title: Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples.
abstract: Routine full characterization ofMycobacterium tuberculosisis 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 forM. tuberculosisWGS. 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. tuberculosiswas 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 culturedMycobacterium bovisstrain 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 ofM. tuberculosisDNA in direct samples.
authors.affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom a.votintseva@gmail.com zam@well.ox.ac.uk.
authors.forename: Antonina A
authors.initials: AA
authors.lastname: Votintseva
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Phelim
authors.initials: P
authors.lastname: Bradley
authors.affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors.forename: Louise
authors.initials: L
authors.lastname: Pankhurst
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Carlos
authors.initials: C
authors.lastname: Del Ojo Elias
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Matthew
authors.initials: M
authors.lastname: Loose
authors.affiliation: Foundation for Medical Research, Mumbai, India.
authors.forename: Kayzad
authors.initials: K
authors.lastname: Nilgiriwala
authors.affiliation: Foundation for Medical Research, Mumbai, India.
authors.forename: Anirvan
authors.initials: A
authors.lastname: Chatterjee
authors.affiliation: Regional Centre for Mycobacteriology, PHE Public Health Laboratory, Birmingham Heartlands Hospital, Birmingham, United Kingdom.|Public Health England, Wellington House, Lambeth, London, United Kingdom.
authors.forename: E Grace
authors.initials: EG
authors.lastname: Smith
authors.affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sanderson
authors.affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Walker
authors.affiliation: Microbiology Laboratory, John Radcliffe Hospital, Oxford University Hospitals NHS Trust, Oxford, United Kingdom.
authors.forename: Marcus R
authors.initials: MR
authors.lastname: Morgan
authors.affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.|Public Health England, Wellington House, Lambeth, London, United Kingdom.|The Jenner Institute, University of Oxford, Oxford, United Kingdom.
authors.forename: David H
authors.initials: DH
authors.lastname: Wyllie
authors.affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.|National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom.
authors.forename: A Sarah
authors.initials: AS
authors.lastname: Walker
authors.affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.|National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom.
authors.forename: Tim E A
authors.initials: TEA
authors.lastname: Peto
authors.affiliation: Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.|Public Health England, Wellington House, Lambeth, London, United Kingdom.|The Jenner Institute, University of Oxford, Oxford, United Kingdom.
authors.forename: Derrick W
authors.initials: DW
authors.lastname: Crook
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom a.votintseva@gmail.com zam@well.ox.ac.uk.
authors.forename: Zamin
authors.identifier: 0000-0001-8466-7547
authors.initials: Z
authors.lastname: Iqbal
mesh_terms: D000995:Antitubercular Agents / Q000627:therapeutic use*; D016680:Genome, Bacterial / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics; D006801:Humans; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics*; D019095:Point-of-Care Systems; D011718:Pyrazinamide / Q000627:therapeutic use; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000188:drug therapy / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D011718:Pyrazinamide
keywords: DNA sequencing; Mycobacterium tuberculosis; antibiotic resistance; diagnostics