b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 373380

http://www.bjmicrobiol.com.br/

Medical Microbiology

Susceptibility and molecular characterization of

Candida species from patients with vulvovaginitis
Gheniffer Fornari a , Vania Aparecida Vicente a , Renata Rodrigues Gomes a ,
Marisol Dominguez Muro b , Rosangela Lameira Pinheiro b , Carolina Ferrari c ,
Patricia Fernanda Herkert a , Marcos Takimura c , Newton Srgio de Carvalho c ,
Flavio Queiroz-Telles a,b,
a

Graduate Program in Microbiology, Parasitology and Pathology, Department of Basic Pathology, Laboratory of Microbiology and
Molecular Biology-LabMicro, Federal University of Paran, Curitiba, Paran, Brazil
b Support and Diagnosis Unit, Mycology Laboratory, Federal University of Paran, Brazil
c Clinical Hospital Federal University of Paran, Brazil

a r t i c l e

i n f o

a b s t r a c t

Article history:

Vulvovaginal candidiasis affects women of reproductive age, which represents approxi-

Received 6 May 2013

mately 1525% of vaginitis cases. The present study aimed to isolate and characterize yeast

Accepted 5 March 2015

from the patients irrespective of the presentation of clinical symptoms. The isolates were

Available online 2 March 2016

subjected to in vitro susceptibility prole and characterization by molecular markers, which

Associate Editor: Carlos Pelleschi

intended to assess the distribution of species. A total of 40 isolates were obtained and iden-

Taborda

tied through the CHROMagar, API20aux and by ITS and D1/D2 regions sequencing of DNAr
gene. Candida albicans strains were genotyped by the ABC system and the isolates were

Keywords:

divided into two genotypic groups. The identity of the C. albicans, C. glabrata, C. guilliermondii,

Vulvovaginal candidiasis

C. kefyr and Saccharomyces cerevisiae isolates was conrmed by the multilocus analysis. The

In vitro susceptibility

strains of Candida, isolated from patients with complications, were found to be resistant

Variability genetic

to nystatin but sensitive to uconazole, amphotericin B and ketoconazole, as observed by

in vitro sensitivity prole. The isolates from asymptomatic patients, i.e., the colonized group,
showed a dose-dependent sensitivity to the anti-fungal agents, uconazole and amphotericin B. However, the isolates of C. albicans that belong to distinct genotypic groups showed
the same in vitro susceptibility prole.
2016 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de
Microbiologia. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Corresponding author.
E-mail: [email protected] (F. Queiroz-Telles).
http://dx.doi.org/10.1016/j.bjm.2016.01.005
1517-8382/ 2016 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Microbiologia. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Introduction
Vulvovaginal candidiasis (VVC) is a primary opportunistic
mycosis or secondary with endogenous or exogenous characteristics. It is also classied as a sexually transmitted disease
(STD) and is caused by different species of Candida.1,2 The disease is characterized by inammation of the genital mucosa
as a response to the yeast proliferation.3
The genus Candida includes approximately 300 heterogeneous species with different morphological and functional
features, and is currently found as a part of the normal ora
in skin, digestive tract and mucous membrane, including the
human genito-urinary tract.4 Predominantly, VVC is caused
by C. albicans and its prevalence can reach 8595%.1 However, infections caused by other species such as C. tropicalis,
C. glabrata, C. krusei, C. parapsilosis, C. kefyr and C. lusitaniae
have been reported as well.1,4,5 According to literature these
species are part of the vaginal mucous microbiota and they
are present in 2080% of healthy adult population, with clinical manifestations in 10% of pre-menopausal patients, 510%
in menopausal and 30% of pregnant women.6,7
Vulvovaginal infection, caused by Candida spp., affects
women of reproductive age representing approximately
1525% of the vaginitis cases.8 These microorganisms usually
remain hosted in the vaginal mucous only as colonizers; however, under inappropriate conditions the yeast reproduction
increases inducing expression of virulence factors, which subsequently affects the mucous membrane, characteristic of the
symptomatic VVC.9
Identication of strains that are isolated from VVC is crucial to clarify the distribution of C. albicans in relation to
other species of Candida genus in different populations with
manifestations of the infection. In clinical practice, the yeast
identication is based on morphological and biochemical
markers, including the automated methods.10,11 However, not
all the species are precisely identied by such procedures.
Therefore, molecular markers based on the sequencing of
variable domain (D1/D2) from the 26S region and internal transcribed spacers (ITS) of the RNA gene were utilized in the
present study to enable identication and detection of various
strains.12
VVC is not a notied disease and generally the drug
treatment is recommended based on the clinical diagnosis.
Epidemiological molecular studies are relevant in context of
establishment of species prevalence, elucidation of virulence
factors and mechanisms of drug resistance so as to support
the treatment protocols.13
A few studies have focused on the correlation of antifungal
susceptibility with clinical results in VVC.14 In spite of a considerable enhancement in the resistance prole among the
various Candida species, uconazole is still widely used for
treatment of VVC.1 Since it has been noticed that C. albicans
displays a variable sensitivity to azoles derivatives, it seems
crucial to identify its sensitivity prole against various drugs
for a better therapeutic conduct.15
In view of such grounds, the current work aimed to evaluate in vitro susceptibility and molecular characterization of
yeast from genus Candida that were isolated from the patients
with infection and the patients with no clinical symptoms,

for elucidation of epidemiological aspects of vulvovaginal candidiasis.

Materials and methods

Test organism
The present study analyzed vaginal material isolates from the
patients assisted by an outpatient clinic of Toco-gynecology
at the Clinical Hospital/UFPR, Paran (Table 1). The study was
conducted from November 2011 to October 2012. The research
work was approved by the Ethical Committee of Federal University of Paran Clinical. Samples from 133 women were
collected with their consent.

Casuistry
The study enrolled women, who were aged between 18 and
56 years, with or without VVC clinical symptoms, and who
had not been administered any drug treatment in the last six
months before collection of the samples. The patients were
divided into two groups: colonized patients (without clinical symptoms) and infected patients.1 The infected patients
presented three or more of the following clinical symptoms: typical discharge, vaginal itching, vulvovaginal burning,
dysuria and dyspareunia. Infected patient group was subdivided into two sub-groups: (i) complicated which included
women with a history of recurrence infection; and (ii) uncomplicated patients with sporadic episodes of the infection. The
exclusion criteria were age (under 18 and over 56), pregnancy
and women with immunosuppressive diseases and under
treatment.

Collection, isolation and phenotypic identication

The samples were collected by swabs, and each sample was
sowed on Sabouraud Dextrose Agar medium followed by incubation at 30 C for a period of 48120 h as per the growth
parameters of each isolate. A presumptive identication of isolates was done by CHROMagar at 37 C for 48 h.16 Some of the
isolates were identied by the API 20 AUX system (BioMrieux,
France).

Molecular characterization of Candida isolates

DNA from the isolates was extracted by physical maceration of the samples in a mixture of silica/celite (2:1)
in CTAB (cetyltrimethylammonium bromide). The isolated DNA was precipitated by CIA (acidic solution of
chloroform-isoamyl alcohol) followed by sequencing on
ABI3500 sequencer.17 For ITS sequencing, the following
primers were used: ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 )
and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) and the reaction
conditions of sequencing were as follows18 : one cycle at
94 C for 2 min, followed by 30 cycles at (94 C for 30 s, 56 C
for 1 min, 72 C for 1 min) and a nal extension at 72 C
for 3 min.17 For amplication of D1/D2 region, the primers
NL-1 (5 -GCATATCAATAAGCGGAGGAAAAG-3 ) and NL-4

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Table 1 List of reference strains and the clinical isolates.

Name

Number of
reference

Substrate

Geographical
indication

GenBank access

ITS
Candida albicans
Candida dubliniensis
Candida inconspcua
Candida intermedia
Candida glabrata
Candida haemulonii
Candida tropicalis
Clavispora lusitaniae
Debaryomyces carsonii
Kluyveromyces marxianus
Kluyveromyces lactis
Meyerozyma guilliermondii
Pichia fermentans
Pichia membranifaciens
Pichia segobiensis
Torulaspora delbrueckii
Saccharomyces cerevisiae
Schizosaccharomyces pombe
Candida glabrata
Saccharomyces cerevisiae
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida glabrata
Candida guilliermondii
Candida kefyr
Candida glabrata
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida dubliniensis
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans
Candida albicans

CBS 562
CBS 7987
CBS 180
CBS 572
CBS 138
CBS 5149
CBS 94
CBS 6986
CBS 2285
CBS 712
CECT1961
CBS 2030
L1B
23
CECT 10-210
CBS 1146
NRRL Y-12632
NRRL Y-12796
HC01
HC02
HC03
HC04
HC05
HC06
HC07
HC08
HC09
HC10
HC11
HC12
HC13
HC14
HC15
HC16
HC17
HC18
HC19
HC20
HC01IC
HC02IC
HC03IC
HC04IC
HC05IC
HC06IC
HC07IC
HC08IC
HC09IC
HC10IC
HC11IC
HC01INC
HC02INC
HC03INC
HC04INC
HC05INC
HC06INC
HC07INC
HC08INC
HC09INC

Clinical isolate
Clinical isolate
Clinical isolate
Clinical isolate

Clinical isolate
Urine
Clinical isolate
Clinical isolate
Environmental
Clinical isolate
Environmental isolates

Clinical isolate

Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents
Cervicovaginal contents

Australia
Japan
Australia
Australia
Ireland
Spain
Japan
Bulgaria
USA
Australia
USA
Australia
UK
Spain
Spain
Australia
USA
USA
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil
Brazil

EF567995
AB035589
AJ853766
EF568011
AY198398
JX459660
AB437068
EF568049
AJ853767
EF568057
AJ401704
EF568003
FJ713081
JQ410476
DQ409166
EF568083
AM900404
AY251633
KJ651873

KJ651903
KJ651904
KJ651905
KJ651906
KJ651907
KJ651908
KJ651909
KJ651910
KJ651874
KJ651875

KJ651876
KJ651877
KJ651878
KJ651879
KJ651880
KJ651881
KJ651882
KJ651883
KJ651884
KJ651885
KJ651886
KJ651887
KJ651888
KJ651889
KJ651890
KJ651891
KJ651892
KJ651893
KJ651894
KJ651895
KJ651896
KJ651897
KJ651898
KJ651899
KJ651900
KJ651901
KJ651902

() data not provided; HC: clinical hospital/UFPR; C: colonized; IC: complicated infection; INC: non-complicated infection.

D1/D1
U45776
U57685
U71062
U44809
U44808
U44812
U45749
U44817
U45743
U94924
U94922
U45709
U75726
AJ508586
U45742
AJ508558
AY048154
U40085
KJ624025
KJ624026
KJ624027
KJ624028
KJ624029
KJ624030
KJ624031
KJ624032
KJ624033
KJ624034
KJ624035
KJ624036
KJ624037
KJ624038
KJ624039
KJ624040
KJ624041
KJ624042
KJ624043
KJ624044
KJ624045
KJ624046
KJ624047
KJ624048
KJ624049
KJ624050
KJ624051
KJ624052
KJ624053
KJ624054
KJ624055
KJ624056
KJ624057
KJ624058
KJ624059
KJ624060
KJ624061
KJ624062
KJ624063
KJ624064

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(5 -GGTCCGTGTTTCAAGACGG-3 ) were used following the

same reaction conditions, as listed above.19
For ABC genotyping of C. albicans, the primers CA-int-L (5 ATAAGGGAAGTCGGCAAAATAGATCCGTAA-3 ) and CA-int-R
(5 -CCTTGGCTGTGGTTTCGCTAGATAGTAGAT-3 ) were used.20
The genotyping was based on the presence or absence of a
DNA insert, which codes for the ribosomal 26S RNA, dividing C. albicans in four groups20 : A (C. albicans 450 bp), B (C.
albicans 840 bp), C (C. stellatoidea 840 bp) and D (C. dubliniensis
1080 bp).

Alignment and phylogenetic construction

The obtained sequences were edited using the Staden program version 1.6, and were compared by the BLAST program
for detection of the similarities using reference sequences
available in the data bank (NCBI, National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/).21,22 The
Mafft program (http://mafft.cbrc.jp/alignment/server/) was
used for the alignment; and visual inspection was done by
MEGA 5.1 version.23 Forty sequences of Candida isolates were
submitted for phylogenetic analysis using Schizosaccharomyces
pombe strain U40085 as outgroup.20 The Maximum Likelihood
phylogenetic tree was built with 100 bootstraps, based on the
evolutionary model Tamura-3 parameters with using 5.1 version of the MEGA software for nal editing.23

In vitro susceptibility tests

The in vitro susceptibility tests were done by micro-dilution
method of broth, as per the Norm M27-A3 recommendations
provided by the Clinical and Laboratory Standards Institute.24
The antifungals used were amphotericin B (SigmaAldrich
110 Qumica, Madrid, Spain), ketoconazole (Pharma Nostra, Brazil), itraconazole (Fragon), uconazole (Pzer, Madrid,
Spain) and nystatin (Pharma Nostra, Brazil). The samples were
diluted in RPMI (Roswell Park Memorial Institute Medium)1640 medium (Sigma) and incubated at 37 C for 48 h.
According to the CLSI criteria, the sensitivity prole is classied as sensitive, dose-dependent sensitivity and resistant.

Results
A total of 40 isolates were obtained from 133 cervicovaginal
samples, which were previously identied by CHROMagar and
API 20AUX systems. On the basis of ITS and D1/D2 sequences,
the isolates could be attributed to the genera Candida and Saccharomyces (Table 1). Among the isolates studied, 20 belonged
to the colonized group, 11 were from the complicated infection
group and 9 were from the uncomplicated infection group.
A tree was constructed using maximum likelihood analysis and the evolutionary model Kimura 2-parameter with 100
bootstraps. A total of 1959 sites were evaluated, of which, 786
were conserved sites, 1092 were variable sites, 712 sites provided parsimonious information (pi), and 361 were unique
sites. The empirical basis frequencies were pi (A): 0.225836
pi (C): 0.283009 pi (G): 0.238533, pi (t) 0.252622. The phylogenetic tree was generated by using 18 strains as references,
which included various types of strains of Candida species,

Kluyveromyces marxianus, K. lactis, Saccharomyces cerevisiae,

Torulaspora delbrueckii, keeping Schizosaccharomyces pombe as an
outgroup.
The evaluated VVC isolates were identied to be C. albicans,
C. dubliniensis, C. guilliermondii, C. kefyr, Saccharomyces cerevisiae
and C. glabrata and were found to be distributed into six clades
supported by bootstrap values (Fig. 1). The phylogenetic data
corroborated with the biochemical data, except for the HC03IC
isolate that was identied as C. albicans by the API 20AUX system, but as C. dubliniensis by the phylogenetic analysis (Fig. 1).
According to the tree, most of the analyzed clinical isolates
were identied to be C. albicans, with 33 isolates clustered in
Albicans clade (bs, 100%). Analysis revealed that C. albicans isolates could not be separated according to the studied groups,
i.e., colonized, complicated, and uncomplicated infection
groups. In Guilliermondii clade (bs, 99%), the clinical isolate
(HC08C) and P. guilliermondii (NRRL Y-2075) type strain were
grouped. The isolate (HC02C) from the colonized group and
Saccharomyces cerevisiae type were clustered in Saccharomyces
clade (bs, 100%). Kefyr clade consisted of Kluyveromyces marxianus (NRRL Y-8281), Candida kefyr teleomorph strain CBS 712,
K. marxianus var. Kluyveromyces lactis strain NRRL Y-8279 and
HC09C isolate of C. kefyr. Three isolates were classied into
the Glabrata clade (HC01C, HC07C and HC10C), belonging to
the colonized group and C. glabrata type (5478) with 100% bootstrap.
Based on the molecular data, amidst the 40 isolates that
were obtained from vaginal samples, the most prevalent
species was C. albicans (82.5%), followed by C. glabrata (7.5%),
C. guilliermondii (2.5%), C. kefyr (2.5%), C. dubliniensis (2.5%) and
Saccharomyces cerevisiae (2.5%). Among the colonized group
alone, a total of 20 isolates belonging to ve different species
C. albicans (60%), C. glabrata (25%), C. guilliermondii (5%), C. kefyr
(5%) and Saccharomyces cerevisiae (5%) were identied. A total
of 9 isolates obtained from the uncomplicated infection group
were C. albicans (100%). In the complicated infection group, 11
isolates were from two different Candida species: C. dubliniensis
(9.1%) and C albicans (90.9%).
Regarding the ABC genotyping of C. albicans, at least two different genotypes (A and B) were observed, although 25 isolates
belonged to type A and 7 isolates to type B, it was not possible
to establish a correlation amidst the genotypes identied and
the susceptibility prole of the tested drugs (Fig. 2).
The susceptibility testing results of the studied isolates
from different patient groups are summarized in Table 2. In
the colonized group (I), all isolates of C. albicans (n = 14) showed
a dose-dependent sensitivity (SDD) to nystatin (8.0 g/mL)
and sensitivity (S) to itraconazole (0.0625 g/mL), uconazole
(0.125 g/mL), amphotericin B (0.031.0 g/mL) and ketoconazole (0.0625 g/mL). Three C. glabrata isolates (HC01C, HC02C
and HC07C) were resistant (R) to itraconazole (4.0 g/mL), SDD
for the uconazole (4.016 g/mL), nystatin (8.0 g/mL) and
sensitive to amphotericin B (0.031.0 g/mL) and ketoconazole (1.04.0 g/mL). The isolate of C. guilliermondii (HC16C)
showed SDD to nystatin (8.0 g/mL), resistance to amphotericin B (2.0 g/mL), sensitivity to itraconazole (0.0625 g/mL),
uconazole (0.125 g/mL), and ketoconazole (0.0625 g/mL).
C. kefyr (HC09C) presented SDD to itraconazole (0.25), nystatin (4.0 g/mL), and sensitivity for uconazole (0.25 g/mL),
amphotericin B (1.0 g/mL) and ketoconazole (0.0625 g/mL).

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15C Candida albicans

14C Candida albicans
11C Candida albicans
09INC Candida albicans
12C Candida albicans
05C Candida albicans
07INC Candida albicans
06IC Candida albicans
04IC Candida albicans
05IC Candida albicans
03INC Candida albicans

CBS562 Candida albicans

05INC Candida albicans
04C Candida albicans
96
13C Candida albicans
17C Candida albicans
01INC Candida albicans
100
100
08IC Candida albicans
11IC Candida albicans
100
06C Candida albicans
01IC Candida albicans
10IC Candida albicans
100
06INC Candida albicans
02IC Candida albicans
19C Candida albicans
20C Candida albicans
100
16C Candida albicans
100
02INC Candida albicans
100
03C Candida albicans
9IC Candida albicans
07IC Candida albicans
100
18C Candida albicans
94 04INC Candida albicans
08INC Candida albicans
03IC Candida dubliniensis
97
100

Clade: Dubliniensis

CBS7987 Candida dubliniensis

CBS94 Candida tropicalis
08C Candida guilliermondii

100

98

Clade: Albicans

Clade: Guilliermondii

CBS2030 Pichia guilliermondii

CBS2285 Debaryomyces carsonii
CECT10-210 Pichia segobiensis

98
100

CBS4159 Candida haemulonii

CBS572 Candida intermedia
100
CBS6986 Clavispora lusitaniae
CBS180 Candida inconspicua
23 Pichia membranifaciens
100
99
L1B Pichia fermentans
100 NRRLY12632 Saccharomyces cerevisiae
100

100

96

02C Saccharomyces cerevisiae

Clade: Saccharomyces

CBS1146 Torulaspora delbrueckii

95 CBS712 Kluyveromyces marxianus
100

100 9C Candida kefyr

Clade: Kefyr

CECT1961 Kluyveromyces lactis

07C Candida glabrata
10C Candida glabrata

100
100

U44808 AY198398 Candida glabrata

Clade: Glabrata

01C Candida glabrata

U40085 Schizosaccharomyces pombe
0.05

Fig. 1 The phylogenetic tree of maximum likelihood based on the alignment of the entire region of its1/its2 and D1/D2 was
built using 100 bootstrap, using the evolutionary model Tamura-3 parameters with program Mega version 5.1.
Schizosaccharomyces pombe was used as an outgroup. The tree showed 6 clades (Albicans; Dubliniensis; Guilliermondii,
Saccharomyces; Kefyr; Glabrata) diversied according to the isolated species. For a thorough understanding, the evaluated
groups in this study are represented by colored squares for discernment: the brown square refers to the colonized group;
one red square indicates isolates from the uncomplicated infection group; the two red squares represent the group with
complicated infection.

378

C
2
H C
C
3
H C
C
4
H C
C
5
H C
C
6
H C
C
1
H 1C
C
1
H 2C
C
1
H 4C
C
1
H 5C
C
1
H 7C
C
1
H 8C
C
1
H 9C
C
2
H 0C
C
1I
H NC
C
2
H INC
C
3
H INC
C
4
H INC
C
5I
H NC
C
6
H INC
C
7
H INC
C
8
H INC
C
9I
H NC
C
1
H IC
C
2
H IC
C
3
H IC
C
4
H IC
C
5
H IC
C
6
H IC
C
7
H IC
C
8I
H C
C
9
H IC
C
10
IC

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 373380

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

2500
2000
1500
1000
750

500

Fig. 2 Agarose electrophoresis ABC genotyping of the C. albicans from the different studied groups, genotype A (C. albicans
450 bp) and B (C. albicans 840 bp). The lanes 214 correspond to the colonized group; 1523 to the uncomplicated infection
group and 2433 to the complicated infection group. Lane 34 represents a blank; the lanes 1 and 35 indicate standard 1 kb
molecular weight markers (Invitrogen, Carlsbad, Ca, USA).

Furthermore, S. cerevisiae isolate (HC02C) was SDD to nystatin

(8.0 g/mL) and sensitive to itraconazole (0.0625 g/mL), uconazole (0.125 g/mL), amphotericin B (0.031.0 g/mL) and
ketoconazole (0.0625 g/mL).
In the complicated infection group (II), one strain of
C. albicans isolate (HC01IC) was found to be SDD to
itraconazole (0.06250.25 g/mL); all isolates (n = 10) were
resistant to nystatin (64 g/mL) and sensitive to uconazole (0.1252.0 g/mL), amphotericin B (1.0 g/mL) and
ketoconazole (0.0625 g/mL). C. dubliniensis isolate (HC03IC)
was resistant to nystatin (64 g/mL) and presented sensitivity toward the itraconazole (0.0625 g/mL), uconazole
(0.1252.0 g/mL), amphotericin B (0.51.0 g/mL) and ketoconazole (0.0625 g/mL). Finally, in the uncomplicated infections group (III), all the isolates (n = 9) of C. albicans were
SDD to nystatin (8.0 g/mL) and sensitive toward itraconazole
(0.0625 g/ml), uconazole (0.1252.0 g/mL), amphotericin B
(0.51.0 g/mL) and ketoconazole (0.0625 g/mL).

Discussion
Identication of Candida species that causes VVC is highly
desirable in microbiological practice, as it may help in clarifying the prevalence and incidence of species that affects

the susceptible population. Moreover, determination of susceptibility of Candida to the antifungal drugs may be crucial
in context of the recurrent clinical forms of VVC. Several
studies have demonstrated the occurrence of vulvovaginitis
due to Candida species, indicating heterogeneity among isolates from different geographical regions. In the present study,
the prevalent species were3,2527 C. albicans, followed by C.
glabrata, C. guilliermondii, C. kefyr, C. dubliniensis and Saccharomyces cerevisiae, thereby suggesting an increase of infection
by non-albicans Candida. An increase in infections that are
caused by non-albicans Candida has been registered, although
C. albicans is still the most isolated species in VVC clinical
cases.13,2831 Furthermore, the cultural and ethnic differences
may also inuence the isolation rate of yeast from vulvovaginitis samples.32,33 The lack of data on epidemiology and genetic
variability reinforces the importance of epidemiological studies by molecular methods.6,27,3436
The colonized group investigated in the current study
presented a wide diversity of species such as C. albicans,
C. glabrata, C. guilliermondii, C. kefyr and Saccharomyces cerevisiae. In addition, C. albicans was found to be prevalent
(n = 19) in the infection group and C. dubliniensis (n = 1) isolates were observed only in the complicated infection group
(Table 2). The microbiota species found in colonized women
are the same as reported in VVC.1,4,27,30,35 Vaginitis caused by

Table 2 Variations in the minimum inhibitory concentration (MIC) of antifungals for the different study groups.
Isolate species
C. albicans (I)
C. albicans (II)
C. albicans (III)
C. glabrata (I)
C. guillermondii (I)
C. Kefyr (I)
S. cerevisiae (I)
C. dubliniensis (II)

Total of samples
14
10
09
03
01
01
01
01

Itraconazole

Fluconazole

Nystatin

Amphotericin B

Ketoconazole

0.06250.0625
0.06250.25 (SDD = 1)
0.1250.125
2.04.0 (R = 3)
0.06250.0625
0.25 (SDD = 1)
0.06250.0625
0.06250.0625

0.1250.125
0.1252.0
0.1250.125
4.016.0 (SDD = 1)
0.1258.0
0.25
0.1250.125
0.1252.0

8.08.0 (SDD=14)
64 (R = 10)
8.08.0 (SDD = 9)
8.08.0 (SDD = 3)
8.0 (SDD = 1)
4.0 (SDD = 1)
8.08.0 (SDD = 1)
64 (R = 1)

0.031.0
0.51.0
1.01.0
0.031.0
0.252.0 (R = 1)
1.0
0.031.0
0.51.0

0.06250.0625
0.06250.25
0.06250.0625
1.04.0
0.06252.5
0.0625
0.06250.0625
0.06250.25

SDD: sensitivity dose dependent; R: resistant; S: sensitivity; I: colonized group; II: complicated infection group; III: uncomplicated group.

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 373380

S. cerevisiae is rare and it has been isolated from an asymptomatic patient.37,38 This corroborates with the ndings of the
present study.
The VVC Candida albicans isolates, analyzed by us, were
clustered into a single clade, indicating a monophyletic group
(Fig. 1), which is in concordance with the data already reported
by several authors.31,39 The strain, identied as C. albicans
(HC03IC) by biochemical test, proved to be C. dubliniensis
according to the phylogenetic analysis. A lack of correlation
between the phenotypic and molecular identication of the
samples can be justied by the limitations of the commercial
identication system, which do not allow distinguishing the
yeast species, which have minor phenotypic differences.40,41
Therefore, multilocus analyses are needed in order to identify
Candida species.31 In the ABC genotyping, two different genotypes among C. albicans isolates were detected. The genotype
A was observed in 75.7% of the isolates, and the isolates of
genotype B were present in all the analyzed groups. However, it
had a higher occurrence in the uncomplicated infected group
(Fig. 2). It has been reported that the candidiasis that is caused
by C. albicans of genotype B has a higher tendency for persistent infections, though further studies with larger populations
for a better assessment are required.36
In the current analysis, all the C. albicans isolates from the
complicated group, regardless of genotype, were resistant to
nystatin and susceptible to other tested antifungals, except
for the isolate HC04IC that showed SDD to uconazole. In the
uncomplicated infections group, the isolates of C. albicans were
SDD to nystatin and susceptible to other tested antifungals.
The same was observed in the isolates from the group of colonized patients. Concerning the non-albicans Candida species,
the isolate of C. dubliniensis was resistant only to nystatin, the
C. glabrata isolates were resistant to itraconazole and SDD to
uconazole and nystatin. These results contrast from the previous reports that regarded C. glabrata isolates as resistant and
SDD to uconazole.42 Besides, C. kefyr strain presented SDD
to itraconazole and nystatin, and a similar susceptibility prole was previously reported.10 In other studies, similar results
were obtained, reporting that VVC species strains of genus
Candida presented resistance and also a high frequency of SDD
for nystatin and sensitivity to others tested drugs.26,35,43
The isolate of C. guilliermondii showed SDD to nystatin
and resistance to amphotericin B. According to the literature,
the treatment is problematical due to a low sensitivity for
some antifungal classes, especially for uconazole, itraconazole and amphotericin B; and VVC infections caused by C.
guilliermondii are rare.2 Furthermore, we obtained an isolate of
S. cerevisiae SDD to nystatin, which differed from the data previously reported for this species, which demonstrated that S.
cerevisiae isolates were resistant to uconazole, posaconazole,
and itraconazole.44
There are several factors that can inuence the clinical response to treatment of VVC, as evident from different
reports showing variation in the in vitro susceptibility and
in vivo response to the drug.39,45 Through in vitro susceptibility
testing, it was observed that all the isolates were sensitive to
ketoconazole, although uconazole remains the drug of choice
for VVC treatment. Such results indicate that the susceptibility
prole for the isolates may not be a factor related to the recurrence of the disease. Therefore, it may be concluded that the

379

molecular analysis provides accurate identication of Candida

species isolated from patients with VVC. Hence, our ndings
demonstrated the importance of molecular tools for identication of the isolates and also to elucidate the epidemiology
of VVC.

Conicts of interest
The authors declare no conicts of interest.

Acknowledgement
We would like to thank the staff of the Diagnostic Unit of
Clinical Hospital and the Microbiology and Molecular Biology
laboratory at UFPR for their technical assistance and the nancial support provided by the Brazilian Federal Agencies: CAPES
(Brazilian Federal Agency for Support and Evaluation of Graduate), CNPq (National Counsel of Technological and Scientic
Development) and the Paranas state agency Fundaco Araucaria.

references

1. Sobel JD. Vulvovaginal candidosis. Lancet.

2007;369:19611971.
2. Barbedo LS, Sgarbi DBG. Candidiasis. DST J Bras Doencas Sex
Transm. 2010;22:2238.
3. Rosa MI, Rumel D. Fatores associados candidase
vulvovaginal: estudo exploratrio. Rev Bras Ginecol Obstet.
2004;26:6570.
4. Mahmoudi RADM, Zafarghandi S, Abbasabadi B, Tavallaee M.
The epidemiology of Candida species associated with
vulvovaginal candidiasis in an Iranian patient population.
Eur J Obstet Gynaecol Reprod Biol. 2010;155:199203.
5. Odds FC, Bernaerts R. CHROMagar Candida, a new differential
isolation medium for presumptive identication of clinically
important Candida species. J Clin Microbiol.
1994;l32:19231929.
6. Corra PR, David PRS, Peres NP, Cunha KC, Almeida MTG.
Caracterizaco fenotpica de leveduras isoladas da mucosa
vaginal em mulheres adultas. Rev Bras Ginecol Obstet.
2009;31:177181.
7. Gondo DCAF, Duarte MTC, Silva MG, Parada CMGL. Abnormal
vaginal ora in low-risk pregnant women cared for by a
public health service: prevalence and association with
symptoms and ndings from gynecological exams. Rev
Latino-Am Enfermagem. 2010;18:919927.
8. Galle LC, Gianinni MJSM. Prevalncia e susceptibilidade de
leveduras vaginais. JBPML. 2004;40:229236.
9. Barrenetxea ZG. Vulvovaginitis candidisica. Rev Iberoam
Micol. 2002;19:2224.
10. Sanguinetti M, Posteraro B, Fiori B, Ranno S, Torelli R, Fadda
G. Mechanisms of azole resistance in clinical isolates of
Candida glabrata collected during a hospital survey of
antifungal resistance. Antimicrob Agents Chemother.
2005;49:668679.
11. Lockhart SR, Messer SA, Pfaller MA, Diekema DJ. Geographic
distribution and antifugal susceptibility of the newly
described species Candida orthopsilosis and Candida
metapsilosis in Comparison to the closely related species
Candida parapsilosis. JCM. 2008;46:26592664.
12. Williams DW, Wilson MJ, Lewis MAO, Potts AJC. Identication
of Candida species by PCR and restriction fragment length

380

13.
14.

15.

16.

17.

18.

19.

20.

21.
22.

23.

24.

25.

26.

27.

28.

29.

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 373380

polymorphism analysis of intergenic spacer regions of

ribosomal DNA. JCM. 1995;33:24762479.
Achkar JM, Fries BC. Candida infections of the genitourinary
tract. Clin Microbiol Rev. 2010;23:253273.
Monroy-Perez E, Sainz-Espunes T, Paniagua-Contreras G,
Negrete-Abascal E, Rodriguez-Moctezuma JR, Vaca S.
Frequency and expression of ALS and HWP1 genotypes in
Candida albicans strains isolated from Mexican patients
suffering from vaginal candidosis. Mycoses. 2012;55:151157.
Dalazen D, Zanrosso D, Wanderley L, Silva NL, Fuentefria
AM. Comparaco do perl de suscetibilidade entre isolados
clnicos de Candida spp. orais e vulvovaginais no Sul do
Brasil. JBPML. 2011;47:3338.
Eckert C, Burghoffer B, Lalande V, Barbuta F. Evaluation of the
chromogenic agar chromID C. difcile. J Clin Microbiol.
2013;l51:10021004.
Vicente VA, Attili-Angelis D, Pie MR, et al. Environmental
isolation of black yeast-like fungi involved in human
infection. Stud Mycol. 2008:137144.
White TJ, Bruns T, Lee S, Taylor J. Amplication and direct
sequencing of fungal ribosomal RNA genes for
phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ,
eds. PCR Protocols: A Guide to Methods and Applications. San
Diego: Academic Press; 1990:315322.
ODonnell K. Fusarium and its near relatives. In: Reynolds
DR, Taylor JW, eds. The Fungal Holomorph: Mitotic, Meiotic and
Pleomorphic Speciation in Fungal Systematics. 1993:225233.
Mccullough MJ, Clemons KV, Stevens DA. Molecular and
phenotypic characterization of genotypic Candida albicans
subgroups and comparison with Candida dubliniensis and
Candida stellatoidea. J Clin Microbiol. 1999;l37:417421.
Boneld J, Beal K, Jordan M, Chen Y, Staden R. The Staden
Package Manual, Cambridge, UK; 2006.
Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST
and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res. 1997;25:33893402.
Tamura K, Dudley J, Nei M, Kumar S. Mega 4: Molecular
Evolutionary Genetics Analysis (MEGA) software version 4.0.
Mol Biol Evol. 2006;24:15961599.
Clinical, Laboratory Standards Institute. Reference method for
broth dilution antifungal susceptibility testing of yeasts; approved
standard third informational supplement. CLSI document
M27-S4. Vol 1. Wayne, PA, USA: Clinical and Laboratory
Standards Institute; 2012:5623856667.
Spinilo A, Capuzzo E, Acciano S, Santolo A, Zara F. Effect of
antibiotic use on the prevalence of symptomatic
vulvovaginal candidiasis. Am J Obstet Gynecol. 1999;180:
1417.
Lopes-Consolaro ME, Albertoni AT, Shizue-Yoshida C, Peralta
RM, Estivalet-Svidzinski TI. Correlation of Candida species
and symptoms among patients with vulvovaginal
candidiasis in Maringa, Parana, Brazil. Rev Iberoam Micol.
2004;21:202205.
Asticcioli S, Sacco L, Daturi R, et al. Trends in frequency and
in vitro antifungal susceptibility patterns of Candida isolates
from women attending the STD outpatients clinic of a
tertiary care hospital in Northern Italy during the years
20022007. Rev Bras Ginecol Obstet. 2009;31:199204.
Linhares LM, Witkin SS, Miranda SD, Fonseca AM, Pinotti JA,
Ledger WJ. Differentiation between women with
vulvovaginal symptoms who are positive or negative for
Candida species by culture. Infect Dis Obstet Gynecol.
2001;l9:221225.
Taverna CG, Bosco-Borgeat ME, Murisengo AO, et al.
Comparative analyses of classical phenotypic method and
ribosomal RNA gene sequencing for identication of

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

medically relevant Candida species. Mem Inst Oswaldo Cruz.

2013;108:178185.
Guzel AB, Jjkit M, Akar T, Burgut R, Demir C. Evaluation of
risk factors in patients with vulvovaginal candidiasis and the
value of chromID Candida agar versus CHROMagar Candida
for recovery and presumptive identication of vaginal yeast
species. Med Mycol. 2007;l49:1625.
Clement KMT, Heide MD, Vincent R, Wieland M.
Re-examining the phylogeny of clinically relevant Candida
species and allied genera based on multigene analyses. FEMS
Yeast Res. 2008;8:651659.
Paulitsch A, Weger W, Ginter-Hanselmayer G, Marth E,
Buzina W. A 5-year (20002004) epidemiological survey of
Candida and non-Candida yeast species causing vulvovaginal
candidiasis in Graz, Austria. Mycoses. 2006;49:471475.
Wei Y, Feng J, Luo C. Isolation and genotyping of vaginal
non-albicans in women from two different ethnic groups in
Lanzhou, China. Int J Gynaecol Obstet. 2010;110:227230.
Chau AS, Mendrick CA, Sabatelli FJ, Loebenberg D,
Mcnicholas PM. Application of real-time quantitative PCR to
molecular analysis of Candida albicans strains exhibiting
reduced susceptibility to azoles. Antimicrob Agents Chemother.
2004;48:21242131.
Ferrazza MHSH, Maluf ML, Consolaro MEL, Shinobu CS,
Svidzinski TIE, Batista MR. Caracterizaco de leveduras
isoladas da vagina e sua associaco com candidase
vulvovaginal em duas cidades do sul do Brasil. Rev Bras
Ginecol Obstet. 2005;27:5863.
Chaves GM, Santos FP, Colombo AL. The persistence of
multifocal colonization by a single ABC genotype of Candida
albicans may predict the transition from commensalism to
infection. Mem Inst Oswaldo Cruz. 2012;107:198204.
Echeverra-Irigoyen MJ, Eraso E, Cano J, Gomriz M, Guarro J,
Quinds G. Saccharomyces cerevisiae vaginitis: microbiology
and in vitro antifungal susceptibility. Mycopathologia.
2011;172:201205.
Pdua RAF, Guilhermetti E, Svidzinski TIE. In vitro activity of
antifungal agents on yeasts isolated from vaginal secretion.
Acta Sci Health Sci. 2003;25:5154.
Diezmann S, Cox CJ, Schnian G, Vilgalys RJ, Mitchell TG.
Phylogeny and evolution of medical species of Candida and
related taxa: a multigenic analysis. J Clin Microbiol.
2004;l42:56245635.
Florez J, Mendez AS, Cano J, Guarro J, Perez RE, Arvalo MP.
Phenotypic and molecular characterization of Candida
nivariensis sp. nov., a possible new opportunistic fungus. J
Clin Microbiol. 2005;43:41074111.
Vijgen SNYSS, Naesens R, Magerman K, Boel A, Cartuyvels R.
Comparison of Vitek identication and antifungal
susceptibility testing methods to DNA sequencing and
Sensitive Yeast One antifungal testing. Med Mycol.
2010;49:107110.
Oliveira PM, Mascarenhas RE, Lacroix C, et al. Candida species
isolated from the vaginal mucosa of HIV-infected women in
Salvador, Bahia, Brazil. Braz J Infect Dis. 2011;15:239244.
Lattif AA. Molecular typing and in vitro uconazole
susceptibility of Candida species isolated from diabetic and
nondiabetic women with vulvovaginal candidiasis in India. J
Microbiol Immunol Infect. 2011;44:166171.
Papaemmanoul V, Georgogiannis N, Plega M, et al. Prevalence
and susceptibility of Saccharomyces cerevisiae causing
vaginitis in Greek women. Anaerobe. 2011;17:298299.
Pfaller MA, Diekema DJ, Sheehan DJ. Interpretative
breakpoints for uconazole and Candida revisited: a
blueprint for the future of antifungal susceptibility testing.
Clin Microbiol Rev. 2006;19:435447.