Evaluation of a biphasic media assay for pyrazinamide drug susceptibility testing of Mycobacterium tuberculosis

Objectives: Pyrazinamide is a key first-line tuberculosis drug. Reliable drug susceptibility testing (DST) data are of clinical importance, but in vitro testing is challenging since the activity of pyrazinamide is pH sensitive. The BACTEC MGIT 960 is considered the principal reference technique, but Wayne's test is an alternative, although it may be difficult to interpret. A further alternative is the use of a biphasic media assay (BMA). The objective of this work was to evaluate the BMA against the MGIT method and with screening of pncA gene mutations. Methods: Twenty strains were inoculated in tubes containing 2 mLof Löwenstein-Jensen (LJ) medium and 2 mL of semi-solid Kirchner medium with a critical concentration of 66 mg/L pyrazinamide at a pH of 5.2 or 5.5, incubated for 2 weeks and visually read. The results obtained were compared with MGIT 960 and DNA sequencing. Results: Resultswere obtained in duplicate for 19 strains. One strain failed to growon two occasions and only one resultwas available. Reproducibilitywas 95%. Eleven of the 19 strainswere susceptible to pyrazinamide, whereas 7 were resistant. One strain was susceptible initially and pyrazinamide resistant on repeat testing. At pH 5.5, two strains reported as susceptible at pH 5.2 gave resistant results. Conclusions: The BMAmight serve as a reliable low-cost DSTalternative for pyrazinamide, particularly in laboratories using locally made solid media for DST. Its major drawback is the time to result. A reliable and affordable test method for the detection of pyrazinamide resistance is needed, especially in settings where multidrug-resistant tuberculosis is increasing. Proficiency testing should be routinely introduced wherever pyrazinamide DST is performed.