Development and validation of a sample-to-result real time multiplex RT-PCR test for Human African Trypanosomiasis and acute febrile illnesses caused by dengue, chikungunya and yellow fever viruses in DRC

Background Human African trypanosomiasis (HAT) is a fatal vector-borne disease caused by Trypanosoma brucei ( T.b.) . Although HAT incidence has declined, meeting the WHO’s eliminating targets remains difficult, particularly due to limited diagnostic sensitivity for low-parasite load infections. Arboviruses such as dengue (DENV 1–4), chikungunya (CHIKV), and yellow fever (YFV) virus, present with nonspecific febrile symptoms similar to HAT and are underdiagnosed in Sub-Saharan Africa. Sample-to-result platforms can reduce hands-on time and infrastructure requirements, making them ideal for peripheral laboratories. We developed a multiplex real-time RT-PCR assay on ARIES®, a sample-to-result molecular diagnostic platform, for simultaneous detection of HAT, DENV, CHIKV and YFV, showing how automated, closed-cartridge PCR can simplify testing. Methods A technical validation and retrospective sample testing (n = 242) was performed at the Institute of Tropical Medicine (ITM). Field validation took place in the Democratic Republic of the Congo (DRC) at two different sites with retrospective samples from a CHIKV outbreak (n = 121) in Institut National pour la Recherche Biomédicale (INRB) Kinshasa and 52 prospective whole blood samples from acute febrile patients in Centre de Recherche en Santé de Kimpese (CRSK) in Kimpese. Results The assay showed a slight loss of sensitivity, evidenced in the technical validation, and the non-detection of retrospective samples at ITM with low pathogen loads. CHIKV samples tested in Kinshasa showed a sensitivity of 89.4%. Although all samples tested in Kimpese were negative for the pathogens of the assay it demonstrated how short training and straightforward workflows can enable robust diagnostics under challenging conditions. Conclusion Ensuring rapid, sensitive molecular diagnostics in resource-limited settings is critical for eliminating HAT and strengthening surveillance of emerging arboviruses. Despite the recent discontinuation of ARIES®, our findings confirm the feasibility and reliability of detecting diverse pathogens with minimal laboratory resources. Notably, the assay aligns with WHO and FIND target-product profiles, underscoring its relevance for neglected diseases in low-resource settings. These results emphasize the ongoing need for open, flexible sample-to-result platforms to support disease surveillance and outbreak preparedness.