In sub-Saharan Africa, malaria is commonly considered the leading cause of febrile illness. However, a substantial proportion of malaria-negative febrile patients suggests the involvement of other infectious agents, particularly arboviruses such as dengue. In Benin, data on circulating dengue virus serotypes in humans remain limited, especially among non-malarial febrile patients. Objective: The main objective of this study was to characterize the dengue virus serotypes circulating among malaria-negative febrile patients attending three hospitals in southern Benin (Abomey, Abomey-Calavi, and Lokossa). A cross-sectional descriptive study was conducted between June 2024 and January 2025 among 1490 febrile patients. After biological testing for malaria, 120 malaria-negative patients were selected for dengue virus detection using nested polymerase chain reaction (nested PCR). Positive samples were further analyzed for serotype identification. Results: Among the 1490 febrile patients, 288 (19.3%) had confirmed malaria, while 1202 (80.7%) tested negative. Of the 120 malaria-negative patients tested by nested PCR, 11 dengue cases were identified, corresponding to a prevalence of 9.16%. Cases were distributed across Abomey-Calavi (5 cases), Abomey (3 cases), and Lokossa (3 cases). Serotype analysis revealed a marked predominance of DENV-2 (10 cases) and the presence of DENV-3 (1 case), indicating co-circulation of multiple serotypes. Young adults aged 20 - 30 years were the most affected group, with a female predominance. Conclusion: This study demonstrates active circulation of DENV-2 and DENV-3 among malaria-negative febrile patients in southern Benin. The predominance of DENV-2 and detection of DENV-3 confirm the co-circulation of dengue virus serotypes in the human population. These findings highlight the importance of integrating dengue testing into the diagnostic workup of non-malarial febrile illnesses and strengthening virological surveillance to prevent potential outbreaks and severe forms of the disease.
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