Tuberculosis (TB), caused by bacteria of the Mycobacterium tuberculosis complex (MTBC), remains a global health challenge, exacerbated by multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains.

This study employs whole-genome sequencing (WGS) to characterise genetic mutations associated with pyrazinamide (PZA) and fluoroquinolone (FQ) resistance in MDR-TB isolates from KPK.

MDR and pre-XDR TB samples were collected and processed at the Provincial Tuberculosis Reference Laboratory under Biosafety Level III conditions. Samples underwent microscopy, GeneXpert MTB/RIF assay, culture, and drug susceptibility testing. DNA was extracted from positive cultures and subjected to WGS. Bioinformatics tools were used to analyse sequencing data, identify resistance-associated mutations, and assess genetic diversity among isolates.

Out of the 78 MTBC isolates analysed, 67 (85.9 %) were identified as MDR-TB, with 48 categorized as pre-XDR, while 11 were drug-susceptible. The isolates predominantly came from young patients (mean age: 29.5 years, SD ±12.64), with a higher proportion of female patients (61.53 %). Mutations in the pncA gene, associated with PZA resistance, were identified in 51 isolates. Resistance to fluoroquinolones was linked to mutations in the gyrA and gyrB genes in 48 isolates. WGS confirmed PZA resistance in 51 isolates, 39 (76.47 %) of which also exhibited FQ resistance.

Phylogenetic analysis revealed that Lineage 3 (L3) was predominant (58.97 %), followed by L4, L2, and L1 strains. The clustering of drug-resistant strains within L3 suggests ongoing localized transmission. These findings underscore the urgent need for targeted interventions, including enhanced molecular surveillance and tailored treatment strategies, to combat MDR-TB in KPK.