Evaluation of colorimetric methods using nicotinamide for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis.
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.
Mirabal
Niuris C
NC
Laboratorio Nacional de Referencia en Tuberculosis y Mycobacteria, PAHO/WHO Collaborating Centre, Instituto de Medicina Tropical Pedro Kouri (IPK), La Habana, Cuba.
Yzquierdo
Sergio L
SL
Lemus
Dihadenys
D
Madruga
Mariela
M
Milián
Yoslaine
Y
Echemendía
Miguel
M
Takiff
Howard
H
Martin
Anandi
A
Van der Stuyf
Patrick
P
Palomino
Juan Carlos
JC
Montoro
Ernesto
E
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
D000995:Antitubercular Agents; D009566:Nitrates; D009942:Organometallic Compounds; C520661:malachite; D009536:Niacinamide; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis