Susceptibility testing of Mycobacterium tuberculosis to pyrazinamide

Heifets, Leonid

doi:10.1099/0022-1317-51-1-11

Cited by 31 publications

(7 citation statements)

References 8 publications

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“…As shown in Table 1, there were six isolates recognized as PZA resistant by the Bactec test, but did not have any mutation in their gene sequences. As suggested before [5], [26], these six isolates might not be resistant to PZA at all. Without considering these 6 isolates, the agreement would be 88% (15/17) for PZA resistant strains.…”

Section: Discussionmentioning

confidence: 59%

“…Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug used in combination with isoniazid, rifampicin and ethambutol in the short-course treatment regimens recommended by the WHO [3]. While the procedures for susceptibility testing of the first-line and second-line drugs have been well standardized in both liquid and solid media, the PZA susceptibility test is challenging because the bactericidal activity of PZA is optimal only in an acid environment (pH 5.5–5.6) that inhibits the growth of M. tuberculosis isolates [4], [5].…”

Section: Introductionmentioning

confidence: 99%

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Rapid Colorimetric Testing for Pyrazinamide Susceptibility of M. tuberculosis by a PCR-Based In-Vitro Synthesized Pyrazinamidase Method

et al. 2011

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Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug. But PZA susceptibility test is challenging because PZA activity is optimal only in an acid environment that inhibits the growth of M. tuberculosis. For current phenotypic methods, inconsistent results between different labs have been reported. Direct sequencing of pncA gene is being considered as an accurate predictor for PZA susceptibility, but this approach needs expensive sequencers and a mutation database to report the results. An in-vitro synthesized Pyrazinamidase (PZase) assay was developed based on PCR amplification of pncA gene and an in vitro wheat germ system to express the pncA gene into PZase. The activity of the synthesized PZase was used as an indicator for PZA susceptibility. Fifty-one clinical isolates were tested along with pncA sequencing and the BACTEC MGIT 960 methods. The in-vitro synthesized PZase assay was able to detect PZA susceptibility of M. tuberculosis within 24 h through observing the color difference either by a spectrometer or naked eyes. This method showed agreements of 100% (33/33) and 88% (14/16) with the pncA sequencing method, and agreements of 96% (27/28) and 65% (15/23) with the BACTEC MGIT 960 method, for susceptible and resistant strains, respectively. The novel in-vitro synthesized PZase assay has significant advantages over current methods, such as its fast speed, simplicity, no need for expensive equipment, and the potentials of being a direct test, predicting resistance level and easy reading results by naked eyes. After confirmation by more clinical tests, this method may provide a radical change to the current PZA susceptibility assays.

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Section: Discussionmentioning

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Rapid Colorimetric Testing for Pyrazinamide Susceptibility of M. tuberculosis by a PCR-Based In-Vitro Synthesized Pyrazinamidase Method

et al. 2011

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“…False resistance results are known to occur with this assay (1,2), which may be the result of alkalinization of the medium due to a high inoculum size or the presence of bovine serum albumin (3). The uses of alternative susceptibility breakpoint concentrations or different media are additional factors that may contribute to disparities in PZA susceptibility results (4)(5)(6).…”

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confidence: 99%

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

et al. 2015

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c Sequencing of the Mycobacterium tuberculosis pncA gene allows for pyrazinamide susceptibility testing. We summarize data on pncA polymorphisms that do not confer resistance at a susceptibility breakpoint of 100 g/ml pyrazinamide in MGIT within a cohort of isolates from South Africa and the U.S. Centers for Disease Control and Prevention. C ulture-based drug susceptibility testing (DST) using Bactec MGIT 960 PZA medium (Becton Dickinson, Sparks, MD) at 100 g/ml is the current gold standard test for pyrazinamide (PZA) resistance (1). False resistance results are known to occur with this assay (1, 2), which may be the result of alkalinization of the medium due to a high inoculum size or the presence of bovine serum albumin (3). The uses of alternative susceptibility breakpoint concentrations or different media are additional factors that may contribute to disparities in PZA susceptibility results (4-6). A further limitation of culture-based methods is the long turnaround time, which can exceed 20 days (7-9). Molecular methods offer an alternative strategy for the detection of PZA susceptibility. These methods detect polymorphisms in the 561-bp pncA gene, which encodes the pyrazinamidase (PZase) enzyme that is responsible for conversion of PZA (prodrug) to pyrazinoic acid (active form) (10). More than 325 polymorphisms (single nucleotide polymorphisms [SNPs], insertions, and deletions) throughout the length of the pncA gene and in the promoter region have been described, complicating molecular detection (11)(12)(13)(14). 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. This study aimed to collate data on pncA polymorphisms in clinical isolates that do not confer resistance at a susceptibility breakpoint of 100 g/ml PZA. To capture the spectrum of pncA mutations not associated with phenotypic PZA resistance, we performed a comprehensive literature search. In the 77 papers reporting genotypic and phenotypic PZA susceptibility (see Table S1 in the supplemental material), 77 different pncA polymorphisms in 71 different codons were reported to have a PZA-susceptible phenotype using either Bactec MGIT 960 PZA (Becton Dickinson, Sparks, MD), Bactec 460 PZA (Becton Dickinson, Sparks, MD) or the Wayne assay (21). Forty-seven (61%) of these polymorphisms have also been reported in PZA-resistant isolates. These inconsistent phenotypic results may be due to technical difficulties of phenotypic PZA assays or MICs that are close to the breakpoint. Another 26 (33.7%...

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“…Negatively charged POA (pK a of 2.9) is then eliminated from the cytoplasm by an unidentified mechanism. Once in the mildly acidic extracellular milieu (typically pH ϳ6 for PZA susceptibility assays [25]), a small proportion of extracellular POA becomes protonated and passively diffuses back into the cytoplasm. In the slightly alkaline cytoplasm (pH 7.4) (26), POA becomes deprotonated, completing the cycle.…”

mentioning

confidence: 99%

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis

Peterson

Rosen

Dillon

et al. 2015

Antimicrob Agents Chemother

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Pyrazinamide (PZA) is a first-line antitubercular drug for which the mode of action remains unresolved. Mycobacterium tuberculosis lacks measurable susceptibility to PZA under standard laboratory growth conditions. However, susceptibility to this drug can be induced by cultivation of the bacilli in an acidified growth medium. Previous reports suggested that the active form of PZA, pyrazinoic acid (POA), operates as a proton ionophore that confers cytoplasmic acidification when M. tuberculosis is exposed to an acidic environment. In this study, we demonstrate that overexpression of the PZA-activating enzyme PncA can confer PZA susceptibility to M. tuberculosis under neutral and even alkaline growth conditions. Furthermore, we find that wild-type M. tuberculosis displays increased susceptibility to POA relative to PZA in neutral and alkaline media. Utilizing a strain of M. tuberculosis that expresses a pH-sensitive green fluorescent protein (GFP), we find that unlike the bona fide ionophores monensin and carbonyl cyanide 3-chlorophenylhydrazone, PZA and POA do not induce rapid uncoupling or cytoplasmic acidification under conditions that promote susceptibility. Thus, based on these observations, we conclude that the antitubercular action of POA is independent of environmental pH and intrabacterial acidification.

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Susceptibility testing of Mycobacterium tuberculosis to pyrazinamide

Cited by 31 publications

References 8 publications

Rapid Colorimetric Testing for Pyrazinamide Susceptibility of M. tuberculosis by a PCR-Based In-Vitro Synthesized Pyrazinamidase Method

Rapid Colorimetric Testing for Pyrazinamide Susceptibility of M. tuberculosis by a PCR-Based In-Vitro Synthesized Pyrazinamidase Method

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

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis

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