title: Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study.
abstract: BACKGROUND
  Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis.
  
  
  METHODS
  Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance.
  
  
  RESULTS
  We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7-93·7) and 98·4% specificity (98·1-98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes.
  
  
  CONCLUSIONS
  A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early.
  
  
  BACKGROUND
  Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.
authors.affiliation: Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK. Electronic address: timothy.walker@ndm.ox.ac.uk.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Walker
authors.affiliation: Molecular Mycobacteriology, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Borstel, Germany.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kohl
authors.affiliation: Centre for Tuberculosis, National Institute for Communicable Diseases, Johannesburg, South Africa.
authors.forename: Shaheed V
authors.initials: SV
authors.lastname: Omar
authors.affiliation: Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
authors.forename: Jessica
authors.initials: J
authors.lastname: Hedge
authors.affiliation: Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
authors.forename: Carlos
authors.initials: C
authors.lastname: Del Ojo Elias
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
authors.forename: Phelim
authors.initials: P
authors.lastname: Bradley
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
authors.forename: Zamin
authors.initials: Z
authors.lastname: Iqbal
authors.affiliation: Molecular Mycobacteriology, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Borstel, Germany; German Center for Infection Research, Borstel Site, Borstel, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Feuerriegel
authors.affiliation: Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, UK.
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Niehaus
authors.affiliation: Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wilson
authors.affiliation: Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, UK.
authors.forename: David A
authors.initials: DA
authors.lastname: Clifton
authors.affiliation: Microbiology Services, Public Health England, London, UK.
authors.forename: Georgia
authors.initials: G
authors.lastname: Kapatai
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
authors.forename: Camilla L C
authors.initials: CLC
authors.lastname: Ip
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
authors.forename: Rory
authors.initials: R
authors.lastname: Bowden
authors.affiliation: Public Health England National Mycobacterial Reference Laboratory, Queen Mary's School of Medicine and Dentistry, London, UK; Department of Infectious Diseases, Imperial College, London, UK.
authors.forename: Francis A
authors.initials: FA
authors.lastname: Drobniewski
authors.affiliation: Genoscreen, Lille, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Allix-Béguec
authors.affiliation: Genoscreen, Lille, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Gaudin
authors.affiliation: Wellcome Trust Sanger Institute, Hinxton, UK.
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Institute for Epidemiology, University Medical Hospital Schleswig-Holstein, Airway Research Center North, Kiel, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Diel
authors.affiliation: Genoscreen, Lille, France; Centre National de la Recherche Scientifique, Lille, France; INSERM, Université de Lille, and Campus de l'Institut Pasteur de Lille, Center for Infection and Immunity of Lille, Lille, France.
authors.forename: Philip
authors.initials: P
authors.lastname: Supply
authors.affiliation: Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK; National Institute of Health Research Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, UK.
authors.forename: Derrick W
authors.initials: DW
authors.lastname: Crook
authors.affiliation: Public Health England West Midlands Public Health Laboratory, Heartlands Hospital, Birmingham, UK.
authors.forename: E Grace
authors.initials: EG
authors.lastname: Smith
authors.affiliation: Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK; National Institute of Health Research Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, UK.
authors.forename: A Sarah
authors.initials: AS
authors.lastname: Walker
authors.affiliation: Centre for Tuberculosis, National Institute for Communicable Diseases, Johannesburg, South Africa; Department of Medical Microbiology, University of Pretoria, Pretoria, South Africa.
authors.forename: Nazir
authors.initials: N
authors.lastname: Ismail
authors.affiliation: Molecular Mycobacteriology, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Borstel, Germany; German Center for Infection Research, Borstel Site, Borstel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Niemann
authors.affiliation: Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK; National Institute of Health Research Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, UK.
authors.forename: Tim E A
authors.initials: TEA
authors.lastname: Peto
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA / Q000379:methods*; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents