Abstract
The diagnosis of Infectious diseases in a typical clinical laboratory is generally based on antigen-antibody reactions, microbiological isolation or molecular methods, such as PCR. Among these testing approaches, molecular methods are more sensitive and specific. The molecular method most commonly used is PCR, but this method is often unavailable in many clinical laboratories in resource-limited settings. As a result, accurate diagnosis may not occur and diseases may not be appropriately treated, which could lead to poor patient care and treatment. The lack or minimal use of PCR for diagnosis in resource-limited settings is mainly associated with infrastructure requirements, cost and required expertise. In order to provide an alternative to PCR, recombinase polymerase amplification technology (RPA) was recently described; this operates isothermally and eliminates many of the complexities associated with PCR. This thesis provides a critical review of this technology since RPA was described. Importantly, it shows that RPA could be used in the diagnosis of Chlamydia trachomatis, Zaire ebolavirus and Mycobacterium tuberculosis. Most of these assays are shown to be comparable with PCR as per analytical and clinical performance (sensitivity and specificity), and provide a pathway towards further clinical validation and commercialisation. In a small pilot study, the C. trachomatis assay indicated a clinical sensitivity of 100.00% (95% CI: 73.54-100.00) and specificity of 100.00% (95% CI: 81.47- 100.00) when compared with Roche Cobas® 4800 CT/NG. The positive and negative predictive values were 100% respectively. The Z. ebolavirus assay produced a sensitivity of 55.00% (95% CI: 31.53-76.94) and specificity of 95.45% (95% CI: 77.16-99.88). The positive and negative predictive values were 91.67% (95% CI: 60.88-98.73) and 70.00% (95% CI: 58.77-79.25) respectively. This assay was compared with RealStar® Filovirus Screen RT-PCR assay. Finally, the M. tuberculosis assay was 100.00% for both clinical sensitivity and specificity when compared with GeneXpert® MTB/RIF. Significantly, this assay could detect all TB-HIV co-infection participants identified in the study. Consequently, the developed assays described in this thesis could be implemented in resource-limited settings and could potentially replace PCR-based assays where that technology is not affordable or available.