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Background Dogs are considered sentinels in areas of Trypanosoma cruzi transmission risk to humans. ELISA is generally the method of choice for diagnosing T. cruzi exposure in dogs, but its performance substantially depends on the antigenic matrix employed. In previous studies, our group has developed four chimeric antigens (IBMP-8.1, 8.2, 8.3, and 8.4) and evaluated their potential for diagnosing T. cruzi exposure in humans. For human sera, these chimeric antigens presented superior diagnostic performances as compared to commercial tests available in Brazil, Spain, and Argentina. Therefore, in this study we have evaluated the potential of these antigenic proteins for detection of anti-T. cruzi IgG antibodies in dog sera. Methodology/Principal findings The IBMP-ELISA assays were optimized by checkerboard titration. Subsequently, the diagnostic potential was validated through analysis of ROC curves and the performance of the tests was determined using double entry tables. Cross-reactivity was also evaluated for babesiosis, ehrlichiosis, dirofilariosis, anaplasmosis, and visceral leishmaniasis. Best performance was shown by IBMP-8.3 and IBMP-8.4, although all four antigens demonstrated a high diagnostic performance with 46 positive and 149 negative samples tested. IBMP-8.3 demonstrated 100% sensitivity, followed by IBMP-8.4 (96.7–100%), IBMP-8.2 (73.3–87.5%), and IBMP-8.1 (50–100%). The highest specificities were achieved with IBMP-8.2 (100%) and IBMP-8.4 (100%), followed by IBMP-8.3 (96.7–97.5%) and IBMP 8.1 (89.1–100%). Conclusions/Significance The use of chimeric antigenic matrices in immunoassays for anti-T. cruzi IgG antibody detection in sera of infected dogs was shown to be a promising tool for veterinary diagnosis and epidemiological studies. The chimeric antigens used in this work allowed also to overcome the common hurdles related to serodiagnosis of T. cruzi infection, especially regarding variation of efficiency parameters according to different strains and cross-reactivity with other infectious diseases., Author summary Despite dogs being considered T. cruzi’s most important domestic sentinel/reservoir and also suffering from the outcomes of the infection, there has never been a commercially available test to diagnose T. cruzi infection in dogs. As such, our group’s objective was to develop a state-of-the-art serological diagnostic test utilizing four chimeric antigens (IBMP-8.1, 8.2, 8.3, and 8.4) in an ELISA platform to accurately identify canine anti-T. cruzi IgG antibodies. The IBMP-ELISA assays were optimized, evaluated for cross-reactivity towards multiple canine parasite’s, including Leishmania spp., its diagnostic potential was validated and the test’s performance was determined using double entry tables. The IBMP-8.3 antigen demonstrated 100% sensitivity, followed by IBMP-8.4, whereas the highest specificities were achieved with IBMP-8.2 (100%) and IBMP-8.4 (100%). Therefore, we’ve concluded that the serodiagnosis through anti-T. cruzi IgG detection in dogs, utilizing chimeric antigenic matrices in immunoassays is a promising tool for veterinary diagnosis and epidemiological surveillance. Furthermore, the use of chimeric antigens efficiently addressed common hurdles related to T. cruzi serodiagnosis, especially regarding efficiency variation in response to different strains and cross-reactivity.

Background and Objectives Serological screening for Chagas’ disease in the blood banks of South America is carried out by using two different assays that generally show a high number of inconclusive results. To establish a combination of two tests that can minimize the number of inconclusive results, we compared a recombinant enzyme-linked immunosorbent assay (ELISA) with two conventional tests. Materials and Methods Serum samples from chagasic patients (n = 112), from non-chagasic individuals (n = 143) and from patients with other diseases (n = 32) were tested using three assays: recombinant ELISA (Rec-ELISA); conventional ELISA (Con-ELISA); and the indirect haemagglutination (IHA) test. Results When we evaluated the data by matching the Rec-ELISA and the IHA test, 52 inconclusive results were obtained. When Rec-ELISA and Con-ELISA were matched, only four inconclusive results were observed. Conclusions Our investigation indicates that the use of two ELISAs with different antigen preparations provides an effective test combination for blood bank screening of Chagas’ disease.

The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.

Com o objetivo de avaliar o desempenho de diferentes tecnicas e antigenos no diagnostico sorologico da doenca de Chagas em diferentes areas endemicas, foram produzidos tres conjuntos diagnosticos (kits) para testes de imunofluorescencia indireta (IFI) e tres para imunoensaio do tipo ELISA, empregando antigenos de T cruzi dos biodemas 1, II e III (cepas Y, 12-SF e Colombiana, respectivamente). Foram testados 541 soros com resultados para anticorpos anti-Tcruzi anteriormente conhecidos atraves dos testes comerciais de IFI e ELISA: 336 positivos e 205 negativos. O painel de soros, constituido por sorteio (tabela de numeros aleatorios), foi composto por 179 amostras de pacientes de Virgem da Lapa-MG, 176 de Jogo Costa-PI e 186 do Sertao da Paraiba-PB. Todos os soros foram submetidos ao teste de IFI quantitativo nas diluic5es 1:40 ate 1:2560, na razao 2, e ao teste de ELISA, na cliluicao de 1:100. A analise dos resultados dos testes de IFI mostrou eficiencia de 100%, 99,8% e 97,6% para os biodemas 1, II e III, respectivamente, enquanto nos testes de ELISA a eficiencia para esses biodemas foi de 93,1%, 94,4% e 93,3%, respectivainente, sem diferenca significativa em relacâo a area de origem dos soros.

In the present report we analyzed the levels of IgG1, IgG2a, IgG2b and IgG3 isotypes from Balb/c mice immunized with cytoplasmic repetitive antigen (CRA), and flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The immunization was done by subcutaneous route three times (20 days apart) and the analysis was performed 14 days after each treatment. CRA-immunized mice produced high levels of all IgG isotypes, mainly IgG3 and IgG1. FRA-immunization elicited only high levels of IgG1.