Rotavirus Prevalence, Genetic Diversity, and Co-Infections During the 2023-2024 Cholera Outbreak in Zambia: Insights from Multi-Pathogen Diagnostics.

During Zambia's 2023-2024 cholera outbreak, reliance on single-pathogen diagnostics risked overlooking co-circulating enteric pathogens. This study estimated the prevalence of rotavirus and described co-detected enteropathogens and rotavirus genotypes among patients admitted with suspected cholera. A sub-analysis was conducted on diarrhoeal stool specimens collected from patients who met the syndromic suspected cholera case definition. Samples were tested using the Bosphore® Gastroenteritis Panel v2, a multiplex PCR enteric panel, to detect rotavirus and other gastrointestinal pathogens. Rotavirus-positive specimens with sufficient viral load were further genotyped by RT-PCR targeting of the VP7 and VP4 genes. Among 319 suspected cholera admissions, rotavirus was detected in 18 patients (5.6%; 95% CI 3.4-8.8%), predominantly in children aged <5 years (27.8%, 5/18) and 6-17 years (27.8%, 5/18). Co-infection was common, with 17/18 (94.4%) of rotavirus-positive samples showing co-infection with at least one additional enteric pathogen, most frequently Campylobacter. Genotyping was successful in five samples and revealed heterogenous circulating strains, including G1P[8], G2P[4], G3P[6], G12P[6], and G1P[6]. Rotavirus accounted for a modest proportion of suspected cholera admissions and was frequently detected in mixed enteric infections, underscoring the value of multi-pathogen diagnostics and continued molecular surveillance during outbreak response.