The Xpert MTB/RIF Ultra assay detects Mycobacterium tuberculosis complex DNA in white rhinoceros (Ceratotherium simum) and African elephants (Loxodonta africana)

Wynand J Goosen,Sven D C Parsons,Peter Buss,David H Persing,Michele A Miller,Léanie Kleynhans,Robin M Warren,Paul D Van Helden,Tanya J Kerr

doi:10.1038/s41598-020-71568-9

Abstract

The study describes the novel use of the Xpert MTB/RIF Ultra assay for detection of Mycobacterium tuberculosis complex (MTBC) DNA in samples from white rhinoceros (Ceratotherium simum) and African elephants (Loxodonta africana). Culture negative respiratory sample matrices were spiked to determine if the Ultra could detect MTBC DNA in rhinoceros and elephant samples. Rhinoceros bronchial alveolar lavage fluid (BALF) was found to have an inhibitory effect on the Ultra. In this study, the limit of detection (LOD) of M. tuberculosis H37Rv in all spiked animal samples were 2 CFU/ml compared to 15.6 CFU/ml for humans, while the LOD for M. bovis SB0121 was 30 CFU/ml compared to 143.4 CFU/ml for M. bovis BCG in humans. Screening was performed on stored tissue and respiratory samples from known MTBC-infected animals and MTBC DNA was detected in 92% of samples collected from six rhinoceros and two elephants. Conversely, 83% of culture-negative tissue and respiratory samples from uninfected animals tested negative on the Ultra. In conclusion, the Ultra assay appears to be a sensitive and rapid diagnostic test for the detection of MTBC DNA from tissue and respiratory samples collected from African elephants and rhinoceros. Furthermore, the Ultra assay could provide a new tool for the detection of MTBC in various sample types from other wildlife species.

Highlights

Mycobacterium tuberculosis complex (MTBC) members, Mycobacterium bovis (M. bovis) and Mycobacterium tuberculosis (M. tuberculosis), are the cause of bovine tuberculosis and human tuberculosis (TB), respectively
Improved features are the inclusion of two new polymerase chain reaction (PCR) targets within the system that target different multicopy genes (IS6110 and IS1081), conversion of assays into fully nested PCRs, and incorporation of a larger PCR reaction tube that doubles the amount of input DNA
The increased sensitivity of the Ultra was observed through the enhanced limit of detection (LOD) for other strains like M. bovis BCG

Summary

Introduction

Mycobacterium tuberculosis complex (MTBC) members, Mycobacterium bovis (M. bovis) and Mycobacterium tuberculosis (M. tuberculosis), are the cause of bovine tuberculosis (bTB) and human tuberculosis (TB), respectively. The risks of MTBC spread to endangered wildlife have been highlighted by the recent discoveries of M. bovis infection in free-ranging white and black rhinoceros’ (Ceratotherium simum, Diceros bicornis) as well as the death of an African elephant (Loxodonta africana) bull shown to have M. tuberculosis infection in the Kruger National Park (KNP)[4,5,7]. Infection of these animals was not unexpected since KNP is endemic for bTB; M. tuberculosis infection in a free-ranging elephant is a novel finding. Initial analytical studies in human sputum have shown the LOD of the Ultra to be 15.6 CFU/ml for M. tuberculosis H37Rv and 143.4 CFU/ ml for M. bovis BCG with a sensitivity of 89% and specificity 98% for M. tuberculosis H37Rv d etection[16]

Methods

Results

Conclusion