MODS-Wayne: A colorimetric adaptation of the MODS assay for the detection of Mycobacterium tuberculosis pyrazinamide resistance from sputum samples.

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 to determine PZA resistance: MODS-Wayne. 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 addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/mL, sensitivity and specificity were evaluated at three time different periods of incubation (Reading 1, reading 2, reading 3)) using a composite standard (MGIT-PZA, pncA sequencing, and the classic Wayne test).MODS-Wayne was able to detect PZA resistance with a sensitivity and specificity of 92.7% and 99.3% respectively at Reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low resource countries, where TB is a major public health problem.