Human tuberculosis (TB) is a leading cause of global mortality and kills around 1.6 million people globally every year (WHO, 2022), with high disease burdens found in West Africa. In low resource settings with high TB burdens, the lack of rapid diagnostic methods for detection and differentiation of the Mycobacterium tuberculosis complex (MTBC) is a major challenge affecting TB management. Treatment efficacy is dependent upon the correct diagnosis of MTBC lineage to inform selection of appropriate medication, e.g. M. bovis is intrinsically resistant to the front-line antibiotic pyrazinamide. This study utilized comparative genomic analyses of MTBC lineages; M. tuberculosis (n = 6802), M. africanum Lineages 5/6 (n = 242) and M. bovis (n = 391) to identify lineage-specific genes. Primers were designed for the development of two assays: conventional multiplex Polymerase Chain Reaction (PCR) and Loop-mediated isothermal amplification assay (LAMP), whose sensitivity (limits of detection) and specificity were further investigated. Validation of the multiplex PCR assay was conducted using sputum DNA extracts from 341 clinically confirmed active TB patients from Ghana. It was observed that 24.9% of cases were caused by M. tuberculosis, while M. africanum L5 & L6 reported 9.0% and 14.4%, respectively. M. bovis infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. The colorimetric LAMP assay equally demonstrated successful detection and differentiation of MTBCs using lineage specific designed LAMP primers.

The application of these assays in low-resource regions will allow rapid TB diagnosis and speciation of MTBC to inform selection of appropriate medication for TB treatment at the earliest possible disease time point, prevent drug resistance and also to identify difficult-to-treat mixed-lineage TB infections.