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Khokhar, F.; Pickard, D.; Dyson, Z.; Jacob John, J.; Pragasam, A.; Veeraraghavan, B.; Iqbal, J.; Qamar, F.; Dougan, G.; MacQueen, H.; Rigas, S.; Holmes, M. and Mutreja, A.
(2022).
URL: https://fems-microbiology.org/opportunities/escmid...
Abstract
Background: Enteric fever infections caused by Salmonella serovars Typhi and Paratyphi A remain a significant public health issue, with up to 20 million infections per year. Increasing rates of antibiotic resistant isolates have rendered many first-line antibiotics potentially ineffective. Current methods of identification require culture for several days followed by whole-genome sequencing (WGS) to determine the specific lineage. Genotype 4.3.1 (H58) is the main circulating lineage of S. Typhi in many South Asian countries and is associated with high levels of antibiotic resistance. The emergence and spread of extensively drug resistant (XDR) typhoid has increased the need for a rapid molecular test to identify and track these high-risk lineages for treatment decisions, surveillance, and vaccine prioritisation.
Methods: Utilising WGS datasets that have identified SNPs specific for H58 and XDR S. Typhi lineages, we designed primers to incorporate these mutations as robust genomic markers of clinical significance and optimised a single multiplex PCR assay. Genomic DNA was extracted from pure cultures of S. Typhi, non-Typhi Salmonella and non-Salmonella samples.
Results: Initial testing of our assay showed 100% specificity of our targets for the identification of S. Paratyphi A and the H58 and Pakistan XDR lineages of S. Typhi, with no false positive amplification with non-Typhi Salmonella or non-Salmonella samples. Our assay also identified 13/75 samples as S. Paratyphi A which had previously been identified as S. Typhi by MALDITOF MS, all of which were confirmed by WGS.
Conclusion: This DNA-based diagnostic assay can offer a cheaper alternative to WGS for sensitive and specific rapid detection of risk stratified S. Typhi and S. Paratyphi A. With further testing planned in typhoid fever endemic regions, this assay could be used directly on both clinical and environmental samples for rapid diagnosis and surveillance.