Development of a camelid single-domain antibody-based antigen detection assay for the pan-specific diagnosis of active human and animal Trypanosoma brucei infections.

Trypanosoma brucei infections cause African trypanosomiasis, also known as sleeping sickness in humans and nagana in animals, presenting a significant global health and economic burden, especially in sub-Saharan Africa. Animal trypanosomiasis also affects the economic development in Asia and South America. Accurate diagnosis of diseases caused by infection with the parasite of the T. brucei group remains a major challenge due to the persistence of infection-induced antibodies long after parasite clearance, complicating serological discrimination between active and past infections. To address this limitation, we developed a sensitive and specific antigen detection assay targeting Trypanosoma brucei enolase ( TbrENO) using a panel of camelid single-domain antibodies (sdAbs also known as nanobodies). Among the candidates, the sdAbsR3-77/sdAbR2-103 sandwich enzyme-linked immunosorbent assay (ELISA) exhibited robust performance in detecting circulating TbrENO in plasma from experimentally infected mice. Additionally, this assay showed strong potential as a "test-of-cure" tool by monitoring real-time antigenemia throughout a chronic T. brucei infection course. We further validated the assay's diagnostic utility in human clinical samples, detecting Trypanosoma brucei rhodesiense infections at both early and advanced stages and Trypanosoma brucei gambiense infections in advanced stage. The sdAbsR3-77H/sdAbR2-103HA ELISA targeting TbrENO shows potential for point-of-care pan-diagnosis of active T. brucei infections (including Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, Trypanosoma brucei evansi, and Trypanosoma brucei equiperdum) in both animals and humans. Therefore, this assay addresses gaps in current diagnostic capabilities by overcoming the key limitations of antibody-based tests, offering a promising tool for improved disease control.IMPORTANCEAfrican trypanosomiasis, commonly known as sleeping sickness in humans and nagana in animals, is a life-threatening disease that remains a major health and economic concern in many parts of the world. One of the key difficulties in managing this disease is detecting ongoing infections, as existing antibody-based tests cannot reliably distinguish between current and past infections. In this study, we developed a novel laboratory test that detects a specific protein released by the parasite during infection. This test uses special antibodies derived from camels, known for their exceptional stability and precision, to accurately identify infections caused by multiple Trypanosoma brucei subspecies. Our approach not only enables accurate diagnosis but also offers a way to monitor treatment success. This work provides a valuable tool for disease control efforts and could help improve the health of both humans and animals in regions where trypanosomiasis is endemic.