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Background: Pleural infection (PI) is a common and complex disease which can be life threatening for immunocompromised and elderly populations. Prior antibiotic use and special bacterial nutritional requirements hamper the accuracy of bacterial identification using current clinical culture-based techniques. Consequently, PI microbiology remains unclear. Next generation sequencing (NGS) has the potential to improve identification of the total bacterial population of a complex sample. Aim: To discover and characterise the microbial patterns of PI using NGS and bioinformatics techniques. Methods: Pleural fluid samples from the “Pleural Infection Longitudinal Outcome Study” (PILOT, ISRCTN50236700, n=243) underwent bacterial DNA extraction followed by 16S rRNA NGS using Illumina MiSeq. Data were analysed with DADA2 and Phyloseq R packages. Results: Analysis showed diverse microbiological patterns for PI as 391 different pathogens were identified up to the genus level. 131 (54%) samples had one pathogen with relative abundance over 50% and 89 (36%) samples had at least three pathogens with relative abundance over 10%, suggesting a polymicrobial infection. Streptococcus pneumoniae was detected in 40 (16%) and Staphylococcus aureus in 20 (8%) samples. Discussion: We established a methodology to extract bacterial DNA from patients with PI and used it as a template to apply NGS. 16S rRNA gene NGS provides a robust method to investigate the bacteriological patterns in pleural fluid of patients with PI. Funding: National Institute for Health Research, Oxford Biomedical Research Centre

Intro: 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. Aims/Methods: 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. Results: In 23 patients with with severe asthma Haemophilus influenzae 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 H.inf plasmid-based RT-qPCR assay. H.inf culture positive patients had sputum neutrophilia and lower FeNO. Conclusions:H.inf 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.

Background: Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. However, whilst they are also activated during human viral infections, it is unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Aims and objectives: To determine whether MAIT cells play a significant role – either protective or detrimental – during influenza A infection in vivo. Methods: We used major histocompatibility complex–related protein 1 (MR1) tetramers and intracellular cytokine staining to track MAIT cell frequencies and activation during in vivo murine experimental challenge with two strains of influenza A virus in immunocompetent (C57BL/6), MAIT-cell deficient (MR1-/-) and immunodeficient (Rag2-/-γC-/-) mice. Results: MAIT cells accumulated and were activated early in infection, with upregulation of CD25, CD69 and Granzyme B peaking at 5 days post infection. Activation was modulated via cytokines interleukin (IL)-12, -15, -18 and type I interferon, independent of MR1. MR1-/- mice, which lack MAIT cells, showed enhanced body weight loss and mortality to severe (H1N1) influenza. This was ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient Rag2-/-γC-/- mice which lack T, B and NK cells. Conclusions: MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.