Nitrate reductase assay for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide

Abstract

Objectives: The purpose of this study was to develop the nitrate reductase assay (NRA) for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide resistance as a marker of pyrazinamide resistance in Lo¨wenstein–Jensen (LJ) medium at neutral pH. Methods: We tested 68 M. tuberculosis isolates using nicotinamide at three different concentrations (1000, 500 and 250 mg/L) by the NRA in LJ medium and compared the results with those obtained with the BACTEC 460-TB or the BACTEC MGIT 960 as reference standard methods. Mutations in the pncA gene were detected by DNA sequencing of the pyrazinamide-resistant isolates. Results: Out of 34 M. tuberculosis pyrazinamide-resistant isolates, 31 were found to be resistant to 1000 and 500 mg/L nicotinamide giving sensitivity and specificity of 91% and 94%, respectively. At 250 mg/L nicotinamide, the sensitivity and specificity decreased to 91% and 71%, respectively. Results were obtained in an average of 10 days. Based on these results, a tentative breakpoint concentration of 500 mg/L nicotinamide was defined. DNA sequencing of the pncA gene detected mutations in 26 out of 34 M. tuberculosis isolates resistant to pyrazinamide. Conclusions: The NRA using nicotinamide to detect resistance to pyrazinamide in LJ medium is a rapid and accurate method that could be useful in limited-resource countries where the BACTEC 460-TB or the BACTEC MGIT 960 system is not available.