Pyrazinamide resistance in Mycobacterium tuberculosis

[摘要] ENGLISH SUMMARY: Pyrazinamide (PZA) is one of the drugs included in the standardized TB treatment regimen for both drug susceptible and resistant TB. PZA has a unique ability to target persister bacilli and since its inclusion has reduced TB treatment duration to 6 months. However, drug susceptible testing (DST) for PZA is not routinely performed due to the challenges associated with the DST (acidic media; false resistance and inoculum size). Significant evidence indicates that mutations in pncA (gene encodes for pyrazinamidase (PZase)) are the primary mechanism of resistance to PZA. Sequencing studies reveal that mutations occur across the entire length of the gene, thus sequencing of the entire gene is required to capture all possible resistance conferring mutations. A rapid molecular diagnostic is required to routinely test for PZA resistance, especially given the continued recommendation of its inclusion in the shortened MTB treatment regimen.PZA is currently being considered for inclusion of new treatment regimens which also include the newer anti-TB drugs. However, we need to better understand the prevalence of PZA resistance globally and across the spectrum of drug resistant groups to determine the utility of this drug in current and future treatment regimens. This PhD thesis aims to further the current knowledge of PZA resistance.The prevalence of PZA resistance regionally and globally is largely unknown. Our systematic review collates the PZA resistance data in the literature from all TB cases (16.2%); in high-risk MDR-TB (41.3%) and in MDR-TB (60.5%). These findings caution against relying on PZA in current and future TB drug regimens, especially in MDR-TB patients. This review also identified more than 600 unique SNPs across the entire length of the pncA gene as the causal mechanism of resistance. This highlights the complexity of the challenge to develop future genetic based PZA resistance tests.From this review it was evident that the prevalence of PZA resistance across a spectrum of drug resistant isolates is unknown. To address this knowledge gap 775 clinical isolates from South Africa were classified into groups according to resistance profile (ranging from pan-susceptible to XDR-TB) and their association with PZA resistance was determined. The prevalence of PZA resistance in each group was: 0% in the pan-susceptible; 2% in the INH-mono; 7.5% in the RIF-mono; 39.3% in the MDR-TB and 96.8% in the XDR-TB group. A statistically significant increase in PZA resistance was observed as the number of resistance markers increases. We concluded that PZA DST should be performed when considering its inclusion in treatment of patients with rifampicin-resistant TB or MDR-TB. This study also showed an excellent correlation between pncA genotype and PZA phenotype. The resulting sensitivity and specificity was 95% and 99%, respectively, thereby confirming the utility of pncA sequencing as a rapid PZA susceptibility test.In an attempt to understanding discordance between genotype and phenotype we characterised pncA mutations which did not confer PZA resistance at a susceptibility breakpoint of 100 μg/ml pyrazinamide in MGIT. A total of 10 non-resistance conferring mutations were identified in collaboration with the U.S. Centers for Disease Control and Prevention. These results will be essential for the interpretation of pncA sequencing results to guide treatment.In a recent paper the authors suggested that pncA mutation have a fitness cost which would impact on the transmission of drug resistant M. tuberculosis strains. To test this hypothesis we compared the growth rates of clinical isolates and in vitro mutants (wild type and SNPs) and clinical isolates which have a large pncA deletion. No difference was observed in the time to positivity in MGIT media when comparing wild type and strains harbouring pncA mutations, thereby suggesting that pncA mutation do not have a significant fitness cost. However, strains harbouring large pncA mutations showed a significant growth deficit (p-value: <0.001) in vitro. However, this fitness cost did not prevent these strains from transmitting in the community.In an attempt to simplify the diagnosis of PZA susceptibility, we investigated the diagnostic utility of a LATE-PCR based technique in combination with fluorescence probes to identify mutations along the entire length of the pncA gene. Using this method the sensitivity and specificity was 98.7% and 98.4%, respective, suggesting that this method could be using in routine laboratories due to the single tube format.Together this body of research has addressed critical knowledge gaps and has introduced a methodology that could simplify the diagnosis of PZA resistance.

[发布机构] Stellenbosch University