Environ Monit Assess (2017) 189: 8

DOI 10.1007/s10661-016-5682-8

Comparison of methods to evaluate the fungal biomass

in heating, ventilation, and air-conditioning (HVAC) dust
Marie-Jeanne Biyeyeme Bi Mve & Yves Cloutier &
Nancy Lacombe & Jacques Lavoie & Maximilien Debia &
Genevive Marchand

Received: 9 February 2016 / Accepted: 7 November 2016 / Published online: 6 December 2016
# Springer International Publishing Switzerland 2016

Abstract Heating, ventilation, and air-conditioning coefficients varied from 0.217 to 0.83. The 18S qPCR
(HVAC) systems contain dust that can be contaminated showed the best sensitivity and precision, as well as the
with fungal spores (molds), which may have harmful best correlation with the culture method. PCR targets
effects on the respiratory health of the occupants of a only molds, and a total count of fungal DNA is obtained.
building. HVAC cleaning is often based on visual in- Among the methods, mold DNA amplification by qPCR
spection of the quantity of dust, without taking the mold is the method suggested for estimating the fungal content
content into account. The purpose of this study is to found in dust of HVAC systems.
propose a method to estimate fungal contamination of
dust in HVAC systems. Comparisons of different analyt- Keywords Indoor air quality . HVAC systems .
ical methods were carried out on dust deposited in a Sampling methods . Dust . Fungal biomass
controlled-atmosphere exposure chamber. Sixty samples
were analyzed using four methods: culture, direct micro-
scopic spore count (DMSC), -N-acetylhexosaminidase
Introduction
(NAHA) dosing and qPCR. For each method, the limit of
detection, replicability, and repeatability were assessed.
In North America, the decision to have a ventilation duct
The Pearson correlation coefficients between the methods
system cleaned is based on a visual assessment of the
were also evaluated. Depending on the analytical method,
amount of dust accumulated on the inner duct surfaces
mean spore concentrations per 100 cm2 of dust ranged
(Lavoie et al. 2010, 2011; NADCA 2006) To address
from 10,000 to 682,000. Limits of detection varied from
the subjective nature of this method of assessment,
120 to 217,000 spores/100 cm2. Replicability and repeat-
various organizations have proposed quantitative
ability were between 1 and 15%. Pearson correlation
criteria for evaluating HVAC cleanliness (Lavoie et al.
2010, 2011; NADCA 2006). However, none of these
M.<J. Biyeyeme Bi Mve : Y. Cloutier : N. Lacombe : criteria takes into account the mycological content of the
J. Lavoie : G. Marchand deposited dust. A number of authors have shown that
Institut de recherche Robert-Sauv en sant et scurit du travail, poorly maintained HVAC systems can be contaminated
505 Boul. de Maisonneuve Ouest, Montral H3A3C2, Canada
by fungal particles (Burge et al. 1985; Krause and
M.<J. Biyeyeme Bi Mve : J. Lavoie : M. Debia : Hammad 2002; Mendell et al. 2003). The dust deposited
G. Marchand (*) in HVAC systems is a source of nutrients that in the right
Dpartement de sant environnementale et sant au travail, cole conditions can support the growth of mold (Foarde et al.
de sant publique, Universit de Montral, Pavillon Marguerite
dYouville, 2375 Chemin de la cte Ste-Catherine, Montral H3T 1996; Mensah-Attipoe et al. 2015). Xerophilic molds
1A8, Canada can grow easily in low water environments such as
e-mail: [email protected] HVAC dust. Krause and Hammad (2002) have shown
8 Page 2 of 10 Environ Monit Assess (2017) 189: 8

that samples taken from a HVAC system that was re- of -N-acetylhexosaminidase (NAHA), and quantita-
cently cleaned and looks clean can still contain high tive polymerase chain reaction (qPCR).
levels of fungal contamination. Aerosolization of The objective is to propose a method for estimating the
HVAC contaminated dust can increase fungal spore mold biomass contained in dust of HVAC systems in
concentrations in the circulated air and can therefore order to determine the state of dirtiness or to assess the
be responsible for the exposure of the occupants cleaning effectiveness. More specifically, this study com-
(Bernstein et al. 1983; Buttner et al. 1999; Kulp 1995). pared four mold analysis methods using samples of de-
Many epidemiological studies have highlighted a link posited dust generated in a laboratory exposure chamber.
between high humidity, molds or their sub-products, and
health effects (CDC-NIOSH 2012; Heseltine and Rosen
2009). Molds are suspected of being responsible for Materials and methods
respiratory tract illnesses, such as rhinitis, humidifier
fever, asthma, and hypersensitivity pneumonitis Experimental design
(Heseltine and Rosen 2009). Symptoms of sick building
syndrome have been reported, the most common being The deposited dust samples were generated in an expo-
lethargy; irritation of the nose, throat, and eyes; head- sure chamber. For each dust/spores generation trial, six
ache; wheezing; shortness of breath; and concentration dust samples were prepared. A total of ten trials was
problems (Burge et al. 1985; Fisk et al. 2010; Garrett performed with different parameters in order to get
et al. 1998; Jarvis and Morey 2001; Meklin et al. 2002; samples with different levels of dust and mold spores.
Mendell et al. 2003; Menzies et al. 2003; Menzies and A common extraction was performed for all four
Bourbeau 1997; Park et al. 2006). A higher prevalence methods and the suspension was aliquoted for each
of symptoms has been found among the occupants of method. This unique extraction protocol allows to min-
buildings affected by mold or humidity. When occu- imizing the variability between samples. This strategy
pants are moved away from contaminated environments allows the observed differences to be attributed directly
or when corrective measures are taken, a significant drop to the analytical methods and not to inter-samples vari-
in symptoms is seen (Jarvis and Morey 2001; Meklin ations. The traditional culture method was used as the
et al. 2005). A dose-response between these illnesses reference method for all statistical test comparisons.
and the presence of molds, their components or indica-
tors of their presence, has been suggested (Jarvis and Mold strains and fungal cultures
Morey 2001; Park et al. 2006; Williamson et al. 1997).
Evaluation of the mold biomass in the deposited dust Six mold strains from the IRSSTs microbial collection
of HVAC should be considered when determining if the were used to contaminate the dust used for the prepara-
system should be cleaned, and after the cleaning to tion of samples in the lab exposure chamber:
evaluate the effectiveness of the work done. It could Cladosporium cladosporoides, Penicillium digitatum,
also be of interest during building mold investigations Ulocladium chartarum, Acremonium recifei,
related to indoor air quality problems. In this regard, the Syncephalastrum racemosum, and Absidia corymbifera.
availability of a reliable analytical method is essential. Fungal spores were harvested from a 710-day old
Analytical methods to evaluate environmental mycolog- culture grown on malt extract agar (MEA) (Oxoid,
ical load have already been reported (Krause et al. 2003; Nepean, ON, Canada) at 25 C. The mold spores were
Mandal and Brandl 2011; Mensah-Attipoe et al. 2015; collected directly from the culture with a wet swab that
Rylander et al. 2010). Some approaches focus on the was rolled all over its surface. The spores on the swab
analysis of the mold-containing particles, while others were then inoculated by immersion in PCR grade water
check for their fragments or mold sub-products. In the to prepare the suspensions. Several back and forth from
present study, four methods previously used to assess the colony to the PCR water is needed to achieve the
the presence of mold in various environments were desired concentrations of spores in all suspensions. The
evaluated (Krause et al. 2003; Krause and Hammad final concentrations of the pure spore suspensions were
2002; Mandal and Brandl 2011; Reeslev et al. 2003; determined using a hemacytometer (Hausser Scientific,
Rylander et al. 2010). The four methods are culture, Horsham, PA, USA). The microscope magnification
direct microscopic spore count (DMSC), enzyme assay used was 500 (40 Plan achromatic objective and
Environ Monit Assess (2017) 189: 8 Page 3 of 10 8

12.5 eyepieces) on a Nikon E400 microscope (Nikon observed when the experiment is duplicated with no
instruments inc., Melville, NY, USA). To calculate the modifications (same analyst, instrument, and day), and
concentrations for the ten suspensions prepared, the 25 the repeatability is the variation observed when at least
large squares of the center sections of both chambers of one change is implemented to the experiment. The
the hemacytometer were counted. The total number of replicability was determined by analyzing four suspen-
spores counted in the 50 squares was used to obtain the sions with different concentrations of spores. Each sus-
concentrations of all pure strain suspensions. The final pension was analyzed three to six times depending on
suspensions used in the generator were prepared by the method. The 95% confidence interval of the coeffi-
combining one to three different pure strain suspensions cients of variation (CV) obtained for the four suspen-
of molds (Table 1). sions was used to calculate the replicability. The same
calculations were performed for repeatability, both are
reported in percentage. For the qPCR method, exclusiv-
Analytical methods validation ity and specificity were also documented. The exclusiv-
ity is the ability of the PCR detection system not to
The limit of detection (LOD), minimum reported value amplify DNA coming from untargeted cells. The selec-
(MRV), replicability, and repeatability were determined tivity is the ability to amplify the entire DNA coming
for all methods. The LOD is the lowest concentration from the targeted cells, in this study, the DNA from
that can be measured by the instrument above the re- molds. The amplification ability of the system was
ported results of a suspension without spores in it tested against 56 different strains of molds for the spec-
(blank). To determine the LODs, a spore suspension ificity and 40 strains of bacteria for the exclusivity.
with a starting concentration of 106 spores/mL was
progressively diluted until no more results could be
achieved. This was performed independently for each Comparison of analytical methods
method. In this study, the LOD corresponds to three
times the standard deviation calculated from 10 readings Simulation of dust contamination in the exposure
performed on a suspension having a concentration of chamber
spores slightly above the no response suspension. The
MRV is the lowest concentration that can be measured The generation of dust and mold spores was accom-
above the background considering the sampling and plished simultaneously inside an exposure chamber
extraction process. The replicability is the variation mimicking an HVAC duct (Fig. 1). Before the

Table 1 Content of the mold spores suspensions used for the aerosolization trials in the exposure chamber and duration of the generation for
the mold and the dust

Concentration of the spore in the suspensions Generation time (hour)

(spores/mL)

Strain Pen.d Ulo.c Clado.c Acre.r Absi.c Sync.r Mold Dust

1 4.0 106 3 3
2 4.0 106 1.0 106 6.0 106 3 3
6
3 6.3 10 4 4
4 2.1 106 6 0.25
5 3.0 106 1.0 106 2.0 106 7 0.5
6 6.2 106 8 0.5
7 110 106 65 106 16 106 7 1
6
8 16 10 7 3
9 8.8 106 8.9 105 8.7 106 7 5
10 6.2 106 8 8

Pen.d Penicillium digitatum, Ulo.c Ulocladium chartarum, Clado.c(s) Cladosporium cladosporode, Acre.r Acremonium recifei, Absi.c
Absidia corymbifera, Sync.r Syncephalastrum racemosum
8 Page 4 of 10 Environ Monit Assess (2017) 189: 8

generations, the dust was passed through multiple sieves 100 cm2 using a 37-mm dust sampling cassette loaded
with a mechanical shaker and sterilized. Only the parti- with a 0.5-m polyvinyl chloride filter and equipped
cles recovered from the stages with a sieve opening of with an integrated angulated nozzle collector having a
38 m and smaller were used in the fluidized bed for the 5 mm inlet diameter (EMS, Charleston, SC, USA). To
generation (model 3400A, TSI, Shoreview, MN, USA). find out the mass of each sample collected, the
The combined flow rate of the bed (8.5 L/min) and the entire cassettes were pre- and post-weighed using
bead purge (3.6 L/min) was 12.1 L/min. The chain an Analytic Plus AP250D (0.01 g) analytical bal-
speed used on the fluidized bed generator was 7 mm3/ ance (OHAUS, Butler, NJ, USA,). Six samples were
min. A 6-jet Collison nebulizer (BGI, Butler, NJ, USA) taken per generation, for a total of 60 samples ana-
was used in parallel with the fluidized bed to aerosolize lyzed by the four methods.
the mold spores suspensions. Its flow rate was set to
20 L/min for all generations. The air in the exposure
chamber was recirculated in a closed loop in order to Extraction of samples
reach the desired concentration in the chamber. After the
generation, the chamber remained closed to give time All samples were extracted on the same day they
for the dust to settle on the duct surface. The results of were collected. The entire content of the cassettes
the ten separate sessions of generation conducted for this was recovered by introducing 3 mL of sterile PCR
study are presented in Table 1. Generation times for water directly into the cassette through the collector
molds and dusts were not always the same in order to spout. The cassettes were then agitated on a maxi-
produce variable levels of dust and mold on the surface. vortex DVX-2500 (VWR, Radnor, PA, USA) at
2500 rpm for 5 min. Each extract was then separated
Samples from the exposure chamber into four aliquots: one for each method. The same
protocol was performed on a cassette not used for
Samples of settled dust containing mold were collected sampling; this was the extraction blank and it was
with a vacuum sampler method and a template of analyzed by the four methods.

Fig. 1 Schematic of the exposure chamber mimicking the duct of an HVAC system used to generate dust and molds samples
Environ Monit Assess (2017) 189: 8 Page 5 of 10 8

Culture calculating the Mycometer value (MV) using the for-

mula provided in the original protocol. The number of
For each sample, 100 L of concentrated extract and of spores/100 cm2 was calculated using Eq. (1) obtained
a 1/10 dilution were plated in triplicates on MEA. One from the regression line of the standard curve for a
hundred microliters of the dilution buffer was also plat- suspension of spores of P. digitatum.
ed as a negative control. After 4 to 7 days of incubation .
at 25 C, the colonies were counted using an SMZ-2T MV 3:428*3 ml 0:0005*0:1 ml 1
stereo microscope (Nikon, Tokyo, Japan) at a magnifi-
cation of 20. For each sample, the number of colonies P. digitatum was chosen to build the standard curves
counted on the three medium having between 10 and for both the NAHA and qPCR methods because it is
200 colony-forming units (CFU) was average and the easily cultivated on MEA and produces high numbers of
result was reported in terms of CFU/100 cm2. spores allowing a good range of concentrations to build
the standard curves.
Microscopy method (DMSC)
qPCR
Before adding 100 L of the concentrate or the 1/10
dilution of the extracted sample, 3 mL of sterile water Genomic DNA was extracted from 500 L of each
was placed in a filtering funnel. To help for the visual- sample suspension using the ZR Fungal Bacterial
ization under the microscope, 100 L of 5% safranin DNA Miniprep kit (Zymo Research, Irvine, CA,
(Becton Dickinson, Sparks Glencoe, MD, USA) was USA). The final volume of elution was 100 L. For
also added. Then, the suspension was filtered on a each batch of extraction, a control was performed using
mixed cellulose ester (MCE) membrane (diameter only PCR water. The primers and probes of the ampli-
25 mm; pore size 0.8 m) (SKC, Eighty Four, PA, fication system used are shown in Table 2. The volume
USA). The filtrate was left to dry at room temperature of the qPCR was 25 L. Each master-mix contained 2.5
for 24 h. Thereafter, MEC membranes were rendered units of HotStarTaq mix (Qiagen, Limburg,
transparent with hot acetone using a Vap-300 vaporizer Netherlands); 1.25 M of each primer (Integrated
(BGI, Waltham, MA, USA). For all samples, analysis DNA Technologies, Coralville, IA, USA); 0.375 M
was performed using a Nikon Eclipse E400 microscope of the probe; 2 mM of MgCl2 (Sigma-Aldrich, St.
at 750 enlargement and a total of 50 random fields Louis, MO, USA); 6.75 L of sterile PCR water; and
were counted regardless of the number of spores per 2 L of DNA. A Master Cycler Realplex2 (Eppendorf,
field. Concentrations are reported in spores/100 cm2. Hamburg, Germany) was used for amplification follow-
ing this program: 15 min at 95 C, followed by forty 15-
NAHA enzyme assay modified method s cycles at 94 C; 30 s at 55.5 C; and 15 s at 72 C. In
with the Mycometer each 96-well plate, a standard curve was incorporated.
The standard curve was made by serial dilutions of
The NAHA enzyme assay was performed on the liquid DNA coming from a suspension of P. digitatum. The
aliquot of the sample following a modified protocol correlation coefficients of the standard curves needed to
based on the assay protocol provided with the be higher than 0.96 and the PCR reaction efficiency
M yc o m e t e r ( M yc o m e t e r h an d bo o k , 20 11 ) needed to be better than 80%. The DNA was extracted
(Mycometer, Tampa, FL, USA). First, the baseline fluo- from a spore suspension to which the concentration was
rescence of the developer, blank value 1 (BV1), was
measured, then an intermediate fluorescence (BV2) was
measured on 100 L of the fluorogenic substrate added Table 2 Probe and primers used in the universal qPCR for the
to 2 mL of the developer. Then, 100 L of the sample amplification of molds DNA in the dust deposited in the exposure
chamber
was incubated in 2 mL of substrate. After 30 min at
ambient temperature, the enzyme activity was quanti- FungiQuant-F GGR AAA CTC ACC AGG TCC AG
fied by measuring the fluorescence analysis value (AV) FungiQuant-R GSW CTA TCC CCA KCA CGA
of the sample. The actual fluorescence, expressed in FungiQuant-Probe TGG TGC ATG GCC GTT
relative fluorescence units (RFUs), was obtained by
8 Page 6 of 10 Environ Monit Assess (2017) 189: 8

previously determined with a hemacytometer. The rela- the qPCR method, selectivity testing showed that all the
tion between the Ct obtained by the qPCR and the bacteria produced Cts greater than 35, whereas for
number of spores in the suspensions was obtained di- specificity, all the molds tested produced much low-
rectly from that standard curve. The concentrations of er Cts, between 17 and 27. Of the 60 samples, 12
the standard curve varied from 3 106 to 30 spores/mL. (20%) were undetected by DMSC method and 2
All amplifications were duplicated and their average (3%) by the NAHA assay.
calculated. Negative and positive controls were added
in all the plates. For the positive control, the 2 L of
sample DNA was replaced by DNA from P. digitatum. Comparison of analytical methods
For the negative control, the DNA was replaced by PCR
water. All results are reported in spores per one hundred Figure 2 presents the box plot of the mass of dust
square centimeters. deposited on the lower horizontal surface of the expo-
sure chamber and of the number of colonies obtained by
Data analysis the culture method for each generation. The weight of
dust sampled varied between 11.50 and 168 mg/
All statistical analyses were performed on log- 100 cm2, while the CFUs values obtained with the
transformed data. A quantile-quantile (Q-Q) plot was reference method ranged between 1080 and 99,000/
used to graphically determinate the lognormal distribu- 100 cm2. Figure 3 presents the concentrations obtained
tion. Variance analyses (ANOVAs) were performed on by each method. The median number of spores reported
the logarithms of spore concentrations reported by each by the other three methods ranged from 10,132 to
method. Dunnetts test was used to identify means sig- 815,262 spores/100 cm2. Dunnetts multiple compari-
nificantly different from those obtained by the culture son procedure showed that qPCR and the NAHA en-
method. Linear association, on the log-transformed data, zyme assay produce statistically higher concentrations
between the different methods was obtained using the than culture (p < 0.05), whereas DMSC is not statisti-
Pearson correlation (r). Results below the detection cally different (p = 0.30). The NAHA enzyme assay
limits were imputed a value of LOD/2 for all statistical yielded significantly higher concentrations than the
analyses. The IBM SPSS Statistics software package for qPCR method, which itself yielded more spores than
Windows, release 21.0 (IBM, Armonk, NY, USA, 2012) the DMSC method (data not shown). Figure 4 shows
was used. the linear associations and the Pearson correlation
coefficients (r) obtained between the different
methods. A significant correlation was observed be-
Results tween culture and qPCR (p < 0.01), and DMSC
method (p < 0.01), but culture has no correlation
Analytical methods validation with the NAHA method (p = 0.169). A significant
correlation was also seen between qPCR and DMSC
Table 3 presents the analytical validation parameters of (p < 0.01) and NAHA (p < 0.05) methods (data not
the four methods. Limits of detection varied from 120 to shown). No significant correlation was observed
217,522 spores/100 cm2. Replicability and repeatability between the NAHA assay and the DMSC method
were between 1 and 15% depending on the method. For (p = 0.571).

Table 3 Validation results for the

four analytical methods Method LOD Replicability (%) Repeatability (%)
spores or CFU/mL

Culture 40 12 11
Microscopy 660 5 10
NAHA assay 72,500 4 9
qPCR 10 4 7
Environ Monit Assess (2017) 189: 8 Page 7 of 10 8

Discussion and conclusion The culture method is often used as a reference in

many fields of microbiology. Its underestimation is
Figure 2 shows that the dust levels generated in the well documented and can reach two orders of mag-
exposure chamber correspond to levels of dustiness nitude (Krause et al. 2003; Mandal and Brandl 2011;
often found in HVAC systems. In fact, Boor et al. re- Schnrer 1993). Even if it has some recognized
ported levels varying from 10 to 1000 mg/100 cm2 and limitations, culture was chosen as the control meth-
Lavoie et al. (2011) described levels ranging from 0.14 od in the present study. In addition to the well-
to 337 mg/100 cm2 in samples taken in a variety of known limitation linked to the inhibition of some
HVAC ducts. The levels of dust generated for this study molds growth in the laboratory conditions, counts
varied from 11 to 160 mg/100 cm2. can be erroneous when confluences of the colonies

Fig. 2 Box plot diagrams

showing on the upper graph the
weight of the dust deposited on
the floor of the exposure chamber
during each generation (mg/
100 cm2) (n = 6) and on the lower
graph the correspondent diagram
showing the concentrations of
molds in the dust obtained by the
culture method (CFU/100 cm2).
The whiskers depicted 1.5 times
the interquartile range, the box
signifies the upper and the lower
quartiles, and the median is
represented by a short black line
within the box. n = 60
8 Page 8 of 10 Environ Monit Assess (2017) 189: 8

** a
a
** a
a
b
b

c b
c b
c c

Fig. 4 Box plot diagrams showing the logarithm of the molds

concentrations (spores or colonies/100cm2) obtained for each
Fig. 3 Box plot diagrams showing the logarithm of the molds method at three different load of dust. The whiskers depicted 1.5
concentrations (spores or colonies/100 cm2) obtained for each times the interquartile range, the box signifies the upper and the
method, n = 60. The whiskers depicted 1.5 times the interquartile lower quartiles, and the median is represented by a short black line
range, the box signifies the upper and the lower quartiles, and the within the box. For each dust load, a, b, and c are significantly
median is represented by a short black line within the box (double different at p < 0.05
asterisk indicates p < 0.05)
yielded higher concentrations of spores. This result is
on the petri dishes make it difficult to distinguish consistent with those reported by Mensah-Attipoe et al.
between two or more colonies. (2015) and Krause and Hammad (2002), who respectively
The NAHA enzyme assay is very easy to perform. It found that the counts of colonies by culture and the counts
does not require a specialist and can be performed directly of spores by microscopy were lower than the one obtained
in the field. In comparison, a trained analyst working in a by the NAHA assay. Mensah-Attipoe et al. (2015) reported
laboratory is required for the other methods. The enzyme a correlation between the colony count and the NAHA
assay proved to be the least sensitive of the methods assay. In their study, in opposition with the present study,
evaluated. The LOD was calculated to be 72,500 spores/ independent samples were analyzed by each analytical
mL. Depending on the measurement unit used, this can be method. The NAHA enzyme is present in viable and
equivalent to 40 RFU/mL or to a minimum reported value non-viable structures, this could explain part of the over-
(MRV) of 120 RFU/100 cm2 when the global method estimation of NAHA when compared to culture (Mensah-
from sampling to analysis is taken into consideration. Attipoe et al. 2015; Rylander et al. 2010). In addition, as it
Despite the lack of sensitivity observed in the present was reported by Rylander, the NAHA enzyme is not
study, it was comparable to the LOD reported by other specific to molds. This enzyme is also produced by bacte-
authors. In fact, Mensah-Attipoe et al. (2015) reported a ria, protozoa, pollen, and some mammalian cells (Rylander
MRVof 216 RFU/100cm2 while Reeslev et al. (2003) and et al. 2010). Because of its lack of specificity to molds, this
Krause and Hammad (2002) reported LODs of 140 and assay is not appropriate. It will overestimate the fungal
120 RFU/mL, respectively. The differences between the contamination of dust in HVAC ducts, mainly because of
reported sensitivities could be attributed to the calculation the presence of high concentrations of bacteria and of other
methods and whether only the analytical or the global types of cells usually found in dust.
process was considered. In this study, the LOD was calcu- In this study, the DMSC method turned out to be less
lated as three times the standard deviation obtained from sensitive than the culture method. Unlike the culture
ten readings taken from a suspension having a concentra- method, the DMSC method is not limited to the culti-
tion slightly above the background level. Other studies did vable cells, so higher concentrations were expected.
not specify their LOD calculation methods. Though less This lack of sensitivity has also been reported by
sensitive than the other methods, the enzyme assay always Mandal and Brandl (2011). In fact, from their point of
Environ Monit Assess (2017) 189: 8 Page 9 of 10 8

Fig. 5 Linear regression and Pearson correlation coefficient (triple asterisk indicates p < 0.01) obtained between the standard culture
method and the three other analytical methods

view, microscopic methods are only semi-quantitative showing that it is a good predictor of the concentrations
methods and we do agree with them. For the DMSC of spores in dust of HVAC systems. Because of all these
method, only 50 out of the 1535 possible random fields advantages, the qPCR method with the universal
were counted during this study. Even though good re- markers is recommended for the estimation of the mold
peatability was demonstrated during the validation pro- biomass in the dust of HVAC.
cess, the low number of fields counted seems to produce As a complement to the gravimetric evaluation of the
in the end only a good approximation of the concentra- dust deposited on the surface of a duct, the mold bio-
tion of mold spores in the dust. In addition, the difficulty mass present in the dust of HVAC system need to be
to distinguish fungal structures from other particles of evaluated. From this study, the qPCR method seems to
dust proved to be another limitation of that method for be the method of choice to perform such an assessment.
dust samples. This study was performed under controlled parameters
The qPCR method showed a LOD of 10 spores/mL with a low diversity of molds. Evaluations in real situ-
or an equivalent of 142 spores/100 cm2. That method ations are needed to confirm the choice of qPCR as the
detects fungal DNA regardless of the spore viability. method for mold biomass evaluation in dust.
This is an important characteristic during indoor air
quality problems investigations since all fungal struc- Acknowledgements This study was supported by the Institut de
tures could conceivably have health effects (Heseltine recherche Robert-Sauv en sant et scurit du travail (IRSST).
and Rosen 2009). The qPCR sensitivity could easily be
improved by many ways: increasing the volume of
sample extracted or lowering the final elution or increas- References
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