title: Evaluation of colorimetric methods using nicotinamide for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis.
abstract: The direct detection of pyrazinamide resistance in Mycobacterium tuberculosis is sufficiently difficult that many laboratories do not attempt it. Most pyrazinamide resistance is caused by mutations that inactivate the pyrazinamidase enzyme needed to convert the prodrug pyrazinamide to its active form. We evaluated two newer and simpler methods to assess pyrazinamidase activity, the nitrate reductase and malachite green microtube assays, using nicotinamide in place of pyrazinamide. A total of 102 strains were tested by these methods and the results compared with those obtained by the classic Wayne assay. Mutations in the pncA gene were identified by sequencing the pncA genes from all isolates in which pyrazinamide resistance was detected by any of the three methods. Both the nitrate reductase and malachite green microtube assays showed sensitivities of 93.75% and specificities of 97.67%. Mutations in the pncA gene were found in 14 of 16 strains that were pyrazinamide resistant and in 1 of 4 strains that were sensitive by the Wayne assay. Both of these simple methods, used with nicotinamide, are promising and inexpensive alternatives for the rapid detection of pyrazinamide resistance in limited-resource countries.
authors.affiliation: Laboratorio Nacional de Referencia en Tuberculosis y Mycobacteria, PAHO/WHO Collaborating Centre, Instituto de Medicina Tropical Pedro Kouri (IPK), La Habana, Cuba.
authors.forename: Niuris C
authors.initials: NC
authors.lastname: Mirabal
authors.forename: Sergio L
authors.initials: SL
authors.lastname: Yzquierdo
authors.forename: Dihadenys
authors.initials: D
authors.lastname: Lemus
authors.forename: Mariela
authors.initials: M
authors.lastname: Madruga
authors.forename: Yoslaine
authors.initials: Y
authors.lastname: Milián
authors.forename: Miguel
authors.initials: M
authors.lastname: Echemendía
authors.forename: Howard
authors.initials: H
authors.lastname: Takiff
authors.forename: Anandi
authors.initials: A
authors.lastname: Martin
authors.forename: Patrick
authors.initials: P
authors.lastname: Van der Stuyf
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Palomino
authors.forename: Ernesto
authors.initials: E
authors.lastname: Montoro
mesh_terms: D000581:Amidohydrolases / Q000235:genetics; D000995:Antitubercular Agents / Q000494:pharmacology*; D003124:Colorimetry / Q000379:methods*; D024881:Drug Resistance, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D009536:Niacinamide / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D011718:Pyrazinamide / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D000995:Antitubercular Agents; D009566:Nitrates; D009942:Organometallic Compounds; C520661:malachite; D009536:Niacinamide; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis