Identification of allergens from Aspergillus fumigatus-Potential association with lung damage in asthma.

BACKGROUND: Component-resolved diagnosis allows detection of IgE sensitization having the advantage of reproducibility and standardization compared to crude extracts. The main disadvantage of the traditional allergen identification methods, 1- or 2-dimensional western blotting and screening of expression cDNA libraries with patients' IgEs, is that the native structure of the protein is not necessarily maintained. METHODS: We used a novel immunoprecipitation technique in combination with mass spectrometry to identify new allergens of Aspergillus fumigatus. Magnetic Dynabeads coupled with anti-human IgE antibodies were used to purify human serum IgE and subsequently allergens from A. fumigatus protein extract. RESULTS: Of the 184 proteins detected by subsequent mass peptide fingerprinting, a subset of 13 were recombinantly expressed and purified. In a panel of 52 A. fumigatus-sensitized people with asthma, 23 non-fungal-sensitized asthmatics and 18 healthy individuals, only the former showed an IgE reaction by immunoblotting and/or ELISA. We discovered 11 proteins not yet described as A. fumigatus allergens, with fructose-bisphosphate aldolase class II (FBA2) (33%), NAD-dependent malate dehydrogenase (31%) and Cu/Zn superoxide dismutase (27%) being the most prevalent. With respect to these three allergens, native versus denatured protein assays indicated a better recognition of the native proteins. Seven of 11 allergens fulfilled the WHO/IUIS criteria and were accepted as new A. fumigatus allergens. CONCLUSION: In conclusion, we introduce a straightforward method of allergen identification from complex allergenic sources such as A. fumigatus by immunoprecipitation combined with mass spectrometry, which has the advantage over traditional methods of identifying allergens by maintaining the structure of the proteins.

Co-exposure to highly allergenic airborne pollen and fungal spores in Europe.

The study is aimed at determining the potential spatiotemporal risk of the co-occurrence of airborne pollen and fungal spores high concentrations in different bio-climatic zones in Europe. Birch, grass, mugwort, ragweed, olive pollen and Alternaria and Cladosporium fungal spores were investigated at 16 sites in Europe, in 2005-2019. In Central and northern Europe, pollen and fungal spore seasons mainly overlap in June and July, while in South Europe, the highest pollen concentrations occur frequently outside of the spore seasons. In the coldest climate, no allergy thresholds were exceeded simultaneously by two spore or pollen taxa, while in the warmest climate most of the days with at least two pollen taxa exceeding threshold values were observed. The annual air temperature amplitude seems to be the main bioclimatic factor influencing the accumulation of days in which Alternaria and Cladosporium spores simultaneously exceed allergy thresholds. The phenomenon of co-occurrence of airborne allergen concentrations gets increasingly common in Europe and is proposed to be present on other continents, especially in temperate climate.

Pollen season trends as markers of climate change impact: Betula, Quercus and Poaceae.

The incidences of respiratory allergies are at an all-time high. Pollen aeroallergens can reflect changing climate, with recent studies in Europe showing some, but not all, pollen types are increasing in severity, season duration and experiencing an earlier onset. This study aimed to identify pollen trends in the UK over the last twenty-six years for a range of pollen sites, with a focus on the key pollen types of Poaceae (grass), Betula (birch) and Quercus (oak) and to examine the relationship of these trends with meteorological factors. Betula pollen seasons show no significant trends for onset, first high day or duration but increasing pollen production in the Midlands region of the UK is being driven by warmer temperatures in the previous June and July. Quercus pollen seasons are starting earlier, due to increasing temperature and sunshine totals in April, but are not becoming more severe. The seasons are lasting longer, although no significant climate drivers for this were identified. The first high day of the Poaceae pollen season is occurring earlier in central UK regions due to an increasing trend for all temperature variables in the previous December, January, April, May and June. Severity and duration of the season show no significant trends and are spatially and temporally variable. Important changes are occurring in the UK pollen seasons that will impact on the health of respiratory allergy sufferers, with more severe Betula pollen seasons and longer Quercus pollen seasons. Most of the changes identified were caused by climate drivers of increasing temperature and sunshine total. However, Poaceae pollen seasons are neither becoming more severe nor longer. The reasons for this included a lack of change in some monthly meteorological variables, or land-use change, such as grassland being replaced by urban areas or woodland.

A systematic review of outdoor airborne fungal spore seasonality across Europe and the implications for health.

Fungal spores make up a significant proportion of organic matter within the air. Allergic sensitisation to fungi is associated with conditions including allergic fungal airway disease. This systematic review analyses outdoor fungal spore seasonality across Europe and considers the implications for health. Seventy-four studies met the inclusion criteria, the majority of which (n = 64) were observational sampling studies published between 1978 and 2020. The most commonly reported genera were the known allergens Alternaria and Cladosporium, measured in 52 and 49 studies, respectively. Both displayed statistically significant increased season length in south-westerly (Mediterranean) versus north-easterly (Atlantic and Continental) regions. Although there was a trend for reduced peak or annual Alternaria and Cladosporium spore concentrations in more northernly locations, this was not statistically significant. Peak spore concentrations of Alternaria and Cladosporium exceeded clinical thresholds in nearly all locations, with median peak concentrations of 665 and 18,827 per m3, respectively. Meteorological variables, predominantly temperature, precipitation and relative humidity, were the main factors associated with fungal seasonality. Land-use was identified as another important factor, particularly proximity to agricultural and coastal areas. While correlations of increased season length or decreased annual spore concentrations with increasing average temperatures were reported in multi-decade sampling studies, the number of such studies was too small to make any definitive conclusions. Further, up-to-date studies covering underrepresented geographical regions and fungal taxa (including the use of modern molecular techniques), and the impact of land-use and climate change will help address remaining knowledge gaps. Such knowledge will help to better understand fungal allergy, develop improved fungal spore calendars and forecasts with greater geographical coverage, and promote increased awareness and management strategies for those with allergic fungal disease.

Abundance of Ganoderma sp. in Europe and SW Asia: modelling the pathogen infection levels in local trees using the proxy of airborne fungal spore concentrations.

Ganoderma comprises a common bracket fungal genus that causes basal stem rot in deciduous and coniferous trees and palms, thus having a large economic impact on forestry production. We estimated pathogen abundance using long-term, daily spore concentration data collected in five biogeographic regions in Europe and SW Asia. We hypothesized that pathogen abundance in the air depends on the density of potential hosts (trees) in the surrounding area, and that its spores originate locally. We tested this hypothesis by (1) calculating tree cover density, (2) assessing the impact of local meteorological variables on spore concentration, (3) computing back trajectories, (4) developing random forest models predicting daily spore concentration. The area covered by trees was calculated based on Tree Density Datasets within a 30 km radius from sampling sites. Variations in daily and seasonal spore concentrations were cross-examined between sites using a selection of statistical tools including HYSPLIT and random forest models. Our results showed that spore concentrations were higher in Northern and Central Europe than in South Europe and SW Asia. High and unusually high spore concentrations (> 90th and > 98th percentile, respectively) were partially associated with long distance transported spores: at least 33% of Ganoderma spores recorded in Madeira during days with high concentrations originated from the Iberian Peninsula located >900 km away. Random forest models developed on local meteorological data performed better in sites where the contribution of long distance transported spores was lower. We found that high concentrations were recorded in sites with low host density (Leicester, Worcester), and low concentrations in Kastamonu with high host density. This suggests that south European and SW Asian forests may be less severely affected by Ganoderma. This study highlights the effectiveness of monitoring airborne Ganoderma spore concentrations as a tool for assessing local Ganoderma pathogen infection levels.

New Perspectives in the Diagnosis and Management of Allergic Fungal Airway Disease.

Allergy to airway-colonising, thermotolerant, filamentous fungi represents a distinct eosinophilic endotype of often severe lung disease. This endotype, which particularly affects adult asthma, but also complicates other airway diseases and sometimes occurs de novo, has a heterogeneous presentation ranging from severe eosinophilic asthma to lobar collapse. Its hallmark is lung damage, characterised by fixed airflow obstruction (FAO), bronchiectasis and lung fibrosis. It has a number of monikers including severe asthma with fungal sensitisation (SAFS) and allergic bronchopulmonary aspergillosis/mycosis (ABPA/M), but these exclusive terms constitute only sub-sets of the condition. In order to capture the full extent of the syndrome we prefer the inclusive term allergic fungal airway disease (AFAD), the criteria for which are IgE sensitisation to relevant fungi in association with airway disease. The primary fungus involved is Aspergillus fumigatus, but a number of other thermotolerant species from several genera have been implicated. The unifying mechanism involves germination of inhaled fungal spores in the lung in the context of IgE sensitisation, leading to a persistent and vigorous eosinophilic inflammatory response in association with release of fungal proteases. Most allergenic fungi, including Alternaria and Cladosporium species, are not thermotolerant and cannot germinate in the airways so only act as aeroallergens and do not cause AFAD. Studies of the airway mycobiome have shown that A. fumigatus colonises the normal as much as the asthmatic airway, suggesting it is the tendency to become IgE-sensitised that is the critical triggering factor for AFAD rather than colonisation per se. Treatment is aimed at preventing exacerbations with glucocorticoids and increasingly by the use of anti-T2 biological therapies. Anti-fungal therapy has a limited place in management, but is an effective treatment for fungal bronchitis which complicates AFAD in about 10% of cases.

Allergic fungal airways disease (AFAD): an under-recognised asthma endotype.

The term allergic fungal airways disease has a liberal definition based on IgE sensitisation to thermotolerant fungi and evidence of fungal-related lung damage. It arose from a body of work looking into the role of fungi in asthma. Historically fungi were considered a rare complication of asthma, exemplified by allergic bronchopulmonary aspergillosis; however, there is a significant proportion of individuals with Aspergillus fumigatus sensitisation who do not meet these criteria, who are at high risk for the development of lung damage. The fungi that play a role in asthma can be divided into two groups; those that can grow at body temperature referred to as thermotolerant, which are capable of both infection and allergy, and those that cannot but can still act as allergens in IgE sensitised individuals. Sensitisation to thermotolerant filamentous fungi (Aspergillus and Penicillium), and not non-thermotolerant fungi (Alternaria and Cladosporium) is associated with lower lung function and radiological abnormalities (bronchiectasis, tree-in-bud, fleeting shadows, collapse/consolidation and fibrosis). For antifungals to play a role in treatment, the focus should be on fungi capable of growing in the airways thereby causing a persistent chronic allergenic stimulus and releasing tissue damaging proteases and other enzymes which may disrupt the airway epithelial barrier and cause mucosal damage and airway remodelling. All patients with IgE sensitisation to thermotolerant fungi in the context of asthma and other airway disease are at risk of progressive lung damage, and as such should be monitored closely.

Predicting the severity of the grass pollen season and the effect of climate change in Northwest Europe.

Allergic rhinitis is an inflammation in the nose caused by overreaction of the immune system to allergens in the air. Managing allergic rhinitis symptoms is challenging and requires timely intervention. The following are major questions often posed by those with allergic rhinitis: How should I prepare for the forthcoming season? How will the season's severity develop over the years? No country yet provides clear guidance addressing these questions. We propose two previously unexplored approaches for forecasting the severity of the grass pollen season on the basis of statistical and mechanistic models. The results suggest annual severity is largely governed by preseasonal meteorological conditions. The mechanistic model suggests climate change will increase the season severity by up to 60%, in line with experimental chamber studies. These models can be used as forecasting tools for advising individuals with hay fever and health care professionals how to prepare for the grass pollen season.

Fungal bronchitis is a distinct clinical entity which is responsive to antifungal therapy.

Chronic productive cough in the context of exacerbations of airway disease can be associated with positive sputum cultures for fungi, in particular Aspergillus fumigatus and Candida spp., suggesting fungal bronchitis, a condition not widely recognised, as a possible cause for the exacerbation. Our objective was to determine the response to antifungal therapy in patients with suspected fungal bronchitis. Retrospective analysis of data extracted from case records of patients under secondary care respiratory clinics who had been treated with triazole therapy for suspected fungal bronchitis between 2010-2017. Primary outcome was lung function response after 1 month of treatment. Nineteen patients with fungal bronchitis due to A. fumigatus and 12 patients due to Candida spp., were included in the study. Most of the patients, particularly in the Aspergillus group, had allergic fungal airway disease on a background of asthma. All but one of the patients in each group were recorded as showing clinical improvement with antifungal therapy. In the majority of patients this was reflected in an improvement in lung function. Aspergillus group: FEV1 (1.44 ± 0.8 L vs 1.6 ± 0.8 L: p < 0.02), FVC (2.49 ± 1.08 L vs 2.8 ± 1.1 L: p = 0.01), and PEF (260 ± 150L/min vs 297 ± 194ml/min: p < 0.02). Candida group: FEV1 (1.6 ± 0.76 L vs 2.0 ± 0.72 L: p < 0.004), FVC (2.69 ± 0.91 L vs 3.13 ± 0.7 L: p = 0.05), and PEF (271± 139L/min vs 333 ± 156 L/min: p = 0.01). Side effects of treatment were common, but resolved on stopping treatment. This service improvement project supports the idea that fungal bronchitis is a distinct clinical entity which is responsive to treatment. Controlled clinical trials to confirm the clinical impression that this is relatively common and treatable complication of complex airway disease are required.

Fungal sensitization and positive fungal culture from sputum in children with asthma are associated with reduced lung function and acute asthma attacks respectively.

BACKGROUND: Sensitization to thermotolerant fungi, including filamentous fungi and Candida albicans, is associated with poor lung function in adults with severe asthma. Data in children are lacking. Environmental exposure to fungi is linked with acute severe asthma attacks, but there are few studies reporting the presence of fungi in the airways during asthma attacks. METHODS: We investigated the association between fungal sensitization and/or positive fungal sputum culture and markers of asthma severity in children with chronic and acute asthma. Sensitization was determined using serum-specific IgE and skin prick testing against a panel of five fungi. Fungal culture was focused towards detection of filamentous fungi from sputum samples. RESULTS: We obtained sensitization data and/or sputum from 175 children: 99 with chronic asthma, 39 with acute asthma and 37 controls. 34.1% of children with chronic asthma were sensitized to thermotolerant fungi compared with no children without asthma (p =< 0.001). These children had worse pre-bronchodilator lung function compared with asthmatics without sensitization including a lower FEV1 /FVC ratio (p < .05). The isolation rate of filamentous fungi from sputum was higher in children with acute compared with chronic asthma. CONCLUSIONS: Fungal sensitization is a feature of children with chronic asthma. Children sensitized to thermotolerant fungi have worse lung function, require more courses of systemic corticosteroids and have greater limitation of activities due to asthma. Asthma attacks in children were associated with the presence of filamentous fungi positive sputum culture. Mechanistic studies are required to establish whether fungi contribute directly to the development of acute asthma.

The airway fungal microbiome in asthma.

BACKGROUND: Fungal involvement in asthma is associated with severe disease. The full spectrum of fungal species in asthma is not well described and is derived largely from insensitive culture techniques. OBJECTIVES: To use high-throughput sequencing to describe the airway mycobiota in asthmatics with and without fungal sensitization and healthy controls; to compare samples representing different airway compartments; to determine whether the mycobiota was influenced by the fungal composition of outdoor air; and to compare findings with clinically relevant outcomes. METHODS: We amplified the internal transcribed spacer region 2 of the nuclear ribosomal operon to identify the fungal species present. Ninety-seven subjects were recruited and provided sputum (83 asthmatics; 14 healthy subjects), with 29 also undergoing a bronchoscopy. A subset of airway samples were compared with matched outdoor air and mouthwash samples. RESULTS: Two hundred and six taxa at the species level were identified in sputum, most at low relative abundance. Aspergillus fumigatus, Candida albicans and Mycosphaerella tassiana had the highest relative abundances and were the most prevalent species across all subjects. The airway mycobiota consisted of a complex community with high diversity between individuals. Notable shifts in the balance of fungi detected in the lung were associated with asthma status, asthma duration and biomarkers of inflammation. Aspergillus tubingensis, a member of the Aspergillus niger species complex, was most prevalent from bronchoscopic protected brush samples and significantly associated with a low sputum neutrophilia. Cryptococcus pseudolongus, from the Cryptococcus humicola species complex, was more abundant from bronchoscopy samples than sputum, and differentially more abundant in asthma than health. CONCLUSIONS AND CLINICAL RELEVANCE: The airway mycobiota was dominated by a relatively small number of species, but was distinct from the oropharyngeal mycobiota and air samples. Members of the A. niger and C. humicola species complexes may play unexpected roles in the pathogenesis of asthma.

Comparative Analysis of Clinical Parameters and Sputum Biomarkers in Establishing the Relevance of Filamentous Fungi in Cystic Fibrosis.

Background: The relationship between fungal culture (FC) positivity and airway inflammation in CF is largely unknown. Identifying the clinical significance of filamentous fungi in CF using both clinical parameters and biomarkers may change our antimicrobial therapeutic strategies. Objectives: To investigate the clinical characteristics and airway biomarker profile in relation to the detection of filamentous fungi in respiratory samples obtained from CF patients. Methods: A prospective cohort study over 24 months, including children and adults with CF. Participants provided sputum and/or bronchoalveolar lavage samples, which underwent processing for bacterial and fungal culture, leukocyte differential cell count and biomarker analysis for neutrophil elastase (NE), interleukin-8 (IL-8), galactomannan and tumor necrosis factor receptor type 2 (TNF-R2). We performed FC using neat sputum plugs, an approach shown to be more sensitive compared to routine laboratory testing. Results: Sixty-one patients provided 76 respiratory samples (72 sputum and 4 BAL). Median age was 17 years (range 6 months-59 years). FC positivity was noted in 49% of the cohort. FC positivity was greater during pulmonary exacerbation compared to the stable state (67 versus 50%). Participants aged 5-30 years had a lower FEV1 within the FC positive group. A significant association between FC positivity and non-tuberculosis mycobacterial (NTM) culture was observed on non-parametric testing (p = 0.022) and regression analysis (p = 0.007). Exposure to indoor mold was a predictor for FC positivity (p = 0.047). There was a trend towards increased lung clearance index (LCI), bronchiectasis and intravenous antibiotic use in the FC positive group. There was no significant difference in biomarkers between FC positive and negative patients. Conclusion: Aspergillus. fumigatus is the commonest filamentous fungi cultured from CF airways. We found no difference in the airway biomarker profile between FC positive and negative patients. The role of galactomannan and TNFR2 as fungal specific biomarkers in CF remains uncertain. FC positivity is associated with a lower FEV1 in younger patients, a lower LCI, NTM positivity, bronchiectasis, and intravenous antibiotic exposure. Larger trials are needed to determine the role of galactomannan and TNF-R2 as potential fungal biomarkers in CF.

Airborne Alternaria and Cladosporium fungal spores in Europe: Forecasting possibilities and relationships with meteorological parameters.

Airborne fungal spores are prevalent components of bioaerosols with a large impact on ecology, economy and health. Their major socioeconomic effects could be reduced by accurate and timely prediction of airborne spore concentrations. The main aim of this study was to create and evaluate models of Alternaria and Cladosporium spore concentrations based on data on a continental scale. Additional goals included assessment of the level of generalization of the models spatially and description of the main meteorological factors influencing fungal spore concentrations. Aerobiological monitoring was carried out at 18 sites in six countries across Europe over 3 to 21 years depending on site. Quantile random forest modelling was used to predict spore concentrations. Generalization of the Alternaria and Cladosporium models was tested using (i) one model for all the sites, (ii) models for groups of sites, and (iii) models for individual sites. The study revealed the possibility of reliable prediction of fungal spore levels using gridded meteorological data. The classification models also showed the capacity for providing larger scale predictions of fungal spore concentrations. Regression models were distinctly less accurate than classification models due to several factors, including measurement errors and distinct day-to-day changes of concentrations. Temperature and vapour pressure proved to be the most important variables in the regression and classification models of Alternaria and Cladosporium spore concentrations. Accurate and operational daily-scale predictive models of bioaerosol abundances contribute to the assessment and evaluation of relevant exposure and consequently more timely and efficient management of phytopathogenic and of human allergic diseases.

Oak pollen seasonality and severity across Europe and modelling the season start using a generalized phenological model.

Oak pollen seasons are relatively unexplored in large parts of Europe despite producing allergens and being a common tree in both continental and northern parts. Many studies are concentrated only on the Iberian Peninsula. In this study, the seasonal pattern of oak pollen in Europe was analysed using 10 observation sites, ranging from Spain to Sweden. The magnitude of peaks and annual pollen integral together with season-length were studied and substantially higher pollen levels and longer seasons were found in Spain. Two northern sites in Denmark and Sweden showed high oak pollen peaks together with two sites in Spain and United Kingdom. The study also tested four common definitions of season start and applied a generalized phenological model for computing the start of the pollen season. The most accurate definition for a European-wide description of the observed oak pollen start was when the cumulative daily average pollen count reached 50 grains per cubic meter. For the modelling of the start a thermal time method based on Growing Degree Day (GDD) was implemented, utilizing daily temperatures and a generalized approach to identify model parameters applicable to all included sites. GDD values varied between sites and generally followed a decreasing gradient from south to north, with some exceptions. Modelled onsets with base temperatures below 7 °C matched well with observed onsets and 76% of the predictions differed ≤4 days compared to observed onsets when using a base temperature of 2 °C. Base temperatures above 7 °C frequently predicted onsets differing >1 week from the observed. This general approach can be extended to a larger area where pollen observations are non-existent. The presented work will increase the understanding of oak pollen variation in Europe and provide knowledge of its phenology, which is a critical aspect both for modelling purposes on large-scale and assessing the human exposure to oak allergens.

Challenges in Laboratory Detection of Fungal Pathogens in the Airways of Cystic Fibrosis Patients.

Study of the clinical significance of fungal colonization/infection in the airways of cystic fibrosis (CF) patients, especially by filamentous fungi, is challenged by the absence of standardized methodology for the detection and identification of an ever-broadening range of fungal pathogens. Culture-based methods remain the cornerstone diagnostic approaches, but current methods used in many clinical laboratories are insensitive and unstandardized, rendering comparative studies unfeasible. Guidelines for standardized processing of respiratory specimens and for their culture are urgently needed and should include recommendations for specific processing procedures, inoculum density, culture media, incubation temperature and duration of culture. Molecular techniques to detect fungi directly from clinical specimens include panfungal PCR assays, multiplex or pathogen-directed assays, real-time PCR, isothermal methods and probe-based assays. In general, these are used to complement culture. Fungal identification by DNA sequencing methods is often required to identify cultured isolates, but matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is increasingly used as an alternative to DNA sequencing. Genotyping of isolates is undertaken to investigate relatedness between isolates, to pinpoint the infection source and to study the population structure. Methods range from PCR fingerprinting and amplified fragment length polymorphism analysis, to short tandem repeat typing, multilocus sequencing typing (MLST) and whole genome sequencing (WGS). MLST is the current preferred method, whilst WGS offers best case resolution but currently is understudied.

Sputum Inflammatory Mediators Are Increased in Aspergillus fumigatus Culture-Positive Asthmatics.

Aspergillus fumigatus sensitization and culture in asthma are associated with disease severity and lung function impairment, but their relationship with airway inflammation is poorly understood. We investigated the profile of 24 sputum inflammatory mediators in A. fumigatus culture-positive or-negative moderate-to-severe asthmatics. Fifty-two subjects were recruited from a single center. A. fumigatus was cultured from 19 asthmatics. Asthma control, symptom score, lung function, and sputum cell count were not significantly different between the asthmatics with and without a positive A. fumigatus culture. All of the sputum mediators were numerically increased in subjects with a positive versus negative sputum A. fumigatus culture. Sputum TNF-R2 was significantly elevated (P=0.03) and the mediator that best distinguished A. fumigatus culture-positive from culture-negative subjects (receiver-operator characteristic area under the curve 0.66 [95% CI: 0.51 to 0.82, P=0.045]). A. fumigates-positive culture in moderate-to-severe asthma is associated with increased inflammatory sputum mediators.