The use of next generation sequencing in the diagnosis and typing of respiratory infections

Thorburn, F., Bennett, S., Modha, S. , Murdoch, D., Gunson, R. and Murcia, P. R. (2015) The use of next generation sequencing in the diagnosis and typing of respiratory infections. Journal of Clinical Virology, 69, pp. 96-100. (doi: 10.1016/j.jcv.2015.06.082) (PMID:26209388) (PMCID:PMC4533236)

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Abstract

Background: Molecular assays are the gold standard methods used to diagnose viral respiratory pathogens. Pitfalls associated with this technique include limits to the number of targeted pathogens, the requirement for continuous monitoring to ensure sensitivity/specificity is maintained and the need to evolve to include emerging pathogens. Introducing target independent next generation sequencing (NGS) could resolve these issues and revolutionise respiratory viral diagnostics. Objectives: To compare the sensitivity and specificity of target independent NGS against the current standard diagnostic test. Study design: Diagnostic RT-PCR of clinical samples was carried out in parallel with target independent NGS. NGS sequences were analyzed to determine the proportion with viral origin and consensus sequences were used to establish viral genotypes and serotypes where applicable. Results: 89 nasopharyngeal swabs were tested. A viral pathogen was detected in 43% of samples by NGS and 54% by RT-PCR. All NGS viral detections were confirmed by RT-PCR. Conclusions: Target independent NGS can detect viral pathogens in clinical samples. Where viruses were detected by RT-PCR alone the Ct value was higher than those detected by both assays, suggesting an NGS detection cut-off – Ct = 32. The sensitivity and specificity of NGS compared with RT-PCR was 78% and 80% respectively. This is lower than current diagnostic assays but NGS provided full genome sequences in some cases, allowing determination of viral subtype and serotype. Sequencing technology is improving rapidly and it is likely that within a short period of time sequencing depth will increase in-turn improving test sensitivity.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Modha, Ms Sejal and Murdoch, Dr David and Gunson, Dr Rory and Thorburn, Dr Fiona and Murcia, Professor Pablo
Authors: Thorburn, F., Bennett, S., Modha, S., Murdoch, D., Gunson, R., and Murcia, P. R.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Journal Name:Journal of Clinical Virology
Publisher:Elsevier B.V.
ISSN:1386-6532
ISSN (Online):1873-5967
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in Journal of Clinical Virology 69:96-100
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
501441Centre for Integrated VirologyMassimo PalmariniMedical Research Council (MRC)G0801822MVLS III - CENTRE FOR VIRUS RESEARCH
501442Centre for Integrated VirologyMassimo PalmariniMedical Research Council (MRC)G0801822MVLS III - CENTRE FOR VIRUS RESEARCH
501443Centre for Integrated VirologyMassimo PalmariniMedical Research Council (MRC)G0801822MVLS III - CENTRE FOR VIRUS RESEARCH