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

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

rAsp f 3 and rAsp f 4 are associated with bronchiectasis in allergic fungal airways disease.

rAsp f 3 and rAsp f 4 are associated with bronchiectasis in allergic fungal airways disease

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

Guidelines on ambient intramural airborne fungal spores

OBJECTIVES: To generate baseline data for indoor airborne fungal spores in noncomplaint residential properties (with no moisture/mold-related problems) and to identify home characteristics indicative of elevated fungal levels. METHODS: Air samples were collected onto petroleum jelly-coated slides from living rooms of 100 residential properties in Leicestershire, United Kingdom, using a Burkard continuous recording air sampler. The slides were examined by microscopy to determine fungal spore concentrations (spores/m3 air/day). RESULTS: Total indoor fungal spore concentrations were approximately 16% of outdoor concentrations. Abundant indoor fungal genera include Cladosporium, Sporobolomyces, Tilletiopsis, and Didymella, all of which followed seasonal patterns of release and detection. No clear association was shown between outdoor-predominant fungi and home characteristics. In contrast, Aspergillus/Penicillium-type (Asp/ Pen-type) spores were common indoors and exceeded outdoor levels, with the highest concentrations detected in properties over 90 years old (P = .006) and terraced properties (P = .003). CONCLUSION: Asp/Pen-type spores are found in noncomplaint UK residential properties and mostly in old terraced houses. This study provides guidelines on acceptable levels of Asp/Pen-type spores and other abundant indoor fungal taxa that can be comparatively used in clinical evaluations of fungal exposure-related disease

Reproducibility between counts of airborne allergenic pollen from two cities in the East Midlands, UK

Historically in the East Midlands, UK, airborne pollen has been monitored in two cities, Derby and Leicester, situated 41 km (25 miles) apart. The aim of the present study was to compare aerobiological data from both sites to determine if a forecast based on data from one site would be sufficient for both, and to address the wider issue of reproducibility between geographically separated sites. Pollen types recorded could be split into two groups according to annual abundance, maximum daily concentration and the number of high count days. Six taxa made up the abundant group; ash, birch, grass, oak, nettle-type and yew-type, representing 90 and 88% of the total air spora for Derby and Leicester, respectively. Three consecutive years of grass and nettle pollen data are presented, supported by one year of abundant tree pollen data. There were highly significant positive correlations between the counts obtained. Line charts showing the average number of pollen grains m−3 air day−1 show similar trends, and Bland–Altman plots show little discrepancy between the amounts of pollen counted on any given day. Each day was classified according to the UK accepted threshold levels for grass. Weighted kappa statistics showed substantial or almost perfect agreement between the forecast classifications. With the caveat that this would not apply in a region with restrictions to air flow such as a mountain range or with extreme fluctuations such as a coastline site, this study suggests that data from a single site is suitable for forecasting a distance of up to 41 km

Isolation of Aspergillus fumigatus from sputum is associated with elevated airborne levels in homes of patients with asthma

Indoor bioaerosols, such as mold spores, have been associated with respiratory symptoms in patients with asthma; however, dose-response relationships and guidelines on acceptable levels are lacking. Furthermore, a causal link between mold exposure and respiratory infections or asthma remains to be established. The aim of this study was to determine indoor concentrations of Aspergillus fumigatus and a subset of clinically relevant fungi in homes of people with asthma, in relation to markers of airways colonization and sensitization. Air and dust samples were collected from the living room of 58 properties. Fungal concentrations were quantified using mold-specific quantitative PCR and compared with traditional microscopic analysis of air samples. Isolation of A. fumigatus from sputum was associated with higher airborne concentrations of the fungus in patient homes (P = 0.04), and a similar trend was shown with Aspergillus/Penicillium-type concentrations analyzed by microscopy (P = 0.058). No association was found between airborne levels of A. fumigatus and sensitization to this fungus, or dustborne levels of A. fumigatus and either isolation from sputum or sensitization. The results of this study suggest that the home environment should be considered as a potential source of fungal exposure, and elevated home levels may predispose people with asthma to airways colonization

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