Detection of Novel Gene Mutations Associated with Pyrazinamide Resistance in Multidrug-Resistant Clinical Isolates in Southern China.

OBJECTIVE

Pyrazinamide (PZA) is a cornerstone of modern tuberculosis regimens. This study aimed to investigate the performance of genotypic testing of upstream region, and genes to add insights for more accurate molecular diagnosis of PZA-resistant (R)

METHODS

Drug susceptibility testing, sequencing analysis of PZA-related genes including the entire operon of () and PZase assay were performed for 448 clinical isolates.

RESULTS

Our data showed that among 448 clinical isolates, 113 were MDR, 195 pre-XDR and 70 XDR TB, while the remaining 70 strains had other combinations of drug-resistance. A total of 60.04% (269/448) clinical isolates were resistant to PZA, of which 78/113 were MDR, 119/195 pre-XDR and 29/70 XDR TB strains. PZA isolates have predominance (83.3%) of Beijing genotype. Genotypic characterization of revealed novel nonsynonymous mutations in with negative PZase activity which led to confer PZA. Compared with phenotypic data, 84.38% (227/269) PZA strains with mutations in upstream region exhibited 83.64% sensitivity but the combined evaluation of the mutations in 2.60% (7/269), 1.48% (4/269), 1.11% (3/269) and 0.74% (2/269) increased the sensitivity to 89.59%. Fifty-seven novel mutations were identified in this study. Interestingly, a frameshift deletion (C-114del) in upstream of nullified the effect of A-11G mutation and induced positive PZase activity, divergent from five PZase negative A-11G PZA mutants. Twenty-six PZA strains having wild-type-sequenced genes with positive or negative PZase suggest the existence of unknown resistance mechanisms.

CONCLUSION

Our study revealed that PZA rate in MDR and pre-XDR TB was markedly higher in southern China. The concomitant evaluation of UFR, and provides more dependable genotypic results of PZA resistance. Fifty-seven novel mutations/indels in this study may play a vital role as diagnostic markers. The upstream region of and PZase regulation are valuable to explore the unknown mechanism of PZA-resistance.