Leishmania (Leishmania) infantum DNA detection in Nyssomyia neivai in Vale do Ribeira, Paraná, Brazil.

BACKGROUND: The incidence of visceral leishmaniasis (VL) has increased in the Southern region of Brazil in recent years, especially in the State of Paraná. New species have been suggested with potential to act as vector in VL endemic areas. OBJECTIVES: Identify the Leishmania species in sand fly specimens collected from 2016 to 2018 in the municipality of Itaperuçu, Vale do Ribeira, Paraná, Brazil. METHODS: Light traps were used for collections and for the analysis of sand fly were used the multiplex polymerase chain reaction (PCR) methodology and subsequent sequencing. FINDINGS: Among the collected specimens, 88.62% were attributed to the species Nyssomyia neivai, which were grouped into 176 pools. Three positive pools were detected: two with Leishmania (Viannia) braziliensis and one with L. (Leishmania) infantum. The positivity rate for the parasite was 0.25% based on the presence of at least one infected insect in the pool. MAIN CONCLUSIONS: The detection of L. infantum in Ny. neivai draws attention due to its abundance and anthropophily in the State of Paraná. Moreover, this finding is considered as an alert and suggests that the vector competence of Ny. neivai and the criteria for its incrimination should be carried out, given its wide distribution in southern of Brazil.

Characterisation of an area of coexistent visceral and cutaneous leishmaniasis transmission in the State of Piauí, Brazil.

BACKGROUND: In Brazil, transmission of visceral and cutaneous leishmaniasis has expanded geographically over the last decades, with both clinical forms occurring simultaneously in the same area. OBJECTIVES: This study characterised the clinical, spatial, and temporal distribution, and performed entomological surveillance and natural infection analysis of a leishmaniasis-endemic area. METHODS: In order to characterise the risk of leishmaniasis transmission in Altos, Piauí, we described the clinical and socio-demographic variables and the spatial and temporal distribution of cases of American visceral leishmaniasis (AVL) and American cutaneous leishmaniasis (ACL) cases and identified potential phlebotomine vectors. FINDINGS: The urban area concentrated almost 54% of ACL and 86.8% of AVL cases. The temporal and spatial distribution of AVL and ACL cases in Altos show a reduction in the number of risk areas, but the presence of permanent disease transmission foci is observed especially in the urban area. 3,808 phlebotomine specimens were captured, with Lutzomyia longipalpis as the most frequent species (98.45%). Of the 35 females assessed for natural infection, one specimen of Lu. longipalpis tested positive for the presence of Leishmania infantum and Leishmania braziliensis DNA. MAIN CONCLUSION: Our results indicate the presence of risk areas for ACL and AVL in the municipality of Altos and highlight the importance of entomological surveillance to further understand a possible role of Lu. longipalpis in ACL transmission.

hsp70 PCR-RFLP as an alternative tool to identify Sauroleishmania species.

Sauroleishmania spp. comprises one of the four Leishmania subgenera, which has been historically considered a non-pathogenic protozoan of reptiles. However, some strains appear to be transiently infective to mammals, and recent findings have detected these parasites in dogs and humans in areas where leishmaniasis is endemic. Herein, the digestion pattern of PCR-RFLP of the 234 bp-hsp70 fragment was evaluated as a simpler and cheaper tool to distinguish the Sauroleishmania species from the other Leishmania subgenera. As a result, the digestion of the 234 bp-hsp70 fragments with HaeIII produced a banding pattern specific to the four Sauroleishmania strains assessed. This technique could contribute to the identification of Leishmania parasites isolated from sandflies, reptiles, or even mammals in fieldworks as an alternative to the use of laborious and expensive methodologies.

Predictors of Trypanosoma cruzi PCR positivity in patients with chronic Chagas disease.

BACKGROUND: A positive Trypanosoma cruzi polymerase chain reaction (PCR) is associated with a worse prognosis in patients with chronic Chagas disease (CD). OBJECTIVES: To study the association of clinical, electrocardiographic, and echocardiographic characteristics and biomarker blood levels with positive T. cruzi PCR in chronic CD. METHODS: This is a single-centre observational cross-sectional study. Positive T. cruzi PCR association with clinical, electrocardiographic, and echocardiographic characteristics, and biomarker blood levels were studied by logistic regression analysis. p values < 0.05 were considered significant. FINDINGS: Among 333 patients with chronic CD (56.4% men; 62 ± 10 years), T. cruzi PCR was positive in 41.1%. Stepwise multivariate logistic regression showed an independent association between positive T. cruzi PCR and diabetes mellitus {odds ratio (OR) 0.53 [95% confidence interval (CI) 0.30-0.93]; p = 0.03}, right bundle branch block [OR 1.78 (95% CI 1.09-2.89); p = 0.02], and history of trypanocidal treatment [OR 0.13 (95% CI 0.04-0.38); p = 0.0002]. Among patients with a history of trypanocidal treatment (n = 39), only four (10%) patients had a positive T. cruzi PCR. MAIN CONCLUSIONS: Among several studied parameters, only diabetes mellitus, right bundle branch block, and history of trypanocidal treatment showed an independent association with positive T. cruzi PCR. History of trypanocidal treatment was a strong protective factor against a positive T. cruzi PCR.

HRM Accuracy and Limitations as a Species Typing Tool for Leishmania Parasites.

High Resolution Melting Analysis (HRM) has been pointed out as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to parasite load-dependent accuracy. In light of these findings and considering the importance of parasite load determination for clinical and research purposes, we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.

A new Trypanosoma cruzi genotyping method enables high resolution evolutionary analyses.

BACKGROUND: Trypanosoma cruzi is an important human pathogen in Latin America with nearly seven million people infected. It has a large degree of genetic diversity, classified into six discrete typing units (DTUs), which probably influences its physiological behavior and clinical manifestations. Several genotyping methods are available, with distinct performance on easiness, cost, resolution and applicability; no method excels in all parameters. OBJECTIVES AND METHODS: To devise a molecular method for T. cruzi genotyping, based on polymerase chain reaction (PCR) amplification of a single target with multiple copies in the nuclear genome by large scale sequencing. We have applied this method to 29 T. cruzi isolates, comprising all described DTUs. FINDINGS: We were able to classify all samples into sub DTU level with high robustness. Evolutionary relationship between DTUs were ascertained, suggesting that TcIII and TcIV DTUs are non-hybrid, and DTU IV is more similar to the common ancestral. CONCLUSION: As the TS-LSS method is based on a single PCR reaction, comprising several copies of the target, it is probably useful for clinical samples, when the amount of DNA is a limiting factor. As large scale sequencing systems become more common, the TS-LSS method can be increasingly applied for T. cruzi genotyping.

Expression and cellular localisation of Trypanosoma cruzi calpains.

BACKGROUND: Calpains are present in almost all organisms and comprise a family of calcium-dependent cysteine peptidases implicated in crucial cellular functions. Trypanosoma cruzi, the causative agent of Chagas disease, presents an expansion on this gene family with unexplored biological properties. OBJECTIVES: Here, we searched for calpains in the T. cruzi genome, evaluated the mRNA levels, calpain activity and the protein expression and determined the cellular localisation in all three parasite life cycle forms. METHODS/FINDINGS: Sixty-three calpain sequences were identified in T. cruzi CL Brener genome, with fourteen domain arrangements. The comparison of calpain mRNA abundance by quantitative polymerase chain reaction (qPCR) revealed seven up-regulated sequences in amastigotes and/or bloodstream trypomastigotes and five in epimastigotes. Western Blotting analysis revealed seven different molecules in the three parasite forms, and one amastigote-specific, while no proteolytic activity could be detected. Flow cytometry assays revealed a higher amount of intracellular calpains in amastigotes and/or trypomastigotes in comparison to epimastigotes. Finally, ultrastructural analysis revealed the presence of calpains in the cytoplasm, vesicular and plasma membranes of the three parasite forms, and in the paraflagellar rod in trypomastigotes. CONCLUSION: Calpains are differentially expressed and localised in the T. cruzi life cycle forms. This study adds data on the calpain occurrence and expression pattern in T. cruzi.

An overview of the sandfly fauna (Diptera: Psychodidae) followed by the detection of Leishmania DNA and blood meal identification in the state of Acre, Amazonian Brazil.

BACKGROUND: In Acre state, Brazil, the dissemination of cutaneous leishmaniasis has increased in recent years, with limited knowledge of the potential Leishmania spp. vectors involved. OBJECTIVES: Here, data concerning the sandfly fauna of Brasiléia municipality, Leishmania DNA-detection rates and the identification of blood meal sources of insects captured in 2013-2015 are presented. METHODS: Parasite detection in female sandflies was performed individually by multiplex polymerase chain reaction (PCR) (Leishmania kDNA/sandfly cacophony-gene), with the identification of Leishmania spp. by hsp70-PCR and sequencing. The identification of blood gut-content from fed females was performed by cyt b-PCR and sequencing. FINDINGS: A total of 4,473 sandflies were captured. A subgroup of 864 non-blood-fed females evaluated for the presence of Leishmania DNA showed 2.9% positivity for Leishmania (Viannia) braziliensis and L. (V.) guyanensis. The identification of blood meal sources was performed in 96 blood-fed females, allowing the identification of 13 vertebrate species. In nine/96 fed females, DNA from L. (V.) shawi, L. (V.) guyanensis, L. (V.) braziliensis and Endotrypanum sp. was detected. MAIN CONCLUSIONS: In Brumptomyia sp. and Evandromyia termitophila, the first report of Leishmania DNA-detection is provided in Acre; Nyssomyia shawi is implicated as potential vector of L. (V.) braziliensis and L. (V.) guyanensis for the first time in Brazil.

Participation of Trypanosoma cruzi gp63 molecules on the interaction with Rhodnius prolixus.

Trypanosoma cruzi is the causative agent of Chagas disease, a vector-borne disease. The parasite molecules involved in vector interaction have been little investigated. Metallopeptidases and gp63 molecules have been implicated in parasite adhesion of several trypanosomatids to the insect midgut. Although gp63 homologues are highly expanded in the T. cruzi genome, and are implicated in parasite-mammalian host interaction, its role in the insect vector has never been explored. Here, we showed that divalent metal chelators or anti-Tcgp63-I antibodies impaired T. cruzi adhesion to Rhodnius prolixus midgut. Parasites isolated after insect colonization presented a drastic enhancement in the expression of Tcgp63-I. These data highlight, for the first time, that Tcgp63-I and Zn-dependent enzymes contribute to the interaction of T. cruzi with the insect vector.

Impact of levamisole in co-administration with benznidazole on experimental Chagas disease.

Levamisole (Lms) is an anthelminthic drug with immunomodulatory activity. Chagas disease (CD) is caused by Trypanosoma cruzi and there is very low access to the drugs available, benznidazole (Bz) and nifurtimox, both far from ideal. In a drug-repurposing strategy to test potential activity as antiparasitic and immunomodulatory agent for CD, Lms was assayed on acute T. cruzi murine infection, alone and in co-administration with Bz. During protocol standardization, 100 and 10 mpk of Bz given for five consecutive days resulted in parasitaemia suppression and 100% animal survival only with the highest dose. Flow cytometry showed that both optimal (100 mpk) and suboptimal (10 mpk) doses of Bz equally decreased the plasma levels of cytokines commonly elevated in this acute infection model. Lms alone (10-0.5 mpk) did not decrease parasitaemia nor mortality rates. Co-administration was investigated using the suboptimal dose of Bz and different doses of Lms. While Bz 10 mpk did not alter parasitaemia, the combo partially reduced it but only slightly promoted animal survival. This effect could be related to Th1-response modulation since interleukin-6 and interferon-γ were higher after treatment with the combo.

In-Depth Quantitative Proteomic Analysis of Trophozoites and Pseudocysts of Trichomonas vaginalis.

Trichomonas vaginalis is a sexually transmitted anaerobic parasite that infects humans causing trichomoniasis, a common and ubiquitous sexually transmitted disease. The life cycle of this parasite possesses a trophozoite form without a cystic stage. However, the presence of nonproliferative and nonmotile, yet viable and reversible spherical forms with internalized flagella, denominated pseudocysts, has been commonly observed for this parasite. To understand the mechanisms involved in the formation of pseudocysts, we performed a mass spectrometry-based high-throughput quantitative proteomics study using a label-free approach and functional assays by biochemical and flow cytometric methods. We observed that the morphological transformation of trophozoite to pseudocysts is coupled to (i) a metabolic shift toward a less glycolytic phenotype; (ii) alterations in the abundance of hydrogenosomal iron-sulfur cluster (ISC) assembly machinery; (iii) increased abundance of regulatory particles of the ubiquitin-proteasome system; (iv) significant alterations in proteins involved in adhesion and cytoskeleton reorganization; and (v) arrest in G2/M phase associated with alterations in the abundance of regulatory proteins of the cell cycle. These data demonstrate that pseudocysts experience important physiological and structural alterations for survival under unfavorable environmental conditions.

Randomized Trial of Benznidazole for Chronic Chagas' Cardiomyopathy.

BACKGROUND: The role of trypanocidal therapy in patients with established Chagas' cardiomyopathy is unproven. METHODS: We conducted a prospective, multicenter, randomized study involving 2854 patients with Chagas' cardiomyopathy who received benznidazole or placebo for up to 80 days and were followed for a mean of 5.4 years. The primary outcome in the time-to-event analysis was the first event of any of the components of the composite outcome of death, resuscitated cardiac arrest, sustained ventricular tachycardia, insertion of a pacemaker or implantable cardioverter-defibrillator, cardiac transplantation, new heart failure, stroke, or other thromboembolic event. RESULTS: The primary outcome occurred in 394 patients (27.5%) in the benznidazole group and in 414 (29.1%) in the placebo group (hazard ratio, 0.93; 95% confidence interval [CI], 0.81 to 1.07; P=0.31). At baseline, a polymerase-chain-reaction (PCR) assay was performed on blood samples obtained from 1896 patients; 60.5% had positive results for Trypanosoma cruzi on PCR. The rates of conversion to negative PCR results (PCR conversion) were 66.2% in the benznidazole group and 33.5% in the placebo group at the end of treatment, 55.4% and 35.3%, respectively, at 2 years, and 46.7% and 33.1%, respectively, at 5 years or more (P<0.001 for all comparisons). The effect of treatment on PCR conversion varied according to geographic region: in Brazil, the odds ratio for PCR conversion was 3.03 (95% CI, 2.12 to 4.34) at 2 years and 1.87 (95% CI, 1.33 to 2.63) at 5 or more years; in Colombia and El Salvador, the odds ratio was 1.33 (95% CI, 0.90 to 1.98) at 2 years and 0.96 (95% CI, 0.63 to 1.45) at 5 or more years; and in Argentina and Bolivia, the odds ratio was 2.63 (95% CI, 1.89 to 3.66) at 2 years and 2.79 (95% CI, 1.99 to 3.92) at 5 or more years (P<0.001 for interaction). However, the rates of PCR conversion did not correspond to effects on clinical outcome (P=0.16 for interaction). CONCLUSIONS: Trypanocidal therapy with benznidazole in patients with established Chagas' cardiomyopathy significantly reduced serum parasite detection but did not significantly reduce cardiac clinical deterioration through 5 years of follow-up. (Funded by the Population Health Research Institute and others; ClinicalTrials.gov number, NCT00123916; Current Controlled Trials number, ISRCTN13967269.).

First description of Leishmania (Viannia) infection in Evandromyia saulensis, Pressatia sp. and Trichophoromyia auraensis (Psychodidae: Phlebotominae) in a transmission area of cutaneous leishmaniasis in Acre state, Amazon Basin, Brazil.

Studies on the sandfly fauna to evaluate natural infection indexes are still limited in the Brazilian Amazon, a region with an increasing incidence of cutaneous leishmaniasis. Here, by using a multiplex polymerase chain reaction directed to Leishmania kDNA and hybridisation, we were able to identify L. (Viannia) subgenus in 12 out of 173 sandflies captured in the municipality of Rio Branco, Acre state, revealing a positivity of 6.94%. By sequencing the Leishmania 234 bp-hsp70 amplified products from positive samples, infection by L. (V.) braziliensis was confirmed in five sandflies: one Evandromyia saulensis, three Trichophoromyia auraensis and one Pressatia sp. The finding of L. (Viannia) DNA in two Ev. saulensis corresponds to the first record of possible infection associated with this sandfly. Moreover, our study reveals for the first time in Brazil, Th. auraensis and Pressatia sp. infected by L. (Viannia) parasites.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells.

BACKGROUND: Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES: In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS: We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS: It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS: Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

Combination Chemotherapy with Suboptimal Doses of Benznidazole and Pentoxifylline Sustains Partial Reversion of Experimental Chagas' Heart Disease.

Chronic chagasic cardiomyopathy (CCC) progresses with parasite persistence, fibrosis, and electrical alterations associated with an unbalanced immune response such as high plasma levels of tumor necrosis factor (TNF) and nitric oxide (NO). Presently, the available treatments only mitigate the symptoms of CCC. To improve CCC prognosis, we interfered with the parasite load and unbalanced immune response using the trypanocidal drug benznidazole (Bz) and the immunoregulator pentoxifylline (PTX). C57BL/6 mice chronically infected with the Colombian strain of Trypanosoma cruzi and with signs of CCC were treated for 30 days with a suboptimal dose of Bz (25 mg/kg of body weight), PTX (20 mg/kg), or their combination (Bz plus PTX) and analyzed for electrocardiographic, histopathological, and immunological changes. Bz (76%) and Bz-plus-PTX (79%) therapies decreased parasite loads. Although the three therapies reduced myocarditis and fibrosis and ameliorated electrical alterations, only Bz plus PTX restored normal heart rate-corrected QT (QTc) intervals. Bz-plus-PTX-treated mice presented complementary effects of Bz and PTX, which reduced TNF expression (37%) in heart tissue and restored normal TNF receptor 1 expression on CD8(+) T cells, respectively. Bz (85%) and PTX (70%) therapies reduced the expression of inducible nitric oxide synthase (iNOS/NOS2) in heart tissue, but only Bz (58%) reduced NO levels in serum. These effects were more pronounced after Bz-plus-PTX therapy. Moreover, 30 to 50 days after treatment cessation, reductions of the prolonged QTc and QRS intervals were sustained in Bz-plus-PTX-treated mice. Our findings support the importance of interfering with the etiological agent and immunological abnormalities to improve CCC prognosis, opening an opportunity for a better quality of life for Chagas' disease (CD) patients.

Iron-modulated pseudocyst formation in Tritrichomonas foetus.

Iron is an essential element for the survival of trichomonads during host-parasite interaction. The availability of this metal modulates several metabolic pathways of the parasites and regulates the expression of virulence factors such as adhesins and proteolytic enzymes. In this study, we investigated the effect of iron depletion on the morphology and life cycle of Tritrichomonas foetus. Scanning and transmission electron microscopy analyses revealed that depletion of iron from the culture medium (named TYM-DIP inducer medium) induces morphological transformation of typical pear-shaped trophozoites into spherical and non-motile pseudocysts. Remarkably, inoculation of pseudocysts into an iron-rich medium (standard TYM medium), or addition of FeSO4 to a TYM-DIP inducer medium reverted the morphological transformation process and typical trophozoites were recovered. These results show that pseudocysts are viable forms of the parasite and highlight the role of iron as a modulator of the parasite phenotype. Although iron is required for the survival of T. foetus, iron depletion does not cause a cellular collapse of pseudocysts, but instead induces phenotypic alterations, probably in order to allow the parasite to survive conditions of nutritional stress. Together, these findings support previous studies that suggest pseudocysts are a resistance form in the life cycle of T. foetus and enable new approaches to understanding the multifactorial role of iron in the cell biology of this protozoan parasite.

Proteomics reveals major components of oogenesis in the reproductive tract of sugar-fed Anopheles aquasalis.

Anopheles (Nyssorhynchus) aquasalis is a malaria vector mainly distributed along the coastal regions of South and Central America. In the absence of an effective vaccine against malaria, strategies for controlling the vector are the main tool for interrupting parasite transmission. Mechanisms of oogenesis and embryogenesis in anautogenous mosquitoes are mainly modulated by blood feeding. However, the expression, at the protein level, of genes involved in such mechanisms in sugar-fed females is unknown. In this work, total protein extracts of the reproductive tract of female An. aquasalis that were fed sugar were analyzed using liquid chromatography followed by mass spectrometry for protein identification and bioinformatic tools for data mining. We identified 922 proteins expressed in the organ, and using several databases, we attributed biological meaning for several of them. Remarkably, nine proteins involved in oogenesis were identified in females fed sugar. Putative vitellogenins, vitellogenin receptor, lipid storage droplet, transferrin, ferritin, and apolipoprotein, identified here, are proteins involved in egg development. Proteins involved in embryonic development, such as paxillin, exuperantia, several growth factors, and dorsal switch protein, were identified. Interestingly, in this study, we identified 15 peptidases of various classes such as aminopeptidases, carboxypeptidases, serine protease, cathepsin, and metalloprotease that could potentially interact with male seminal components. Here, we demonstrated that the reproductive tract of female An. aquasalis fed on sugar expresses proteins involved in oogenesis and embryonic development. These findings reveal unknown aspects of the physiology of this organ under the given nutritional conditions.

Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains.

We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30min of incubation in lysis buffer at 4°C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS(E) approach. ProteinLynx Global Server (version 2.5) with Expression(E) analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.

Tumor necrosis factor is a therapeutic target for immunological unbalance and cardiac abnormalities in chronic experimental Chagas' heart disease.

BACKGROUND: Chagas disease (CD) is characterized by parasite persistence and immunological unbalance favoring systemic inflammatory profile. Chronic chagasic cardiomyopathy, the main manifestation of CD, occurs in a TNF-enriched milieu and frequently progresses to heart failure. AIM OF THE STUDY: To challenge the hypothesis that TNF plays a key role in Trypanosoma cruzi-induced immune deregulation and cardiac abnormalities, we tested the effect of the anti-TNF antibody Infliximab in chronically T. cruzi-infected C57BL/6 mice, a model with immunological, electrical, and histopathological abnormalities resembling Chagas' heart disease. RESULTS: Infliximab therapy did not reactivate parasite but reshaped the immune response as reduced TNF mRNA expression in the cardiac tissue and plasma TNF and IFNγ levels; diminished the frequency of IL-17A(+) but increased IL-10(+) CD4(+) T-cells; reduced TNF(+) but augmented IL-10(+) Ly6C(+) and F4/80(+) cells. Further, anti-TNF therapy decreased cytotoxic activity but preserved IFNγ-producing VNHRFTLV-specific CD8(+) T-cells in spleen and reduced the number of perforin(+) cells infiltrating the myocardium. Importantly, Infliximab reduced the frequency of mice afflicted by arrhythmias and second degree atrioventricular blocks and decreased fibronectin deposition in the cardiac tissue. CONCLUSIONS: Our data support that TNF is a crucial player in the pathogenesis of Chagas' heart disease fueling immunological unbalance which contributes to cardiac abnormalities.