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
(HVAC) systems contain dust that can be contaminated
with fungal spores (molds), which may have harmful
effects on the respiratory health of the occupants of a
building. HVAC cleaning is often based on visual in-
spection of the quantity of dust, without taking the mold
content into account. The purpose of this study is to
propose a method to estimate fungal contamination of
dust in HVAC systems. Comparisons of different analyt-
ical methods were carried out on dust deposited in a
controlled-atmosphere exposure chamber. Sixty samples
were analyzed using four methods: culture, direct micro-
scopic spore count (DMSC), -N-acetylhexosaminidase
(NAHA) dosing and qPCR. For each method, the limit of
detection, replicability, and repeatability were assessed.
The Pearson correlation coefficients between the methods
were also evaluated. Depending on the analytical method,
mean spore concentrations per 100 cm2 of dust ranged
from 10,000 to 682,000. Limits of detection varied from
120 to 217,000 spores/100 cm2. Replicability and repeat-
ability were between 1 and 15%. Pearson correlation
M.<J. Biyeyeme Bi Mve : Y. Cloutier : N. Lacombe :
J. Lavoie : G. Marchand
Institut de recherche Robert-Sauv en sant et scurit du travail,
505 Boul. de Maisonneuve Ouest, Montral H3A3C2, Canada
M.<J. Biyeyeme Bi Mve : J. Lavoie : M. Debia :
G. Marchand (*)
Dpartement de sant environnementale et sant au travail, cole
de sant publique, Universit de Montral, Pavillon Marguerite
dYouville, 2375 Chemin de la cte Ste-Catherine, Montral H3T
1A8, Canada
e-mail: marchand.genevieve@irsst.qc.ca
coefficients varied from 0.217 to 0.83. The 18S qPCR
showed the best sensitivity and precision, as well as the
best correlation with the culture method. PCR targets
only molds, and a total count of fungal DNA is obtained.
Among the methods, mold DNA amplification by qPCR
is the method suggested for estimating the fungal content
found in dust of HVAC systems.
Keywords Indoor air quality . HVAC systems .
Sampling methods . Dust . Fungal biomass
Introduction
In North America, the decision to have a ventilation duct
system cleaned is based on a visual assessment of the
amount of dust accumulated on the inner duct surfaces
(Lavoie et al. 2010, 2011; NADCA 2006) To address
the subjective nature of this method of assessment,
various organizations have proposed quantitative
criteria for evaluating HVAC cleanliness (Lavoie et al.
2010, 2011; NADCA 2006). However, none of these
criteria takes into account the mycological content of the
deposited dust. A number of authors have shown that
poorly maintained HVAC systems can be contaminated
by fungal particles (Burge et al. 1985; Krause and
Hammad 2002; Mendell et al. 2003). The dust deposited
in HVAC systems is a source of nutrients that in the right
conditions can support the growth of mold (Foarde et al.
1996; Mensah-Attipoe et al. 2015). Xerophilic molds
can grow easily in low water environments such as
HVAC dust. Krause and Hammad (2002) have shown
8 Page 2 of 10
that samples taken from a HVAC system that was re-
cently cleaned and looks clean can still contain high
levels of fungal contamination. Aerosolization of
HVAC contaminated dust can increase fungal spore
concentrations in the circulated air and can therefore
be responsible for the exposure of the occupants
(Bernstein et al. 1983; Buttner et al. 1999; Kulp 1995).
Many epidemiological studies have highlighted a link
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
respiratory tract illnesses, such as rhinitis, humidifier
fever, asthma, and hypersensitivity pneumonitis
(Heseltine and Rosen 2009). Symptoms of sick building
syndrome have been reported, the most common being
lethargy; irritation of the nose, throat, and eyes; head-
ache; wheezing; shortness of breath; and concentration
problems (Burge et al. 1985; Fisk et al. 2010; Garrett
et al. 1998; Jarvis and Morey 2001; Meklin et al. 2002;
Mendell et al. 2003; Menzies et al. 2003; Menzies and
Bourbeau 1997; Park et al. 2006). A higher prevalence
of symptoms has been found among the occupants of
buildings affected by mold or humidity. When occu-
pants are moved away from contaminated environments
or when corrective measures are taken, a significant drop
in symptoms is seen (Jarvis and Morey 2001; Meklin
et al. 2005). A dose-response between these illnesses
and the presence of molds, their components or indica-
tors of their presence, has been suggested (Jarvis and
Morey 2001; Park et al. 2006; Williamson et al. 1997).
Evaluation of the mold biomass in the deposited dust
of HVAC should be considered when determining if the
system should be cleaned, and after the cleaning to
evaluate the effectiveness of the work done. It could
also be of interest during building mold investigations
related to indoor air quality problems. In this regard, the
availability of a reliable analytical method is essential.
Analytical methods to evaluate environmental mycolog-
ical load have already been reported (Krause et al. 2003;
Mandal and Brandl 2011; Mensah-Attipoe et al. 2015;
Rylander et al. 2010). Some approaches focus on the
analysis of the mold-containing particles, while others
check for their fragments or mold sub-products. In the
present study, four methods previously used to assess
the presence of mold in various environments were
evaluated (Krause et al. 2003; Krause and Hammad
2002; Mandal and Brandl 2011; Reeslev et al. 2003;
Rylander et al. 2010). The four methods are culture,
direct microscopic spore count (DMSC), enzyme assay
Environ Monit Assess (2017) 189: 8
of -N-acetylhexosaminidase (NAHA), and quantita-
tive polymerase chain reaction (qPCR).
The objective is to propose a method for estimating the
mold biomass contained in dust of HVAC systems in
order to determine the state of dirtiness or to assess the
cleaning effectiveness. More specifically, this study com-
pared four mold analysis methods using samples of de-
posited dust generated in a laboratory exposure chamber.
Materials and methods
Experimental design
The deposited dust samples were generated in an expo-
sure chamber. For each dust/spores generation trial, six
dust samples were prepared. A total of ten trials was
performed with different parameters in order to get
samples with different levels of dust and mold spores.
A common extraction was performed for all four
methods and the suspension was aliquoted for each
method. This unique extraction protocol allows to min-
imizing the variability between samples. This strategy
allows the observed differences to be attributed directly
to the analytical methods and not to inter-samples vari-
ations. The traditional culture method was used as the
reference method for all statistical test comparisons.
Mold strains and fungal cultures
Six mold strains from the IRSSTs microbial collection
were used to contaminate the dust used for the prepara-
tion of samples in the lab exposure chamber:
Cladosporium cladosporoides, Penicillium digitatum,
Ulocladium chartarum, Acremonium recifei,
Syncephalastrum racemosum, and Absidia corymbifera.
Fungal spores were harvested from a 710-day old
culture grown on malt extract agar (MEA) (Oxoid,
Nepean, ON, Canada) at 25 C. The mold spores were
collected directly from the culture with a wet swab that
was rolled all over its surface. The spores on the swab
were then inoculated by immersion in PCR grade water
to prepare the suspensions. Several back and forth from
the colony to the PCR water is needed to achieve the
desired concentrations of spores in all suspensions. The
final concentrations of the pure spore suspensions were
determined using a hemacytometer (Hausser Scientific,
Horsham, PA, USA). The microscope magnification
used was 500 (40 Plan achromatic objective and
Environ Monit Assess (2017) 189: 8
12.5 eyepieces) on a Nikon E400 microscope (Nikon
instruments inc., Melville, NY, USA). To calculate the
concentrations for the ten suspensions prepared, the 25
large squares of the center sections of both chambers of
the hemacytometer were counted. The total number of
spores counted in the 50 squares was used to obtain the
concentrations of all pure strain suspensions. The final
suspensions used in the generator were prepared by
combining one to three different pure strain suspensions
of molds (Table 1).
Analytical methods validation
The limit of detection (LOD), minimum reported value
(MRV), replicability, and repeatability were determined
for all methods. The LOD is the lowest concentration
that can be measured by the instrument above the re-
ported results of a suspension without spores in it
(blank). To determine the LODs, a spore suspension
with a starting concentration of 106 spores/mL was
progressively diluted until no more results could be
achieved. This was performed independently for each
method. In this study, the LOD corresponds to three
times the standard deviation calculated from 10 readings
performed on a suspension having a concentration of
spores slightly above the no response suspension. The
MRV is the lowest concentration that can be measured
above the background considering the sampling and
extraction process. The replicability is the variation
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
(spores/mL)
Strain Pen.d Ulo.c Clado.c Acre.r
1 4.0 106
2 4.0 106 1.0 106 6.0 106
6
3 6.3 10
4 2.1 106
5 3.0 106 1.0 106 2.0 106
6 6.2 106
7 110 106
8 16 10
9 8.8 106 8.9 105 8.7 106
10 6.2 106
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
Page 3 of 10 8
observed when the experiment is duplicated with no
modifications (same analyst, instrument, and day), and
the repeatability is the variation observed when at least
one change is implemented to the experiment. The
replicability was determined by analyzing four suspen-
sions with different concentrations of spores. Each sus-
pension was analyzed three to six times depending on
the method. The 95% confidence interval of the coeffi-
cients of variation (CV) obtained for the four suspen-
sions was used to calculate the replicability. The same
calculations were performed for repeatability, both are
reported in percentage. For the qPCR method, exclusiv-
ity and specificity were also documented. The exclusiv-
ity is the ability of the PCR detection system not to
amplify DNA coming from untargeted cells. The selec-
tivity is the ability to amplify the entire DNA coming
from the targeted cells, in this study, the DNA from
molds. The amplification ability of the system was
tested against 56 different strains of molds for the spec-
ificity and 40 strains of bacteria for the exclusivity.
Comparison of analytical methods
Simulation of dust contamination in the exposure
chamber
The generation of dust and mold spores was accom-
plished simultaneously inside an exposure chamber
mimicking an HVAC duct (Fig. 1). Before the
Generation time (hour)
Absi.c Sync.r Mold Dust
3 3
3 3
4 4
6 0.25
7 0.5
8 0.5
65 106 16 106 7 1
6
7 3
7 5
8 8
8 Page 4 of 10
generations, the dust was passed through multiple sieves
with a mechanical shaker and sterilized. Only the parti-
cles recovered from the stages with a sieve opening of
38 m and smaller were used in the fluidized bed for the
generation (model 3400A, TSI, Shoreview, MN, USA).
The combined flow rate of the bed (8.5 L/min) and the
bead purge (3.6 L/min) was 12.1 L/min. The chain
speed used on the fluidized bed generator was 7 mm3/
min. A 6-jet Collison nebulizer (BGI, Butler, NJ, USA)
was used in parallel with the fluidized bed to aerosolize
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
reach the desired concentration in the chamber. After the
generation, the chamber remained closed to give time
for the dust to settle on the duct surface. The results of
the ten separate sessions of generation conducted for this
study are presented in Table 1. Generation times for
molds and dusts were not always the same in order to
produce variable levels of dust and mold on the surface.
Samples from the exposure chamber
Samples of settled dust containing mold were collected
with a vacuum sampler method and a template of
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
100 cm2 using a 37-mm dust sampling cassette loaded
with a 0.5-m polyvinyl chloride filter and equipped
with an integrated angulated nozzle collector having a
5 mm inlet diameter (EMS, Charleston, SC, USA). To
find out the mass of each sample collected, the
entire cassettes were pre- and post-weighed using
an Analytic Plus AP250D (0.01 g) analytical bal-
ance (OHAUS, Butler, NJ, USA,). Six samples were
taken per generation, for a total of 60 samples ana-
lyzed by the four methods.
Extraction of samples
All samples were extracted on the same day they
were collected. The entire content of the cassettes
was recovered by introducing 3 mL of sterile PCR
water directly into the cassette through the collector
spout. The cassettes were then agitated on a maxi-
vortex DVX-2500 (VWR, Radnor, PA, USA) at
2500 rpm for 5 min. Each extract was then separated
into four aliquots: one for each method. The same
protocol was performed on a cassette not used for
sampling; this was the extraction blank and it was
analyzed by the four methods.
Environ Monit Assess (2017) 189: 8
Culture
For each sample, 100 L of concentrated extract and of
a 1/10 dilution were plated in triplicates on MEA. One
hundred microliters of the dilution buffer was also plat-
ed as a negative control. After 4 to 7 days of incubation
at 25 C, the colonies were counted using an SMZ-2T
stereo microscope (Nikon, Tokyo, Japan) at a magnifi-
cation of 20. For each sample, the number of colonies
counted on the three medium having between 10 and
200 colony-forming units (CFU) was average and the
result was reported in terms of CFU/100 cm2.
Microscopy method (DMSC)
Before adding 100 L of the concentrate or the 1/10
dilution of the extracted sample, 3 mL of sterile water
was placed in a filtering funnel. To help for the visual-
ization under the microscope, 100 L of 5% safranin
(Becton Dickinson, Sparks Glencoe, MD, USA) was
also added. Then, the suspension was filtered on a
mixed cellulose ester (MCE) membrane (diameter
25 mm; pore size 0.8 m) (SKC, Eighty Four, PA,
USA). The filtrate was left to dry at room temperature
for 24 h. Thereafter, MEC membranes were rendered
transparent with hot acetone using a Vap-300 vaporizer
(BGI, Waltham, MA, USA). For all samples, analysis
was performed using a Nikon Eclipse E400 microscope
at 750 enlargement and a total of 50 random fields
were counted regardless of the number of spores per
field. Concentrations are reported in spores/100 cm2.
NAHA enzyme assay modified method
with the Mycometer
The NAHA enzyme assay was performed on the liquid
aliquot of the sample following a modified protocol
based on the assay protocol provided with the
M yc o m e t e r ( M yc o m e t e r h an d bo o k , 20 11 )
(Mycometer, Tampa, FL, USA). First, the baseline fluo-
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
to 2 mL of the developer. Then, 100 L of the sample
was incubated in 2 mL of substrate. After 30 min at
ambient temperature, the enzyme activity was quanti-
fied by measuring the fluorescence analysis value (AV)
of the sample. The actual fluorescence, expressed in
relative fluorescence units (RFUs), was obtained by
Page 5 of 10 8
calculating the Mycometer value (MV) using the for-
mula provided in the original protocol. The number of
spores/100 cm2 was calculated using Eq. (1) obtained
from the regression line of the standard curve for a
suspension of spores of P. digitatum.
.
MV 3:428*3 ml 0:0005*0:1 ml 1
P. digitatum was chosen to build the standard curves
for both the NAHA and qPCR methods because it is
easily cultivated on MEA and produces high numbers of
spores allowing a good range of concentrations to build
the standard curves.
qPCR
Genomic DNA was extracted from 500 L of each
sample suspension using the ZR Fungal Bacterial
DNA Miniprep kit (Zymo Research, Irvine, CA,
USA). The final volume of elution was 100 L. For
each batch of extraction, a control was performed using
only PCR water. The primers and probes of the ampli-
fication system used are shown in Table 2. The volume
of the qPCR was 25 L. Each master-mix contained 2.5
units of HotStarTaq mix (Qiagen, Limburg,
Netherlands); 1.25 M of each primer (Integrated
DNA Technologies, Coralville, IA, USA); 0.375 M
of the probe; 2 mM of MgCl2 (Sigma-Aldrich, St.
Louis, MO, USA); 6.75 L of sterile PCR water; and
2 L of DNA. A Master Cycler Realplex2 (Eppendorf,
Hamburg, Germany) was used for amplification follow-
ing this program: 15 min at 95 C, followed by forty 15-
s cycles at 94 C; 30 s at 55.5 C; and 15 s at 72 C. In
each 96-well plate, a standard curve was incorporated.
The standard curve was made by serial dilutions of
DNA coming from a suspension of P. digitatum. The
correlation coefficients of the standard curves needed to
be higher than 0.96 and the PCR reaction efficiency
needed to be better than 80%. The DNA was extracted
from a spore suspension to which the concentration was
Table 2 Probe and primers used in the universal qPCR for the
amplification of molds DNA in the dust deposited in the exposure
chamber
FungiQuant-F GGR AAA CTC ACC AGG TCC AG
FungiQuant-R GSW CTA TCC CCA KCA CGA
FungiQuant-Probe TGG TGC ATG GCC GTT
8 Page 6 of 10
previously determined with a hemacytometer. The rela-
tion between the Ct obtained by the qPCR and the
number of spores in the suspensions was obtained di-
rectly from that standard curve. The concentrations of
the standard curve varied from 3 106 to 30 spores/mL.
All amplifications were duplicated and their average
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.
For the negative control, the DNA was replaced by PCR
water. All results are reported in spores per one hundred
square centimeters.
Data analysis
All statistical analyses were performed on log-
transformed data. A quantile-quantile (Q-Q) plot was
used to graphically determinate the lognormal distribu-
tion. Variance analyses (ANOVAs) were performed on
the logarithms of spore concentrations reported by each
method. Dunnetts test was used to identify means sig-
nificantly different from those obtained by the culture
method. Linear association, on the log-transformed data,
between the different methods was obtained using the
Pearson correlation (r). Results below the detection
limits were imputed a value of LOD/2 for all statistical
analyses. The IBM SPSS Statistics software package for
Windows, release 21.0 (IBM, Armonk, NY, USA, 2012)
was used.
Results
Analytical methods validation
Table 3 presents the analytical validation parameters of
the four methods. Limits of detection varied from 120 to
217,522 spores/100 cm2. Replicability and repeatability
were between 1 and 15% depending on the method. For
Table 3 Validation results for the
four analytical methods Method LOD
spores or CFU/mL
Culture 40
Microscopy 660
NAHA assay 72,500
qPCR 10
Environ Monit Assess (2017) 189: 8
the qPCR method, selectivity testing showed that all the
bacteria produced Cts greater than 35, whereas for
specificity, all the molds tested produced much low-
er Cts, between 17 and 27. Of the 60 samples, 12
(20%) were undetected by DMSC method and 2
(3%) by the NAHA assay.
Comparison of analytical methods
Figure 2 presents the box plot of the mass of dust
deposited on the lower horizontal surface of the expo-
sure chamber and of the number of colonies obtained by
the culture method for each generation. The weight of
dust sampled varied between 11.50 and 168 mg/
100 cm2, while the CFUs values obtained with the
reference method ranged between 1080 and 99,000/
100 cm2. Figure 3 presents the concentrations obtained
by each method. The median number of spores reported
by the other three methods ranged from 10,132 to
815,262 spores/100 cm2. Dunnetts multiple compari-
son procedure showed that qPCR and the NAHA en-
zyme assay produce statistically higher concentrations
than culture (p < 0.05), whereas DMSC is not statisti-
cally different (p = 0.30). The NAHA enzyme assay
yielded significantly higher concentrations than the
qPCR method, which itself yielded more spores than
the DMSC method (data not shown). Figure 4 shows
the linear associations and the Pearson correlation
coefficients (r) obtained between the different
methods. A significant correlation was observed be-
tween culture and qPCR (p < 0.01), and DMSC
method (p < 0.01), but culture has no correlation
with the NAHA method (p = 0.169). A significant
correlation was also seen between qPCR and DMSC
(p < 0.01) and NAHA (p < 0.05) methods (data not
shown). No significant correlation was observed
between the NAHA assay and the DMSC method
(p = 0.571).
Replicability (%) Repeatability (%)
12 11
5 10
4 9
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

Figure 2 shows that the dust levels generated in the
exposure chamber correspond to levels of dustiness
often found in HVAC systems. In fact, Boor et al. re-
ported levels varying from 10 to 1000 mg/100 cm2 and
Lavoie et al. (2011) described levels ranging from 0.14
to 337 mg/100 cm2 in samples taken in a variety of
HVAC ducts. The levels of dust generated for this study
varied from 11 to 160 mg/100 cm2.
many fields of microbiology. Its underestimation is
well documented and can reach two orders of mag-
nitude (Krause et al. 2003; Mandal and Brandl 2011;
Schnrer 1993). Even if it has some recognized
limitations, culture was chosen as the control meth-
od in the present study. In addition to the well-
known limitation linked to the inhibition of some
molds growth in the laboratory conditions, counts
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
**
**
Fig. 3 Box plot diagrams showing the logarithm of the molds
concentrations (spores or colonies/100 cm2) obtained for each
method, n = 60. 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 (double
asterisk indicates p < 0.05)
on the petri dishes make it difficult to distinguish
between two or more colonies.
The NAHA enzyme assay is very easy to perform. It
does not require a specialist and can be performed directly
in the field. In comparison, a trained analyst working in a
laboratory is required for the other methods. The enzyme
assay proved to be the least sensitive of the methods
evaluated. The LOD was calculated to be 72,500 spores/
mL. Depending on the measurement unit used, this can be
equivalent to 40 RFU/mL or to a minimum reported value
(MRV) of 120 RFU/100 cm2 when the global method
from sampling to analysis is taken into consideration.
Despite the lack of sensitivity observed in the present
study, it was comparable to the LOD reported by other
authors. In fact, Mensah-Attipoe et al. (2015) reported a
MRVof 216 RFU/100cm2 while Reeslev et al. (2003) and
Krause and Hammad (2002) reported LODs of 140 and
120 RFU/mL, respectively. The differences between the
reported sensitivities could be attributed to the calculation
methods and whether only the analytical or the global
process was considered. In this study, the LOD was calcu-
lated as three times the standard deviation obtained from
ten readings taken from a suspension having a concentra-
tion slightly above the background level. Other studies did
not specify their LOD calculation methods. Though less
sensitive than the other methods, the enzyme assay always
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
method at three different load of dust. 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. For each dust load, a, b, and c are significantly
different at p < 0.05
yielded higher concentrations of spores. This result is
consistent with those reported by Mensah-Attipoe et al.
(2015) and Krause and Hammad (2002), who respectively
found that the counts of colonies by culture and the counts
of spores by microscopy were lower than the one obtained
by the NAHA assay. Mensah-Attipoe et al. (2015) reported
a correlation between the colony count and the NAHA
assay. In their study, in opposition with the present study,
independent samples were analyzed by each analytical
method. The NAHA enzyme is present in viable and
non-viable structures, this could explain part of the over-
estimation of NAHA when compared to culture (Mensah-
Attipoe et al. 2015; Rylander et al. 2010). In addition, as it
was reported by Rylander, the NAHA enzyme is not
specific to molds. This enzyme is also produced by bacte-
ria, protozoa, pollen, and some mammalian cells (Rylander
et al. 2010). Because of its lack of specificity to molds, this
assay is not appropriate. It will overestimate the fungal
contamination of dust in HVAC ducts, mainly because of
the presence of high concentrations of bacteria and of other
types of cells usually found in dust.
In this study, the DMSC method turned out to be less
sensitive than the culture method. Unlike the culture
method, the DMSC method is not limited to the culti-
vable cells, so higher concentrations were expected.
This lack of sensitivity has also been reported by
Mandal and Brandl (2011). In fact, from their point of
Environ Monit Assess (2017) 189: 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
methods and we do agree with them. For the DMSC
method, only 50 out of the 1535 possible random fields
were counted during this study. Even though good re-
peatability was demonstrated during the validation pro-
cess, the low number of fields counted seems to produce
in the end only a good approximation of the concentra-
tion of mold spores in the dust. In addition, the difficulty
to distinguish fungal structures from other particles of
dust proved to be another limitation of that method for
dust samples.
The qPCR method showed a LOD of 10 spores/mL
or an equivalent of 142 spores/100 cm2. That method
detects fungal DNA regardless of the spore viability.
This is an important characteristic during indoor air
quality problems investigations since all fungal struc-
tures could conceivably have health effects (Heseltine
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-
ing the amount of DNA in the qPCR reaction. The
universal qPCR detection system (Liu et al. 2012) used
for this study demonstrated excellent specificity as only
the molds and none of the bacteria tested were ampli-
fied. As seen on Fig. 5, qPCR also demonstrated a
significant correlation with all three tested methods
Page 9 of 10 8
showing that it is a good predictor of the concentrations
of spores in dust of HVAC systems. Because of all these
advantages, the qPCR method with the universal
markers is recommended for the estimation of the mold
biomass in the dust of HVAC.
As a complement to the gravimetric evaluation of the
dust deposited on the surface of a duct, the mold bio-
mass present in the dust of HVAC system need to be
evaluated. From this study, the qPCR method seems to
be the method of choice to perform such an assessment.
This study was performed under controlled parameters
with a low diversity of molds. Evaluations in real situ-
ations are needed to confirm the choice of qPCR as the
method for mold biomass evaluation in dust.
Acknowledgements This study was supported by the Institut de
recherche Robert-Sauv en sant et scurit du travail (IRSST).
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