Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection

Background: Diversified etiology of lower respiratory tract infection renders diagnosis challenging. The mainstay microbial culture is time-consuming and constrained by variable growth requirements. In this study, we explored the use of Nanopore sequencing as a supplementary tool to alleviate this diagnostic bottleneck.

Methods: We developed a targeted Nanopore method based on amplification of bacterial 16S rRNA gene and fungal internal transcribed spacer region. The performance was compared with routine infectious disease workups on 43 respiratory specimens.

Results: Nanopore successfully identified majority of microbes (47/54, 87.04%) and 7 possible pathogens not detected by routine workups, which were attributable to the content of microbiological investigations (n = 5) and negative culture (n = 2). The average sequencing time for first target reads was 7 min (1-43 min) plus 5 h of pre-sequencing preparation.

Conclusions: The Nanopore method described here was rapid, economical and hypothesis-free, which might provide valuable hints to further microbiological follow-up for opportunistic pathogens missed or not detectable by conventional tests.