Teija Meklin

Analysis of Fungal Flora in Indoor Dust by Ribosomal DNA Sequence Analysis, Quantitative PCR, and Culture

ABSTRACT In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed.

Prevalence of moisture problems in Finnish houses

Abstract A random sample of 450 houses representing various decades was surveyed by trained civil engineers for signs of water leaks or condensation. In 80% of the houses, signs of current or previous moisture fault were observed. In most houses, the surveyors made at least two separate observations of moisture but the occupants missed one in every two of these signs of moisture damage. Part of the faults were caused by flaws in design or construction, and part were due to aging of materials. Approximately 55% of the Finnish houses (i.e., a total of nearly 500 000 houses), were assessed to be in need of repair or more thorough inspection. The observations were equally frequent in the houses of different age, but certain types of moisture damage were characteristic of certain types of buildings or constructional methods used in different eras. Roof geometry was a significant factor accounting for roof leakages. The majority of the faults could be repaired at reasonable cost.

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.

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

BackgroundIndoor 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).ResultsCulture-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).ConclusionsWe 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.

Effect of moisture-damage intervention on the immunotoxic potential and microbial content of airborne particles and on occupants' upper airway inflammatory responses

This intervention study evaluated the effect of moisture-damage repairs on the exposure and on the upper airway inflammatory responses of the occupants. The airborne microbial exposure was followed by quantitative PCR analyses of 13 microbial species in repeated long-term indoor air samples before (N = 26) and after (N = 28) repairs of the school building. Airborne particulate matter was collected similarly from the same premises (before N = 25, after N = 34) for determination of nitric oxide (NO), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6), measured in the cell culture medium of mouse macrophages. NO, TNFα, IL-6, and IL-4 were also analyzed in the nasal lavage (NAL) samples of the occupants (N = 13) to characterize their upper airway inflammatory responses during the exposure and after its cessation. After the repairs, concentrations of the measured airborne microbes decreased, the difference being significant for six of 13 species. After renovation, airborne particulate matter also caused significantly lower production of IL-6 and TNF-α in mouse macrophages than the material collected before the renovation. The concentration of IL-4 in the NAL samples was significantly lower after the renovation. These results show that the inflammatory potential of the airborne material decreases after intensive repair of the moisture damage.

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.