Development of a Luminex assay and NGS workflow for geno-serotyping for national Shigella surveillance

The phylogenetically closely related Shigella and enteroinvasive Escherichia coli (EIEC) species are responsible for millions of episodes of bacterial dysenteriae worldwide. Given its distinct epidemiology and public health relevance, only Shigellae are subject to mandatory reporting and follow-up by public health authorities. However, many clinical laboratories struggle to differentiate non-EIEC, EIEC and Shigella in their current workflows, leading to inaccuracies in surveillance and rising numbers of misidentified E. coli samples at the National Reference Centre (NRC). In this paper, we describe two novel tools to enhance Shigella surveillance. First, we developed a low-cost Luminex-based multiplex assay combining five genetic markers for species identification with 11 markers for serotype prediction for S. sonnei and S. flexneri isolates. Using a test panel of 254 clinical samples, this assay has a sensitivity of 100% in differentiation of EIEC/Shigella pathotype from non-EIEC strains, and 68.7% success rate in distinction of Shigella and EIEC. A novel, and particularly successful marker was a Shigella-specific deletion in the spermidine acetyltransferase gene speG, reflecting its metabolic decay. For Shigella serotype prediction, the multiplex assay scored a sensitivity and specificity of 96.6% and 98.4%, respectively. All discrepancies were analysed with whole-genome sequencing and shown to be related to causative mutations (stop codons, indels and promoter mutations) in glycosyltransferase genes. This observation spurred the development of an in silico workflow which extracts the Shigella serotype from NGS data, taking into account gene functionality. Both tools with will be implemented in the workflow of the NRC, and will play a major role in the shift from phenotypic to genotyping based surveillance of shigellosis in Belgium.