The Importance of Binomial Nomenclature for the Identification of Pollen Aeroallergens.

The diagnosis and treatment of atopic disorders associated with specific aerobiological triggers require basic botanical training. However, the identification of specific pollen can often be confounded by broad naming conventions that range from categorized colloquial to scientific names based on either higher taxonomic levels or, in some cases, binomial nomenclature. Physicians specializing in allergy often lack a comprehensive understanding with respect to plant taxonomy and botanical nomenclature that are critical skills required for clinical practice and research programs evaluating pollen and airborne fungal spores. In addition, binomial and current family designation and synonyms, including author citation are often misused, causing a misinterpretation of existing plants species or pollen types. It is critical that the correct botanical name is linked to a validated specimen and scientific naming conventions are used where possible by the clinician and researcher. In relation to pollen identification, we propose that clinicians and researchers should provide the currently accepted binomial nomenclature, offer relevant synonyms, and use the Angiosperm Phylogeny Group names.

Severe lung disease characterized by lymphocytic bronchiolitis, alveolar ductitis, and emphysema (BADE) in industrial machine-manufacturing workers.

BACKGROUND: A cluster of severe lung disease occurred at a manufacturing facility making industrial machines. We aimed to describe disease features and workplace exposures. METHODS: Clinical, functional, radiologic, and histopathologic features were characterized. Airborne concentrations of thoracic aerosol, metalworking fluid, endotoxin, metals, and volatile organic compounds were measured. Facility airflow was assessed using tracer gas. Process fluids were examined using culture, polymerase chain reaction, and 16S ribosomal RNA sequencing. RESULTS: Five previously healthy male never-smokers, ages 27 to 50, developed chest symptoms from 1995 to 2012 while working in the facility's production areas. Patients had an insidious onset of cough, wheeze, and exertional dyspnea; airflow obstruction (mean FEV1 = 44% predicted) and reduced diffusing capacity (mean = 53% predicted); and radiologic centrilobular emphysema. Lung tissue demonstrated a unique pattern of bronchiolitis and alveolar ductitis with B-cell follicles lacking germinal centers, and significant emphysema for never-smokers. All had chronic dyspnea, three had a progressive functional decline, and one underwent lung transplantation. Patients reported no unusual nonoccupational exposures. No cases were identified among nonproduction workers or in the community. Endotoxin concentrations were elevated in two air samples; otherwise, exposures were below occupational limits. Air flowed from areas where machining occurred to other production areas. Metalworking fluid primarily grew Pseudomonas pseudoalcaligenes and lacked mycobacterial DNA, but 16S analysis revealed more complex bacterial communities. CONCLUSION: This cluster indicates a previously unrecognized occupational lung disease of yet uncertain etiology that should be considered in manufacturing workers (particularly never-smokers) with airflow obstruction and centrilobular emphysema. Investigation of additional cases in other settings could clarify the cause and guide prevention.

Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy.

Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-µm fragments, compared to 100% of >2-µm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.

Profile and Morphology of Fungal Aerosols Characterized by Field Emission Scanning Electron Microscopy (FESEM).

Fungal aerosols consist of spores and fragments with diverse array of morphologies; however, the size, shape, and origin of the constituents require further characterization. In this study, we characterize the profile of aerosols generated from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum grown for 8 weeks on gypsum boards. Fungal particles were aerosolized at 12 and 20 L min-1 using the Fungal Spore Source Strength Tester (FSSST) and the Stami particle generator (SPG). Collected particles were analyzed with field emission scanning electron microscopy (FESEM). We observed spore particle fraction consisting of single spores and spore aggregates in four size categories, and a fragment fraction that contained submicronic fragments and three size categories of larger fragments. Single spores dominated the aerosols from A. fumigatus (median: 53%), while the submicronic fragment fraction was the highest in the aerosols collected from A. versicolor (median: 34%) and P. chrysogenum (median: 31%). Morphological characteristics showed near spherical particles that were only single spores, oblong particles that comprise some spore aggregates and fragments (<3.5 µm), and fiber-like particles that regroup chained spore aggregates and fragments (>3.5 µm). Further, the near spherical particles dominated the aerosols from A. fumigatus (median: 53%), while oblong particles were dominant in the aerosols from A. versicolor (68%) and P. chrysogenum (55%). Fiber-like particles represented 21% and 24% of the aerosols from A. versicolor and P. chrysogenum, respectively. This study shows that fungal particles of various size, shape, and origin are aerosolized, and supports the need to include a broader range of particle types in fungal exposure assessment.

Submicronic fungal bioaerosols: high-resolution microscopic characterization and quantification.

Submicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in mold-contaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min(-1) airflow. The overall median numbers of aerosolized submicronic particles were 2 × 10(5) cm(-2), 2.6 × 10(3) cm(-2), and 0.9 × 10(3) cm(-2) for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.

Bioaerosol exposure assessment in the workplace: the past, present and recent advances.

Louis Pasteur described the first measurements of airborne microorganisms in 1861. A century later, the inhalation of spores from thermophilic microorganisms was shown to induce attacks of farmers' lung in patients with this disease, while endotoxins originating from Gram-negative bacteria were identified as causal agents for byssinosis in cotton workers. Further epidemiological and toxicological studies have demonstrated inflammatory, respiratory, and pathogenic effects following exposure to bioaerosols. Exposure assessment is often confounded by the diversity of bioaerosol agents in the environment. Microorganisms represent a highly diverse group that may vary in toxicity. Fungi and bacteria are mainly quantified as broad groups using a variety of viable and nonviable assessment methods. Endotoxins and ß(1 → 3)-glucans are mainly measured by their activity in the Limulus amebocyte lysate assay, enzymes by immuno-chemical methods and mycotoxins by liquid chromatography-mass spectrometry. Few health-based occupational exposure limits (OELs) are available for risk assessment. For endotoxins, a health-based OEL of 90 endotoxin units m(-3) has been proposed in the Netherlands. A criteria document for fungal spores recently proposed a lowest observed effect level of 100,000 spores m(-3) for non-pathogenic and non-mycotoxin producing species based on inflammatory respiratory effects. Recent developments in bioaerosol assessment were presented at the Organic Dust Tromsø Symposium including molecular biological methods for infectious agents and organisms that are difficult to cultivate; studies of submicronic and hyphal fragments from fungi; the effect of biodiversity of microorganisms in asthma studies; and new/improved measurement methods for fungal antigens, enzymes and allergens. Although exposure assessment of bioaerosol agents is complex and limited by the availability of methods and criteria, the field is rapidly evolving.

Analysis of airborne betula pollen in Finland; a 31-year perspective.

In this 31-year retrospective study, we examined the influence of meteorology on airborne Betula spp. (birch) pollen concentrations in Turku, Finland. The seasonal incidence of airborne birch pollen in Turku occurred over a brief period each year during spring (April 30 - May 31). Mean peak concentrations were restricted to May (May 5 to 13). Statistically significant increases in the annual accumulated birch pollen sum and daily maximum values were observed over the study period. Birch pollen counts collected in April were retrospectively shown to increase over the duration of the study. Increases in April temperature values were also significantly associated with the earlier onset of the birch pollen season. Furthermore, the number of days where daily birch pollen concentrations exceeded 10 and 1,000 grains/m(3) also increased throughout the study period. These data demonstrate that increases in temperature, especially during months preceding the onset of the birch pollen season, favor preseason phenological development and pollen dispersal. Birch pollen derived from other geographical locations may also contribute to the aerospora of Turku, Finland. To date, the public health burden associated with personal exposure to elevated birch pollen loads remains unclear and is the focus of future epidemiological research.

Halogen immunoassay, a new method for the detection of sensitization to fungal allergens; comparisons with conventional techniques.

BACKGROUND: Accurate diagnosis of allergy to specific fungal species is confounded by the variability in allergens occurring with different diagnostic systems. We compared the halogen immunoassay (HIA), which uses allergens expressed by freshly germinated spores that are bound to protein binding membranes (PBM), with the commercial Pharmacia UniCap assay (CAP) and with skin prick tests (SPT). METHODS: Serum from 60 subjects was used; 30 were SPT positive and sensitized to at least one of Alternaria alternata or Aspergillus fumigatus and the other 30 were SPT negative to these fungi but known to be sensitized to non-fungal allergens. All sera were analyzed by CAP against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum purpurascens. For HIA, spores from reference cultures belonging to these four species were germinated on PBM, laminated and then probed with each serum. Two independent observers using an ordinal ranking system quantified the intensity and occurrence of the resultant immunoglobulin E (IgE) immunostained haloes around spores and this was statistically compared with the results of the two conventional immunodiagnostic techniques. RESULTS: Germinated conidia of each species expressed detectable allergen in the HIA. The agreement between the ordinal rank scores assigned by the pair of observers was very good (k >or= 0.8) and only differed for A. fumigatus (k = 0.66) . Between 3% and 7% of SPT negative sera was identified by HIA to have specific IgE towards A. fumigatus and A. alternata. For all four species tested there were strong correlations between HIA and CAP (P < 0.0001). However the correlation of both HIA and CAP to SPT was weaker for A. alternata (r(s) = 0.44, P < 0.0153) and absent for A. fumigatus. CONCLUSIONS: Overall, the HIA is a new immunodiagnostic technique for the detection of sensitization to fungal allergens that correlates significantly with CAP and to a lesser extent with SPT. This may be due to extract variability and system differences. The significance of this derives from the unique ability of the HIA to measure IgE antibodies to the undegraded allergens that are actively secreted by germinating conidia and hyphae. These are the natural agents of exposure to fungi, and as such, are most likely to be relevant to clinical disease.

Measurement of personal exposure to outdoor aeromycota in northern New South Wales, Australia.

Aerobiological sampling traditionally uses a volumetric spore trap located in a fixed position to estimate personal exposure to airborne fungi. In this study, the number and identity of fungi inhaled by human subjects (n=34), wearing Intra-nasal air samplers (INASs), was measured over 2-hour periods in an outdoor community setting, and compared to fungal counts made with a Burkard spore trap and Institute of Occupational Medicine personal filter air samplers (IOMs). All sampling devices were in close proximity and located in an outdoor environment in Casino, northern New South Wales, Australia. Using INASs, the most prevalent fungi inhaled belonged to soil or vegetation borne spores of Alternaria, Arthrinium, Bipolaris, Cladosporium, Curvularia, Epicoccum, Exserohilum, Fusarium, Pithomyces, Spegazzinia and Tetraploa species, Xylariaceae ascospores, in addition to hyphal fragments. These results showed that inhaled fungal exposure in most people varied in a 2-fold range with 10-fold outliers. In addition, the INASs and personal air filters agreed more with each other than with Burkard spore trap counts (r=0.74, p < 0.0001). These findings further support a new paradigm of personal fungal exposure, which implicates the inhalation of a spectrum of fungi more closely associated with soil or vegetation borne mycoflora and hyphal fragments than what is collected by stationary spore traps in the same geographic region.

Enumeration and detection of aerosolized Aspergillus fumigatus and Penicillium chrysogenum conidia and hyphae using a novel double immunostaining technique.

The identification of collected airborne unicellular fungal conidia and hyphae using nonviable techniques is subjective and an imprecise process. Similarly, to determine whether an individual is allergic to a particular genus requires a separate immunodiagnostic analysis. This study demonstrates the development of a novel double immunostaining halogen assay, which enables (1) the simultaneous identification of collected airborne fungal conidia and hyphae of Aspergillus fumigatus and Penicillium chrysogenum using monoclonal antibodies and (2) the demonstration of patient-specific allergy to the same particles using human serum IgE. The results demonstrate that when conidia were ungerminated the binding of antibodies was homogeneous and localized in close proximity around the entire conidia for both species. However, when conidia were germinated, the proportion expressing antigen increased (P < 0.0001) for both species and the sites of binding of the two antibodies changed with double immunostaining restricted to the hyphal tips for A. fumigatus, in addition to the sites of germination for P. chrysogenum. The described immunoassay has the potential to identify fungal particles in personal environmental air samples, provided species-specific monoclonal antibodies are available, while simultaneously demonstrating allergic sensitization to the same particles by co-staining the samples with the patient's own serum. Such an immunoassay can use those fungi that the patient is actually exposed to and potentially avoids many problems associated with extract variability based on the performance of current diagnostic techniques for fungal allergy.

Detection of aerosolized Alternaria alternata conidia, hyphae, and fragments by using a novel double-immunostaining technique.

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.

Fungal fragments and undocumented conidia function as new aeroallergen sources.

BACKGROUND: More than 100 genera of fungal conidia are currently recognized as sources of allergens. The contribution of other fungal genera plus airborne fungal hyphae and fragmented conidia to allergic diseases is poorly understood. OBJECTIVE: We sought to investigate the expression of allergens from airborne wild-type fungi using the Halogen immunoassay, which uses allergic serum IgE to immunostain immobilized allergens extracted from individual fungal particles. METHODS: Airborne fungi were collected onto mixed cellulose ester protein-binding membranes for 2.5 hours with volumetric air pumps. Collected fungi were incubated overnight in a humid chamber to promote the germination of conidia. The membranes were laminated with an adhesive cover slip and immunostained with an Alternaria species-sensitive serum IgE pool. The samples were examined by means of light microscopy, and positively immunostained fungal particles were classified and counted. RESULTS: All air samples contained fungal hyphae that expressed soluble allergens and were significantly higher in concentration than counts of conidia of individual well-characterized allergenic genera (P < .05). Resultant immunostaining of fungal hyphae was heterogeneous, and approximately 25% of all hyphae expressed detectable allergen compared with nonstained hyphae (P < .05). Fungal conidia of 10 genera that were previously uncharacterized as allergen sources were shown to demonstrate IgE binding to expressed antigens and accounted for 8% of the total airborne conidia count. CONCLUSIONS: Our analysis of wild-type fungi collected indoors presents a new paradigm of natural fungal exposure, which, in addition to commonly recognized species, implicates airborne hyphae, fragmented conidia, and the conidia of a much more diverse range of genera as airborne allergens.

Atmospheric Poaceae pollen frequencies and associations with meteorological parameters in Brisbane, Australia: a 5-year record, 1994-1999.

Grass pollen is an important risk factor for allergic rhinitis and asthma in Australia and is the most prevalent pollen component of the aerospora of Brisbane, accounting for 71.6% of the annual airborne pollen load. A 5-year (June 1994-May 1999) monitoring program shows the grass pollen season to occur during the summer and autumn months (December-April), however the timing of onset and intensity of the season vary from year to year. During the pollen season, Poaceae counts exceeding 30 grains m(-3) were recorded on 244 days and coincided with maximum temperatures of 28.1 +/- 2.0 degrees C. In this study, statistical associations between atmospheric grass pollen loads and several weather parameters, including maximum temperature, minimum temperature and precipitation, were investigated. Spearman's correlation analysis demonstrated that daily grass pollen counts were positively associated (P < 0.0001) with maximum and minimum temperature during each sampling year. Precipitation, although considered a less important daily factor (P < 0.05), was observed to remove pollen grains from the atmosphere during significant periods of rainfall. This study provides the first insight into the influence of meteorological variables, in particular temperature, on atmospheric Poaceae pollen counts in Brisbane. An awareness of these associations is critical for the prevention and management of allergy and asthma for atopic individuals within this region.

Quantitative trends in airborne loads of Celtis sinensis pollen and associations with meteorological variables in a subtropical Australian environment.

Celtis sinensis is an introduced plant species to the southeastern region of Queensland that has had a destructive affect on indigenous plant communities and its pollen has been identified as an allergen source. Pollen belonging to C. sinensis was sampled during a 5-year (June 1994-May 1999) atmospheric pollen-monitoring programme in Brisbane, Australia, using a Burkard 7-day spore trap. The seasonal incidence of airborne C. sinensis pollen (CsP) in Brisbane occurred over a brief period each year during spring (August-September), while peak concentrations were restricted to the beginning of September. Individual CsP seasons were heterogeneous with daily counts within the range 1-10 grains m(-3) on no more than 60 sampling days; however, smaller airborne concentrations of CsP were recorded out of each season. Correlation co-efficients were significant each year for temperature (p < 0.05), but were less consistent for precipitation (p > 0.05) and relative humidity (p > 0.05). A significant relationship (r2 =0.81, p=0.036) was established between the total CsP count and pre-seasonal average maximum temperature; however, periods of precipitation (> 2 mm) were demonstrated to significantly lower the daily concentrations of CsP from the atmosphere. Given the environmental and clinical significance of CsP and its prevalence in the atmosphere of Brisbane, a clinical population-based study is required to further understand the pollen's importance as a seasonal sensitizing source in this region.

Allergen detection from 11 fungal species before and after germination.

BACKGROUND: Allergens dispersed by airborne fungal spores play an important but poorly understood role in the underlying cause and exacerbation of asthma. Previous studies suggest that spores of Alternaria and Aspergillus release greater quantities of allergen after germination than before germination. It is unknown whether this is true of other allergenic fungi. OBJECTIVE: Our purpose was to investigate the release of allergen from a range of individual fungal spores before and after germination. METHODS: Allergen expression from spores of Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus, Botrytis cinerea, Epicoccum nigrum, Exserohilum rostratum, Penicillium chrysogenum, Stemphylium botryosum, Curvularia lunata, Trichoderma viride, and Bipolaris spicifera was examined by halogen immunoassays through the use of pooled serum IgE from patients allergic to fungus. Spores were deposited onto protein-binding membranes direct from culture. To germinate spores, samples were incubated in high humidity at room temperature for 48 hours. Ungerminated and germinated samples were then laminated with an adhesive film and immunostained by the halogen assay. The samples were examined by light microscopy, and positive counts (haloed particles) were expressed as percentages of total spores. RESULTS: For 9 of 11 species, between 5.7% and 92% of spores released allergen before germination. Spores of Penicillium and Trichoderma did not release detectable allergen. After germination, all spores that germinated had allergen elution from their hyphae. Eight of 11 species showed a significant increase (P <.05) in the percentage of spores eluting detectable allergen. Localization of allergen along the hyphae varied with species, such that some eluted allergen mainly from hyphal tips and septal junctions whereas others eluted allergen along the entire length. CONCLUSIONS: Increased elution of allergen after germination might be a common feature of many species of allergenic fungi. Although allergens from both spores and hyphae were recognized by human IgE, the extent to which human exposure occurs to allergens eluted from inhaled spores or from hyphae that germinate after deposition in the respiratory tract remains to be explored. The patterns of allergen expression might affect the clinical response to such exposure.