Background <p>Human African trypanosomiasis (HAT) is a fatal vector-borne disease caused by <i>Trypanosoma brucei</i>. Although HAT incidence has declined, meeting WHO’s elimination targets remain 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. Due to this overlap in symptoms and a suspected geographical overlap of vectors and pathogens in the Democratic Republic of the Congo (DRC) the pathogens were combined in a multiplex-PCR panel. Sample-to-result platforms (S2R) can reduce hands-on time and infrastructure requirements, making them ideal for peripheral laboratories. We developed a multiplex real-time RT-PCR assay on the ARIES^® platform, for simultaneous detection of HAT, DENV, CHIKV and YFV, showing how automated, closed-cartridge PCR can simplify testing.</p> Methods <p>A technical validation and retrospective sample testing (<i>n</i> = 242) were performed at the Institute of Tropical Medicine (ITM). Field validation took place in the DRC with retrospective samples from a CHIKV outbreak (<i>n</i> = 121) in <i>Institut National pour la Recherche Biomédicale</i> (INRB) Kinshasa and 52 prospective whole blood samples from acute febrile patients in <i>Centre de Recherche en Santé de Kimpese</i> (CRSK) in Kimpese.</p> Results <p>The assay showed a slight loss of sensitivity, evidenced in the technical validation, and the non-detection of some <i>T.b.gambiense</i> and retrospective arboviral 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, it demonstrated how just a few days of training and the simplified workflows of a S2R-platform can enable robust diagnostics under challenging conditions.</p> Conclusion <p>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. The assay aligns with WHO and FIND target-product profiles, highlighting its relevance for neglected diseases in low-resource settings. These results emphasize the ongoing need for open, flexible S2R platforms to support disease surveillance and outbreak preparedness.</p> Clinical trial <p>Clinicaltrials.gov NCT04760678, registered on February 17th, 2021.</p>