Evolution of anti-Trypanosoma cruzi antibody production in patients with chronic Chagas disease: Correlation between antibody titers and development of cardiac disease severity

Abstract

Chagas disease is one of the most important endemic infections in Latin America affecting around 6-7 million people. About 30-50% of patients develop the cardiac form of the disease, which can lead to severe cardiac dysfunction and death. In this scenario, the identification of immunological markers of disease progression would be a valuable tool for early treatment and reduction of death rates. In this observational study, the production of anti-Trypanosoma cruzi antibodies through a retrospective longitudinal follow-up in chronic Chagas disease patients´ cohort and its correlation with disease progression and heart commitment was evaluated. Strong inverse correlation (ρ = -0.6375, p = 0.0005) between anti-T. cruzi IgG1 titers and left ventricular ejection fraction (LVEF) in chronic Chagas cardiomyopathy (CCC) patients were observed after disease progression. Elevated levels of anti-T. cruzi IgG3 titers were detected in all T. cruzi-infected patients, indicating a lack of correlation of this IgG isotype with disease progression. Furthermore, low levels of anti-T. cruzi IgG2, IgG4, and IgA were detected in all patients through the follow-up. Although without statistical significance anti-T. cruzi IgE tends to be more reactive in patients with the indeterminate form (IND) of the disease (p = 0.0637). As this study was conducted in patients with many years of chronic disease no anti-T. cruzi IgM was detected. Taken together, these results indicate that the levels of anti-T. cruzi IgG1 could be considered to seek for promising biomarkers to predict the severity of chronic Chagas disease cardiomyopathy.

Fig 1. Study flowchart.

Strategy used to define groups and sub-groups in the study.

Fig 2. Kinetics of anti-T. cruzi IgG₁ during the course of infection in Chagas disease patients.

(A) and (B) represent the kinetics of anti-T. cruzi IgG₁ titers during the follow-up ordered from first to sixth serum collection for each patient in IND and CCC(S) groups, respectively. Blood samples were obtained sequentially with a minimum of one-year interval between each other. Dashed lines delimitate the range of the antibody titer, represented in the vertical axis. (C) and (D) represent the kinetics of anti-T. cruzi IgG₁ titers during the follow-up from 48 months before to 48 months after disease progression for each patient in CCC(P-WD/MD) and CCC(P-MOD/SD) sub-groups, respectively. The time 0 corresponds to the titer measured at the time of disease progression. Open and filled circles represent CCC(P-WD/MD) patients without and with mild LVEF dysfunction, respectively, while open and filled squares represent CCC(P-MOD/SD) patients with moderate and severe LVEF dysfunction, respectively.

Fig 3. Reactivity of IgG₁ for anti-T. cruzi antigen and correlation with ventricular ejection fraction in patients with Chagas disease.

(A) Inverse of the anti-T. cruzi IgG₁ titers in sera of patients with chronic form of Chagas Disease in the indeterminate form of disease (IND), stable cardiomyopathy (CCC(S)), progressive cardiomyopathy (CCC(P)) before disease progression (BP) and progressive cardiomyopathy after disease progression (AP), respectively. The statistical analysis was calculated using ANOVA-one way plus Kruskal-Wallis post-test for multiple comparisons. (B) Inverse of anti-T. cruzi IgG₁ titers in sera of patients with progressive cardiac form of Chagas disease (CCC(P) group) before and after disease progression sub-grouped according to the severity of cardiac commitment. Patients with progressive cardiac disease without (circles) or mild (squares) (CCC(P-WD/MD)) and moderate (triangles) or severe (inverted triangles) (CCC(P-MOD/SD)) cardiac dysfunction before (open symbols) and after (filled symbols) disease progression were represented. The statistical analysis was calculated using ANOVA-two way of repeated measures with Sidak’s multiple comparisons test. The data of A and B were plotted as the mean ± standard deviation (SD). (C, D, and E) Correlation between anti-T. cruzi IgG₁ titers and ventricular ejection fraction (LVEF) in patients with the cardiac form of Chagas disease. (C) represents the correlation in patients with stable cardiac disease. (D) represents the correlation in patients with progressive cardiac disease before disease progression. Open circles, squares, triangles, and inverted triangles, represent CCC(P-WD), CCC(P-MD), CCC(P-MOD) and CCC(P-SD) patients, respectively. (E) represents the correlation in patients with progressive cardiac disease after disease progression. Filled circles, squares, triangles, and inverted triangles, represent CCC(P-WD), CCC(P-MD), CCC(P-MOD) and CCC(P-SD) patients, respectively. Spearman correlation was used to identify association between LVEF and anti-T. cruzi IgG₁ levels.

Fig 4. Reactivity of IgG₂ for anti-T. cruzi antigen and correlation with ventricular ejection fraction in patients with Chagas disease.

(A) Inverse of the anti-T. cruzi IgG₂ titers in sera of patients with chronic form of Chagas Disease in the indeterminate form of disease (IND), stable cardiomyopathy (CCC(S)), progressive cardiomyopathy (CCC(P)) before disease progression (BP) and progressive cardiomyopathy after disease progression (AP), respectively. The statistical analysis was calculated using ANOVA-one way plus Kruskal-Wallis post-test for multiple comparisons. (B) Inverse of anti-T. cruzi IgG₂ titers in sera of patients with progressive cardiac form of Chagas disease (CCC(P) group) before and after disease progression sub-grouped according to the severity of cardiac commitment. Patients with progressive cardiac disease without (circles) or mild (squares) (CCC(P-WD/MD)) and moderate (triangles) or severe (inverted triangles) (CCC(P-MOD/SD)) cardiac dysfunction before (open symbols) and after (filled symbols) disease progression were represented. The statistical analysis was calculated using ANOVA-two way of repeated measures with Sidak’s multiple comparisons test. The data of A and B were plotted as the mean ± standard deviation (SD). (C, D, and E) Correlation between anti-T. cruzi IgG₂ titers and ventricular ejection fraction (LVEF) in patients with the cardiac form of Chagas disease. (C) represents the correlation in patients with stable cardiac disease. (D) represents the correlation in patients with progressive cardiac disease before disease progression. Open circles, squares, triangles, and inverted triangles, represent CCC(P-WD), CCC(P-MD), CCC(P-MOD) and CCC(P-SD) patients, respectively. (E) represents the correlation in patients with progressive cardiac disease after disease progression. Filled circles, squares, triangles, and inverted triangles, represent CCC(P-WD), CCC(P-MD), CCC(P-MOD) and CCC(P-SD) patients, respectively. Spearman correlation was used to identify association between LVEF and anti-T. cruzi IgG₂ levels.

Fig 5. Reactivity of IgG₃ for anti-T. cruzi antigen and correlation with ventricular ejection fraction in patients with Chagas disease.

(A) Inverse of the anti-T. cruzi IgG₃ titers in sera of patients with chronic form of Chagas Disease in the indeterminate form of disease (IND), stable cardiomyopathy (CCC(S)), progressive cardiomyopathy (CCC(P)) before disease progression (BP) and progressive cardiomyopathy after disease progression (AP), respectively. The statistical analysis was calculated using ANOVA-one way plus Kruskal-Wallis post-test for multiple comparisons. (B) Inverse of anti-T. cruzi IgG₃ titers in sera of patients with progressive cardiac form of Chagas disease (CCC(P) group) before and after disease progression sub-grouped according to the severity of cardiac commitment. Patients with progressive cardiac disease without (circles) or mild (squares) (CCC(P-WD/MD)) and moderate (triangles) or severe (inverted triangles) (CCC(P-MOD/SD)) cardiac dysfunction before (open symbols) and after (filled symbols) disease progression were represented. The statistical analysis was calculated using ANOVA-two way of repeated measures with Sidak’s multiple comparisons test. The data of A and B were plotted as the mean ± standard deviation (SD). (C, D, and E) Correlation between anti-T. cruzi IgG₃ titers and ventricular ejection fraction (LVEF) in patients with the cardiac form of Chagas disease. (C) represents the correlation in patients with stable cardiac disease. (D) represents the correlation in patients with progressive cardiac disease before disease progression. Open circles, squares, triangles, and inverted triangles, represent CCC(P-WD), CCC(P-MD), CCC(P-MOD) and CCC(P-SD) patients, respectively. (E) represents the correlation in patients with progressive cardiac disease after disease progression. Filled circles, squares, triangles, and inverted triangles, represent CCC(P-WD), CCC(P-MD), CCC(P-MOD) and CCC(P-SD) patients, respectively. Spearman correlation was used to identify association between LVEF and anti-T. cruzi IgG₃ levels.

Fig 6. Reactivity of IgG₄ for anti-T. cruzi antigen in patients with Chagas disease.

(A) Inverse of the anti-T. cruzi IgG₄ titers in sera of patients with chronic form of Chagas Disease in the indeterminate form of disease (IND), stable cardiomyopathy (CCC(S)), progressive cardiomyopathy (CCC(P)) before disease progression (BP) and progressive cardiomyopathy after disease progression (AP), respectively. The statistical analysis was calculated using ANOVA-one way plus Kruskal-Wallis post-test for multiple comparisons. (B) Inverse of anti-T. cruzi IgG₄ titers in sera of patients with progressive cardiac form of Chagas disease (CCC(P) group) before and after disease progression sub-grouped according to the severity of cardiac commitment. Patients with progressive cardiac disease without (circles) or mild (squares) (CCC(P-WD/MD)) and moderate (triangles) or severe (inverted triangles) (CCC(P-MOD/SD)) cardiac dysfunction before (open symbols) and after (filled symbols) disease progression were represented. The statistical analysis was calculated using ANOVA-two way of repeated measures with Sidak’s multiple comparisons test. The data of A and B were plotted as the mean ± standard deviation (SD).

Fig 7. Disease classification in CCC(P) patients before and after disease progression.

Disease classification from entry into the study (BP) to after disease progression (AP) in CCC(P) sub-groups are represented (horizontal axis). The vertical axis represents the levels of disease severity according to LVEF values as described in Methods. Connecting lines link the patients’ disease classification BP and AP disease progression. Open squares represent CCC(P-WD/MD) patients, while filled triangles represent CCC(P-MOD/SD) patients. *Statistical difference between CCC(P-WD/MD) and CCC(MOD/SD) sub-groups BP calculated by chi-square test, p = 0.033). **** Statistical difference between CCC(P-WD/MD) and CCC(MOD/SD) sub-groups calculated by paired ANOVA-two way with Sidak’s multiple comparisons test, p<0.0001. To perform this calculation the vertical axes were transformed in arbitrary units where: CCC(P-WD) = 4; CCC(P-MD) = 3; CCC(P-MOD) = 2; and CCC(P-SD) = 1.