Quantitative PCR analysis of molds in the dust from homes of asthmatic children in North Carolina

The vacuum bag (VB) dust from the homes of 19 asthmatic children in North Carolina (NC) was analyzed by mold specific quantitative PCR. These results were compared to the analysis of the VB dust from 176 homes in the HUD, American Healthy Home Survey of homes in the US. The Environmental Relative Moldiness Index (ERMI) was calculated for each of the homes. The mean and standard deviation (SD) of the ERMI values in the homes of the NC asthmatic children was 16.4 (6.77), compared to the HUD survey VB ERMI value mean and SD of 11.2 (6.72), and was significantly greater (t-test, p = 0.003) in the NC asthmatic children's homes. The molds Chaetomium globosum, Aspergillus fumigatus, and the Eurotium Group were the primary species in the NC homes of asthmatics, making the ERMI values significantly higher (p < 0.02 for each). Vacuum bag dust analysis may be a useful method for estimating the mold burden in a home.     

Utilizing pyrosequencing and quantitative PCR to characterize fungal populations among house dust samples

Molecular techniques are an alternative to culturing and counting methods in quantifying indoor fungal contamination. Pyrosequencing offers the possibility of identifying unexpected indoor fungi. In this study, 50 house dust samples were collected from homes in the Yakima Valley, WA. Each sample was analyzed by quantitative PCR (QPCR) for 36 common fungi and by fungal tag-encoded flexible (FLX) amplicon pyrosequencing (fTEFAP) for these and additional fungi. Only 24 of the samples yielded amplified results using fTEFAP but QPCR successfully amplified all 50 samples. Over 450 fungal species were detected by fTEFAP but most were rare. Twenty-two fungi were found by fTEFAP to occur with at least an average of ≥0.5% relative occurrence. Many of these fungi seem to be associated with plants, soil or human skin. Combining fTEFAP and QPCR can enhance studies of fungal contamination in homes.     

Passive airborne dust sampling to assess mite antigen exposure in farming environments

The aim of this study was to estimate mite antigen exposure in farming environments by passive sampling of airborne dust. Antigen concentrations were measured with enzyme immunoassays specific for three storage mites (SM): Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae and the house dust mite (HDM) Dermatophagoides pteronyssinus. Dust samples were collected with electrostatic dust fall collectors (EDCs) in three different areas of cattle farms. EDCs were placed in cow stables (working area), in changing rooms (transit area) and in different rooms of farmer dwellings (living area). Mite concentrations in the living area of farm homes were compared to those of urban homes. In dust samples from stables, antigens of all four mite species could be detected. The highest exposure level was to L. destructor (median 56.7 μg/m(2)), the lowest to A. siro (median 14.4 μg/m(2)). Mite concentrations of different species showed no correlation within the cow stables. In comparison to stables, the median mite concentrations in farm homes were significantly lower, ranging from below the detection limit to 1.5 μg/m(2). Antigens of SM were predominantly found in changing rooms and kitchens, and HDM antigens were mainly detected in bedrooms. Antigens of all mites were measured the least often in living rooms. T. putrescentiae was the most prevalent mite in all room types, and the exposure levels correlated strongly between different rooms. The number of SM positive samples in farm homes was considerably higher than in urban homes, while the percentage of HDM positive samples did not differ significantly.     

Organic dust and gaseous contaminants at wood working shops

Airborne dust bioaerosols, ammonia and formaldehyde levels were determined inside two different (ventilated and unventilated) wood working shops. Airborne dust was found at mean values of 4.3 and 3.01 mg m(-3). These levels were higher than that recommended by Egyptian environmental law [1 mg m(-3) indoor maximum allowable concentration (MAC) for hard wood]. The highest frequency of aerodynamic size distribution of airborne wood dust was detected at a diametre of 4.9 microm which was recorded during a machining operation. Total viable bacteria were recorded at a mean value of 10(4) colony-forming units (cfu) m(-3), whereas Gram-negative bacteria were found at very low counts (10(1) cfu m(-3)). Fungi levels were recorded at mean values of 10(3) and 10(2) cfu m(-3) in ventilated and unventilated shops, respectively. Penicillium, Aspergillus, Cladosporium and yeast species were dominant isolates. Moreover, actinomycetes were found at a mean value of 10(3) cfu m(-3) at both workshops. Ammonia was detected in relatively low concentrations (mean values of 457 and 623 microg m(-3)), whereas formaldehyde was found in relatively moderate concentrations (mean values of 0.42 and 0.64 ppm).     

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.     

Seasonal variability of endotoxin in ambient fine particulate matter

Endotoxin is a toxic, pro-inflammatory compound that has been detected in indoor air and dust in homes and occupational settings, and also in outdoor air. Data on the outdoor sampling of endotoxin are limited. Currently, little is known about the seasonal variation and influence of temperature on outdoor endotoxin levels. In the present study, we report endotoxin levels in fine fraction particulate matter with a 50% aerodynamic cutoff diameter of 2.5 microm (PM2.5) and describe the seasonal variation of endotoxin in Munich, Germany. In 1999-2000, PM2.5 was collected at forty outdoor monitoring sites across Munich. Approximately four samples were collected at each site for a total of 158 samples. Endotoxin concentrations in the PM2.5 samples were determined using the kinetic chromogenic Limulus Amebocyte Lysate (LAL) assay. The geometric mean endotoxin concentration was 1.07 EU mg PM2.5(-1) (95% C.I.: 0.915-1.251) or 0.015 EU m(-3) of sampled air (95% C.I.: 0.013-0.018). Munich endotoxin levels were significantly related to ambient temperature (p < 0.0001) and percent relative humidity (p < 0.0001). Sampling periods with higher average temperatures yielded higher levels of endotoxin in PM2.5 (r = 0.641), whereas decreases in percent relative humidity were associated with increased endotoxin levels in PM2.5 (r = -0.388). Endotoxin levels were significantly higher during the warmer seasons of spring [means ratio (MR): 2.5-2.7] and summer (MR: 2.1-3.0) than during winter. Although temperature and relative humidity do not explain all of the variability in endotoxin levels, their effects were significant in our data set. Temperature effects and seasonal variation of endotoxin should be considered in future studies of outdoor endotoxin.     

Nitrous acid concentrations in homes and offices in residential areas in Greater Cairo

Indoor and outdoor measurements of nitrous acid and nitrogen dioxide were conducted at four homes and two offices in residential areas in Greater Cairo during winter (2000-2001) and summer (2001) seasons. Indoor nitrogen dioxide concentrations were higher than outdoor levels at the four homes, whereas indoor concentrations of nitrogen dioxide were lower than outdoor levels at the two offices, during both seasons. Indoor nitrous acid concentrations were higher than outdoor levels at all homes and offices during the period of study. The mean indoor nitrous acid concentrations were 6.8 ppb and 3.67 ppb in the four homes, whereas they were 1.42 ppb and 1.24 ppb in the two offices, during the winter and summer seasons, respectively. Indoor/outdoor ratios of nitrous acid concentration were 6.94 in the winter and 5.03 in the summer for all of the homes. However, the ratios were 1.31 and 1.61 during the winter and summer seasons, respectively, for the two offices. Insignificant positive correlation coefficients were found between indoor and outdoor concentrations of nitrous acid at homes and offices. The maximum outdoor nitrous acid concentrations were recorded during the winter season. Significant positive correlation coefficients were found between nitrous acid and nitrogen dioxide and relative humidity in homes and offices. The ratios of nitrous acid to nitrogen dioxide concentrations ranged from 0.045 to 0.16, with a mean of 0.1, in the four homes, whereas the ratios ranged from 0.026 to 0.09, with a mean of 0.059, in the two offices.     

Quantitative measurement of streptomycetes using real-time PCR

A quantitative real-time PCR method was developed and used for determination of streptomycetes in indoor dust samples of five homes collected during three years. The specificity of the method was tested with 14 Streptomyces and ten non-streptomycetous species, revealing a high specificity for mesophilic streptomycetes. Thermophilic species and S. albus were not efficiently detected. The method gave reproducible results in replicate analyses of the same dust DNA as well as of duplicate DNA isolations. The amount of streptomycetes in house dust was lowest in winter, followed by summer, and highest in spring and fall. The greatest variation in Streptomyces-concentrations was observed in the spring and fall samples.     

Concentrations of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in vacuum cleaner dust collected in Japanese homes

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are shown to be globally distributed, environmentally persistent and bioaccumulative. Although there is evidence that these compounds exist in the serum of non-occupationally exposed humans, the pathways leading to the presence of PFOS and PFOA are not well characterized. The concentrations of PFOS and PFOA in the vacuum cleaner dust collected in Japanese homes were measured. The compounds were detected in all the dust samples and the ranges were 11-2500 ng g(-1) for PFOS and 69-3700 ng g(-1) for PFOA. It was ascertained that PFOS and PFOA were present in the dust in homes, and that the absorption of the dust could be one of the exposure pathways of the PFOS and PFOA to humans. With regard to risk management, it is important to consider the usage of PFOS and PFOA in the indoor environment in order to avoid further pollution.     

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.     

Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status

Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study, we used a culture-independent method, PCR-denaturing gradient gel electrophoresis (PCR-DGGE), to investigate the composition of the Streptomyces community in house dust. Twenty-three dust samples each from two sets of homes categorized as high-mold and low-mold based on mold-specific quantitative PCR analysis were used in the study. Taxonomic identification of prominent bands was performed by cloning and sequencing. Associations between DGGE amplicon band intensities and home mold status were assessed using univariate analyses as well as multivariate recursive partitioning (decision trees) to test the predictive value of combinations of bands intensities. In the final classification tree, a combination of two bands was significantly associated with mold status of the home (p?=?0.001). The sequence corresponding to one of the bands in the final decision tree matched a group of Streptomyces species that included Streptomyces coelicolor and Streptomyces sampsonii, both of which have been isolated from moisture-damaged buildings previously. The closest match for the majority of sequences corresponding to a second band consisted of a group of Streptomyces species that included Streptomyces hygroscopicus, an important producer of antibiotics and immunosuppressors. Taken together, the study showed that DGGE can be a useful tool for identifying bacterial species that may be more prevalent in mold-damaged buildings.     

Exposure to inhalable dust and endotoxin among Danish livestock farmers: results from the SUS cohort study

Studies on personal dust and endotoxin concentrations among animal farmers have been either small or limited to a few sectors in their investigations. The present study aimed to provide comparable information on the levels and variability of exposure to personal dust and endotoxin in different types of animal farmers. 507 personal inhalable dust samples were collected from 327 farmers employed in 54 pig, 26 dairy, 3 poultry, and 3 mink farms in Denmark. Measurements in pig and dairy farmers were full-shift and performed during summer and winter, while poultry and mink farmers were monitored during 4 well-defined production stages. The collected samples were measured for dust gravimetrically and analyzed for endotoxin by the Limulus amebocyte lysate assay. Simple statistics and random-effect analysis were used to describe the levels and the variability in measured dust and endotoxin exposure concentrations. Measured inhalable dust levels had an overall geometric mean of 2.5 mg m(-3) (range 相似文献   

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.     

Pentachlorophenol in indoor environments. Does a single measurement of air and dust concentrations represent the contamination?

In order to be able to make a decision, as to whether a room or building has a health-endangering pentachlorophenol (PCP) concentration, usually the PCP concentrations in air and settled dust are measured. The variability of the PCP concentration in indoor air and dust was studied. Air and dust samples were taken from 75 rooms in 30 buildings with suspicion of application of PCP-containing wood preservatives. Sampling was repeated four times within 18 months. Thirty-six rooms were reconstructed within the study; 39 rooms had unchanged contamination status during the study. The four times repeated measurements of PCP concentrations in air and dust in these rooms showed large variations of the measured values. The variability of the results is to a large extent in the same range as the measured values. The observed relative standard deviation of the PCP concentrations in air and dust does not depend on the average PCP concentration detected in the individual rooms.     

Simultaneous detection of enteric bacteria from surface waters by QPCR in comparison with conventional bacterial indicators

A rapid quantitative polymerase chain reaction (QPCR) method was developed for simultaneous detection of enteric bacteria from surface waters by utilizing a pair of universal primers which targeted four bacteria strains, namely Shigella dysenteriae, Vibrio cholerae, Salmonella typhimurium, and Escherichia coli. It was estimated that the QPCR method had a 94% confidence, and a detection limit as 2.7 E. coli cells per sample in undiluted DNA extracts. The QPCR method was applied for the bacteriological examination of several surface waters in the urban area of Xi'an, China and comparison was made with the conventional bacteria indicators determined by conventional membrane filter (MF) method. As a result, the calibrator cell equivalents (CCE) determined by QPCR was 2.2 to five times of the total coliform CFU, and the characteristics of the bacterial quality of different waters could be well presented by the QPCR results with a higher sensitivity. The coefficient of variation (CV) of data obtained by QPCR was smaller than that by traditional MF method, indicating a more stable analysis result. The QPCR method could thus be used as a supplement of the conventional culture method for more sensitive detection of pathogenic enteric bacteria from water.     

Influence of home characteristics on airborne and dustborne endotoxin and β-D-glucan

The aim of this study was to assess the associations between airborne and dustborne microbial contaminants (endotoxin and β-D-glucan) and estimate the effects of home characteristics on exposure levels of these microbial contaminants. Endotoxin and β-D-glucan concentrations in airborne inhalable particles, airborne PM1 (<1 μm) and vacuumed dust from 184 residential homes were determined using specific Limulus amebocyte assays. Home characteristics were recorded by visual inspection and questionnaires. Linear regression and correlation analyses were performed. Inhalable endotoxin correlated with dust endotoxin (r = 0.34, p < 0.001) and PM1 endotoxin (r = 0.33, p < 0.001). Inhalable β-D-glucan correlated with dust β-D-glucan (r = 0.18, p < 0.01), but not with PM1 β-D-glucan. Significant correlation was also found between PM1 and dust concentrations for endotoxin (r = 0.26, p < 0.001), but not for β-D-glucan. Multivariate regression analyses showed only one significant association between airborne contaminants and environmental characteristics: inhalable β-D-glucan was positively associated with relative humidity with an effect size (change in the dependent variable corresponding to a unit increase in the independent variable) of 2.32 and p < 0.05. In contrast, several associations were found between dust concentrations and environmental characteristics. Dust endotoxin was positively associated with temperature (2.87, p < 0.01) and number of inhabitants (2.76, p < 0.01), whereas dust β-D-glucan was inversely associated with the presence of dogs (-2.24, p < 0.05) and carpet (-3.05, p < 0.01) in the home. In conclusion, dustborne contaminants were more strongly affected by home characteristics than airborne contaminants. Furthermore, even though statistically significant, the correlations between airborne and dustborne contaminants were weak. This indicates that airborne concentrations cannot be reliably predicted based on dustborne concentrations.     

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.     

Fungi Associated with Degradation of Wastes from Rubber Processing Industry

A study of fungi associated with wastes from a rubber processing factory was carried out. Rubber processing wastes, natural rubber waste serum (NRWS) and washing effluents were obtained from Greenpark Rubber Industries Ltd, Umutu, Delta State, Nigeria. The NRWS was collected from seepage from starks of coagulated rubber in the factory and effluents were collected from the four tanks used for washing the coagulated rubber. Three fungal species, Mucor racemous, Mucor sp. and Aspergilus niger, were isolated from the NRWS. From the effluents collected from the four wash tanks, five fungal species were isolated. Mucor recemous, Mucor sp., Aspergillus niger, Aspergillus, sp. and Rhizopus sp. Of all the species isolated, the Mucor species were found capable of utilizing NRWS as a growth substrate. Optimal growth of Mucor in NRWS was achieved at near-neutral pH of 7.1. It was also observed that as biomass of Mucor increased in NRWS, the BOD of NRWS decreased. Pollution strength of the wastes as determined by biochemical oxygen demand (BOD) and total solids were found to be highest with NRWS (440 and 4520) and wash tank (A) (370 and 3520) respectively.     

Heavy Metal Concentrations in and Around Households Near a Secondary Lead Smelter

Housedusts and garden soils were sampled in 14 houses in the vicinity of a secondary Pb smelter and analysed for concentrations of Pb, Zn, Cu, Cd, As, and Hg. Sixty-one topsoil samples were also taken from a 2 km² grid covering the smelter grounds and surrounding residential areas and analysed for concentrations of Pb, Zn, Cd and Cu. Contour maps generated from the grid data indicate significant contamination in the area (maximum Pb concentration 58 500 g g^-1), particularly down-wind of the smelter grounds. A geometric mean Pb concentration of 2225 g g^-1 was recorded in garden soil and similarly elevated levels were recorded for Zn, Cd, As and Sb. In housedusts, a geometric mean Pb concentration of 1668 g g^-1 was observed. Whilst housedust metal concentrations were generally elevated, compared to other urban or residential areas, there appears to be a large degree of attenuation of the metals between the exterior and interior environments of the homes studied. A significant correlation was not recorded between metal concentrations of garden soils and housedusts. There were significant correlations for: distance from the smelter against garden soil metal concentrations; garden soil metal concentrations against each other; housedust metal concentrations against each other; and house age against garden soil metal concentrations.