Development and validation of a sample-to-result real time multiplex RT-PCR test for human African trypanosomiasis and arboviral febrile illnesses in the Democratic Republic of the Congo

Abstract

Background: Human African trypanosomiasis (HAT) is a fatal vector-borne disease caused by Trypanosoma brucei. 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. Methods: A technical validation and retrospective sample testing (n = 242) were performed at the Institute of Tropical Medicine (ITM). Field validation took place in the DRC 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 some T.b.gambiense 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. 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. 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. Clinical trial: Clinicaltrials.gov NCT04760678, registered on February 17th, 2021.