Identification and molecular characterization of Mycobacterium bovis DNA in GeneXpert® MTB/RIF ultra-positive, culture-negative sputum from a rural community in South Africa

Abstract

This study investigated the presence of <i>Mycobacterium bovis</i> (<i>M. bovis</i>) DNA in archived human sputum samples previously collected from residents who reside adjacent to the <i>M. bovis</i>-endemic Hluhluwe-iMfolozi wildlife park, South Africa (SA). Sixty-eight sputum samples were GeneXpert MTB/RIF Ultra-positive for <i>M. tuberculosis</i> complex (MTBC) DNA but culture negative for <i>M. tuberculosis</i>. Amplification and Sanger sequencing of <i>hsp65</i> and <i>rpoB</i> genes from DNA extracted from stored heat-inactivated sputum samples confirmed the presence of detectable amounts of MTBC from 20 out of the 68 sputum samples. Region of difference PCR, spoligotyping and <i>gyrB</i> long-read amplicon deep sequencing identified <i>M. bovis</i> (<i>n</i> = 10) and <i>M. tuberculosis</i> (<i>n</i> = 7). Notably, <i>M. bovis</i> spoligotypes <i>SB0130</i> and <i>SB1474</i> were identified in 4 samples, with <i>SB0130</i> previously identified in local cattle and wildlife and <i>SB1474</i> exclusively in African buffaloes in the adjacent park. <i>M. bovis</i> DNA in sputum, from people living near the park, underscores zoonotic transmission potential in SA. Identification of spoligotypes specifically associated with wildlife only and spoligotypes found in livestock as well as wildlife, highlights the complexity of TB epidemiology at wildlife-livestock-human interfaces. These findings support the need for integrated surveillance and control strategies to curb potential spillover and for the consideration of human <i>M. bovis</i> infection in SA patients with positive Ultra results.