Phenotypic drug resistance and genetic mutations linked to resistance among extrapulmonary tuberculosis patients in Ethiopia: Insights from Whole Genome Sequencing

Abstract Globally, drug-resistant tuberculosis (DR-TB) is responsible for 13% of mortality attributable to antimicrobial resistance. In Ethiopia, extrapulmonary tuberculosis (EPTB) is a significant public health challenge, and drug resistance (DR) in EPTB is often overlooked. In a cross-sectional study conducted between August 2022 and October 2023, we aimed to explore the magnitude of phenotypic drug resistance and identify genetic mutations linked to resistance using 189 Mycobacterium tuberculosis (MTB) isolates cultured from extrapulmonary clinical specimens. Additionally, we assessed the agreement of the phenotypic and whole genome sequencing (WGS) based genotypic drug resistance detection. We performed phenotypic drug sensitivity testing (pDST) using liquid culture BD BACTECTM MGITTM 960 system and WGS using Illumina NextSeq500/550. The genomic data analysis pipelines MTBSeq and TBProfiler were used to predict drug resistance-conferring mutations. The agreement between the pDST and WGS was analyzed using SPSS version 29.0 software. Our result demonstrated phenotypic resistance to at least one anti-TB drug was detected in 16.9% (32/189) of the study participants. Isoniazid-resistant rifampicin-susceptible-TB (Hr-TB) and multi-drug-resistant TB (MDR-TB) phenotypes accounted for 2.6% (5/189) and 4.2% (8/189) respectively. Prevalence of MDR-TB was 2.4% (4/170) among newly diagnosed and 21.1% (4/19) among previously treated cases. WGS identified more (14/160, 8.75%) rifampicin-resistant genotypes (RR-TB) compared to pDST (8/189, 4.2%). We have identified a putative compensatory mutation for rifampicin (rpoBSer450Leu, rpoCAsp747Ala) for the first time from an EPTB clinical specimen in Ethiopia. Overall, there was a 3.75% rifampicin mono-resistant-TB(RMR-TB) genotype, which remains undetected using the conventional pDST and represented 42.9% (6/14) of the identified RR-TB genotypes. Mutations conferring rifampicin resistance-interim (rpoB.Ser450Ala) represented the majority (83.3%) of RMR-TB. Changes in ethA genes associated with ethionamide resistance were the most common resistance (n=7, 87.5%) in MDR-TB cases. There was a strong agreement between the pDST and WGS-TB Profiler pipeline to detect RR-TB (kappa=0.8) compared to the MTBSeq pipeline (k=0.58). In conclusion, MDR-TB, Hr-TB, and interim-RMR-TB are equally important public health challenges in the realm of EPTB in Ethiopia. The role of WGS is tremendous in detecting borderline/interim RMR-TB, which will help for tailored, personalized treatment strategies.