoformans strains (mean 16.8 lm 9 5.6 lm,
drolipoyl dehydrogenase was the most effective recombinant antigens P = 0.002) when incubated in the induction medium. Capsule size
for discriminating FLD patients from controls, with AUC = 0.82 and was inversely related to MS quality. All replicates with capsule size
with sensitivity and specificity of 81% and 77%, respectively. ELISA above 10 lm had unreliable species identification. The mean capsule
using proteasome a also showed AUC above 0.80, with sensitivity of size of Cryptococcus strains with correct species identification was
88%, but sensitivity was only 65%. 2.18 lm, whereas for those assigned with log score below 2.000
Conclusion Involvement of L. corymbifera in FLD has been described was 5.77 lm (P = 0.03).
mainly in East of France and Finland. Combining recombinant anti- Conclusion The capsule of Cryptococcus negatively interfered with
gens from L corymbifera with recombinant antigens from other the performance of MALDI-TOF MS species identification. Capsule size
micro-organisms (Saccharopolyspora rectivirgula, Aspergillus) involved reduction is recommended to achieve a reliable laboratory identifica-
in FLD would be probably helpful to produce a standardized ELISA tion of Cryptococcus isolates by this technology.
kit effective for diagnosing FLD whatever the geographic location of
the patient. A prospective study, using such a test combining most
effective recombinant antigens from S rectivirgula1and from Aspergil-
lus2 with dihydrolipoyl dehydrogenase and proteasome alpha from L. P475
corymbifera, is ongoing in our lab to assess diagnosis performance
with patients from different geographic origins.
1. Barrera C, Millon L, et al. Immunoreactive proteins of Saccha- Dermatophyte infections in the Lisbon and Tagus valley
ropolyspora rectivirgula for farmer’s lung serodiagnosis Proteomics Clin C. Verı́ssimo,1 J. C. Brand~ao,1 H. L. Simo
~ es2 and R. F. P. Sabino1
Appl. 2014: 8(11–12):971–81 1
National Institute of Health Dr. Ricardo Jorge, Lisboa, Portugal
2. Millon, Roussel et al. Aspergillus recombinant antigens for sero-
and 2INSA, Lisboa, Portugal
diagnosis of farmer’s lung disease J Allergy Clin Immunol, 2012,
130(3):803–805.
Objective A retrospective study was done in the Portuguese
National Institute of Health, in order to establish the prevalence and
characterize dermatophytic infections within the NUTSII region of
Lisbon and Tagus Valley.
P471 Material and methods This retrospective study included 4193 bio-
logical samples from patients with medical suspicion of fungal infec-
Variation in the polysaccharide capsule size interferes with tion, collected from 2004 to 2013. Samples were obtained by
identification of Cryptococcus neoformans and C. gattii by extracting hairs and scraping skin and nails using a sterile curette
MALDI-TOF mass spectrometry over the affected areas. Samples were microscopically examined after
D. Y. Thomaz,1 M. S. M. Vidal,2 R. C. Grenfell,3 M. C. Giudice,2 30% W/V for 20 min potassium hydroxide preparation and cultured
on Sabouraud dextrose agar with cicloheximide. Species were identi-
L. Juliano Neto,3 G. Benard,4 G. M. B. del Negro5 and
fied by the observation of morphological features that included col-
J. N. Almeida Jr5 ony pigmentation, texture, growth rate and distinctive microscopic
1
School of Medicine - University of Sa~o Paulo, Sa~o Paulo, Brazil; structures. Physiological tests (urea, vitamin and amino acid test
2
Institute of Tropical Medicine, University of Sa~o Paulo, Sa~o agars) were used whenever necessary.
Paulo, Brazil; 3School of Medicine, Federal University of Sa~o Results The average frequency of dermatophyte infections was 21%,
Paulo, Sa~o Paulo, Brazil; 4Medical School, University of Sa~o ranging from 18% to 26%; 841 individuals (425 female and 410
Paulo, Sa~o Paulo, Brazil and 5Clinics Hospital, School of male) had positive cultures. Tinea capitis was confirmed in 236 (28%)
Medicine, University of Sa~o Paulo, Sa~o Paulo, Brazil patients and was more prevalent in children from the group of 1–
9 years old. In scalp dermatomycosis, Microsporum audouinii was the
most frequently isolated species (N = 120, 51%), followed by T.
Objectives to investigate the interference of Cryptococcus’s capsule soudanense (N = 44, 19%). Males were more affected (58%) than
size in MALDI-TOF MS species identification. females (42%).
Methods four C. neoformans reference strains, WM 148 (VNI), WM Onychomycosis caused by dermatophytes were confirmed in 385
626 (VNII), WM 628 (VNIII), WM 629 (VNIV), and four C. gattii, cases (46%). Other fungi recognized as cause of onychomycosis were
WM 179 (VGI), WM 178 (VGII), WM 161 (VGIII), and WM 779 not considered for this report. Skin samples were positive in 220
(VGIV) were used for capsule growth experiments. Initially the yeast cases (26%).
cells of each strain were incubated overnight in acid Sabouraud The most prevalent dermatophytes isolated in nail and skin sam-
broth at 30 °C with shaking. After centrifugation, the pellets were ples were T. rubrum (206 and 93 isolates, respectively) and T. menta-
incubated in the capsule growth inducing medium (Sabouraud broth grophytes with (49 and 39 isolates respectively).
diluted 10 times, pH 7.3) at 37 °C with shaking. The capsule induc- E. floccosum was the species less frequently found in skin samples
tion was carried out replacing medium every 48 h during a period of (1%).

ª 2015 The Authors

220 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

Conclusion The frequency of infections by dermatophytes has

revealed itself steady for the last 10 years. The spectrum of dermato-
phytes is similar to those reported in other studies for tinea corporis
and onychomycosis, being T. rubrum the main aetiological agent.
However, our results differed from several studies that describe a
raise, during the recent years, in the number of cases of tinea capitis
caused by M. canis, especially in other Mediterranean countries. Our
results showed a high prevalence of anthropophilic species (M.
audouinii and T. soudanense) in the region of Lisbon and Tagus valley,
where the number of foreign citizens from African countries is
higher.
Like in other studies the number of infections by E. floccosum was
very low suggesting that there was a replacement of this aetiological
agent by other dermatophytes like T. rubrum.

P476

In treatment of primary cutaneous Aspergillosis, systemic

antifungal therapy and surgical management; Presentation
of two cases
V. Avkan-Oguz,1 I. S. Satoglu,1 M. Celik,1 A. E. Acan1 and
N. Yapar2
1
Dokuz Eylul University Faculty of Medicine, Izmir, Turkey and
2
Dokuz Eylul University, Izmir, Turkey

Objectives Aspergillus skin involvement occurs by hematogenous

spread or local inoculation. Primary cutaneous aspergillosis that
appears with local inoculation is rarely determined on immunocom-
petent patient. However, it is an important cause of morbidity and
mortality in surgical patients. Treatment is controversial and differ-
ent medical and/or surgical approaches can be applied. Here, two
cases of primary cutaneous aspergillosis diagnosed with the growth
of Aspergillus flavus on tissue samples are presented. In the treatment
of patients, voriconazole and surgical treatment was applied together.
Different methods were used for the wound care.
thought that slow progressive massive necrosis might be guiding for
Methods Patient 1: 53 years old female, who had a motorcycle acci-
the opportunistic fungus infection in both cases. In treatment of
dent resulted with left leg injury. She had a wound with a large tis-
cases, repeated debridement/ different wound care and systemic
sue defect on her left cruris with tibia and fibula fracture. Together
voriconazole treatment was administered together. In slow but pro-
with fracture treatment repeated surgical debridement were applied
gressively destructive post-traumatic wound infections, primary cuta-
on her wounds that had wide tissue defects. But in 3 weeks of surgi-
neous aspergillosis should be considered in differential diagnosis.
cal treatment due to the appearance of infected necrotic tissue, the
wounds were debrided again for the tissue cultures. A. flavus has
grown in culture. For the distinction of colonisation and infection,
tissue cultures were repeated for twice. A. flavus has also grown in
both. Fungal spores and hyphae structure branching with narrow P477
angle were demonstrated. Because of having necrotic areas with the
continuing infected appearance and growing of A. flavus in 3 conse-
Fusarium solani isolated from patients with
quent tissue cultures she was treated with recurrent surgical debride-
onychomycosis: nail infection potential and biofilm
ment and iv voriconazole. Antibacterial absorbent polymer dressing -
Sorbact absorption dressing; SB was also used for the wound care. formation ability
After third week of the treatment there was no fungus growth in cul- J. Galletti,1 F. K. Tobaldini,2 S. Silva,3 E. S. Kioshima,4 M. Negri4
tures. Reconstruction was made with the skin graft taken from lat- and T. I. E. Svidzinski4
eral side of right thigh. Voriconazole therapy was completed in 1
Universidade Estadual de Maringa, Maringa, Brazil;
12 weeks (Figure 1). 2
Universidade Estadual de Maringa/ Universidade do Minho,
Patient 2: 39-years old male patient applied to emergency service Maringa, Brazil; 3University of Minho, Braga, Portugal and
with burn and carbon monoxide intoxication. Because of the develop- 4
Universidade Estadual de Maringa, Maringa, Brazil
ment of right forearm, right thigh and cruris compartment syndrome,
fasciotomies were performed for both extremities. He stayed in ICU for
16 days. Infected necrotic wounds were debrided for twice. A. flavus Objective The aim of the present study was to perform an epidemio-
has grown in the tissue culture. He was treated with voriconazole logical study, evaluate the ability of Fusarium solani to use the
and tigecycline. Vacuum-assisted closure (VAC) therapy was used for human nail as a single source of nutrients and its potential for bio-
wound care. There was not any growth in the tissue cultures taken film formation.
in the second week of the antifungal treatment. Following negativity Methods We first performed an epidemiological study to determine
in the tissue cultures, wound was primarily sutured. He was treated the frequency of F. solani in patients with onychomycosis. The
successfully with voriconazole for 8 weeks (Figure 2). study included data from all patients who attended the Teaching
Discussion Primary cutaneous aspergillosis may develop at intra- and Research Laboratory of Clinical Analysis (LEPAC), Division of
venous catheter insertion sites and under adhesive tape dressings Mycology, Universidade Estadual de Maring a (UEM), between Jan-
and bandages or in cases whose skin integrity is impared. Both of uary and December 2013 with suspected onychomycosis. This
the reported cases are primary cutaneous aspergillosis. It has been descriptive, retrospective, cross-sectional, observational study was

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 221
Poster Presentations

were clinically diagnosed as tinea cruris, as 39 were affected by tinea

corporis and 18 had both. Amongst the dermatophytes, 14 were T.
rubrum, 18 were T. mentagrophytes, 3 were T. tonsurans, 2 were T.
verrucosum, while 16 isolates belonged to other Trichophyton species.
Five isolates were identified as Microsporum gypseum, 1 was M. distor-
tum, 3 was M. canis and 2 were other Microsporum species. The mini-
mum inhibitory concentration (MIC) ranged from 0.313–32.0
mcg ml
1 for fluconazole, 0.313–0.5 mcg ml
1 for voriconazole,
0.313–1.0 mcg ml
1 for ketoconazole, 0.313–4.0 mcg ml
1 for
itraconazole.
Conclusions Amongst dermatophytes, lowest MICs were obtained
with voriconazole. Around 80% of the isolates revealed high MIC
with fluconazole. In vitro analysis of antifungal activity of these
performed in Maring a, Parana, Brazil, and approved by the agents would allow for comparison between different systemic anti-
Research Ethics Committee of UEM (approval no. 615.643/2014). fungal agents which may clarify reasons for lack of clinical response
To in vitro study, it was randomly chosen one clinical isolate of F. or serve as an effective therapy in patients with chronic infection.
solani that was subcultured on Potato Dextrose Agar for 7 days at Since the MIC breakpoints of antifungal agents have not been estab-
25 °C. In vitro nail infection was performed with 5 9 104 coni- lished for dermatophytes, more number of large scale studies are
dia ml
1 in 0.85% sterile saline on sterilized nail fragments (col- warranted to predict their clinical outcome to assist clinicians in
lected from healthy female volunteer) and incubated for 30 days at treatment strategies.
25 °C. After incubation, nail fragments were clarified with 40%
KOH and observed under a light microscope. Biofilm formation was
carried out on nails containing a cellular suspension of 1x106 coni-
dia ml
1 in Sabouraud Dextrose Broth and incubated for 3 days at
P479
37 °C. The ability of F. solani to form biofilm on nail was evaluated
by scanning electron microscopy.
Results It was possible to observe that in the period of January to Clinical signs associated with dermatophyte
December 2013, 695 patients were attended in the LEPAC-UEM with onychomycosis
clinical suspicion of dermatomycoses. Of these, onychomycosis was S. Ranque,1 C. l’Ollivier,1 P. Griffon,2 R. Piarroux1 and
confirmed by culture in 131 clinical samples. Fusarium spp. were iso- J. P. Weber2
lated in 26 (19.8%) cases of onychomycosis, and it was the most iso- 1
lated among nondermatophyte molds. F. solani was the most Aix Marseille University, Marseille, France and 2Ecole de
frequently (48%) isolated species. Patients with onychomycosis Podologie de Marseille, Marseille, France
caused by this species had an average age of 47  9 years, more-
over women were more frequently infected than men. Concerning Objectives The diagnostics of tinea unguis (dermatophyte ony-
in vitro nail infection, the growth was observed in 3 days and micro- chomycosis) is complex because the clinical signs are non-specific
scopic analysis revealed evidence of hyaline, thick and septate and the mycological analyses have a relatively high false-negative
hyphae in the entire nail, chlamydospores, and microconidia. With rate. This prospective study aimed to evaluate which clinical signs
regard to the ability to form biofilm on nail, F. solani showed great are significantly associated with tinea unguis.
potential of biofilm formation (Figure 1). Methods From September 2011 to June 2013, outpatients at the
Conclusion The frequency of onychomycosis caused by Fusarium Podiatric School of Marseille in whom a tinea pedis and/or tinea
spp. in our region is high and F. solani is able to use nail as a single unguis was suspected were prospectively included in the study.
source of nutrient and it has the potential to invade the nail by itself. Demographic data, personal history, topical and systemic treatment
Moreover, F. solani was able to form biofilm on nail. Thus, the model and clinical signs were collected on a standardized form. Dermato-
of nail infection proposed herein may be useful for future studies on phyte culture (Sabouraud medium with antibacterials and cyclohex-
the pathogenesis of onychomycosis. imide) and dermatophyte-specific real-time PCR was performed on
each nail sample at the Parasitology-Mycology of the University
Hospital of Marseille. The tinea unguis diagnostic criteria were a pos-
itive dermatophyte culture and/or a positive dermatophyte PCR. The
P478 association between each clinical sign and tinea unguis diagnosis
was tested via univariate and multivariate logistic regression
analysis.
Clinico-mycological and antifungal susceptibility profile of Results A total of 468 nail samples were collected from 234
tinea corporis and tinea cruris cases in India patients. Tinea unguis diagnosis was positive in 86 (18.4%). In the
N. Kamra,1 M. R. Capoor,1 G. Kaushik,2 B. Sharma,2 D. Rynga,2 multivariate analysis, lesions of the distal free margin (OR = 7.71,
R. Gaind2 and V. Ramesh2 95%IC[3.37 -17.61], P<10
4) and proximal matrix (OR = 2.34,
1
Vardhman mahavir medical college and Safdarjung hospital, 95% IC [1.38 -3.96], P = 0.0016) of the nail, and skin maceration
of toe web spaces (OR = 3.71, 95% IC [1.37–10.03], P = 0.0100)
Delhi, India and 2VMMC and Safdarjung Hospital, Delhi, India
were independently associated with tinea unguis.
Conclusion In the multivariate analysis, conventional clinical signs
Objectives The study was carried out to know the clinico-mycologi- of tinea unguis, such as nail color, nail thickening, debris and distal
cal profile and antifungal susceptibility profile of tinea corporis and lateral subungual localization did not significantly discriminate
tinea cruris cases. between onychomycosis and other nail diseases. Unexpectedly,
Methods Skin scrapings were collected from the margins of the among all dependent clinical signs of nail disease, the most strongly
lesions and a 10% KOH mount was prepared. Various dermatophyte associated with tinea unguis was lesion of the distal free margin of
species were tested for antifungal susceptibility to antifungal agents the nail.
viz. fluconazole, voriconazole, itraconazole and ketoconazole, as per
protocols of the Clinical and Laboratory Standards Institute (CLSI
M38-A) for molds by broth-microdilution and E-strip methods.
Results A total of 122 patients consented to participate in the study.
Samples of 68 patients revealed fungal hyphae in the KOH mount
and revealed growth on culture. Among these, 64 were dermato-
phytes. Of these patients, 45 were males and 19 were females. Seven

ª 2015 The Authors

222 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P480 Immunohistochemical parameters were characterized by a decreased

of CD3 + and CD68 + cells and increased CD4 + and CD8 + cells
in the epithelium of the esophagus and the underlying tissues.
Maintenance antifungal therapy of recurrent esophageal Conclusions Immunological parameters, identified in this trial may
candidiasis in HIV-negative patients be of interest in the pathogenesis of recurrent esophageal candidiasis
J. E. Melekhina, E. V. Frolova, M. A. Shevyakov, J. L. Avdeenko, in HIV-negative patients.
T. S. Bogomolova, I. V. Vybornova, N. Vasilieva and N. Klimko
Metchnikov North-West State Medical University, Saint
Petersburg, Russia
P482
Objective The efficiency of maintenance antifungal therapy of recur-
rent esophageal candidiasis (REC) in HIV-negative patients is not well Validation of the DermaGenius Nail plus multiplex assay, a
established. new commercial PCR assay developed for the detection
Methods In prospective single-center study (2004–2014) we include and identification of dermatophyte and Candida in nails
124 HIV-negative patients with REC. Median age was 53 y (46–65), M. P. Hayette,1 H. Graide,1 C. Adjetey,1 J. Arrese,2
women 91. Endoscopic examination with a biopsy, microscopy and G. Gaajetaan,3 D. van Tegelen,3 T. Kampermann,3 G. Simons3
culture was made in all patients. Identification of the pathogen was
and G. Dingemans3
made with MALDI-TOF Mass Spectrometry. Determination of suscep-
tibility of patogens to fluconazole and voriconazole was made with
1
CHU Liege, Liege, Belgium; 2University Hospital of Liege, Liege,
the disco-diffusion CLSI M44-A method. Criteria of RCE was ≥ 1 Belgium and 3PathoNostics B.V., Maastricht, the Netherlands
relapse in a year.
Results Risk factors were: thyroid disease (43%) with hypothy- Objectives Superficial dermatophytosis is the most common fungal
roidism (17%), bronchial asthma (12%), diabetes mellitus (9%), infection in humans. Diagnosis of dermatophytosis is currently based
smoking (100%), inhaled (90%) and per os (52%) corticosteroids, on microscopy or histology associated with culture on specific agar
and very hot food use (67%). The clinical signs were dysphagia media. However, direct microscopy lacks specificity and culturing has
(90%), retrosternal discomfort (50%), and odinophagia (18%). Endo- a long turn-around-time of 2–4 weeks. These limitations can be pre-
scopic features were hyperemia (100%) and contact sensitivity vented by the use of molecular diagnostics. The DermaGenius (DG)
(100%) of esophagus mucosa, and white fibrin plaques (75%). Main multiplex kit (PathoNostics, the Netherlands) is a new commercial
etiology agent was C. albicans (99%), sensitive in vitro to fluconazole realtime-PCR kit, which can differentiate various dermatophytes spe-
and voriconazole (100%). Treatment of relapse fluconazole cies including the nail pathogens T. rubrum, T. mentagrophytes, T.
150 mg day
1 for 3–4 weeks was effective in 100% patients. The interdigitale and 2 Candida species (C. albicans and C. parapsilosis). This
patients were divided into two groups: with or without maintenance study aimed at the validation of the kit on nails clippings. Results
antifungal therapy
50 mg fluconazole once a week for 6 months. were compared with histology and culture data.
If patients did not receive maintenance therapy rate of relapse in Methods A set of 76 nail clippings was collected from 76 patients
6 months was 57%. All patients with maintenance therapy were in attending the dermatology consultation at the University Hospital
remission of REC in 6 months (P < 0.005). No side effects or drug- of Liege on suspicion of onychomycosis. All nails were divided in
drug interactions in patients with maintenance antifungal therapy three pieces for histology, culture and the PCR multiplex assay.
were detected. Histologic preparations were stained with PAS staining. Cultures
Conclusions Maintenance antifungal therapy with 150 mg flucona- were performed on 2 different Sabouraud agar medium slants
zole once a week for 6 months is effective and safe in HIV-negative (bioMerieux). The DNA extraction protocol used a proteinase K
patients with recurrent esophageal candidiasis. pre-treatment followed by an automated DNA-extraction (EasyMag,
bioMerieux). An Internal Control (IC) was included to monitor for
PCR inhibition or manual errors. The realtime-PCR amplification
was performed with the DG kit on a Rotor-Gene Q instrument (Qi-
P481 agen) by using quantitative amplification and melting curve
analysis.
Results In total, 35 of 76 cases (46%) were classified as confirmed
Immunological and immunohistochemical features of onychomycosis based on positive microscopy (M+) with or without
recurrent esophageal candidiasis in HIV-negative patients. positive culture (C+) or just by positive culture of a confirmed patho-
J. E. Melekhina, E. V. Frolova, L. V. Filippova, A. E. Uchevatkina, gen. Based on negative microscopy (M-) and negative culture of a
M. A. Shevyakov, J. L. Avdeenko, N. Vasilieva and N. Klimko confirmed pathogen, 41 cases (54%) were reported as non-fungal
Metchnikov North-West State Medical University, Saint onychodystrophy. Agreement between DermaGenius (DG) and cul-
Petersburg, Russia ture was found in 52% of the cases while 86% agreement was
reported when comparing positive DG with confirmed onychomyco-
sis. Three positive cultures (microscopy negative) were not detected
Objectives The immunological and immunohistochemical features by DG (2 T. rubrum, 1 C. albicans). However, DG could detect 7 addi-
of a reNAAAAA urrent esophageal candidiasis (REC) in HIV-negative tional infections (9%). Eleven discrepancies DG+/C+ were determined
patients are not well understood. which could be positively confirmed in favour of DG result by ITS
Methods We examined immunological and immunohistochemical sequence analysis. Most discrepancies could be explained by fungal/
parameters in 78 patients with REC. The median age of patients was yeast species overgrowing the agar slant, including species of Can-
53 years. Criteria of REC was ≥1 relapse in a year. Subpopulations of dida, Fusarium, Trichosporon, which were not considered as the source
lymphocytes were determined by immunocytochemical method using of infection.
a monoclonal antibody «DAKO». Blood cell supernatants were tested Conclusion The DermaGenius Nail plus multiplex was able to
for IFN-g and IL-10 by using an ELISA test ‘Cytokine’. The antibodies detect the most prevalent pathogenic dermatophytes species in clin-
used for immunohistochemical staining were monoclonal mouse ical nail specimens and proved to be more sensitive and specific
anti-human CD3 + , CD4 + , CD8 + , CD68 + («DAKO»). Statistical than culture and direct microscopy. The DNA extraction procedure
analysis was done using Mann-Whitney U-test (P < 0.05). has been shown to work efficiently in diagnostics which enables
Results Our study showed that in patients with REC was a reduction the physician in charge of the patient to start a dedicated treat-
of the absolute numbers of CD3 + (1.216*109/L), CD4 + ment rapidly.
(0.735*109/L), CD8 + (0.483*109/L), CD16 + (0.157*109/L)
lymphocytes, and the increase the spontaneously secreting IFN-c
(63.0 pg ml
1) and IL-10 (144.0 pg ml
1) (P < 0.05).

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 223
Poster Presentations

P483 predominant organisms causing dermatophytosis in Urmia, North

West of Iran were identified.
Methods The study samples were collected from clinically suspected
Onychomycosis caused by uncommon agents in cutaneus lesions. All the specimens were transported to Medical
immunocompetent patients: two case reports Mycology Center, UMS University of Medical Sciences. First of all, a
F. J. R. Mota,1 I. Raposo,1 V. M. Lopes,1 G. C. Velho,1 I. Lobo,1 conventional diagnosis was carried out which included microscopic
S. Machado,1 H. Ramos2 and M. Selores1 examination and culture on sabouraud dextrose agar medium with
1 antibiotics: chloramphenicol and cyclohexamide. All the dermato-
Centro Hospitalar do Porto, Porto, Portugal and 2Microbiology
phyte isolates were then identified at the level of species by the
Department, Centro Hospitalar do Porto, Porto, Portugal molecular method of PCR-RFLP.
Results From the tested 357 clinical specimens, 30 dermatophytic
Objective Onychomycosis is a fungal infection of nails and accounts isolates were identified. The dermatophyte species Trichophyton menta-
for up to 50% of nail disorders. Dermatophytes are the most fre- grophytis (36%), Microsporum canis (32%), Microsporum gypseum
quently causative agents in onychomycosis (nearly 70% of the cases) (16%), Trichophyton rubrum (4%),and Epidermophyton floccosum (12%)
whereas non-dermatophytic molds cause 1.5–6% of the cases. We were included, respectively.
report two cases of onychomycosis of the halux in immunocompetent Conclusion The results showed that some dermatophyte species
patients due to uncommon agents in this setting: Aspergillus niger and including T. rubrum and T. mentagrophytes were mainly isolated from
Paecilomyces lilacinus. foot, whereas E. fluccosum was predominantly found in the scrapping
Methods In the first case a 45-year-old caucasian male was referred of groin and hand.
to the Dermatology department due to changes in the coloration and
form of the left halux nail. Clinical examination revealed onycholysis
involving one of the lateral nail margins and black pigmentation of
the nail plate, with paranoquia. Other nails were normal. The second P486
case is related to a 25-year-old caucasian woman with progressive
right great toenail dystrophy since 1 year. Clinical examination
revealed onycholysis which involved both lateral nail margins, sub- Co-existence of Aspergillus niger and Trichophyton
ungual hyperkeratosis and yellowish discoloration of the nail plate. interdigitale toenail onychomycosis in an
Other nails were all normal. In both cases nail scrapings were col- immunocompetent 70-years-old patient
lected and sent to the Microbiology laboratory. M. A. Saracli,1 R. Gumral,1 S. T. Yildiran,1 A. Akar1 and
Results Direct microscopy of nail scrapings of the male patient M. M. Ilkit2
showed thick septated hyaline hyphae and Aspergillus niger was iso- 1
lated in all cultures of three different samples collected. The patient Gulhane Military Medical Academy, Ankara, Turkey and
2
was treated with oral itraconazol in combination with topical Cukurova Uni. School of Medicine, Adana, Turkey
sertaconazol for 4 months with clinical resolution of the infection.
Direct microscopy of nail scrapings of the female patient showed Objectives Onychomycosis is the most frequently encountered nail
septated hyaline hyphae. Paecilomyces lilacinus was isolated on sev- disease. To our knowledge, this study represents an unusual case of
eral cultures of samples collected with one month interval. Suscepti- toenail onychomycosis caused by the co-existence of Aspergillus niger
bility tests to antifungal agents revealed high MIC0 s to itraconazole and Trichophyton interdigitale in an immunocompetent patient.
and fluconazole, but low MIC0 s to ketoconazole and voriconazole. Methods Samples obtained from two totally dystrophic toenails of
She initiated a treatment with oral ketoconazole combined with the patient were processed separately. The patient was sampled two
applications of 40% urea cream and amorolfine lacquer on the nail times, 3 weeks apart. Microscopic examination by KOH method and
bed. This treatment was performed during 9 months without culture-based identification of the causative agents from scrapings of
marked benefits. Clinical and microbiological resolution was reached the two toenails were performed. Phenotypic identification of the
with surgical removal of the nail plate and amorolfine cream strains was confirmed by sequencing of the beta tubuline gene and
application in the nail bed. the ITS region. The patient was prescribed terbinafine 250 mg p.o.
Conclusions Factors such as ageing, immunodeficiency, trauma, daily for three months.
hyperhidrosis, socio-economic status, climatic conditions and parony- Results The macroscopic appearances of the two toenails were
chia predispose to onychomycosis. This mycosis can significantly totally different. The black-dotted nail was more dystrophic than the
impair the quality of life of a patient and may create a number of yellowish one. The microscopic features of the infecting strains from
clinical and occupational complications. These successfully treated the two toenails were also different on KOH examination: while the
cases of onychomycosis caused by two uncommon agents document sample from the black-dotted toenail had hyphae presumptive for an
the fact that the identification of the causative fungi is therefore Aspergillus species, the yellowish one was for a dermatophyte species.
essential in all suspected cases of onychomycosis due to epidemio- Also, the growth characteristics of the strains were different from
logic purposes and because the type and duration of treatment varies each other, and compatible with the presumptive microscopic identi-
when the pathogen is a non-dermatophyte agent. fication. The two strains were phenotypically identified as Aspergillus
niger and Trichohpyton interdigitale and the final identifications were
confirmed by sequencing.
Conclusion An exact diagnosis of onychomycosis relies on clinical
P485 examination, microscopic investigation, culture identification and
molecular methods when necessary. At least half of the causative
agents of onychomycosis are dermatophytes, as Trichophyton interdigi-
Molecular Identification of Dermatophyte species causing tale is a common worldwide dermatophytic fungus. Additionally, in
cutaneus infections in West Azarbayjan of Iran cases of co-isolation of molds, aspergilli infections should not be
K. Diba,1 A. Namaki2 and A. Gheibi3 easily considered saprophytic.
1
Urmia University of Medical Sciences, Urmia, Iran; 2Arefian
General Hospital, Urmia, Iran and 3University of Istanbul,
Istanbul, Turkey

Objectives The most common cutaneus fungal infections are caused

by dermatophyte fungi including Microsporum, Trichophyton, and Epi-
dermophyton species. In this study, the epidemiologic trends and the

ª 2015 The Authors

224 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226
 Poster Presentations

P487 mentagrophytes was detected after routine diagnostics. The PCR meth-
ods confirm this identification but RAPD results shows strains differ-
entiation among the T. mentagrophytes isolates.
The first Turkish case of onychomycosis caused by Conclusion The results obtain by molecular methods confirm the
Chaetomium globosum in an immunocompetent patient dermatophyte infection even in symptomless animals and suggests
F. Ozakkas,1 R. Altinbas,2 H. Sav,2 M. A. Kuskucu,2 K. Midilli2 that infections may be due different strains of T. mentagrophytes.
and N. Kiraz2
1
Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey
and 2Istanbul University, Istanbul, Turkey
P490
Objectives Onycomycosis is a chronic fungal infections of toenail and
fingernail. The most causative agents are observed as dermatophyte, Protein profile of the main species of the Sporothrix
yeast and nondermatophyte species. Several types of nondermatophyte complex associated with cases of sporotrichosis.
mould such as Aspergillus spp.,Fusarium spp., Chaetomium spp may M. M. E. Oliveira,1 M. A. Almeida,1 C. V. Pizzini,1 R. Almeida-
cause these infections. The genus Chaetomium, which belongs to (fam- Paes,1 C. M. A. Soares2 and R. M. Zancope-Oliveira1
ily Chaetomiaceae, class Sordariomycetes, phylum Ascomycota), is demati-
aceous nondermatophyte fungus that are commonly found in
1
Fundac
a~o Oswaldo Cruz- Instituto Nacional de Infectologia
deteriorating wood products, soil and cellulosic substrates. Evandro Chagas, Rio de Janeiro, Brazil and 2Universidade Federal
Methods We report a case of distal subungual onychomycosis of the de Goias- Instituto de Cie^ncias Biologicas, Goia^nia, Brazil
thumb of the right foot of a 25-year-old female. In direct microbio-
logical examination septate hyphae was observed by using % 20 Sporotrichosis, a subcutaneous mycosis caused by Sporothrix spp., is
KOH. The same brown colonies were produced on repeated cultures cosmopolitan and more frequent in Latin America and East Asia. In
by using Sabouraud’s dextrose agar without cycloheximide. Antifun- last years the number of sporotrichosis cases has significantly
gal susceptibiltiy test was performed and microplates were prepared increased in Brazil, especially in the metropolitan area of Rio de
as described in document M38-A2. Janeiro. At the last decade, it has been proposed that S. schenckii
Results After growth of colony, slide cultures were prepared and should not be considered a single taxon but a complex including the
brown-colored septated hyphae, perithecia, lemon- shaped ascospores species S. brasiliensis, S. globosa, S. mexicana, S. pallida and S. luriei.
were observed by light microscopy. The causative agent was identi- The phylogenetic characterization of strains around of the world
fied as Chaetomium globosum based on DNA sequencing andmycologi- from patients treated with sporotrichosis has been reported that the
cal examination. Using CLSI M38-A2 microdilution method, the species often identified using phenotypic and genotypic tests were
minimum inhibitory concentration values of amfotericin B,flucona- S. brasiliensis, S. schenckii, S. globosa, S. mexicana and S. pallida.
zole, itracanozole, miconazole, ketoconazole, flucytosine voriconazole Objectives The objective of this study is to characterize the protein
were determined as 4, >64, 1, 0.125, 0.125, >64, 0.5 lg ml
1 profile of these species of the Sporothrix complex.
respectively. Fluorocytosine and fluconazole were determined as resis- Methods Five Sporothrix isolates of control strains of S. brasiliensis
tant for Chaetomium globosum and the best effective antifungal was (IPEC16490), S. schenckii (IPEC27722), S. globosa (IPEC27135), S.
determined as miconazole and ketoconazole. The patient was treated mexicana (MUM11.02) and S. pallida (SPA8) were used in this study
by using oral itraconazole (250 mg/a day) and local application of for characterization of the protein profile of these species. For analysis
amorolfine 5% nail lacquer for 12 weeks. of these exoantigens from the mycelial phase the SDS-PAGE tech-
Conclusion we report the onychomycosis caused by C. globosum in an nique was applied. Briefly, fungi were growth in Sabouraud Dextrose
immunocompetent patient which was confirmed by mycological exam- medium with constant stirring for 14 days at 28 °C.
ination and molecular analysis. Antifungal susceptibility was per- Results The exoantigens of S. brasiliensis, S. schenckii, S. globosa, S.
formed and the most effective agent was determined as ketoconazole mexicana and S. pallida showed protein complex profiles, composed of
and miconazole, but clinicalrecovery was provided by using itraconazole. molecules with molecular weight ranging from 10-160 kDa.
Conclusion The characteristic profiles observed for each species of
Sporothrix spp. allowed the clear distinction of the control strains
suggesting that this tool can be used in the implementation of the
P488 polyphasic taxonomy of this complex and in the development of new
molecular diagnostic methods with higher accuracy.

Dermatophytes isolating from foxes from private farm

B. Dworecka-Kaszak,1 M. Brzozowski,2 I. Dabrowska1 and
A. Brillowska-Dabrowska3 P491
1
Faculty of Veterinary Medicine, Warsaw, Poland; 2Warsaw
University of Life Sciences-SGGW, Warsaw, Poland and 3Gdansk Insight into the intricate taxonomy of Aspergillus
University of Technology, Gdansk, Poland viridinutans complex (section Fumigati) comprising
opportunistic human and animal pathogens
The aim of this study was to determine the prevalence of dermato- V. Hubka,1 Z. Dudova,1 T. Yaguchi,2 Y. Horie,3 T. Matsuzawa,2
phytes colonization in breeding foxes (Polish line of Vulpes vulpes A. Novakova,4 L. Svobodova,5 P. Hamal6 and M. Kolarik7
with white neck). 1
Methods In this study, the routine method of isolation and identifi- Charles University in Prague, Prague, Czech Republic; 2Chiba
cation of dermatophyte was supported by a method patented by Bril- University, Chiba, Japan; 3Natural History Museum and Institute,
lowska-Dabrowska and co-workers [2007]. This method involves Chiba, Japan; 4Institute of Microbiology ASCR, Prague, Czech
extraction of fungal DNA and PCR amplification with pan-dermato- Republic; 5Palacky University Olomouc, Olomouc, Czech
phyte primers. To confirm the presence of dermatophytes in samples Republic; 6Faculty of Medicine and Dentistry, Palacky University,
took from 5 foxes with typical ringworm lesions and 70 healthy farm Olomouc, Czech Republic and 7Institute of Microbiology, Czech
animals (hair), after confirmation by culture, another two PCR Academy of Science, Prague, Czech Republic
assays were performed; with primers specific for Trichophyton genus
and T. mentagrophytes complex. To investigate the strains differentia-
Objectives The aim of this study was to determine the species
tion GACA primers were used.
boundaries within the Aspergillus viridinutans complex. These species
Results the frequency of isolation of dermatophytes in breeding of
are increasingly isolated as opportunistic human and animal
foxes was 20% among all investigated animals. Only Trichophyton

ª 2015 The Authors

Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226 225
Poster Presentations

pathogens. Their differentiation from each other and from A. fumiga- Methods Recently we received some atypical cultures of Sporothrix
tus is clinically relevant due species-specific susceptibility patterns. recovered from a clinical case as well as from environmental origins
Methods A total number of ~100 isolates from various substrates in Vi~na del Mar, Chile. Morphological and molecular research sug-
and countries was subjected to morphological, physiological and gested that an undescribed taxon was concerned. The aim of the pre-
molecular analysis (DNA sequences of RPB2, calmodulin, beta-tubu- sent study was to characterize these atypical Sporothrix isolates using
lin and actin genes). Mating experiments were provided between iso- a polyphasic approach incorporating morphology, virulence and
lates representing opposite mating types within and between all multigene data to recognize species boundaries among phylogeneti-
major clades of combined phylogenetic tree. The viability of ascos- cally related species, and to develop diagnostic barcodes for the spe-
pores was tested by flow-cytometry. cies concerned.
Results Phylogenetic analysis based on combined dataset supported Results Multigene analyses based on ITS1/2 + 5.8s region, beta-
recognition of at least eight species within A. viridinutans complex tubulin, calmodulin and translation elongation factor 1a revealed
including one undescribed species sister to A. aureolus. Aspergillus that S. chilensis is a member of the Sporothrix pallida complex, and
aureolus and A. siamensis represent homothallic species whereas other the nearest taxon is Sporothrix mexicana, a rare soil-borne species,
species are heterothallic and isolates of both mating types were non-pathogenic to humans. The ITS region serves as a primary bar-
detected. Fertile ascomata were observed in three heterothallic spe- code marker, while each one of the protein-coding loci easily recog-
cies, A. udagawae, A. felis and A. wyomingensis. The morphology of nized species boundaries providing sufficient information for species
ascospores and their viability was stable within major clades. Proba- identification. A disseminated model of murine sporotrichosis
ble interspecies hybrides detected between isolates from four clades revealed a mild-pathogenic potential, with lung invasion.
(representing phylogenetic species) can be distinguished by unstable Conclusion We provided evidences based on a polyphasic approach,
morphology of ascospores and their decreased viability. including examination of cultures, morphology and virulence in
Conclusion Aspergillus viridinutans complex comprises at least eight murine model combined with phylogenetic analyses of protein coding
species, four of them are clinically relevant - A. udagawae, A. felis, A. loci that S. chilensis is distinct from S. pallida and allied species. There-
wyomingensis and A. aureolus. The taxonomy of this complex is com- fore, a new taxa is proposed to accommodate Sporothrix chilensis sp.
plicated by relativelly simple induction of interspecies hybrides during nov., and CAL is recommended as barcoding gene. Although S.
in-vitro mating experiments. The stability of ascospore morphology chilensis is not a primary pathogen, accidental infection may have an
and the viability of ascospores seem to be crucial features in defini- impact in the immunosuppressed population. With the introduction
tion of species boundaries. Flow-cytometry is a promissing tool to of distinct species with similar routes of transmission but different
assess the percentage of viable ascopores in samples. virulence, identification of Sporothrix agents at the species level is
mandatory.
Financial support S~ ao Paulo Research Foundation (FAPESP 2009/
54024-2 and FAPESP 2011/07350-1), National Council for Scien-
P492 tific and Technological Development (CNPq 472600/2011-7), and
Coordination for the Improvement of Higher Education Personnel
(CAPES).
Sporothrix chilensis sp. nov. (Ascomycota:
Ophiostomatales), a soil-borne agent of human
sporotrichosis with mild-pathogenic potential to mammals.
A. M. Rodrigues,1 R. Cruz Choappa,2 G. F. Fernandes,1 G. S. de P493
Hoog3 and Z. P. de Camargo1
1
Federal University of Sao Paulo, Sa~o Paulo, Brazil; 2Universidad The Jabuti method for Candida species identification
de Valparaso, Valparaa~so, Chile and 3CBS-KNAW Fungal R. B. Caligiorne,1 M. Gontijo,2 C. A. Rosa,3 T. Tirone3 and
Biodiversity Centre, Utrecht, the Netherlands S. Johan3
1
Santa Casa Hospital, Belo Horizonte, Brazil; 2Nucelo de Pos-
Sporotrichosis, a cutaneous and subcutaneous mycosis affecting graduac
a~o Santa Casa Hospital, Mg, Brazil, Belo Horizonte, Brazil
humans and animals worldwide, is caused by several members of the
and 3Federal University of Minas Gerais, Belo Horizonte, Brazil
genus Sporothrix. Diagnostic characteristics of the genus comprise
single-celled, hyaline conidia which are sympodially arranged on
clusters of denticles in a bouquet-like fashion at the ends of conidio- Several species of the genus Candida are common saprophytes which
phores. This morphology is expressed in numerous environmental reside on the skin and mucous membranes in healthy persons.
members of the order Ophiostomatales. A combination of phylogeny, Opportunistic pathogen of Candida species, such as C. albicans, C. glab-
evolution, morphologies and ecologies has enabled major advances rata, C. krusei, C. parapsilosis and C. tropicalis are ordinarily weak
in understanding the taxonomy of Sporothrix species, including mem- pathogens which produce mild superficial infection, but occasionally
bers exhibiting distinct lifestyles such as saprobes, human/animal may cause systemic diseases with involvement of the lung, kidney,
pathogens, and insect symbionts. Phylogenetic analyses of ITS1/ endocardium, brain, or reticuloendothelial system. In this study we
2 + 5.8s sequences split Sporothrix genus in two well-defined groups propose a new methodology for identification of Candida species and
with dissimilar ecologies. Species embedded in the Sporothrix schenckii compare this approach with the system Vitec for identification, that
complex are frequently agents of human and animal sporotrichosis, has been validated, and the molecular biology profiles. The new
and some of these are responsible for large sapronoses and zoonoses methodology proposed is faster and presents an affordable cost. The
around the warmer temperate regions of world. At the other results of the three methods were above 95% concordant, demon-
extreme, basal saprophytic species evolved in association with decay- strating that the new methodology for identification of Candida can
ing wood and soil, and are rarely found to cause human disease. be applied; favoring the laboratories that have limited financial
Objectives We propose to create new taxa, Sporothrix chilensis sp. resources, to oppose such hospitals network publishes health. This
nov., to accommodate strains collected from a clinical case of ony- methodology is already Patented at the Brazilian government.
chomycosis as well as from environmental origins in Chile.

ª 2015 The Authors

226 Mycoses ª 2015 Blackwell Verlag GmbH, 58 (Suppl. 4), 51–226