Potential for Erroneous Results Indicating Resistance When Using the Bactec MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to Pyrazinamide

Abstract

During susceptibility testing of 743 isolates of Mycobacterium tuberculosis to pyrazinamide (PZA) using the Bactec 960 system, 57 (7.7%) isolates showed PZA resistance. Repeat testing of resistant isolates with the Bactec 460 reference method confirmed 33 (4.4%) of these isolates as resistant, and 24 (3.2%) were susceptible. Erroneous results for resistance with the Bactec 960 were confirmed by testing the 24 discordant isolates for pyrazinamidase and mutations in the pncA gene.Pyrazinamide (PZA) is an important component of the multidrug regimen used to treat tuberculosis (TB). With increasing worldwide prevalence of drug-resistant TB, it is vital for laboratories to accurately detect resistance to first-line antimicrobials.The CLSI-recommended method for PZA testing (4) is the Bactec 460TB radiometric system (Becton Dickinson, Sparks, MD). Most laboratories have now replaced the 460TB system with the nonradiometric Bactec MGIT 960 (BT960) system (Becton Dickinson, Sparks, MD). Both methods utilize an acidified Middlebrook broth and a critical concentration of 100 g/ml.PZA is a prodrug which in Mycobacterium tuberculosis is converted to its active form, pyrazinoic acid (POA), by the enzyme pyrazinamidase (PZase) (5, 7). The absence of a functional PZase enzyme in an M. tuberculosis strain therefore indicates resistance to PZA. The pncA gene coding for PZase in M. tuberculosis has been sequenced, and mutations in this gene have been shown to be responsible for resistance to PZA (5,8,12). Tests both for PZase activity and for the detection of mutations in pncA may be utilized as alternative methods for the detection of PZA resistance in M. tuberculosis.All new M. tuberculosis isolates are tested for susceptibility to first-line drugs, including PZA, at the Public Health Laboratory, Toronto. During the report period, any isolate demonstrating PZA resistance by the BT960 was retested using the 460TB. If the 460TB PZA result was discordant, these isolates were further tested for the presence of PZase activity and mutations in the pncA gene, and testing in the BT960 was repeated.PZA susceptibility testing in the BT960 system was performed according to the manufacturer's instructions (2). Briefly, isolates of M. tuberculosis in Mycobacteria Growth Indicator Tubes (MGITs) were used as the test inocula. A drug-free control tube was inoculated with a 1:10 dilution of the inoculum, and the PZA test tube was inoculated with 0.5 ml of the inoculum and 0.1 ml of PZA. The tubes were monitored with the BT960 instrument until the growth control tube flagged positive. At that time, the instrument read the PZA test tube as either resistant (growth unit [GU] Ն 100) or susceptible (GU Ͻ 100). A blood agar purity plate from the inoculum was incubated for 3 days.PZA testing in the BT 460TB system was performed according to the manufacturer's instructions (13). Briefly, a drug-free control vial and the PZA test vial were each inoculated with 0.1 ml of inoculum from an actively growing culture. The vials were incubated and were read on...

Rapid Sequencing of the Mycobacterium tuberculosis pncA Gene for Detection of Pyrazinamide Susceptibility

“…Review of the current literature shows that 87 to 95% of the isolates determined phenotypically to be PZA resistant harbor nonsynonymous mutations in the pncA gene (5). PZA resistance in the remaining 5 to 13% of the isolates may occur through mutations in other genes, including the rpsA gene (11,12) or could be false positives of the phenotypic assays (2,13). Inclusion of the rpsA gene may improve the assay.…”

“…T uberculosis (TB) clinical trials are planned for the evaluation of new treatment regimens that include pyrazinamide (PZA); however, culture-based methods for the detection of PZA resistance are complicated because: pyrazinamidase (PZase) is active at low pH only, results are influenced by inoculum size, and the assays are difficult and time-consuming (1,2). Alternative methods to rapidly screen for PZA susceptibility need to be evaluated.…”

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

“…For PZA Radiometric Bactec MGIT 960 method that utilize an acidified Middlebrook broth with a critical concentration of 100 µg/ml [19] is commonly used method in developed countries. However, PZase enzyme activity assay using Wayne's method can be used as an alternative to BACTEC especially in areas where BACTEC is not available [29].…”

“…However, reliable testing of the susceptibility to MTB to PZA in vitro is challenging [11]. The Bactec 460 TB radiometric system [Becton Dickinson (BD), Sparks MD,USA] and Bactec MGIT 960 are the only two methods are recommended by the Clinical and Laboratory Standards Institute (CLSI) [19] and WHO for susceptibility testing to PZA, due to the difficulty in standardizing its susceptibility on solid media [11]. Most laboratories have now replaced the 460 TB system with no radiometric Bactec MGIT 960 system.…”

“…Most laboratories have now replaced the 460 TB system with no radiometric Bactec MGIT 960 system. Both methods utilize an acidified Middle brook broth and a critical concentration of 100 µg/ml [19]. DST in such liquid media is costly, especially in some regions that do not have enough economic capabilities [20].…”