Applying Modern Molecular Microbiological Techniques to Identify Treatable Chronic Bacterial Airway Infection in Severe Asthma

<b>Intro:</b> Prior metagenomic studies in asthma are limited by inconsistent clinical phenotyping and inadequate sequencing depth for species-level bacterial identification. We hypothesise chronic bacterial infection is i) a ‘treatable trait’ whose prevalence, clinical phenotype and reliable biomarkers need definition and ii) is best characterised using Nanopore sequencing. <b>Aims/Methods:</b> To compare performance of Illumina MiSeq, Nanopore sequencing, and RT-qPCR on total DNA extracts against culture/MALDI-TOF for analysis of induced sputum samples from well-phenotyped severe asthma. <b>Results:</b> In 23 patients with with severe asthma <i>Haemophilus influenzae</i> was commonly cultured (n=8/23), and identified as the dominant bacterial species by metagenomic sequencing using MiSeq and Nanopore. Nanopore provided superior operational characterisitics and longer read lengths allowing whole bacterial genome reconstruction and greater resolution between species. Clinically significant infection was confirmed with validated <i>H.inf</i> plasmid-based RT-qPCR assay. <i>H.inf</i> culture positive patients had sputum neutrophilia and lower FeNO. <b>Conclusions:</b><i>H.inf</i> is a clinically-relevant pathogen in severe ashma and is identified reliably using molecular microbiological methods. Application of these optimised protocols in ongoing analysis of 3 large patient cohorts is allowing full characterisation of this clinical phenotype.