Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease. 1 - Comparative xenodiagnosis with nine triatomine species of animals with acute infections by Trypanosoma cruzi

In search of a suitable vector species for xenodiagnosis of humans and animals with chronic Chagas' disease we first investigated the reactions of different vector species to acute infection with Trypanosoma cruzi. Vector species utilized in this study were: Triatoma infestans, Rhodnius prolixus and Triatoma dimidiata, all well adapted to human habitats; Triatoma rubrovaria and Rhodnius neglectus both considered totally wild species; Panstrongylus megistus, Triatoma sordida, Triatoma pseudomaculata and Triatoma brasiliensis, all essentially sylvatic but some with domiciliary tendencies and others restricted to peridomestic biotopes with incipient colonization of human houses after successful eradication of T. infestans. Results summarized in Table IV suggest the following order of infectivity among the 9 studied vector species: P. megistus with 97.8% of infected bugs, T. rubrovaria with 95% of positive bugs a close second followed by T. Pseudomaculata with 94.3% and R. neglectus with 93.8% of infected bugs, almost identical thirds. R. prolixus, T. infestans and T. dimidiata exhibited low infection rates of 53.1%, 51.6% and 38.2% respectively, coupled with sharp decreases occuring with aging of infection (Fig. 1). The situation was intermediate in T. brasiliensis and T. sordida infection rates being 76.9% and 80% respectively. Results also point to the existence of a close correlation between prevalence and intensity of infection in that, species with high infection rates ranging from 93.8% to 97.8% exhibited relatively large proportions of insects (27.3% - 33.5%) harbouring very dense populations of T. cruzi. In species with low infection rates ranging from 38.2% to 53.1% the proportion of bugs demonstrating comparable parasite densities was at most 6%. No differences attributable to blood-meal size or to greater susceptibility of indigenous vector species to parasites of their own geographical area, as suggested in earlier...

Evaluation of the rabbit as a model for chagas' disease: I. Parasitological studies

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.

Evaluation of Chagas' disease transmission through breast-feeding

On hundred milk or colostrum samples from 78 mothers with chronic Chagas' disease were parasitologically studied for Trypanosoma cruzi infection by means of direct examination and inoculation of mice. The mice were submitted to direct bllod examination three times a week. At the end of 45 days, xenodiagnosis and indirect immunofluorescent test (IFAT) for T. cruzi antibodies were carried out in the animals. No parasitized sample was observed even though five mothers had parasitemia at milk collection. In addition, 97 breast-fed children of chronic chagasic mothers, born free of infection, were tested for IgG antibodies to T. cruzi using IFAT. No case of T. cruzi infection was detected. The authors conclude that breast-feeding should not be avoided for children for chronic chagasic women. However, as these mothers had intermittent parasitemia, they should avoid nursing when there is nipple bleeding.

Serum uric acid levels in Chagas' disease

Uricemia was studied in a sample of 192 individuals from a highly endemic site for Chagas' disease (Bambuí, State of Minas Gerais, Brazil). The sample had serologically negative individuals (controls) and the positive ones were classified on the basis of the presence of electrocardiographic alterations (63), altered esophageal emptying (16), or without any sign on sympton of the disease (76). Only the individuals with the digestive form of chronic Chagas' disease showed hyperuricemia, when compared with the appropriate controls. Family data suggest that hyperuricemia is an effect of the digestive pathology, rather than a cause, since the non-infected sibs of the megaesophagous patients did not show elevated levesl of serum uric acid. Possible mechanisms responsible for these findings are postulated.

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the refered kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.

Increased plasma levels of tumor necrosis factor-alpha in asymptomatic/"indeterminate" and Chagas disease cardiomyopathy patients

We compared plasma tumor necrosis factor-alpha (TNF-alpha) levels among asymptomatic/"indeterminate" Chagas disease patients (ASY) and patients across the clinical spectrum of chronic Chagas disease cardiomyopathy (CCC). Idiopathic dilated cardiomyopathy (DCM) patients and normal controls (NC) were included as controls. ASY Chagas disease patients had significantly higher plasma TNF-alpha levels than NC. TNF-alpha levels among severe CCC patients with significant left ventricular (LV) dysfunction were similar to those of DCM patients, showing average 2-fold higher levels than CCC patients without LV dysfunction and ASY patients, and 8-fold higher levels than NC. In Chagas disease, chronic TNF-a production prior to heart failure may play a role in CCC progression.

Treatment and seroconversion in a cohort of children suffering from recent chronic Chagas infection in Yoro, Honduras

Between 1999-2002, Médécins Sans Frontières-Spain implemented a project seeking to determine the efficacy and safety of benznidazole in the treatment of recent chronic Chagas disease in a cohort of seropositive children in the Yoro Department, Honduras. A total of 24,471 children were screened for Trypanosoma cruzi IgG antibodies through conventional enzyme-linked immunosorbent assays (ELISA) on filter paper. Recombinant ELISA (0.93% seroprevalence) showed 256 initially reactive cases, including 232 confirmed positive cases. Of these, 231 individuals were treated with benznidazole (7.5 mg/kg/day) for 60 days and were followed with a strict weekly medical control and follow-up protocol. At the end of the project, 229 patients were examined by the Honduras Secretariat of Health for post-treatment serological assessments; 88.2% seroconverted after 18 months and 93.9% seroconverted after three years. No differences were found in the seroconversion rates according to age or sex. Most of the side effects of the treatment were minor. These results support the argument that in areas where T. cruzi I is predominant and in areas affected by T. cruzi II, when vector transmission has been interrupted, Chagas disease diagnosis and treatment are feasible, necessary and ethically indisputable.

Back to the future in Chagas disease: from animal models to patient cohort studies, progress in immunopathogenesis research

Despite the wealth of information generated by trans-disciplinary research in Chagas disease, knowledge about its multifaceted pathogenesis is still fragmented. Here we review the body of experimental studies in animal models supporting the concept that persistent infection by Trypanosoma cruzi is crucial for the development of chronic myocarditis. Complementing this review, we will make an effort to reconcile seemingly contradictory results concerning the immune profiles of chronic patients from Argentina and Brazil. Finally, we will review the results of molecular studies suggesting that parasite-induced inflammation and tissue damage is, at least in part, mediated by the activities of trans-sialidase, mucin-linked lipid anchors (TLR2 ligand) and cruzipain (a kinin-releasing cysteine protease). One hundred years after the discovery of Chagas disease, it is reassuring that basic and clinical research tends to converge, raising new perspectives for the treatment of chronic Chagas disease.

Ergosterol biosynthesis and drug development for Chagas disease

This article presents an overview of the currently available drugs nifurtimox (NFX) and benznidazole (BZN) used against Trypanosoma cruzi, the aetiological agent of Chagas disease; herein we discuss their limitations along with potential alternatives with a focus on ergosterol biosynthesis inhibitors (EBI). These compounds are currently the most advanced candidates for new anti-T. cruzi agents given that they block de novo production of 24-alkyl-sterols, which are essential for parasite survival and cannot be replaced by a host's own cholesterol. Among these compounds, new triazole derivatives that inhibit the parasite's C14± sterol demethylase are the most promising, as they have been shown to have curative activity in murine models of acute and chronic Chagas disease and are active against NFX and BZN-resistant T. cruzi strains; among this class of compounds, posaconazole (Schering-Plough Research Institute) and ravuconazole (Eisai Company) are poised for clinical trials in Chagas disease patients in the short term. Other T. cruzi-specific EBI, with in vitro and in vivo potency, include squalene synthase, lanosterol synthase and squalene epoxidase-inhibitors as well as compounds with dual mechanisms of action (ergosterol biosynthesis inhibition and free radical generation), but they are less advanced in their development process. The main putative advantages of EBI over currently available therapies include their higher potency and selectivity in both acute and chronic infections, activity against NFX and BZN-resistant T. cruzi strains, and much better tolerability and safety profiles. Limitations may include complexity and cost of manufacture of the new compounds. As for any new drug, such compounds will require extensive clinical testing before being introduced for clinical use, and the complexity of such studies, particularly in chronic patients, will be compounded by the current limitations in the verification of true parasitological cures for T. cruzi infections.

Current status and perspectives of cell therapy in Chagas disease

One century after its discovery, Chagas disease, caused by the protozoan, Trypanosoma cruzi, remains a major health problem in Latin America. Mortality and morbidity are mainly due to chronic processes that lead to dysfunction of the cardiac and digestive systems. About one third of the chronic chagasic individuals have or will develop the symptomatic forms of the disease, with cardiomyopathy being the most common chronic form. This is a progressively debilitating disease for which there are no currently available effective treatments other than heart transplantation. Like in other cardiac diseases, tissue engineering and cell therapy have been investigated in the past few years as a means of recovering the heart function lost as a consequence of chronic damage caused by the immune-mediated pathogenic mechanisms elicited in individuals with chronic chagasic cardiomyopathy. Here we review the studies of cell therapy in animal models and patients with chronic Chagas disease and the perspectives of the recovery of the heart function lost due to infection with T. cruzi.

Cytokine production profile of heart-infiltrating T cells in Chagas' disease cardiomyopathy

The hallmark of chronic Chagas' disease cardiomyopathy (CCC) is the finding of a T cell-rich inflammatory mononuclear cell infiltrate in the presence of extremely few parasites in the heart lesions. The scarcity of parasites in affected heart tissue casts doubt on the direct participation of Trypanosoma cruzi in CCC heart tissue lesions, and suggests the possible involvement of autoimmunity. The cells in the infiltrate are presumably the ultimate effectors of tissue damage, and there is evidence that such cells recognize cardiac myosin in molecular mimicry with T. cruzi proteins rather than primary reactivity to T. cruzi antigens (Cunha-Neto et al. (1996) Journal of Clinical Investigation, 98: 1709-1712). Recently, we have studied heart-infiltrating T cells at the functional level. In this short review we summarize the studies about the role of cytokines in human and experimental T. cruzi infection, along with our data on heart-infiltrating T cells in human Chagas' cardiomyopathy. The bulk of evidence points to a significant production of IFN-g and TNF-a which may be linked to T. cruzi-induced IL-12 production

Proteomic inventory of myocardial proteins from patients with chronic Chagas' cardiomyopathy

Chronic Chagas' disease cardiomyopathy (CCC) is an often fatal outcome of Trypanosoma cruzi infection, with a poorer prognosis than other cardiomyopathies. CCC is refractory to heart failure treatments, and is the major indication of heart transplantation in Latin America. A diffuse myocarditis, plus intense myocardial hypertrophy, damage and fibrosis, in the presence of very few T. cruzi forms, are the histopathological hallmarks of CCC. To gain a better understanding of the pathophysiology of CCC, we analyzed the protein profile in the affected CCC myocardium. Homogenates from left ventricular myocardial samples of end-stage CCC hearts explanted during heart transplantation were subjected to two-dimensional electrophoresis with Coomassie blue staining; protein identification was performed by MALDI-ToF mass spectrometry and peptide mass fingerprinting. The identification of selected proteins was confirmed by immunoblotting. We demonstrated that 246 proteins matched in gels from two CCC patients. They corresponded to 112 distinct proteins. Along with structural/contractile and metabolism proteins, we also identified proteins involved in apoptosis (caspase 8, caspase 2), immune system (T cell receptor ß chain, granzyme A, HLA class I) and stress processes (heat shock proteins, superoxide dismutases, and other oxidative stress proteins). Proteins involved in cell signaling and transcriptional factors were also identified. The identification of caspases and oxidative stress proteins suggests the occurrence of active apoptosis and significant oxidative stress in CCC myocardium. These results generated an inventory of myocardial proteins in CCC that should contribute to the generation of hypothesis-driven experiments designed on the basis of the classes of proteins identified here.

Serum uric acid levels in Chagas' disease

A uricemia foi estudada em uma amostra de 192 indivíduos de uma região altamente endêmica para a doença de Chagas (Bambuí, Estado de Minas Gerais, Brasil). A amostra continha 50 indivíduos sorologicamente negativos (controles) e os positivos foram classificados na base da presença de alterações eletrocardiográficas (63), esvaziamento esofagiano alterado (16), ou ausência de sinais ou sintomas da doença (76). Somente os indivíduos com a forma digestiva da doença de Chagas crônico mostraram hiperuricemia, quando comparados com controles adequados. Dados familiares sugerem que a hiperuricemia é um efeito da patologia digestiva em vez de causa, uma vez que os irmãos não afetados dos pacientes com megaesôfago não apresentaram níveis elevados de ácido úrico sérico. São postulados alguns mecanismos possivelmente responsáveis pelos achados.

Deficient Regulatory T Cell Activity and Low Frequency of IL-17-Producing T Cells Correlate with the Extent of Cardiomyopathy in Human Chagas' Disease

Background: Myocardium damage during Chagas' disease results from the immunological imbalance between pro-and production of anti-inflammatory cytokines and has been explained based on the Th1-Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas' disease. Methodology/Principal Findings: First, we observed CD4(+)IL-17(+) T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-alpha, IFN-gamma and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4(+)IL-17(+) cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas' disease patients presented the same frequency of CD4(+)CD25(+) regulatory T cells. However, CD4(+)CD25(+) T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-gamma levels during chronic Chagas' disease are inversely correlated to the LVEF (P = 0.007, r = -0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500). Conclusion/Significance: These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-gamma and TNF-alpha is correlated with the severity of the Chagas' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.