Comparative Evaluation of Löwenstein-Jensen Proportion Method, BacT/ALERT 3D System, and Enzymatic Pyrazinamidase Assay for Pyrazinamide Susceptibility Testing of Mycobacterium tuberculosis

Abstract

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

Validation of pncA Gene Sequencing in Combination with the Mycobacterial Growth Indicator Tube Method To Test Susceptibility of Mycobacterium tuberculosis to Pyrazinamide

“…We repeated analyses to minimize this error. We did not use the Wayne method to assess pyrazinamide activity to determine whether the mutations led to an impairment of pyrazinamidase activity, because at the time we undertook our study this assay was considered less sensitive for diagnosing pyrazinamide resistance (22). Moreover, a large quantity of bacilli is mandatory for this assay, leading to falsely resistant results.…”

Wayne’s Assay: A Screening Method for Indirect Detection of Pyrazinamide Resistance in Mycobacterium Tuberculosis Clinical Isolates

“…Authors showed that 9.5% (16/169) of the PZAresistant strains were determined to be positive to PZase activity testing, indicating that, for these strains, PZA resistance was not caused by the inactivation of PZase but by some other unknown cause(s) [11]. Other studies also detected PZase activity in PZAresistant strains [24,36,37]. Cui Z study showed only 1% (3/260) of the PZA-susceptible strains negative according to the Wayne's assay, probably due to the weak activity of PZase or to the small quantity of MTB, which makes it difficult to observe the Wayne assay color change with the naked eye at 10 days [11].…”

“…DST in such liquid media is costly, especially in some regions that do not have enough economic capabilities [20]. Several other DST methods have been developed, including the molecular drug susceptibility test (mDST) based on the detection of a pncA mutation, the PZase activity assay, and colorimetric methods based on a MIC or redox indicator [21][22][23][24][25][26].…”

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“…The need for rapid methods of the diagnosis and determination of drug susceptibility testing (DST) is very important. While the procedures for DST of the most of the first-line and second-line antituberculosis drugs have been well standardized in both liquid and solid media, the main problem comes with the PZA DST, is the requirement of acidic pH for PZA activity [17]. It also becomes difficult because of the acidic pH of culture medium restricts the growth of organism.…”

“…In addition, the use of large inoculum sizes results in the release of NH 3 that leads to increased pH and inactivated PZA [15]. The application of various rapid phenotypic methods such as radiometric BACTEC 460, flourimetric Mycobacteria Growth Indicator Tube (MGIT) 960 (Becton Dickinson), ESP Culture System II (Trek Diagnostic Systems, West-lake, OH), and the colorimetric BacT/ALERT 3D system (bioMerieux Inc., Durham, NC), previously designated MB/BACT (Organon Teknika, Boxtels, The Netherlands) has been reported to be very useful for rapid and reliable susceptibility testing of M. tuberculosis isolates [17]. Presently many laboratories performing PZA liquid DST by the non-radiometric, fully automated, continuous-monitoring MGIT 960 system (Becton Dickinson) that gives reliable result with the turnaround time of 15-22 days [18,19].…”