Comparative evaluation of Lowenstein-Jensen proportion method, BacT/ALERT 3D system, and enzymatic pyrazinamidase assay for pyrazinamide susceptibility testing of Mycobacterium tuberculosis

Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug because of its sterilizing activity against semi-dormant tubercle bacilli. In spite of very high in vivo activity, in vitro activity is not apparent unless acidic environment is available, which makes the PZA susceptibility testing difficult by conventional methods. The present study was, therefore, planned to assess the performance of colorimetric BacT/ALERT 3D system and compare the results with those by conventional tests i.e. Lowenstein-Jensen (LJ) proportion method (pH 4.85) and Wayne's Pyrazinamidase (PZase) assay, using 107 clinical isolates. The concordance among all these tests was 89.71% after first round of testing which reached 92.52% after resolving the discordant results by retesting. Prolonged incubation of PZase tube upto 10 days was found to increase the specificity of PZase test. The concordance between LJ proportion-BacT/ALERT 3D, LJ proportion-PZase assay, and BacT/ALERT 3D-PZase assay was found to be 99.06, 93.46, and 92.52% respectively. Taking LJ results as the gold standard, the sensitivities of BacT/ALERT 3D and PZase assay were 100 and 82.85% respectively while specificity was 98.61% for both the tests. The difference between the sensitivities of BacT/ALERT 3D and PZase assay was significant (P value 0.025). The mean turnaround time for detection of resistant and susceptible results by BacT/ALERT 3D was 8.04 and 11.32 days respectively. While the major limitations associated with PZase assay and LJ proportion method are lower sensitivity in previously treated patients and longer time requirement respectively, the BacT/ALERT 3D system was found to be rapid, highly sensitive and specific