<p>Detection of Novel Gene Mutations Associated with Pyrazinamide Resistance in Multidrug-Resistant <em>Mycobacterium tuberculosis</em> Clinical Isolates in Southern China</p>

Objective: Pyrazinamide (PZA) is a cornerstone of modern tuberculosis regimens. This study aimed to investigate the performance of genotypic testing of pncA + upstream region, rpsA, panD, Rv2783c, and clpC1 genes to add insights for more accurate molecular diagnosis of PZA-resistant (R) Mycobacterium tuberculosis. Methods: Drug susceptibility testing, sequencing analysis of PZA-related genes including the entire operon of pncA ( Rv2044c-pncA-Rv2042c ) and PZase assay were performed for 448 M. tuberculosis clinical isolates. Results: Our data showed that among 448 M. tuberculosis 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) M. tuberculosis clinical isolates were resistant to PZA, of which 78/113 were MDR, 119/195 pre-XDR and 29/70 XDR TB strains. PZA R isolates have predominance (83.3%) of Beijing genotype. Genotypic characterization of Rv2044c-pncA-Rv2042c revealed novel nonsynonymous mutations in Rv2044c with negative PZase activity which led to confer PZA R . Compared with phenotypic data, 84.38% (227/269) PZA R strains with mutations in pncA + upstream region exhibited 83.64% sensitivity but the combined evaluation of the mutations in rpsA 2.60% (7/269), panD 1.48% (4/269), Rv2783c 1.11% (3/269) and Rv2044c 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 pncAwt nullified the effect of A− 11G mutation and induced positive PZase activity, divergent from five PZase negative A− 11G PZA R mutants. Twenty-six PZA R strains having wild-type-sequenced genes with positive or negative PZase suggest the existence of unknown resistance mechanisms. Conclusion: Our study revealed that PZA R rate in MDR and pre-XDR TB was markedly higher in southern China. The concomitant evaluation of pncA + UFR, rpsA, panD, Rv2783c, and Rv2044c 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 pncA and PZase regulation are valuable to explore the unknown mechanism of PZA-resistance. Keywords: tuberculosis, pyrazinamidase, drug resistance, molecular diagnosis, novel mutations, frameshift deletion