Beyond Pilot Data: Reframing Tc‐24 ELISA in the Diagnostic Ecology of Canine Chagas Disease

Dear Editor: We read with interest the pilot study by Mejia et al. (2025) introducing the Tc-24 recombinant antigen ELISA for canine Chagas disease. The assay's reported sensitivity and specificity are encouraging, but we believe the broader implications of this tool require deeper scrutiny. Strain Diversity and Antigenic Conservation: while Tc-24 is described as highly conserved across T. cruzi lineages, field studies demonstrate that serologic test performance can vary substantially across discrete typing units (DTUs) (Truyens et al. 2021; Fontes et al. 2024). In Louisiana, for example, Dumonteil et al. (2020) reported differential reactivity in dogs infected with TcII and TcVI strains. It remains uncertain whether Tc-24 truly provides pan-DTU coverage in practice. We suggest future studies adopt geographically comparative validation cohorts to test the robustness of Tc-24 across the Americas. Serology versus Infection Status: the study acknowledges the limitation of relying solely on antibody detection, but the implications warrant further consideration. Seropositivity may reflect prior exposure rather than active infection, leading to potential overestimation of prevalence (Ascanio et al. 2024). Integration of dual-platform diagnostics—for example, Tc-24 ELISA combined with PCR or antigen detection—could better define infection dynamics. Without molecular confirmation, seroepidemiologic estimates risk misinforming surveillance and control strategies. Cross-Reactivity in Co-Endemic Settings: although the authors report minimal cross-reactivity with Leishmania in Texas, canine leishmaniasis is increasingly reported in North America (Rodrigues et al. 2022). Moreover, multiplex microsphere assays (Rodriguez et al. 2023) have shown that multi-antigen panels can better discriminate between infections in co-endemic areas. We recommend that Tc-24 ELISA be evaluated within multi-pathogen diagnostic frameworks, where its added diagnostic value can be measured relative to existing platforms. By situating Tc-24 within a diagnostic ecology framework—where tools are complementary rather than standalone—its translational value could be more fully appreciated. In summary, Mejia et al. (2025) provided important pilot data, but the next phase should emphasize cross-DTU validation, integration with molecular tools and deployment strategies within real-world surveillance systems. Only through this broader perspective can Tc-24 ELISA move from proof-of-concept towards meaningful impact in veterinary and zoonotic Chagas disease control. N.K.R. conceived and drafted the letter. C.N.G., K.L.S. and S.K.R. contributed to the writing, critical revision, and editing of the manuscript. All authors approved the final version. We thank Mejia et al. (2025) for their important contribution in developing the Tc-24 ELISA, which provides the foundation for our commentary. The authors have nothing to report. The authors declare no conflicts of interest. The authors have nothing to report. The authors have nothing to report. The peer review history for this article is available at https://doi.org/10.1002/vms3.70855.