The existence of an imperfect reference standard presents complications when evaluating the unbiased performance of novel diagnostic techniques. This is especially true in the absence of a gold standard, as is the case in chronic Chagas disease (CD) diagnosis. To circumvent this constraint, we elected to use latent class analysis (LCA). Previously, our group demonstrated the high performance of four Trypanosoma cruzi–chimeric proteins (Molecular Biology Institute of Paraná [IBMP]-8.1, -8.2, -8.3, and -8.4) for CD diagnosis using several distinct immunoassays. Although commercial tests had previously been established as a reference standard, the diagnostic performance of these chimeric antigens could present bias because these tests fail to produce 100% accurate results. Thus, we used LCA to assess the performance of these IBMP chimeric antigens in chronic CD diagnosis. Using the LCA model as a gold standard, sensitivity and specificity values ranged from 93.5% to 99.4% and 99.6% to 100%, respectively. The accuracy values were 96.2% for IBMP-8.2, approximately 98% for IBMP-8.1 and IBMP-8.3, and nearly 100% for IBMP-8.4. For IBMP-8.1 and IBMP-8.2, higher positive predictive values were associated with increases in hypothetical prevalence. Similarly, higher hypothetical prevalence resulted in lower negative predictive values for IBMP-8.1, IBMP-8.2, and IBMP-8.3. In addition, samples with serodiscordant results from commercial serological tests were analyzed using LCA. Molecular Biology Institute of Paraná -8.1 demonstrated potential for use in confirmatory testing with regard to samples with inconsistent results. Moreover, our findings further confirmed the remarkable performance of the IBMP-8.4 antigen to diagnose chronic CD in both endemic and non-endemic areas.

Chronic Chagas Disease (CD) diagnosis is based on serological methods employing crude, semipurified or recombinant antigens, which may result in low sensitivity or cross-reactivity. To reduce these restrictions, we developed a strategy involving use of molecules containing repetitive fragments of Trypanosoma cruzi conserved proteins. Diagnostic performance of IBMP-8.1 and IBMP-8.4 chimeric antigens (Molecular Biology Institute of Paraná - IBMP in Portuguese acronym) was assessed to diagnose T. cruzi-infected and non-infected immigrants living in Barcelona (Spain), a non-endemic setting for Chagas disease. Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house automated ELISA with 347 positive and 331 negative individuals to Chagas disease. Antigenic cross-reactivity was measured with sera samples from pregnant women with Toxoplasma gondii (n = 98) and Zika virus (n = 75) antibodies. The area under the curve values was 1 and 0.99 for the IBMP-8.1 and IBMP-8.4 proteins, respectively, demonstrating excellent diagnostic accuracy. The reactivity index was higher for IBMP-8.1 than IBMP-8.4 in positive samples and no significant difference in reactivity index was observed in negative samples. Sensitivity ranged from 99.4% for IBMP-8.1 to 99.1% for IBMP-8.4 and was not statistically different. Specificity for IBMP-8.1 reached 100 and 99.7% for IBMP-8.4, both nearly 100% accurate. No antigenic cross-reactivity was observed and reactivity index was similar to that for negative Chagas disease individuals. Our results showed an outstanding performance of IBMP-8.1 and IBMP-8.4 chimeric antigens by ELISA and suggest both chimeric antigens could also be used for Chagas disease diagnosis in immigrants living in non-endemic settings.