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

Authors Hameed HMA, Tan Y, Islam MM, Lu Z, Chhotaray C, Wang S, Liu Z, Fang C, Tan S, Yew WW, Zhong N, Liu J, Zhang T

Received 12 September 2019

Accepted for publication 20 December 2019

Published 22 January 2020 Volume 2020:13 Pages 217—227


Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Dr Eric Nulens

Video abstract presented by HM Adnan Hameed.

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HM Adnan Hameed, 1, 2,* Yaoju Tan, 3,* Md Mahmudul Islam, 1, 2 Zhili Lu, 1 Chiranjibi Chhotaray, 1, 2 Shuai Wang, 1, 2 Zhiyong Liu, 1 Cuiting Fang, 1, 2 Shouyong Tan, 3 Wing Wai Yew, 4 Nanshan Zhong, 5 Jianxiong Liu, 3 Tianyu Zhang 1, 2, 5 

1State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Guangzhou, People’s Republic of China; 2University of Chinese Academy of Sciences (UCAS), Beijing, People’s Republic of China; 3State Key Laboratory of Respiratory Disease, Guangzhou Chest Hospital, Guangzhou, People’s Republic of China; 4Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Hong Kong, People’s Republic of China; 5National Clinical Research Center for Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Tianyu Zhang
Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Room A132, 190 Kaiyuan Ave, Science Park, Huangpu District, Guangzhou 510530, People’s Republic of China
Tel +86-2032015270
Jianxiong Liu
Guangzhou Chest Hospital, 62 Hengzhigang Road, Yuexiu District, Guangzhou 510095, People’s Republic of China
Tel +86-2083595977

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. PZAR 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 PZAR. Compared with phenotypic data, 84.38% (227/269) PZAR 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 PZAR mutants. Twenty-six PZAR strains having wild-type-sequenced genes with positive or negative PZase suggest the existence of unknown resistance mechanisms.
Conclusion: Our study revealed that PZAR 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

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