Molecular detection of salmonella typhi and paratyphi A with genomic markers of resistance

Khokhar, Fahad; Pickard, DDJ; Dyson, ZA; Iqbal, J; Pragasam, A; Jacob, John J; Veeraraghavan, B; Qamar, FN; Dougan, G; MacQueen, Hilary; Rigas, Sushila; Holmes, MA and Mutreja, A (2021). Molecular detection of salmonella typhi and paratyphi A with genomic markers of resistance. In: 12th International Conference on Typhoid & Other Invasive Salmonelloses., 06-08 Dec 2021, Online.

URL: https://www.coalitionagainsttyphoid.org/typhoid-co...

Abstract

Background: Enteric fever infections remain a significant public health issue, with up to 20 million infections per year. Increasing rates of antibiotic resistant strains have rendered many first-line antibiotics potentially ineffective. Current methods of identification require samples to be cultured for several days followed by DNA extraction and sequencing 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 strains has increased the need for a rapid molecular test to identify and track these high-risk lineages for treatment decisions, surveillance and vaccine prioritisation.

Methodology: Through screening of thousands of publicly available bacterial genomes, we have identified robust genomic markers at SNP resolution for targeting S. Paratyphi A as well as the H58 and XDR lineages of S. Typhi. Custom primers were designed to incorporate these markers of clinical significance and have been optimised to work in a single multiplex PCR reaction. Reactions were carried out using a standard PCR thermocycler with results visualised by gel-electrophoresis.

Results: Initial testing of our assay using DNA extracted from 32 pure cultures showed 100% specificity for our targets for the identification of H58 and Pakistan XDR lineage of S. Typhi and S. Paratyphi A and produced no false positive reactions with non-Typhi Salmonella or non-Salmonella strains.

Conclusions: We envision that this multiplex assay would be used in routine laboratory diagnosis from DNA extracted after blood culture incubation, and eventually directly in combination with enrichment methods. This will serve in the development of a cheap DNA-based diagnostics for sensitive and specific rapid detection of risk stratified Salmonella Typhi and Paratyphi A serovars directly from both clinical and environmental samples.

Viewing alternatives

No digital document available to download for this item

Item Actions

Export

About