Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis. However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 _g/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, c...

Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 μg/ml, sensitivity and specificity were evaluated at three different periods of incub...