Detlef Schmechel

Fungal Fragments as Indoor Air Biocontaminants

ABSTRACT 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.

Airborne fungal fragments and allergenicity

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 in combination with a fluorescent-confocal HIA technique should, for the first time, enable the speciation of morphologically indiscernible fungal fragments. The application of this novel method will help to characterize the contribution of fungal fragments to adverse health effects due to fungi and provide patient-specific exposure and sensitization profiles.

Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI-TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI-TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI-TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI-TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.

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.

Prevalence of allergic sensitization to indoor fungi in West Virginia

Exposure to indoor fungi is of growing concern in residential and occupational environments in the United States. The purpose of this study was to determine the prevalence of sensitization to common indoor fungal species in an atopic population. We evaluated 102 patients (73 female and 29 male patients) for immunoglobulin E (IgE) reactivity to a panel of skin-prick test (SPT) reagents used for routine allergy testing. Patients also were tested for six additional fungi that are common indoor contaminants. All patients had symptoms consistent with allergic rhinitis or asthma. The presence of specific IgE against the fungal species was determined using immunoblotting. Of the 102 eligible patients, 68% had at least one positive skin test. The most prevalent positive SPTs were to dust mites, cats, vernal grass, and short ragweed. Overall, 21/102 (21%) patients with asthma or allergic rhinitis were skin test positive to at least one fungal extract. Of the patients with a positive SPT to fungi, 12/21 (58%) showed sensitivity to one or more of the newly tested species; most notably Trichoderma viride (8%), Chaetomium globosum (7%), Paecilomyces variotii (7%), and Acremonium strictum (6%). Immunoblotting revealed specific IgE against a number of protein bands belonging to these fungal species. The prevalence of fungal sensitization was common, particularly for indoor fungal contaminants that are not routinely included in SPT panels. Cross-reactivity with other fungi may partially explain our results; however, skin testing for these indoor fungi may provide useful diagnostic information.

A Multi-Center Ring Trial of Allergen Analysis using Fluorescent Multiplex Array Technology

BACKGROUND Consistent performance of allergen assays is essential to ensure reproducibility of exposure assessments for investigations of asthma and occupational allergic disease. This study evaluated intra- and inter-laboratory reproducibility of a fluorescent multiplex array, which simultaneously measures eight indoor allergens in a single reaction well. METHODS A multi-center study was performed in nine laboratories in the US and Europe to determine the inter-laboratory variability of an 8-plex array for dust mite, cat, dog, rat, mouse and cockroach allergens. Aliquots of 151 dust extract samples were sent to participating centers and analyzed by each laboratory on three separate occasions. Agreement within and between laboratories was calculated by the concordance correlation coefficient (CCC). RESULTS Results were obtained for over 32,000 individual allergen measurements. Levels covered a wide range for all allergens from below the lower limit of detection (LLOD = 0.1-9.8 ng/ml) to higher than 6800 ng/ml for all allergens except Mus m 1, which was up to 1700 ng/ml. Results were reproducible within as well as between laboratories. Within laboratories, 94% of CCC were ≥ 0.90, and 80% of intra-laboratory results fell within a 10% coefficient of variance (CV%). Results between laboratories also showed highly significant positive correlations for all allergens (~0.95, p<0.001). Overall means of results were comparable, and inter-laboratory CV% for all allergens except Rat n 1 ranged between 17.6% and 26.6%. CONCLUSION The data indicate that performance criteria for fluorescent multiplex array technology are reproducible within and between laboratories. Multiplex technology provides standardized and consistent allergen measurements that will streamline environmental exposure assessments in allergic disease.

Detection of Aerosolized Alternaria alternata Conidia, Hyphae, and Fragments by Using a Novel Double-Immunostaining Technique

ABSTRACT A double-immunostaining halogen immunoassay was developed to identify aerosolized conidia, hyphae, and fragments of Alternaria alternata by using an anti-Alternaria polyclonal antiserum, while, simultaneously, allergy to these components was concurrently determined by using human immunoglobulin E antibodies.

Aerosolized fungal fragments

Airborne fungal conidia derived from environmentally abundant and morphologically discernible fungal genera, including Alternaria Nees, Aspergillus P. Micheli ex Link, Cladosporium Link and Penicillium Link have been traditionally acknowledged as the etiological agents responsible for personal fungal exposure. The contribution of these fungal conidia to indoor, outdoor and occupational environments is well documented. Exposure assessment studies have shown that airborne particulate concentrations vary widely and are strongly influenced by climatic conditions, geographical location and the type of disturbance (Luoma and Batterman 2001, Mitakakis et al. 2001b, Ferro et al. 2004, Tovey and Green 2004, Chen and Hildemann 2009). Epidemiological studies have identified associations between personal exposure to fungal conidia and exacerbations of respiratory disease in persons suffering from seasonal rhinitis (Li and Kendrick 1995a) and asthma (Fung et al. 2000, Downs et al. 2001, Zureik et al. 2002). Even mortality (Licorish et al. 1985, Targonski et al. 1995) has been attributed to conidial exposure in subjects previously sensitized to fungi. As a result of these studies and the conclusions of a recent report on damp indoor spaces and health by the Institute of Medicine (IOM 2004), the indoor biodeterioration and health effects associated with fungal contamination in indoor environments has become a research priority.

A New Field-Compatible Methodology for the Collection and Analysis of Fungal Fragments

A field-compatible collection system was developed and tested for the collection and analysis of fungal fragments. The new collection system consists of two types of Sharp-Cut cyclone samplers (PM 2.5 and PM 1.0 ) and an after-filter. Fungal particles are collected into three size fractions: (1) spores ( > 2.5 μ m); (2) a fragment-spore mixture (1.0–2.5 μ m); and (3) submicrometer-sized fragments ( < 1.0 μ m). The system was laboratory-tested using polystyrene latex (PSL) particles and particulate matter aerosolized from sporulating Aspergillus versicolor and Stachybotrys chartarum cultures. In addition to the particle count measured with direct-reading instruments, the (1 → 3)- β -D-glucan content in each size fraction was determined with the Limulus Amebocyte Lysate (LAL) assay. Experiments conducted with PSL particles showed that the 50% cut-off values of the two cyclone samplers under the test conditions were 2.25 μ m and 1.05 μ m, respectively. No particle bounce onto the after-filter was observed when the total particle number entering the collection system was kept below 1.6 × 10 8 . The (1 → 3)- β -D-glucan assay of samples aerosolized from both fungal species suggested that surface area is an important factor for determining the (1 → 3)- β -D-glucan content in the entire size-range of particles. In conclusion, the new methodology is a promising tool for separating and analyzing fungal fragment samples.

Murine models of airway fungal exposure and allergic sensitization

Inhalation of common indoor filamentous fungi has been associated with the induction or exacerbation of allergic respiratory disease. The understanding of fungal inhalation and allergic sensitization has significantly advanced with the use of small animal models, especially mouse models. Numerous studies have employed different animal exposure and sensitization techniques, each with inherent advantages and disadvantages that are addressed in this review. In addition, most studies involve exposure of animals to fungal spores or spore extracts while neglecting the influence of hyphal or subcellular fragment exposures. Recent literature examining the potential for hyphae and fungal fragments to induce or exacerbate allergy is discussed. Innate immune recognition of fungal elements and their contribution to lung allergic inflammation in animal models are also reviewed. Though physical properties of fungi play an important role following exposure, host immune development is also critical in airway inflammation and allergy. We discuss the importance of environmental factors that influence early immune development and subsequent susceptibility to allergy. Murine studies that examine the role of intestinal microflora and prenatal or early life environmental factors that promote allergic sensitization are also evaluated. Future studies will require animal models that accurately reflect natural fungal exposures and identify environmental factors that influence immune development and thus promote respiratory fungal allergy and disease.