Rajendran Ananthi, Refaya Ahmed Kabir, Subramanyam Balaji, Karunaianantham Ramesh, RaviKumar Dhandapani, Haribabu Hemalatha, Gopalaswamy Radha, Golla Radhika, Senthildevi Vadivel, Gomathi Narayanan Sivaramakrishnan, Shanmugam Sivakumar, Palaniyandi Kannan
Department of Immunology, ICMR-National Institute for Research in Tuberculosis, Chennai, India.
Department of Bacteriology, ICMR-National Institute for Research in Tuberculosis, Chennai, India.
Curr Res Microb Sci. 2025 Aug 23;9:100462. doi: 10.1016/j.crmicr.2025.100462. eCollection 2025.
Pyrazinamide (PZA) plays a crucial role in the treatment of both active and latent tuberculosis, particularly in regimens designed to treat drug-resistant TB. However, diagnosing resistance to PZA poses challenges for managing TB, highlighting the need for accurate detection methods. This study aims to address the challenges in detecting PZA resistance by modifying the standard MGIT960 PZA drug susceptibility testing method by optimizing the inoculum dilution. Briefly, three MGIT DST versions were evaluated: the standard method, the reduced inoculum (RI) method employing a 1:20 inoculum dilution and the sparse dilution (SD) method using a 1:50 dilution of the inoculum for growth control tube, while the undiluted MGIT positive culture was used for the PZA test tube. The SD MGIT DST approach minimized the number of false-resistant PZA results to (31/401) 7.7 % against 27 % by standard MGIT DST and 11.7 % by RI MGIT DST approach, thereby reducing the false-positivity rate by 19.3 %. Targeted sequencing of gene identified mutations in only 14/401 isolates (3.5 %). Whole genome sequencing (WGS) of the 31 phenotypically resistant isolates identified resistance -associated mutations in gene (45 %), (9.6 %), (12.9 %), (3.2 %), and (3.2 %), and others efflux associated genes like (3.2 %) (3.2 %), and (6.45 %), except for 4 isolates, for which no mutations were detected in the target genes. These genes are involved in various resistance mechanisms including cell wall synthesis, metabolic pathways, and drug tolerance, which are essential for PZA efficacy. Notably, new mutations in and were detected in isolates with wild-type and were absent in the sensitive isolates. Our study substantiates the improvement of phenotypic testing methods and enhances the detection of PZA resistance even in resource-limited settings and direct research towards improving the diagnostic accuracy in TB drug resistance management.
吡嗪酰胺（PZA）在活动性和潜伏性结核病的治疗中都起着关键作用，尤其是在用于治疗耐多药结核病的方案中。然而，诊断对PZA的耐药性给结核病管理带来了挑战，凸显了准确检测方法的必要性。本研究旨在通过优化接种物稀释来改进标准MGIT960 PZA药敏试验方法，以应对检测PZA耐药性的挑战。简而言之，评估了三种MGIT DST版本：标准方法、采用1:20接种物稀释的低接种量（RI）方法和用于生长对照管的接种物1:50稀释的稀疏稀释（SD）方法，而未稀释的MGIT阳性培养物用于PZA试管。SD MGIT DST方法将假耐药PZA结果的数量降至（31/401）7.7%，标准MGIT DST为27%，RI MGIT DST方法为11.7%，从而将假阳性率降低了19.3%。对基因的靶向测序仅在14/401株分离株（3.5%）中鉴定出突变。对31株表型耐药分离株进行全基因组测序（WGS），在基因（45%）、（9.6%）、（12.9%）、（3.2%）和（3.2%）以及其他外排相关基因如（3.2%）（3.2%）和（6.45%）中鉴定出耐药相关突变，除了4株分离株在靶基因中未检测到突变。这些基因参与了包括细胞壁合成、代谢途径和药物耐受性在内的各种耐药机制，这些机制对PZA的疗效至关重要。值得注意的是，在野生型的分离株中检测到了和的新突变，而敏感分离株中不存在这些突变。我们的研究证实了表型检测方法的改进，并提高了即使在资源有限的环境中对PZA耐药性的检测能力，同时直接推动了结核病耐药管理中诊断准确性的研究。
