Virome sequencing maps viral diversity in Zambian children with diarrhea

A cross-sectional study at nine provincial health facilities in Zambia found that VirCapSeq-VERT, a virome-wide targeted sequencing method, detected a diverse set of viral pathogens in stool samples from children younger than five years with acute diarrheal illness. Among 247 participants analyzed by the sequencing method, rotavirus A was the most frequently detected virus, followed by enterovirus, human herpesvirus, adenovirus, astrovirus and norovirus. The study also identified frequent coinfections, with 31.9% of participants carrying evidence of two viruses.

Diarrheal disease remains a leading cause of morbidity and mortality among children under five, particularly in low- and middle-income countries and sub-Saharan Africa. Although rotavirus vaccination has reduced disease burden, the researchers noted that a substantial proportion of pediatric diarrhea remains unattributed because of limited diagnostic capacity. Their cross-sectional, facility-based study was designed to characterize viral detections and coinfection patterns among children presenting with diarrhea and to examine associations between detected pathogens, pathogen combinations and clinical severity.

It was an observational diagnostic study, not an intervention trial, so it did not establish whether wider viral testing improves clinical outcomes or disease control. Of the 247 participants whose samples were analyzed by VirCapSeq-VERT, 48.6% were between zero and 12 months old and 52.6% were male. Investigators also tested 233 samples using quantitative polymerase chain reaction, or qPCR, allowing prevalence estimates from the two platforms to be compared within the same subset.

A VirCapSeq-VERT result was considered positive when the viral sequence represented at least 25% of the full coding reference sequence. Diarrhea severity was assessed with the Vesikari scoring system and divided into mild or moderate-to-severe categories. The investigators analyzed overall and age-specific viral prevalence, as well as the patterns and severity associations of mono-infections and coinfections.

VirCapSeq-VERT detected rotavirus A in 42.5% of the full set of samples, enterovirus in 36%, human herpesvirus in 30%, adenovirus in 25.5%, astrovirus in 23.9% and norovirus in 22.7%. In the 233 samples tested by both methods, the sequencing approach yielded higher prevalence estimates for two viruses. Adenovirus was detected in 24.5% of these samples by VirCapSeq-VERT compared with 10.7% by qPCR; for norovirus, the respective estimates were 21.9% and 15.9%.

Rotavirus A and astrovirus prevalence was comparable across the platforms. Age patterns varied among the detected viruses. Rotavirus prevalence decreased as age increased, while adenovirus and enterovirus predominated among children older than 24 months.

These were cross-sectional differences within the study population, not longitudinal changes in individual children. Mono-infections were identified in 38.5% of participants, and 8.9% had no detectable virus. No individual viral pathogen was significantly associated with diarrhea severity.

The exception in the analysis involved a particular coinfection: children with rotavirus and sapovirus together had a higher prevalence of moderate-to-severe disease than the comparison group, corresponding to an adjusted prevalence ratio of 1.39, a 95% confidence interval of 1.03–1.88 and a p-value of 0.029. The result is an association measured in the study, not evidence that sapovirus caused the increase in severity. The results show that virome-wide targeted sequencing can provide a broad characterization of viral detections and coinfections in pediatric diarrheal samples.

Those proposed applications were not directly tested in this research. The findings are limited by the cross-sectional design, the focus on symptomatic children attending nine Zambian facilities and the sample of 247 participants. They therefore provide a detailed snapshot of viral diversity and severity patterns in the enrolled population rather than a population-wide estimate of all pediatric diarrhea in Zambia or evidence of clinical benefit from expanded sequencing.

Subject of Research: Viral pathogen detection and coinfection patterns in children with acute diarrhea in Zambia

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

Article References: Musukuma-Chifulo, K., Silwamba, S., Ngosa, D., Chibesa, K., Luchen, C. C., Mwape, K., Collins, J., Nzangwa, B. T., Kuntawala, D. H., Chilengi, R., Yingst, S., Wickiser, J. K., Chisenga, C. C., & Simuyandi, M. (2026). Application of VirCapSeq-VERT for viral pathogen detection in children under five with acute diarrheal illness in Zambia. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14170-0

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14170-0

Keywords: VirCapSeq-VERT, pediatric diarrhea, viral sequencing, coinfections, rotavirus, norovirus, adenovirus, Zambia, qPCR, disease severity, Application, viral