CONICET | Buscador de Institutos y Recursos Humanos

DEVELOPMENT AND EVALUATION OF A IMMUNODIAGNOSTIC TEST FOR CYSTIC ECHINOCOCCOSIS IN INTERMEDIATE HOSTSSerafino, Juan; Gallo Calderón Marina B ; Jensen Oscar ; Larrieu E, Heath D, Poggio T Verónica1.Centro de Virología Animal ? CEVAN-Instituto de Ciencia y Tecnología ?Cesar Milstein? (CONICET). Saladillo 2468, C1440FFX. Bs As, Argentina.vpoggiocevan@centromilstein.org.ar Echinococcosis Cystic is one of the most prevalent zoonosis in Argentina. There is not availability of serological diagnostic tools for detection of antibodies against Echinococcus granulosus antigens in sheep and goats to support the epidemiological monitoring and surveillance over time.Analysis of the E granulosus genome shows that the 8-kDa glycoprotein family from taeniid cestodes comprises widely distributed members with high molecular diversity either for cross- reaction or specific reaction among metacestodes. For these reason, some variants of the 8-kDa protein can be used for the specific diagnosis of metacestode infections.AimDevelopment and validation of a new diagnostic technology for detection of E. granulosus antibodies in serum of sheep and goat by an immunoenzymatic assay.Materials and MethodsGene coding for 8-kDa protein of E. granulosus was amplified by the polymerase chain reaction and cloned into pGEX-1 lamba T vector. Nucleotide sequence analysis was performed.The recombinant 8-kDa protein was expressed as inclusion bodies, purified by affinity chromatography and immunologically characterized by Western blot.Pool of sera from experimentally or naturally infected sheep from endemic zone, and non vaccinated animals living in a hydatid free area, which revealed no cysts and non reactive Western blot, were involved into the direct ELISA standarization technique. All reagents, plate design, dilution of sera and controls, the determination of the cut off and the acceptance and rejection criteria were performed according to requierements from OIE Manual of diagnostic test.ResultsThe recombinant 8 kDa protein (90 aminoacids) was expressed in bacteria according to the expected size and the immunological characterization after purification was achieved by monoclonal antibodies anti-Glutathione S-transferase (GST) and anti Histidine (6XHis). The recombinant antigen was captured to the plate at optimal coating concentration, and secondary antibody worked optimally at suggested dilutions. No evidence of cross reactivity with other related parasitic infections was dectected. These results minimized the risk of obtaining false positive immune response.Sensitivity, specificity, and positive or negative predictive values above to 90% have been estimated comparing our ELISA test with Western Blot. To date, this thecnology is being submitted to a specific set of essential validation criteria from OIE for infecious disease.ConclusionsThere is an urgent need for specific, inexpensive, rapid and highly sensitive screening diagnostic tests that can be used for epidemiological studies, control programs, surveillance and identification of echinococcosis infected intermediate hosts.Progress has being made in development of a novel immunodiagnostic kit using the recombinant 8kDa- protein of E. granulosus as antigen capture ELISA method. This technology including a well-characterized recombinante antigen has been successfully standardized and tested with a promising specificity, sensitivity and positive/negative predictive values as an accurate diagnostic tool.

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