Identification and Molecular Characterization of Mycobacterium bovis DNA in GeneXpert® MTB/RIF Ultra-Positive, Culture-Negative Sputum Samples from a Rural Community in South Africa.

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Abstract This study investigated the presence of Mycobacterium bovis (M. bovis) DNA in human sputum samples collected from residents who reside adjacent to the M. bovis-endemic Hluhluwe-iMfolozi wildlife park, South Africa (SA). Sixty-eight sputum samples were GeneXpert MTB/RIF Ultra-positive for M. tuberculosis complex (MTBC) DNA but culture negative for M. tuberculosis. Amplification and Sanger sequencing of hsp65 and rpoB genes from residual frozen sputum confirmed the presence of detectable amounts of MTBC in DNA extracted from 20 out of the 68 sputum samples. Region of difference PCR, spoligotyping and gyrB long-read amplicon deep sequencing together identified M. bovis (n = 10) from the 20 samples from which MTBC DNA could be detected. Notably, M. bovis spoligotypes SB0130 and SB1474 were identified in 4 samples, with SB0130 previously identified in local cattle and wildlife and SB1474 exclusively in African buffaloes in the adjacent park. M. bovis 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 emphasize the need for integrated surveillance and control strategies to curb potential spillover and for the consideration of human M. bovis infection in SA patients with positive Ultra results.

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