Enhancing diagnostic efficiency of pyrazinamide resistance in Mycobacterium tuberculosis via modified MGIT assay and genotypic correlation

Abstract

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 pncA 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 pncA gene (45 %), panD (9.6 %), mas (12.9 %), glpK (3.2 %), and lprG (3.2 %), and others efflux associated genes like Rv1258c (3.2 %), Rv0191c (3.2 %), and Rv3008 (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 glpK and mas were detected in isolates with wild-type pncA 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.