Molecular profiling of fungal communities in moisture damaged buildings before and after remediation--a comparison of culture-dependent and culture-independent methods.

BACKGROUND: Indoor microbial contamination due to excess moisture is an important contributor to human illness in both residential and occupational settings. However, the census of microorganisms in the indoor environment is limited by the use of selective, culture-based detection techniques. By using clone library sequencing of full-length internal transcribed spacer region combined with quantitative polymerase chain reaction (qPCR) for 69 fungal species or assay groups and cultivation, we have been able to generate a more comprehensive description of the total indoor mycoflora. Using this suite of methods, we assessed the impact of moisture damage on the fungal community composition of settled dust and building material samples (n = 8 and 16, correspondingly). Water-damaged buildings (n = 2) were examined pre- and post- remediation, and compared with undamaged reference buildings (n = 2). RESULTS: Culture-dependent and independent methods were consistent in the dominant fungal taxa in dust, but sequencing revealed a five to ten times higher diversity at the genus level than culture or qPCR. Previously unknown, verified fungal phylotypes were detected in dust, accounting for 12% of all diversity. Fungal diversity, especially within classes Dothideomycetes and Agaricomycetes tended to be higher in the water damaged buildings. Fungal phylotypes detected in building materials were present in dust samples, but their proportion of total fungi was similar for damaged and reference buildings. The quantitative correlation between clone library phylotype frequencies and qPCR counts was moderate (r = 0.59, p < 0.01). CONCLUSIONS: We examined a small number of target buildings and found indications of elevated fungal diversity associated with water damage. Some of the fungi in dust were attributable to building growth, but more information on the material-associated communities is needed in order to understand the dynamics of microbial communities between building structures and dust. The sequencing-based method proved indispensable for describing the true fungal diversity in indoor environments. However, making conclusions concerning the effect of building conditions on building mycobiota using this methodology was complicated by the wide natural diversity in the dust samples, the incomplete knowledge of material-associated fungi fungi and the semiquantitative nature of sequencing based methods.

Concentrations and diversity of microbes from four local bioaerosol emission sources in Finland.

Microbial particles can readily be released into the air from different types of man-made sources such as waste operations. Microbiological emissions from different biological sources and their dispersion may be an issue of concern for area planning and for nearby residents. This study was designed to determine the concentrations and diversity of microbiological emissions from four different man-made source environments: waste center with composting windrows, sewage treatment plant, farming environment, and cattle manure spreading. Samples of airborne particles were collected onto polyvinyl chloride filters at three distances along the prevailing downwind direction, from each source environment during a period of approximately 1 week. These samples were analyzed for 13 species or assay groups of fungi, bacterial genus Streptomyces, and Gram-positive and -negative bacteria using quantitative polymerase chain reaction (PCR). Samples for determining the concentrations of viable fungi and bacteria were collected from all environments using a six-stage impactor. The results show that there were variations in the microbial diversity between the source environments. Specifically, composting was a major source for the fungal genera Aspergillus and Penicillium, particularly for Aspergillus fumigatus, and for the bacterial genus Streptomyces. Although the microbial concentrations in the sewage treatment plant area were significantly higher than those at 50 or 200 m distance from the plant area, in the farming environment or cattle manure spreading area, no significant difference was observed between different distances from the source. In summary, elevated concentrations of microbes that differ from background can only be detected within a few hundred meters from the source. This finding, reported earlier for culturable bacteria and fungi, could thus be confirmed using molecular methods that cover both culturable and nonculturable microbial material.

Immunotoxicological properties of airborne particles at landfill, urban and rural sites and their relation to microbial concentrations.

We investigated the immunotoxicological activity of airborne particles in three different environments during 11 months. Specifically, we analyzed the relation of the immunotoxicological activity to microbial concentrations. During the study period, samples from a landfill, an urban and a rural site were collected on filters once a month. The immunotoxicological characteristics of collected particle samples were studied by exposing mouse macrophages (RAW264.7), and measuring the viability and production of inflammatory mediators i.e. nitric oxide (NO), tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 after 24 hours. In addition, the results were compared to corresponding microbial concentrations measured with quantitative polymerase chain reaction (qPCR) in the same sites. The particles collected from the landfill caused significantly more toxicity and inflammatory responses than the particles from other sites. The immunotoxicological activity of the samples changed according to the season, being the lowest in all study sites in the winter. In the rural and urban sites the responses peaked during the spring, whereas at the landfill the highest responses were detected towards the fall. All immunotoxic responses correlated strongly with airborne microbial concentrations at the landfill, whereas on the other sites the correlations were weaker. These results indicate that the overall immunotoxicological activity of the particles is increased in waste handling area with a heavy microbial load. The activity of the samples collected from different sites changes according to the season being at their lowest at wintertime. At the waste center, the immunotoxicological responses are related with concentrations of microbes, whereas at rural area and city center other factors seem to govern the toxicity of the sample.

The effect of ozonization on furniture dust: microbial content and immunotoxicity in vitro.

Moisture and mold problems in buildings contaminate also the furniture and other movable property. If cleaning of the contaminated furniture is neglected, it may continue to cause problems to the occupants even after the moisture-damage repairs. The aim of this study was to determine the effectiveness of high-efficiency ozone treatment in cleaning of the furniture from moisture-damaged buildings. In addition, the effectiveness of two cleaning methods was compared. Samples were vacuumed from the padded areas before and after the treatment. The microbial flora and concentrations in the dust sample were determined by quantitative cultivation and QPCR-methods. The immunotoxic potential of the dust samples was analyzed by measuring effects on cell viability and production of inflammatory mediators in vitro. Concentrations of viable microbes decreased significantly in most of the samples after cleaning. Cleaning with combined steam wash and ozonisation was more effective method than ozonising alone, but the difference was not statistically significant. Detection of fungal species with PCR showed a slight but nonsignificant decrease in concentrations after the cleaning. The immunotoxic potential of the collected dust decreased significantly in most of the samples. However, in a small subgroup of samples, increased concentrations of microbes and immunotoxicological activity were detected. This study shows that a transportable cleaning unit with high-efficiency ozonising is in most cases effective in decreasing the concentrations of viable microbes and immunotoxicological activity of the furniture dust. However, the method does not destroy or remove all fungal material present in the dust, as detected with QPCR analysis, and in some cases the cleaning procedure may increase the microbial concentrations and immunotoxicity of the dust.

Microbial content of house dust samples determined with qPCR.

This study was designed to produce information about microbial concentrations using qPCR and their variation in different seasons and home environments with analyses of two types of house dust samples. Also the correlations between the two types of samples and the reproducibility of the parallel subsamples were studied. Two types of vacuumed house dust samples, rug dust and vacuum cleaner bag dust, were collected in 5 normal urban homes in four different seasons (N=20+20). From all dust samples, five parallel subsamples were subjected to qPCR analyses of 17 microbial species or assay groups of microbes. The highest fungal concentrations were found for the Penicillium/Aspergillus/Paecilomyces variotii group, and for the species Aspergillus penicillioides, Aureobasidium pullulans, Cladosporium cladosporioides and Cladosporium herbarum. These species/groups were present in almost all samples. The two types of dust samples gave similar results for most microbial species or groups analyzed, but in general, concentrations were slightly higher in rug dust than in dust from vacuum cleaner bag. Microbial concentrations varied significantly between different seasons and hence the similarity of samples within home was mainly low. The concentrations varied significantly also between different home environments. The reproducibility of the parallel subsamples was good or moderate for most of the analyzed species or assay groups. However, further studies are needed to fully understand the factors causing variation in these methods. Nevertheless, in order to show actual differences in fungal concentrations between urban homes with no known microbial sources, all dust samples to be compared should be taken during the same season.

Indoor air particles and bioaerosols before and after renovation of moisture-damaged buildings: the effect on biological activity and microbial flora.

Many building-related health problems coincide with moisture damage and mold growth within a building. Their elimination is assumed to improve indoor air quality. The aim of this study was to follow the success of remediation in two individual buildings by analyzing the microbial flora and immunotoxicological activity of filter samples. We compare results from samples collected from indoor air in the moisture-damaged buildings before and after renovation and results from matched reference buildings and outdoor air. The microbial characteristics of the samples were studied by analyzing ergosterol content and determining the composition of fungal flora with quantitative polymerase chain reaction (QPCR). In addition, the concentrations of particles were monitored with optical particle counter (OPC). The immunotoxicological activity of collected particle samples was tested by exposing mouse macrophages (RAW264.7) for 24 h to particle suspension extracted from the filters, and measuring the viability of the exposed cells (MTT-test) and production of inflammatory mediators (nitric oxide, IL-6 and TNF*) in cell culture medium by enzyme-linked immunoassay (ELISA). The results show that for Location 1 the renovation decreased the immunotoxicological activity of the particles collected from damaged building, whereas no difference was detected in the corresponding samples collected from the reference building. Interestingly, only slight differences were seen in the concentration of fungi. In the Location 2, a decrease was seen in the concentration of fungi after the renovation, whereas no effect on the immunotoxicological responses was detected. In this case, the immunotoxicological responses to the indoor air samples were almost identical to those caused by the samples from outdoor air. This indicates that the effects of remediation on the indoor air quality may not necessarily be readily measurable either with microbial or toxicological parameters. This may be associated with different spectrum of harmful agents in different mold and moisture-damaged buildings.

Comparison of mold concentrations quantified by MSQPCR in indoor and outdoor air sampled simultaneously.

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 m(3) of indoor air and 827 per m(3) of outdoor air samples were significantly different (por=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.

Effects of moisture damage and renovation on microbial conditions and pupils' health in two schools--a longitudinal analysis of five years.

Airborne microbes and pupils' symptoms were monitored in a moisture-damaged (index) school and a reference school for five consecutive years. These surveys were carried out in two separate years before the renovation of the index school, during the renovation, and one and two years after the renovation. Microbial concentrations were higher in the index school than those in the reference school before and during renovation, but afterwards, the levels decreased to the level of the reference school. The effect of remediation was seen as an altered mycobiota in the index school. Year-to-year variation of microbial concentrations, probably due to climatic factors, caused a peak in both schools but their difference remained. Several symptoms were more prevalent in the moisture-damaged school than in the reference school, but the differences disappeared during the renovations. These results emphasize the importance of using a reference building in assessing the microbial conditions of a moisture damaged building. Furthermore, microbial concentrations reflected well the technical condition of the construction, but the reported symptoms of the occupants did not strictly follow the timely fluctuation in microbial conditions.

Mold damage in homes and wheezing in infants.

BACKGROUND: In most studies that investigate the association of mold or water damage and respiratory disorders in infants, the analysis is not adjusted for exposure to house dust mite (HDM), which is also a known cause of respiratory illnesses. OBJECTIVE: To investigate the relationship between visually observable mold or water damage and HDM (Der f 1) levels and the prevalence of lower respiratory tract symptoms and allergen sensitization in infants of atopic parents as part of a prospective birth cohort study. METHODS: On-site home visits (at the infants' age of 8 months) were performed to evaluate observable mold or water damage and HDM exposure. At a clinic visit near the infant's first birthday, medical histories, including parent-reported wheezing episodes, and a skin prick test to food and 15 common aeroallergens were conducted in 640 infants. RESULTS: More than half of the homes were found to have mold or water damage, and 5% had major mold or water damage with visible mold at 0.2 m2 or more. Only 16% of homes had a HDM allergen (Der f 1) concentration of more than 2 microg/g. Major mold or water damage increased the risk of recurrent wheezing nearly 2 times in infants, 5 times in food or aeroallergen-sensitized infants, and 6 times in aeroallergen-sensitized infants. Neither visible mold or water damage nor HDM exposure was associated with sensitization to either mold or aeroallergens. CONCLUSIONS: Visible mold was shown to be a significant risk factor for recurrent wheezing in infants at high risk of developing atopic disorders, whereas HDM exposure did not significantly increase the risk.

Mycobacteria and fungi in moisture-damaged building materials.

In contrast to the growth of fungi, the growth of mycobacteria in moisture-damaged building materials has rarely been studied. Environmental mycobacteria were isolated from 23% of samples of moisture-damaged materials (n = 88). The occurrence of mycobacteria increased with increasing concentrations of fungi. Mycobacteria may contribute to indoor exposure and associated adverse health effects.

Microbial exposure, symptoms and inflammatory mediators in nasal lavage fluid of kitchen and clerical personnel in schools.

OBJECTIVES: The aim of this study was to investigate how the microbial conditions of kitchen facilities differ from those in other school facilities. The health status of the personnel was also studied. MATERIALS AND METHODS: The microbial investigations were conducted in six moisture-damaged schools and two reference schools. The symptoms of the kitchen personnel were surveyed with questionnaires and inflammatory responses in nasal lavage (NAL) fluid were measured. RESULTS: The total concentrations of airborne microbes were lower in kitchens than in other facilities of the schools. However, the occurrence of moisture damage increased the airborne microbial concentrations both in kitchens, and in other facilities. Bacterial concentrations were high on surfaces in the damaged kitchens. Gram-negative bacteria predominated, but also thermophilic bacteria and mycobacteria were detected. Respiratory and general symptoms were prevalent both among kitchen workers and clerical personnel in the moisture-damaged environments. Reported allergies and repeated respiratory infections were connected with high IL-4 concentrations in NAL fluid. Median concentrations of studied inflammatory mediators (NO, IL-4, IL-6 and TNF-alpha) were slightly higher in NAL samples of kitchen workers than among the clerical personnel. CONCLUSIONS: Kitchen facilites differ from other facilities of the school building for their moisture conditions and microbial contamination. Thus, they represent a specific type of environment that may affect the health status of the personnel.

Effects of mould remediation on school teachers' health.

The follow-up study of the health of teachers (n = 56) of three mould damage schools were done with self-administered symptom questionnaire before and 1 year after the remediation of school buildings. Technical and microbiological investigations were done parallel at the same time. In the beginning of the study symptoms of allergic rhinitis, sinusitis, conjunctivitis and fatigue were high compared to normal population and 1 year after the intervention a decrease in fatigue (OR = 0.4) and headache (OR = 0.2) was observed. An association between female gender and sinusitis was found before the remediation (OR = 8.1). Age over 40 years was a risk factor for voice problems and more than 10 working years at the same school were associated with increased risk for conjunctivitis (OR = 8.5) and headache (OR = 5.4). Other exposure situations such as mould problems at home and mould exposure during leisure time also have an effect on teachers' health. Significant reduction was found in symptoms of fatigue and headache after the cessation of exposure, while respiratory symptoms need much longer time to relieve after the remediation. Age, female gender, atopy, long-term exposure time and other exposure situations might be the risk factors for prolonged symptoms among mould exposed teachers.

Quantitative PCR analysis of house dust can reveal abnormal mold conditions.

Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.

Exposure to Aspergillus fumigatus spores induces chemokine expression in mouse macrophages.

Inhalation of fungal spores may cause inflammation and respiratory diseases, such as bronchitis, allergic alveolitis, and asthma. Alveolar macrophages provide the first line of defense in the respiratory tract. To examine the cellular mechanisms involved in Aspergillus fumigatus-induced airway inflammation, mouse macrophage cell line (RAW 264.7) cells were exposed for 2 h or 6 h to graded doses of A. fumigatus spores that were either alive or heat-killed. Furthermore, the ability of the cells to phagocytize the spores was visualized by electron microscopy. Expression of selected cytokines and chemokines was assessed by a real time quantitative PCR method and by enzyme-linked immunoabsorbent assay (ELISA) after exposure. A significant increase in mRNA expression of TNF-alpha, MIP-1alpha, MIP-1beta, and MCP-1 was observed with a maximal induction at 6h after exposure to the highest (1 x 10(7)) concentration of live spores. Similar response was not detected with heat-killed spores in the expression of chemokines and cytokines, even though there were no differences between the phagocytosis of live and heat-killed spores. These results suggest that exposure to live spores of A. fumigatus can modulate the expression of proinflammatory cytokines and chemokines in mouse macrophages and thus influence the development of inflammatory processes in the airways.

Effect of building frame and moisture damage on microbiological indoor air quality in school buildings.

The effect of building frame and moisture damage on microbial indoor air quality was characterized in 17 wooden and 15 concrete or brick school buildings. Technical investigations to detect visible moisture and mold damage were performed according to a standardized protocol. Viable airborne microbes were determined by using a six-stage impactor (Andersen 10-800). Mean concentrations of viable airborne fungi were significantly higher in wooden schools than in concrete schools, showing that the frame material was a determinant of concentrations of airborne fungi. Moisture damage of the building did not alter the fungal concentrations in wooden school buildings. In contrast, in concrete schools the effect of moisture damage was clearly seen as higher concentrations compared with the reference schools. Aspergillus versicolor, Stachybotrys, and Acremonium were detected only in samples from moisture damaged buildings, and can be considered marker fungi of such damage in school buildings. In addition, the presence of Oidiodendron as well as elevated concentrations of Cladosporium and actinobacteria were associated with moisture damage in concrete schools.

Microbial exposure and mold-specific serum IgG levels among children with respiratory symptoms in 2 school buildings.

In this study, the authors determined the association between serum mold-specific immunoglobulin G (IgG) levels in primary school children (N = 181) with asthma, wheezing, or cough symptoms and exposure to indoor mold in 2 schools, with and without mold damage. Microbial exposure was determined with environmental sampling. Serum IgG antibody concentrations against 24 microbial strains were determined with enzyme-linked immunosorbent assay. Children's microbe-specific IgG levels were often higher in the reference school. There was an association between elevated serum IgG antibody levels to Penicillium notatum and moisture damage in the school. In addition, moisture damage both in school and at home was associated with Penicillium notatum and Eurotium amstelodami IgG levels. These observations comported with microbial findings in the index school. No other positive associations between IgG antibodies and microbial findings for the school buildings were observed. IgG levels in sera of school children did not provide a specific means for assessing the microbial exposure associated with moisture and microbial problems in the school buildings. Thus, IgG analysis cannot be readily suggested as a routine method for the evaluation of these exposures.