Desenvolvimento e padronização de novas metodologias aplicadas ao diagnóstico e monitoração de cura da esquistossomose mansoni na fase inicial (aguda) e crônica (2012)

In this project, various candidate antigens for standardization of new diagnostic methods were tested for use at different phases of schistosome infection. Initially, the crude antigens of the parasite were analyzed, since their low cost and ease of obtaining deserve new approaches. Thus, antigens of adult worms, egg antigens, and tegument antigens of schistosomula were analyzed by means of sera of patients submitted to a rigorous parasitological diagnosis for determination of infection. When applied to the indirect method of ELISA, these antigens presented high sensitivity and specificity levels at different phases of murine infection. Based on these results, it was possible to confirm that the use of antigens obtained at different evolutive phases of the parasite acts as a potential tool for analysis of the chronological evolution of infection. When applied to human samples, the use of adult worm antigens was promising for diagnosis of patients living in endemic areas, and presenting low worm burden, with sensitivity and specificity levels of 95%. Under these conditions, they were considered superior than the egg antigens. The study related to tourists presenting acute phase of infection allowed the validation of the indirect technique of ELISA, with tegument antigens of schistosomula. This technique showed a significant sensitivity for identification of a large part of recently infected patients. Another approach used in this study involved the use of Circulating Cathodic Antigen (CCA), which was secreted/excreted by juvenile and adult worms, that were directed to development of new and promissing diagnostic methodologies. For this purpose, the CCA was used at different forms of antigens: as purified glycoprotein obtained from worms, as recombinant protein and as immunogenic peptides of 20 aminoacids. These antigens were used for standardization of the Immunomagnetic Separation Method, named IMS. The use of recombinant CCA in the indirect method of IMS showed the most significant levels of sensitivity and specificity, and no false-negative results could be detected. On the other hand, the use of purified glycoproteinCCA demonstrated to be superior for diagnosis of cure control. Based on these results, which were more promissing than the conventional ELISA, we started the final standardization of this technique for the direct detection of CCA in the same samples, in order to allow only the identification of active infections. For this purpose, specific monoclonal antibodies for glycoprotein CCA were produced and conjugated to merchandises. The choice of the clone was based on the lack of its specificity by the portion responsible for the cross-reactions of CCA, the glicidic portion Lewis x, and in order that a low level of cross-reactivity could be detected by this method, in future analyses. The new method IMS demonstrated high levels of sensitivity (94%) and specificity (100%), reaching superior levels than the ones showed by the current immunological methods, as well as presenting a direct correlation with those patients´worm burdens, which were obtained by fecal egg counts. The excellent results obtained in the present study regarding the detection of circulating antigen, that did not corroborate the results obtained in other published papers, are due to the new methodology used, that utilizes the concentration of these antigens and not the dilution of samples. Finally, we planned another method, named FluoIMS, for the qualitative identification of the presence of CCA by means of fluorescence microscopy. This method, offering direct detection and ease of execution, showed significant levels of sensitivity, when three individual glass-plates for each sample were analyzed. Our results offer great expectancy for the improvement of diagnosis of infected patients with low Schistosoma mansoni burdens, at different phases of infection, and indicate new perspectives for application of these methods in the post-treatment cure control