Robust performance of culture, real-time PCR, and genomic approaches for shigella serotyping in a pediatric surveillance cohort

BACKGROUND: Shigella causes severe diarrheal disease, and S. flexneri and S. sonnei are the targets for multivalent vaccine development. Culture-based agglutination has been the gold standard for serotyping, but it is limited by logistics, subjectivity, and the availability of antisera for emerging serotypes. Newer methods, including a real-time PCR-based approach and whole-genome sequencing offer alternatives, but their performance in Shigella endemic populations are not well documented. METHODS: Shigella isolates obtained from the Enterics for Global Health (EFGH) study in Iquitos, Peru were simultaneously serotyped using four methods: culture-based agglutination, isolate-based real-time PCR serotyping, stool-based real-time PCR serotyping and WGS using the in-silico tool ShigaPass. The definitive adjudicated serotype was established by an expert analysis of the WGS data, involving the mapping of sequence reads to known O-antigen biosynthesis and modification genes to identify key mutations. RESULTS: Results from all four serotyping methods were available for 107/114 isolates. Accuracy for vaccine subtypes S. flexneri 1b, 2a, 3a, 6, and S. sonnei, ranged from 93.3-100% for all methods. Complete concordance between methods was noted in 83/107 isolates, while 24/107 (22.4%) exhibited at least one discrepancy. Most discrepancies derived from S. flexneri serotypes Y, Yv and 1a. Agglutination misclassified eight Y/Yv isolates as 4a, and six isolates correctly classified as 1a by agglutination were classified as 1b by the other methods, a discrepancy associated with a nonsense mutation in the oac gene. CONCLUSION: All four serotyping methods achieved acceptable accuracy for Shigella vaccine efficacy evaluation. Although discrepancies are infrequent, WGS provides information of their genomic basis.