Diagnostic performance of a baculovirus-expressed Tc-CPC-polyhedrin fusion protein in chronic Chagas disease

Diagnosis of chronic Chagas disease relies on serological assays and usually requires concordant results from tests based on different antigenic preparations because assay performance may be affected by parasite diversity and cross-reactivity. The development of well-characterized recombinant antigens therefore remains a priority. Here, we evaluated Trypanosoma cruzi cysteine peptidase C (Tc-CPC) as a candidate diagnostic antigen by integrating sequence- and structure-based analyses, expression in a eukaryotic system, and immunoassay assessment. In silico analyses identified features consistent with a secreted papain-like cysteine peptidase, including an N-terminal signal peptide, conserved catalytic residues, predicted glycosylation sites, disulfide-compatible cysteine residues, and a surface-accessible conformational B-cell epitope. Structural comparisons predicted that the principal epitope-bearing region remained accessible in the Tc-CPC-polyhedrin-6×His fusion. The recombinant protein was produced in baculovirus-infected insect cells, and expression was confirmed by SDS-PAGE and immunoblotting. In an indirect ELISA, the fusion antigen discriminated samples from patients with chronic Chagas disease (n = 115) from those of healthy seronegative individuals (n = 18), with an area under the receiver operating characteristic curve of 0.976 (95% confidence interval, 0.952-1.000). At the selected cutoff (OD > 0.509), sensitivity was 94.78% and specificity against this seronegative control group was 100.00%, with reactivity detected across cardiac, digestive, mixed, and indeterminate clinical forms. Inter-assay variability was low (median coefficient of variation, 0.62%). These findings identify baculovirus-expressed Tc-CPC-polyhedrin-6×His as a promising candidate antigen for chronic Chagas disease serodiagnosis in this single-center Brazilian cohort. Cross-reactivity with sera from individuals with leishmaniasis, T. rangeli exposure, and other potentially cross-reactive infections was not assessed and remains a priority for future validation.