Chronic Chagasic Patients Research Articles

KDNA gene signatures of Trypanosoma cruzi in blood and oesophageal mucosa from chronic chagasic patients

Trypanosoma cruzi presents a high degree of intraspecific variability, with possible implications for the pathogenesis of Chagas disease. The aim of this study was to evaluate T. cruzi kDNA minicircle gene signatures using the low-stringency single-specific-primer PCR technique in both peripheral blood and oesophageal mucosa from chronic chagasic patients, with or without megaesophagus, alone or in combination with cardiopathy and megacolon. It was not possible to identify a uniform pattern of shared bands between blood and oesophageal mucosa samples from individuals with the same clinical form or mixed forms, suggesting multiple T. cruzi infections with differential tissue tropism. Thus, the results indicate that there is an intense intraspecific variability in the hypervariable regions of T. cruzi kDNA, which has so far made it impossible to correlate the genetic profile of this structure with the clinical manifestations of Chagas disease.

Different serological cross-reactivity of Trypanosoma rangeli forms in Trypanosoma cruzi-infected patients sera.

BackgroundAmerican Trypanosomiasis or Chagas disease is caused by Trypanosoma cruzi which currently infects approximately 16 million people in the Americas causing high morbidity and mortality. Diagnosis of American trypanosomiasis relies on serology, primarily using indirect immunofluorescence assay (IFA) with T. cruzi epimastigote forms. The closely related but nonpathogenic Trypanosoma rangeli has a sympatric distribution with T. cruzi and is carried by the same vectors. As a result false positives are frequently generated. This confounding factor leads to increased diagnostic test costs and where false positives are not caught, endangers human health due to the toxicity of the drugs used to treat Chagas disease.ResultsIn the present study, serologic cross-reactivity between the two species was compared for the currently used epimastigote form and the more pathologically relevant trypomastigote form, using IFA and immunoblotting (IB) assays. Our results reveal an important decrease in cross reactivity when T. rangeli culture-derived trypomastigotes are used in IFA based diagnosis of Chagas disease. Western blot results using sera from both acute and chronic chagasic patients presenting with cardiac, indeterminate or digestive disease revealed similar, but not identical, antigenic profiles.ConclusionThis is the first study addressing the serological cross-reactivity between distinct forms and strains of T. rangeli and T. cruzi using sera from distinct phases of the Chagasic infection. Several T. rangeli-specific proteins were detected, which may have potential as diagnostic tools.

Evidence of Trypanosoma cruzi II infection in Colombian chagasic patients

Trypanosoma cruzi is genetically classified into at least two major lineages named T. cruzi I (also named Tc I) and T. cruzi II (also named Tc IIb). T. cruzi II is associated with Chagas' disease in the southern cone of South America, while T. cruzi I is the only one so far identified in chagasic patients of Central America and in the northern part of South America. Herein we identified T. cruzi IIb directly in 9.9% of blood of chronic chagasic patients of Colombia. This finding establishes that in this region, the two T. cruzi lineages are associated with the pathology of Chagas' disease and have implications in the morbidity and epidemiology of the disease.

Antibodies with beta-adrenergic activity from chronic chagasic patients modulate the QT interval and M cell action potential duration

The aim of this study was to investigate whether the sera from chronic chagasic patients (CChPs) with beta-1 adrenergic activity (Ab-beta) can modulate ventricular repolarization. Beta-adrenergic activity has been described in CChP. It increases the L-type calcium current and heart rate in isolated hearts, but its effects on ventricular repolarization has not been described. In isolated rabbit hearts, under pacing condition, QT interval was measured under Ab-beta perfusion. Beta-adrenergic activity was also tested in guinea pig ventricular M cells. Furthermore, the immunoglobulin fraction (IgG-beta) of the Ab-beta was tested on Ito, ICa, and Iks currents in rat, rabbit, and guinea pig myocytes, respectively. Beta-adrenergic activity shortened the QT interval. This effect was abolished in the presence of propranolol. In addition, sera from CChP without beta-adrenergic activity (Ab-beta) did not modulate QT interval. The M cell action potential duration (APD) was reversibly shortened by Ab-beta. Atenolol inhibited this effect of Ab-beta, and Ab- did not modulate the AP of M cells. Ito was not modulated by isoproterenol nor by IgG-beta. However, IgG-beta increased ICa and IKs. The shortening of the QT interval and APD in M cells and the increase of IKs and ICa induced by IgG-beta contribute to repolarization changes that may trigger malignant ventricular arrhythmias observed in patients with chronic chagasic or idiopathic cardiomyopathy.

Autoantibodies Enhance Agonist Action and Binding to Cardiac Muscarinic Receptors in Chronic Chagas' Disease

Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M2-muscarinic acetylcholine receptors (M2AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M2AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M2AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [3H]-N-methyl scopolamine ([3H]-NMS) in allosterism binding assays. A peptide corresponding to the M2AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [3H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [3H]-NMS dissociation right shifted from an IC50 of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 × 10− 8, 1.33 × 10− 7, and 2.0 × 10− 7 mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M2AChRs as a positive cooperativity effect on acetylcholine action.

Diversidade genética de populações naturais do <em>Trypanosoma</em> cruzi no Paraná, Sul do Brasil

The genetic characteristics of twelve Trypanosoma cruzi strains isolated from chronic chagasic patients, triatomines and sylvatic reservoirs from Paraná state, Southern Brazil, were studied using the randomly amplified polymorphic DNA (RAPD) and simple sequence repeat-anchored polymerase chain reaction amplification (SSR-PCR) techniques. Sylvio and Esmeraldo stocks were used as reference for T. cruzi I and II, respectively. The DNA was amplified using three different primers for RAPD and (CA)8RY primer for SSRPCR. Gel analyses were scored by eye for a numerical taxonomy analysis based on the proportion of shared bands and by employing computer programs. The phenograms showed the existence of two distinct genetic groups: one containing the strains from chronic chagasic patients grouped with Esmeraldo and the other with the strains from triatomines and sylvatic reservoirs grouped with Sylvio. The existence of two phylogenetic groups of T. cruzi in Paraná state, Southern Brazil, is described for the first time.

QRS slopes for assessment of myocardial damage in chronic chagasic patients

In this study the slopes of the QRS complex are evaluated for determination of the degree of myocardial damage in chronic chagasic patients. Previous studies have demonstrated the ability of the slope indices to reflect alterations in the conduction velocity of the cardiac impulse. Results obtained in the present study show that chronic chagasic patients have significantly flatter QRS slopes as compared to healthy subjects. Not only that but the extent of slope lessening turns out to be proportional to the degree of myocardial damage caused by the disease. Additionally, when incorporating the slope indices into a classification analysis together with other indices indicative of the presence of ventricular late potentials obtained from high resolution electrocardiography, results show that the percentages of correct classification increase up to 62.5%, which means eight points above the percentages obtained prior to incorporation of the slope indices. It can be concluded that QRS slopes have great potential for assessing the degree of severity associated with Chagas' disease.

Expresión de marcadores en células dendríticas de pacientes chagásicos crónicos estimuladas con la proteína KMP-11 y el péptido K1 de Trypanosoma cruzi

The kinetoplastid membrane protein 11, KMP-11, from Trypanosoma cruzi elicits humoral and cellular immunity in mice that protects them from infection against further parasite challenge. To characterize the expression of surface markers on dendritic cells from chronic chagasic patients and healthy individuals, in response to the KMP-11 protein from Trypanosoma cruzi and its N-terminal peptide K1. Monocyte-derived dendritic cells from seven chronic chagasic patients and seven healthy individuals were stimulated with the KMP-11 protein and the K1 peptide. Seven days after culturing, the CD83, CD86, and HLA-DR membrane expression as well as the production of cytokines were evaluated by flow cytometry. Neither KMP-11 protein nor K1 peptide elicited the expression of the maturation marker CD83 on dendritic cells of patients or healthy control individuals. Dendritic cells from chronic chagasic patients exposed to K1 and LPS at the same time presented a significant reduction in CD86 and CD83 membrane expression in contrast to the cells exposed to LPS alone, whereas dendritic cells from healthy individuals did not show this behavior. The secretion of interleukin-12 was decreased in the cultures of dendritic cells from chronic chagasic patients but not from healthy controls. KMP-1 1 protein does not affect the maturation of dendritic cells, but in the presence of LPS the K1 peptide leads to a decreased expression of CD86 and CD83 as well as interleukin-12 production, This phenomenon may be associated with an impaired T cell stimulation.

Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples

The aim of this study was to develop a real-time PCR technique to detect Trypanosoma cruzi DNA in blood of chagasic patients. Analytical sensitivity of the real-time PCR was assessed by two-fold serial dilutions of T. cruzi epimastigotes in seronegative blood (7.8 down to 0.06 epimastigotes/mL). Clinical sensitivity was tested in 38 blood samples from adult chronic chagasic patients and 1 blood sample from a child with an acute congenital infection. Specificity was assessed with 100 seronegative subjects from endemic areas, 24 seronegative subjects from non-endemic area and 20 patients with Leishmania infantum-visceral leishmaniosis. Real-time PCR was designed to amplify a fragment of 166 bp in the satellite DNA of T. cruzi. As internal control of amplification human RNase P gene was coamplified, and uracil- N-glycosylase (UNG) was added to the reaction to avoid false positives due to PCR contamination. Samples were also analysed by a previously described nested PCR (N-PCR) that amplifies the same DNA region as the real-time PCR. Sensitivity of the real-time PCR was 0.8 parasites/mL (50% positive hit rate) and 2 parasites/mL (95% positive hit rate). None of the seronegative samples was positive by real-time PCR, resulting in 100% specificity. Sixteen out of 39 patients were positive by real-time PCR (41%). Concordance of results with the N-PCR was 90%. In conclusion, real-time PCR provides an optimal alternative to N-PCR, with similar sensitivity and higher throughput, and could help determine ongoing parasitaemia in chagasic patients.

Lesões epicárdicas na cardiopatia chagásica são reflexo de processo inflamatório

Furnish a more detailed anatomicopathological description of the epicardial lesions in chronic chagasic cardiopathy, evaluate their incidence and discuss their probable pathogenesis We examined the hearts of 39 chronic chagasic cardiopathy patients who underwent autopsies and submitted to histological analysis the epicardial lesions. Milk spots, characterized by well defined white areas in the epicardium were found in 80% of the cases, mainly on the anterior face of the right ventricle. Histological analysis revealed abrupt fibrous thickening of the epicardium, with no elastic fibers, inflammation or blood vessels. Chagasic rosary, characterized by small round whitish granules deposited sequentially along the coronary vessels were present in 23% of the hearts. They presented the same histological structure as the milk spots, but interestingly were only found immediately above the coronary artery branches. Villous plaque was found on the apex or anterior face of 21% of the hearts. It is characterized by an exophytic aspect, probably due to previous localized pericardial adhesion. Microscopic analysis revealed foci of inflammatory infiltrate and vascular proliferation, typical of epicarditis still under organization. In addition to the lesions described above, the vast majority of cases presented sparse mononuclear inflammatory cells with occasional foci. We concluded that epicardial lesions related to chronic chagasic heart disease are probably a result of epicardial reactions to chronic inflammatory process.

Quantitative analysis of fibrosis and mast cells in the tongue of chronic chagasic patients: autopsy study

Objective Recently, an orally transmitted outbreak of Chagas disease was reported in Santa Catarina, Brazil, after ingestion of sugar cane juice (garapa). This disease is caused by Trypanosoma cruzi, a parasite that stimulates the development of chronic inflammatory response, characterized by fibrous connective tissue neoformation (fibrosis). As the density of tissue mast cells (MC) may be an index of fibroblast proliferation and development of local fibrosis, the purpose of this autopsy study was to quantify the fibrosis rate and the number of MC in the tongues of chronic chagasic (CC) patients, compared with a non-chagasic (NC) control group. Methodology Twenty-four evaluations, with a quantitative assessment of fibrosis percentage and MC density were performed. Results The percentage of fibrosis in the tongue was higher among CC than in the control group. In the CC group, a positive and significant correlation was found when the fibrosis rate was compared with the MC density. Conclusions These morphometric findings suggest that tongue biopsy may be useful to study specific changes associated with Chagas disease. They also suggest that the systematic analysis of oral cavity, including tongue histopathology changes, could be useful in forensic pathology of the orally acquired chronic Chagas disease.

Antioxidant therapy attenuates oxidative stress in chronic cardiopathy associated with Chagas' disease

Oxidative stress is common in inflammatory processes of many diseases, including the Chagas' disease, which is characterized by chronic inflammation. The present study is a sequence of a related publication [Oliveira TB, Pedrosa RC, Wilhelm Filho D. Oxidative stress in chronic cardiopathy associated with Chagas' disease. Int J Cardiol in press.] on the same subjects, which showed an increase in oxidative stress associated with the progression of the severity of the disease. Components of the antioxidant system and oxidative biomarkers present in the blood were measured in the same chronic chagasic patients ( n = 40), before and after vitamin E (800 IU/day) and vitamin C (500 mg/day) supplementation for 6 months. Antioxidant enzymes and contents of reduced glutathione in erythrocytes and plasma TBARS contents were analyzed in four groups of patients in different stages of chronic Chagas heart disease ( n = 10 each group, groups I, II, III, and IV) according to the Los Andes classification. After the combined vitamin supplementation, TBARS and protein carbonyl levels were decreased in plasma, whilst red cell GSH contents were increased in group I. The vitamin E contents found in the plasma were inversely related to the severity of the disease. No differences in gamma-glutamiltransferase activities were detected but the myeloperoxidase levels were decreased in patients at the initial stages, whilst seric nitric oxide levels were increased in groups II and III. After the antioxidant supplementation, CAT activity was increased in group II, GPx activity was increased in group I, GR activity was increased in groups I and II, whilst the GST activity was decreased in groups II, III and IV. The results clearly indicate that the antioxidant supplementation was able to counteract the progressive oxidative stress associated with the disease. New perspectives for the treatment of Chagas' disease might include an antioxidant therapy in order to attenuate the consequences of oxidative insult related to this disease.

Morphometric study of eosinophils, mast cells, macrophages and fibrosis in the colon of chronic chagasic patients with and without megacolon

The mechanisms involved in the pathogenesis of chagasic megacolon are not completely characterized. Although autoimmunity may play a role in the pathogenesis of Chagas' disease, recent studies suggest a positive association of tissue parasitism, inflammation, and severity of lesions. The aim of this study was to evaluate the role of inflammatory cells and the occurrence of fibrosis in the colon of chagasic patients with and without megacolon. Samples from 26 patients were randomly selected and paraffin-embedded tissue blocks were sectioned and evaluated by histology and immunohistochemistry to analyse the occurrence and relation among eosinophils, mast cells, macrophages and fibrosis. Section analyses showed that the presence of eosinophils and mast cells in the analysed inflammatory cells has a direct correlation with fibrosis density in the chagasic megacolon. These data suggest that the megacolon's pathogenesis is based on a continuous process of cell damage. Our data propose that eosinophils, mast cells and macrophages may have a direct connection with the occurrence of fibrosis in the colon of chagasic patients. We believe that potential therapeutic agents against these cells could avoid the fibrosis process and contribute to prevent the development of chagasic megacolon.

Human antibodies with muscarinic activity modulate ventricular repolarization: Basis for electrical disturbance

Introduction In chronic chagasic patients sudden death has been reported when QT interval dispersion is increased and antibodies with muscarinic-like activity have been demonstrated to trigger arrhythmias. The aims were to investigate, in vivo and in vitro, relation between these antibodies and heterogeneity of ventricular repolarization and to identify predictors of cardiac death in chronic chagasic patients. Methods and results Clinical, electrocardiograph and echocardiograph variables from 32 chronic chagasic patients with moderate to severe left ventricular dysfunction, followed-up for 10 years were analyzed. Sera from chronic chagasic patients with or without muscarinic activity were tested in isolated rabbit hearts to study ventricular repolarization. Stepwise multivariate logistic analysis was applied to identify independent predictors of cardiac death. QT interval dispersion of patients with muscarinic activity (75.9 ± 5.5 ms) was larger than that of patients without muscarinic activity (51.3 ± 4.0 ms, p < 0.001). Maximum uncorrected and corrected QT intervals were not significantly different between groups of patients. Sera from patients with muscarinic activity significantly and reversibly increased QT interval in isolated rabbit hearts ( p = 0.002). This effect was abolished in the presence of the muscarinic antagonist atropine. Multivariate analysis identified maximum corrected QT intervals and left ventricular end diastolic index as independent predictors of cardiac death ( p = 0.03 and p = 0.02, respectively). Conclusions Sera with muscarinic activity from chagasic patients have a strong contribution to evoke ventricular repolarization rhythm disorder. In these patients, ventricular repolarization heterogeneity is increased significantly. In vitro, muscarinic sera reversibly increased repolarization duration. Maximum corrected QT intervals and left ventricular end diastolic index are independent predictors of cardiac death.

Comparison of Trypanosoma cruzi detection by PCR in blood and dejections of Triatoma infestans fed on patients with chronic Chagas disease

In this study, we compare the sensitivity of detecting Trypanosoma cruzi in dejections of Triatoma infestans nymphs that had fed on the blood of chronic chagasic patients, with detection of T. cruzi in peripheral blood, using a polymerase chain reaction assay (PCR-D and PCR-B, respectively). Fifty-seven chronic patients were evaluated who were positive (group I) or negative (group II) by xenodiagnosis (XD). Patients showed 84.8 and 75% positive PCR results in both kinds of samples in groups I and II, respectively. Six cases (10.5%) showed positive PCR-D and negative PCR-B, five of them belonged to group I. In contrast, five cases of group II showed negative PCR-D and positive PCR-B. Overall, the PCR-D assay gave positive results in 52 out of 57 samples (91.2%), while 51 out of 57 (89.5%) were positive by PCR-B. In comparison, only 57.9% were positive by XD ( p = 0.0001). In conclusion, PCR performed in dejection or blood was more sensitive for the parasite detection than xenodiagnosis. All patients (100%) were detected positive when both, PCR-D and PCR-B, were applied.

Short communication: Trypanosoma cruzi lineage I in endomyocardial biopsy from a north‐eastern Brazilian patient at end‐stage chronic chagasic cardiomyopathy

Trypanosoma cruzi, the agent of Chagas disease, is genetically classified into two major evolutionary lineages, T. cruzi I and T. cruzi II. In Southern American Cone countries it is T cruzi II which causes most cases of severe chronic Chagas disease. Contrary to this, we isolated T. cruzi I nested in endomyocardial biopsies of a chronic chagasic patient with end-stage heart failure. Our finding should alert clinicians to the possibility of severe Chagas disease in all regions where T. cruzi circulates, regardless of its lineage.

Interstitial cells of Cajal in patients with chagasic megacolon originating from a region of old endemicity

Megacolon is the second most frequent and most important digestive manifestation of Chagas’ disease. It is characterized by motor disorders and dilatation of the distal segments of the colon. Several theories have been presented to explain the physiopathology of chagasic megacolon, e.g. the plexus theory. Objective: In the present study the distribution of interstitial cells of Cajal (ICC) was evaluated in colon biopsies from chronic chagasic patients originating from a region of old endemicity for Trypanosoma cruzi and for comparison in subjects with other colon disorders. The chagasic patients had been submitted to colectomy for the investigation of other possible mechanisms underlying the physiopathogenesis of megacolons. Design study: Twenty-two colon biopsies (15 from chagasic patients and 7 from nonchagasic patients) were examined. ICC were identified by immunohistochemistry by using the anti-CD117 antibody. The number of ICC was determined in longitudinal and circular muscle layers and in the myenteric plexus, and the results were analyzed by the Kruskal–Wallis and Student t-tests. Results: A reduced number of ICC was observed in all layers and in the myenteric plexus of patients with chagasic megacolon ( P < 0.05). Conclusions: We conclude that the physiopathological manifestations observed in the large bowel of chagasic patients originate from alterations that occur in the ICC, which play an important role in the control of gut motility.

Random amplified polymorphic DNA profiles of Trypanosoma cruzi isolates from chagasic patients with different clinical forms

The genetic variability of 61 Trypanosoma cruzi isolates from 47 chronic chagasic patients of Minas Gerais state was analyzed by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) using M13-40, lambdagt11-F, and L15996 primers. Cluster analysis by unweighted pair group method analysis was applied to RAPD profiles, and cluster analysis used to verify a possible correlation among different clinical forms of the disease from these patients. The T. cruzi isolates showed distinct grouping on tree topology, with the isolates not being possible to establish a correlation to the clinical forms of Chagas' disease. These data showed that the T. cruzi isolates from these patients would compose a group of populations well correlated genetically.

Estudos eletrofisiológicos seriados do sistema éxcito-condutor do coração de pacientes com cardiopatia chagásica crônica

To study the evolution of lesions in conduction system using invasive electrophysiological studies (EPS), the impact of these alterations on cardiovascular events (CVE), and the frequency of these events in chronic chagasic patients. Prospective study, initiated in 1979 with clinical follow-up until 2000, with the participation of 28 chronic chagasic patients that were 18 to 65 years old, with bundle branch and/or atrioventricular blocks. The patients were submitted to two EPS with a minimum interval of four years between the studies in order to assess five electrophysiologists parameters that were correlated with CVE. A 24-hour Holter and echocardiogram were performed. The average follow-up time after the first EPS was 154.5 months while the interval between EPS was 107.5 months. Ages ranged from 25 to 65 years. Twenty seven patients presented complete right bundle branch block associated with a left anterior hemiblock. The echocardiogram showed alteration in twelve patients. During the 24-hour Holter and ventricular stimulation, only one patient presented sustained ventricular tachycardia (SVT). Nine presented CVE during the study and only the HV interval > or = 70 ms presented a significant statistical relationship with CVE. a) The chronic form of Chagas disease presents different progressive abnormality percentages for the electrophysiological variables and WP alterations are the most common. b) Among the electrophysiological findings, only the HV interval > or = 70 ms was associated with CVE. c) The incidence of CVE was 31.1% during the average follow-up period of 154.5 months.

Evaluation of IFN‐γ production by CD8+ T lymphocytes in response to the K1 peptide from KMP‐11 protein in patients infected with Trypanosoma cruzi

The cellular response mediated by MHC class I restricted CD8+ T cells has been shown to be crucial in the control of Chagas disease. The K1 peptide derived from T. cruzi KMP-11 protein has a high binding affinity to the HLA-A*0201 molecule. Nevertheless, it is not known whether this peptide is processed and displayed as an MHC class I epitope during natural infection by T. cruzi. The aim of this study was to evaluate, by ELISPOT assay, the ability of K1 peptide to activate CD8+ T lymphocytes to produce IFN-gamma. Therefore, CD8+ T lymphocytes from 22 HLA-A*0201+ individuals, 12 chronic chagasic patients and 10 uninfected controls, were analysed. The results revealed that two of the chagasic patients had IFN-gamma-secreting CD8+ T cells that were able to respond to K1 peptide with a relative frequency of 110 and 230 per million CD8+ T cells. In contrast, none of HLA-A*0201+ uninfected controls responded to K1 peptide. Responses to HLA-A*0201 restricted peptide from the influenza matrix protein were found in six chagasic patients and four uninfected controls with an average frequency of 175 and 111 cells per million CD8+ T cells, respectively. Moreover, a flow cytometric assay for degranulation showed that chagasic responders had K1-specific cytotoxic CD8+ T cells. It is shown here for the first time that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of Chagas disease.