MODS-Wayne, a Colorimetric Adaptation of the Microscopic-Observation Drug Susceptibility (MODS) Assay for Detection of Mycobacterium tuberculosis Pyrazinamide Resistance from Sputum Samples

Abstract

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 incubation (reading 1, reading 2, and reading 3) using a composite reference 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, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an 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.

Keywords: MODS; pyrazinamide; pyrazinoic acid; sputum; tuberculosis.

FIG 1

MODS-Wayne plate design. Three samples were evaluated in each plate. One control well was included for every sample for each reading (R1, R2, and R3). Red arrows represent the 3 days of incubation after pyrazinamide (PZA) addition. Ferrous ammonium sulfate (SAF) was added after the incubation with PZA.

FIG 2

MODS-Wayne results. The intensity of the color is enough to allow discrimination between a positive (presence of pink color) result (A), and a negative (absence of pink color) result (B), indicating pyrazinamide (PZA) susceptibility and resistance, respectively.

FIG 3

Sample distribution by PZA susceptibility profile. Distribution displayed according to the results from Bactec MGIT 960 PZA, pncA sequencing, and classic Wayne test. For stacked numerals, the number of PZA-susceptible isolates is given above the number of PZA-resistant isolates.