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Detection of Pyrazinamide Heteroresistance in Mycobacterium tuberculosis.

ABSTRACT: Heteroresistance is defined as the coexistence of both susceptible and resistant bacteria in a bacterial population. Previously published data show that it may occur in 9 to 57% of Mycobacterium tuberculosis isolates for various drugs. Pyrazinamide (PZA) is an important first-line drug used for treatment of both drug-susceptible and PZA-susceptible multidrug-resistant TB. Clinical PZA resistance is defined as a proportion of resistant bacteria in the isolate exceeding 10%, when the drug is no longer considered clinically effective. The ability of traditional drug susceptibility testing techniques to detect PZA heteroresistance has not yet been evaluated. The aim of this study was to compare the capacity of Bactec MGIT 960, Wayne's test, and whole-genome sequencing (WGS) to detect PZA-resistant subpopulations in bacterial suspensions prepared with different proportions of mutant strains. Both Bactec MGIT 960 and WGS were able to detect the critical level of 10% PZA heteroresistance, whereas Wayne's test failed to do so, with the latter falsely reporting highly resistant samples as PZA susceptible. Failure to detect drug-resistant subpopulations may lead to inadvertently weak treatment regimens if ineffective drugs are included, with the risk of treatment failure with the selective growth of resistant subpopulations. We need clinical awareness of heteroresistance as well as evaluation of new diagnostic tools for their capacity to detect heteroresistance in TB.

SUBMITTER: Werngren J

PROVIDER: S-EPMC8370246 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZA(r)). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZA(r) strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZA(r) strains, (iii) mutations with an unclear role found in less than 70% of PZA(r) strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZA(r); the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%. Importance: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.

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Detection of Pyrazinamide Heteroresistance in Mycobacterium tuberculosis.

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