**Background:** Diarrheal diseases remain a major health threat to children in low- and middle-income countries, attributed to approximately 500,000 deaths annually among children under 5 years and ranking as the third highest cause of disability-adjusted life-years in children under 10 years. The Vaccine Impact on Diarrhea in Africa (VIDA) study was a 36-month, prospective, matched case-control study designed to estimate the etiology, incidence, and adverse clinical consequences of moderate-to-severe diarrhea (MSD) in children aged 0–59 months, conducted following rotavirus vaccine introduction at 3 censused sites in sub-Saharan Africa (The Gambia, Mali, Kenya) that had participated in the Global Enteric Multicenter Study (GEMS) approximately 10 years earlier. Rotavirus vaccine had been introduced prior to study commencement: RotaTeq in The Gambia (14 August 2013) and Mali (15 January 2014), and Rotarix in Kenya (1 July 2014).
**Methods:** The study aimed to enroll 8–9 MSD cases every 2 weeks from sentinel health centers across 3 age strata (0–11, 12–23, 24–59 months), totaling approximately 220 MSD cases per stratum per year or 1,980 cases over the 36-month enrollment period. For each enrolled case, 1–3 controls were randomly selected from the demographic surveillance system database using a computerized algorithm matching by age (plus or minus 2 months for 0–11 months, plus or minus 4 months for 12–59 months), sex, date of case enrollment (within 14 days), and village. The required number of controls was determined by tracking enrollment every 2 weeks: 1:1 matching if 7–9 cases were enrolled, 1:2 if 4–6 cases, and 1:3 if 3 or fewer cases. Clinical, epidemiological, and anthropometric data were collected at enrollment and at a follow-up visit 50–90 days later. A single fresh whole stool specimen was collected within 12 hours of arrival at the sentinel health center. Pathogens were detected using both conventional methods (bacterial culture, multiplex PCR, RT-PCR, immunoassay) and quantitative PCR (qPCR) via a custom TaqMan Array Card, with a positivity threshold of Ct < 35. Unlike GEMS where qPCR was performed retrospectively on a random subset, VIDA performed qPCR for all cases and their first matched control. Giardia testing by qPCR was omitted during the third year due to a supplier error, though immunoassay testing continued.
**Key Results:** The primary analytic approach used conditional logistic regression to estimate the association between pathogen quantity (Ct value) and case-control status. Pathogens detected in at least 2% of all case and control stool samples were included in the models. A separate model was created for each pathogen of interest, with interaction terms for study site and age stratum included only for that pathogen, and a quadratic term allowed when statistically significant (P < .05). The pathogen-specific attributable fraction (AF) was estimated by categorizing continuous Ct values, with Ct ≥ 35 as the reference (no pathogen detected). The AF for each pathogen was calculated as the sum of individual AFs for each case child. Pathogen-specific attributable incidence per 100 child-years was estimated using the formula: AI_j = (AF_j × M) / (r × N × 3), where M is the total number of eligible children, r is the proportion of children with MSD seeking care at sentinel health centers (from Health Care Utilization and Coverage Surveys), N is the number of children in the demographic surveillance system, and 3 is the number of study years. Confidence intervals for AF and AI were estimated using 5,000 bootstrap samples, taking the 2.5th and 97.5th percentiles. A key methodological change from the GEMS re-analysis was that pathogen-specific odds ratios below 1 were not truncated to null, allowing for the possibility that some putative enteropathogens may lower the risk of acute diarrhea. For assessing clinical consequences, a linear mixed-effects model with restricted maximum likelihood was used to analyze repeated height-for-age z-score (HAZ) measurements at enrollment and follow-up, incorporating random effects for both matched case-control sets and longitudinal repeated measurements. Only interactions significant at P < .1 were retained in the final model, and Scheffé-adjusted 95% CIs were calculated for the difference in HAZ between MSD cases and controls.
**Clinical Implications:** GEMS and VIDA together comprise the largest and most comprehensive assessment of MSD conducted to date in sub-Saharan African populations at highest risk for morbidity and mortality from diarrhea. The statistical methods used in VIDA maximize the use of available data to produce more robust estimates of pathogen-specific disease burden that might be prevented by effective interventions. The nested prospective cohort design provides a unique opportunity to examine the impact of MSD on linear growth over time, a feature not typically available from standard case-control studies. The study's findings are particularly relevant for the sub-Saharan Africa region, which continues to report more than half of global deaths among children younger than 5 years.