Isothermal amplification assays for point of-care diagnostics of bovine brucellosis and tuberculosis in South Africa

Abstract

Bovine brucellosis, caused by Brucella abortus, is a disease of global socio-economic importance. Bovine tuberculosis (bovine TB) is a chronic wasting disease of cattle caused primarily by Mycobacterium bovis. Complete eradication of these diseases is reliant on the removal of diseased animals from herds; however, this is often challenging due to the absence of reliable, quick, and cost-effective diagnostic tools. This study aimed to conduct systematic reviews on molecular diagnostic techniques for bovine brucellosis and tuberculosis. Furthermore, this study sought to develop South African-specific molecular diagnostic assays based on PCR and LAMP technology for improved laboratory and fieldfriendly diagnostics of bovine brucellosis and bovine TB. A systematic review and meta analysis of peer-reviewed journal articles published between 1990 and 2020 revealed enhanced diagnostic performance with LAMP (sensitivity of 92.0% [95% CI 78.0–98.0%] and specificity of 100.0% [95% CI 97.0–100.0%]). The analysis further suggested that sample type and target gene choice influence assay performance, thus recommending the use of tissue (sensitivity 92.7% [95% CI 82.0–98.0%]) and serum (sensitivity 91.3% [95% CI 86.0–95.0%]) for brucellosis detection. The BruAb2_0168 gene PCR assay had a sensitivity of 92.3% [95% CI 87.0–96.0%], and specificity of 99.3% [95% CI 98.0100.0%]. Overall, depending on specific needs and available resources published LAMP, conventional PCR and qPCR assays reported by some literature may preferentially be used where applicable. Conventional PCR, nPCR and LAMP assays were designed based a unique region of the B. abortus genome. Primer specificity was ensured by testing against closely related species. The respective sensitivities were determined by serial 10-fold dilutions of synthetic plasmid DNA. The nPCR can detect down to 1 x 10-7 of the diluted cPCR’s limit of detection and was therefore more sensitive than both cPCR (pathogen DNA detectable at 1 x 103 copies) and LAMP (detectable at 1 x 100 copies), which, in turn, outperformed cPCR. When evaluated on 100 field samples, the nPCR displayed a high positivity rate of 68% surpassing cPCR (13%) and LAMP (59%). Limitation encountered with the cPCR led to its exclusion from further consideration. Therefore, only the nPCR and LAMP achieved technology readiness level 6. Based on the findings of this study, the adoption of the nPCR by reference laboratories is recommended due to its robustness and superior performance. Concurrently, LAMP testing conducted on serum samples, presents promising prospects for optimization and integration into a portable testing device, positioning it ideally for efficient point-of-care diagnostics for brucellosis. Controlling bovine TB requires highly sensitive, specific, quick, and reliable diagnostic methods. A systematic review and meta-analysis were conducted to evaluate molecular diagnostic tests for M. bovis detection to inform test selection by authorities. Despite showing promising potential in diagnostic accuracy, the existing tests for detecting M. bovis lack the capability to accurately differentiate it from other species within the Mycobacterium tuberculosis complex (MTC). Their design, based on non-specific gene targets, poses limitations in achieving accurate and reliable diagnosis. However, the findings attribute improvement in diagnostic test performance to the choice of sample type and target gene. Test sensitivity was higher with milk (90.9% [95% CI: 56.0%-98.7%]), while assays developed on the RD4 gene among others demonstrated improved specificity (90.7% [95% CI: 52.2%-98.9%]). Since the RD4 region is the only target identified by genome sequence data as being distinctive for recognizing M. bovis it is recommended for future assay development. The availability of a sufficiently accurate diagnostic test combined with the routine screening of milk samples holds the potential to mitigate the risk of zoonotic transmissions of M. bovis, presenting a significant advancement in controlling its spread. To develop molecular diagnostic assays for detection of M. bovis a region of the M. bovis genome that allows distinction between M. bovis and other species within the MTC was targeted. Conventional PCR, and LAMP assays were designed and optimized for reaction sensitivity and specificity, followed by a nPCR assay. All assays were specific. The cPCR exhibited a sensitivity of 1 x 103 copies in the lab and classified 24% of field samples as positive for M. bovis. LAMP with a laboratory sensitivity of 1 x 101 copies was consistently more sensitive in the field with a positivity rate of 56% and the nPCR exhibited greater sensitivity than both method both in the lab and with field samples with a positivity rate of 85%. While the cPCR assay was excluded from further consideration due to limitations encountered in this study, the designed LAMP assay shows great potential for use as a field-friendly screening test for M. bovis particularly with milk samples. Though the test had moderate sensitivity, it can help inform decision making in the field and the adoption of rapid containment measures while confirmatory tests are conducted. These results further suggest that the nPCR is a robust and specific test that can be used for the routine laboratory confirmation of M. bovis and is therefore recommended for adoption by reference laboratories in South Africa. Overall, both these technologies are at technology readiness level 6 and have the potential to be licensed and commercialized.