The findings from this study show that the Ultra assay combined with tissue swabs that were inactivated in PrimeStore® MTM can accurately identify culture-confirmed M. bovis positive tissues from African buffaloes. The swabs taken directly from dissected tissues and later tested on the Ultra assay performed similar to the Ultra on tissue homogenates prepared prior to culture. Recent studies have shown that Ultra provides accurate, rapid diagnosis of MTBC infection in livestock and wildlife when performed on tissue homogenates 13− 15. However, the combined use of PrimeStore® MTM and tissue swabs with the Ultra assay have the added advantage of providing more rapid test results, since it can directly be used in the Ultra assay without the extra steps of tissue processing; results may even be acquired in-field if portable Ultra equipment is available (such as Cepheid’s GeneXpert® Edge system), providing same day results. Typically, tissue samples are kept frozen at -20°C and transported to a biosafety level 3 (BSL-3) facility before being homogenised to perform the Ultra test. PrimeStore® MTM incubated swabs do not require refrigeration and since PrimeStore® MTM inactivates pathogens, samples could even be transported through standard postal services without biosafety concerns. Furthermore, a BSL-3 facility is not required for sample processing of swabs in PrimeStore® MTM and samples can be handled safely without the risk of infection to other animals and humans.
There was a greater number of positive Ultra results using swabs than tissue homogenates. However, the difference was not significant and there was only fair agreement between the two methods. There could be various reasons for the discordant Ultra results between tissue swabs and matching tissue homogenate samples. PrimeStore® MTM has been shown to inactivate nucleases in samples. Since the tissue homogenate samples were not incubated in stabilising media (PrimeStore® MTM), there may have been degradation of DNA in some samples during freeze-thaw cycles, which could result in a negative Ultra result, especially if the samples were paucibacillary. Tissue swabs, however, did not undergo a freeze-thaw cycle prior to testing with Ultra. Also, since Ultra results are influenced by bacillary load 16, swab samples from tissues with visible lesions are more targeted than taking an aliquot of the tissue homogenate for Ultra testing, which may result in some dilution of mycobacteria in a larger homogenate volume than the swabs.
Studies performed with human specimens have shown that Ultra results provide a measure of the bacillary load in a specimen, with a positive correlation between the semi-quantitative Ultra results and detection of acid-fast bacilli with smear-microscopy 17. The “MTB trace detected” and “MTB not detected” Ultra results have been shown to be correlated with no acid-fast bacilli detected, whereas Ultra results “MTB detected high” and “medium” typically correlate with positive smear-microscopy results 17. The Ultra test results in this study included “MTB detected low”, “MTB detected very low”, “MTB trace detected” and “MTB not detected”. Since the “MTB trace detected” Ultra results from tissue swabs and homogenates were seen in culture-confirmed M. bovis positive specimens, it may indicate greater sensitivity of this method compared to smear microscopy. However, varying copy numbers of IS6110 and IS1081 between MTBC members and between strains have also been shown to influence the sensitivity and may not entirely relate to the performance of the Ultra assay 11,13. The bacillary load in samples in which no MTBC DNA were detected (“MTB not detected”) was likely below the threshold for detection by the Ultra assay, although the homogenates from the same tissue sample had a positive M. bovis result by mycobacterial culture and PCR speciation. This could be due to a low but viable bacillary load that would have increased to a detectable level during the 56 days of mycobacterial culture, which could further explain the discordant results between mycobacterial culture (all M. bovis positive) and Ultra results from tissue homogenates. Difference in sample volume between mycobacterial culture and Ultra from tissue swabs may also contribute to discordant results between these two methods.
The oral swabs from the M. bovis-unexposed buffaloes were all negative on the Ultra assay. These results are consistent with the reported high specificity of the Ultra assay 11. Interestingly, although these swabs were used as negative controls, the swabs would have had a greater likelihood of contamination than those collected post-mortem, since these animals were sampled in a dusty, natural environment, where they were likely exposed to various environmental organisms.
The study had several limitations. Firstly, there was a small sample size of culture-confirmed M. bovis-positive buffaloes and oral swab samples available from the M. bovis-unexposed buffaloes, which did not allow for direct comparison of the same sample type tested in the Ultra. Secondly, since the Ultra assay was only performed in the laboratory, we were unable to compare laboratory and in-field testing with the Ultra assay and PrimeStore® sampling platform. Finally, since tissue samples for Ultra were specifically selected based on visible lesions, it is unknown how the Ultra would perform using M. bovis culture-positive tissue samples with no visible lesions and therefore presumed low bacillary load. Future studies should include a larger sample size with buffaloes at various stages of infection. The application of field testing should also be investigated, which would allow more rapid sample processing and the use of fresh samples.
The Ultra assay with samples stored in PrimeStore® MTM shows promise as a rapid post-mortem screening test, which could be used to identify MTBC DNA in tissue samples and ultimately identify infected herds more rapidly than mycobacterial culture. This test can also be used to decrease the number of samples sent for mycobacterial culture by identifying those that contain mycobacterial DNA. Pooling swab samples from each animal will further decrease the number of samples tested. In addition, the use of PrimeStore® MTM to safely store and transport infectious samples will safeguard human and animal health as well as permit greater use of samples that may otherwise be restricted from being transported to diagnostic laboratories.