Robust Performance of Culture, qPCR, and Genomic Approaches for Shigella Serotyping in a Pediatric Surveillance Cohort

Abstract 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 qPCR-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 qPCR serotyping, stool-based qPCR 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. Importance Shigella serotyping is critically important for the evaluation of future multivalent vaccines, of which there are several in advanced stages of development, as well as for monitoring of emerging Shigella serotypes. Culture based agglutination is the most widely used serotyping method, yet its successful implementation is associated with key logistical constraints. This study compares culture-based, qPCR-based, and whole-genome sequencing serotyping methods using isolates from a Shigella -endemic population in Peru. The study demonstrates that molecular and genomic approaches achieve high accuracy for vaccine-relevant serotypes and identifies the genomic basis of serotyping discrepancies. These methods would also reduce variation and improve data quality for future vaccine trials and epidemiologic surveillance. Ultimately, this work informs clinical microbiology laboratories and public health programs that seek a reliable and scalable alternative to traditional serotyping methods.