Colorimetric Methods for Rapid Determination of Pyrazinamide Resistance

Abstract

Background: Because pyrazinamide (PZA) is only effective for Mycobacterium tuberculosis at an acidic pH, susceptibility tests are more difficult to perform than those for other anti-tuberculosis (TB) drugs. The purpose of our work was to investigate the effectiveness of colorimetric methods to detect PZA susceptibility and to detect pncA gene mutations in resistant isolates by sequence analysis. Methods: In this study, 30 clinical isolates and 2 reference isolates were used, 15 of which were resistant to PZA. The PZA susceptibility of all the isolates was determined by the BACTEC MGIT 960 reference method. As colorimetric methods, Resazurin Microtiter Assay (REMA), Nitrate Reductase Assay (NRA), Malachite Green Decolorization Assay (MGDA), and Crystal Violet Decolorization Assay (CVDA) methods were included in the study. In addition, mutations in the pncA gene were investigated using sequence analysis in PZA-resistant isolates. Results: As a result of the comparison of the colorimetric methods with the reference method, agreement was determined as 93.3% in REMA and NRA, 90% in MGDA, and 93.3% in CVDA. In 13 of 15 resistant isolates, the pncA gene mutation was detected by sequence analysis. Conclusions: As a result of the work, the results from the colorimetric methods were found to be at a high level of concordance with the reference method. They are also inexpensive and easily applicable methods.