A new multiplex assay platform was evaluated to detect Trypanosoma cruzi
infection using the recombinant antigens
CRA, FRA, CRAFRA fusion and parasite lysate. The antigens presented different sensitivity and specificity in a singleplex test when compared to a serial dilution of two pools comprising 10 positive serum samples and one pool of 10 negative samples. The recombinant protein CRA presented lower sensitivity (55%) in contrast to the 100% specificity
and sensitivity of FRA, CRAFRA and T. cruzi
lysate. These antigens also showed good results in a duplex test and the duplex test with CRAFRA/T. cruzi
lysate showed better performance with 100% specificity and sensitivity, as well as a lower cut-off value in comparison to the other duplex test, FRA/T. cruzi
lysate. Hence, when the antigens were used in duplex format, both tests showed decreased cut-off values and no interference between different bead sets, resulting in increasing sensitivity and specificity. The results of these multiplex tests show that they could be an alternative to singleplex detection for Chagas disease, and also indicate the necessity of using multiplex diagnostic tools to increase the sensitivity and specificity for diagnostic tests. Emerging data from the T. cruzi
genome and from its ORFeome project will also allow the identification of new antigens for this disease detection application.