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

Abstract

Pyrazinamide is important in the treatment of tuberculosis. Unfortunately, the diagnosis of pyrazinamide resistance is hampered by technical difficulties. We hypothesized that mutation analysis combined with the mycobacterial growth indicator tube (MGIT) phenotypic method would be a good predictor of pyrazinamide resistance. We prospectively analyzed 1,650 M. tuberculosis isolates referred to our tuberculosis reference laboratory in 2008 and 2009. In our laboratory, the MGIT 960 system was used for pyrazinamide resistance screening. If a pyrazinamide-resistant strain was detected, we performed a pncA gene mutation analysis. A second MGIT 960 susceptibility assay was performed afterwards to evaluate the accuracy of the pncA mutation analysis to detect true-or false-positive MGIT results. We observed pyrazinamide resistance in 69 samples using the first MGIT 960 analysis. In a second MGIT 960 analysis, 47 of the 69 samples proved susceptible (68% false positivity). Sensitivity of nonsynonymous pncA mutations for detecting resistant isolates was 73% (95% confidence interval [CI], 61% to 73%), and specificity was 100% (95% CI, 95% to 100%). A diagnostic algorithm incorporating phenotypic and molecular methods would have a 100% positive predictive value for detecting pyrazinamide-resistant isolates, indicating that such an algorithm, based on both methods, is a good predictor for pyrazinamide resistance in routine diagnostics.

Role of pncA and rpsA Gene Sequencing in Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from Southern China

“…None of the isolates with identical pncA mutations were epidemiologically linked, so the PZA-resistant strains might not have been obtained from transmission. For the mutations in pncA, 23 types of point mutations had been reported previously (8,9,21,(26)(27)(28)(29), and to our best knowledge, 10 novel types of mutations (13 strains) were first found in this study. The latter included all six types of the deletion mutations (L27, 91E, 111E, 122Q, 130-132VVG, and 131V) and four types of amino acid substitutions (D8A, S18P, F58S, and E174GϩM175R double mutation) in PZases as summarized in Table 1.…”

Mycobacterium tuberculosis pncA Polymorphisms That Do Not Confer Pyrazinamide Resistance at a Breakpoint Concentration of 100 Micrograms per Milliliter in MGIT

“…A good correlation (sensitivity of Ͼ90%) between pncA polymorphisms in circulating isolates and phenotypic susceptibility results have been observed for PZA (15)(16)(17)(18). Incomplete correlation of pncA molecular results with culture-based PZA testing has been ascribed to poor reproducibility of the phenotypic test or the presence of alternative genetic mechanisms for resistance, including polymorphisms in the rpsA gene (19,20). Additionally, a few pncA mutations have been reported in the absence of phenotypic resistance (15), but the role of such mutations in PZA resistance has not been thoroughly investigated.…”

Prevention of False Resistance Results Obtained in Testing the Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Bactec MGIT 960 System Using a Reduced Inoculum

“…In this context, the Bactec 460 radiometric system (Becton, Dickinson and Company, Sparks, MD), currently the "gold standard" method for PZA susceptibility (13), has been withdrawn from the market, and most clinical laboratories have already replaced or soon will replace it with the nonradiometric Bactec MGIT 960 (M960) system (Becton, Dickinson and Company, Sparks, MD). Although both methods utilize an acidified, 7H9-like broth and a modified proportion method with a critical concentration of 100 g/ml, some recently published papers have reported unprecedented false resistance rates with the M960 system (14)(15)(16). Alternatively, PZA susceptibility may also be determined by testing cultured strains for PZase activity or by detecting pncA mutations.…”