Standardized Recombinase Polymerase Amplification (RPA) Assay for Detecting Active Trypanosoma brucei evansi Infections.

The causative agent of "Surra", Trypanosoma brucei evansi (T. b. evansi), is thought to have originated from Trypanosoma brucei brucei (T. b. brucei) and primarily causes trypanosomosis in a wide range of wild and domesticated animals. The disease inflicts significant economic damage to farmers and the livestock industry. Additionally, T. b. evansi is considered a potential zoonotic pathogen, as atypical human infections have been reported. Unlike T. brucei, which requires the tsetse fly as a biological vector, T. b. evansi can be transmitted mechanically by various biting flies, leading to a broader and less predictable global distribution. The economic impact and the global presence of T. b. evansi increase the need for rapid, accurate, and field-deployable diagnostic tests. While polymerase chain reaction (PCR)-based tests are widely used for direct pathogen detection, they generally require skilled personnel and a laboratory environment to ensure proper protocol execution. In contrast, recombinase polymerase amplification (RPA) offers an alternative approach using isothermal nucleic acid amplification that is simple, fast, cost-effective, and well-suited for use in minimally equipped laboratories (and even in field settings). The results of RPA can be visualized using different methods, such as agarose gel electrophoresis (RPA-AGE), lateral flow assay (RPA-LFA), and real-time fluorescence (RPA-RT). In this chapter, we describe the procedures that are used for specifically detecting active T. b. evansi infections. The choice of procedure to be used is determined by several key factors, including the intended application, available resources, and the required sensitivity.