| Summary: | Since certain <i>Mycobacterium tuberculosis</i> complex (MTBC) members, such as <i>M. bovis</i>, are endemic in specific South African wildlife reserves and zoos, cases of clinically important nontuberculous mycobacteria (NTM) in wildlife may be neglected. Additionally, due to the inability of tests to differentiate between the host responses to MTBC and NTM, the diagnosis of MTBC may be confounded by the presence of NTMs. This may hinder control efforts. These constraints highlight the need for enhanced rapid detection and differentiation methods for MTBC and NTM, especially in high MTBC burden areas. We evaluated the use of the GeneXpert MTB/RIF Ultra, the Hain CM<i>direct</i> V1.0 line probe assay, and novel amplicon sequencing PCRs targeting the mycobacterial <i>rpoB</i> and <i>ku</i> gene targets, directly on antemortem African elephant (<i>n</i> = 26) bronchoalveolar lavage fluid (BALF) (<i>n</i> = 22) and trunk washes (<i>n</i> = 21) and rhinoceros (<i>n</i> = 23) BALF (<i>n</i> = 23), with known MTBC culture-positive and NTM culture-positive results. Our findings suggest that the Ultra is the most sensitive diagnostic test for MTBC DNA detection directly in raw antemortem respiratory specimens and that the <i>rpoB</i> PCR is ideal for <i>Mycobacterium</i> genus DNA detection and species identification through amplicon sequencing.
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