Whole-genome sequencing analysis of phylogenetic relationships and lineage diversity of Mycobacterium tuberculosis in Western Kenya

Abstract

Western Kenya remains a high-burden tuberculosis (TB) region, yet genomic data describing the diversity and phylogenetic relationships of Mycobacterium tuberculosis complex (MTBC) strains are limited. Understanding sub-lineage patterns and the phylogenetic relationships is essential for effective TB surveillance and control strategies. This was a cross-sectional whole genome sequencing (WGS) study using archived MTBC isolates from 12 counties in Western Kenya. Genomic DNA was extracted using a modified CTAB protocol and sequenced using the Element AVITI platform. Reads were processed using DuffyNGS/DuffyTools pipeline and aligned to the H37Rv reference genome. Lineages were assigned using TBProfiler v4.3.0 based on single nucleotide polymorphisms (SNP). Phylogenetic relationships were reconstructed using Nextstrain and interpreted using tree topology. Of 316 MTBC isolates processed, 290 met quality criteria for WGS. Lineage 4 (L4) (42%) and Lineage 3 (L3) (38%) predominated, with significant county-level heterogeneity observed in Kisii and Homa Bay (L4), and Siaya and Migori (L3) ( p < 0.0296). Sub-lineage analysis revealed widespread circulation of L2.2.1 and L3.1.1 strains, alongside substantial L4 diversity. Rare sub-lineages showed limited geographic distribution and one Mycobacterium bovis isolate was identified. Phylogenetic analysis revealed geographically structured relatedness, with a localized circulation in Kisii, broader regional distribution in Homa Bay, and additional clades showing intermediate regional patterns. Western Kenya harbors genetically diverse and spatially MTBC population with distinct patterns of phylogenetic relatedness. These findings demonstrate the value of WGS for characterizing population structure and informing targeted TB control in high-burden settings.