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J Clin Microbiol. 2017 May;55(5):1285-1298. doi: 10.1128/JCM.02483-16. Epub 2017 Mar 8.

Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.

Author information

1
Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom a.votintseva@gmail.com zam@well.ox.ac.uk.
2
Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
3
Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
4
School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
5
Foundation for Medical Research, Mumbai, India.
6
Regional Centre for Mycobacteriology, PHE Public Health Laboratory, Birmingham Heartlands Hospital, Birmingham, United Kingdom.
7
Public Health England, Wellington House, Lambeth, London, United Kingdom.
8
Microbiology Laboratory, John Radcliffe Hospital, Oxford University Hospitals NHS Trust, Oxford, United Kingdom.
9
The Jenner Institute, University of Oxford, Oxford, United Kingdom.
10
National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom.
11
Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom a.votintseva@gmail.com zam@well.ox.ac.uk.

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.

KEYWORDS:

DNA sequencing; Mycobacterium tuberculosis; antibiotic resistance; diagnostics

PMID:
28275074
PMCID:
PMC5405248
DOI:
10.1128/JCM.02483-16
[Indexed for MEDLINE]
Free PMC Article

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