title: Whole Genome Sequence Analysis of a Large Isoniazid-Resistant Tuberculosis Outbreak in London: A Retrospective Observational Study.
abstract: BACKGROUND
  A large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster.
  
  
  RESULTS
  We sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998-22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation.
  
  
  CONCLUSIONS
  Our study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases.
authors.affiliation: Department of Infectious Diseases and Immunity, Imperial College London, London, United Kingdom.|Centre for Immunology and Infectious Disease, Blizard Institute, Queen Mary University of London, London, United Kingdom.
authors.forename: Nicola
authors.initials: N
authors.lastname: Casali
authors.affiliation: Department of Infectious Diseases and Immunity, Imperial College London, London, United Kingdom.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Broda
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.
authors.forename: Simon R
authors.initials: SR
authors.lastname: Harris
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.
authors.forename: Julian
authors.identifier: http://orcid.org/0000-0002-7069-5958
authors.initials: J
authors.lastname: Parkhill
authors.affiliation: Public Health England National Mycobacterium Reference Laboratory, London, United Kingdom.
authors.forename: Timothy
authors.initials: T
authors.lastname: Brown
authors.affiliation: Department of Infectious Diseases and Immunity, Imperial College London, London, United Kingdom.|Public Health England National Mycobacterium Reference Laboratory, London, United Kingdom.|Departments of Microbiology and Respiratory Medicine, Barts Health NHS Trust, London, United Kingdom.
authors.forename: Francis
authors.initials: F
authors.lastname: Drobniewski
mesh_terms: D000328:Adult; D002648:Child; D004269:DNA, Bacterial; D004196:Disease Outbreaks; D016680:Genome, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008131:London / Q000453:epidemiology; D018598:Minisatellite Repeats; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D020641:Polymorphism, Single Nucleotide; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA; D014376:Tuberculosis / Q000188:drug therapy / Q000453:epidemiology* / Q000382:microbiology / Q000635:transmission; D014397:Tuberculosis, Pulmonary / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology / Q000635:transmission
chemical_list: D004269:DNA, Bacterial; D007538:Isoniazid