Background: Shigella spp. and enteroinvasive Escherichia coli (EIEC) are prominent enteric pathogens and estimates of prevalence are limited by suboptimal sensitivity of current detection methods. Recent studies have suggested common co-infection and frequent asymptomatic pathogen detection. Objective: The objective of this research is to examine the association between the quantity of Shigella/EIEC identified in stool and moderate-to-severe diarrhea (MSD) and how this association is modified by different factors such as age, location, co-infecting pathogens and the gut microbiota. Methods: The Global Enteric Multicenter Study (GEMS) estimated the etiology and population-based burden of MSD in sub-Saharan Africa and South Asia in children under the age of five years. Here, we conducted a case-control study sampled from GEMS utilizing quantitative polymerase chain reaction (QPCR) for the identification of the Shigella/EIEC ipaH gene in stool samples and compared the results of conventional culture to those of QPCR. Using multivariate logistic regression modeling we tested for effect modification and interaction and examined the effects of co-infecting pathogens identified through traditional microbiologic techniques and 16s rRNA sequencing. Results: A cut-point of 1.4x104 ipaH gene copies results in an increased burden of MSD cases attributable to Shigella/EIEC. Both site and age were identified as significant effect modifiers of the relationship between ipaH quantity and MSD, with the greatest association seen in children 24-59 months of age (Odds Ratio 8.2; 95% CI 4.3-15.7) and from Bangladesh (OR13.2; 95% CI 7.3-23.8). After adjusting for age and site, rotavirus exhibited a significant negative association with high ipaH quantities in cases (OR=0.31, 95% CI 0.17-0.55) and resulted in large combined effects (OR=28.85; 95% CI 3.77-220). Five Lactobacillus taxa exhibited significant additive antagonistic effects, decreasing the association between Shigella and MSD, consistent with a possible protective role. Conclusions: QPCR detection of the ipaH gene substantially increases the fraction of MSD attributable to Shigella/EIEC above that attributed by culture and identifies similar age and site effects. Our results suggest that Lactobacillus may specifically inhibit the pathogenicity of Shigella/EIEC and conclude that future studies should continue to consider the effects of significant characteristics such as age and site as well as co-occurring species.