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

Wynand J Goosen; Sashen Moodley; Giovanni Ghielmetti; Yumna Moosa; Thando Zulu; Theresa Smit; Vukuzazi Team; Leanie Kleynhans; Tanya J Kerr; Elizabeth M Streicher; Willem A Hanekom; Robin M Warren; Emily B Wong; Michele A Miller

doi:10.1016/j.onehlt.2024.100702

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

One Health. 2024 Mar 3:18:100702. doi: 10.1016/j.onehlt.2024.100702. eCollection 2024 Jun.

Authors

Wynand J Goosen¹, Sashen Moodley², Giovanni Ghielmetti^{1

3}, Yumna Moosa², Thando Zulu², Theresa Smit²; Vukuzazi Team; Leanie Kleynhans^{1

4}, Tanya J Kerr¹, Elizabeth M Streicher¹, Willem A Hanekom^{2

5}, Robin M Warren¹, Emily B Wong^{2

6}, Michele A Miller¹

Affiliations

¹ Department of Science and Innovation - National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, PO Box 241, Cape Town 8000, South Africa.
² Africa Health Research Institute, KwaZulu-Natal, South Africa.
³ Section of Veterinary Bacteriology, Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270, 8057 Zurich, Switzerland.
⁴ Mater Research Institute - The University of Queensland, Translational Research Institute, Brisbane, QLD 4102, Australia.
⁵ Division of Infection and Immunity, University College London, London, UK.
⁶ Division of Infectious Diseases, Department of Medicine, Heersink School of Medicine, University of Alabama Birmingham, Birmingham, AL, USA.

Abstract

This study investigated the presence of Mycobacterium bovis (M. bovis) DNA in archived human sputum samples previously 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 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 gyrB long-read amplicon deep sequencing identified M. bovis (n = 10) and M. tuberculosis (n = 7). 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 support 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.

Keywords: Hluhluwe-iMfolozi park; Mycobacterium bovis; Spoligotyping; Tuberculosis control; Wildlife-livestock-human interface; Zoonotic transmission.