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

Figures

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  Figure 4. Differential antigen recognition in cardiopathic and non-cardiopathic Chagas sera. (A) Representative Western blots of sera from three patients with Chagasic cardiomyopathy tested against TS, ES, TU, and EU extracts. (B) Representative Western blots of sera from three seropositive non-cardiopathic individuals tested against the same extracts. (C) Individual distribution of the number of recognized bands according to clinical group and antigenic extract. Each dot represents one individual serum sample. Green dots indicate sera from patients with Chagasic cardiomyopathy, whereas orange dots indicate sera from seropositive non-cardiopathic individuals. Horizontal red lines and error bars indicate the mean ± SD. (D) Recognition frequencies of the 18, 22, 24, and 26 kDa bands. Green bars represent cardiopathic sera, and orange bars represent non-cardiopathic seropositive sera. Cardiopathic and non-cardiopathic sera were analyzed across separate gels and blots. The comparisons shown are qualitative and were not used for densitometric comparisons of band intensity across gels. TS, sonicated metacyclic trypomastigotes; ES, sonicated epimastigotes; TU, urea-extracted metacyclic…

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  Figure 1. Progressive in vitro metacyclogenesis of Trypanosoma cruzi under nutritional stress. (A) Percentage of metacyclogenesis at days 4, 7, and 12, determined by complement lysis assay. ****, p < 0.0001, ns: not significant, (B–D) Representative Giemsa-Wright-stained parasites at day 4 (B), day 7 (C), and day 12 (D), illustrating the progressive transition from epimastigote to trypomastigote forms during metacyclogenesis. Arrows indicate epimastigotes, in which the kinetoplast is located between the nucleus of the parasite and the flagellum, whereas asterisks indicate trypomastigotes, in which the kinetoplast is located between the nucleus and the posterior end of the parasite.

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  Figure 2. Comparative protein banding patterns of T. cruzi antigenic extracts. (A) SDS-PAGE of sonicated and urea-extracted preparations obtained from metacyclic trypomastigotes and epimastigotes, using 10 µg of total protein per lane. (B) Binary presence/absence matrix summarizing detectable protein bands across the four antigenic preparations according to apparent molecular weight. Yellow denotes the presence of a detectable band, and black denotes its absence. This matrix was used to compare the qualitative band distribution and the total number of bands. TS, sonicated metacyclic trypomastigotes; ES, sonicated epimastigotes; TU, urea-extracted metacyclic trypomastigotes; EU, urea-extracted epimastigotes.

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  Figure 3. Representative immunoblot and ELISA analysis of sera with discordant Chagas serology. (A) Western blot profiles of two ELISA-negative/Western blot-positive sera tested against TS, ES, TU, and EU extracts. (B) Western blot profiles of two ELISA-positive/Western blot-negative sera tested against TS, ES, TU, and EU extracts. (C) ELISA reactivity, expressed as an OD490-to-cutoff ratio, for discordant sera challenged with the four antigenic preparations, including negative and positive control sera. Statistical comparison among TS, ES, TU, and EU was performed using the Friedman test followed by Dunn’s multiple-comparisons test because the same initially discordant sera were evaluated under all four conditions. Positive and negative control sera are shown as contextual references and were not included in the inferential analysis. The dotted line indicates the cutoff value. TS, sonicated metacyclic trypomastigotes; ES, sonicated epimastigotes; TU, urea-extracted metacyclic trypomastigotes; EU, urea-extracted epimastigotes. ****, p < 0.0001.

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  Figure 5. Representative Western blot evaluation of specificity in seronegative control sera. (A) Immunoblot profiles of two seronegative control sera and one T. cruzi-positive control serum tested against TS, ES, TU, and EU using 3.5 µg of antigen. (B) Immunoblot profiles of the same sera tested under increased antigen-loading conditions (10 µg of antigen). The sonicated metacyclic trypomastigote extract retained lower nonspecific reactivity than the remaining preparations. TS, sonicated metacyclic trypomastigotes; ES, sonicated epimastigotes; TU, urea-extracted metacyclic trypomastigotes; EU, urea-extracted epimastigotes.

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  Figure 6. Representative Western blot analysis of cross-reactivity with heterologous parasitic infections. Western blot strips prepared with the sonicated metacyclic trypomastigote extract (TS) were incubated with sera from a seronegative control, a T. cruzi-seropositive control, and sera from patients with toxoplasmosis, taeniasis/cysticercosis, and leishmaniasis. No immunoreactivity was observed in the toxoplasmosis or taeniasis/cysticercosis sera, whereas a subset of leishmaniasis sera recognized a limited number of bands, consistent with partial cross-reactivity. Molecular weight markers (kDa) are shown on the left.