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

During cholera outbreaks in Zambia, diagnostic strategies that rely on single-plex or targeted assays risk overlooking concomitant infections with other clinically important enteric pathogens. We estimated the prevalence of rotavirus and described co-detected enteropathogens and rotavirus genotypes among patients admitted with clinically suspected cholera during Zambia’s 2023–2024 cholera outbreak.We conducted a sub-analysis of diarrhoeal specimens collected from patients admitted to five cholera treatment centres who met the syndromic suspected cholera case definition. Stool samples were tested using the Bosphore® Gastroenteritis Panel v2, a multiplex PCR enteric panel, to detect rotavirus and other gastrointestinal pathogens. Rotavirus-positive specimen with sufficient viral load were further characterised by RT-PCR genotyping and Sanger sequencing targeting VP7 and VP4 genes. Among 319 suspected cholera admissions, rotavirus was detected in 18 patients, yielding a prevalence of 5.6% (95% CI 3.4%, 8.8%). Rotavirus detections occurred predominantly in children aged < 5 years (87.5%) and 6-15 years (80.0%). Co-infection was common - 93.7%, (15/16) of rotavirus-positive samples showed co-infection with at least one additional enteric pathogen, primarily Campylobacter. Genotyping was successful in five samples and showed heterogenous circulating strains, including G1P[8], G2P[4], G3P[6], G12P[6], and a rare G1P[6] reassortant. During a large 2023–2024 cholera outbreak in Zambia, rotavirus accounted for a modest but clinically important fraction of the suspected cholera admissions and was typically identified within mixed enteric infections. These findings highlight the limitations of syndromic diagnosis in outbreak settings and support integrating multi-pathogen diagnostics and sustained molecular surveillance to improve case management, antimicrobial stewardship, and vaccine-era monitoring.