Genomic insights into extrapulmonary tuberculosis reveal enrichment of high drug- resistant lineage 2 in specific clinical phenotypes

Extrapulmonary tuberculosis (EPTB) poses unique challenges due to poor drug penetration at certain anatomical sites and the potential for lineage-specific differences in disease manifestation and resistance. Identifying resistance profiles and Mycobacterium tuberculosis (MTB) lineages is critical for guiding personalized treatment and improving outcomes in EPTB.In this study, we analyzed 117 culture-confirmed MTB isolates from seven EPTB sites collected at PGIMER, Chandigarh, India. Specimens included cerebrospinal fluid (CSF), pus, fine-needle aspiration cytology (FNAC), tissue, ileocaecal biopsy, synovial, and vitreous fluids. Phenotypic drug susceptibility testing (DST) was performed using the MYCOTB assay, and whole genome sequencing (WGS) was conducted on the Illumina XTen platform. An in-house pipeline was used to assess resistance mutations, lineages, and mixed infections.WGS identified resistance in 29.9% of isolates, compared to 23.9% by phenotypic DST, with 93.0% concordance. WGS also detected resistant cases missed by DST (p = 0.039). Resistance was highest in CSF samples (40.0%) and was significantly associated with sample type (p = 0.0446). Lineage 2 (Beijing strain) showed the highest resistance rate (76.5%), predominantly from CSF. Mixed infections were identified in 6.8% of cases, mostly involving Lineages 2 and 3. These were significantly associated with heteroresistance (p = 0.0139), underscoring the risk of underestimating resistance with conventional DST.Our findings highlight WGS as a reliable tool for detecting resistance, lineage, and strain diversity in EPTB. Its ability to identify minority resistant variants and lineage-specific patterns makes it particularly valuable in low-culture-yield, drug-restricted compartments such as CSF. Integrating WGS or targeted sequencing into routine diagnostics could enable timely, personalized therapy and enhance clinical outcomes in EPTB.