Abstract
In an effort to better understand the relationship between different fungal sampling methods in the indoor environment, four methods were used to quantify mold contamination in 13 homes with visible mold. Swab, fungal spore source strength tester (FSSST), and air samples (total of 52 samples) were analyzed using both the microscopic (total spore count) and culture-based (CFU count) enumeration techniques. Settled dust samples were analyzed for culturable fungi only, as the microscopic enumeration was restricted by the masking effect. The relationships between the data obtained with the different sampling methods were examined using correlation analysis. Significant relationships were observed between the data obtained from swab and FSSST samples both by the total counting (r = 0.822, p <0.05) and by the CFU counting (r = 0.935, p <0.01). No relationships were observed between air and FSSST samples or air and settled dust samples. Percentage culturability of spores for each sampling method was also calculated and found to vary greatly for all three methods (swab: 0.03% to 63%, FSSST: 0.1% to >100%, air: 0.7% to 79%). These findings confirm that reliance on one sampling or enumeration method for characterization of an indoor mold source might not provide an accurate estimate of fungal contamination of a microenvironment. Furthermore, FSSST sampling appears to be an effective measurement of a mold source in the field, providing an upper bound estimate of potential mold spore release into the indoor air. Because of the small sample size of this study, however, further research is needed to better understand the observed relationships in this study.
Keywords: air sampling, indoor fungi, settled dust
It has been estimated that 20% to 40% of homes in Northern Europe and Canada have mold contamination.(1) This number is likely to be much higher in tropical and subtropical countries.(2,3) In the United States, as many as 40% of homes have mold problems.(1,4) Various health effects, such as respiratory symptoms, allergic rhinitis, asthma, and hypersensitivity pneumonitis, are associated with mold exposure.(5–12) A case control study conducted in Europe suggested a relationship between increases in symptoms in asthmatic patients and increased mold and moisture problems in the home.(13)
Other studies have shown that exposure to visible mold, or excessive moisture, which promotes mold growth, leads to an increase in allergic symptoms.(6,8,14–20) Toxicity caused by exposure to the metabolites of certain molds have also been linked to health effects.(6,21) However, the relationship between specific health effects and the mold spore concentration has not been well defined.(6) It has been criticized that the methodologies for sampling and analysis are neither standardized nor definitive.(22) Available quantitative methods are used in combination with a comprehensive qualitative assessment.(23–25) Jarvis and Morey(22) have suggested that lack of a standard methodology is a primary cause for the poorly understood relationship between fungal exposures and health outcomes. Therefore, it is important to be able to identify and quantify the mold contamination levels in indoor environments using validated methods for sampling and analysis.
One of two approaches is typically used to assess mold contamination with respect to fungal spore identification and enumeration: culture-based analysis (the colony forming unit [CFU] count) and the microscopic analysis (the total spore count). The culture-based analysis, which is more common, gives the ability to identify colonies to the species level and a large reference database is available for proper identification of colonies.(26) Species-level identification is useful in detecting “indicator fungi” that are commonly found in moldy buildings. For many years, the culture-based methods have tended to be the dominant choice of both the practicing indoor air quality professionals and the research community since the Andersen sampler was used as “the gold standard” for bioaerosol sampling.
However, several disadvantages of the CFU analysis are also apparent. The incubation period is usually long (over 7 days for some fungal species),(27,28) and CFU analysis can overlook fungal species that are not easily culturable. Furthermore, it might underrepresent those fungal types that grow slowly because they are overtaken by faster growing colonies.(26,28–32) Kozak et al.(33) demonstrated that although the level of culturable spores may be below the limit of detection, the total number of spores may be sufficient to cause respiratory symptoms.
Some fungal species, such as the spores from Stachybotrys chartartum, have been found to lose their culturability soon after they become airborne; however, this does not appear to affect their allergenicity or toxicity.(3,33,34) Furthermore, some health effects, especially respiratory allergies, have been shown to be associated with the total spore count rather than with the CFU count.(33,35)
Similar to the CFU count, there are some advantages and disadvantages of the total spore count method. Two advantages are that (1) both viable and nonviable spores can be included, and (2) the total count is less time-consuming than the CFU analysis (can be performed within hours of sample collection). Among disadvantages of this enumeration method, there are (a) masking effect, when the background matrix may mask small spores; (b) high data variability when spore density is low; (c) overestimation of large pigmented spores; and (d) impossibility of performing the species-level identification.(26,36)
Other methods for fungal analysis include the use of surrogate markers that measure quantitative loads of fungal biomass, such as β-glucan and ergosterol. These indicator methods are useful for providing general information about the total amount of fungi in the environment but are often not specific enough to relate to health outcomes because of their surrogate nature.(29) Recently, polymerase chain reaction (PCR) and immunochemical methods have become available for fungal analysis.(34,36–41) There is currently, however, very little reference data available with these techniques.
Currently, there are numerous sampling methods available to measure fungal concentrations in the environment. Source sampling, which includes methods such as swab, tape, bulk, and dust, is commonly used to identify indoor fungi. These source sampling methods have been cited by the American Industrial Hygiene Association (AIHA)(42) as “necessary adjuncts” to air sampling, especially under conditions of low air movement, or when air sampling might result in false-negative findings. However, these surface-based methods cannot identify hidden sources of mold.(43) Swab and tape sampling are common methods of fungal exposure assessment through the source characterization, partially because of ease of collection. They are often used as tools for identification of fungi but do not provide measures of exposure to airborne spores. Bulk samples include pieces of material such as wallboard, carpet, or return air filter, that are collected from the contaminated area to identify and find the relative concentration of mold in the sample.(43)
Fungal spores can also be measured in settled dust sampled from the floor.(44,45) This method is usually attempted to evaluate long-term respiratory exposure to fungi, though the stability of microorganisms over time is questionable.(46–48) Flannigan(49) indicated that dust may not adequately reflect human inhalation exposure, evidenced by his research findings that only a very small amount of reaerosolized dust particles is of respirable size. Furthermore, Chew et al.(46) found that culturable air and dust samples represent differing types of potential mold exposure and, thus, are not related indicators of exposure to mold. Settled dust can be analyzed by various techniques, such as CFU, PCR, and biochemical methods for β-glucan and ergosterol. However, it is difficult to conduct the microscopic enumeration from dust samples, in part, because fungi in dust is masked by other particles.(40,46,50) Some investigators have managed to overcome this problem using a two-phase technique.(51)
Air sampling is one of the most common methods used to assess fungal levels in indoor environments. Many studies have related human health effects, such as increases in allergic and asthmatic respiratory symptoms, to airborne fungal spores.(22,33,52–56) As the health effects of fungal exposure are mainly respiratory, air sampling is believed to be adequate to represent the exposure. However, fungal spores have been found to exhibit varying patterns in their release into the air depending on several environmental factors.(22,46,52–56)
In an effort to link the mold source characterization and assessment of exposure to airborne fungal spores, several recent studies addressed the conditions necessary for fungal spore release from a mold source.(57–58) Two devices have been developed to measure the aerosolization potential of a visible fungal source: (1) the fungal spore source strength tester (FSSST)(59–61) and (2) the particle field and laboratory emission cell (PFLEC).(62) Both of these devices use portable aerosolization chambers in which spores are aerosolized from a fungal source and immediately collected into an air sampler.
The relationship between different fungal assessment methods has not been extensively characterized. Very little information is available on the comparison of the data obtained with the microscopic and culture-based enumeration of samples collected by a specific method, as well as the data collected by different sampling methods. Thus, a pilot study was conducted to compare the data collected using four sampling methods in mold contaminated homes. These methods include swab, FSSST, air, and settled dust sampling, and the first three were used to generate the total spore data and CFU data.
METHODS
Twenty-six homes with self-reported mold contamination were screened for this study in the greater Cincinnati, Ohio, metropolitan area. Thirteen homes were selected for evaluation by a trained indoor air quality researcher based on the size of the visible mold contamination (>144 cm2). Four types of sampling were performed on the selected homes: swab and settled dust (representing the sources of sporulation [the former] and resuspension [the latter]), FSSST (representing the source potential for aerosolization from the growth surface), and air sampling (representing the actual air contamination). Swab, FSSST and air methods were used as outlined by Sivasubramani et al.(60)
Prior to the mold sampling, both relative humidity and indoor temperature were recorded using a traceable humidity/temperature pen (Fisher Scientific Company, Pittsburgh, Pa.). The surface moisture content of the test surface was measured with a Protimeter (BLD 5800, GE Protimeter, Wilmington, Del.) and expressed as a percentage of the mass of water in a given volume of a material [(wet mass – dry mass) × 100/(dry mass)]. For a specific material, this percentage is calculated as a wood-equivalent value.
Swab sampling was performed on a 1-cm2-area of the mold contaminated surface (usually a wall). The 1-cm2-area was chosen to be negligibly small as compared with the FSSST sampling area of 90.25 cm2 (internal cross-section that covered the former). The surface was thoroughly swabbed with a sterile wet swab (Fisher Scientific) to remove as much of the mold as possible and collected in a 0.05% Tween 80 solution (Sigma Chemicals Co., St. Louis, Mo.). The FSSST sampling unit is a closed, two-pump aerosolization chamber that is held tightly on the contaminated surface during sampling. A push-vacuum (air supply) pump (11.5 L/min) produces airflow that first passes through a HEPA filter (1244 HEPA capsule filter, PALL Gelman Laboratory, Ann Arbor, Mich.), then is directed through a 112-hole orifice stage, passing over the mold-contaminated surface.
The air is then drawn through a center orifice into an SKC BioSampler (SKC, Inc., Eighty Four, Pa.) at a rate of 12.5 L/min, using another vacuum pump. Each FSSST sample was collected for 10 min, which was shown to be sufficient to determine the spore aerosolization potential.(59–61) Simultaneously with the FSSST samples, the air samples were collected at least 1 m away from the source into the BioSampler (0.05% Tween 80 solution) using a vacuum-pull (air sampling) pump operating at a flow rate of 12.5 L/min for 10 min (a short-term sampling).
Dust sampling was performed using a canister-type vacuum cleaner (Filterqueen Majestic, HMI Industries Inc., Seven Hills, Ohio) fitted with a nozzle filter bag (HEPA) (Filtration Group Inc., Joliet, Ill.). In every home, the dust sample was collected in the same room where the visible mold contamination was identified. Samples were vacuumed from a 2-m2-area of carpet for 4 min. For noncarpeted floors, the settled dust sample was taken at a rate of 1 m2/min, as described by Meklin et al.(40)
The swab, air, and FSSST samples were analyzed for both culturable and total fungal spores. An aliquot of each sample was cultured on malt extract agar (MEA), supplemented with streptomycin sulfate to inhibit bacterial growth.(63) Each sample was plated in triplicate, incubated for 7 days and identified to the species level, whenever possible, based on the colony morphology. The dust samples were analyzed by the culture-based method only, since the total fungal spore count is restricted by masking of spores by other dust particles. For these samples, dust was suspended in a buffer solution containing 0.0425 g l−1 KH2PO4, 0.25 g MgSO4 × 7 H2O l−1, 0.008 g NaOH, and (0.02% v/v) Tween 80. An aliquot of this solution (0.1 mL) was cultured in triplicate on MEA treated with an antibiotic agent to inhibit bacterial growth. The samples were incubated for 7 days and then identified to genus level.
The procedures used for the total count of fungal spores in the swab, air, and FSSST samples have been fully described by Sivasubramani et al.(60) Briefly, an aliquot of each sample was filtered onto a 13-mm mixed cellulose ester filter (0.8 μm pore size; Fisher Scientific) and then placed on a glass slide. Filters were dried overnight, then cleared by acetone vapor using a modified instant acetone-vaporizing unit (Quickfix, Environmental Monitoring Systems, Charleston, S.C.). A 25 × 25-mm cover glass was mounted on the slide using glycerin jelly (gelatin: 20 g, Phenol crystals: 2.4 g, glycerol: 60 mL, water: 70 mL). A light microscope (model Leitz Laborlux S, Leica Mikroskopie und Systeme GmbH, Wetzler, Germany) at a magnification of 400× was used to identify and enumerate the collected fungal spores. For slides with a relatively high number of spores (>50 spores per microscopic field), spores were enumerated in 20 microscopic fields; for the slides with sparse deposit (<50 spores per field), 40 microscopic fields were counted. The spores were microscopically identified to genus/group level. The limits of detection for the total spore count were 82 spores per cm2 for swab samples, 659 spores per m3 for air samples, and 0.9 spores per cm2 for the FSSST samples.
Correlation analyses were used to relate the number of spores collected by the different methods. Multiple comparisons were made between each collection type for both culturable and total spore counts. Scatterplots were generated using Sigma Plot (SPSS Inc.) and a correlation coefficient was calculated and tested for significance at α = 0.05 for each relationship. The statistical significances of the correlation results were calculated using SPSS. The percentage of culturable spores was determined for swab, FSSST, and air samples. Indoor air concentrations of fungi were compared by utilizing the data on the outdoor levels determined on the same day around the greater Cincinnati metropolitan area through the regional ambient monitoring campaign carried out using an SKC Button Aerosol Sampler (24-hour samples). The latter collected particles on a mixed cellulose ester filter at a flow rate of 4 L/min (the method has been fully described by Adhikari et al.(64)). The sampling efficiencies of the air samplers used for indoor (BioSampler) and outdoor (Button Sampler) fungal spore collection are about the same for the size range of fungal spores.
RESULTS
Three types of surfaces with mold contamination were observed in the 13 homes in the study. Mold contamination on concrete surfaces occurred in five of the homes. Contamination of wood surfaces, including wood paneling and wood joists, occurred in four homes. Contamination of drywall also occurred in four homes. Relative humidity ranged from 23% to 74% among the homes. Only four homes had relative humidity values over 50%. Surface moisture values ranged from 5.0% to 18.4% among the homes. The highest surface moisture (18.4%) occurred in the home that was found to be contaminated with Stachybotrys. Temperatures in the homes ranged from 18.8° to 26.1°C.
Figure 1 presents boxplots for the concentration of spores with respect to the microscopic and culture-based fungal enumeration obtained from swab, FSSST, air (indoor and outdoor), and dust samples across all 13 homes. For one home, CFU analysis was not performed from any of the sampling methods due to an oversight during the sampling period. In addition, swab samples from two homes and an air sample from one home were contaminated with bacteria, so CFU counts for fungal spores could not be determined. Lastly, culture samples taken from one home did not grow for either the swab or air samples. In all of these homes, the total spore count was still obtained.
Percentile and median values of concentrations of spores and colony forming units across 13 homes. The boxplot shows the following: horizontal lines from left, 5%, 25%, 50%, 75%, 95%, percentiles; symbol • shows the range of data; n = number of (more ...)
Figure 1 shows that the median spore concentrations over all the homes were an order of magnitude higher for both swab and FSSST in the total count method, as compared with the culturable count. The CFU count for these methods, however, had much higher variability. The median spore level for indoor air was also higher for the total count method (by a factor of 4). The variabilities of the indoor air spore count levels were similar for both microscopic and culture-based methods. The median value measured in outdoor air (the sampling methodology utilized in the outdoor monitoring campaign included only the total count) was approximately an order of magnitude lower than the one determined in indoor air. The median culturable dust level fell between the median levels of culturable counts obtained from the FSSST and swab samples and had slightly lower variability.
Figures 2 and 3 show the number of homes in which each specific spore type (genus or group) was identified, with the total spore count (Figure 2) and CFU count (Figure 3). The abovementioned number of homes is referred to as “occurrence” in the x-axis of each figure (some investigators would define it as “frequency”). Not all spore types were found for each sampling method. Aspergillus, Penicillium, and Cladosporium were the most common fungal types identified in both the total and culture-based spore counts.
Swab sampling from the visible mold sources (collected from contaminated walls) in 13 homes revealed 8 different types of fungal spores, as well as unidentified spores. For CFU analysis of swab samples taken from 9 homes, 7 spore types were identified. FSSST sampling from the visible mold sources in 13 homes revealed 7 different types of fungal spores present as unidentified spores in the total spore population as well. For CFU analysis of FSSST samples taken from 12 homes, 8 spore types were identified. Short-term air sampling conducted simultaneously with FSSST sampling in each of the 13 homes revealed 10 different types of fungal spores present for the total spore population, as well as unidentified spores. For CFU analysis of the air samples taken from 10 homes, 9 spore types were identified. The settled dust sampling in 12 homes with visible mold contamination revealed 16 different fungal spore types (including nonsporulating colonies) through the CFU enumeration.
Correlations between different collection methods were calculated for both the total spore count and the CFU count (Figure 4). For total spore counts, the correlation analyses were performed comparing the data obtained with the FSSST technique to both short-term air and swab measures. Since multiple types of fungi were collected in each home, comparisons were made wherever data points could be matched by fungal types. A significant correlation was observed between the FSSST and swab collection techniques (Figure 4B). No significant relationship was observed between the short-term air and FSSST techniques for total spore count .
Correlations between the data obtained by different measurement methods for both total spore (A,B) and CFU enumerations (C,D,E)
Correlations were also determined wherever data points could be matched by fungal type with respect to the CFU count. Again, the only significant correlation was observed between the FSSST and swab collection techniques (Figure 4D). There was no significant relationship observed between the short-term air and dust techniques nor between air and FSSST techniques for CFU count .
The percentage of culturable spores among the total counts obtained with swab, FSSST, and air sampled was calculated for each home. The results of this calculation are presented in Table I. The culturable fungal fraction ranged from 0.03% to 63% in swab samples; FSSST samples revealed the culturability range of 0.1% to over 100%; and short-term air samples showed culturability of 0.7% to 78.7%. No information on the spore culturability in dust samples was obtained because the total count was not available.
A comparison was made within homes to determine which type of sampling produced the highest percentage of culturable spores. In 6/11 (55%) homes, the air samples showed the highest spore culturability. The FSSST samples showed the highest culturability in four homes (36%). Swab samples revealed the highest culturablity in only one home (9%).
DISCUSSION
The results comparing the swab and FSSST methods showed that the median levels of spores collected by the swab method (with respect to both the microscopic and culture-based counts) were much greater than those collected by the FSSST. These ranges are similar to those that we observed in our recent study of the FSSST performance.(60) Furthermore, the median indoor total spore counts in this study were about one order of magnitude higher than the outdoor levels. Shelton et al.,(65) who collected both indoor and outdoor mold samples with the Andersen sampler and analyzed the samples using a culture-based analysis, found that the indoor levels were lower than those determined outdoors. Our indoor levels were most likely higher due to the presence of visible mold in housing. Though the indoor and outdoor air samples were collected with different methods (BioSampler and Button Sampler, respectively), both methods have been found to have high collection efficiency in the size range of fungal spores (primarily <5μm).(64,66,67)
The swab samples showed greater variability of the CFU count compared with the total count. This large variation in the enumeration type was seen in neither of the other two methods (FSSST or air). This is possibly due to a limited representativeness of a single swab sample taken in a 1-cm2-area. Perhaps the size of the swab sample led to greater variability of culturable spores determined by this method. In future studies, swab samples should be taken from a larger area when comparing swab with other methods. The surface properties (e.g., the roughness) may also affect the data variability.(58)
Aspergillus, Penicillium, and Cladosporium spores were the most common types of fungi found in this study with both enumeration methods. These spore types are among the most predominant in the United States and are generally considered to have indoor origins.(65,68) In the total spore count method, Cladosporium spores were found in approximately the same number of homes by each sampling method.
Aspergillus/Penicillium spores were identified in 6 of 13 homes for the swab method and in 8 of 13 homes for the FSSST method. In both cases when Aspergillus/Penicillium spores were identified in the FSSST samples but not in the swab, these spores represented a small fraction of the total spore count obtained by the former method. Perhaps these spores were also present in the swab samples, but were masked by the more prominent spore types. Aspergillus/Penicillium spores were also identified in 12 of the 13 homes during air sampling. Furthermore, all of the spore types identified by the total count were found in either equal amounts or, more often, in the air samples as compared with the swab and FSSST samples. Hyvarinen et al.(69) reported similar results, namely, that more fungal species were identified using air sampling than by swab sampling. The investigators suggested that this could be due to the influence of unidentified indoor sources, or outdoor mold sources.
For the culture-based analysis, the FSSST showed a greater number of homes containing both Aspergillus and Cladosporium culturable spores than did the swab or air sampling methods. Aspergillus colonies appeared in the FSSST samples in five more homes, when compared with the swab. For three of these homes, however, the swab CFU samples were either contaminated or did not grow. For the other two homes, Aspergillus spores represented a small fraction of the CFU count in the FSSST samples. It is possible that these spores were present in the swab samples but did not grow due to the much higher concentration of other spore types, which might have overgrown the Aspergillus spores. As previously mentioned, it is also possible that the small size of the swab sample was not fully representative of the source contamination when compared with the FSSST, which samples a much larger area. This explanation is also valid when comparing the samples from two homes, among which the FSSST revealed Cladosporium but the swab method did not.
When comparing the culturable FSSST and air samples, one should remember that the FSSST is designed to assess a “worst-case scenario” for spore aerosolization.(59,60) Thus the FSSST induced culturable spore release in the homes where sporulation was not yet occurring by natural means and therefore was not detected in the air. In the settled dust, culturable spore types were usually found in either equal or greater amounts compared with the other three methods. This supports the hypothesis that dust acts as a long-term sink for fungal spores.(46) Furthermore, three different mold types (Fusarium, Mucor, and Alternaria) were found in five or more homes in the dust but appeared only one time or less in the other sampling methods. Perhaps these fungal types represent the outdoor sources, suggesting the spore penetration and subsequent deposition on the floor.
When investigating the relationship between the different sampling methods, a statistically significant relationship was observed between the FSSST and swab for both the microscopic and culture-based analyses results. This was an expected result, since both techniques measure the fungal source. There was no observed relationship between the FSSST and air level of fungi for either of the enumeration methods. Duchaine and Meriaux(70) reported that the number of mold sources in a home was significantly related to air CFU levels, suggesting an association between air and source samples in homes with visible mold contamination, and recommended that both sampling types are necessary for a complete assessment of molds. Our study went a step further to quantitatively compare mold levels at the source with those found in the air. We did not observe a relationship between these two methods. There are three possible explanations for this. First, spore release from fungal colonies is sporadic, and short-term air sampling might not accurately represent airborne levels.(46,60) Second, fungal spores sampled from the indoor air represent a mixture of spores from other potential indoor sources (other mold contamination, including nonidentified growth on indoor surfaces and inside the ventilation system, as well as from the reaerosolized dust). Outdoor sources may also contribute considerably, as the presence of outdoor spores may lead to underrepresentation of those released from identified sources in indoor air samples. Third, the visibly mold-contaminated areas were different in different homes. Only data points for which fungal types could be matched across sampling types were included in the analysis. It would be expected that correlation values would be less if all fungal types had been taken together.
Results on dust sampled from the carpet (or floor) were also compared with the results on air samples taken in the room with mold contamination. This comparison was made because settled dust is often used as a measure of exposure to fungi, due to the potential reaerosolization of dust particles into the air.(44–46) Again, no relationship was observed between these measures. Similar to the findings reported by Chew et al.,(46) many more types of fungi were identified in the dust, as compared with the indoor air. Although dust has been recognized as a long-term reservoir for fungi, there appears to be little potential for reaerosolization of fungi from indoor dust, as evidenced by this study and other investigations.(46,49,71) One potential factor that has not been considered thus far is the difficulty to analyze dust by the total spore count method. The dust samples analyzed only by CFU method leave the nonculturable fraction unknown. As stated previously, it has been suggested that allergic reaction to fungi is generally independent of culturability of spores.(33) It is then imperative that an appropriate total spore enumeration method for fungal spores in dust be used.
The data collected in this study allowed us to examine the relationship between total and CFU spore counts obtained by each of the three sampling methods (swab, FSSST, and air). Each method resulted in a wide range of culturability of spores both between and within homes (swab: 0.03% to 63%, FSSST: 0.1% to >100%, air: 0.7% to 78.7%). This finding is of particular interest because a number of fungal sampling methods rely solely on the culture-based enumeration technique. The results of this study support previous reports that this reliance might grossly underestimate the number of spores present in a sample, which might potentially lead to an underestimation of the severity of mold contamination.(26) The culturability of spores is dependent on a number of factors, including spore type, temperature, and type of agar. Furthermore, since culturability is not generally linked to allergenic respiratory symptoms, these symptoms may still occur when the culture-based enumeration technique generates results below the limit of detection.(33) In one FSSST sample, the percent culturable spores was found to be greater than 100% (125%). It has been hypothesized, though not tested, that this is due to the release of mycelial fragments, which could potentially grow to form new colonies but would not be counted as spores. The limitations associated with the accuracy and precision of the microscopic spore count might also have contributed to the above discrepancy.
Selecting appropriate methods for sampling fungal spores in indoor environments is crucial in order to link the human exposure and disease caused by fungi. It has been argued that the lack of standardized and definitive methods for mold sampling is a primary cause for the poorly understood relationship between mold exposure and health outcome.(22) Generally, air sampling has been a commonly used method to assess fungal exposure and has also been described as the most representative of human respiratory exposure.(35,72–76) However, this study has demonstrated that short-term air sampling may not be an indicative measure of mold contamination in the indoor environment, as the number of spores released by the source (FSSST) did not relate to the airborne spore concentration. This was the case even though the indoor mold contamination levels were approximately an order of magnitude higher than the outdoor levels. All of the environments chosen in this study had visible mold contamination, and multiple sampling methods were used for its quantification.
It can be argued that in this type of environment, source testing would be the obvious choice for a sampling method. Furthermore, it could be suggested that if the mold source has been identified, there is no reason to sample but, instead, to simply clean the contaminated area. At the same time, if the exposure to fungal spores is to be assessed in the presence of an identified mold source short-term air sampling does not seem to be predictive of the source even when the air sample is taken in the same room where the source was identified.
CONCLUSIONS
The results of this study confirm that reliance on one sampling or enumeration method for characterization of an indoor mold source might not provide an accurate estimate of fungal contamination of a microenvironment. As shown by other investigations, multiple sampling techniques are suggested when attempting to assess indoor mold contamination. The exclusive use of a culture-based enumeration technique must be performed with the understanding that it might drastically underestimate the quantity of mold in the indoor environment. Additionally, culturable spores alone are not responsible for adverse health effects associated with mold exposure.
The relationships between the data obtained with the four different sampling methods were examined using correlation analysis. Significant relationships were observed between the data from swab and FSSST samples both by the microscopic counting and by the CFU counting. No relationships were observed between the data from air and FSSST samples or air and settled dust samples. Percentage culturability of spores for each sampling method was also calculated and found to vary greatly for all three methods (swab: 0.03% to 63%, FSSST: 0.1% to >100%, air: 0.7% to 79%). FSSST sampling appears to be an effective way to assess the mold source in the field, providing an upper bound estimate of potential mold spore release into the indoor air. However, because of the small sample size of this study, further research is needed to better understand the observed relationships in this study.
RECOMMENDATIONS
Multiple sampling methods are recommended to assess indoor mold contamination (which is consistent with the American Conference of Governmental Industrial Hygienists (ACGIH®) and AIHA recommendations).
The total spore count technique is recommended for analysis over CFU technique for assessing indoor mold contamination.
Further research efforts should be pursued to better characterize the relationship between indoor fungal spore concentration and the factors that affect the spore release into the air.
Acknowledgments
Mr. Niemeier was supported in part by the University of Cincinnati through its Education and Research Center from the Centers for Disease Control and Prevention/National Institute for Occupational Safety and Health. All the above support is deeply appreciated.
The authors are indebted to Taehkee Lee and Sung-Chul Seo for their assistance in collecting the field samples, and to Maureen Niemeier for her help in editing the manuscript.
This study was supported by the U.S. Department of Housing and Urban Development and by the National Institute of Environmental Health Sciences (NIEHS) Center for Environmental Genetics Pilot Project Program.
The contents are solely the responsibility of the authors and do not necessarily represent the official views of the institutions that sponsored this study.
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Written By: R. Todd Niemeier, Satheesh K. Sivasubramani, Tiina Reponen, and Sergey A. Grinshpun
Date Posted: 2008-07-22
Simple Prevention
An ounce of prevention is worth a pound of cure.
Lower the humidity in your home by using a dehumidifier.
Keep your spaces well ventilated.
Trap spores and prevent them spreading by installing electrostatic filters in your AC.
Install exhaust fans in humid areas like bathrooms.
Keep an eye on water pipes to make sure they’re not leaking.
Vaccuum carpets and rugs regularly.
Disinfect bathrooms, with cleaners that have mold killing properties.
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Written By: staff
Date Posted: 2008-07-22
Microbiological contamination with molds in work environment in libraries and archive storage facilities.
Summary of pending article
Nofer Institute of Occupational Medicine, Department of Environmental Health Hazards, Sw. Teresy 8, 91-348 Lodz, Poland. anias@imp.lodz.pl.
Microbiological contamination with fungi, including moulds, can pose a significant health hazard to those working in archives or museums. The species involved include Aspergillus, Penicillium, Geotrichum, Alternaria, Cladosporium, Mucor, Rhizopus, Trichoderma, Fusarium which are associated mostly with allergic response of different types. The aim of the study was to analyse, both in quantitative and qualitative terms, workplace air samples collected in a library and archive storage facilities. Occupational exposure and the related health hazard from microbiological contamination with moulds were assessed in three archive storage buildings and one library. Air samples (total 60) were collected via impact method before work and at hourly intervals during work performance. Surface samples from the artifacts were collected by pressing a counting (RODAC) plate filled with malt extract agar against the surface of the artifacts. The air sample and surface sample analyses yielded 36 different mould species, classified into 19 genera, of which Cladosporium and Penicillium were the most prevalent. Twelve species were regarded as potentially pathogenic for humans: 8 had allergic and 11 toxic properties, the latter including Aspergillus fumigatus. Quantitative analysis revealed air microbiological contamination with moulds at the level ranging from 1.8 x 10(2)-2.3 x 10(3) cfu/m(3). In surface samples from library and archive artifacts, 11 fungal species were distinguished; the number of species per artifact varying from 1-6 and colony count ranging from 4 x 10(1) to 8-10(1) cfu/100 cm(2). Higher contamination levels were found only for Cladosporium cladosporioides (1.48 x 10(3) cfu/100 cm(2)) and Paecillomyces varioti (1.2 x 10(2) cfu/100 cm(2)). At the workposts examined, although no clearly visible signs of mould contamination could be found, the study revealed abundant micromycetes, with the predominant species of Cladosporium and Penicillium. The detected species included also potentially pathogenic microorganisms which can cause allergic and toxic effects, such as Aspergillus fumigatus, that could be hazardous to workers' health. For some species, the concentration levels exceeded the values considered the proposed hygienic standards for total microscopical fungi in occupational settings. The findings of the study point to unsatisfactory hygienic conditions at the worksites examined, resulting in microbiological contamination with moulds, as well as the necessity for prompt remedial activities on the part of the employers.
Written By: Zielinska-Jankiewicz K, Kozajda A, Piotrowska M, Szadkowska-Stanczyk I.
Date Posted: 2008-07-14
The fundamentals of mold-related illness
When to suspect the environment is making a patient sick
To review current medical evidence about the link between mold exposure and clinical illness
To recognize the clinical implications of indoor mold exposure
To understand the basic management of mold-related medical conditions
The authors disclose no financial interests in this article and no unlabeled uses of any product mentioned.
Preview: Disorders related to indoor air quality have become a major concern for primary care physicians, who often are asked to evaluate patients whose symptoms may be caused or aggravated by indoor exposure to mold. In this article, the authors review the common types of indoor mold and discuss the management of mold exposure and related illnesses. Fung F, Hughson WG. The fundamentals of mold-related illness: when to suspect the environment is making a patient sick. Postgrad Med 2003;113(6)
The public, healthcare professionals, and engineers are becoming more aware of the possible medical consequences of indoor mold exposure (1). It is not uncommon for primary care physicians to encounter patients such as the woman cited in the following case report.
Hypothetical case
A patient has noted recent upper respiratory symptoms (eg, nasal congestion, sinus headache, episodic dyspnea). She works in an office building that has flower planters along the outside walls. Water from the planters has leaked into the building and stained the wallboard and adjacent carpet. The office smells musty, and other workers have complained of the odor.
What is the diagnosis? Has the water leak had a role in causing the symptoms? What should be done for the patient? Should the patient be considered disabled? What is the prognosis? What should be done to correct the problem in the building?
What is mold?
Mold belongs to the kingdom Fungi, one of five classifications of living organisms. The others are Monera (eg, bacteria), Protista (eg, protozoa), Plantae, and Animalia. Fungi cannot photosynthesize and therefore are described as heterotrophic; they obtain nutrients from other organisms, either living or dead (2). Fungi are regarded as decomposers, whereas plants are producers and animals are consumers.
Molds are classified into three groups: microfungi, mushrooms, and dry-rot fungi. Mold spores are analogous to plant seeds. They can remain dormant for months or years and can withstand extreme conditions. Whenever nutrients and moisture are present in sufficient amounts, spores germinate to form hyphae. Fungal growth continues as long as moisture is present.
Types of mold in indoor environments
Mold is ubiquitous in the environment. Typically, levels of viable and nonviable spores in a modern, air-conditioned structure are about 60% of those outdoors, and the distribution of mold species is similar. The number and types of molds inside homes are comparable with those outdoors because domestic structures are leakier than modern, tightly sealed office buildings.
Increased levels of mold or a distortion in the distribution of mold species in a building compared with outdoors suggests the presence of excessive moisture and possible mold amplification. In general, when the level of a mold species in a building is at least one order of magnitude greater than the outdoor concentration, a person's symptoms are probably related to indoor mold exposure.
Indoor mold can be classified into two groups. The first group requires low to moderate moisture and includes Penicillium, Cladosporium, and Alternaria. The second group requires high moisture and includes Stachybotrys, Chaetomium, Trichoderma, and Aspergillus niger. Some species of fungi are prone to growing in certain substrates. Table 1 lists the common indoor molds.
Table 1. List of molds commonly found in indoor environments
Mucous membrane discomfort (ie, eye, nose, and throat irritation), headache, and fatigue are the most common symptoms caused by poor indoor air quality. The term sick building syndrome has been used to describe these building-related symptoms when a specific diagnosis (eg, asthma, rhinitis) cannot be established.
In contrast to sick building syndrome, the term building-related illness is used when a disease that affects one or more building occupants has been proven to be clinically and causally linked to the indoor environment. With proper therapy and avoidance, the medical condition usually resolves.
Mold-related illnesses
In general, mold can produce four types of human illness: allergy, infection, irritation, and toxic effects (table 2).
Table 2. Health effects of exposure to some types of fungi
Allergy
Mycosis
Irritation
Mycotoxicosis
Alternaria
+
+/-
+
+/-
Aspergillus
+
+
+
+
Cladosporium
+
+/-
+
-
Fusarium
+
+/-
+
+
Penicillium
+
+
+
+
Stachybotrys
+
+/-
+
+
Trichoderma
+
+/-
+
+/-
+, reported; +/-, possible; -, not reported.
Allergy and hypersensitivity pneumonitis
Allergic reactions to mold can range from mild to severe and from transitory to chronic. Detailed descriptions of allergic diseases are available in a textbook by Middleton and associates (3). Briefly, allergic rhinitis and asthma are associated with responses mediated by immunoglobulin E (IgE); hypersensitivity pneumonitis is associated with T-cell responses and responses mediated by immunoglobulin G (IgG). Allergic rhinitis and sinusitis can be diagnosed using patient history, physical examination findings, the presence of eosinophils in nasal smears, and the results of skin prick tests and radioallergosorbent tests to detect specific IgE antibodies.
Asthma due to fungal allergens is characterized by chest tightness, wheezing, cough, and dyspnea that worsen with exposure to the allergen (4,5). Symptoms typically occur within 1 hour of exposure. Diagnosis is made on the basis of patient history, physical evidence of bronchospasm, pulmonary function tests demonstrating reversible air flow obstruction, or a positive methacholine chloride (Provocholine) challenge test.
In its early stages, hypersensitivity pneumonitis is characterized by recurrent symptoms of fever, cough, and chest tightness and the presence of pulmonary infiltrates on a chest radiograph. Chronic hypersensitivity pneumonitis features progressive dyspnea, fatigue, interstitial pneumonitis, and pulmonary fibrosis. This condition occurs mainly in farmers, pigeon breeders, cheese makers, wood processors, and mushroom growers exposed to high levels of organic dust and fungal antigens. Diagnosis is made on the basis of patient history, physical examination findings, an abnormal chest radiograph or computed tomographic (CT) scan (figure 1a), a restrictive pattern on pulmonary function tests, reduced diffusion capacity, and the presence of numerous lymphocytes on bronchoalveolar lavage. Open lung biopsy may be needed to confirm the diagnosis.
Infection
The most common fungal disease is a superficial mycosis, such as tinea infection, that is not linked to indoor air quality or building-related illness. Bronchopulmonary aspergillosis, or allergic bronchopulmonary aspergillosis, is an inflammatory disease caused by an immunologic response to an Aspergillus species, usually Aspergillus fumigatus, growing in the bronchi of patients with asthma. It has been reported in immunocompromised patients and in patients with chronic obstructive pulmonary disease and has been linked to building-related illness.
Systemic fungal infections such as histoplasmosis, coccidioidomycosis, and cryptococcosis have occurred after contaminated bird droppings or construction dusts were disseminated in an indoor environment. Detailed descriptions of mycoses can be found in standard textbooks, such as the publication by Mandell and colleagues on infectious diseases (6).
Irritation
Mold exposure may aggravate existing allergic rhinitis or asthma because of its irritant effects. Mold can produce a variety of organic chemicals, including alcohols and sulfur-containing compounds that exude musty and pungent odors. Irritation may cause mucous membrane symptoms such as conjunctivitis and rhinitis with trigeminal nerve stimulation. Pungent odors may initiate avoidance reactions, a generalized feeling of discomfort, breath holding, and a burning sensation on the skin.
Under unusual circumstances, volatile organic chemicals may reach levels sufficient to produce central nervous system symptoms such as headache, inability to concentrate, or dizziness. Glucans are glucose polymers that are components of most fungal cell walls, and exposure to airborne 13-beta-D-glucan has been known to cause irritation symptoms due to airway inflammation (7). However, these irritant effects are transient and self-limiting.
Mycotoxicosis
Molds produce antibiotics and mycotoxins to gain a competitive advantage over bacteria and other mold species. Mycotoxins, which typically are cytotoxic, disrupt cell membranes and interfere with the synthesis of protein, RNA, and DNA. Not all molds produce mycotoxins, however. Toxigenic molds vary in their mycotoxin production depending on the substrate, environmental factors (eg, temperature, relative humidity, light, presence of oxygen and carbon dioxide), and seasonal and life cycle stages. The presence of a toxigenic mold in an indoor environment does not prove that the occupants have been exposed to mycotoxins.
The only well-documented human mycotoxicoses have been the result of ingestion rather than inhalation. Although these cases are unrelated to indoor mold exposure, they are important to the understanding of the toxin-production mechanism, pathophysiologic factors, and clinical syndrome of mycotoxicosis.
One of the earliest documented examples of mycotoxicosis is ergotism from eating rye contaminated with Claviceps purpurea (2). The major clinical syndromes of ergotism are classified as either gangrenous or convulsive. The toxic effects of aflatoxins, which are produced by Aspergillus flavus, were collectively called turkey X disease when they were first documented in 1960 (2). Aflatoxins, which probably are the most extensively studied mycotoxins, cause such diseases as acute fatty liver syndrome, hepatic necrosis, and an encephalopathy similar to Reye's syndrome. Chronic exposure to aflatoxins can lead to hepatocellular carcinoma (8).
Stachybotrys chartarum (also called Stachybotrys atra) typically is slimy and not easily aerosolized. Stachybotrys mycotoxin had been thought to cause acute pulmonary hemorrhage and death in infants (9). However, the Centers for Disease Control and Prevention recently issued a report stating that the association has not been proved (10). The controversy over airborne Stachybotrys mycotoxins originated from extrapolation of data from case series that did not have specific medical diagnoses. About 10 ng of mycotoxins are produced for every 1 million mold spores (11). Assuming the exposure is cumulative, inhalation of 109 spores per hour would be required for toxic effects. Therefore, it is unlikely that inhalation of fungal parts presumed to contain mycotoxins could produce significant human illness--particularly in a nonagricultural setting (12,13).
Identification of a serum fungal antibody means only that the patient has been exposed at one time; it does not prove that a disease was caused by the fungus or its metabolites. Interpretation of antibody tests is complicated by cross-reactivity with other fungi that have similar antigenic properties. Also, the level of antibody at any point in time is not predictable (14). Therefore, S chartarum IgG antibodies cannot be used to establish the date or source of the last exposure.
Management of mold exposure
The first step in the management of mold exposure is to establish a diagnosis using the previously described clinical tools. A medical history of the chronology of symptoms and the duration of exposure should be obtained. Open-ended questions about a building's water intrusion, leaks, musty odor, and water stains and the presence of visible mold on the walls, carpets, or clothes should be asked.
Physical examination should focus on the evaluation of rhinitis, sinusitis, asthma, and hypersensitivity pneumonitis. Laboratory studies, radioallergosorbent tests, radiographs, pulmonary function tests, cultures, and other specialized tests (eg, CT, bronchoscopy) may be indicated when clinical symptoms and signs suggest allergy, asthma, hypersensitivity pneumonitis, or sinus infection.
The issue of disability depends on the specific diagnosis. Allergy can be managed with antihistamines and avoidance of mold exposure. Asthma can be treated and controlled with a beta-agonist and a corticosteroid inhaler. Infection can be treated with antifungal medications, and recovery is usually complete. Hypersensitivity pneumonitis, however, may require prolonged prednisone therapy, and the patient may have significant disability due to pulmonary impairment.
Unfortunately, many patients have nonspecific signs and symptoms, and the diagnostic label may be imprecise. Nevertheless, the presence of similar problems in coworkers and the temporal relationship between when the patient entered the building and the onset of symptoms suggest a causal relationship, even when a specific diagnosis is not possible.
Typically, the environmental problem that caused the mold has been present for some time, and corporate management may be aware of employee complaints. To avoid creating adversarial work relationships, it is wise for the physician to discuss the situation with the patient's supervisor. In some cases, an environmental assessment already has been done and data are available for review. If not, the physician can recommend an industrial hygiene inspection to detect water intrusion and mold amplification.
Industrial hygiene evaluation is useful only when a patient has symptoms that suggest mold exposure. Without medical complaints, results of industrial hygiene assessment are too nonspecific to be useful. In general, an industrial hygienist is able to perform a visual inspection and to obtain surface, bulk, and air samples of molds and mold spores to confirm the presence and source of mold growth. Once identified, these problems usually can be corrected in a relatively straightforward manner. Currently, because of a lack of sensitivity and specificity in laboratory tests, sampling for mycotoxins is performed only in the research setting.
Patient symptoms usually improve or disappear when the mold contamination is eliminated. In rare instances, health problems (eg, asthma, hypersensitivity pneumonitis) may persist, requiring long-term healthcare, permanent work restrictions, or a determination of disability.
Summary and conclusion
Health effects of bioaerosol exposure from fungal sources include allergy, infection, irritation, and toxicity. The first three categories have well-established mechanisms, but dose-response data are lacking and the degree of individual susceptibility is highly variable.
Specific toxic effects due to inhaled mycotoxins are not well documented and therefore remain controversial. In the absence of specific illness and respiratory symptoms in the occupants of a home or office, responding to visible mold with knee-jerk advice is not advised. Such statements as "Move out of your home" or "Evacuate the building" have significant psychosocial and economic consequences.
Excessive moisture is the fundamental cause of mold proliferation. Before starting expensive and often low-yield environmental investigations, the first step is to eliminate excess moisture (15). Proper building design and construction, combined with periodic preventive maintenance to avoid water intrusion, are fundamental in preventing the adverse health effects of mold exposure.
References
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Halsey J. Performance of a Stachybotrys chartarum serology panel. (Abstr) Presented at the Western Society of Allergy, Asthma and Immunology Annual Meeting. Allergy Asthma Proc 2000;21(3):174-5
Macher J, ed. Bioaerosols: assessment and control. Cincinnati: American Conference of Governmental Industrial Hygienists, 1999
Web sites for further information on mold-related illness
Dr Fung is chief toxicologist, Sharp Rees-Stealy Medical Group, San Diego, and medical toxicology consultant, University of California, San Diego, School of Medicine. Dr Hughson is director and professor, Center for Occupational and Environmental Health, University of California, San Diego, School of Medicine. Correspondence: Frederick Fung, MD, MS, Sharp Rees-Stealy Medical Group, 2001 4th Ave, San Diego, CA 92101. E-mail: fred.fung@sharp.com.
Terre Haute North treated for mold
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3-beta-D-glucan has been known to cause irritation symptoms due to airway inflammation (7). However, these irritant effects are transient and self-limiting.