Abstract
Mold specific quantitative PCR (MSQPCR) was used to measure the concentrations of the 36 mold species in indoor and outdoor air samples that were taken simultaneously for 48 h in and around 17 homes in Cincinnati, Ohio. The total spore concentrations of 353 per m3 of indoor air and 827 per m3 of outdoor air samples were significantly different (p≤0.05). However, only the concentrations of Aspergillus penicillioides, Cladosporium cladosporioides types 1 and 2 and Cladosporium herbarum were correlated in indoor and outdoor air samples (p-value≤0.05 and sufficient data for estimate and absolute value rho estimate ≥0.5). These results suggest that interpretation of the meaning of short-term (<48 h) mold measurements in indoor and outdoor air samples must be made with caution.
Keywords: Mold, Indoor air, Outdoor air, Mold specific quantitative PCR
1. Introduction
One common procedure used to determine if a building has an abnormal mold condition is the comparison of indoor and outdoor mold concentrations in air samples (Gots et al., 2003). Often the comparison is based on the ratio of the total number of spores, or totals for a few genera, quantified by either spore counting or culturing on one or two media. These methods, however, ignore three basic problems. First, not all species grow on the same media and/or at the same rate. Second, not identifying molds at the species level reduces the relevance of mold diversity. Third, air sampling times are usually short, often less than 5 min, because of overgrowth on culture plates or masking in spore traps. In this study, longer sampling times and a DNA-based method for identifying and quantifying species of molds were tested to examine the relationship between indoor and outdoor mold populations in air samples.
2. Materials and methods
2.1. Selection of homes
Seventeen homes were selected as part of the Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) (Ryan et al., 2005). The homes were inspected for visible mold/water damage and classified groups: 1) no mold or water damage, 2) minor mold or water damage, and 3) major mold damage (Cho et al., 2006). Among the seventeen homes, six had no mold or water damage and eleven had minor mold–water damage that appeared as visible signs of water and/or mold damage <0.2 m2, moldy odor or water mold damage history reported by the occupants (Cho et al., 2006). The homes were built between 1928 and 1998. Sixteen homes had central air conditioning. Fibrous HVAC filters were used in seven homes and an electrostatic filter in four homes. No filters were found in the other homes.
2.2. Indoor air and outdoor air sample collection
All homes were sampled in the March to May period of 2003. Indoor and outdoor air samples were taken simultaneously using Button Personal Inhalable Aerosol Samplers (SKC Inc., Eighty Four, PA) loaded with 25 mm polycarbonate filters (1 μm pore size, Osmonics Inc., Minnetonka, MN, USA). Each sampling device was operated at a sampling flow rate of 4 l per min, which was maintained by a small vacuum pump (Model 224-PCXR4; SKC Inc.) and the flow rate verified with dryCal® DC-Lite Calibrator (Bios International Corporation, Butler, NJ, USA) before and after each 48-h measurement. The outdoor samplers were fixed on a tripod with a rain shield at a height of 1.5 m. Each indoor sampler was placed in the room with the sampler pumps housed inside a noise-insulated enclosure. The residents stayed at home and carried-on with their normal activities.
2.3. Mold specific quantitative PCR (MSQPCR) analysis of air samples
Methods have been reported previously for extracting DNA and performing MSQPCR analyses (Haugland et al., 2002; Brinkman et al., 2003; Haugland et al., 2004). All primer and probe sequences, as well as known species comprising the assay groups, were published at the website: http://www.epa.gov/microbes/moldtech.htm.
2.4. Statistical analyses
Mold concentration data having a minimum detection limit of 1 cell per m3 for air samples were treated as left-censored data with appropriate statistical methods applied (Helsel, 2005). Procedurally, non-detections were set at 1/2 the limit of detection (LOD). Thus, non-detections were given equal and lowest rank for non-parametric rank-based analyses (Helsel, 2005). The Wilcoxon Signed Rank Test was used for comparisons of mold spore concentrations between paired samples taken at each of the seventeen homes with p-values adjusted for multiple comparisons using the method of Benjamini and Hochberg (1995). Correlation between mold species in the different sample types was tested using the rank based Spearman’s Rho test. An agreement ratio reflecting the number of identical species isolated from both of two sample types and relative to the total number of species identified in both sample types was calculated by equation R=2W/(A+ B),where W=number of species both samples have in common, A=total number of species in sample 1 and B=total number of species in sample 2. Statistical analyses were performed using SAS (SAS Institute Inc., Cary, NC) and the R Software environment for statistical computing and graphics (http://www.r-project.org/).
3. Results
Table 1 shows the average concentrations of the 36 mold species measured by MSQPCR in the indoor (I) or outdoor (O) air samples. The total spore concentrations of 353 per m3 of indoor air and 827 per m3 of outdoor air samples were significantly different (p≤0.05). Comparison of the concentration of specific molds in indoor and outdoor air samples showed that many were statistically significantly different in concentrations (p≤0.05 based on the Wilcoxon Signed Rank Test) (Table 2). The I/O ratios for the species varied from 0.15 to 1.23 (Table 2) compared to the total spore I/O of 0.43. The correlation between mold species in indoor and outdoor air samples was evaluated with the Spearman’s Rho test (Table 2). Only Aspergillus penicillioides, Cladosporium cladosporioides types 1 and 2 and Cladosporium herbarum were correlated between indoor air and outdoor air samples.
Table 1.
Averages of the number of spores per m3 of indoor or outdoor air followed by percent of homes with mold concentrations above the limit of detection (LOD) of 1 cell per m3 of air
| Averages (% above LOD of 17 homes)
|
||
|---|---|---|
| Indoor | Outdoor | |
| Group 1 molds | ||
| Aspergillus flavus | <LOD (12%) | 1 (35%) |
| Aspergillus fumigatus | 1 (29%) | 3 (82%) |
| Aspergillus niger | 1 (35%) | 1 (59%) |
| Aspergillus ochraceus | 1 (18%) | <LOD (24%) |
| Aspergillus penicillioides | 7 (94%) | 9 (82%) |
| Aspergillus restrictus | 1 (47%) | 3 (41%) |
| Aspergillus sclerotiorum | 1 (24%) | <LOD (6%) |
| Aspergillus sydowii | 1 (12%) | 35 (18%) |
| Aspergillus unguis | <LOD (12%) | <LOD (6%) |
| Chaetomium globosum | <LOD (0%) | <LOD (6%) |
| Aspergillus versicolor | 3 (35%) | 5 (65%) |
| Eurotium chevalieri | 6 (94%) | 34 (100%) |
| Penicillium brevicompactum | 20 (65%) | 40 (71%) |
| Cladosporium sphaerospermum | 2 (71%) | 2 (47%) |
| Penicillium corylophilum | 3 (24%) | <LOD (12%) |
| Penicillium purpurogenum | <LOD (6%) | <LOD (6%) |
| Penicillium spinulosum | 4 (47%) | 10 (65%) |
| Penicillium variabile | 2 (29%) | 5 (41%) |
| Paecilomyces variotii | 1 (29%) | 1 (41%) |
| Penicillium crustosum | 2 (12%) | 4 (41%) |
| Aureobasidium pullulans | 104 (94%) | 264 (100%) |
| Scopulariopsis brevicaulis | <LOD (6%) | <LOD (18%) |
| Scopulariopsis chartarum | <LOD (6%) | 1 (53%) |
| Stachybotrys chartarum | <LOD (0%) | <LOD (0%) |
| Trichoderma viride | <LOD (0%) | <LOD (12%) |
| Wallemia sebi | 4 (82%) | 13 (100%) |
| Group 2 molds | ||
| Acremonium strictum | 21 (59%) | 17 (53%) |
| Alternaria alternata | 1 (47%) | 3 (94%) |
| Penicillium chrysogenum Type 2 | 1 (59%) | 1 (12%) |
| Aspergillus ustus | <LOD (6%) | <LOD (0%) |
| Cladosporium cladosporioides Type 1 | 103 (100%) | 165 (100%) |
| Cladosporium cladosporioides Type 2 | 2 (59%) | 13 (77%) |
| Cladosporium herbarum | 37 (94%) | 114 (100%) |
| Epicoccum nigrum | 25 (88%) | 84 (100%) |
| Mucor racemosus | <LOD (24%) | <LOD (6%) |
| Rhizopus stolonifer | <LOD (0%) | <LOD (0%) |
Table 2.
Evaluation of the difference in each mold species’ concentration in indoor or outdoor air are based on Wilcoxon Signed Rank Test (significant in bold)
| Wilcoxon test p-values a |
Spearman’s Rho test
|
Proportion >LOD
|
||||
|---|---|---|---|---|---|---|
| In vs out doors | I/O ratios | Rho | p-values | Indoor | Outdoor | |
| Group 1 | ||||||
| A. flavus | 0.424 | NA | 0.177 | 0.49 | 0.118 | 0.35 |
| A. fumigatus | 0.093 | 0.33 | 0.412 | 0.10 | 0.294 | 0.82 |
| A. niger | 0.067 | 1.00 | −0.083 | 0.75 | 0.353 | 0.59 |
| A. ochraceus | 0.231 | NA | 0.463 | 0.06 | 0.176 | 0.24 |
| A. penicillioides | 0.003 | 0.78 | 0.544 | 0.03 | 0.941 | 0.82 |
| A. restrictus | 0.030 | 0.33 | 0.373 | 0.14 | 0.471 | 0.41 |
| A. sclerotiorum | 0.140 | NA | −0.137 | 0.60 | 0.235 | 0.06 |
| A. sydowii | 0.424 | 0.03 | 0.336 | 0.19 | 0.118 | 0.18 |
| A. unguis | 0.424 | NA | −0.091 | 0.73 | 0.118 | 0.06 |
| C. globosum | NA b | NA | NA | NA | 0.000 | 0.06 |
| A. versicolor | 0.067 | 0.60 | 0.656 | 0.01 | 0.353 | 0.65 |
| E. chevalieri | 0.003 | 0.18 | 0.350 | 0.17 | 0.941 | 1.00 |
| P. brevicompactum | 0.013 | 0.50 | 0.363 | 0.15 | 0.647 | 0.71 |
| C. sphaerospermum | 0.010 | 1.00 | 0.176 | 0.50 | 0.706 | 0.47 |
| P. corylophilum | 0.140 | NA | 0.176 | 0.49 | 0.235 | 0.12 |
| P. purpurogenum | 1.000 | NA | −0.062 | 0.81 | 0.059 | 0.06 |
| P. spinulosum | 0.030 | 0.40 | 0.073 | 0.78 | 0.471 | 0.65 |
| P. variabile | 0.093 | 0.40 | 0.096 | 0.71 | 0.294 | 0.41 |
| P. variotii | 0.093 | 1.00 | 0.451 | 0.07 | 0.294 | 0.41 |
| P. crustosum | 0.424 | 0.50 | −0.292 | 0.26 | 0.118 | 0.41 |
| A. pullulans | 0.003 | 0.39 | 0.432 | 0.08 | 0.941 | 1.00 |
| S. brevicaulis | 1.000 | NA | 0.576 | 0.02 | 0.059 | 0.18 |
| S. chartarum | 1.000 | NA | 0.168 | 0.52 | 0.059 | 0.53 |
| S. chartarum | NA | NA | NA | NA | 0.000 | 0.00 |
| T. viride | NA | NA | NA | NA | 0.000 | 0.12 |
| W. sebi | 0.005 | 0.31 | 0.273 | 0.29 | 0.824 | 1.00 |
| Group 2 | ||||||
| A. strictum | 0.014 | 1.23 | −0.136 | 0.60 | 0.588 | 0.53 |
| A. alternata | 0.021 | 0.33 | 0.447 | 0.07 | 0.471 | 0.94 |
| P. chrysogenum 2 | 0.015 | 1.00 | −0.143 | 0.58 | 0.588 | 0.12 |
| A. ustus | 1.000 | NA | NA | NA | 0.059 | 0.00 |
| C. cladosporioides 1 | 0.003 | 0.62 | 0.616 | 0.01 | 1.000 | 1.00 |
| C. cladosporioides 2 | 0.015 | 0.15 | 0.585 | 0.02 | 0.588 | 0.77 |
| C. herbarum | 0.003 | 0.32 | 0.531 | 0.03 | 0.941 | 1.00 |
| E. nigrum | 0.004 | 0.30 | 0.279 | 0.28 | 0.882 | 1.00 |
| M. racemosus | 0.110 | NA | −0.139 | 0.60 | 0.235 | 0.06 |
| R. stolonifer | NA | NA | NA | NA | 0.000 | 0.00 |
Correlations in concentrations are based on Spearman’s Rho test. Species in bold have p-value ≤0.5 and sufficient data for estimate of absolute value Rho ≥0.5.
Adjusted for multiple comparisons.
NA = All values below limit of detection.
4. Discussion
Spearman’s Rho analysis showed that the concentrations of only four individual species in indoor and outdoor air samples were correlated with each other. This is consistent with our previous results in which the fungal genera clusters Aspergillus/Penicillium and Cladosporium were correlated in indoor and outdoor air samples (Lee et al., 2006). This may be explained by the fact that A. penicillioides is the dominant member of the Aspergillus/Penicillium genus cluster and C. cladosporioides types 1 and 2 and C. herbarum are the dominant members of the Cladosporium genus cluster (Vesper et al., 2004; Meklin et al., 2004; Vesper et al., 2006). The limitations of air samples have been observed by others.
O’Connor et al. (2004) noted that air samples have often been limited to 5 min or less with the resulting limitations in understanding the environmental source of the mold. Verhoeff et al. (1992) used N6-Andersen samplers in combination with DG-18 agar with a sampling time of 5 min to compare indoor and outdoor air samples. They stated that “the number of CFU/m3 in the indoor and outdoor air varied widely” and the “low predictive value …limits their use in epidemiological studies.” Spicer and Gangloff (2005) showed that “the levels of fungi in the outdoor air varied significantly between morning and afternoon …with no pattern by species, time of day or location.”
In our study, the lack of species specific concentration correlations may be due to the relatively small number of homes, most without significant water damage. Also, most homes had central air conditioning with either a fibrous or electrostatic filter. However, our results suggest that evaluating the mold burden indoors by a simple genus level comparison to the outdoors may be misleading.
Acknowledgments
The authors are grateful to all parents and children who participated as well as to all home visit teams, subject recruitment teams, and clinic personnel of the CCAAPS. This research was supported by the National Institute of Environmental Health Sciences (NIEHS) Grant No. RO1 ES11170 awarded to the University of Cincinnati. Additional, support was provided by the US EPA Asthma Initiative. Also, support for the lead author (Meklin) was from the Postgraduate Research Participation Program administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US DOE and the US EPA.
Footnotes
Notice
The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development, funded and collaborated in the research described here. It has been subjected to the Agency's peer review and has been approved as an EPA publication. Mention of trade names or commercial products does not constitute endorsement or recommendation by the EPA for use. The US EPA has a financial interest in the commercial application of MSQPCR technology.
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