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.

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.

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 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.

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.

The long distance transport of airborne Ambrosia pollen to the UK and the Netherlands from Central and south Europe.

The invasive alien species Ambrosia artemisiifolia (common or short ragweed) is increasing its range in Europe. In the UK and the Netherlands, airborne concentrations of Ambrosia pollen are usually low. However, more than 30 Ambrosia pollen grains per cubic metre of air (above the level capable to trigger allergic symptoms) were recorded in Leicester (UK) and Leiden (NL) on 4 and 5 September 2014. The aims of this study were to determine whether the highly allergenic Ambrosia pollen recorded during the episode could be the result of long distance transport, to identify the potential sources of these pollen grains and to describe the conditions that facilitated this possible long distance transport. Airborne Ambrosia pollen data were collected at 10 sites in Europe. Back trajectory and atmospheric dispersion calculations were performed using HYSPLIT_4. Back trajectories calculated at Leicester and Leiden show that higher altitude air masses (1500 m) originated from source areas on the Pannonian Plain and Ukraine. During the episode, air masses veered to the west and passed over the Rhône Valley. Dispersion calculations showed that the atmospheric conditions were suitable for Ambrosia pollen released from the Pannonian Plain and the Rhône Valley to reach the higher levels and enter the airstream moving to northwest Europe where they were deposited at ground level and recorded by monitoring sites. The study indicates that the Ambrosia pollen grains recorded during the episode in Leicester and Leiden were probably not produced by local sources but transported long distances from potential source regions in east Europe, i.e. the Pannonian Plain and Ukraine, as well as the Rhône Valley in France.

Allergic fungal airway disease: pathophysiologic and diagnostic considerations.

PURPOSE OF REVIEW: Fungal spores are ubiquitously present in indoor and outdoor air. A number can act as aeroallergens in Immunoglobulin E (IgE)-sensitized individuals and some thermotolerant fungi germinate in the lung where they can cause a combined allergic and infective stimulus leading to a number of clinical presentations characterized by evidence of lung damage. We discuss which biomarkers are useful in helping to guide diagnosis, prognosis and treatment of allergic fungal airway disease (AFAD). RECENT FINDINGS: Diagnostic biomarkers, such as specific IgEs and fungal culture, for AFAD are limited by sensitivity, although this may be improved with novel agents such as specific IgEs to fungal components and quantitative PCR. Total IgE and hypereosinophilia are nonspecific and do not clearly relate to disease activity. High attenuation mucus and proximal bronchiectasis are specific, albeit insensitive markers of AFAD. Biomarkers that predict prognosis and treatment response are yet to be defined. SUMMARY: This review summarizes the fungi involved and the current debate regarding the diagnostic criteria to define fungal-associated lung disease. We advocate the phasing out of the term allergic bronchopulmonary aspergillosis and the use of a more inclusive term such as AFAD, together with a more liberal set of criteria based largely on IgE sensitization to thermotolerant fungi, which identifies those patients at risk of developing lung damage.

Fungal culture and sensitisation in asthma, cystic fibrosis and chronic obstructive pulmonary disorder: what does it tell us?

Collectively asthma, chronic obstructive pulmonary disorder (COPD) and cystic fibrosis (CF) are very common, important causes of disease and ill health. Filamentous fungal colonisation of the airways can occur in all three disease groups, although the clinical relevance is unclear. Allergic bronchopulmonary aspergillosis (ABPA) is a well-recognised severe complication of airway colonisation associated primarily with Aspergillus fumigatus. Fungal colonisation may have a deleterious effect without fulfilling all the diagnostic criteria of ABPA; however, a lack of standardisation in processing respiratory samples hampers comparisons. Whilst mycology laboratory accreditation programs are common, most countries have no national standard guidelines for processing respiratory samples. Fungal recovery from sputum in CF, asthma and COPD can be around 40, 54 and 49%, respectively. Isolation of fungi from sputum has been associated with reduced lung function in asthma and CF, although no such associations have been found in COPD. It is unclear whether fungal colonisation contributes to lower lung function or is a marker of more severe lung disease and aggressive therapy. Fungal sensitisation may contribute to the persistence of active respiratory symptoms; however, the exact prevalence is unclear. Sensitisation to A. fumigatus has been associated with reduced lung function in asthma, COPD and CF. It has suggested that both skin prick tests and specific IgE measurement by the ImmunoCAP system should be used in diagnoses of allergy, due to discordance in test results; however, there is currently no widely adopted consensus as to which fungi to test for.

Amplicon-based metagenomic analysis of mixed fungal samples using proton release amplicon sequencing.

Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific "conserved" primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.

Aspergillus fumigatus during stable state and exacerbations of COPD.

Bacteria are often isolated in stable chronic obstructive pulmonary disease (COPD). Whether fungi are also commonly present and associated with clinical and pathological features of disease is uncertain. We investigated the frequency of filamentous fungal culture and IgE sensitisation to Aspergillus fumigatus and the relationship to clinical outcomes in COPD subjects. COPD subjects were recruited to enter a 1-year observational study. Assessments of lung function, allergen testing and sputum analysis for inflammation, bacteria and fungus were undertaken in COPD subjects and healthy smoking and nonsmoking controls. Filamentous fungi were cultured at baseline in 49% (63 out of 128) of COPD subjects, of which 75% (47 out of 63) were A. fumigatus. Fungus was cultured in three out of 22 controls (two were A. fumigatus). The total sputum cell count and inhaled corticosteroid dosage were significantly increased in COPD patients with a positive filamentous fungal culture at baseline (p<0.05). Sensitisation to A. fumigatus was present in 13% of COPD subjects and was associated with worse lung function (forced expiratory volume in 1 s 39% predicted versus 51% predicted; p=0.01), but not related to filamentous fungal culture. A. fumigatus sensitisation is related to poor lung function. Positive filamentous fungal culture is a common feature of COPD. The clinical significance of this remains uncertain.

Effectiveness of voriconazole in the treatment of Aspergillus fumigatus-associated asthma (EVITA3 study).

BACKGROUND: IgE sensitization to Aspergillus fumigatus and a positive sputum fungal culture result are common in patients with refractory asthma. It is not clear whether these patients would benefit from antifungal treatment. OBJECTIVES: We sought to determine whether a 3-month course of voriconazole improved asthma-related outcomes in patients with asthma who are IgE sensitized to A fumigatus. METHODS: Asthmatic patients who were IgE sensitized to A fumigatus with a history of at least 2 severe exacerbations in the previous 12 months were treated for 3 months with 200 mg of voriconazole twice daily, followed by observation for 9 months, in a double-blind, placebo-controlled, randomized design. Primary outcomes were improvement in quality of life at the end of the treatment period and a reduction in the number of severe exacerbations over the 12 months of the study. RESULTS: Sixty-five patients were randomized. Fifty-nine patients started treatment (32 receiving voriconazole and 27 receiving placebo) and were included in an intention-to-treat analysis. Fifty-six patients took the full 3 months of medication. Between the voriconazole and placebo groups, there were no significant differences in the number of severe exacerbations (1.16 vs 1.41 per patient per year, respectively; mean difference, 0.25; 95% CI, 0.19-0.31), quality of life (change in Asthma Quality of Life Questionnaire score, 0.68 vs 0.88; mean difference between groups, 0.2; 95% CI, -0.05 to -0.11), or any of our secondary outcome measures. CONCLUSION: We were unable to show a beneficial effect of 3 months of treatment with voriconazole in patients with moderate-to-severe asthma who were IgE sensitized to A fumigatus on either the rate of severe exacerbations, quality of life, or other markers of asthma control.

DNA analysis of outdoor air reveals a high degree of fungal diversity, temporal variability, and genera not seen by spore morphology.

Fungi are ubiquitous with many capable of causing disease by direct infection, toxicoses, or allergy. Fungal spores are present in outdoor air throughout the year, yet airborne diversity is poorly characterised. Airborne fungal spores are routinely counted by microscopy, enabling identification to genera at best. We generated traditional microscopic counts over a year, then used environmental sequencing techniques to assess and compare 3 d selected from the main fungal spore season. The days selected corresponded to one with a high quantity of spores unidentifiable by microscopy, and two representing dry and wet summer periods. Over 86 % of genera detected by sequencing were not routinely identifiable by microscopy. A high degree of temporal variability was detected, with the percentage of clones attributed to Basidiomycota or Ascomycota, and composition of genera within each phylum varying greatly between days. Throughout the year Basidiomycota spores were found at higher levels than Ascomycota, but levels fluctuated daily with Ascomycota comprising 11-84 % of total spores and Basidiomycota 7-81 %. No significant difference was found between the proportion of clones attributed to each morphological group detected by sequencing to that counted by microscopy (P = 0.477, 0.985, and 0.561). The majority of abundant genera detected by DNA analysis are not routinely identified by microscopy (>75 %). Of those, several are known human and plant pathogens, and may represent unrecognised aeroallergens.