Stage-Informed Trypanosoma cruzi Antigen Selection Enhances Assay Concordance in the Evaluation of Discordant Chagas Disease Serology

Chagas disease is difficult to diagnose in the chronic phase, especially when serological assays disagree. We examined whether Trypanosoma cruzi metacyclic trypomastigotes’ antigenic extracts may increase ELISA/Western blot concordance in initially discordant serum compared to epimastigotes. A total of 178 human blood samples were studied, including seropositive persons, Chagasic cardiomyopathy patients, discordant sera, seronegative controls, and sera from various parasitic illnesses. Antigenic extracts from epimastigotes and metacyclic trypomastigotes were extracted using sonication or urea, and Western blot and ELISA were used to compare their behavior. Sonicated metacyclic trypomastigote extracts produced concordant ELISA/Western blot results in 31/35 originally discordant sera, compared to 19/35 for sonicated epimastigotes, 20/35 for urea-extracted trypomastigotes, and 17/35 for urea-extracted epimastigotes. All extracts in the serum panel previously classified using the standardized in-house serological platform showed 100% positive percent agreement, but negative percent agreement varied between 93.3% for sonicated metacyclic trypomastigotes and 66.7% for urea-extracted epimastigotes. These estimates reflect agreement with the previous serological classification, not diagnostic accuracy versus an independent reference standard. Cross-reactivity was mainly observed in sera from patients with leishmaniasis, while sera from the cardiopathic group showed an exploratory preferential recognition pattern with bands of 18, 22, 24, and 26 kDa in the sonicated metacyclic trypomastigote preparation. The comparison did not account for potential clinical variables or molecularly identify these bands. These data demonstrate that stage-informed antigen selection, particularly using sonicated metacyclic trypo-mastigote extracts, can affect serum panel assay concordance and nonspecific reactivity.