Development of new Recombinase Polymerase Amplification (RPA) and antigen-based diagnostic tools for the detection of **Trypanosoma evansi** infections

Animal trypanosomosis (AT) is a neglected tropical disease affecting domestic and wild animals, which severely impairs the socio-economic development of endemic areas. The most widespread causative agent of AT is Trypanosoma evansi. This parasite has a lifecycle that allows transmission by a range of different insect vectors and has spread beyond the African continent to large parts of the world including the Middle East, the Mediterranean, Asia and South America. Disease control mainly relies on chemotherapy, as there are no effective vaccines available. In order to optimize treatment, reliable and sensitive point-of-care (POC) tests are needed to detect infected animals. Hence, the work presented in this thesis has focused on two diagnostic development approaches. First, a new technology called Recombinase Polymerase Amplification (RPA) was adopted for the molecular diagnosis of T. evansi infections by targeting the gene encoding T. evansi specific RoTat1.2 VSG. The technique is an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories. RPA was subsequently combined with lateral flow (LF) technology, providing a simple test readout suitable for use in resource-limited settings and field conditions. Secondly, the PhD work focused on the development of a serological antigen-based assay format, which would be suitable for the detection of active infections and could be used as a test-of-cure device to monitor anti-trypanosome treatment success in animals. The test we developed is based on Nanobody technology, as this allows antigen capturing through cryptic epitopes on parasite molecules that are hard to target with much larger conventional monoclonal or polyclonal antibodies. The use of an “unbiased” alpaca immunization strategy with T. evansi secretome resulted in the identification of the glycolytic enzyme enolase (TevENO) as a potential biomarker for the Nb-based detection of T. evansi infections. As our approach indicated that TevENO is actively released by the parasite in detectable quantitates, we initiated work that aims at the future elucidation of the role this secreted enzyme could play in the context of the host-pathogen interface. Initial results show the ability of TevENO to bind to human plasminogen (PLG), the precursor of plasmin, which itself an important factor in fibrinolysis and the prevention is of blood clotting.

Jaar van publicatie:2020

Trefwoorden:Doctoral thesis

Toegankelijkheid:Closed