The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.Water damage in buildings is common and is often associated with mold problems. In North America, cross-sectional questionnaire studies have found that 27 to 36% of homes have mold problems (9, 51). Studies that included indoor air quality measurements have shown even higher numbers, from 42 to 56% (8, 11). In Europe, the prevalence of damp and moldy homes has been reported to be 17 to 46% for Great Britain (5,23,34,42), 15 to 18% for The Netherlands (3, 55,56), and 15% for Finland (41). Alarmingly, signs of present or previous moisture-related defects were found in 80% of randomly selected private homes investigated by civil engineers trained to recognize the signs of water leaks or condensation (37).Increased prevalence of water-damaged buildings and subsequent fungal contamination may contribute to the noted increase in allergic diseases. Fungi can affect human health in a variety of ways. Possible reactions generally fall into one of three groups: allergic reactions (sensitization and immune responses, i.e., asthma, allergic rhinitis, or hypersensitivity pneumonitis), infections (growth of the fungus in or on the body, e.g., aspergillosis), and toxic responses (24,30,45). The toxic reactions are mainly connected with the secondary fungal metabolites, i.e., mycotoxins, but the role of cell wall components, such as ␤-(133)-D-glucans, has also been reported (4,26,29,45,47,50). In addition, expos...

Exposure to fungi, particularly in water damaged indoor environments, has been thought to exacerbate a number of adverse health effects, ranging from subjective symptoms such as fatigue, cognitive difficulties or memory loss to more definable diseases such as allergy, asthma and hypersensitivity pneumonitis. Understanding the role of fungal exposure in these environments has been limited by methodological difficulties in enumerating and identifying various fungal components in environmental samples. Consequently, data on personal exposure and sensitization to fungal allergens are mainly based on the assessment of a few select and easily identifiable species. The contribution of other airborne spores, hyphae and fungal fragments to exposure and allergic sensitization are poorly characterized. There is increased interest in the role of aerosolized fungal fragments following reports that the combination of hyphal fragments and spore counts improved the association with asthma severity. These fragments are particles derived from any intracellular or extracellular fungal structure and are categorized as either submicron particles or larger fungal fragments. In vitro studies have shown that submicron particles of several fungal species are aerosolized in much higher concentrations (300-500 times) than spores, and that respiratory deposition models suggest that such fragments of Stachybotrys chartarum may be deposited in 230-250 fold higher numbers than spores. The practical implications of these models are yet to be clarified for human exposure assessments and clinical disease. We have developed innovative immunodetection techniques to determine the extent to which larger fungal fragments, including hyphae and fractured conidia, function as aeroallergen sources. These techniques were based on the Halogen Immunoassay (HIA), an immunostaining technique that detects antigens associated with individual airborne particles >1 microm, with human serum immunoglobulin E (IgE). Our studies demonstrated that the numbers of total airborne hyphae were often significantly higher in concentration than conidia of individual allergenic genera. Approximately 25% of all hyphal fragments expressed detectable allergen and the resultant localization of IgE immunostaining was heterogeneous among the hyphae. Furthermore, conidia of ten genera that were previously uncharacterized could be identified as sources of allergens. These findings highlight the contribution of larger fungal fragments as aeroallergen sources and present a new paradigm of fungal exposure. Direct evidence of the associations between fungal fragments and building-related disease is lacking and in order to gain a better understanding, it will be necessary to develop diagnostic reagents and detection methods, particularly for submicron particles. Assays using monoclonal antibodies enable the measurement of individual antigens but interpretation can be confounded by cross-reactivity between fungal species. The recent development of species-specific monoclonal antibodies, used...

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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.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral 'fingerprints' for twelve Penicillium species. Prior to MALDI-TOF MS analysis, eight replicate cultures of each Penicillium species were subjected to three one-minute bead-beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contained abundant peaks in the range of m/z 5000-20 000, and allowed unambiguous discrimination between species. In addition, a biomarker common to all Penicillium mass spectra was observed at m/z 13 900. Discriminant analysis using the MALDI-TOF MS data yielded classification error rates of 0% (i.e. 100% correct identification), indicating that MALDI-TOF MS data may be a useful diagnostic tool for the objective identification of Penicillium species of environmental and clinical importance.

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