Same-day diagnostic and surveillance data for tuberculosis via whole-genome sequencing of direct respiratory samples

Antonina A. Votintseva*, Phelim Bradley, Louise Pankhurst, Carlos Del Ojo Elias, Matthew Loose, Kayzad Nilgiriwala, Anirvan Chatterjee, E. Grace Smith, Nicolas Sanderson, Timothy M. Walker, Marcus R. Morgan, David Wyllie, A. Sarah Walker, Tim E.A. Peto, Derrick W. Crook, Zamin Iqbal

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

204 Citations (Scopus)

Abstract

Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

Original languageEnglish
Pages (from-to)1285-1298
Number of pages14
JournalJournal of Clinical Microbiology
Volume55
Issue number5
DOIs
Publication statusPublished - May 2017

Bibliographical note

Publisher Copyright:
Copyright © 2017 American Society for Microbiology. All Rights Reserved.

Keywords

  • Antibiotic resistance
  • DNA sequencing
  • Diagnostics
  • Mycobacterium tuberculosis

Fingerprint

Dive into the research topics of 'Same-day diagnostic and surveillance data for tuberculosis via whole-genome sequencing of direct respiratory samples'. Together they form a unique fingerprint.

Cite this