Integrated surveillance of arboviruses in febrile patients from the Brazilian Amazon reveals complex co-circulation dynamics and hidden viral diversity

Background: Arboviral infections continue to be a significant public health challenge in the Brazilian Amazon. Overlapping symptoms, limited laboratory access, and the circulation of multiple arboviruses hamper clinical diagnosis. This study aimed to characterize the epidemiological, clinical, laboratory and genomic profiles of arboviral infections in febrile patients in Manaus, Brazil, and explore additional viral agents using metagenomic sequencing. Methods: A cross-sectional study was conducted between February 2021 and February 2023 at a tertiary reference center in Manaus, Brazil. Patients aged ≥ 5 years of age presenting with a rash and either a fever or a history of fever lasting <7 days and a negative thick blood smear for malaria were enrolled. Serum samples were tested for dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus (CHIKV), yellow fever virus (YF), Oropouche virus (OROV), and Mayaro virus (MAYV) using ELISA and RT-qPCR. Positive samples were subjected to amplicon-based genome sequencing for phylogenetic analysis. A subset of RT-qPCR negative samples was analyzed using de novo shotgun metagenomic sequencing. Results: Among the 708 enrolled participants, 243 (34.3%) had a laboratory-confirmed arboviral infection: 92 (37.9%) DENV, 64 (26.3%) CHIKV, and 4 (1.6%) ZIKV, while 83 (34.2%) laboratory profiles were compatible with coinfection, predominantly DENV+CHIKV (55/83; 66.3%). Circulation of DENV-1 genotype V and DENV-2 genotypes III (Asian American) and II (Cosmopolitan) was identified. Metagenomic analysis of 35 samples detected Pegivirus hominis and Erythroparvovirus primate 1. Conclusions: These findings demonstrate complex arbovirus co-circulation in Manaus and support integrated surveillance strategies combining molecular, serological, and genomic approaches.