Application of VirCapSeq-VERT for viral pathogen detection in children under five with acute diarrheal illness in Zambia

Diarrheal disease remains a leading cause of morbidity and mortality among children under five years, especially in low- and middle-income countries, particularly sub-Saharan Africa. Although rotavirus vaccination has reduced burden in children, a substantial proportion of diarrhea remains unattributed due to limited diagnostic capacity. We applied VirCapSeq-VERT (VCS), a virome-wide targeted sequencing technique, to characterize viral detections and co-infection patterns in stool samples from children with diarrhea and to explore their comparison with disease severity. A cross-sectional, facility-based study across nine provincial health facilities in Zambia. Stool collected from children U5 years presenting with diarrhea was analyzed using qPCR and VCS. Positivity by VCS was defined as ≥ 25% in relation to full coding reference sequence. Severity was assessed using the Vesikari scoring system and categorized as mild or moderate-to-severe. Descriptive and comparative analyses were performed to evaluate viral prevalence, age-specific distribution, and co-infection patterns. Samples from 247 participants were analyzed by VCS with 48.6% aged 0–12 months; males accounting for 52.6%. Using VCS to interrogate the stool samples, rotavirus A was the most detected virus (42.5%), followed by enterovirus (36%), human herpesvirus (HHV) (30%), adenovirus (25.5%), astrovirus (23.9%), and norovirus (22.7%). VCS detected a broader range of viral pathogens among the subset of 233 participant samples that were also tested using qPCR, with higher prevalence measurements for adenovirus (24.5% by VCS vs. 10.7% by qPCR) and norovirus (21.9% by VCS vs. 15.9% by qPCR) whereas rotavirus A and astrovirus showed comparable prevalence across both platforms. Age-specific trends revealed decreasing prevalence of rotavirus with increasing age, while adenovirus and enterovirus predominated in children aged > 24 months. No individual viral pathogen was significantly associated with diarrhea severity; however, rotavirus co-infections modified outcomes, with rotavirus-sapovirus associated with increased severity (aPR = 1.39, 95% CI 1.03–1.88, p = 0.029). Dual infections were common (31.9%), while mono-infections accounted for 38.5% of cases, and 8.9% had no detectable virus. VCS enables broad detection of pathogenic viruses, revealing diversity and co-infections in pediatric diarrheal disease. Expanded viral surveillance may improve pathogen detection, guide clinical management, reduce inappropriate antibiotic use, assess vaccine strategies, and strengthen disease control in high-burden settings.