Diagnostic value of two domains derived from a novel Trypanosoma cruzi protein for chronic Chagas disease in Brazilian endemic regions by ELISA assay

Abstract

Introduction: Chronic Chagas disease (CCD) is a potentially life-threatening illness caused by the Trypanosoma cruzi parasite. In the absence of a definitive "gold-standard" laboratory assay, the WHO recommends using at least two distinct IgG-based tests for a reliable diagnosis. Recently, we demonstrated the diagnosis performance of two domains (TcD3 and TcD6) derived from a hypothetical T. cruzi protein, named Tc323, which has no homologs in T. brucei or Leishmania genomes. For this first study, we used plasma of patients with CCD and non-infected donors from Argentina, Bolivia, and Paraguay. Here, we extended our work by testing plasma of individuals with CCD or infected with unrelated diseases living in endemic regions of Brazil.Methodology: After the optimization of our indirect ELISA, recombinant (rTcD3 and rTcD6) proteins were assessed by employing plasma of 405 T. cruzi-positive and 530 T. cruzi-negative individuals from distinct areas of Brazil. Additionally, 748 samples from donors with unrelated diseases were included to evaluate cross-reactivity. The receiver operating characteristic (ROC) curve was used to define the cut-off value with the best diagnostic performance for each ELISA assay using pooled plasma samples from positive and negative donors. The results were expressed as reactivity index (RI), calculated as the ratio between the optical density (OD) of the samples and the OD of the cut-off, where RI >1.00 were considered positive. To evaluate the overall precision for each antigen, areas under the ROC curve (AUC) were performed.Findings: The AUC values were 92.89% for rTcD3 and 92.11% for rTcD6, while both exhibited a specificity of 97.92%, with rTcD3 demonstrating a sensitivity of 79.51% and TcD6 showing a sensitivity of 81.48%. The accuracy of rTcD6 was 90.8%, slightly higher than rTcD3's 89.95%. Assuming an inconclusive range of RI values within 1.0 ± 0.10, 13 (3.95%) and 16 (4.86%) true positive samples tested against rTcD3 and rTcD6, respectively, yielded false results. Among the true negative samples, 4 (0.75%) fell into the inconclusive range with rTcD3, compared to only 1 (0.18%) with rTcD6. Cross-reactivity was minimal: only 4 out of 748 plasma samples showed seroreactivity with rTcD3, and 5 samples generated false positives with rTcD6. In both cases, these samples were from patients infected with Leptospira or Leishmania spp. However, no reactivity was detected with plasma from individuals with dengue, filariasis, HBV, HCV, HIV, rubella, schistosomiasis, syphilis, COVID, leprosy and toxoplasmosis.Conclusion: Our results demonstrated a noteworthy performance of rTcD3 and rTcD6 for the diagnosis of CCD individuals from endemic areas of Brazil. Evenmore, both proteins showed insignificant cross-reactivity with non-related infectious diseases