Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates.

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a chagasic mother to her offspring, but the male partner's contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody. Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.

American trypanosomiasis and Chagas disease: Sexual transmission.

OBJECTIVE: To contribute to the discussion on the research findings indicating the sexual transmission of American trypanosomiasis and Chagas disease in humans. METHODS: A review of the literature was performed to investigate the routes of transmission of Trypanosoma cruzi parasites and to evaluate the distribution of Chagas disease, which is now found across five continents. RESULTS: The epidemiological profile of American trypanosomiasis, which is still considered a neglected disease of the poor people of Latin America, has changed over time. A family-based study demonstrated that the blood protozoan T. cruzi can be transmitted sexually from infected males and females to naïve mates. CONCLUSIONS: Evidence that Chagas disease can be transmitted sexually, coupled with the migration of individuals with Chagas disease to previously non-endemic countries and increased travel to endemic countries, has implications for public health. Improved screening of blood supplies and prenatal care are required to prevent congenital spread.

Parasite induced genetically driven autoimmune Chagas heart disease in the chicken model.

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases show Chagas disease up to three decades or later. Autoimmunity and parasite persistence are competing theories to explain the pathogenesis of Chagas disease. To separate roles played by parasite persistence and autoimmunity in Chagas disease we inoculate the T. cruzi in the air chamber of fertilized eggs. The mature chicken immune system is a tight biological barrier against T. cruzi and the infection is eradicated upon development of its immune system by the end of the first week of growth. The chicks are parasite-free at hatching, but they retain integrated parasite mitochondrial kinetoplast DNA (kDNA) minicircle within their genome that are transferred to their progeny. Documentation of the kDNA minicircle integration in the chicken genome was obtained by a targeted prime TAIL-PCR, Southern hybridizations, cloning, and sequencing. The kDNA minicircle integrations rupture open reading frames for transcription and immune system factors, phosphatase (GTPase), adenylate cyclase and phosphorylases (PKC, NF-Kappa B activator, PI-3K) associated with cell physiology, growth, and differentiation, and other gene functions. Severe myocarditis due to rejection of target heart fibers by effectors cytotoxic lymphocytes is seen in the kDNA mutated chickens, showing an inflammatory cardiomyopathy similar to that seen in human Chagas disease. Notably, heart failure and skeletal muscle weakness are present in adult chickens with kDNA rupture of the dystrophin gene in chromosome 1. Similar genotipic alterations are associated with tissue destruction carried out by effectors CD45+, CD8γδ+, CD8α lymphocytes. Thus this protozoan infection can induce genetically driven autoimmune disease.

Interactome: Smart hematophagous triatomine salivary gland molecules counteract human hemostasis during meal acquisition.

Human populations are constantly plagued by hematophagous insects' bites, in particular the triatomine insects that are vectors of the Trypanosoma cruzi agent in Chagas disease. The pharmacologically-active molecules present in the salivary glands of hematophagous insects are injected into the human skin to initiate acquisition of blood meals. Sets of vasodilators, anti-platelet aggregators, anti-coagulants, immunogenic polypeptides, anesthetics, odorants, antibiotics, and detoxifying molecules have been disclosed with the aid of proteomics and recombinant cDNA techniques. These molecules can provide insights about the insect-pathogen-host interactions essential for understanding the physiopathology of the insect bite. The data and information presented in this review aim for the development of new drugs to prevent insect bites and the insect-transmitted endemic of Chagas disease.

Real-time PCR in HIV/Trypanosoma cruzi coinfection with and without Chagas disease reactivation: association with HIV viral load and CD4 level.

BACKGROUND: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. METHODOLOGY: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. PRINCIPAL FINDINGS: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. CONCLUSIONS: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

Diversity of anti-haemostatic proteins in the salivary glands of Rhodnius species transmitters of Chagas disease in the greater Amazon.

The triatomines in the tribe Rhodniini are the main vectors of the Trypanosoma cruzi to humans in recent outbreaks of acute Chagas disease in the Amazon. These insects dwelling in palm trees do not colonize the human domicile. Their success to transmit the infection relies partially on the efficacy of their salivary gland apparatuses. Here we show the transcriptome of the Rhodnius brethesi and Rhodnius robustus salivary glands, comprising 56 and 122 clusters, respectively. Approximately one third of these clusters are described for the first time. The LC-MS/MS analysis identified 123 and 111 proteins in R. brethesi and R. robustus sialome, respectively. Noteworthy, lipocalin platelet aggregation inhibitors, inositol polyphosphate 5-phosphatases, and Kazal domain proteins, which are essential for the insect's successful acquisition of blood meals, were found in our analysis. Moreover, glutathione S transferase and antigen-5, which play roles in the insect's defense and resistance against insecticide, were also observed.

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.

Redundancy of proteins in the salivary glands of Panstrongylus megistus secures prolonged procurement for blood meals.

Panstrongylus megistus, a vector for the Chagas disease parasite Trypanosoma cruzi, is a hematophagous bug widely distributed in South America. This ubiquitous triatomine is known to colonize different wild life habitats. Additionally, P. megistus synanthropy, preying upon mammals, birds, reptiles, and eventually being predators upon insect's hemolymph probably increases its ability to survive after prolonged fasting. It was suspected that the P. megistus mechanisms of adaptation to survival might include a salivary gland complex tool-box with a diversity of pharmacologically active proteins for obtaining blood meals. Herein we describe comprehensive proteome and transcriptome of the P. megistus salivary gland. The proteomic analysis led to the identification of 159 proteins, and the transcriptome revealed 47 complete cDNAs. A diversity of protein functions associated to blood feeding was identified. The most prevalent proteins were related to blood clotting, anti-platelet aggregation and anti-vasoconstriction activities, which correlate with the insect's ability to obtain meals from different sources. Moreover, a gene of resistance to insecticides was identified. These features augments the comprehension towards P. megistus enormous capacity to survive in adverse wild life-changing habitats.

Trypanosoma cruzi in the chicken model: Chagas-like heart disease in the absence of parasitism.

BACKGROUND: The administration of anti-trypanosome nitroderivatives curtails Trypanosoma cruzi infection in Chagas disease patients, but does not prevent destructive lesions in the heart. This observation suggests that an effective treatment for the disease requires understanding its pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To understand the origin of clinical manifestations of the heart disease we used a chicken model system in which infection can be initiated in the egg, but parasite persistence is precluded. T. cruzi inoculation into the air chamber of embryonated chicken eggs generated chicks that retained only the parasite mitochondrial kinetoplast DNA minicircle in their genome after eight days of gestation. Crossbreeding showed that minicircles were transferred vertically via the germ line to chicken progeny. Minicircle integration in coding regions was shown by targeted-primer thermal asymmetric interlaced PCR, and detected by direct genomic analysis. The kDNA-mutated chickens died with arrhythmias, shortness of breath, cyanosis and heart failure. These chickens with cardiomyopathy had rupture of the dystrophin and other genes that regulate cell growth and differentiation. Tissue pathology revealed inflammatory dilated cardiomegaly whereby immune system mononuclear cells lyse parasite-free target heart fibers. The heart cell destruction implicated a thymus-dependent, autoimmune; self-tissue rejection carried out by CD45(+), CD8γδ(+), and CD8α lymphocytes. CONCLUSIONS/SIGNIFICANCE: These results suggest that genetic alterations resulting from kDNA integration in the host genome lead to autoimmune-mediated destruction of heart tissue in the absence of T. cruzi parasites.

2-DE-based proteomic investigation of the saliva of the Amazonian triatomine vectors of Chagas disease: Rhodnius brethesi and Rhodnius robustus.

The triatomine bugs are obligatory haematophagous organisms that act as vectors of Chagas disease by transmitting the protozoan Trypanosoma cruzi. Their feeding success is strongly related to salivary proteins that allow these insects to access blood by counteracting host haemostatic mechanisms. Proteomic studies were performed on saliva from the Amazonian triatomine bugs: Rhodnius brethesi and R. robustus, species epidemiologically relevant in the transmission of T. cruzi. Initially, salivary proteins were separated by two-dimensional gel electrophoresis (2-DE). The average number of spots of the R. brethesi and R. robustus saliva samples were 129 and 135, respectively. The 2-DE profiles were very similar between the two species. Identification of spots by peptide mass fingerprinting afforded limited efficiency, since very few species-specific salivary protein sequences are available in public sequence databases. Therefore, peptide fragmentation and de novo sequencing using a MALDI-TOF/TOF mass spectrometer were applied for similarity-driven identifications which generated very positive results. The data revealed mainly lipocalin-like proteins which promote blood feeding of these insects. The redundancy of saliva sequence identification suggested multiple isoforms caused by gene duplication followed by gene modification and/or post-translational modifications. In the first experimental assay, these proteins were predominantly phosphorylated, suggesting functional phosphoregulation of the lipocalins.

Inheritance of DNA transferred from American trypanosomes to human hosts.

Interspecies DNA transfer is a major biological process leading to the accumulation of mutations inherited by sexual reproduction among eukaryotes. Lateral DNA transfer events and their inheritance has been challenging to document. In this study we modified a thermal asymmetric interlaced PCR by using additional targeted primers, along with Southern blots, fluorescence techniques, and bioinformatics, to identify lateral DNA transfer events from parasite to host. Instances of naturally occurring human infections by Trypanosoma cruzi are documented, where mitochondrial minicircles integrated mainly into retrotransposable LINE-1 of various chromosomes. The founders of five families show minicircle integrations that were transferred vertically to their progeny. Microhomology end-joining of 6 to 22 AC-rich nucleotide repeats in the minicircles and host DNA mediates foreign DNA integration. Heterogeneous minicircle sequences were distributed randomly among families, with diversity increasing due to subsequent rearrangement of inserted fragments. Mosaic recombination and hitchhiking on retrotransposition events to different loci were more prevalent in germ line as compared to somatic cells. Potential new genes, pseudogenes, and knockouts were identified. A pathway of minicircle integration and maintenance in the host genome is suggested. Thus, infection by T. cruzi has the unexpected consequence of increasing human genetic diversity, and Chagas disease may be a fortuitous share of negative selection. This demonstration of contemporary transfer of eukaryotic DNA to the human genome and its subsequent inheritance by descendants introduces a significant change in the scientific concept of evolutionary biology and medicine.

Environment, interactions between Trypanosoma cruzi and its host, and health.

An epidemiological chain involving Trypanosoma cruzi is discussed at the environmental level, and in terms of fine molecular interactions in invertebrate and vertebrate hosts dwelling in different ecosystems. This protozoan has a complex, genetically controlled plasticity, which confers adaptation to approximately 40 blood-sucking triatomine species and to over 1,000 mammalian species, fulfilling diverse metabolic requirements in its complex life-cycle. The Tr. cruzi infections are deeply embedded in countless ecotypes, where they are difficult to defeat using the control methods that are currently available. Many more field and laboratory studies are required to obtain data and information that may be used for the control and prevention of Tr. cruzi infections and their various disease manifestations. Emphasis should be placed on those sensitive interactions at cellular and environmental levels that could become selected targets for disease prevention. In the short term, new technologies for social mobilization should be used by people and organizations working for justice and equality through health information and promotion. A mass media directed program could deliver education, information and communication to protect the inhabitants at risk of contracting Tr. cruzi infections.

Environment, interactions between Trypanosoma cruzi and its host, and health: [review] / Meio-ambiente, interações entre Trypanosoma cruzi e seu hospedeiro e saúde humana: [revisão]

An epidemiological chain involving Trypanosoma cruzi is discussed at the environmental level, and in terms of fine molecular interactions in invertebrate and vertebrate hosts dwelling in different ecosystems. This protozoan has a complex, genetically controlled plasticity, which confers adaptation to approximately 40 blood-sucking triatomine species and to over 1,000 mammalian species, fulfilling diverse metabolic requirements in its complex life-cycle. The Tr. cruzi infections are deeply embedded in countless ecotypes, where they are difficult to defeat using the control methods that are currently available. Many more field and laboratory studies are required to obtain data and information that may be used for the control and prevention of Tr. cruzi infections and their various disease manifestations. Emphasis should be placed on those sensitive interactions at cellular and environmental levels that could become selected targets for disease prevention. In the short term, new technologies for social mobilization should be used by people and organizations working for justice and equality through health information and promotion. A mass media directed program could deliver education, information and communication to protect the inhabitants at risk of contracting Tr. cruzi infections.

Uma rede epidemiológica envolvendo o Trypanosoma cruzi foi discutida nos níveis ambientais e de interações moleculares nos hospedeiros que habitam em 19 diferentes ecossistemas. O protozoário tem uma enorme plasticidade controlada geneticamente que confere sua adaptação a cerca de quarenta espécies de triatomíneos e mais de mil espécies de mamíferos. Essas infecções estão profundamente embutidas em inúmeros ecótopos, onde elas estão inacessíveis aos métodos de controle utilizados. Muito mais estudos de campo e de laboratório são necessários à obtenção de dados e informação pertinentes ao controle e prevenção das infecções pelo Tr. cruzi e as várias manifestações da doença. Ênfase deve ser dada àquelas interações que ocorrem nos níveis celulares e ambientais que se poderiam tomar como alvos seletivos para prevenção da doença. Novas tecnologias para mobilização social devem ser disponibilizadas para os que trabalham pela justiça e pela igualdade, mediante informação para a promoção da saúde. Um programa direcionado de educação de massa pode prover informação e comunicação necessárias para proteger os habitantes atualmente expostos ao risco de contrair as infecções pelo Tr. cruzi.

Trypanosoma cruzi alkaline 2-DE: Optimization and application to comparative proteome analysis of flagellate life stages.

BACKGROUND: Trypanosoma cruzi, a flagellate protozoan, is the etiological agent of Chagas disease, a chronic illness that causes irreversible damage to heart and digestive tract in humans. Previous 2-DE analyses of T. cruzi proteome have not focused on basic proteins, possibly because of inherent difficulties for optimizing 2-DE in the alkaline pH range. However, T. cruzi wide pH range 2-DE gels have shown few visible spots in the alkaline region, indicating that the parasite either did not have an appreciable amount of alkaline proteins or that these proteins were underrepresented in the 2-DE gels. RESULTS: Different IEF conditions using 6-11 pH gradient strips were tested for separation of T. cruzi alkaline proteins. The optimized methodology described here was performed using anodic "paper bridge" sample loading supplemented by increased concentration of DTT and Triton X-100 on Multiphor II (GE Healthcare) equipment and an electrode pad embedded in DTT- containing solution near the cathode in order to avoid depletion of reducing agent during IEF. Landmark proteins were identified by peptide mass fingerprinting allowing the production of an epimastigote 2-DE map. Most identified proteins corresponded to metabolic enzymes, especially those related to amino acid metabolism. The optimized 2-DE protocol was applied in combination with the "two-in-one gel" method to verify the relative expression of the identified proteins between samples from epimastigote and trypomastigote life stages. CONCLUSION: High resolution 2-DE gels of T. cruzi life forms were achieved using the optimized methodology and a partial epimastigote alkaline 2-DE map was built. Among 700 protein spots detected, 422 were alkaline with a pI above 7.0. The "two-in-one gel" method simplified the comparative analysis between T. cruzi life stages since it minimized variations in spot migration and silver-stained spot volumes. The comparative data were in agreement with biological traits of T. cruzi life forms and also corroborated previous T. cruzi proteomic studies. For instance, enzymes related to amino acid metabolism and dehydrogenases were more abundant in epimastigote 2-DE gel whilst trans-sialidase and a paraflagellar protein were found specifically in the trypomastigote 2-DE profile.

Exposure to mixed asymptomatic infections with Trypanosoma cruzi, Leishmania braziliensis and Leishmania chagasi in the human population of the greater Amazon.

Lack of conservation of the Amazon tropical rainforest has imposed severe threats to its human population living in newly settled villages, resulting in outbreaks of some infectious diseases. We conducted a seroepidemiological survey of 1100 inhabitants of 15 villages of Paço do Lumiar County, Brazil. Thirty-five (3%) individuals had been exposed to Trypanosoma cruzi (Tc), 41 (4%) to Leishmania braziliensis (Lb) and 50 (4.5%) to Leishmania chagasi (Lc) infections. Also, 35 cases had antibodies that were cross-reactive against the heterologous kinetoplastid antigens. Amongst these, the Western blot assays revealed that 11 (1%) had Tc and Lb, that seven (0.6%) had Lc and Tc, and that 17 (1.6%) had Lb and Lc infections. All of these cases of exposures to mixed infections with Leishmania sp, and eight of 11 cases of Tc and Lb were confirmed by specific PCR assays and Southern hybridizations. Two cases had triple infections. We consider these asymptomatic cases showing phenotype and genotype markers consistent with mixed infections by two or more kinetoplastid flagellates a high risk factor for association with Psychodidae and Triatominae vectors blood feeding and transmitting these protozoa infections. This is the first publication showing human exposure to mixed asymptomatic kinetoplastid infections in the Amazon.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Triatoma infestans chooses to feed upon immune prey.

Blood-feeding Triatoma infestans obtained its fills from immune chickens in 15 min, but it needed 40 min for feeding upon non-immune chickens. High-titer specific IgGs and skin reactivity against T. infestans saliva antigens were elicited in immune chickens. Fluorescence-labeled leukocytes from non-immune or immune chickens were used to determine sources of blood drawn by equal numbers of triatomines distributed in separate compartments of a hut-like box. It was shown that 64.4 +/- 4.7% of the reduviids were captured in the immune chicken room; 35.6 +/- 4.5% were present in the non-immune chicken dwelling, and these differences were statistically significant (P < 0.001). Furthermore, T. infestans feeding upon immune birds reached the adult stage 40 days before those feeding upon non-immune birds, and differences were statistically significant. These results appear to have a broad epidemiologic significance as for spreading enzootics; hence, the immunologic status of vertebrate host populations appears to favor T. infestans as the main transmitter of Trypanosoma cruzi.

Evolution and pathology in chagas disease--a review.

Trypanosoma cruzi acute infections often go unperceived, but one third of chronically infected individuals die of Chagas disease, showing diverse manifestations affecting the heart, intestines, and nervous systems. A common denominator of pathology in Chagas disease is the minimal rejection unit, whereby parasite-free target host cells are destroyed by immune system mononuclear effectors cells infiltrates. Another key feature stemming from T. cruzi infection is the integration of kDNA minicircles into the vertebrate host genome; horizontal transfer of the parasite DNA can undergo vertical transmission to the progeny of mammals and birds. kDNA integration-induced mutations can enter multiple loci in diverse chromosomes, generating new genes, pseudo genes and knock-outs, and resulting in genomic shuffling and remodeling over time. As a result of the juxtaposition of kDNA insertions with host open reading frames, novel chimeric products may be generated. Germ line transmission of kDNA-mutations determined the appearance of lesions in birds that are indistinguishable from those seen in Chagas disease patients. The production of tissue lesions showing typical minimal rejection units in birds' refractory to T. cruzi infection is consistent with the hypothesis that autoimmunity, likely triggered by integration-induced phenotypic alterations, plays a major role in the pathogenesis of Chagas disease.