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 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

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

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

Details of the two mock fungal populations.

<p>Concentration refers to the number of spores used for DNA extraction. ATCC - American Type Culture Collection, CABI - CAB International fungal culture collection, EGS - E.G. Simmons Culture Collection, FRR - Culture collection of CSIRO Sydney Australia, NCPF - National Collection of Pathogenic Fungi, NCYC - National Collection of Yeast Cultures, NRRL - Northern Regional Research Laboratory, UAMH - University of Alberta Microfungus Collection and Herbarium</p

Characteristics of OTUs formed by the USEARCH algorithm at an identity of 0.97. Only OTUs with a minimum of 25 reads were retained.

<p>Characteristics of OTUs formed by the USEARCH algorithm at an identity of 0.97. Only OTUs with a minimum of 25 reads were retained.</p

Fusion oligonucleotide primers containing target specific sequences (White, Bruns et al., 1990, Issakainen, Jalava et al., 1999; emboldened), sequencing adapters (plain text) and the Ion Torrent key sequence (underlined).

<p>Fusion oligonucleotide primers containing target specific sequences (White, Bruns et al., 1990, Issakainen, Jalava et al., 1999; emboldened), sequencing adapters (plain text) and the Ion Torrent key sequence (underlined).</p

Next generation sequencing read statistics.

<p>Raw Reads - the total number of reads generated by each sequencing run; Full Length Reads - the total number of reads representing a full length amplicon; Dereplicated Reads - the number of unique read clusters once all identical reads were collapsed; Denoised Reads - the number of unique read clusters once reads within 1% sequence identity were combined; Total Reads Available - the total number of reads (the sum of all clusters) available for further analysis.</p