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Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis Isolates.

ABSTRACT: Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the Bactec MGIT 960 system is unreliable, and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA-resistant Mycobacterium tuberculosis isolates. Sanger sequencing and/or whole-genome sequencing were performed to detect mutations in pncA, rpsA, panD, and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed using the Bactec MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA-resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in the pncA, rpsA, and panD genes. Of the 40 isolates, 16 (40%) were found to be susceptible using the reduced inoculum method (i.e., false resistance). No mutations were detected in two PZA-resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, East African Indian strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.

SUBMITTER: Mok S

PROVIDER: S-EPMC8284449 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis isolates

Project description:Pyrazinamide Susceptibility Testing in M. tuberculosis

| PRJEB42011 | ENA

Evaluation of methods for testing the susceptibility of clinical Mycobacterium tuberculosis isolates to pyrazinamide.

Project description:Pyrazinamide (PZA) is a first-line antituberculosis (anti-TB) drug capable of killing nonreplicating, persistent Mycobacterium tuberculosis. However, reliable testing of the susceptibility of M. tuberculosis to PZA is challenging. Using 432 clinical M. tuberculosis isolates, we compared the performances of five methods for the determination of M. tuberculosis susceptibility to PZA: the MGIT 960 system, the molecular drug susceptibility test (mDST), the pyrazinamidase (PZase) activity assay, the resazurin microtiter assay (REMA), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction test. The sensitivities of the MGIT 960 system, the PZase activity assay, the mDST, the REMA, and the MTT assay were 98.8%, 88.8%, 90.5%, 98.8%, and 98.2%, respectively. The sensitivities of the PZase activity assay and the mDST were lower than those of the other three methods (P < 0.05). The specificities of the MGIT 960 system, the PZase activity assay, the mDST, the REMA and the MTT assays were 99.2%, 98.9%, 90.9%, 98.5%, and 100%, respectively. The specificity of the mDST was lower than those of the other four methods (P < 0.05). In conclusion, the MGIT 960 system, the MTT assay, and the REMA are superior to the PZase activity assay and the mDST in determining the susceptibility of M. tuberculosis to PZA. The MTT assay and the REMA might serve as alternative methods for clinical laboratories without access to the MGIT 960 system. For rapid testing in well-equipped laboratories, the mDST might be the best choice, particularly for small quantities of M. tuberculosis. The PZase activity assay has no obvious advantage in the assessment of M. tuberculosis susceptibility to PZA, as it is less accurate and requires larger quantities of bacteria.

| S-EPMC3647927 | biostudies-literature

Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis.

Project description:Pyrazinamide (PZA) plays the important role in shortening the tuberculosis treatment period and in treating MDR-TB. Phenotypic PZA susceptibility methods are limited because they require specialized acidified media, which increases costs and complexity. In this study we developed a genotypic high resolution melt (HRM) analysis technique to detect pncA mutations associated with PZA resistant Mycobacterium tuberculosis. Seven overlapping primer pairs were designed to cover the entire pncA gene and upstream regions. Each gene segment was individually amplified by real-time PCR followed by HRM analysis. The assay was evaluated on 98 clinical M. tuberculosis isolates (41 PZA susceptible by MGIT method, 55 PZA resistant, 2 undetermined). HRM was 94% concordant to full-length sequencing results, with most discrepancies attributable to mixed populations per HRM or transversions. Sequencing and HRM yielded 82% and 84% concordance, respectively, to phenotypic PZA susceptibilities by MGIT, with most discrepancies attributable to isolates with wild-type pncA but phenotypic PZA resistance. This HRM technique is a simple and high-throughput method for screening clinical M. tuberculosis samples for PZA resistance.

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Pyrazinamide susceptibility testing in Mycobacterium tuberculosis: a systematic review with meta-analyses.

Project description:Standard culture-based testing of the susceptibility of Mycobacterium tuberculosis to pyrazinamide is difficult to perform. This systematic review with meta-analyses evaluated the roles of molecular assays targeting pncA and of pyrazinamidase assays. PubMed and Embase were searched for relevant publications in English. Sensitivity and specificity were estimated in bivariate random-effects models. Of 128 articles identified, 73 sets of data involving culture isolates were initially included in meta-analyses. Summary estimates of sensitivity and specificity, respectively, were 87% and 93% for PCR-DNA sequencing (n = 29), 75% and 95% for PCR-single-stranded conformation polymorphism (SSCP) (n = 5), 96% and 97% for a mixture of other molecular assays (n = 6), and 89% and 97% for pyrazinamidase assays using the Wayne method (n = 33). The median prevalence (range) of pyrazinamide resistance was 51% (31% to 89%) in multidrug-resistant M. tuberculosis isolates and 5% (0% to 9%) in non-multidrug-resistant isolates. Excluding studies with possibly considerable false resistance in the reference assay gave the following estimates of sensitivity and specificity, respectively: 92% and 93% for PCR-DNA sequencing (n = 20), 98% and 96% for other molecular assays (n = 5), and 91% and 97% for the Wayne assay (n = 27). The Wayne assay had significant funnel plot asymmetry, so the test performance might have been overestimated. Considering the prevalence of pyrazinamide resistance in different clinical settings, PCR-DNA sequencing, and possibly other molecular assays targeting pncA, can detect pyrazinamide resistance in multidrug-resistant M. tuberculosis isolates, with predictive values largely exceeding 90%, and rule out pyrazinamide resistance in non-multidrug-resistant isolates, with predictive values exceeding 99%. Molecular assays are probably the way forward for detecting pyrazinamide resistance.

| S-EPMC3186960 | biostudies-literature

Association between pncA gene mutations, pyrazinamidase activity, and pyrazinamide susceptibility testing in Mycobacterium tuberculosis.

Project description:We determined MICs for, confirmed the presence of pncA mutations in, and performed pyrazinamidase testing on colonies (subclones) obtained from seven isolates that exhibited differential pyrazinamide (PZA) susceptibility. Six of the seven strains were found to exhibit characteristics resulting from the mixture of strains possessing different properties. In addition, our analysis revealed large pncA-spanning deletions (1,565 bp, 4,475 bp, and 6,258 bp) in three strains that showed high PZA resistance.

| S-EPMC4136016 | biostudies-literature

Systematic analysis of pyrazinamide-resistant spontaneous mutants and clinical isolates of Mycobacterium tuberculosis.

Project description:Pyrazinamide (PZA) is a first-line antitubercular drug known for its activity against persistent Mycobacterium tuberculosis bacilli. We set out to systematically determine the PZA susceptibility profiles and mutations in the pyrazinamidase (pncA) gene of a collection of multidrug-resistant tuberculosis (MDR-TB) clinical isolates and PZA-resistant (PZA(r)) spontaneous mutants. The frequency of acquired resistance to PZA was determined to be 10(-5) bacilli in vitro. Selection at a lower concentration of PZA yielded a significantly larger number of spontaneous mutants. The methodical approach employed allowed for determination of the frequency of the PZA(r) phenotype correlated with mutations in the pncA gene, which was 87.5% for the laboratory-selected spontaneous mutants examined in this study. As elucidated by structural analysis, most of the identified mutations were foreseen to affect protein activity through either alteration of an active site residue or destabilization of protein structure, indicating some preferential mutation site rather than random scattering. Twelve percent of the PZA(r) mutants did not have a pncA mutation, strongly indicating the presence of at least one other mechanism(s) of PZA(r).

| S-EPMC3457413 | biostudies-literature

Systematic review of mutations in pyrazinamidase associated with pyrazinamide resistance in Mycobacterium tuberculosis clinical isolates.

Project description:Pyrazinamide (PZA) is an important first-line drug in the treatment of tuberculosis (TB) and of significant interest to the HIV-infected community due to the prevalence of TB-HIV coinfection in some regions of the world. The mechanism of resistance to PZA is unlike that of any other anti-TB drug. The gene pncA, encoding pyrazinamidase (PZase), is associated with resistance to PZA. However, because single mutations in PZase have a low prevalence, the individual sensitivities are low. Hundreds of distinct mutations in the enzyme have been associated with resistance, while some only appear in susceptible isolates. This makes interpretation of molecular testing difficult and often leads to the simplification that any PZase mutation causes resistance. This systematic review reports a comprehensive global list of mutations observed in PZase and its promoter region in clinical strains, their phenotypic association, their global frequencies and diversity, the method of phenotypic determination, their MIC values when given, and the method of MIC determination and assesses the strength of the association between mutations and phenotypic resistance to PZA. In this systematic review, we report global statistics for 641 mutations in 171 (of 187) codons from 2,760 resistant strains and 96 mutations from 3,329 susceptible strains reported in 61 studies. For diagnostics, individual mutations (or any subset) were not sufficiently sensitive. Assuming similar error profiles of the 5 phenotyping platforms included in this study, the entire enzyme and its promoter provide a combined estimated sensitivity of 83%. This review highlights the need for identification of an alternative mechanism(s) of resistance, at least for the unexplained 17% of cases.

| S-EPMC4538510 | biostudies-literature

Transcriptome analysis of Mycobacterium tuberculosis clinical isolates.

Project description:Bacterial persister cells are phenotypic variants of regular cells that are tolerant to antibiotics. Analysis of clinical isolates of M. tuberculosis showed that strains vary substantially in their tolerance to antibiotics. The level of persisters was very high is some isolates, suggesting that these are hip mutants. We investigated gene expression differences in eight clinical isolates, four of which we characterized as high-persister strains and four as low-persister, or regular, strains. Comparison of gene expression patterns may provide clues as to the genetic mechanisms underlying persister formation.

2015-01-01 | GSE62025 | GEO

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Project description:Pyrazinamide (PZA) is a key antibiotic in current anti-tuberculosis regimens. Although the WHO has stressed the urgent need to obtain data on PZA resistance, in high tuberculosis burden countries, little is known about the level of PZA resistance, the genetic basis of such resistance or its link with Mycobacterium tuberculosis families. In this context, this study assessed PZA resistance through the molecular analysis of 260 Vietnamese M. tuberculosis isolates. First-line drug susceptibility testing, pncA gene sequencing, spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing were performed. Overall, the pncA mutation frequency was 38.1% (99 out of 260 isolates) but was higher than 72% (89 out of 123 isolates) in multidrug and quadruple-drug resistant isolates. Many different pncA mutations (71 types) were detected, of which 55 have been previously described and 50 were linked to PZA resistance. Among the 16 novel mutations, 14 are likely to be linked to PZA resistance because of their mutation types or codon positions. Genotype analysis revealed that PZA resistance can emerge in any M. tuberculosis cluster or family, although the mutation frequency was the highest in Beijing family isolates (47.7%, 62 out of 130 isolates). These data highlight the high rate of PZA resistance-associated mutations in M. tuberculosis clinical isolates in Vietnam and bring into question the use of PZA for current and future treatment regimens of multidrug-resistant tuberculosis without PZA resistance testing.

| S-EPMC5658769 | biostudies-literature

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2005-08-26 | GSE3201 | GEO

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