Doctor of Philosophy(PhD), / Airborne fungi are ubiquitous in the environment and human exposure is inevitable. Such fungi differ greatly in their taxonomic, physical, ecological and pathogenic characteristics. Currently, 69 000 species have been taxonomically classified and more than 80 of these are recognised to be aeroallergen sources. Many strategies have evolved to sample, identify and interpret fungal exposure to these species, however no strategy serves all purposes as exposure is a complex and dynamic process confounded by spatial, temporal and geographic variations in airborne counts, in addition to the inadequacies of the immunodiagnostic techniques available. To date, the interpretation of personal exposure and sensitisation to fungal allergens has been restricted to a few select species and the contribution of other genera, airborne hyphae and fragmented conidia to allergic disease are all poorly understood. The aim of the thesis was to utilize the Halogen Immunoassay (HIA) to diagnose fungal allergic sensitisation, to investigate the distribution and factors influencing allergens of fungi in the air and to understand what is actually inhaled in exposure settings. The novelty of the HIA derives from its unique ability to provide allergen sources that are actively secreted by the collected fungal spores and hyphae, which are bound to protein binding membranes (PBM) and then immunoprobed. In Chapter 2, the HIA was compared to the commercial in vitro Pharmacia UniCap assay (CAP) and the in vivo skin prick test (SPT), using 30 sera from subjects SPT positive to Aspergillus fumigatus and/or Alternaria alternata and 30 who were SPT negative to these fungi but sensitised to non-fungal allergens. Sera were analysed by CAP and the HIA against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum purpurascens and compared statistically. Between 3% and 7% of SPT negative sera were identified to have specific IgE towards A. fumigatus and A. iv alternata, respectively. For the SPT positive sera, significant associations were found between the HIA and CAP scores for all fungal species tested (P<0.0001). Correlations between the HIA and SPT however, were weakly correlated for A. alternata (rs = 0.44, P<0.05) but not for A. fumigatus. In Chapter 3, personal exposure to indoor fungal aerosols was examined using the HIA to identify the fungal components that people were allergic to. Personal air sampling pumps (PASs) collected airborne fungal propagules onto PBMs for 2.5 hours indoors (n=21). Collected fungi were incubated overnight in a humid chamber to promote the germination of conidia. The membranes were then immunostained with pooled human Alternaria species-positive sera. All air samples contained fungal hyphae that expressed soluble allergens and were significantly higher in concentration than counts of conidia of individual well-characterised allergenic genera. Approximately 25% of all hyphae expressed detectable allergen compared to non-stained hyphae (P<0.05) and the resultant localisation of immunostaining was heterogeneous among hyphae. Fungal conidia of ten genera that were previously uncharacterised as allergen sources accounted for 8% of the total conidia that demonstrated IgE binding. In Chapter 4, the number and identity of fungi inhaled by 34 adults in an outdoor community setting was measured over 2 hour periods by people wearing Intra-nasal air samplers (INASs) and compared to fungal counts made with a Burkard spore trap and filter air samplers worn on the lapel. Using INAS, the most prevalent fungi inhaled belonged to soil borne spores of Alternaria, Arthrinium, Bipolaris, Cladosporium, Curvularia, Epicoccum, Exserohilum, Fusarium, Pithomyces, Spegazzinia, Tetraploa and Xylariaceae species, in addition to hyphal fragments. These results showed that inhaled exposure in most people varied in a 2-fold range with 10-fold outliers. In addition, the INAS and personal air filters agreed more with each other than with Burkard spore trap counts. The analysis was further confounded by different sampling efficiencies, locations of devices and ability to visualise and count fungal propagules. In Chapter 5, a double immunostaining technique based on the HIA was developed and applied to the conidia, hyphae and fungal fragments of A. alternata, A. fumigatus and Penicillium chrysogenum to discriminate between sources of allergens, v using IgE and to identify the fungi, using a fungal-specific antibody. The localisation of immunostaining was heterogeneous between both conidia and the state of germination with greater concentrations of double immunostaining detected following germination for each fungal species (P<0.0001). Fragmented A. alternata hyphae and morphologically indiscernible fragments could be identified for the first time using this technique. In Chapter 6, the factors affecting the release of allergen from the spores of eleven different species were studied. For nine of eleven species, between 5.7% and 92% of spores released allergen before germination. Ungerminated spores of P. chrysogenum and Trichoderma viride did not release detectable allergen. After germination, all spores that germinated eluted allergen from their hyphae. Upon germination there was a significant increase in the percentage of spores eluting detectable allergen (P<0.0001) and the localisation of allergen along the hyphae varied between species. Increased elution of allergen post germination might be a common feature of many species of allergenic fungi following inhalation. Additionally, Chapter 6 explored the extent to which inhaled spores or hyphae germinate after deposition in the nasal cavity and thus cause exposure to allergens. Twenty subjects had their noses lavaged at three separate intervals, (1) at the beginning of the experiment, (2) after one hour indoors and (3) after one hour outdoors. The recovery of spores and hyphal fragments from the nasal cavity varied between individuals and was significantly greater after outdoor exposures. Germinated fungal spores were recovered often in high concentrations for Aspergillus-Penicillium species, however the proportion between ungerminated and germinated spores were much lower for other genera recovered. Conclusions: Our analysis of cultured and wild-type fungi 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. Exposure is heterogeneous between individuals in the same geographic locality and the spectrum of fungal genera inhaled differs with the method of analysis. Many of the spores inhaled are likely to be allergenic, however upon germination there is an increased elution of allergen and this might be a common vi feature of many fungal species following inhalation. This project also provides novel techniques to diagnose fungal allergy by immunostaining wild-type fungi to which a patient is exposed with the patient’s own serum. Such an immunoassay combines environmental with serological monitoring on a patient specific basis and potentially avoids many problems associated with extract variability, based on the performance of current diagnostic techniques for fungal allergy.

Doctor of Philosophy(PhD), / Airborne fungi are ubiquitous in the environment and human exposure is inevitable. Such fungi differ greatly in their taxonomic, physical, ecological and pathogenic characteristics. Currently, 69 000 species have been taxonomically classified and more than 80 of these are recognised to be aeroallergen sources. Many strategies have evolved to sample, identify and interpret fungal exposure to these species, however no strategy serves all purposes as exposure is a complex and dynamic process confounded by spatial, temporal and geographic variations in airborne counts, in addition to the inadequacies of the immunodiagnostic techniques available. To date, the interpretation of personal exposure and sensitisation to fungal allergens has been restricted to a few select species and the contribution of other genera, airborne hyphae and fragmented conidia to allergic disease are all poorly understood. The aim of the thesis was to utilize the Halogen Immunoassay (HIA) to diagnose fungal allergic sensitisation, to investigate the distribution and factors influencing allergens of fungi in the air and to understand what is actually inhaled in exposure settings. The novelty of the HIA derives from its unique ability to provide allergen sources that are actively secreted by the collected fungal spores and hyphae, which are bound to protein binding membranes (PBM) and then immunoprobed. In Chapter 2, the HIA was compared to the commercial in vitro Pharmacia UniCap assay (CAP) and the in vivo skin prick test (SPT), using 30 sera from subjects SPT positive to Aspergillus fumigatus and/or Alternaria alternata and 30 who were SPT negative to these fungi but sensitised to non-fungal allergens. Sera were analysed by CAP and the HIA against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum purpurascens and compared statistically. Between 3% and 7% of SPT negative sera were identified to have specific IgE towards A. fumigatus and A. iv alternata, respectively. For the SPT positive sera, significant associations were found between the HIA and CAP scores for all fungal species tested (P<0.0001). Correlations between the HIA and SPT however, were weakly correlated for A. alternata (rs = 0.44, P<0.05) but not for A. fumigatus. In Chapter 3, personal exposure to indoor fungal aerosols was examined using the HIA to identify the fungal components that people were allergic to. Personal air sampling pumps (PASs) collected airborne fungal propagules onto PBMs for 2.5 hours indoors (n=21). Collected fungi were incubated overnight in a humid chamber to promote the germination of conidia. The membranes were then immunostained with pooled human Alternaria species-positive sera. All air samples contained fungal hyphae that expressed soluble allergens and were significantly higher in concentration than counts of conidia of individual well-characterised allergenic genera. Approximately 25% of all hyphae expressed detectable allergen compared to non-stained hyphae (P<0.05) and the resultant localisation of immunostaining was heterogeneous among hyphae. Fungal conidia of ten genera that were previously uncharacterised as allergen sources accounted for 8% of the total conidia that demonstrated IgE binding. In Chapter 4, the number and identity of fungi inhaled by 34 adults in an outdoor community setting was measured over 2 hour periods by people wearing Intra-nasal air samplers (INASs) and compared to fungal counts made with a Burkard spore trap and filter air samplers worn on the lapel. Using INAS, the most prevalent fungi inhaled belonged to soil borne spores of Alternaria, Arthrinium, Bipolaris, Cladosporium, Curvularia, Epicoccum, Exserohilum, Fusarium, Pithomyces, Spegazzinia, Tetraploa and Xylariaceae species, in addition to hyphal fragments. These results showed that inhaled exposure in most people varied in a 2-fold range with 10-fold outliers. In addition, the INAS and personal air filters agreed more with each other than with Burkard spore trap counts. The analysis was further confounded by different sampling efficiencies, locations of devices and ability to visualise and count fungal propagules. In Chapter 5, a double immunostaining technique based on the HIA was developed and applied to the conidia, hyphae and fungal fragments of A. alternata, A. fumigatus and Penicillium chrysogenum to discriminate between sources of allergens, v using IgE and to identify the fungi, using a fungal-specific antibody. The localisation of immunostaining was heterogeneous between both conidia and the state of germination with greater concentrations of double immunostaining detected following germination for each fungal species (P<0.0001). Fragmented A. alternata hyphae and morphologically indiscernible fragments could be identified for the first time using this technique. In Chapter 6, the factors affecting the release of allergen from the spores of eleven different species were studied. For nine of eleven species, between 5.7% and 92% of spores released allergen before germination. Ungerminated spores of P. chrysogenum and Trichoderma viride did not release detectable allergen. After germination, all spores that germinated eluted allergen from their hyphae. Upon germination there was a significant increase in the percentage of spores eluting detectable allergen (P<0.0001) and the localisation of allergen along the hyphae varied between species. Increased elution of allergen post germination might be a common feature of many species of allergenic fungi following inhalation. Additionally, Chapter 6 explored the extent to which inhaled spores or hyphae germinate after deposition in the nasal cavity and thus cause exposure to allergens. Twenty subjects had their noses lavaged at three separate intervals, (1) at the beginning of the experiment, (2) after one hour indoors and (3) after one hour outdoors. The recovery of spores and hyphal fragments from the nasal cavity varied between individuals and was significantly greater after outdoor exposures. Germinated fungal spores were recovered often in high concentrations for Aspergillus-Penicillium species, however the proportion between ungerminated and germinated spores were much lower for other genera recovered. Conclusions: Our analysis of cultured and wild-type fungi 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. Exposure is heterogeneous between individuals in the same geographic locality and the spectrum of fungal genera inhaled differs with the method of analysis. Many of the spores inhaled are likely to be allergenic, however upon germination there is an increased elution of allergen and this might be a common vi feature of many fungal species following inhalation. This project also provides novel techniques to diagnose fungal allergy by immunostaining wild-type fungi to which a patient is exposed with the patient’s own serum. Such an immunoassay combines environmental with serological monitoring on a patient specific basis and potentially avoids many problems associated with extract variability, based on the performance of current diagnostic techniques for fungal allergy.