Cross-Reactivity Using Chimeric Trypanosoma cruzi Antigens: Diagnostic Performance in Settings Where Chagas Disease and American Cutaneous or Visceral Leishmaniasis Are Coendemic.

Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Here, we validated our findings using serum samples from different Brazilian geographic areas reporting endemic Chagas disease, endemic visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and with only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2 and 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions where T. cruzi and Leishmania spp. are coendemic.

Immune reactivity to Trypanosoma cruzi chimeric proteins for Chagas disease diagnosis in immigrants living in a non-endemic setting.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Highly Accurate Chimeric Proteins for the Serological Diagnosis of Chronic Chagas Disease: A Latent Class Analysis.

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.

Acute experimental trypanosoma cruzi infection in mice-alterations of lysossomal profiles in non-parasitized myocardial cells

O parasitismo celular e a miocardite têm sido considerados os mecanismos mais significativos para o dano cardíaco na fase aguda da doença de Chagas. todavia, degeneração e morte de células não parasitadas também ocorrem, contribuindo para o surgimento de alterações funcionais cardíacas. Para melhor elucidar a patogênese dessas lesões, lisossomos de células não parasitada foram analisados por estudo citoquímico ultrastrutural da fosfatase ácida. Comudongos Swiss foram inoculados intra peritonealmente com a cepa colombiana do T. cruzi e examinados até a terceira semana após a infecção. Áreas contendo células não parasitadas foram selecionadas em cortes semi-finos. A partir da segunda semana, foi observado um acentuado aumento no número dos perfis lisossômicos em células miocárdicas e macrofágicas não infectadas. essas alterações foram coincidentes com o desenvolvimento de miocardite. Os resultados sugerem que, na fase aguda de doença de chagas experimental, o aumento na atividade lisossomica, secundário a fenõmenos inflamatórios e /ou hipóxicos, pode desempenhar um papel importante no processo de dano das células miocardicas não parasitadas