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

Thorburn, Fiona; Bennett, Susan; Modha, Sejal; Murdoch, David; Gunson, Rory; Murcia, Pablo R.

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.