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A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.
Published on: November 7, 2018
Robust Performance of Culture, qPCR, and Genomic Approaches for Shigella Serotyping in a Pediatric Surveillance
Francesca Schiaffino1,2, Craig T Parker3, Lucero Romaina Cachique4
1Faculty of Veterinary Medicine, Universidad Peruana Cayetano Heredia, San Martin de Porres, Lima, Peru.
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.
Insights
Newer methods like qPCR and whole-genome sequencing (WGS) accurately serotype Shigella strains, crucial for vaccine development. WGS offers genomic insights into discrepancies, improving surveillance and diagnostics.
Area of Science:
- Clinical Microbiology
- Infectious Diseases
- Genomics
- Vaccinology
Background:
- Shigella causes severe diarrheal disease, with S. flexneri and S. sonnei being key targets for multivalent vaccine development.
- Traditional culture-based agglutination for Shigella serotyping faces logistical, subjective, and antisera availability limitations, especially for emerging serotypes.
- Performance of newer methods like qPCR and whole-genome sequencing (WGS) in Shigella-endemic populations requires further documentation.
Purpose of the Study:
- To compare the accuracy and performance of culture-based agglutination, isolate-based qPCR, stool-based qPCR, and WGS (ShigaPass) for Shigella serotyping.
- To evaluate these methods in a Shigella-endemic population in Iquitos, Peru, focusing on vaccine-relevant serotypes.
- To identify the genomic basis of any observed discrepancies in serotyping results.
Main Methods:
- Shigella isolates from the Enterics for Global Health (EFGH) study in Peru were serotyped using four distinct methods.
- Quantitative PCR (qPCR) was performed on both isolates and stool samples.
- Whole-genome sequencing (WGS) was utilized with the ShigaPass in-silico tool, with expert analysis of O-antigen genes for definitive serotyping.
Main Results:
- All four methods demonstrated high accuracy (93.3-100%) for key vaccine subtypes (S. flexneri 1b, 2a, 3a, 6, and S. sonnei).
- Complete concordance was observed in 83/107 isolates; however, 22.4% showed discrepancies, primarily involving S. flexneri serotypes Y, Yv, and 1a.
- Specific discrepancies included agglutination misclassifying Y/Yv as 4a and misclassifying 1a isolates due to a mutation in the oac gene.
Conclusions:
- All evaluated methods (culture-based agglutination, qPCR, WGS) achieve acceptable accuracy for Shigella vaccine efficacy studies.
- Whole-genome sequencing (WGS) provides valuable genomic context for understanding and resolving serotyping discrepancies.
- Molecular and genomic approaches offer scalable, reliable alternatives to traditional methods, improving data quality for surveillance and vaccine trials.
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