Study setting

Individuals followed at the Evandro Chagas National Institute of Infectious Diseases (INI) of the Oswaldo Cruz Foundation (Fiocruz) will be invited during a medical appointment to participate in the AMIO-CHAGAS study. INI-Fiocruz, located in Rio de Janeiro, Brazil, is a national referral center for the treatment and research of infectious and tropical diseases within the framework of the Brazilian Unified Health System (SUS). INI-Fiocruz receives patients from across the country and provides comprehensive, multidisciplinary care for individuals with CD [21]. Patients currently living in the state of Rio de Janeiro with suspected or confirmed CD are referred to INI for diagnostic investigation or specialized healthcare. Many of these patients are migrants from endemic rural areas. The INI staff includes infectious disease specialists, cardiologists, gastroenterologists, nurses, nutritionists, pharmacists, and physical therapists or exercise physiologists. Patients routinely undergo blood tests, echocardiography (ECHO), electrocardiogram (ECG), 24-hour Holter monitoring, exercise testing, and cardiopulmonary exercise testing, all of which are available at this center. Data from medical records will be collected using standardized protocols. In the event of inconsistencies, records will be reviewed independently by two examiners. Only data with confirmed consistency will be included in the analyses.

Study design

This is a mixed (retrospective and prospective) longitudinal observational study designed to evaluate the association between chronic use of amiodarone (AMIO) and the following:

1. (I). Primary outcome: inflammatory profile, serologic reactivity to anti-T. cruzi antibody, and parasitic load;
2. (II). Secondary outcomes: cardiovascular events (new sustained atrial arrhythmias, new sustained or non-sustained ventricular tachyarrhythmias, stroke, heart failure, atrioventricular block, and cardiovascular hospital admissions), CCC progression, internal cardioverter-defibrillator (ICD) implantation, heart transplantation, all-cause death, and a composite endpoint (including the aforementioned cardiovascular events, disease progression, ICD implantation, heart transplantation, and death).

Patients with CCC who take AMIO (exposed group) will be compared with those who do not take this drug (non-exposed group). To minimize potential confounding, patients will be matched based on the stage of CCC according to the Brazilian Consensus on Chagas Disease (A and B1 versus B2 and C), age (<50 years versus >50 years), and sex (female versus male) [15].

Clinical and laboratory data including blood tests, ECG, transthoracic two-dimensional echocardiography (ECHO), and 24-hour Holter monitoring, will be evaluated at three different time points:

1. (i). Retrospective data: from up to 3 years before the start of the study;
2. (ii). Baseline/recruitment: at the time of inclusion in the study;
3. (iii). Prospective data: during the 2 years of follow-up after recruitment;

The duration of the study, totaling five years of clinical follow-up, was established considering the CCC progresses at a rate of approximately 2% per year [22]. At recruitment, blood samples will be collected for biochemical and molecular analyses to obtain information related to the study’s primary outcome (Table 1) (Fig 1).

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Table 1. AMIO-CHAGAS’s study Chronogram: Enrollment, Interventions and Outcome Assessments.

https://doi.org/10.1371/journal.pone.0347305.t001

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Fig 1. Study flow diagram for data collection at recruitment.

ECG: Electrocardiogram; ECHO: Echocardiography; ICD: Implantable cardioverter-defibrillator; PBMCs: Peripheral blood mononuclear cells.

https://doi.org/10.1371/journal.pone.0347305.g001

Study population

Participant recruitment began on March 7, 2023, and is expected to be completed by December 2025. Participants are being recruited sequentially during their routine medical visits. Patients using AMIO are identified from the database of individuals monitored by the Clinical Research Laboratory on Chagas Disease at INI-Fiocruz. Only patients who have been receiving AMIO for at least three months will be included, ensuring adequate tissue impregnation with a cumulative dose of 10 g [23].

Inclusion criteria are as follows:

1. Confirmed diagnosis of CD, based on two serological tests with different methodologies (enzyme-linked immunosorbent assay [ELISA] and chemiluminescence) [14,15];
2. Individuals over 18 years of age;
3. Patients classified within the following CCC stages: A, B1, B2, and C [15].

Exclusion criteria are as follows:

1. Co-infection (HIV, hepatitis B and C, HTLV, Hansen’s disease, leishmaniasis and paracoccidioidomycosis);
2. Immunosuppression of any nature;
3. Cancer;
4. Patients with stage D of CCC will be excluded due to their complex clinical management, including frequent hospitalizations and medication adjustments, which could affect the study’s feasibility.

Patients meeting the inclusion criteria will be invited to participate in the study, and written informed consent will be obtained from all individuals who agree to enroll.

Importantly, AMIO use was prescribed at medical discretion to treat atrial or ventricular arrhythmias [12], independently of this study, characterizing an observational study design.

Blood collection and isolation of plasma, serum and peripheral blood mononuclear cells (PBMCs)

Venous blood will be collected by hospital staff during the routine consultation of patients enrolled in the study. Two samples of 5 mL each will be collected: one in BD Vacutainer® Plus Plastic Serum tubes and kept for at least 30 minutes at room temperature to obtain serum, and one in BD Vacutainer® Plus Plastic EDTA K3 tubes (Becton, Dickinson, New Jersey, USA), for the separation of PBMCs and plasma by Histopaque 1077 gradient (Sigma Aldrich™, St. Louis, USA) following the manufacturer’s protocol (GE Healthcare). At the end of the separation procedure, the cells will be resuspended in 100 μL of TRI reagent (Sigma-Aldrich) and stored at −80º C until further analysis, such as plasma and serum. Biological samples will be stored during the study in accordance with established technical, ethical, and operational regulations, under institutional responsibility and the supervision of the principal investigator. Samples will be stored for up to five years after study completion and then discarded according to institutional biosafety regulations. During this period, samples may be used in additional subprojects subject to appropriate ethical approval.

Analysis of gene expression of CCC progression biomarkers in PBMCs

The assessment of gene expression for key inflammatory and regulatory markers, including TNF, IFN-ɣ, IL-10, TGF-β, iNOS, and NF-κβ, in PBMCs provides valuable insights for monitoring systemic immune responses associated with chronic Chagasic cardiomyopathy (CCC) progression [24,25]. For this purpose, RNA will be extracted from collected PBMCs using TRI reagent (Sigma-Aldrich). cDNA synthesis will be performed with the SuperScript III First-Strand Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s specifications. Gene expression will be measured by real-time quantitative PCR (RT-qPCR), using the following TaqMan gene expression assays: TNF (tumor necrosis factor; Assay ID: Hs00174128_m1), IFN-ɣ (interferon-gamma; Assay ID: Hs03044218_g1), IL-10 (interleukin; Assay ID: Hs00961622_m1), TGF-β (transforming growth factor beta; Assay ID: Hs00998133_m1), iNOS (inducible nitric oxide synthase; Assay ID: Hs01075529_m1) and NF-κβ (nuclear factor kappa β; Assay ID: Hs00765730_m1). Experiments will be conducted on a QuantiStudio 7 Pro Real-Time PCR System (Applied Biosystems), according to the manufacturer’s instructions. Gene expression will be calculated by relative quantification, using the Expression Suite software (V1.3), on the Molecular Analysis Platform RTP09J (FIOCRUZ/RJ).

Determination of serum levels of cytokines and nitric oxide

In addition to gene expression analysis of inflammatory markers in PBMCs, quantification of cytokine levels and nitric oxide in the serum of patients with chronic Chagasic cardiomyopathy (CCC) has previously been associated with the progression of symptomatic forms of the disease [26]. Accordingly, detection of IL-2, IL-17, IL-4, IL-6, IL-10, TNF, and IFN-ɣ will be performed using the Cytometric Bead Array Human Th1/Th2/Th17 kit (Becton Dickinson), following the manufacturer’s protocol. Furthermore, nitrate and nitrite concentrations will be quantified in patient serum samples using the Griess reaction (Sigma-Aldrich), according to the manufacturer’s specifications.

Determination of anti-T. cruzi antibody titers and antigen quantification

Previous studies have indicated that reduced serological reactivity and lower anti-T. cruzi antibody titers may be associated with improved prognosis in CCC, as these changes have been correlated with improvements in patients’ electrocardiographic profiles [27,28]. Based on this evidence, in the present study, the serological reactivity of anti-T. cruzi antibodies will be determined using the commercial Chagastest-Wienner kit (ELISA recombinant v.4.0), following the manufacturer’s recommendations. Positive and negative controls will be included to ensure test functionality. The cut-off value will be calculated as the mean absorbance of the negative controls, with the indeterminate zone defined as within ±10% of the cut-off value. In parallel, antigen quantification will be performed to enable subsequent correlation between these two parameters. For the detection and quantification of T. cruzi in patient samples, the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) (IBMP, Curitiba, Brazil) will be used. This kit, based on a duplex TaqMan assay targeting the nuclear satellite DNA of T. cruzi and an exogenous internal amplification control, offers a quantification range from 10⁴ to 0.5 parasite equivalents per milliliter of blood. The assays will be performed according to the manufacturer’s instructions.

Electrocardiogram, two-dimensional transthoracic echocardiogram, 24-hour Holter and blood pressure

All analyses will be performed annually by Lapclin-Chagas (INI), by qualified professionals. The 12-lead electrocardiogram will be conducted with patients at rest, using a long recording on D2 to assess arrhythmias. The presence or absence of electrocardiographic changes will be determined according to the criteria recommended by the II Brazilian Consensus on Chagas Disease [15]. Electrocardiographic analysis will be performed using the Holter system (portable three-channel recorder and analyzer; Cardio Light® and CardioSmart® 5.0, Cardio System, São Paulo, Brazil), with three channels connected continuously for 24 hours at a rate of 200 samples per second, to detect arrhythmias predictive of mortality and to obtain the arrhythmogenic profile [29,30]. Two-dimensional transthoracic echocardiography will be performed using a phased-array ultrasound system (Philips CVx, USA) equipped with an M4S transducer, following the recommendations of the World Heart Federation [31]. Left ventricular systolic ejection fraction (LVEF), as well as left ventricular diastolic and systolic volumes, will be quantified using Simpson’s biplane method of discs. Assessments will also include left ventricular diastolic function, right ventricular (RV) systolic function, and strain parameters of the left atrium (LA), RV, and left ventricle (LV), to provide a comprehensive evaluation of cardiac mechanics. All measurements will be performed by experienced cardiologists blinded to the clinical data to minimize bias. Blood pressure will be measured on the left arm with participants seated in a quiet room after 5 minutes of rest, using an Omron digital sphygmomanometer.

Sample size

A minimum sample size of 90 volunteers was estimated, with 45 using AMIO and 45 not using AMIO. The sample size calculation for the primary endpoint was based on a study by Rodriguez-Angulo et al., 2017 [26] which evaluated differences in inflammatory markers in individuals with CD treated with AMIO (IFN-γ: 43.75 ± 24.83; TNF-α: 27.08 ± 13.81) compared to untreated individuals (IFN-γ: 84.37 ± 35.08; TNF-α: 52.08 ± 23.38). For the secondary endpoint, sample size calculation was based on a study by Saraiva et al., 2022 [22], which compared LVEF at different stages of CCC (LVEV; Stage A: 68.0 ± 6.4; Stage B: 54.7 ± 9.1; Stage C: 36.7 ± 10.7). Sample size calculations assumed a two-sided significance level of α = 0.05, a statistical power of 80% (1 − β = 0.8), and standard deviations and mean differences derived from these studies. The largest sample requirement among the two endpoints was adopted, and the sample size was increased by 20% to account for potential losses or refusals, resulting in a total of 90 participants.

Statistical analysis

Descriptive statistics were used to summarize the characteristics of the study population. Continuous variables will be presented as medians and interquartile ranges (IQR), and categorical variables as absolute and relative frequencies. Missing data will be assessed for their pattern and mechanism (i.e., missing completely at random (MCAR), missing at random (MAR), or missing not at random (MNAR)). The primary approach to handling missing data will be determined once the dataset is available. Depending on the extent and nature of missingness, appropriate strategies may include multiple imputation, complete case analysis, or sensitivity analyses to evaluate the robustness of the findings. The Shapiro-Wilk test will be used to assess the normality of continuous variables. When distributions deviate from normality, appropriate transformations or non-parametric tests will be applied.

Comparisons of patient characteristics according to AMIO use will be evaluated using the independent Student’s t-test for normally distributed continuous variables or the Mann-Whitney U-test for non-normally distributed data. Categorical variables will be compared using the chi-square test or Fisher’s exact test, as appropriate. Correlations between continuous variables related to the primary outcome will be assessed using Pearson or Spearman correlation coefficients, depending on data distribution. AMIO use and longitudinal changes in continuous variables will be analyzed using mixed linear models, adjusted for the duration of amiodarone use. Analyses of time course outcomes (e.g., cardiovascular events, disease progression, all-cause mortality) will be conducted using Cox proportional hazards regression models adjusted for potential confounders such as age, sex, comorbidities and previous etiologic treatment. Hazard ratios (HRs) and their 95% confidence intervals (CI) will be reported. A two-sided p-value ≤ 0.05 will be considered statistically significant.

Data collection

Data collection included clinical, immunological, molecular, and imaging parameters, categorized as either categorical or continuous variables. The data generated and analyzed during this study are available from the corresponding author upon reasonable request, subject to ethical and privacy considerations.

Continuous variables included:

1. (i). Gene expression levels of TNF, IFN-γ, IL-10, TGF-β, iNOS, and NF-κB in PBMCs, quantified by RT-qPCR and expressed as fold change (arbitrary units);
2. (ii). Serum cytokine concentrations—including IL-2, IL-4, IL-6, IL-10, IL-17, TNF, and IFN-γ—quantified in pg/mL using a Cytometric Bead Array (CBA);
3. (iii). Serum levels of nitrate and nitrite (μM), determined by the Griess reaction;
4. (iv). Anti-Trypanosoma cruzi IgG antibody titers, quantified by ELISA and reported as absorbance values;
5. (v). Parasitic DNA load (parasite equivalents/mL), quantified by duplex TaqMan qPCR using the NAT Chagas kit;
6. (vi). Left ventricular systolic ejection fraction (LVEF, %), as well as left ventricular diastolic and systolic volumes (mL), quantified using Simpson’s biplane method of discs via two-dimensional transthoracic echocardiography;
7. (vii). Left ventricular diastolic function (graded by established echocardiographic criteria), right ventricular (RV) systolic function (expressed as fractional area change or TAPSE in mm), and strain parameters (%) of the left atrium (LA), right ventricle (RV), and left ventricle (LV), evaluated to provide a comprehensive assessment of cardiac mechanics;
8. (viii). Electrocardiographic parameters: heart rate (beats per minute, BPM) and electrocardiographic intervals, including PR interval (ms), QRS duration (ms), QT interval (ms), and corrected QT interval (QTc, ms);
9. (ix). Systolic and diastolic blood pressure (mmHg), measured on the left arm with participants seated in a quiet room after 5 minutes of rest, using an Omron digital sphygmomanometer;

Categorical variables included:

1. (i). Echocardiographic parameters: preserved left ventricular systolic function, left ventricular akinesia, left ventricular hypokinesia, mitral regurgitation, tricuspid regurgitation, pulmonary insufficiency, aortic insufficiency, septal hypertrophy, thrombus observation and aneurysm observation.
2. (ii). Electrocardiographic parameters: sinus bradycardia; atrial fibrillation, atrial flutter, supraventricular premature beats (airv), left atrial enlargement, right atrial enlargement, right ventricular enlargement, ventricular extrasystoles, isolated ventricular extrasystoles, first-degree atrioventricular block, second-degree atrioventricular block, incomplete right bundle branch block, complete right bundle branch block, incomplete left bundle branch block, complete left bundle branch block, left anterior hemiblock, and left posterior hemiblock.

Ethical aspects

This study received approval from the Institutional Review Board of the Evandro Chagas National Institute of Infectious Disease in September 2022 (Protocol No. 61674422.7.0000.5262). All investigators are committed to maintaining the confidentiality and privacy of participants’ data. Study results will be disseminated through scientific channels, ensuring the anonymity of all participants. No conflicts of interest exist among the researchers or collaborators, and no restrictions have been placed on the public dissemination of findings, regardless of their concordance with the initial hypotheses. Inclusion criteria require that all participants provide written informed consent, adhering to Good Clinical Practice (GCP) guidelines. Patients will be instructed to read – or, when necessary, listen to the consent form without time constraints and all questions regarding the study’s aims and procedures will be clearly addressed. Participants will be informed that enrollment is voluntary, and refusal to participate will not affect the quality or continuity of their treatment at the institution. Furthermore, they will be made aware of their prerogative to withdraw from study at any point without any penalty or consequence.