title: Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing.
abstract: OBJECTIVE
  To evaluate Pyrazinamide (PZA) susceptibility results obtained by phenotypic MGIT 960 TB system against enzymatic Pyrazinamidase assay and genotypic pncA gene sequencing. To find the prevalence of infections caused by M. bovis in PZA resistant M. tuberculosis complex isolates.
  
  
  METHODS
  33 consecutive PZA resistant and 30 consecutive PZA susceptible isolates reported for PZA susceptibility testing by MGIT 960 TB system were included in this study. Presence of active pyrazinamidase enzyme was sought by using the Wayne assay. The pncA gene was amplified by PCR and then sequenced to screen mutations. All the PZA resistant isolates were further spoligotyped to identify M. bovis, if present.
  
  
  RESULTS
  Of 33 PZA resistant strains by MGIT 960, 31 were Wayne assay negative and two were positive. Of the 30 susceptible PZA strains six were Wayne assay negative reporting false resistance. PncA gene sequencing revealed that 32 of the 33 MGIT PZA resistant isolates had diverse nucleotide changes scattered throughout the pncA gene (one isolate did not show any mutation). Of the 30 phenotypically susceptible isolates, 21 were wild types whilst nine isolates showed the presence of a silent mutation C-T at codon 195. Fifteen mutations found in this study has not been described earlier. Not a single isolate of M. bovis was detected among PZA resistant M. tuberculosis complex isolates.
  
  
  CONCLUSIONS
  MGIT 960 showed better concordance with sequencing results in comparison with Wayne assay. In present study, a high proportion (85%) of MDR-TB isolates from patients receiving anti-TB treatment were found to be resistant to PZA.
authors.affiliation: P.D. Hinduja National Hospital and Medical Research Centre. Mahim, Mumbai-16.
authors.forename: Shubhada
authors.initials: S
authors.lastname: Shenai
authors.forename: Camilla
authors.initials: C
authors.lastname: Rodrigues
authors.forename: Meeta
authors.initials: M
authors.lastname: Sadani
authors.forename: Nesline
authors.initials: N
authors.lastname: Sukhadia
authors.forename: Ajita
authors.initials: A
authors.lastname: Mehta
mesh_terms: D000581:Amidohydrolases / Q000187:drug effects / Q000235:genetics*; D000995:Antitubercular Agents / Q000627:therapeutic use; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D056726:Genetic Association Studies; D005838:Genotype; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D010641:Phenotype; D011718:Pyrazinamide / Q000627:therapeutic use*; D014376:Tuberculosis / Q000188:drug therapy* / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis