title: Genome sequencing reveals novel deletions associated with secondary resistance to pyrazinamide in MDR Mycobacterium tuberculosis.
abstract: OBJECTIVE
  Detection of pyrazinamide resistance in Mycobacterium tuberculosis isolates presents significant challenges in settings with no dominant clonal lineages, such as Australia. We assessed the utility of WGS versus standard PCR amplification assays for the characterization of pyrazinamide resistance in MDR-TB isolates identified in New South Wales, Australia, over an 8 year period.
  
  
  METHODS
  PCR amplicon sequencing was used to identify molecular markers associated with antibiotic resistance in pyrazinamide-resistant MDR-TB isolates recovered by the New South Wales Mycobacterium Reference Laboratory between 2007 and 2014. WGS was subsequently performed on two isolates for which pncA amplification failed.
  
  
  RESULTS
  WGS identified two novel genomic deletions associated with in vitro resistance to pyrazinamide in MDR-TB. One isolate also carried a second deletion involving the genes dfrA and thyA associated with resistance to para-aminosalicylic acid.
  
  
  CONCLUSIONS
  Steadily decreasing sequencing costs are increasing the appeal of WGS as an alternative approach for detecting complex patterns of pyrazinamide resistance in MDR-TB.
authors.affiliation: Centenary Institute, Sydney, Australia Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, Australia NSW Mycobacterium Reference Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research-Pathology West, Sydney, Australia Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Western Sydney Local Health District, Sydney, Australia e.martinez@centenary.org.au maria.martinezdiaz@sydney.edu.au.
authors.forename: Elena
authors.initials: E
authors.lastname: Martinez
authors.affiliation: Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, Australia NSW Mycobacterium Reference Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research-Pathology West, Sydney, Australia Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Western Sydney Local Health District, Sydney, Australia.
authors.forename: Nadine
authors.initials: N
authors.lastname: Holmes
authors.affiliation: NSW Mycobacterium Reference Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research-Pathology West, Sydney, Australia Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Western Sydney Local Health District, Sydney, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Jelfs
authors.affiliation: Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, Australia NSW Mycobacterium Reference Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research-Pathology West, Sydney, Australia Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Western Sydney Local Health District, Sydney, Australia.
authors.forename: Vitali
authors.initials: V
authors.lastname: Sintchenko
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D009517:New South Wales; D016133:Polymerase Chain Reaction; D011718:Pyrazinamide / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D011718:Pyrazinamide
keywords: multidrug-resistant tuberculosis; pyrazinamide resistance; whole-genome sequencing