title: Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study.
abstract: BACKGROUND
  Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows.
  
  
  METHODS
  We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics.
  
  
  RESULTS
  Compared with routine results, WGS predicted species with 93% (95% CI 90-96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91-95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10-26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6-10), a median of 21 days (IQR 14-32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21-44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows.
  
  
  CONCLUSIONS
  We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis.
  
  
  BACKGROUND
  National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.
authors.affiliation: Microbiology and Infectious Diseases, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK. Electronic address: louise.pankhurst@ndm.ox.ac.uk.
authors.forename: Louise J
authors.initials: LJ
authors.lastname: Pankhurst
authors.affiliation: Microbiology and Infectious Diseases, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: Carlos
authors.initials: C
authors.lastname: Del Ojo Elias
authors.affiliation: Microbiology and Infectious Diseases, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: Antonina A
authors.initials: AA
authors.lastname: Votintseva
authors.affiliation: Microbiology and Infectious Diseases, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Walker
authors.affiliation: Brighton and Sussex University Hospitals NHS Trust, Brighton, UK.
authors.forename: Kevin
authors.initials: K
authors.lastname: Cole
authors.affiliation: Department of Computer Science, University of Oxford, Oxford, UK.
authors.forename: Jim
authors.initials: J
authors.lastname: Davies
authors.affiliation: Health Economics Research Centre, Nuffield Department of Population Health, University of Oxford, Oxford, UK.
authors.forename: Jilles M
authors.initials: JM
authors.lastname: Fermont
authors.affiliation: Leeds Teaching Hospitals NHS Trust, Leeds, UK.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Gascoyne-Binzi
authors.affiliation: Molecular Mycobacteriology, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Schleswig-Holstein, Germany.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kohl
authors.affiliation: British Columbia Public Health Microbiology and Reference Laboratory, Vancouver, Canada.
authors.forename: Clare
authors.initials: C
authors.lastname: Kong
authors.affiliation: Université de Lille, Centre national de la recherche scientifique Unité mixte de recherche 8204, Institut national de la santé et de la recherche médicale U1019, Centre Hospitalier Universitaire, and Center for Infection and Immunity of Lille, Institut Pasteur de Lille, Lille, France.
authors.forename: Nadine
authors.initials: N
authors.lastname: Lemaitre
authors.affiliation: Molecular Mycobacteriology, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Schleswig-Holstein, Germany; German Center for Infection Research, Borstel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Niemann
authors.affiliation: Brighton and Sussex University Hospitals NHS Trust, Brighton, UK.
authors.forename: John
authors.initials: J
authors.lastname: Paul
authors.affiliation: Department of Clinical Microbiology Trinity College Dublin and Irish Mycobacteria Reference Laboratory, St James's Hospital, Dublin, Ireland.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Rogers
authors.affiliation: Department of Clinical Microbiology Trinity College Dublin and Irish Mycobacteria Reference Laboratory, St James's Hospital, Dublin, Ireland.
authors.forename: Emma
authors.initials: E
authors.lastname: Roycroft
authors.affiliation: Public Health England Regional Centre for Mycobacteriology, Birmingham Heartlands Hospital NHS Foundation Trust, Birmingham, UK.
authors.forename: E Grace
authors.initials: EG
authors.lastname: Smith
authors.affiliation: Université de Lille, Centre national de la recherche scientifique Unité mixte de recherche 8204, Institut national de la santé et de la recherche médicale U1019, Centre Hospitalier Universitaire, and Center for Infection and Immunity of Lille, Institut Pasteur de Lille, Lille, France; Genoscreen, Lille, France.
authors.forename: Philip
authors.initials: P
authors.lastname: Supply
authors.affiliation: British Columbia Public Health Microbiology and Reference Laboratory, Vancouver, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: Tang
authors.affiliation: Leeds Teaching Hospitals NHS Trust, Leeds, UK.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Wilcox
authors.affiliation: Health Economics Research Centre, Nuffield Department of Population Health, University of Oxford, Oxford, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Wordsworth
authors.affiliation: Microbiology and Infectious Diseases, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK; Public Health England, Oxford, UK.
authors.forename: David
authors.initials: D
authors.lastname: Wyllie
authors.affiliation: Public Health England Regional Centre for Mycobacteriology, Birmingham Heartlands Hospital NHS Foundation Trust, Birmingham, UK.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: Microbiology and Infectious Diseases, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: Derrick W
authors.initials: DW
authors.lastname: Crook
mesh_terms: D000995:Antitubercular Agents; D002170:Canada; D015331:Cohort Studies; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D061345:Early Medical Intervention; D005602:France; D005858:Germany; D006801:Humans; D007494:Ireland; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011446:Prospective Studies; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis*; D006113:United Kingdom
chemical_list: D000995:Antitubercular Agents