Myocardial gene and protein expression profiles after autoimmune injury in Chagas' disease cardiomyopathy

One third of the 16 million of individuals infected by the protozoan Trypanosoma cruzi in Latin America eventually develop chronic Chagas' disease cardiomyopathy (CCC), an inflammatory dilated cardiomyopathy with shorter survival than non-inflammatory cardiomyopathies. The presence of a T cell-rich mononuclear inflammatory infiltrate and the relative scarcity of parasites in the heart suggested that chronic inflammation secondary to the autoimmune recognition of cardiac proteins could be a major pathogenetic mechanism. Sera from CCC patients crossreactively recognize cardiac myosin and T. cruzi protein B13. T cell clones elicited from peripheral blood with T. cruzi B13 protein or its peptides could crossreactively recognize epitopes from cardiac myosin heavy chain. Likewise, CD4+ T cell clones infiltrating CCC myocardium crossreactively recognize cardiac myosin and T. cruzi protein B13, and intralesional T cell lines produce the inflammatory cytokines IFN-γ and TNF-α. Conversely, IFN-γ-induced genes and chemokines were found to be upregulated in CCC heart samples, and IFN-γ is able to induce cardiomyocyte expression of atrial natriuretic factor, a key member of the hypertrophy/heart failure signature. Proteomic analysis of CCC heart tissue showed reduced expression of the energy metabolism enzymes. It can be hypothesized that cytokine-induced modulation of cardiomyocyte gene/protein expression may be a novel disease mechanism in CCC, in addition to direct inflammatory damage.     

Captopril increases the intensity of monocyte infection by Trypanosoma cruzi and induces human T helper type 17 cells

The anti-hypertensive drug captopril is used commonly to reduce blood pressure of patients with severe forms of Chagas disease, a cardiomyopathy caused by chronic infection with the intracellular protozoan Trypanosoma cruzi. Captopril acts by inhibiting angiotensin-converting enzyme (ACE), the vasopressor metallopeptidase that generates angiotensin II and promotes the degradation of bradykinin (BK). Recent studies in mice models of Chagas disease indicated that captopril can potentiate the T helper type 1 (Th1)-directing natural adjuvant property of BK. Equipped with kinin-releasing cysteine proteases, T. cruzi trypomastigotes were shown previously to invade non-professional phagocytic cells, such as human endothelial cells and murine cardiomyocytes, through the signalling of G protein-coupled bradykinin receptors (B(2) KR). Monocytes are also parasitized by T. cruzi and these cells are known to be important for the host immune response during infection. Here we showed that captopril increases the intensity of T. cruzi infection of human monocytes in vitro. The increased parasitism was accompanied by up-regulated expression of ACE in human monocytes. While T. cruzi infection increased the expression of interleukin (IL)-10 by monocytes significantly, compared to uninfected cells, T. cruzi infection in association with captopril down-modulated IL-10 expression by the monocytes. Surprisingly, studies with peripheral blood mononuclear cells revealed that addition of the ACE inhibitor in association with T. cruzi increased expression of IL-17 by CD4(+) T cells in a B(2) KR-dependent manner. Collectively, our results suggest that captopril might interfere with host-parasite equilibrium by enhancing infection of monocytes, decreasing the expression of the modulatory cytokine IL-10, while guiding development of the proinflammatory Th17 subset.     

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.     

Regulation of extracellular matrix expression and distribution in Trypanosoma cruzi-infected cardiomyocytes

Alterations in the extracellular matrix have been observed in the cardiomyopathy of Chagas disease caused by Trypanosoma cruzi infection. However, the mechanism of extracellular matrix regulation in T. cruzi-infected cultured cardiomyocytes (CMs) is unclear. Using confocal laser microscopy, we demonstrated that treatment of these cultures with transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha) leads to an enhancement of the fibronectin matrix only in uninfected CMs, while infected myocytes displayed low fibronectin expression. Digital image analysis also revealed low superposition of the fibronectin signal with parasite nests in cytokine treated and untreated cultures. Cytochalasin D treatment resulted in microfilament disarray that induced a disturbance in the fibronectin network of CMs, suggesting that cytoskeleton disruption caused by T. cruzi infection disorganizes the fibronectin matrix. Western blot analysis revealed a 2-fold increase in the fibronectin expression in CM cultures after cytokine treatment, whereas T. cruzi infection significantly reduced fibronectin levels in all conditions. In contrast, no change in the laminin expression was detected after cytokine treatment. Laminin distribution was altered in T. cruzi-infected CMs, with intense laminin labeling only at the cell periphery even after cytokine treatment. Our observations indicate that TGF-beta and TNF-alpha stimulates fibronectin expression only in uninfected cells of the T. cruzi-infected cultures, whereas the cells harboring the parasites display low or no fibronectin fibrils.     

Curcumin treatment provides protection against Trypanosoma cruzi infection

Trypanosoma cruzi, the etiologic agent of Chagas disease, causes an acute myocarditis and chronic cardiomyopathy. The current therapeutic agents for this disease are not always effective and often have severe side effects. Curcumin, a plant polyphenol, has demonstrated a wide range of potential therapeutic effects. In this study, we examined the effect of curcumin on T. cruzi infection in vitro and in vivo. Curcumin pretreatment of fibroblasts inhibited parasite invasion. Treatment reduced the expression of the low density lipoprotein receptor, which is involved in T. cruzi host cell invasion. Curcumin treatment of T. cruzi-infected CD1 mice reduced parasitemia and decreased the parasitism of infected heart tissue. This was associated with a significant reduction in macrophage infiltration and inflammation in both the heart and liver; moreover, curcumin-treated infected mice displayed a 100% survival rate in contrast to the 60% survival rate commonly observed in untreated infected mice. These data are consistent with curcumin modulating infection-induced changes in signaling pathways involved in inflammation, oxidative stress, and apoptosis. These data suggest that curcumin and its derivatives could be a suitable drug for the amelioration of chagasic heart disease.     

Pivotal role of interleukin-12 and interferon-gamma axis in controlling tissue parasitism and inflammation in the heart and central nervous system during Trypanosoma cruzi infection.

The role of cytokines in the control of tissue parasitism and pathogenesis of experimental Chagas' disease was investigated. Wild-type and different cytokine as well as inducible nitric oxide synthase (iNOS) knockout mice were infected with the Colombian strain of Trypanosoma cruzi, and the kinetics of tissue parasitism, inflammatory reaction, parasitemia, and mortality were determined. We demonstrate the pivotal role of the interleukin (IL)-12/interferon (IFN)-gamma/iNOS axis and the antagonistic effect of IL-4 in controlling heart tissue parasitism, inflammation, and host resistance to acute infection with T. cruzi. Further, the heart and central nervous system were shown the main sites of reactivation of T. cruzi infection in mice lacking functional genes for IFN-gamma and IL-12, respectively. Our results also show that in contrast to IFN-gamma knockout (KO) mice, splenocytes from IL-12 KO mice infected with T. cruzi produced low levels of IFN-gamma upon stimulation with antigen. Consistently, high levels of anti-T. cruzi IgG2a antibodies were detected in the sera from IL-12 KO, but not from IFN-gamma KO mice, infected with the Colombian strain of T. cruzi. Thus, our results suggest that the level of IFN-gamma deficiency is a major determinant of the site of reactivation of T. cruzi infection in immunocompromised host.     

Temporal expression of pro-inflammatory cytokines and inducible nitric oxide synthase in experimental acute Chagasic cardiomyopathy.

To characterize the kinetics of myocardial cytokine and inducible nitric oxide synthase (iNOS) expression in acute Chagasic cardiomyopathy, we studied a rat model of acute Trypanosoma cruzi infection. Rats were euthanized 36 hours and 5, 10, and 15 days after infection, and hearts were collected for histology, mRNA, and protein analyses. Histological analysis of myocardium showed a progressive increase in the number of amastigotes and mononuclear inflammatory cells. Organisms were first detected 5 days after intraperitoneal inoculation as isolated nests and became numerous by day 15. Northern blot analysis of total RNA revealed no signal for interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha and a weak signal for IL-6 in control hearts. High levels of expression for the three genes were detected in the infected animals at 36 hours after infection. Although IL-1beta and IL-6 levels increased steadily up to 10 days, TNF-alpha levels were the highest at 5 days, remained high at 10 days, and declined thereafter. Western blot analysis showed similar results to that of mRNA expression. No signal was detected for iNOS in the controls, but both its mRNA and protein were found in the infected animals, with levels being highest at 15 days after infection. Immunohistochemistry revealed no iNOS immunoreactivity in uninfected animals, but intense iNOS staining was detected in blood vessels of infected animals, which decreased progressively with period of infection. Positive staining for iNOS in cardiomyocytes was first detected at 36 hours after infection (at a time when there was no histological inflammatory reaction), which steadily increased, being the highest at 15 days after infection. These results indicate that, in addition to mechanical damage by T. cruzi, substantial pro-inflammatory cytokine production within the myocardium is likely to participate in the pathophysiology of acute Chagasic cardiomyopathy.     

MHC class I and class II genes in Mexican patients with Chagas disease

Chagas' disease contributes significantly to cardiovascular morbidity and mortality in several Latin-American countries. Previous studies have reported the effect of the human leukocyte antigen (HLA) molecules in the immune response regulation of Trypanosoma cruzi infection, and the association of HLA antigens with heart damage. We studied the major histocompatibility complex (MHC) class I (HLA-A and HLA-B), and class II (HLA-DR) genes in a sample of 66 serologically positive individuals with and without cardiomyopathy, and in 127 healthy controls. The total group of seropositive individuals revealed increased frequencies of HLA-B39 (pc=4.3x10(-5), odds ratio [OR]=3.35) and DR4 (pc=1.8x10(-5), OR=2.91) when compared to healthy controls. Increased frequencies of HLA-A68 and HLA-B39 were found in asymptomatic individuals when compared to patients with cardiomyopathy (pc=0.014, OR=4.99 and pc=0.001, OR=4.46, respectively). Also, patients with cardiomyopathy exhibited increased frequency of HLA-B35 when compared to healthy controls (pc=0.048, OR=2.56). The HLA-DR16 frequency was increased in patients with cardiomyopathy compared with asymptomatic individuals (pc=0.05, OR=No determined) and healthy controls (pc=0.02, OR=5.0). The results suggest that MHC alleles might be associated with the development of chronic infection and with heart damage in Chagas' disease. HLA-DR4 and HLA-B39 could be associated directly with the infection by T. cruzi, whereas, HLA-DR16 could be marker of susceptibility to heart damage and HLA-A68 might confer protection to develop cardiomyopathy.     

Pathology affects different organs in two mouse strains chronically infected by a Trypanosoma cruzi clone: a model for genetic studies of Chagas' disease

Chagas' disease is a chronic infection caused by Trypanosoma cruzi and represents an important public health burden in Latin America. Frequently the disease evolves undetectable for decades, while in a significant fraction of the affected individuals it culminates in death by heart failure. Here, we describe a novel murine model of the chronic infection with T. cruzi using a stable clone isolated from a human patient (Sylvio X10/4). The infection in the C3H/HePAS mouse strain progresses chronically and is mainly characterized by intense cardiac inflammatory lesions that recapitulate the chronic cardiac pathology observed in the human disease. Moderate striated muscle lesions are also present in C3H/HePAS mice. Viable parasites are detected and recovered from the chronic heart lesions of C3H/HePAS mice, supporting the current notion that development of heart pathology in Chagas' disease is related to parasite persistence in the inflamed tissue. By contrast, in infected A/J mice, chronic inflammatory lesions are targeted to the liver and the skeletal muscle, while pathology and parasites are undetectable in the heart. The phenotypic analysis of F(1) (A/J x C3H/HePAS) and F(2) (A/J x C3H/HePAS) mice suggests that the genetic predisposition to develop the inflammatory lesions caused by T. cruzi (Sylvio X10/4 clone) is heterogeneous because the heart and liver pathology segregate in the F(2) generation. These findings raise the hypothesis that the pathology heterogeneity observed in humans with Chagas' disease (absence and presence of cardiac or digestive chronic lesions) may be attributable to host genetic factors.     

The CC chemokine receptor 5 is important in control of parasite replication and acute cardiac inflammation following infection with Trypanosoma cruzi

Infection of susceptible mice with the Colombiana strain of Trypanosoma cruzi results in an orchestrated expression of chemokines and chemokine receptors within the heart that coincides with parasite burden and cellular infiltration. CC chemokine receptor 5 (CCR5) is prominently expressed during both acute and chronic disease, suggesting a role in regulating leukocyte trafficking and accumulation within the heart following T. cruzi infection. To better understand the functional role of CCR5 and its ligands with regard to both host defense and/or disease, CCR5(-/-) mice were infected with T. cruzi, and the disease severity was evaluated. Infected CCR5(-/-) mice develop significantly higher levels of parasitemia (P < or = 0.05) and cardiac parasitism (P < or = 0.01) during acute infection that correlated with reduced survival. Further, we show that CCR5 is essential for directing the migration of macrophages and T cells to the heart early in acute infection with T. cruzi. In addition, data are provided demonstrating that CCR5 does not play an essential role in maintaining inflammation in the heart during chronic infection. Collectively, these studies clearly demonstrate that CCR5 contributes to the control of parasite replication and the development of a protective immune response during acute infection but does not ultimately participate in maintaining a chronic inflammatory response within the heart.     

Gamma Interferon Modulates CD95 (Fas) and CD95 Ligand (Fas-L) Expression and Nitric Oxide-Induced Apoptosis during the Acute Phase of Trypanosoma cruzi Infection: a Possible Role in Immune Response Control

We have previously shown that splenocytes from mice acutely infected with Trypanosoma cruzi exhibit high levels of nitric oxide (NO)-mediated apoptosis. In the present study, we used the gamma interferon (IFN-gamma)-knockout (IFN-gamma(-/-)) mice to investigate the role of IFN-gamma in modulating apoptosis induction and host protection during T. cruzi infection in mice. IFN-gamma(-/-) mice were highly susceptible to infection and exhibited significant reduction of NO production and apoptosis levels in splenocytes but normal lymphoproliferative response compared to the infected wild-type (WT) mice. Furthermore, IFN-gamma modulates an enhancement of Fas and Fas-L expression after infection, since the infected IFN-gamma(-/-) mice showed significantly lower levels of Fas and Fas-L expression. The addition of recombinant murine IFN-gamma to spleen cells cultures from infected IFN-gamma(-/-) mice increased apoptosis levels, Fas expression, and NO production. In the presence of IFN-gamma and absence of NO, although Fas expression was maintained, apoptosis levels were significantly reduced but still higher than those found in splenocytes from uninfected mice, suggesting that Fas-Fas-L interaction could also play a role in apoptosis induction in T. cruzi-infected mice. Moreover, in vivo, the treatment of infected WT mice with the inducible nitric oxide synthase inhibitor aminoguanidine also led to decreased NO and apoptosis levels but not Fas expression, suggesting that IFN-gamma modulates apoptosis induction by two independent and distinct mechanisms: induction of NO production and of Fas and Fas-L expression. We suggest that besides being of crucial importance in mediating resistance to experimental T. cruzi infection, IFN-gamma could participate in the immune response control through apoptosis modulation.     

Pathogenesis of chagas' disease: parasite persistence and autoimmunity

Acute Trypanosoma cruzi infections can be asymptomatic, but chronically infected individuals can die of Chagas' disease. The transfer of the parasite mitochondrial kinetoplast DNA (kDNA) minicircle to the genome of chagasic patients can explain the pathogenesis of the disease; in cases of Chagas' disease with evident cardiomyopathy, the kDNA minicircles integrate mainly into retrotransposons at several chromosomes, but the minicircles are also detected in coding regions of genes that regulate cell growth, differentiation, and immune responses. An accurate evaluation of the role played by the genotype alterations in the autoimmune rejection of self-tissues in Chagas' disease is achieved with the cross-kingdom chicken model system, which is refractory to T. cruzi infections. The inoculation of T. cruzi into embryonated eggs prior to incubation generates parasite-free chicks, which retain the kDNA minicircle sequence mainly in the macrochromosome coding genes. Crossbreeding transfers the kDNA mutations to the chicken progeny. The kDNA-mutated chickens develop severe cardiomyopathy in adult life and die of heart failure. The phenotyping of the lesions revealed that cytotoxic CD45, CD8(+) γδ, and CD8α(+) T lymphocytes carry out the rejection of the chicken heart. These results suggest that the inflammatory cardiomyopathy of Chagas' disease is a genetically driven autoimmune disease.     

Immunotherapy of Trypanosoma cruzi infection with DNA vaccines in mice

The mechanisms involved in the pathology of chronic chagasic cardiomyopathy are still debated, and the controversy has interfered with the development of new treatments and vaccines. Because of the potential of DNA vaccines for immunotherapy of chronic and infectious diseases, we tested if DNA vaccines could control an ongoing Trypanosoma cruzi infection. BALB/c mice were infected with a lethal dose (5 x 10(4) parasites) as a model of acute infection, and then they were treated with two injections of 100 microg of plasmid DNA 1 week apart, beginning on day 5 postinfection. Control mice had high levels of parasitemia and mortality and severe cardiac inflammation, while mice treated with plasmid DNA encoding trypomastigote surface antigen 1 or Tc24 had reduced parasitemia and mild cardiac inflammation and >70% survived the infection. The efficacy of the immunotherapy also was significant when it was delayed until days 10 and 15 after infection. Parasitological analysis of cardiac tissue of surviving mice indicated that most mice still contained detectable parasite kinetoplast DNA but fewer mice contained live parasites, suggesting that there was efficient but not complete parasite elimination. DNA vaccine immunotherapy was also evaluated in CD1 mice infected with a low dose (5 x 10(2) parasites) as a model of chronic infection. Immunotherapy was initiated on day 70 postinfection and resulted in improved survival and reduced cardiac tissue inflammation. These results suggest that DNA vaccines have strong potential for the immunotherapy of T. cruzi infection and may provide new alternatives for the control of Chagas' disease.     

Role of SOCS2 in modulating heart damage and function in a murine model of acute Chagas disease

Infection with Trypanosoma cruzi induces inflammation, which limits parasite proliferation but may result in chagasic heart disease. Suppressor of cytokine signaling 2 (SOCS2) is a regulator of immune responses and may therefore participate in the pathogenesis of T. cruzi infection. SOCS2 is expressed during T. cruzi infection, and its expression is partially reduced in infected 5-lipoxygenase-deficient [knockout (KO)] mice. In SOCS2 KO mice, there was a reduction in both parasitemia and the expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-10, SOCS1, and SOCS3 in the spleen. Expression of IFN-γ, TNF-α, SOCS1, and SOCS3 was also reduced in the hearts of infected SOCS2 KO mice. There was an increase in the generation and expansion of T regulatory (Treg) cells and a decrease in the number of memory cells in T. cruzi-infected SOCS2 KO mice. Levels of lipoxinA(4) (LXA(4)) increased in these mice. Echocardiography studies demonstrated an impairment of cardiac function in T. cruzi-infected SOCS2 KO mice. There were also changes in calcium handling and in action potential waveforms, and reduced outward potassium currents in isolated cardiac myocytes. Our data suggest that reductions of inflammation and parasitemia in infected SOCS2-deficient mice may be secondary to the increases in Treg cells and LXA(4) levels. This occurs at the cost of greater infection-associated heart dysfunction, highlighting the relevance of balanced inflammatory and immune responses in preventing severe T. cruzi-induced disease.     

Immunopathology of experimental Chagas'' disease: binding of T cells to Trypanosoma cruzi-infected heart tissue.

The immunopathology of Chagas' disease was studied in the experimental model of chronic infection in C57BL/10JT or mice. Sublethal infection with Trypanosoma cruzi, Y strain, induced specific antibodies and a delayed hypersensitivity response to parasite antigens. Mice developed chronic chagasic myocarditis but not skeletal muscle myositis. Binding of T cells to infected heart tissue was investigated during short-term cocultivation of lymphocytes with heart cryostat sections. T cells from infected mice and from normal controls bound equally to myocardium and liver sections from both infected and normal mice. A search in depth was attempted with cells heavily tagged with 99mTc. Labeled T cells from chagasic mice bound to both normal and infected myocardium slices. 99mTc-labeled T cells from controls gave the same binding values. Glass-adherent spleen cells behaved identically to T cells. Prior treatment of the tissue with serum from chronically infected mice did not increase the number of binding cells. Peritoneal macrophages tagged with 99mTc-sulfur colloid also bound to infected myocardium slices. The binding of macrophages was not changed by pretreatment of infected tissue with anti-T, cruzi antibodies. In short, this work did not detect any population of T cells or macrophages which could bind specifically to infected heart tissue to initiate an autoreactive process.     

Trypanosoma cruzi infection in pregnant mice induces a cellular immune response with citokines production in their fetuses

The objective of this study was to detect the cytokines IFN-gamma, IL-4 and IL-10 expressed by CD4+ T cells in tissues of fetal mice with acute chagasic infection. For this, we examined the fetuses of NMRI mice whose mothers were infected with 22x10(3) metacyclic trypomastigotes of the M/HOM/BRA/53/Y strain of T. cruzi and made pregnant during the acute phase of infection. For the detection and localization of inflammatory infiltrates, nest parasites, antigens of T. cruzi a nd cytokines w eused hematoxylin-eosin techniques, peroxidase-anti-peroxidase and immunofluorescence. The immunohistochemical study revealed the presence of inflammatory infiltrates and antigens with amastigote nests in fetal skeletal muscle. CD4 + T cells producing IFN-gamma, as well as deposits of IFN-gamma and IL-10, were detected in sections of placenta, heart and skeletal muscle of fetuses of mice infected, while CD4+/IL-10+ was found only in skeletal muscle; in addition, deposits of IL-4 were detected only in placentas of healthy mice. These results indicate that fetuses are capable of generating their own immune response to antigens transmitted by their mother, which induces the secretion of cytokines and that, acting in synergy with the maternal antibodies, confer them a state of protection against infection; and that the transmission of the parasite depends on factors specific to each mother, which may modify its ability to control such transmission at the placental or systemic levels.     

Heart-infiltrating and peripheral T cells in the pathogenesis of human Chagas' disease cardiomyopathy

Heart tissue destruction in chronic Chagas' disease cardiomyopathy (CCC), occurring in 30% of individuals chronically infected by the protozoan parasite Trypanosoma cruzi, may be caused by autoimmune recognition of patients' heart tissue by a T cell rich inflammatory infiltrate. Recently, our group demonstrated that T cells infiltrating the heart of CCC patients crossreactively recognize cardiac myosin heavy chain and tandemly repetitive T. cruzi antigen B13, and possess an inflammatory T1-type cytokine profile. Susceptibility factors leading 30% of infected patients to develop CCC, while the rest of the patients remain largely asymptomatic (ASY), are still obscure. We compared immunological phenotypes of CCC and ASY patients, who have distinct clinical outcomes despite bearing a similar chronic T. cruzi infection. Preliminary observations indicate that PBMC from CCC patients recognize a set of B13 and cardiac myosin epitopes distinct from that recognized by ASY patients. Moreover, the IFN-gamma response of CCC patients is more intense than that of ASY, both at qualitative and quantitative levels. Taken together, results suggest that heart damage in Chagas' disease cardiomyopathy may be secondary to inflammatory cytokines and a delayed-type hypersensitivity process started and/or maintained by heart-crossreactive T cells. Furthermore, the distinct recognition repertoire and the high frequency of IFN-gamma producing among CCC patients could be important factors leading to the differential development of CCC among T. cruzi infected individuals.