Rapid Detection of Pnca Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Clinical isolates

Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this study PCR mediated automated DNA sequencing was used to check for prevalence of Pyrazinamide (PZA) resistance among treatment failure cases of pulmonary tuberculosis. A total of 50 clinical isolates from treatment failures cases were isolated by culture and were analyzed for mutation in pncA gene by automated DNA sequencing. 39 clinical strains showed mutation in pncA gene out of 50 clinical isolates of Mycobacterium tuberculosis. The identified mutations were dispersed along the pncA gene, but some degrees of clustering of mutations were found at the following regions: Gly132, Lys96, Lys72, Pro69 (7.7%) Asp12 (10.3%) and Leu85 (15.4%). We found a significant proportion of resistance to PZA among treatment failure cases. If we are to prevent an epidemic of drug-resistant tuberculosis we must take steps to ensure that all treatment failure patients are diagnosed early and effectively treated. This will reduce treatment cost and potentially limit the spread of PZA drug-resistance.