a Fast-track Diagnostics, Esch-sur-Alzette/LU b Fondation Mérieux, Lyon/FR c Institut Pasteur, Paris/FR d ICDDR,B, Dhaka/BD e Fast-track Diagnostics, Luxembourg/LU Purpose: Enteric fever due to Salmonella remains a significant public health problem, predominantly in children in developing countries. Typhoid fever affects more than 20 million people per year associated to about 300,000 deaths. These high rates are mainly due to the lack of fast, reliable and inexpensive diagnostic assays. Diagnosis of typhoid remains a challenge because a low bacterial load can be responsible of an illness and also because the volume of blood that can be collected from children is limited.To overcome the problem of sensitivity, the first goal of this study was to define an appropriate pre-enrichment method of the collected sample before nucleic-acid extraction. The second objective was to develop a sensitive multiplex real-time PCR assay for detection of the common etiological agents of Enteric fever: Salmonella enterica spp., Salmonella typhi and Salmonella paratyphi A.Methods & Materials: To determine the best pre-enrichment medium, we spiked S. paratyphi A within blood samples, added the enrichment medium, cultured for 5 h at 37 • C, performed nucleic acid extraction and determined the yield of specific nucleic acids by real-time PCR. We tested several commercial enrichment broths and a home-made TSB medium supplemented with Oxgall (inhibitor of Gram+ bacteria). The results show that the Selenite broth (Copan) and the TSB-Oxgall medium are the most efficient pre-enrichment methods of free bacteria in blood samples.Results: In parallel we developed a multiplex real-time PCR assay that distinguish between infection by S. enterica spp., S. typhi and/or S. paratyphi A (+ internal control). This assay shows a limit of detection between 1E+02 and 1E+03 copies/ml and a linearity from 1E+09 to 1E+03 copies/ml for all pathogens. The specificity of the assay was validated with various negative samples which did not generate any positive signals. Furthermore, different positive materials containing bacteria, parasites, and viruses were evaluated with this assay and no other than the expected bacteria were detected.Conclusion:In conclusion, this study shows the development of an efficient diagnostic pipeline that combines a quick preenrichment of infected blood samples with a sensitive real-time multiplex PCR assay that could be used as a reference diagnostic method for Enteric fever infectious agents. http://dx.

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