Hameed H M Adnan, Fang Cuiting, Liu Zhiyong, Gao Yamin, Wang Shuai, Chen Xinwen, Zhong Nanshan, Aung Htin Lin, Hu Jinxing, Zhang Tianyu
State Key Laboratory of Respiratory Disease, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou Chest Hospital, Guangzhou 510530, China.
China-New Zealand Joint Laboratory of Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
Microorganisms. 2025 Jun 16;13(6):1401. doi: 10.3390/microorganisms13061401.
Pyrazinamide (PZA) is a key component of tuberculosis treatment, with drug resistance (PZA) primarily related to mutations. However, discordance between phenotypic resistance and conventional -based molecular diagnostics challenges diagnostic accuracy. This study investigates discrepancies between phenotypic and genotypic resistance profiles among (Mtb) clinical isolates. Fifty-three Mtb isolates from Guangzhou Chest Hospital were tested for PZA resistance using the BACTEC MGIT 960 system and PZase activity assay. Thirty-one phenotypically PZA strains were genetically assessed by Sanger sequencing of PZA-associated customary genes. Five -wild-type PZA strains were investigated through whole-genome sequencing. ClpC1P1P2 activity was evaluated by proteolytic degradation assay. Notably, 26/31 of the PZA strains harbored mutations in and/or its upstream region, aligning PZase activity and phenotypic profiles. However, five PZA strains lacked mutations. The WGS of five discordant strains revealed four novel mutations (Gly58Ser, Val63Ala, Ala567Val, and Pro796Leu) across ClpC1 domains. Incorporating mutations improved molecular diagnostic sensitivity and accuracy from 48.3% and 69.8% ( alone) to 100%. This is the first report from southern China that identifies novel mutations in wild-type PZA Mtb isolates. Our findings underscore the limitations of -targeted diagnostics and support the integration of WGS and analysis in molecular diagnostics to prevent false-negative diagnoses and improve clinical outcomes.
吡嗪酰胺（PZA）是结核病治疗的关键组成部分，耐药性（PZA）主要与突变有关。然而，表型耐药性与传统分子诊断之间的不一致性对诊断准确性提出了挑战。本研究调查了结核分枝杆菌（Mtb）临床分离株中表型和基因型耐药谱之间的差异。使用BACTEC MGIT 960系统和PZase活性测定法对广州胸科医院的53株Mtb分离株进行PZA耐药性检测。通过对PZA相关常规基因进行Sanger测序，对31株表型PZA耐药菌株进行基因评估。通过全基因组测序对5株野生型PZA耐药菌株进行研究。通过蛋白水解降解测定法评估ClpC1P1P2活性。值得注意的是，31株PZA耐药菌株中有26株在pncA及其上游区域存在突变，使PZase活性与表型谱一致。然而，有5株PZA耐药菌株缺乏pncA突变。对5株不一致菌株的全基因组测序揭示了ClpC1结构域中的4个新突变（Gly58Ser、Val63Ala、Ala567Val和Pro796Leu）。纳入pncA突变将分子诊断的敏感性和准确性从单独检测pncA时的48.3%和69.8%提高到了100%。这是中国南方首次报道在野生型PZA Mtb分离株中发现新的pncA突变。我们的研究结果强调了靶向pncA诊断的局限性，并支持在分子诊断中整合全基因组测序和pncA分析，以防止假阴性诊断并改善临床结果。
