Optimizing <i>Shigella</i> isolation: a multi-site evaluation of laboratory culture methods for <i>Shigella</i> detection, speciation, and serotyping with different transport media and sample types in the Enterics for Global Health study

<i>Shigella</i> is a leading cause of diarrhea and dysentery in children younger than 5 years of age in low-resource settings, and several vaccines are in development. Due to its fastidious nature, <i>Shigella</i> can be difficult to culture, and eventual vaccine trials will need to optimize the isolation of <i>Shigella</i> to ensure efficient sample sizes. In the recently concluded Enterics for Global Health (EFGH) <i>Shigella</i> Surveillance study, we compared <i>Shigella</i> culture isolation rates between rectal swab vs. whole stool, between two swabs vs. one, and between Cary Blair (CB) vs. modified-Buffered Glycerol Saline (mBGS) transport media to identify the optimal methods for <i>Shigella</i> recovery by microbiologic culture. Among 9,476 children aged 6-35 months enrolled in the EFGH study from seven country sites, <i>Shigella</i> isolation rates did not differ significantly between CB (7.8%) and mBGS (7.9%) (<i>P</i> > 0.99). Using two swabs improved the detection rates (9.3%), compared with one swab (7.9%) (<i>P</i> < 0.001). Among the 2,048 children from Bangladesh and The Gambia, where both rectal swabs and whole stool were collected from the same children, rectal swabs were found to be non-inferior to whole stool for <i>Shigella</i> culture (12.4% and 12.7%, respectively, with a difference of -0.29% [95% CI: -0.83% to 0.24%]). To optimize <i>Shigella</i> recovery for future multi-country vaccine trials, we recommend collecting two flocked rectal swabs in CB or mBGS media with strict adherence to transit conditions-an approach proven feasible across EFGH sites.Clinical TrialsThis study is registered with ClinicalTrials.gov as NCT06047821.IMPORTANCE<i>Shigella</i> is difficult to isolate by culture, making optimized sampling and transport essential for microbiologic confirmation. As <i>Shigella</i> vaccines are tested for efficacy and eventual licensure, it is critical that the laboratory methods are optimized to avoid missing children with shigellosis. In the multi-country Enterics for Global Health (EFGH) study, which enrolled over 9,000 children with diarrhea, rectal swabs and whole stool recovered a similar number of <i>Shigella</i> isolates, with two swabs improving detection over a single swab. Cary Blair (CB) and modified-Buffered Glycerol Saline (mBGS) transport media also resulted in similar isolation rates. To maximize outcome ascertainment in future vaccine trials, two flocked rectal swabs with transport in either medium are recommended to balance sensitivity, feasibility, and scalability.