Fungal DNA and pet allergen levels in Swedish day care centers and associations with building characteristics

Pet allergens and mold growth related to damp are common phenomena in day care centers in Sweden but exposure measurements of these factors are lacking. The aim of this study was to investigate the relationship between building construction and indoor environment quality in Swedish day care centers and the potential for exposure to fungi (analyzed by quantitative PCR) and animal allergens (analyzed by ELISA). Measurements were performed in 21 day care centers (103 rooms) from one municipality in Sweden, which were identified as constructions at risk of dampness (85% of the buildings) and with visible damage and mold growth (54% of the buildings). Dust samples were collected using cotton swab and Petri dishes. Total fungal DNA was detected in 99% and 100%, Aspergillus/Penicillium DNA in 54% and 68%, and Stachybotrys chartarum DNA in 4% and 9% of the investigated rooms in cotton swab and Petri dish samples, respectively. The total fungal DNA levels (Geometric Mean, GM) were 4.2 × 10(6) cell equivalents per m(2) and 2.9 × 10(5) cell equivalents per m(2) per day in the swab and Petri dish samples, respectively. The concentrations (GM) of cat (Fel d1), dog (Can f1), and horse (Equ cx) allergens were 9.4, 7.2 ng m(-2) day(-1), and 5.0 unit per m(2) per day, respectively. Total fungal DNA levels were higher in risk construction buildings (p = 0.01), in rooms with linoleum flooring material (p = 0.003), and in buildings with rotating heat exchangers (p = 0.02). There were associations between total fungal DNA levels and cat (p = 0.02), dog (p < 0.001), and horse (p = 0.001) allergens. In conclusion, risk constructions, damp constructions, mould growth, fungal DNA, and animal allergens were common exposure factors in Swedish day care centers. Building constructions that represent a high risk for internal dampness should be avoided in the future, and measures to reduce allergen levels should be considered to protect pet-allergic children from asthmatic problems.     

Quantitative PCR analysis of fungi and bacteria in building materials and comparison to culture-based analysis

Prolonged moisture on building materials can lead to microbial growth on them. Microbes can emit spores, metabolites and structural parts into the indoor air and thus, cause adverse health effects of people living and working in these buildings. So far, culture methods have been used for assessment of microbial contamination of building materials. In this work, we used quantitative PCR (qPCR) for the detection of selected fungal and bacterial groups in 184 building materials of different types and compared the results with culture-based analysis. Nine either commonly found species, genera or groups of fungi, or those considered as moisture damage indicators, and one bacterial genus, Streptomyces, were determined using qPCR. Fungi and mesophilic actinomycetes were also cultivated using standard media and conditions of the routine analysis. The bacterial genus Streptomyces and the fungal group Penicillium/Aspergillus/Paecilomyces were the most prevalent microbial groups in all building material types, followed by Stachybotrys chartarum and Trichoderma viride/atroviride/koningii. The highest prevalences, concentrations and species diversity was observed on wooden materials. In general, the results of the two methods did not correlate well, since concentrations of fungi and streptomycetes were higher and their occurrence more prevalent when determined by qPCR compared to culture-based results. However, with increasing concentrations, the correlation generally increased. The qPCR assay did not detect Aspergillus versicolor and Acremonium strictum as often as culture.     

Remediation of mould damaged building materials--efficiency of a broad spectrum of treatments

We compared the efficiency of some commercially available products and methods used for remediation of mould-contaminated building materials. Samples of gypsum board and pinewood were artificially contaminated with toxin-producing isolates of Stachybotrys chartarum and Aspergillus versicolor, respectively, then, ten different remediation treatments were applied according to the manufacturers' instructions. Microbial and chemical analyses of the infested materials were carried out both immediately before and after treatment, after six weeks of drying at room temperature, and after another six weeks of remoistening. The aim of the study was to determine whether the investigated methods could inhibit the mould growth and destroy some selected mycotoxins produced by the moulds. None of the decontamination methods tested could completely eliminate viable moulds. Some methods, especially boron based chemicals, ammonium based chemicals, and oxidation reduced the contents of mycotoxins produced by S. chartarum (satratoxin G and H, verrucarol), whereas the one which uses an ammonium based chemical reduced the amount of sterigmatocystin produced by A. versicolor with statistical significance. No remediation treatment eliminated all the toxins from the damaged materials. These results emphasize the importance to work preventively with moisture safety throughout the construction processes and management to prevent mould growth on building materials.     

A two-stage cyclone using microcentrifuge tubes for personal bioaerosol sampling

Personal aerosol samplers are widely used to monitor human exposure to airborne materials. For bioaerosols, interest is growing in analyzing samples using molecular and immunological techniques. This paper presents a personal sampler that uses a two-stage cyclone to collect bioaerosols into disposable 1.5 ml Eppendorf-type microcentrifuge tubes. Samples can be processed in the tubes for polymerase chain reaction (PCR) or immunoassays, and the use of multiple stages fractionates aerosol particles by aerodynamic diameter. The sampler was tested using fluorescent microspheres and aerosolized fungal spores. The sampler had first and second stage cut-off diameters of 2.6 microm and 1.6 microm at 2 l min(-1)(geometric standard deviation, GSD = 1.45 and 1.75), and 1.8 microm and 1 microm at 3.5 l min(-1)(GSD = 1.42 and 1.55). The sampler aspiration efficiency was >or=98% at both flow rates for particles with aerodynamic diameters of 3.1 microm or less. For 6.2 microm particles, the aspiration efficiency was 89% at 2 l min(-1) and 96% at 3.5 l min(-1). At 3.5 l min(-1), the sampler collected 92% of aerosolized Aspergillus versicolor and Penicillium chrysogenum spores inside the two microcentrifuge tubes, with less than 0.4% of the spores collecting on the back-up filter. The design and techniques given here are suitable for personal bioaerosol sampling, and could also be adapted to design larger aerosol samplers for longer-term atmospheric and indoor air quality sampling.     

Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing

Traditional methods of assessing fungal exposure have been confounded by a number of limiting variables. The recent utilization of molecular methods such as internal transcribed spacer (ITS) sequencing of ribosomal RNA genes has provided improved insight into the diversity of fungal bioaerosols in indoor, outdoor and occupational environments. However, ITS analyses may also be confounded by a number of methodological limitations. In this study, we have optimized this technology for use in occupational or environmental studies. Three commonly used DNA extraction methodologies (UltraClean Soil kit, High Pure PCR Template kit, and EluQuik/DNeasy kit) were compared in terms of sensitivity and susceptibility to PCR inhibitors in dust for three common fungal bioaerosols, Aspergillus versicolor, Rhizopus microsporus and Wallemia sebi. Environmental dust samples were then studied using each extraction methodology and results were compared to viable culture data. The extraction methods differed in terms of their ability to efficiently extract DNA from particular species of fungi (e.g. Aspergillus versicolor). In addition, the ability to remove PCR inhibitors from dust samples was most effective using the soil DNA extraction kit. The species composition varied greatly between ITS clone libraries generated with the different DNA extraction kits. However, compared to viable culture data, ITS clone libraries included additional fungal species that are incapable of growth on solid culture medium. Collectively, our data indicated that DNA extraction methodologies used in ITS sequencing studies of occupational or environmental dust samples can greatly influence the fungal species that are detected.     

Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust

Advances in polymerase chain reaction (PCR) have permitted accurate, rapid and quantitative identification of microorganisms in pure cultures regardless of viability or culturability. In this study, a simple sample processing method was investigated for rapid identification and quantification of fungal spores from dust samples using both conventional and real-time PCR. The proposed method was evaluated for susceptibility to interference from environmental dust samples. Stachybotrys chartarum and Aspergillus fumigatus were used as test organisms. The sensitivity of detection in pure culture was 0.1 spore DNA equivalents per PCR reaction corresponding to 20 spores ml(-1) in the sample. However, 1 spore DNA equivalent per PCR reaction corresponding to 200 spores ml(-1) in the sample was the lowest amount of spores tested without interference in dust samples spiked with spores of either fungal species. The extent of inhibition was calculated using conventional and real-time PCR reactions containing fungal spores, specific primers, specific probes (for real-time PCR) and various amounts of dust. The results indicate that the extent of inhibition by dust on PCR varies with the type and amount of dust, and number of spores. No interference in the analysis of spiked samples was detected from 0.2 mg ml(-1) of four real-life dust samples at p-value >0.05 using 2 x 10(4) spores for conventional PCR and 2 x 10(5) spores for real-time PCR. However, samples containing >0.2 mg ml(-1) real-life dust compromised the PCR assay. These results suggest the potential usefulness of a simple sample processing method in conjunction with PCR for monitoring the fungal content of aerosols collected from indoor environments.     

Assessment of multi-contaminant exposure in a cancer treatment center: a 2-year monitoring of molds,mycotoxins, endotoxins,and glucans in bioaerosols

Indoor air quality in health care facilities is a major public health concern, particularly for immunocompromised patients who may be exposed to microbiological contaminants such as molds, mycotoxins, endotoxins, and (1,3)-ß-D-glucans. Over 2 years, bioaerosols were collected on a monthly basis in a cancer treatment center (Centre F. Baclesse, Normandy, France), characterized from areas where there was no any particular air treatment. Results showed the complexity of mycoflora in bioaerosols with more than 100 fungal species identified. A list of major strains in hospital environments could be put forward due to the frequency, the concentration level, and/or the capacity to produce mycotoxins in vitro: Aspergillus fumigatus, Aspergillus melleus, Aspergillus niger, Aspergillus versicolor, Cladosporium herbarum, Purpureocillium lilacinum, and Penicillium brevicompactum. The mean levels of viable airborne fungal particles were less than 30.530 CFU per m³ of air and were correlated to the total number of 0.30 to 20 μm particles. Seasonal variations were observed with fungal particle peaks during the summer and autumn. Statistical analysis showed that airborne fungal particle levels depended on the relative humidity level which could be a useful indicator of fungal contamination. Finally, the exposure to airborne mycotoxins was very low (only 3 positive samples), and no mutagenic activity was found in bioaerosols. Nevertheless, some fungal strains such as Aspergillus versicolor or Penicillium brevicompactum showed toxigenic potential in vitro.     

Fungal flora in indoor and outdoor air of different residential houses in Tekirdag City (Turkey): Seasonal distribution and relationship with climatic factors

This study was investigated the density and monthly distribution of indoor and outdoor microfungi in six different residential houses in Tekirdag City through the exposure of Petri dishes containing Rose-Bengal Streptomycin Agar media. Samples were collected in 1-month intervals over a period of 12 months between March, 2001, and February, 2002. We used 432 Petri dishes and counted a total of 4,205 microfungi colonies, 1,790 from indoor air and 2,415 from outdoor air. As a result, 42 species belonging to 12 genera were identified. The most frequent fungal genera were Penicillium (28.61%), Cladosporium (16.08%) and Alternaria (15.98%). While Penicillium (40.61%) and Cladosporium (15.92%) were the dominant genera of indoor air, Alternaria (20.62%) and Penicillium (19.71%) were isolated most frequently from outdoor air (Table 3). Alternaria citri (10.15%) and Penicillium brevicompactum (10.15%) were found to be the most frequent among the 42 identified species. While P. brevicompactum (19.55%) and Aspergillus niger (6.37%) were the most frequent indoor species, A. citri (13.37%) and Cladosporium cladosporioides (8.20%) were the most frequent outdoor species. Linear Regression Analysis was applied to determine whether or not there was a relationship between the number of colonies of isolated fungal genera and meteorological factors during the research period. Correlations between the presence of Aspergillus and temperature, relative humidity, duration of sunny periods and agents of air pollution such as SO(2) and PM were statistically significant. No significant correlations, however, were found between other fungal genera and environmental variables.     

Volatile metabolites from microorganisms grown on humid building materials and synthetic media

Growth of different microorganisms is often related to dampness in buildings. Both fungi and bacteria produce complicated mixtures of volatile organic compounds that include hydrocarbons, alcohols, ketones, sulfur- and nitrogen-containing compounds etc. Microbially produced substances are one possible explanation of odour problems and negative health effects in buildings affected by microbial growth. A mixture of five fungi, Aspergillus versicolor, Fusarium culmorum, Penicillium chrysogenum, Ulocladium botrytis and Wallemia sebi were grown on three different humid building materials (pinewood, particle board and gypsum board) and on one synthetic medium. Six different sampling methods were used, to be able to collect both non-reactive volatile organic compounds and reactive compounds such as volatile amines, aldehydes and carboxylic acids. Analysis was performed using gas chromatography, high-performance liquid chromatography and ion chromatography, mass spectrometry was used for identification of compounds. The main microbially produced metabolites found on pinewood were ketones (e.g. 2-heptanone) and alcohols (e.g. 2-methyl-1-propanol). Some of these compounds were also found on particle board, gypsum board and the synthetic medium, but there were more differences than similarities between the materials. For example, dimethoxymethane and 1,3,5-trioxepane and some nitrogen containing compounds were found only on particle board. The metabolite production on gypsum board was very low, although some terpenes (e.g. 3-carene) could be identified as fungal metabolites. On all materials, except gypsum board, the emission of aldehydes decreased during microbial growth. No low molecular weight carboxylic acids were identified.     

Microbial secondary metabolites in school buildings inspected for moisture damage in Finland, The Netherlands and Spain

Secondary metabolites produced by fungi and bacteria are among the potential agents that contribute to adverse health effects observed in occupants of buildings affected by moisture damage, dampness and associated microbial growth. However, few attempts have been made to assess the occurrence of these compounds in relation to moisture damage and dampness in buildings. This study conducted in the context of the HITEA project (Health Effects of Indoor Pollutants: Integrating microbial, toxicological and epidemiological approaches) aimed at providing systematic information on the prevalence of microbial secondary metabolites in a large number of school buildings in three European countries, considering both buildings with and without moisture damage and/or dampness observations. In order to address the multitude and diversity of secondary metabolites a large number of more than 180 analytes was targeted in settled dust and surface swab samples using liquid chromatography/mass spectrometry (LC/MS) based methodology. While 42%, 58% and 44% of all samples collected in Spanish, Dutch and Finnish schools, respectively, were positive for at least one of the metabolites analyzed, frequency of detection for the individual microbial secondary metabolites - with the exceptions of emodin, certain enniatins and physcion - was low, typically in the range of and below 10% of positive samples. In total, 30 different fungal and bacterial secondary metabolites were found in the samples. Some differences in the metabolite profiles were observed between countries and between index and reference school buildings. A major finding in this study was that settled dust derived from moisture damaged, damp schools contained larger numbers of microbial secondary metabolites at higher levels compared to respective dust samples from schools not affected by moisture damage and dampness. This observation was true for schools in each of the three countries, but became statistically significant only when combining schools from all countries and thus increasing the sample number in the statistical analyses.     

Indoor air quality during renovation actions: a case study

A temporary renovation activity releases considerably high concentrations of particulate matter, viable and non-viable, into air. These pollutants are a potential contributor to unacceptable indoor air quality (IAQ). Particulate matter and its constituents lead, sulfate, nitrate, chloride, ammonium and fungi as well as fungal spores in air were evaluated in a building during renovation action. Suspended dust was recorded at a mean value of 6.1 mg m(-3) which exceeded the Egyptian limit values for indoor air (0.15 mg m(-3)) and occupational environments (5 mg m(-3)). The highest particle frequency (23%) of aerodynamic diameter (dae) was 1.7 microm. Particulate sulfate (SO(4)(2-)), nitrate (NO(3)(-)), chloride (Cl(-)), ammonium (NH(4)(+)) and lead components of suspended dust averaged 2960, 28, 1350, 100 and 13.3 microg m(-3), respectively. Viable fungi associated with suspended dust and that in air averaged 1.11 x 10(6) colony forming unit per gram (cfu g(-1)) and 92 colony forming unit per plate per hour (cfu p(-1) h(-1)), respectively. Cladosporium(33%), Aspergillus(25.6%), Alternaria(11.2%) and Penicillium(6.6%) were the most frequent fungal genera in air, whereas Aspergillus(56.8%), Penicillium(10.3%) and Eurotium(10.3%) were the most common fungal genera associated with suspended dust. The detection of Aureobasidium, Epicoccum, Exophiala, Paecilomyces, Scopulariopsis, Ulocladium and Trichoderma is an indication of moisture-damaged building materials. Alternaria, Aureobasidium, Cladosporium, Scopulariopsis and Nigrospora have dae > 5 microm whereas Aspergillus, Penicillium and Verticillium have dae < 5 microm which are suited to penetrate deeply into lungs. Particulate matter from the working area infiltrates the occupied zones if precautionary measures are inadequate. This may cause deterioration of IAQ, discomfort and acute health problems. Renovation should be carefully designed and managed, in order to minimize degradation of the indoor and outdoor air quality.     

Airborne fungi in child day care centers in Edirne City, Turkey

The purpose of this study was to determine the concentration, in terms of monthly and seasonal distribution and in relation to meteorological factors, of indoor and outdoor microfungi at selected sites in several child day care centers in the city of Edirne, Turkey. Samples were collected at one month intervals over a period of 12 months between January-December 2004, by exposing petri plates containing Peptone Dextrose Agar with Rose-Bengal and Streptomycin medium to the air for 10-15 min. A total of 2,071 microfungal colonies were counted on 192 petri plates. Thirty microfungal genera (Acremonium, Alternaria, Arthrinium, Aspergillus, Bahusakala, Beauveria, Ceuthospora, Chaetomium, Cladosporium, Curvularia, Drechslera, Epicoccum, Eurotium, Fusarium, Mycotypha, Myrotechium, Paecilomyces, Penicillium, Pestalotiopsis, Phoma, Ramichloridium, Rhizopus, Scopulariopsis, Stachybotrys, Stemphylium, Torula, Trichoderma, Trichothecium, Ulocladium, Verticillium) and 75 microfungal species were isolated from the air indoor and outdoor of the day care centers. The dominant microfungal genera were Cladosporium, Penicillium and Alternaria (44.11%, 18.94%, 14.67% of the total respectively), while the genus with the most species richness was Penicillium (26 species). Alternaria, Cladosporium, Penicillium and non-sporulating microfungi were found every month. Cladosporium was the dominant genus in both indoor and outdoor air. Although the predominant genus was the same in both indoor and outdoor air, Cladosporium was followed by Penicillium, Alternaria and Aspergillus genera in indoor air and by Alternaria, Penicillium and Aspergillus genera in outdoor air. While a positive correlation was found between the concentration of monthly outdoor microfungi and monthly average temperature, a negative correlation was found between the concentration of monthly outdoor microfungi and monthly average wind velocity. Also, some relationships were found between the monthly concentrations of the most predominant microfungal genera (Cladosporium, Penicillium and Alternaria) and various meteorological factors.     

Relationship between airborne fungal allergens and meteorological factors in Manisa City, Turkey

In this study, the effect of relative humidity, temperature, and wind on airborne fungal allergens in the 11 different districts of Manisa City was investigated from January 2004 to December 2005. The aim of this study was to conduct a survey to get to know the relation between wind, temperature, and relative humidity and population of allergenic fungal spores in the atmosphere. A total of 792 samples were observed by using the Merck MAS100 air sampler and 12,988 fungal colonies were counted. Fourteen fungal genera could be determined; Cladosporium that was generally found as the predominant genus followed by Penicillium, Aspergillus, and Alternaria. During the entire study, seasonal variation was found to be related to atmospheric conditions especially. The optimal conditions of meteorological factors for the fungi growth resulted in the increased number of mycoflora, qualitatively and quantitatively.     

Monitoring and assessment of airborne fungi in Kolkata, India, by viable and non-viable air sampling methods

The composition and variability of airborne fungal spores were studied using two complementary sampling methods in an outdoor environment in Kolkata suburb for 2 years, from November 2002 to October 2004. For monitoring the total fungal spore burden in the air, Burkard 7-day volumetric sampler was used, whereas Andersen two-sage viable sampler was used for isolating the cultivable airborne fungi. Among the 37 fungal spore types identified in the air samples, the predominant ones were Cladosporium, unidentified ascospores, unidentified basidiospores, Aspergilli/Penicilli, Nigrospora, Periconia, Chaetomium, Drechslera, Alternaria, Coprinus, Ganoderma, Pithomyces, and rust spores. Only six fungal spore types (Alternaria, Aspergilli/Penicilli, Cladosporium, Curvularia, Drechslera, and Nigrospora) were recovered in common by the two samplers. For Aspergilli/Penicilli, Drechslera, and Nigrospora, the spore concentration was underestimated in the non-viable sampling method (Burkard sampler). In general, higher spore count was recorded in winter. The highest fungal species variability was observed in early monsoon (June). Relative humidity could significantly predict the seasonal periodicity of the maximum number of airborne spores. The total airborne fungi concentration recorded in the study (15-16?×?10(3) spores m(-3) of air) was lower than the proposed threshold limit value for clinical significance, suggesting apparently no or less airborne-fungi-exposure-related health risk in the sampling area. Cladosporium cladosporioides was recorded beyond the proposed threshold limit value in January 2003 and March 2004; Aspergillus fumigatus and Aspergillus nidulans in winter that might have posed considerable health risk to sensitized individuals.     

Viable fungi and bacteria in personal exposure samples in relation to microenvironments

Personal exposures to viable fungi and bacteria were compared with the concentrations being assessed by stationary samplers in home and workplace microenvironments. A random sample of 81 elementary school teachers in eastern Finland performed two 24-hour measurement periods in wintertime. Concentrations and prevalences of viable fungi and bacteria on the collection filters were determined by cultivation method. The geometric mean concentration was 3-12 cfu m(-3) for total viable fungi, 0.6-3.7 cfu m(-3) for Penicillium and mainly under 1 cfu m(-3) for other fungi. The samples with higher fungal concentrations also had higher diversity of fungi than samples with lower concentrations. The total number of fungal genera recovered was 39 for personal, 34 for home and 23 for work samples. The variation in concentration of Penicillium explained even 25-95% of the variations of total fungal concentration in personal exposure, home and workplace environments. There was an association between personal exposure and home concentration of viable fungi and between personal exposure and home and work concentrations of viable bacteria. Personal exposure and home concentrations of fungi were higher in rural areas than in urban areas. Our results also indicate that presence of a certain fungus in a microenvironment does not necessarily mean similar findings in personal exposure samples.     

Air sampling of fungal spores on filters. An investigation on passive sampling and viability

In this study, glycerol was tested as a collection substrate for passive bioaerosol sampling. Filters (mixed cellulose acetate and nitrate) were soaked in glycerol and exposed for an aerosol from three different fungal species: Penicillum commune, Aspergillus versicolor and Paecilomyces variotii. The passive sampling method was compared with a closed-face polycarbonate filter sampling method. Exposure was performed in an exposure chamber. The total number of spores was determined by microscopic techniques, and the cultivable number was determined by cultivation on Malt Extract Agar dishes. The glycerol soaked filter demonstrated a good correlation with the closed-face sampler with regard to the total count. Spores stored in a pumped filter cassette were not affected by storage for up to 7 days. On the other hand, the culturability of the spores was markedly decreased after 1 day when stored on glycerol soaked filters.     

A small change in the design of a slit bioaerosol impactor significantly improves its collection characteristics

While several methods are available for bioaerosol monitoring, impaction remains the most common one, particularly for collecting fungal spores. Earlier studies have shown that the collection efficiency of many conventional single-stage bioaerosol impactors falls below 50% for spores with an aerodynamic diameter between 1.7 and 2.5 microm because their cut-off size is 2.5 microm or greater. The cut-off size reduction is primarily done by substantially increasing the sampling flow rate or decreasing the impaction jet size, W, to a fraction of a millimetre, with both measures often impractical to implement. Some success has recently been reported on the utilization of an ultra-low jet-to-plate distance, S (S/W < 0.1), in circular impactors. This paper describes a laboratory evaluation and some field testing of two single-stage, single-nozzle, slit bioaerosol impactors, Allergenco-D and Air-O-Cell, which feature the same jet dimensions and flow rate but have some design configuration differences that were initially thought to be of low significance. The collection efficiency and the spore deposit characteristics were determined in the laboratory using real-time aerosol spectrometry and different microscopic enumeration methods as the test impactors were challenged with the non-biological polydisperse NaCl aerosol and the aerosolized fungal spores of Cladosporium cladosporioides, Aspergillus versicolor, and Penicillium melinii. The tests showed that a relatively small reduction in the jet-to-plate distance of a single-stage, single-nozzle impactor with a tapered inlet nozzle, combined with adding a straight section of sufficient length, can significantly decrease the cut-off size to the level that is sufficient to efficiently collect spores of all fungal species. Furthermore, it appears that the slit jet design may improve the application of partial spore counting methodologies with respect to those applied to circular deposits. Data from a demonstration field study, conducted with the two samplers in environments containing a variety of fungal species, supported the laboratory findings.     

Real-time PCR for detection of the Aspergillus genus

Aspergillus is a genus of mold that has strong indoor sources, including several species capable of acting as opportunistic pathogens. Previous studies suggest that Aspergillus could serve as an indicator for abnormal mold growth or moisture, making it an important genus for environmental monitoring. Here, a quantitative polymerase chain reaction (qPCR, or real-time PCR) assay is presented for Aspergillus. The assay shows good specificity for the genus, detecting all Aspergillus species tested, although a few non-Aspergillus species are also amplified. Sensitivity testing demonstrates that DNA representing one conidium can be detected. A validation study compared qPCR results against direct microscopy counts using A. fumigatus conidia aerosolized into a laboratory chamber. The assay was then used to quantify Aspergillus in indoor air samples, demonstrating its utility for environmental monitoring. Analysis of a small number of clinical sputum samples showed complete agreement with culturing results.     

Airborne and allergenic fungal spores of the Karachi environment and their correlation with meteorological factors

Airborne fungal spores are well known to cause respiratory allergic diseases particularly bronchial asthma, allergic rhinitis, rhino-conjunctivitis and allergic broncho-pulmonary aspergillosis in both adults and children. In order to monitor and analyze airborne fungal flora of the Karachi environment, an aeromycological study was conducted using a Burkard 7-Day Recording Volumetric Spore Trap from January to December 2010. The data recorded from the Spore Trap was further analyzed for percent catch determination, total spores concentration, seasonal periodicities and diurnal variations. Cladosporium spp (44.8%), Alternaria spp. (15.5%), Periconia spp (6.1%), Curvularia spp (2.1%), Stemphylium spp (1.3%) and Aspergillus/Penicillium type (1%) emerged to be major components constituting more than 70% of the airborne fungal flora. Cladosporium, Curvularia and Stemphylium displayed a clear seasonal trend, while there were no clear seasonal trends for other fungal spore types. Diurnal variations were observed to be mainly having daytime maxima. Spearman Rank Correlation Coefficient analysis was conducted using various weather parameters. The various fungal types showed a negative correlation with heat index, dew point, wind velocity and wind chill. However, a positive correlation was found with humidity, rain and barometric pressure. In fact, Alternaria, Bipolaris and Periconia showed a negative correlation with temperature, while Cladosporium and Periconia showed a negative correlation with heat index, dew point, wind velocity and wind chill. The barometric pressure was positively correlated with Cladosporium. On the basis of these findings, it can be concluded that a number of fungal spores are present in the atmosphere of Karachi throughout the year, with certain atmospheric conditions influencing the release, dispersion, and sedimentation processes of some genera. It is expected that clinicians will use the identified fungal flora for diagnosis and treatment and/or adopt preventative measures for allergic individuals.     

Volatile organic compounds (VOCs) emitted from materials collected from buildings affected by microorganisms

In this study mould damaged materials, including carpet, concrete, gypsum board, insulation, plastic, sand and wood, from 20 different buildings with moisture problems were collected. To study emissions from these materials both conventional methods for sampling, such as collection on Tenax TA, were used as well as complementary methods for sampling a wider spectrum of compounds, such as more volatile VOCs, amines and aldehydes. Analysis was carried out using gas chromatography and high-performance liquid chromatography. Mass spectrometry was used for identification of compounds. Alcohols and ketones were almost exclusively emitted from the materials after they had been wet for a week. Acids were also emitted in large quantities from wet gypsum board and plastic. No primary or secondary amines could be identified, but two tertiary amines, trimethylamine and triethylamine, were emitted from sand contaminated by Bacillus. The most common moulds found were Penicillium and Aspergillus. A multivariate method (partial least squares, PLS) was used to investigate the emission patterns from the materials. Materials with bacterial growth had a different VOC profile to those with only mould growth.