Evaluating Nanopore Sequencing as a Respiratory Virus Diagnostic Tool for the Prehospital Setting.

Abstract

Introduction: Upper respiratory tract infections are a strain on military that results in lost duty days and an overall reduced readiness of the force. Improved diagnostic testing would enable better force health protection measures and earlier treatment of illness. Lightweight portable devices are preferred for diagnostic testing in austere environments where they are sometimes needed during military deployment. Current diagnostic testing is targeted to specific pathogens despite multiple pathogens that present with similar symptoms. In practice the pathogens that cause upper respiratory tract infections often go unidentified, which could be improved using agnostic or semi-agnostic diagnostic testing. Here, we performed an evaluation of shotgun metagenomic sequencing using the Oxford Nanopore Technologies (ONT) Rapid Sequencing Kit as a method for diagnostic testing of upper respiratory tract infections. This sequencing library preparation kit was chosen because of its ease of use and compatibility with the ONT MinION, a lightweight portable sequencer.

Materials and methods: Samples from patients with symptoms of upper respiratory tract infections were collected at Wilford Hall Ambulatory Surgical Center under an approved IRB protocol. Nasal rinse samples from 59 study participants were tested using the BioFire FilmArray Respiratory 2.1 Panel as well as shotgun metagenomic sequencing using ONT Rapid Sequencing Kit and ONT R9.4.1 flow cells.

Results: A mixture of various viral pathogens was present among the 59 samples used in this study. We observed high specificity and modest sensitivity to detect the identified pathogens using shotgun metagenomic sequencing. Shotgun metagenomic sequencing detected additional pathogens that were missed by the BioFire FilmArray Respiratory 2.1 Panel, which are discussed. Lastly, we observe modest evidence of nonuniformity of the proportion of reads belonging to the pathogen during the duration of sequencing runs, which has implications for improving sensitivity by increasing the amount of sequencing performed.

Conclusions: Overall, ONT Rapid Sequencing Kit combined with alignment to a known panel of pathogens has shown great potential utility in our hands for quickly and accurately identifying viral respiratory pathogens. This, combined with its ease of use and portability, makes it a great candidate for further research and development toward a deployable agnostic diagnostic testing platform.