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Elevated IL-17 levels and echocardiographic signs of preserved myocardial function in benznidazole-treated individuals with chronic Chagas’ disease

Open AccessPublished:December 05, 2018DOI:https://doi.org/10.1016/j.ijid.2018.11.369

      Highlights

      • The benznidazole (Bz) treatment for chronic Chagas’ disease (CCD) may improve immune response and myocardial function.
      • Higher levels of IL-17 were observed in treated CCD patients, specially after adjustment for time of disease and NYHA class.
      • The treated group of patients exhibited better parameters of myocardial function by tissue Doppler than the untreated group.

      Abstract

      Background

      The immunological and clinical impact of trypanocidal treatment in chronic Chagas’ disease (CCD) is unclear.

      Methodology and findings

      Several cytokines were measured in plasma of 66 patients with CCD. Thirty-three patients had been previously treated with benznidazole and 33 had never been treated. The treated group exhibited higher levels of IL-17 (median 142.45 × 1.22 pg/ml, P = 0.025), which was the only one significantly associated with Bz treatment, especially after adjusting for time of disease and NYHA class (P = 0.024; OR 1.006, 95% CI 1.001-1.010). Compared to untreated patients, the treated group exhibited higher median values of mitral annular E’ lateral (13.0 × 10.0 cm/s, P = 0.038), S’ infero-lateral (8.9 × 7.6 cm/s, P = 0.013), S’ septal (8.5 × 7.4 cm/s, P = 0.034), mean S’ (9.0 × 7.9 cm/s, P = 0.013) and tricuspid annular S’ (13.3 × 11.1 cm/s, P = 0.001) and lower values of E/E’ septal (7.2 × 9.5 cm/s, P = 0.049). After adjustment for time of disease and NYHA class, S’ infero-lateral (P = 0.031), mean S’ (P = 0.049) and S’ tricuspid (P = 0.024) persisted as significantly associated with treatment.

      Conclusion

      The present findings suggest that the group of CCD patients treated with Bz displayed increased plasma levels of IL-17 and preserved myocardial function, reinforcing the idea that Bz treatment may be beneficial.

      Keywords

      Introduction

      Chagas’ disease (CD) is a serious health problem, affecting 6 to 7 million people worldwide and leading to roughly ten thousand deaths every year (
      • World Health Organization (WHO)
      Chagas disease (American trypanosomiasis).
      ). Although mortality due to CD has decreased (
      • Da Nóbrega A.A.
      • De Araújo W.N.
      • Vasconcelos A.M.
      Mortality due to chagas disease in Brazil according to a specific cause.
      ), it still causes several irreversible consequences to the cardiovascular systems of 20–30% of infected individuals long after the initial phase, including arrhythmia, heart failure, thromboembolism, stroke, heart block and sudden death (
      • Rassi Jr, A.
      • Rassi A.
      • Marcondes de Rezende J.
      American trypanosomiasis (Chagas disease).
      ). It is estimated that over $1 billion/year is spent worldwide on morbidity associated with Chagas’ cardiomyopathy (
      • Dutra W.O.
      • Menezes C.A.
      • Magalhães L.M.D.
      • Gollob K.J.
      Immunoregulatory networks in human Chagas disease.
      ).
      Chagas’ cardiomyopathy is characterized by diffuse myocarditis in the presence of few parasites (
      • Andrade Z.A.
      • Andrade S.G.
      The pathology of Chagas’ disease (cardiac chronic form).
      ) but with the persistence of Trypanosoma cruzi antigens and nuclear DNA, suggesting occult T. cruzi infection (
      • Jones E.M.
      • Colley D.G.
      • Tostes S.
      • Lopes E.R.
      • Vnencak-Jones C.L.
      • McCurley T.L.
      Amplification of a Trypanosoma cruzi DNA sequence from inflammatory lesions in human chagasic cardiomyopathy.
      ). The recognition of either T. cruzi antigens or myocardial antigens stimulates T cell proliferation (
      • Añez N.
      • Carrasco H.
      • Parada H.
      • Crisante G.
      • Rojas A.
      • Fuenmayor C.
      • et al.
      Myocardial parasite persistence in chronic chagasic patients.
      ,
      • Cunha-Neto E.
      • Coelho V.
      • Guilherme L.
      • Fiorelli A.
      • Stolf N.
      • Kalil J.
      Autoimmunity in Chagas’ disease: identification of cardiac myosin-B13 Trypanosoma cruzi protein crossreactive T cell clones in heart lesions of a chronic Chagas’ cardiomyopathy patient.
      ) consequently triggering a persistent inflammatory response with a dominant Th1 cytokine pattern characterized by a high number of cells expressing TNF and IFN-gamma, associated with heart failure (
      • Rocha Rodrigues D.B.
      • dos Reis M.A.
      • Romano A.
      • Pereira S.A.
      • Teixeira V.P.
      • Tostes S.
      • et al.
      In situ expression of regulatory cytokines by heart inflammatory cells in Chagas’ disease patients with heart failure.
      ). As persistence of T. cruzi may be implicated in persistent inflammatory activation, the etiologic treatment of CD during the chronic phase may have some impact on disease progression.
      Anti-parasite treatment for CD is limited to benznidazole (Bz) or nifurtimox, and Bz is considered the first-line treatment due to better tolerance and fewer side effects (
      • Bern C.
      • Montgomery S.P.
      • Herwaldt B.L.
      • Rassi Jr, A.
      • Marin-neto J.A.
      • Dantas R.O.
      • et al.
      Evaluation and treatment of Chagas disease in the United States: a systematic review.
      ). These trypanocidal drugs are effective during the acute and early chronic phases of CD with a high cure rate (
      • Cançado J.R.
      Long term evaluation of etiological treatment of Chagas disease with benznidazole.
      ,
      • de Andrade A.L.
      • Zicker F.
      • de Oliveira R.M.
      • Silva S.A.
      • Luquetti A.
      • Travassos L.R.
      • et al.
      Randomised trial of efficacy of benznidazole in treatment of early Trypanosoma cruzi infection.
      ,
      • Sosa Estani S.
      • Segura E.L.
      • Ruiz A.M.
      • Velazquez E.
      • Porcel B.M.
      • Yampotis C.
      Efficacy of chemotherapy with benznidazole in children in the indeterminate phase of Chagas’ disease.
      ). Despite an unclear indication of the effectiveness of trypanocidal drugs (
      • Fabbro D.L.
      • Streiger M.L.
      • Arias E.D.
      • Bizai M.L.
      • Del Barco M.
      • Amicone N.A.
      Trypanocide treatment among adults with chronic Chagas disease living in Santa Fe City (Argentina), over a mean follow-up of 21 years: parasitological, serological and clinical evolution.
      ,
      • Morillo C.A.
      • Marin-Neto J.A.
      • Avezum A.
      • Sosa-Estani S.
      • Rassi Jr, A.
      • Rosas F.
      • et al.
      Randomized trial of benznidazole for chronic Chagas’ cardiomyopathy.
      ,
      • Viotti R.
      • Vigliano C.
      • Lococo B.
      • Bertocchi G.
      • Petti M.
      • Alvarez M.G.
      • et al.
      Long-term cardiac outcomes of treating chronic Chagas disease with benznidazole versus no treatment.
      ), the WHO recommends the etiological treatment of chronic Chagas’ disease (CCD) patients, primarily those with no symptoms (
      • World Health Organization (WHO)
      Chagas disease (American trypanosomiasis).
      ). It has been shown that Bz treatment in CCD may have an impact on B and T cell responses (
      • Laucella S.A.
      • Mazliah D.P.
      • Bertocchi G.
      • Alvarez M.G.
      • Cooley G.
      • Viotti R.
      • et al.
      Changes in Trypanosoma cruzi-specific immune responses following treatment: surrogate markers of treatment efficacy.
      ,
      • Sathler-Avelar R.
      • Vitelli-Avelar D.M.
      • Elói-Santos S.M.
      • Gontijo E.D.
      • Teixeira-Carvalho A.
      • Martins-Filho O.A.
      Blood leukocytes from benznidazole-treated indeterminate Chagas disease patients display an overall type-1-modulated cytokine profile upon short-term in vitro stimulation with Trypanosoma cruzi antigens.
      ,
      • Sathler-Avelar R.
      • Vitelli-Avelar D.M.
      • Massara R.L.
      • de Lana M.
      • Pinto Dias J.C.
      • Teixeira-Carvalho A.
      • et al.
      Etiological treatment during early chronic indeterminate Chagas disease incites an activated status on innate and adaptive immunity associated with a type 1-modulated cytokine pattern.
      ,
      • Sathler-Avelar R.
      • Vitelli-Avelar D.M.
      • Massara R.L.
      • Borges J.D.
      • Lana M.
      • Teixeira-Carvalho A.
      • et al.
      Benznidazole treatment during early-indeterminate Chagas’ disease shifted the cytokine expression by innate and adaptive immunity cells toward a type 1-modulated immune profile.
      ,
      • Vallejo A.
      • Monge-Maillo B.
      • Gutiérrez C.
      • Norman F.F.
      • López-Vélez R.
      • Pérez-Molina J.A.
      Changes in the immune response after treatment with benznidazole versus no treatment in patients with chronic indeterminate Chagas disease.
      ), but these findings have not been related to clinical improvement. As the clinical advantage of the etiological treatment of CCD remains undefined, further investigation of its efficacy and its potentially protective mechanisms may help in establishing its usefulness.
      The present study compared untreated to Bz-treated CD patients with long-term follow-up and used an analytical approach that integrated clinical features with electrocardiography and echocardiography parameters, and immune markers.

      Methods

      Study design and participants

      Outpatients with CCD were randomly recruited between 2012 and 2013 from two reference centers in Bahia state, Brazil (University Hospital Prof. Edgard Santos and Climecar, an outpatient clinic in an endemic region for CD), to participate in a prospective cohort study, to investigate biomarkers of prognosis in Chagas’ disease. In the current paper, we will compare patients treated with Benznidazole to those untreated in relation to cytokines and echocardiographic data, in a cross-sectional study. All patients underwent a detailed medical interview with a standard questionnaire, and their medical records were assessed to obtain clinical data. The treatment was considered complete if the patient had taken five mg/Kg/day for sixty days. Only patients with a complete treatment course confirmed by their medical records and those without prior treatment with benznidazole were included in the study. The treatment was performed several months before inclusion in the study (median 38 months, IQR 30.8–45.7 mo.). Most of the individuals from the treated and untreated groups were female (67% and 70%, respectively); no differences were observed in terms of gender or age (Table 1). The untreated group had been diagnosed with CD for a longer period than had the treated group (median 15 and 6 years, respectively, P = 0.001, Table 1). Furthermore, more dyspnea was reported in the untreated group vs. treated group (64% × 33%, P = 0.003), and more class I NYHA (New York Heart Association) was observed in the treated group than in the untreated group (63% x 33%, P = 0.014) (Table 1). Some NYHA class III patients were included in the study, although all of our patients, except one, had LV ejection fraction greater than 50% and none had advanced heart failure. No differences were observed in terms of other clinical parameters (palpitation, syncope, edema, hypertension, diabetes II and stroke) according to previous Bz treatment (Table 1). The electrocardiogram (ECG) obtained at the time of admission in the study indicated that the untreated group had more ventricular premature beats than did the treated group (27% x 0%, P = 0.016, Table 2); however, when adjusted for time of disease and NYHA class, the difference was not statistically significant. The other ECG parameters were similar between the study groups (Table 2). CD was confirmed by IgG and ELISA.
      Table 1Clinical and baseline characteristics of patients according to previous Chagas’ disease treatment.
      TreatedUntreatedP
      n = 33n = 33
      Female – n (%)22 (67)23 (70)0.79
      Age – median years (IQR)51.0 (47–55)51.5 (49–56)0.537
      Time of disease – median years (IQR)6 (410.5)15 (8–20)0.001
      Interval between treatment and inclusion in the study – median months (IQR)38 (30.8–47.5)
      Dyspnea – n (%)11 (33)21 (64)0.003
      Palpitation – n (%)19 (57)22 (67)0.35
      Syncope – n (%)11 (21)12 (36)0.196
      Lower limb edema – n (%)13 (39)6 (18)0.08
      Hypertension – n (%)19 (58)21 (64)0.61
      Type II diabetes – n (%)1 (3)3 (9)0.61
      Previous stroke – n (%)0 (0.00)2 (6)0.28
      NYHA class – n(%)0.029
      I21 (63)11 (33)0.014
      II10 (30)17 (52)0.080
      III2 (5)5 (15)0.230
      ACEI/ARB14 (42)20 (61)0.112
      Calcium channel blockers5 (15)9 (27)0.206
      Statins14 (42)18 (55)0.273
      IQR: interquartile range; NYHA: New York Heart Association.
      Table 2Electrocardiogram parameters according to previous treatment.
      TreatedUntreatedPAdjusted P#
      n = 33n = 33
      ECG normal21 (64)10 (30)0.6430.554
      Heart rate – mean (SD)65 (13.1)69 (13.6)0.2350.218
      Left ventricular hypertrophy – n (%)3 (9)3 (9)0.5000.500
      Supraventricular premature beats – n (%)1 (3)0 (0.00)1.000.235
      Ventricular premature beats – n (%)0 (0.00)9 (27)0.0160.513
      Atrioventricular block (grade I) – n (%)0 (0.00)2 (6)0.5920.307
      Right bundle branch block – n (%)7 (21)10 (30)0.5890.791
      Left bundle branch block – n (%)0 (0.00)1 (3)1.000.321
      Left anterior hemiblock – n (%)4 (12)8 (24)0.5330.649
      Left posterior hemiblock – n (%)0 (0.00)0 (0.00)1.0001.000
      ST-T wave changes – n (%)14 (42)14 (42)1.000.466
      #P adjusted for time of disease and NYHA class.
      Individuals older than 70 years; those treated for more than eight years since the beginning of the study; patients with incomplete Bz treatment; patients with cardiac rhythm other than sinus, such as atrial fibrillation or atrial flutter; advanced atrioventricular block (> grade I); pacemaker users; heart disease other than Chagas; and patients with cancer or other chronic inflammatory diseases (such as connective tissue diseases and rheumatic diseases), were not included in the study.

      Ethics statement

      All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants before enrolling in the study. This study was approved by the Ethics Committee of the Complexo Hospitalar Professor Edgard Santos (protocol number: 77/10).

      Plasma measurements

      The collection of clinical and laboratory data began at a median of 38 months (IQR 30.8–47.5) after treatment with benznidazole. Blood was obtained by venipuncture, and heparinized plasma was separated by centrifugation and stored at −70° C until use in immunoassays. We measured circulating levels of several cytokines and chemokines, including IL-1β (assay sensitivity: 0.6 pg/ml), IL-2 (1.6 pg/ml), IL-4 (0.7 pg/ml), IL-5 (0.6 pg/ml), IL-6 (2.6 pg/ml), IL-7 (1.1 pg/ml), IL-8 (1.0 pg/ml), IL-10 (0.3 pg/ml), IL-12 p70 (3.5 pg/ml), IL-13 (0.7 pg/ml), IL-17 (3.3 pg/ml), IFN-γ (6.4 pg/ml), TNF (6.0 pg/ml), CCL2 (1.1 pg/ml), CCL4 (2.4 pg/ml), GCSF (1.7 pg/ml), and GMCSF (0.2 pg/ml). Samples were measured in duplicate using a single multiplex assay according to the manufacturer’s protocol (BIO-RAD, Hercules, CA, USA).

      Electrocardiogram (ECG) and Doppler echocardiography (ECHO)

      A 12-lead electrocardiogram (ECG) was performed at admission to the study and it was read by two independent and blinded cardiologists. Two-dimensional echocardiograms with conventional Doppler and tissue Doppler (2D Echo) systematized for the study, without knowledge to which group of treatment the patient belonged, were performed in 37 (56%) of the patients (20 treated and 17 untreated group) with a Samsung Sonoview machine, soon after inclusion in the study. Previously performed echocardiograms, not standardized for this study, were not considered for statistical analysis. The 2D Echo parameters were obtained according to the American Society of Echocardiography recommendations (
      • Lang R.M.
      • Badano L.P.
      • Mor-Avi V.
      • Afilalo J.
      • Armstrong A.
      • Ernande L.
      • et al.
      Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American society of echocardiography and the European association of cardiovascular imaging.
      ). Left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV) and ejection fraction (EF) were obtained using the biplane Simpson’s method. E and A waves and the E/A ratio were measured from the mitral inflow by pulsed Doppler. E’(diastole), and S’(systole) velocities were obtained by tissue Doppler at the medial (septal), lateral and infero-lateral mitral annulus and the lateral tricuspid annulus. The E/E’ ratio from each mitral annulus, the mean E/E’ ratio from the three mitral annular measurements, and the mean S’ were calculated (
      • Lang R.M.
      • Badano L.P.
      • Mor-Avi V.
      • Afilalo J.
      • Armstrong A.
      • Ernande L.
      • et al.
      Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American society of echocardiography and the European association of cardiovascular imaging.
      ).

      Data analysis

      In the exploratory analysis of the data, frequency tables were constructed and Chi-square or Fisher exact tests were applied to evaluate the association between qualitative variables. The quantitative variables were tested for Gaussian distribution within the total sample using D’Agostino and Pearson omnibus normality tests. Mann-Whitney test was used to assess the differences between the two clinical groups. Receiver operator characteristic (ROC) curves for immune and Doppler echocardiography measurements were employed to test the ability to distinguish the different groups. Binary regression analysis (stepwise forward method) was performed to test for independent association of the cytokines and Doppler echocardiography markers with treatment, controlled by the time of disease and by NYHA class. Each of the cytokines and each marker of the echocardiogram were tested separately. The statistical analyses were performed using the programs GraphPad Prism 6.0 (GraphPad Software Inc., USA), SPSS 19.0 (IBM, Armonk, NY, USA), and JMP 11.0 (SAS, Cary, NC, USA). A two-tailed P value less than 0.05 was considered statistically significant.

      Results

      Levels of immune markers according to previous Bz treatment

      Compared to the untreated group, treated patients exhibited significantly higher levels of IL-17 (median, IQR 142.45, 1.22–287.13 versus 1.22, 1.22–142.45, P = 0.025).
      Significant differences between study groups, in terms of time of disease and NYHA class, could be influencing the results due to different stages of CD. Than, the statistical significance for each of the cytokines/chemokines was adjusted for time of disease and for NYHA Class, using binary logistic regression analysis, stepwise forward method.
      After adjustment, IL-17 persisted as the only immune marker exhibiting an independent association with Bz treatment. This cytokine was higher in the treated group than in the untreated group (OR: 1.006, 95% CI: 1.001–1.010, P = 0.024) – Table 3, Figure 1.
      Table 3Cytokine and chemokine levels according to previous treatment.
      Treated pg/ml – median (IQR)Untreated pg/ml – median (IQR)P#P adjO.R.95% C.I.
      IL-1β45.74 (1.27–77.96)1.27 (1.27–54.64)0.2840.212
      IL-238.03 (0.46–81.90)0.46 (0.46–56.83)0.1350.590
      IL-437.20 (23.29–49.94)30.44 (19.39–49.94)0.4230.597
      IL-584.52 (7.66–111.88)54.41 (1.44–95.70)0.4390.407
      IL-690.81 (1.04–143.86)41.93 (1.04–153.86)0.4950.501
      IL-784.31 (28.35–127.93)57.98 (28.35–90.65)0.1970.082
      IL-867.64 (8.60–125.50)48.16 (0.77–125.50)0.6870.990
      IL-1013.80 (6.11–23.87)13.80 (6.11–23.87)0.8820.874
      IL-12p7079.54 (8.36–241.48)79.54 (8.36–156.31)0.6270.512
      IL-13204.60 (77.65–354.3)204.62 (103.13–346.27)0.7560.794
      IL-17142.45 (1.22–287.13)1.22 (1.22–142.45)0.025
      P<0.05.
      0.024
      P<0.05.
      1.0061.001–1.010
      IFN-γ1888.76 (854.6–2781.4)1888.76 (854.62–3236.27)0.8210.962
      TNF195.57 (77.98–382.02)143.67 (58.29–252.85)0.2320.119
      CCL275.30 (42.43–107.69)42.43 (8.38–115.73)0.3160.178
      CCL462.13 (16.94–80.18)41.95 (1.05–75.77)0.0590.165
      GCSF243.31 (133.85–438.43)198.04 (154.55–338.35)0.1880.0691.0030.999–1.007
      GMCSF0.51 (0.51–59.48)0.51 (0.51–3.24)0.1860.249
      IQR: interquartile range; #P Adjusted for time of disease and NYHA class. Binary logistic regression analysis (stepwise forward method) was used.
      * P < 0.05.
      Figure 1
      Figure 1Scatter plot for IL-17: A- according to treatment; B- according to treatment and time of disease.
      ROC curves of the several cytokines, with or without treatment, are shown in Table 4. IL-17 had a larger AUC and was the only cytokine with statistical significance (AUC 0.655, C.I. 0.522–0.787, P = 0.031).
      Table 4ROC curves of cytokines.
      VariablesAUC (area under curve)Standard modelP95% C.I.
      Inferior limitSuperior limit
      GCSF.594.071.191.454.733
      GMCSF.615.070.108.479.752
      IFN-G.516.072.822.374.658
      IL1-B.573.071.308.434.712
      IL-2.601.070.158.463.739
      IL-4.557.072.427.416.698
      IL-5.555.071.445.415.694
      IL-6.548.072.505.406.689
      IL-7.592.072.200.451.733
      IL-8.528.072.691.387.670
      IL-10.511.072.883.370.651
      IL-12.534.073.631.392.677
      IL-13.522.072.758.381.663
      IL-17.655.068.031
      P<0.05.
      .522.787
      MCP-1.571.071.320.432.710
      MIP-1B.634.069.062.499.768
      TNF-A.585.072.236.444.726
      Results of ROC curves of the cytokines and chemokines, with or without treatment, are shown.
      * P < 0.05.

      ECHO parameters according to previous Bz treatment

      Echocardiograms were performed in 37 patients (56%), 20 of the treated group and 17 of the untreated group, with similar NYHA class and clinical data, as well as time of disease and interval between treatment and inclusion in the study.
      The measures of the left atrium (LA), the E/E’ septal ratio (E/E’ SEP), the mean of the E/E’ septal and E/E’ lateral ratios (MEAN E/E’), the E/E’ lateral ratio (E/E’ LAT), and the A-wave on mitral (A-WAVE), exhibited higher values in the untreated group, suggesting worse myocardial function in this group (Table 5). By contrast, the septal S’-wave (S’ SEP), lateral S’ wave (S’ LAT), infero-lateral S’ wave (S’ INF-LAT) on the mitral annulus (MA), mean of the septal, lateral and infero-lateral S’-waves (MEAN S’), and the S’-wave on basal tricuspid annulus (S’ TRIC), exhibited higher values in the treated group, suggesting better myocardial function in this group (Table 5).
      Table 5Doppler echocardiogram parameters in patients according to previous Chagas’ disease treatment.
      Treated median (IQR)Untreated median (IQR)P value#Adjusted POR95% CI
      LA (mm)33.0 (29.0–35.3)36.0 (32.0–37.0)0.0730.615
      LVEDD (mm)48.8 (46.8–50.0)47.0 (44.0–52.0)0.4860.301
      LVEDDind.28.6 (25.5–30.8)27.6 (25.5–30.4)0.6300.508
      LVESD (mm)29.0 (27.0–31.6)27.5 (25.0-31.6)0.3610.483
      Ejection fraction72.5 (70.0–76.0)69.0 (66.0–74.0)0.1450.201
      E-wave80.0 (69.0–87.5)78.0 (70.0–96.3)0.8520.279
      A-wave65.9 (58.0–77.0)70.0 (60.0–90.5)0.1700.102
      E/A1.17 (1.10–1.35)1.14 (0.88–1.29)0.3630.471
      E’ septal11.0 (9.8–12.0)10.0 (7.0–12.0)0.1140.436
      E’ lateral13.0 (11.2–14.9)10.0 (8.0–13.5)0.0380.409
      E/E’ septal7.2 (6.0–8.4)9.5 (7.5–10.0)0.0490.0510.570.33–1.00
      E/E’ lateral5.7 (5.2–7.4)7.1 (5.7–9.3)0.0940.070
      Mean E/E’6.4 (5.5–8.0)8.0 (6.4–9.6)0.0830.0580.500.24–1.02
      S’ Septal8.5 (7.7–9.5)7.4 (6.1–8.7)0.0340.0612.300.96–5.51
      S’ Lateral8.9 (8.4–12.1)8.2 (7.2–10.5)0.0740.115
      S’ Infero-lateral8.9 (8.5–9.3)7.6 (6.5–8.7)0.0130.031
      P<0.05
      3.991.14–13.98
      MEAN S’9.0 (8.2–10.4)7.9 (6.0–8.2)0.0130.049
      P<0.05
      3.051.00–9.30
      S’ tricuspid13.3 (12.7–14.2)11.1 (9.8–12.2)0.0010.024
      P<0.05
      2.551.13-.5.74
      #Adjusted P value for time of disease and NYHA class. Binary logistic regression analysis was used. LA = left atrium; LV = left ventricle; RV = right ventricle; E wave, A wave and E/A ratio from mitral inflow with pulsed Doppler; E’ and S’ from mitral annular velocities with tissue Doppler; S’ tricuspid from tricuspid annular velocities with tissue Doppler; mean E/E’ = the mean of septal plus lateral E/E’; mean S’ = mean of septal plus lateral plus infero-lateral S’.
      ## P < 0.05
      Because the statistically significant differences between study groups in terms of time of disease and NYHA class could possibly influence results because of different stages of CD, Doppler echocardiogram and tissue Doppler variables were adjusted for these two variables using binary logistic regression analysis, forward conditional method. After adjustment, LVEDD, LVESD, S’ TRIC, S’ INF-LAT and MEAN S’ were independently associated with treatment (Table 5).
      ROC curves of the mitral and tricuspid annular S’ waves from tissue Doppler, with or without treatment, are shown in Table 6. S’ tricuspid had the largest AUC (0.866, C.I. 0.689–1.000, P = 0.003), followed by mean S’ (AUC 0.765, C.I. 0.550–0.979, P = 0.029), S’ infero-lateral (AUC 0.761, 0.552–0.971, P = 0.031) and S’ septal (AUC 0.755, C.I. 0.544–0.966, P = 0.036).
      Table 6ROC curves of mitral annular S’ from tissue Doppler analysis.
      VariablesAUC (area under curve)Standard modelP95% C.I.
      Inferior limitSuperior limit
      S’ tric.866.090.003
      P<0.05.
      .6891.000
      S’ sept.755.108.036
      P<0.05.
      .544.966
      S’ lat.709.122.085.470.949
      S’ in_lat.761.107.031
      P<0.05.
      .552.971
      Mean S’.765.110.029
      P<0.05.
      .550.979
      Results of ROC curves of the mitral and tricuspid annular S’ waves from tissue Doppler, with or without treatment, are shown.
      * P < 0.05.

      Discussion

      In the current study, we observed better myocardial function and interestingly higher IL-17 levels in Chagas’ disease patients treated with benznidazole compared to untreated ones, even after adjusting for the disease stage. This appears to argue for the notion that Bz treatment may be beneficial in CD patients. To the best of our knowledge, this is the first study to compare several cytokines and chemokines in the indeterminate and chronic cardiac phase of Chagas’ disease in patients treated with benznidazole versus those untreated, and simultaneously to investigate myocardial function in this population with tissue Doppler.
      IL-17 is a proinflammatory cytokine and is the central component of the Th17 response. It stimulates production of GM-CSF, IL-6, and TNF and induces neutrophil migration (
      • Miyazaki Y.
      • Hamano S.
      • Wang S.
      • Shimanoe Y.
      • Iwakura Y.
      • Yoshida H.
      IL-17 is necessary for host protection against acute-phase Trypanosoma cruzi infection.
      ). IL-17 has a protective function in experimental mice BALB/c infected with T. cruzi. IL-17 neutralization resulted in increased levels of IL-12, IFN and TNF, increased myocarditis and premature mortality, despite parasitological reduction (
      • Da Matta Guedes P.M.
      • Gutierrez F.R.S.
      • Maia F.L.
      • Milanezi C.M.
      • Silva G.K.
      • Pavanelli W.R.
      • et al.
      IL-17 produced during Trypanosoma cruzi infection plays a central role in regulating parasite-induced myocarditis.
      ). Similarly, mice without the receptor subunit for IL17 also showed an amplified inflammatory response and increased mortality (
      • Boari J.T.
      • Vesely M.C.A.
      • Bermejo D.A.
      • Ramello M.C.
      • Montes C.L.
      • Cejas H.
      • et al.
      IL-17RA signaling reduces inflammation and mortality during Trypanosoma cruzi infection by recruiting suppressive IL-10-producing neutrophils.
      ). Furthermore, high IL-17 expression was related to improved cardiac function as determined by left ventricular ejection fraction and left ventricular end-diastolic diameter (
      • Magalhães L.M.
      • Villani F.N.
      • Nunes M.C.
      • Gollob K.J.
      • Rocha M.O.
      • Dutra W.O.
      High interleukin 17 expression is correlated with better cardiac function in human Chagas disease.
      ). In another study in humans, higher numbers of CD4 + IL-17+ cells were found in patients without or with mild cardiomyopathy than in subjects with severe disease (
      • Guedes P.M.
      • Gutierrez F.R.
      • Silva G.K.
      • Dellalibera-Joviliano R.
      • Rodrigues G.J.
      • Bendhack L.M.
      • et al.
      Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas’ disease.
      ). A previous study with a follow-up of 18 months examining 14 Bz-treated CD patients in indeterminated phase reported increased numbers of Th17 cells in the peripheral circulation following treatment (
      • Vallejo A.
      • Monge-Maillo B.
      • Gutiérrez C.
      • Norman F.F.
      • López-Vélez R.
      • Pérez-Molina J.A.
      Changes in the immune response after treatment with benznidazole versus no treatment in patients with chronic indeterminate Chagas disease.
      ). The present study confirmed that IL-17 serum levels were higher in the treated group than in the untreated group. We expanded these observations by showing that IL-17 was the only cytokine that remained significantly different after adjustment for the disease stage, identified here by the time of disease and the NYHA functional class. In a different way, an article with children with chronic CD in Mexico (different population from our study) showed that Th17 circulating cytokines (IL-17, IL-6 and IFN) are increased in children with severe cardiac manifestations (
      • Alba-Alvarado M.
      • Salazar-Schttino P.M.
      • Jiménez-Álvarez L.
      • Cabrera-Bravo M.
      • García-Sancho C.
      • Zenteno E.
      • et al.
      Th-17 cytokines are associated with severity of Trypanosoma cruzi chronic infection in pediatric patients from endemic areas of Mexico.
      ).
      Previous studies showed that in CD, TNF correlated with both trypanocidal action (
      • Lima E.C.
      • Garcia I.
      • Vicentelli M.H.
      • Vassalli P.
      • Minoprio P.
      Evidence for a protective role of tumor necrosis factor in the acute phase of Trypanosoma cruzi infection in mice.
      ) and cardiac tissue injury (
      • Ferreira R.C.
      • Ianni B.M.
      • Abel L.C.
      • Buck P.
      • Mady C.
      • Kalil J.
      • et al.
      Increased plasma levels of tumor necrosis factor-alpha in asymptomatic/“indeterminate” and Chagas disease cardiomyopathy patients.
      ,
      • Pérez-Fuentes R.
      • Guégan J.F.
      • Barnabé C.
      • López-Colombo A.
      • Salgado-Rosas H.
      • Torres-Rasgado E.
      • et al.
      Severity of chronic Chagas disease is associated with cytokine/antioxidant imbalance in chronically infected individuals.
      ). However, the TNF levels in the current study were not different between the treated and untreated CD groups after adjustment for time of disease and NYHA class.
      Some studies comparing the long-term outcomes of Bz-treated patients with nonacute disease, indicated that the treatment prevents the progression of disease, and may lead to negative seroconversion (
      • Fabbro D.L.
      • Streiger M.L.
      • Arias E.D.
      • Bizai M.L.
      • Del Barco M.
      • Amicone N.A.
      Trypanocide treatment among adults with chronic Chagas disease living in Santa Fe City (Argentina), over a mean follow-up of 21 years: parasitological, serological and clinical evolution.
      ,
      • Viotti R.
      • Vigliano C.
      • Lococo B.
      • Bertocchi G.
      • Petti M.
      • Alvarez M.G.
      • et al.
      Long-term cardiac outcomes of treating chronic Chagas disease with benznidazole versus no treatment.
      ,
      • Lana M.
      • Lopes L.A.
      • Martins H.R.
      • Bahia M.T.
      • Machado-de-Assis G.F.
      • Wendling A.P.
      • et al.
      Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment.
      ). However, the effectiveness of trypanocidal treatment remained unclear in other research groups. A Brazilian study showed no effectiveness of nitroderivative therapy for CCD during a follow-up period of 10 years (
      • Lauria-Pires L.
      • Braga M.S.
      • Vexenat A.C.
      • Nitz N.
      • Simões-Barbosa A.
      • Tinoco D.L.
      • et al.
      Progressive chronic Chagas heart disease ten years after treatment with anti-Trypanosoma cruzi nitroderivatives.
      ). The BENEFIT study (a trial involving 2854 patients in 5 countries in Latin America), despite showing a reduction in serum parasite detection and seronegative conversion 5 years after treatment with Bz, did not show significant clinical impact (
      • Morillo C.A.
      • Marin-Neto J.A.
      • Avezum A.
      • Sosa-Estani S.
      • Rassi Jr, A.
      • Rosas F.
      • et al.
      Randomized trial of benznidazole for chronic Chagas’ cardiomyopathy.
      ). However, in the BENEFIT trial there were geographical differences, and in Brazil (country with a different strain of T. cruzi) the efficacy of Bz-treatment differed significantly from other countries (
      • Rassi Jr, A.
      • Marin Neto J.A.
      • Rassi A.
      Chronic Chagas cardiomyopathy: a review of the main pathogenic mechanisms and the efficacy of aetiological treatment following the Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial.
      ). In Brazil, there was a strong trend to avoid an overall primary clinical endpoint (based in morbidity and mortality), as seen in BENEFIT trial (
      • Rassi Jr, A.
      • Marin Neto J.A.
      • Rassi A.
      Chronic Chagas cardiomyopathy: a review of the main pathogenic mechanisms and the efficacy of aetiological treatment following the Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial.
      ). Additionally, in a post-hoc analysis there were fewer hospital admissions due to cardiovascular causes in Bz-treated patients (
      • Morillo C.A.
      • Marin-Neto J.A.
      • Avezum A.
      • Sosa-Estani S.
      • Rassi Jr, A.
      • Rosas F.
      • et al.
      Randomized trial of benznidazole for chronic Chagas’ cardiomyopathy.
      ,
      • Rassi Jr, A.
      • Marin Neto J.A.
      • Rassi A.
      Chronic Chagas cardiomyopathy: a review of the main pathogenic mechanisms and the efficacy of aetiological treatment following the Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial.
      ). Thus the BENEFIT study is not enough to abolish the trypanocidal treatment in non-acute phase, especially in Brazil. More recently, another study which observed in a follow up of two years 1,959 patients with chronic CD in Brazil, found that patients who previously received Bz treatment had less mortality, lower rates of parasitemia and less ECG abnormalities, suggesting a clinical and parasitological benefit of etiological treatment in the early phase chronic CD in Brazil (
      • Cardoso C.S.
      • Ribeiro A.L.P.
      • Oliveira C.D.L.
      • Oliveira L.
      • Ferreira A.M.
      • Bierrenbach A.L.
      • et al.
      Beneficial effects of benznidazole in Chagas disease: NIH SaMi-Trop cohort study.
      ). In the perspective of no new treatments for CD, and also knowing that Bz is a safe drug, with tolerable side effects and has an affordable price, Bz-treatment is still recommended for the treatment of nonacute CD by the WHO (
      • World Health Organization (WHO)
      Chagas disease (American trypanosomiasis).
      ). Further studies should address the trypanocidal clinical efficacy of Bz during CCD.
      Chagas’ disease patients with no or only mild cardiac involvement did not differ significantly from controls using classical echocardiographic parameters including ejection fraction, chamber dimensions and Doppler (
      • Barbosa M.M.
      • Rocha M.O.C.
      • Vidigal D.F.
      • Carneiro R.C.B.
      • Araújo R.D.
      • Palma M.C.
      • et al.
      Early detection of left ventricular contractility abnormalities by two-dimensional speckle tracking strain in Chagas’ disease.
      ,
      • Lima M.S.
      • Villarraga H.R.
      • Abduch M.C.
      • Lima M.F.
      • Cruz C.B.
      • Bittencourt M.S.
      • et al.
      Comprehensive left ventricular mechanics analysis by speckle tracking echocardiography in Chagas disease.
      ,
      • Moreira H.T.
      • Volpe G.J.
      • Marin-Neto J.A.
      • Nwabuo C.C.
      • Ambale-Venkatesh B.
      • Gali L.G.
      • et al.
      Right ventricular systolic dysfunction in Chagas disease defined by speckle-tracking echocardiography: a comparative study with cardiac magnetic resonance imaging.
      ,
      • Regueiro A.
      • García-Álvarez A.
      • Sitges M.
      • Ortiz-Pérez J.T.
      • De Caralt M.T.
      • Pinazo M.J.
      • et al.
      Myocardial involvement in Chagas disease: insights from cardiac magnetic resonance.
      ). More recently, tissue Doppler and speckle-tracking strain demonstrated the possibility of early detection of left ventricular contractility abnormalities (
      • Barbosa M.M.
      • Rocha M.O.C.
      • Vidigal D.F.
      • Carneiro R.C.B.
      • Araújo R.D.
      • Palma M.C.
      • et al.
      Early detection of left ventricular contractility abnormalities by two-dimensional speckle tracking strain in Chagas’ disease.
      ,
      • Barros M.V.
      • Rocha M.O.
      • Ribeiro A.L.P.
      • Machado F.S.
      Doppler tissue imaging to evaluate early myocardium damage in patients with undetermined form of Chagas’ disease and normal echocardiogram.
      ,
      • García-Álvarez A.
      • Sitges M.
      • Regueiro A.
      • Poyatos S.
      • Jesus Pinazo M.
      • Posada E.
      • et al.
      Myocardial deformation analysis in Chagas heart disease with the use of speckle tracking echocardiography.
      ,
      • Gomes V.A.
      • Alves G.F.
      • Hadlich M.
      • Azevedo C.F.
      • Pereira I.M.
      • Santos C.R.F.
      • et al.
      Analysis of regional left ventricular strain in patients with Chagas disease and normal left ventricular systolic function.
      ,
      • Lima M.S.
      • Villarraga H.R.
      • Abduch M.C.
      • Lima M.F.
      • Cruz C.B.
      • Bittencourt M.S.
      • et al.
      Comprehensive left ventricular mechanics analysis by speckle tracking echocardiography in Chagas disease.
      ,
      • Moreira H.T.
      • Volpe G.J.
      • Marin-Neto J.A.
      • Nwabuo C.C.
      • Ambale-Venkatesh B.
      • Gali L.G.
      • et al.
      Right ventricular systolic dysfunction in Chagas disease defined by speckle-tracking echocardiography: a comparative study with cardiac magnetic resonance imaging.
      ,
      • Nascimento C.A.
      • Gomes V.A.
      • Silva S.K.
      • Santos C.R.
      • Chambela M.C.
      • Madeira F.S.
      • et al.
      Left atrial and left ventricular diastolic function in chronic Chagas disease.
      ,
      • Regueiro A.
      • García-Álvarez A.
      • Sitges M.
      • Ortiz-Pérez J.T.
      • De Caralt M.T.
      • Pinazo M.J.
      • et al.
      Myocardial involvement in Chagas disease: insights from cardiac magnetic resonance.
      ,
      • Sánchez-Montalvá A.
      • Salvador F.
      • Rodríguez-Palomares J.
      • Sulleiro E.
      • Sao-Avilés A.
      • Roure S.
      • et al.
      Chagas cardiomyopathy: usefulness of EKG and echocardiogram in a non-endemic country.
      ). Our study compared untreated patients and patients treated with a trypanocidal drug using tissue Doppler, and showed that treated patients had better preservation of left and right ventricular function. Our findings of increased levels of IL-17, a protective factor in the same population, together with better myocardial function by tissue Doppler parameters, reinforced the notion of beneficial effects of Bz in the treatment of Chagas’ disease, even in patients with mild cardiac abnormalities.
      This was not a randomized and placebo-controlled study, and treated patients had less time of disease evolution than did untreated subjects, a confounder effect for which we made statistical adjustment. In addition, detection of blood parasite DNA was not performed, and thus efficacy of Bz treatment was not assessed; therefore a direct relationship of variables with reduced T. cruzi parasitism could not be ascertained.
      The present findings suggested that the group of CD patients that were subject to trypanocidal treatment with Bz displayed increased plasma levels of IL-17, less severe NYHA class and improved myocardial function as displayed by the analysis with tissue Doppler, reinforcing the idea that Bz treatment may be beneficial to CD patients.

      Acknowledgments

      The authors would like to thank the Instituto Gonçalo Moniz for technical support The authors also thank Adorielze Leite for logistical support.

      Conflict of interest

      The authors declare that they have no conflict of interests.

      Funding source

      This work has no financial support. MBN is a senior investigator from Conselho Nacional de Pesquisa e Tecnologia (CNPq), Brazil. VRRM and LCLS were supported by CNPq.

      Ethical approval

      This study was approved by the Ethics Committee of the Complexo Hospitalar Professor Edgard Santos (protocol number: 77/10).

      Appendix A. Supplementary data

      The following is Supplementary data to this article:

      References

        • Alba-Alvarado M.
        • Salazar-Schttino P.M.
        • Jiménez-Álvarez L.
        • Cabrera-Bravo M.
        • García-Sancho C.
        • Zenteno E.
        • et al.
        Th-17 cytokines are associated with severity of Trypanosoma cruzi chronic infection in pediatric patients from endemic areas of Mexico.
        Acta Trop. 2018; 178: 134-141
        • Andrade Z.A.
        • Andrade S.G.
        The pathology of Chagas’ disease (cardiac chronic form).
        Bol Fund G Moniz. 1955; 6: 1-53
        • Añez N.
        • Carrasco H.
        • Parada H.
        • Crisante G.
        • Rojas A.
        • Fuenmayor C.
        • et al.
        Myocardial parasite persistence in chronic chagasic patients.
        Am J Trop Med Hyg. 1999; 60: 726-732
        • Barbosa M.M.
        • Rocha M.O.C.
        • Vidigal D.F.
        • Carneiro R.C.B.
        • Araújo R.D.
        • Palma M.C.
        • et al.
        Early detection of left ventricular contractility abnormalities by two-dimensional speckle tracking strain in Chagas’ disease.
        Echocardiography. 2014; 31: 623-630
        • Barros M.V.
        • Rocha M.O.
        • Ribeiro A.L.P.
        • Machado F.S.
        Doppler tissue imaging to evaluate early myocardium damage in patients with undetermined form of Chagas’ disease and normal echocardiogram.
        Echocardiography. 2001; 18: 131-136
        • Bern C.
        • Montgomery S.P.
        • Herwaldt B.L.
        • Rassi Jr, A.
        • Marin-neto J.A.
        • Dantas R.O.
        • et al.
        Evaluation and treatment of Chagas disease in the United States: a systematic review.
        JAMA. 2007; 298: 2171-2181
        • Boari J.T.
        • Vesely M.C.A.
        • Bermejo D.A.
        • Ramello M.C.
        • Montes C.L.
        • Cejas H.
        • et al.
        IL-17RA signaling reduces inflammation and mortality during Trypanosoma cruzi infection by recruiting suppressive IL-10-producing neutrophils.
        PLoS Pathog. 2012; 8e1002658
        • Cançado J.R.
        Long term evaluation of etiological treatment of Chagas disease with benznidazole.
        Rev Inst Med Trop Sao Paulo. 2002; 44: 29-37
        • Cardoso C.S.
        • Ribeiro A.L.P.
        • Oliveira C.D.L.
        • Oliveira L.
        • Ferreira A.M.
        • Bierrenbach A.L.
        • et al.
        Beneficial effects of benznidazole in Chagas disease: NIH SaMi-Trop cohort study.
        PLoS Negl Trop Dis. 2018; 12e0006814
        • Cunha-Neto E.
        • Coelho V.
        • Guilherme L.
        • Fiorelli A.
        • Stolf N.
        • Kalil J.
        Autoimmunity in Chagas’ disease: identification of cardiac myosin-B13 Trypanosoma cruzi protein crossreactive T cell clones in heart lesions of a chronic Chagas’ cardiomyopathy patient.
        J Clin Invest. 1996; 98: 1709-1712
        • Da Matta Guedes P.M.
        • Gutierrez F.R.S.
        • Maia F.L.
        • Milanezi C.M.
        • Silva G.K.
        • Pavanelli W.R.
        • et al.
        IL-17 produced during Trypanosoma cruzi infection plays a central role in regulating parasite-induced myocarditis.
        PLoS Negl Trop Dis. 2010; 4: e604
        • Da Nóbrega A.A.
        • De Araújo W.N.
        • Vasconcelos A.M.
        Mortality due to chagas disease in Brazil according to a specific cause.
        Am J Trop Med Hyg. 2014; 91: 528-533
        • de Andrade A.L.
        • Zicker F.
        • de Oliveira R.M.
        • Silva S.A.
        • Luquetti A.
        • Travassos L.R.
        • et al.
        Randomised trial of efficacy of benznidazole in treatment of early Trypanosoma cruzi infection.
        Lancet. 1996; 348: 1407-1413
        • Dutra W.O.
        • Menezes C.A.
        • Magalhães L.M.D.
        • Gollob K.J.
        Immunoregulatory networks in human Chagas disease.
        Parasite Immunol. 2014; 36: 377-387
        • Fabbro D.L.
        • Streiger M.L.
        • Arias E.D.
        • Bizai M.L.
        • Del Barco M.
        • Amicone N.A.
        Trypanocide treatment among adults with chronic Chagas disease living in Santa Fe City (Argentina), over a mean follow-up of 21 years: parasitological, serological and clinical evolution.
        Rev Soc Bras Med Trop. 2007; 40: 1-10
        • Ferreira R.C.
        • Ianni B.M.
        • Abel L.C.
        • Buck P.
        • Mady C.
        • Kalil J.
        • et al.
        Increased plasma levels of tumor necrosis factor-alpha in asymptomatic/“indeterminate” and Chagas disease cardiomyopathy patients.
        Mem Inst Oswaldo Cruz. 2003; 98: 407-411
        • García-Álvarez A.
        • Sitges M.
        • Regueiro A.
        • Poyatos S.
        • Jesus Pinazo M.
        • Posada E.
        • et al.
        Myocardial deformation analysis in Chagas heart disease with the use of speckle tracking echocardiography.
        J Card Fail. 2011; 17: 1028-1034
        • Gomes V.A.
        • Alves G.F.
        • Hadlich M.
        • Azevedo C.F.
        • Pereira I.M.
        • Santos C.R.F.
        • et al.
        Analysis of regional left ventricular strain in patients with Chagas disease and normal left ventricular systolic function.
        J Am Soc Echocardiogr. 2016; 29: 679-688
        • Guedes P.M.
        • Gutierrez F.R.
        • Silva G.K.
        • Dellalibera-Joviliano R.
        • Rodrigues G.J.
        • Bendhack L.M.
        • et al.
        Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas’ disease.
        PLoS Negl Trop Dis. 2012; 6: e1630
        • Jones E.M.
        • Colley D.G.
        • Tostes S.
        • Lopes E.R.
        • Vnencak-Jones C.L.
        • McCurley T.L.
        Amplification of a Trypanosoma cruzi DNA sequence from inflammatory lesions in human chagasic cardiomyopathy.
        Am J Trop Med Hyg. 1993; 48: 348-357
        • Lana M.
        • Lopes L.A.
        • Martins H.R.
        • Bahia M.T.
        • Machado-de-Assis G.F.
        • Wendling A.P.
        • et al.
        Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment.
        Mem Inst Oswaldo Cruz. 2009; 104: 1139-1147
        • Lang R.M.
        • Badano L.P.
        • Mor-Avi V.
        • Afilalo J.
        • Armstrong A.
        • Ernande L.
        • et al.
        Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American society of echocardiography and the European association of cardiovascular imaging.
        J Am Soc Echocardiogr. 2015; 28 (1–39.e14)
        • Laucella S.A.
        • Mazliah D.P.
        • Bertocchi G.
        • Alvarez M.G.
        • Cooley G.
        • Viotti R.
        • et al.
        Changes in Trypanosoma cruzi-specific immune responses following treatment: surrogate markers of treatment efficacy.
        Clin Infect Dis. 2009; 49: 1675-1684
        • Lauria-Pires L.
        • Braga M.S.
        • Vexenat A.C.
        • Nitz N.
        • Simões-Barbosa A.
        • Tinoco D.L.
        • et al.
        Progressive chronic Chagas heart disease ten years after treatment with anti-Trypanosoma cruzi nitroderivatives.
        Am J Trop Med Hyg. 2000; 63: 111-118
        • Lima E.C.
        • Garcia I.
        • Vicentelli M.H.
        • Vassalli P.
        • Minoprio P.
        Evidence for a protective role of tumor necrosis factor in the acute phase of Trypanosoma cruzi infection in mice.
        Infect Immun. 1997; 65: 457-465
        • Lima M.S.
        • Villarraga H.R.
        • Abduch M.C.
        • Lima M.F.
        • Cruz C.B.
        • Bittencourt M.S.
        • et al.
        Comprehensive left ventricular mechanics analysis by speckle tracking echocardiography in Chagas disease.
        Cardiovasc Ultrasound. 2016; 14: 20
        • Magalhães L.M.
        • Villani F.N.
        • Nunes M.C.
        • Gollob K.J.
        • Rocha M.O.
        • Dutra W.O.
        High interleukin 17 expression is correlated with better cardiac function in human Chagas disease.
        J Infect Dis. 2013; 207: 661-665
        • Miyazaki Y.
        • Hamano S.
        • Wang S.
        • Shimanoe Y.
        • Iwakura Y.
        • Yoshida H.
        IL-17 is necessary for host protection against acute-phase Trypanosoma cruzi infection.
        J Immunol. 2010; 185: 1150-1157
        • Moreira H.T.
        • Volpe G.J.
        • Marin-Neto J.A.
        • Nwabuo C.C.
        • Ambale-Venkatesh B.
        • Gali L.G.
        • et al.
        Right ventricular systolic dysfunction in Chagas disease defined by speckle-tracking echocardiography: a comparative study with cardiac magnetic resonance imaging.
        J Am Soc Echocardiogr. 2017; 30: 493-502
        • Morillo C.A.
        • Marin-Neto J.A.
        • Avezum A.
        • Sosa-Estani S.
        • Rassi Jr, A.
        • Rosas F.
        • et al.
        Randomized trial of benznidazole for chronic Chagas’ cardiomyopathy.
        N Engl J Med. 2015; 373: 1295-1306
        • Nascimento C.A.
        • Gomes V.A.
        • Silva S.K.
        • Santos C.R.
        • Chambela M.C.
        • Madeira F.S.
        • et al.
        Left atrial and left ventricular diastolic function in chronic Chagas disease.
        J Am Soc Echocardiogr. 2013; 26: 1424-1433
        • Pérez-Fuentes R.
        • Guégan J.F.
        • Barnabé C.
        • López-Colombo A.
        • Salgado-Rosas H.
        • Torres-Rasgado E.
        • et al.
        Severity of chronic Chagas disease is associated with cytokine/antioxidant imbalance in chronically infected individuals.
        Int J Parasitol. 2003; 33: 293-299
        • Rassi Jr, A.
        • Rassi A.
        • Marcondes de Rezende J.
        American trypanosomiasis (Chagas disease).
        Infect Dis Clin North Am. 2012; 26: 275-291
        • Rassi Jr, A.
        • Marin Neto J.A.
        • Rassi A.
        Chronic Chagas cardiomyopathy: a review of the main pathogenic mechanisms and the efficacy of aetiological treatment following the Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) trial.
        Mem Inst Oswaldo Cruz. 2017; 112: 224-235
        • Regueiro A.
        • García-Álvarez A.
        • Sitges M.
        • Ortiz-Pérez J.T.
        • De Caralt M.T.
        • Pinazo M.J.
        • et al.
        Myocardial involvement in Chagas disease: insights from cardiac magnetic resonance.
        Int J Cardiol. 2013; 165: 107-112
        • Rocha Rodrigues D.B.
        • dos Reis M.A.
        • Romano A.
        • Pereira S.A.
        • Teixeira V.P.
        • Tostes S.
        • et al.
        In situ expression of regulatory cytokines by heart inflammatory cells in Chagas’ disease patients with heart failure.
        Clin Dev Immunol. 2012; 2012361730
        • Sathler-Avelar R.
        • Vitelli-Avelar D.M.
        • Massara R.L.
        • Borges J.D.
        • Lana M.
        • Teixeira-Carvalho A.
        • et al.
        Benznidazole treatment during early-indeterminate Chagas’ disease shifted the cytokine expression by innate and adaptive immunity cells toward a type 1-modulated immune profile.
        Scand J Immunol. 2006; 64: 554-563
        • Sathler-Avelar R.
        • Vitelli-Avelar D.M.
        • Massara R.L.
        • de Lana M.
        • Pinto Dias J.C.
        • Teixeira-Carvalho A.
        • et al.
        Etiological treatment during early chronic indeterminate Chagas disease incites an activated status on innate and adaptive immunity associated with a type 1-modulated cytokine pattern.
        Microbes Infect. 2008; 10: 103-113
        • Sathler-Avelar R.
        • Vitelli-Avelar D.M.
        • Elói-Santos S.M.
        • Gontijo E.D.
        • Teixeira-Carvalho A.
        • Martins-Filho O.A.
        Blood leukocytes from benznidazole-treated indeterminate Chagas disease patients display an overall type-1-modulated cytokine profile upon short-term in vitro stimulation with Trypanosoma cruzi antigens.
        BMC Infect Dis. 2012; 12: 123
        • Sosa Estani S.
        • Segura E.L.
        • Ruiz A.M.
        • Velazquez E.
        • Porcel B.M.
        • Yampotis C.
        Efficacy of chemotherapy with benznidazole in children in the indeterminate phase of Chagas’ disease.
        Am J Trop Med Hyg. 1998; 59: 526-529
        • Sánchez-Montalvá A.
        • Salvador F.
        • Rodríguez-Palomares J.
        • Sulleiro E.
        • Sao-Avilés A.
        • Roure S.
        • et al.
        Chagas cardiomyopathy: usefulness of EKG and echocardiogram in a non-endemic country.
        PLoS One. 2016; 11e0157597
        • Vallejo A.
        • Monge-Maillo B.
        • Gutiérrez C.
        • Norman F.F.
        • López-Vélez R.
        • Pérez-Molina J.A.
        Changes in the immune response after treatment with benznidazole versus no treatment in patients with chronic indeterminate Chagas disease.
        Acta Trop. 2016; 164: 117-124
        • Viotti R.
        • Vigliano C.
        • Lococo B.
        • Bertocchi G.
        • Petti M.
        • Alvarez M.G.
        • et al.
        Long-term cardiac outcomes of treating chronic Chagas disease with benznidazole versus no treatment.
        Ann Intern Med. 2006; 144: 724-734
        • World Health Organization (WHO)
        Chagas disease (American trypanosomiasis).
        2017 (http://www.who.int/mediacentre/factsheets/fs340/en/#. [Accessed 3 May 2017])
        • World Health Organization (WHO)
        Chagas disease (American trypanosomiasis).
        2018 (http://www.who.int/news-room/fact-sheets/detail/chagas-disease-(american-trypanosomiasis). [Accessed 28 October 2018])