Phylogenetic lineages of tuberculosis isolates and their association with patient demographics in Tanzania

Drug resistant Mycobacterium tuberculosis (M.tb) is an emerging threat for TB control Whole genome sequencing (WGS) has emerged as a new diagnostic to reliably predict drug resistance within a clinically relevant time and its use can have great impact on TB control in highly endemic regions. This project was set to determine the occurrence of phylogenetic lineages of M.tb complex and to examine their relationship with patient demographic characteristics and multidrug-resistant TB (MDR-TB) among pulmonary TB patients enrolled during the second national anti-TB drug resistance survey in Tanzania. A cross-sectional survey was conducted involving a sample of new smear-positive pulmonary TB and previously treated individuals. Sputum samples were collected and transported to the Central TB Reference Laboratory (CTRL) in Dar es Salaam, Tanzania for smear microscopy, culture, strain identification and susceptibility testing following standard National TB and Leprosy Programme (NTLP) guidelines. For WGS, all culture positive isolates were shipped to the National TB Reference Laboratory/Supranational Tuberculosis Reference Laboratory in Uganda. The isolates were sub-cultured on selective Middlebrook 7H11 agar. High quality genomic DNA was extracted using an in-house cetyl trimethylammonium bromide (CTAB) method. Genomic libraries were sequenced using the Illumina MiSeq V3 cartridge. Tools for resistance profiling and lineage inference (Kvarq v0.12.2, Mykrobe v0.8.1 and TBprofiler v3.0.5) were ran. De novo genome assembly of all samples was done using Unicycler v0.4.8. The resulting trees were plotted, annotated and visualized using ggtree v2.0.4