Characterization of Rpob gene mutations outside the rifampicin resistance-determining region in Mycobacterium tuberculosis isolates from North-West Zonal TB Reference Laboratory, Kano, Nigeria.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a global health priority, particularly in resource-limited settings. Rifampicin resistance, a cornerstone of multidrug-resistant TB (MDR-TB), is primarily associated with mutations within the rifampicin resistance-determining region (RRDR) of the rpoB gene. However, emerging studies demonstrate that mutations outside the RRDR also contribute to resistance but remain undetected by routine diagnostics. This study investigated rpoB mutations beyond the RRDR among rifampicin-resistant MTB isolates from North-west Zonal TB Reference Laboratory (NZTRL) in Aminu Kano Teaching Hospital (AKTH), Kano, Nigeria. Forty-one rifampicin-resistant isolates were sequenced, and bioinformatics analysis was performed to characterize mutations and assess evolutionary relationships. Mutations outside the RRDR were identified in several isolates, leading to amino acid substitutions including Pro→Ser (22.2%), Pro→Phe (22.2%), Pro→Leu (11.1%), and Arg→Cys (11.1%). Phylogenetic analysis revealed close clustering of Nigerian isolates with strains from India, Egypt, China, and Mexico, suggesting potential links with global transmission dynamics. These findings highlight the importance of expanding molecular diagnostic tools to capture resistance-associated mutations beyond the RRDR, which is critical for accurate diagnosis, timely treatment, and strengthening TB control strategies in high-burden settings.