title: pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil.
abstract: OBJECTIVE
  To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined.
  
  
  METHODS
  All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Löwenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products.
  
  
  RESULTS
  Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity.
  
  
  CONCLUSIONS
  These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.
authors.affiliation: Department of Clinical Analysis and Toxicology, University of São Paulo, São Paulo, Brazil.
authors.forename: P
authors.initials: P
authors.lastname: Barco
authors.forename: R F
authors.initials: RF
authors.lastname: Cardoso
authors.forename: R D C
authors.initials: RD
authors.lastname: Hirata
authors.forename: C Q F
authors.initials: CQ
authors.lastname: Leite
authors.forename: J R
authors.initials: JR
authors.lastname: Pandolfi
authors.forename: D N
authors.initials: DN
authors.lastname: Sato
authors.forename: M L
authors.initials: ML
authors.lastname: Shikama
authors.forename: F Fiúza
authors.initials: FF
authors.lastname: de Melo
authors.forename: E M
authors.initials: EM
authors.lastname: Mamizuka
authors.forename: P A Z
authors.initials: PA
authors.lastname: Campanerut
authors.forename: M H
authors.initials: MH
authors.lastname: Hirata
mesh_terms: D000581:Amidohydrolases / Q000187:drug effects* / Q000235:genetics*; D000995:Antitubercular Agents / Q000494:pharmacology*; D001938:Brazil; D004351:Drug Resistance / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D011718:Pyrazinamide / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis; C022490:pyrazinamide deamidase