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Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, is a vector-borne zoonotic parasite which has been little studied regarding its infection in domestic animals. In this study, we evaluated the occurrence of natural infection by T. cruzi in farm animals using molecular markers and phylogenetic analysis in blood clot samples of 60 sheep (Ovis aires), 22 goats (Capra hircus), and 14 horses (Equus caballus) in eight municipalities located in an infection risk area in the state of Rio Grande do Norte (RN), Northeast Region of Brazil. Trypanosoma spp. infection was identified by amplifying the rRNA 18S SSU gene in 48.9% of the samples. The SH022 sample showed 99.8% similarity with the Y strain of T. cruzi in phylogeny, grouped in the DTU II clade. Blood clots of sheep, goats, and horses detected T. cruzi kDNA in 28.3% (17/60), 22.7% (5/22), and 15.4% (2/14) of the samples, respectively. These animals were distributed in the three studied mesoregions throughout the state of RN. The identification of natural infection in domestic animals contributes to expand the epidemiological transmission scenario in an area where T. brasiliensis is the main vector., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Components of the antioxidant defense system in Trypanosoma cruzi are potential targets for new drug development. Superoxide dismutases (SODs) constitute key components of antioxidant defense systems, removing excess superoxide anions by converting them into oxygen and hydrogen peroxide. The main goal of the present study was to investigate the genes coding for iron superoxide dismutase (FeSOD) in T. cruzi strains from an evolutionary perspective., Methods: In this study, molecular biology methods and phylogenetic studies were combined with drug assays. The FeSOD-A and FeSOD-B genes of 35 T. cruzi strains, belonging to six discrete typing units (Tcl-TcVI), from different hosts and geographical regions were amplified by PCR and sequenced using the Sanger method. Evolutionary trees were reconstructed based on Bayesian inference and maximum likelihood methods. Drugs that potentially interacted with T. cruzi FeSODs were identified and tested against the parasites., Results: Our results suggest that T. cruzi FeSOD types are members of distinct families. Gene copies of FeSOD-A (n = 2), FeSOD-B (n = 4) and FeSOD-C (n = 4) were identified in the genome of the T. cruzi reference clone CL Brener. Phylogenetic inference supported the presence of two functional variants of each FeSOD type across the T. cruzi strains. Phylogenetic trees revealed a monophyletic group of FeSOD genes of T. cruzi TcIV strains in both distinct genes. Altogether, our results support the hypothesis that gene duplication followed by divergence shaped the evolution of T. cruzi FeSODs. Two drugs, mangafodipir and polaprezinc, that potentially interact with T. cruzi FeSODs were identified and tested in vitro against amastigotes and trypomastigotes: mangafodipir had a low trypanocidal effect and polaprezinc was inactive., Conclusions: Our study contributes to a better understanding of the molecular biodiversity of T. cruzi FeSODs. Herein we provide a successful approach to the study of gene/protein families as potential drug targets., (© 2022. The Author(s).)

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qβ-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue., Competing Interests: The authors have declared that no competing interests exist. Author Professor Egler Chiari was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

Vector transmission of Trypanosoma cruzi occurs in several areas of Brazil, including the northeastern region, and domestic animals can serve as reservoirs of the parasite. The aim of this study was to monitor dogs as domestic reservoirs for infection by T. cruzi, and the main triatomine species involved in parasite transmission in rural areas of municipalities in the State of Rio Grande do Norte, in northeastern, Brazil. Blood samples from dogs (n = 40) and manual triatomine capture were performed in domiciliary and peridomiciliary environments in rural areas of the towns of Acari, Caraúbas and Marcelino Vieira, between 2013 and 2016. Subsequently, infection of dogs was determined by Polymerase Chain Reaction (PCR), Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of IgM and IgG isotypes and Indirect Immunofluorescence (IIF) reactions for detection of IgG. Triatomine infection was determined by PCR. Forty (16/40) percent of the dogs were seropositive for T. cruzi; 20.0% (8/40) of such reactivity indicated the acute phase, and 20.0% (8/40), the chronic phase. PCR was positive in 42.5% (17/40) of the dogs' blood samples. Specimens of Triatoma brasiliensis, Triatoma pseudomaculata, Rhodnius nasutus and Panstrongylus lutzi were found to be infected; however only T. brasiliensis nymphs and adults were infected in both environments. Triatomines evaluation showed 82.5% (94/114) of PCR positivity. Taken together, our results confirm that dogs are domestic reservoirs of T. cruzi in northeastern Brazil and T. brasiliensis is the main triatomine species correlated with parasite transmission in domiciliary environments. There is a continuing need to control peridomiciliary populations of triatomines and to implement continuous surveillance strategies for reservoirs with the help from the community., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Herein we report a fatal case of donor-derived transmission of XDR-resistant carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) in cardiac transplantation. A 59-year-old male patient with non-obstructive hypertrophic cardiomyopathy underwent heart transplantation. On day 5 post-operation, blood cultures from the donor were positive for colistin-resistant carbapenemase-producing K. pneumoniae (ColR KPC-Kp) susceptible only to amikacin. Recipient blood cultures were also positive for ColR KPC-Kp with the same sensitivity profile as the donor isolate with an identical PFGE pattern. The patient was treated with double-carbapenems and amikacin. The patient evolved to pericarditis, osteomyelitis, and pulmonary necrosis, all fragment cultures positive for the same agent. The patient developed septic shock, multiple organ failure and died on day 50 post-transplantation. Based on current microbiological scenario worldwide the possibility of transmitting multidrug resistant (MDR) organisms should be considered., (Copyright © 2018 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.)

The α-Gal antigen [Galα(1,3)Galβ(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.

Chagas disease is a neglected chronic condition with a high burden of morbidity and mortality. It has considerable psychological, social, and economic impacts. The disease represents a significant public health issue in Brazil, with different regional patterns. This document presents the evidence that resulted in the Brazilian Consensus on Chagas Disease. The objective was to review and standardize strategies for diagnosis, treatment, prevention, and control of Chagas disease in the country, based on the available scientific evidence. The consensus is based on the articulation and strategic contribution of renowned Brazilian experts with knowledge and experience on various aspects of the disease. It is the result of a close collaboration between the Brazilian Society of Tropical Medicine and the Ministry of Health. It is hoped that this document will strengthen the development of integrated actions against Chagas disease in the country, focusing on epidemiology, management, comprehensive care (including families and communities), communication, information, education, and research .

Chagas disease is a neglected chronic condition that presents high morbidity and mortality burden, with considerable psychological, social, and economic impact. The disease represents a significant public health issue in Brazil, with different regional patterns. This document presents the evidence that resulted in the Brazilian Consensus on Chagas Disease. The objective was to review and standardize strategies for diagnosis, treatment, prevention, and control of Chagas disease in the country, based on the available scientific evidence. The consensus is based on collaboration and contribution of renowned Brazilian experts with vast knowledge and experience on various aspects of the disease. It is the result of close collaboration between the Brazilian Society of Tropical Medicine and the Ministry of Health. This document shall strengthen the development of integrated control measures against Chagas disease in the country, focusing on epidemiology, management, comprehensive care (including families and communities), communication, information, education, and research.

Ischemic strokes have been implicated as a cause of death in Chagas disease patients. Inflammation has been recognized as a key component in all ischemic processes, including the intravascular events triggered by vessel interruption, brain damage and repair. In this study, we evaluated the association between inflammatory markers and the death risk (DR) and stroke risk (SR) of patients with different clinical forms of chronic Chagas disease. The mRNA expression levels of cytokines, transcription factors expressed in the adaptive immune response (Th1, Th2, Th9, Th17, Th22 and regulatory T cell), and iNOS were analyzed by real-time PCR in peripheral blood mononuclear cells of chagasic patients who exhibited the indeterminate, cardiac, digestive and cardiodigestive clinical forms of the disease, and the levels of these transcripts were correlated with the DR and SR. Cardiac patients exhibited lower mRNA expression levels of GATA-3, FoxP3, AHR, IL-4, IL-9, IL-10 and IL-22 but exhibited higher expression of IFN-γ and TNF-α compared with indeterminate patients. Digestive patients showed similar levels of GATA-3, IL-4 and IL-10 than indeterminate patients. Cardiodigestive patients exhibited higher levels of TNF-α compared with indeterminate and digestive patients. Furthermore, we demonstrated that patients with high DR and SR exhibited lower GATA-3, FoxP3, and IL-10 expression and higher IFN-γ, TNF-α and iNOS mRNA expression than patients with low DR and SR. A negative correlation was observed between Foxp3 and IL-10 mRNA expression and the DR and SR. Moreover, TNF-α and iNOS expression was positively correlated with DR and SR. Our data suggest that an inflammatory imbalance in chronic Chagas disease patients is associated with a high DR and SR. This study provides a better understanding of the stroke pathobiology in the general population and might aid the development of therapeutic strategies for controlling the morbidity and mortality of Chagas disease.

INTRODUCTION Natural and artificial ecotope infestation by the kissing bug triatomines and their colonization and infection by Trypanosoma cruzi , the Chagas disease agent, were evaluated in nine municipalities of the State of Rio Grande do Norte, Brazil. METHODS Following identification, triatomine intestinal contents were analyzed by direct microscopic examination, xenoculture, and polymerase chain reaction (PCR) for parasite detection. Trypanosoma cruzi isolates were genotyped using three different markers. RESULTS Of 842 triatomines captured, 65% were Triatoma brasiliensis , 17.8% Triatoma pseudomaculata , 12.5% Panstrongylus lutzi , and 4.7% Rhodnius nasutus . Triatoma brasiliensis and P. lutzi adults were found in the intradomicile. T. brasiliensis, T. pseudomaculata , and R. nasutus nymphs and adults were found in the peridomicile and wild environment. Intradomiciliary and peridomiciliary infestation indexes were 5.6% and 33.7%, respectively. In the peridomicile, chicken coops were the most infested ecotope. The T. cruzi triatomine infection rate was 30.2%, of which PCR detected 29%. P . lutzi (78.1%), T . brasiliensis (24.5%), and T . pseudomaculata (22.7%) were the most infected species. TcII and III genotypes were detected in T. brasiliensis and TcIII in P. lutzi . CONCLUSIONS T. brasiliensis was found in all environments and most ecotopes with high T. cruzi infection rates. High infection rates were also detected in T . pseudomaculata and P. lutzi , suggesting their role in the interchange between the wild and peridomestic transmission cycles. The combination of PCR, microscopic examination, and xenoculture contributed to improving T. cruzi infection evaluation in triatomine bugs. The TcII and TcIII genotypes were predominant in the study area.

Dogs are the primary urban reservoir of Leishmania infantum and play a crucial role in the transmission of this parasite to man via sandflies. The spleen and liver are the main target organs of L. infantum infection, but few studies have evaluated the immune response to this infection in the canine liver. To identify the immunological mediators involved in resistance and/or susceptibility to canine visceral leishmaniosis (CVL), we selected 21 dogs naturally infected by L. infantum and classified as asymptomatic or symptomatic. Immunological parameters were analysed and correlations with clinical signs were determined. Symptomatic dogs showed higher numbers of parasites and less leucocyte infiltration in the liver compared with asymptomatic dogs. The progression of this disease was characterized not only by the down regulation of T helper (Th) 1-related cytokines, such as interferon (IFN)-γ and tumour necrosis factor (TNF)-α, but also by the down regulation of genes encoding interleukin (IL)-17A, inducible nitric oxide synthase (iNOS) and IL-10 in the spleen and liver in symptomatic dogs compared with asymptomatic dogs. Importantly, IL-17A gene transcription level was positively correlated with mRNA expression for iNOS and IFN-γ. Th1- and Th17-related cytokines therefore appear to play a role in restricting parasite growth via iNOS activation and decrease susceptibility of dogs to CVL., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: This study evaluated the clinical forms and manifestation severities of Chagas disease among serologically reactive individuals from Western Rio Grande do Norte (Northeastern Brazil)., Methods: This cross-sectional study included 186 adults who were evaluated using electrocardiography, echocardiography, chest radiography, and contrast radiography of the esophagus and colon. A clinical-epidemiological questionnaire was also used., Results: The indeterminate, cardiac, digestive, and cardiodigestive clinical forms of Chagas disease were diagnosed in 51.6% (96/186), 32.2% (60/186), 8.1% (15/186) and 8.1% (15/186) of the participants, respectively. Heart failure (functional classes I-IV) was detected in 7.5% (14/186) of the participants, and 36.4% (24/66), 30.3% (20/66), 15.2% (10/66), 13.6% (9/66), and 4.5% (3/66) of the patients were at stage A, B1, B2, C, and D, respectively. Dilated cardiomyopathy and electrocardiographic changes were detected in 10.2% (19/186) and 48.1% (91/186) of the participants, respectively. Apical aneurysm was diagnosed in 10.8% (20/186) of the participants, and other changes in the segmental myocardial contractility of the left ventricle were diagnosed in 33.9% (63/186) of the participants. Megaesophagus (groups I-IV) was observed in 7% (13/186) of the participants, megacolon (grades 1-3) was detected in 12.9% (24/186) of the participants, and both organs were affected in 29.2% (7/24) of the megacolon cases., Conclusions: We detected various clinical forms of Chagas disease (including the digestive form). Our findings indicate that clinical symptoms alone may not be sufficient to exclude or confirm cardiac and/or digestive damage, and the number of patients with symptomatic clinical forms may be underestimated.

Objective: Trypanosoma cruzi is subdivided into six discrete typing units (DTUs), TcI-TcVI. The precise identification of each can contribute to tracking wild DTUs that invade the domiciliary environment., Methods: Twenty T. cruzi stocks isolated from 16 chagasic patients, two Panstrongylus lutzi, one Galea spixii, and one Euphractus sexcinctus, from different localities in the State of Rio Grande do Norte, Brazil, were characterized by genotyping the 3' region of the 24Sα rRNA gene, the mitochondrial cytochrome oxidase subunit 2 gene, and the spliced leader intergenic region., Results: TcIII was identified in 18.7% (3/16) of patients from different municipalities, as well as in P. lutzi, G. spixii, and E. sexcinctus, indicating the connection between the sylvatic and domestic cycles in this Brazilian semi-arid region. TcI and TcII were also detected, in 37.5% (6/16) and 43.8% (7/16) of patients, respectively. These DTUs were associated with cardiac, digestive, and indeterminate clinical forms, while TcIII was identified only in patients with the indeterminate form., Conclusions: The occurrence of these DTUs reveals important phylogenetic diversity in T. cruzi isolates from humans. TcIII is reported for the first time in northeastern Brazil. These findings appear to indicate an overlap between the sylvatic and domestic transmission cycles of the parasite in this region., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Trypanosoma cruzi strains are currently classified into six discrete typing units (DTUs) named TcI to VI. It is known that these DTUs have different geographical distribution, as well as biological features. TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively. Our hypothesis is that upon infection of human peripheral blood cells, Y strain (Tc II) and Col cl1.7 (Tc I), cause distinct immunological changes, which might influence the clinical course of Chagas disease., Methodology/principal Findings: We evaluated the infectivity of CFSE-stained trypomastigotes of Col cl1.7 and Y strain in human monocytes for 15 and 72 hours, and determined the immunological profile of lymphocytes and monocytes exposed to the different isolates using multiparameter flow cytometry. Our results showed a similar percentage and intensity of monocyte infection by Y and Col cl1.7. We also observed an increased expression of CD80 and CD86 by monocytes infected with Col cl1.7, but not Y strain. IL-10 was significantly higher in monocytes infected with Col cl1.7, as compared to Y strain. Moreover, infection with Col cl1.7, but not Y strain, led to an increased expression of IL-17 by CD8+ T cells. On the other hand, we observed a positive correlation between the expression of TNF-alpha and granzyme A only after infection with Y strain., Conclusion/significance: Our study shows that while Col cl1.7 induces higher monocyte activation and, at the same time, production of IL-10, infection with Y strain leads to a lower monocyte activation but higher inflammatory profile. These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression.

The protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that afflicts approximately 8 million people in Latin America. Diagnosis of chronic Chagas disease is currently based on serological tests because this condition is usually characterized by high anti-T. cruzi IgG titers and low parasitemia. The antigens used in these assays may have low specificity due to cross reactivity with antigens from related parasite infections, such as leishmaniasis, and low sensitivity caused by the high polymorphism among T. cruzi strains. Therefore, the identification of new T. cruzi-specific antigens that are conserved among the various parasite discrete typing units (DTUs) is still required. In the present study, we have explored the hybrid nature of the T. cruzi CL Brener strain using a broad genome screening approach to select new T. cruzi antigens that are conserved among the different parasite DTUs and that are absent in other trypanosomatid species. Peptide arrays containing the conserved antigens with the highest epitope prediction scores were synthesized, and the reactivity of the peptides were tested by immunoblot using sera from C57BL/6 mice chronically infected with T. cruzi strains from the TcI, TcII or TcVI DTU. The two T. cruzi proteins that contained the most promising peptides were expressed as recombinant proteins and tested in ELISA experiments with sera from chagasic patients with distinct clinical manifestations: those infected with T. cruzi from different DTUs and those with cutaneous or visceral leishmaniasis. These proteins, named rTc_11623.20 and rTc_N_10421.310, exhibited 94.83 and 89.66% sensitivity, 98.2 and 94.6% specificity, respectively, and a pool of these 2 proteins exhibited 96.55% sensitivity and 98.18% specificity. This work led to the identification of two new antigens with great potential application in the diagnosis of chronic Chagas disease.

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagas disease and the proximity of parasitised glial cells and neurons in damaged myenteric ganglia is a frequent finding. Glial cells have crucial roles in many neuropathological situations and are potential sources of NO. Here, we investigate peripheral glial cell response to Trypanosoma cruzi infection to clarify the role of these cells in the neuronal lesion pathogenesis of Chagas disease. We used primary glial cell cultures from superior cervical ganglion to investigate cell activation and NO production after T. cruzi infection or lipopolysaccharide (LPS) exposure in comparison to peritoneal macrophages. T. cruzi infection was greater in glial cells, despite similar levels of NO production in both cell types. Glial cells responded similarly to T. cruzi and LPS, but were less responsive to LPS than macrophages were. Our observations contribute to the understanding of Chagas disease pathogenesis, as based on the high susceptibility of autonomic glial cells to T. cruzi infection with subsequent NO production. Moreover, our findings will facilitate future research into the immune responses and activation mechanisms of peripheral glial cells, which are important for understanding the paradoxical responses of this cell type in neuronal lesions and neuroprotection.

Gold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Due to the scarcity of evidence of sexuality in Trypanosoma cruzi, the causative agent of Chagas disease, it has been general accepted that the parasite reproduction is essentially clonal with infrequent genetic recombination. This assumption is mainly supported by indirect evidence, such as Hardy-Weinberg imbalances, linkage disequilibrium and a strong correlation between independent sets of genetic markers of T. cruzi populations. However, because the analyzed populations are usually isolated from different geographic regions, the possibility of population substructuring as generating these genetic marker imbalances cannot be eliminated. To investigate this possibility, we firstly compared the allele frequencies and haplotype networks using seven different polymorphic loci (two from mitochondrial and five from different nuclear chromosomes) in two groups of TcII strains: one including isolates obtained from different regions in Latin America and the other including isolates obtained only from patients of the Minas Gerais State in Brazil. Our hypothesis was that if the population structure is essentially clonal, Hardy-Weinberg disequilibrium and a sharp association between the clusters generated by analyzing independent markers should be observed in both strain groups, independent of the geographic origin of the samples. The results demonstrated that the number of microsatellite loci in linkage disequilibrium decreased from 4 to 1 when only strains from Minas Gerais were analyzed. Moreover, we did not observed any correlation between the clusters when analyzing the nuclear and mitochondrial loci, suggesting independent inheritance of these markers among the Minas Gerais strains. Besides, using a second subset of five physically linked microsatellite loci and the Minas Gerais strains, we could also demonstrate evidence of homologous recombination roughly proportional to the relative distance among them. Taken together, our results do not support a clonal population structure for T. cruzi, particularly in TcII, which coexists in the same geographical area, suggesting that genetic exchanges among these strains may occur more frequently than initially expected., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA., Methodology: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain., Findings and Conclusions: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

Objective: To evaluate the potential involvement of anti-Trypanosoma cruzi and cardiac protein antibody (IgG total and isotypes) production and their possible association with different clinical forms of human chronic Chagas disease., Methods: IgG total and isotypes were measured by ELISA, using epimastigote and trypomastigote forms of T. cruzi as antigens and human cardiac proteins (myosin and troponin T) in sera of patients with indeterminate (IND, n = 72), cardiac (CARD, n = 47) and digestive/cardiodigestive (DIG/CARD-DIG, n = 12) clinical forms of the disease. Samples from uninfected health individuals (CONT, n = 30) and patients with ischaemic cardiomyopathy (ISCH, n = 15) were used as controls. Autoantibody levels were correlated with parameters of cardiac function obtained by electrocardiographic, radiographic and echocardiographic examinations., Results: Fifty five per cent of patients were classified as IND, 35.9% as CARD and 9.1% as DIG/CARD-DIG. Greater total IgG production was observed in IND, CARD and DIG/CARD-DIG chagasic patients than in CONT and ISCH, using trypomastigote, epimastigote and cardiac antigens. Moreover, patients with CARD and DIG/CARD-DIG presented greater total IgG production (trypomastigote and epimastigote antigen) than IND, and a negative correlation was determined between total IgG and left ventricular ejection fraction (LVEF). Patients with IND and CARD presented similar higher levels of total IgG specific to troponin T and myosin than CONT and ISCH individuals. Patients with chronic Chagas disease presented a negative correlation between left ventricular ejection fraction (LVEF) and the production of anti-myosin and troponin T autoantibodies. When grouped as low and high antibody producers and compared with LVEF, we observed that high anti-troponin T (P = 0.042) and myosin (P = 0.013) producers presented lower LVEF than low producers. Moreover, there was a positive correlation (r = 0.9508, P = 0.0001) between the production of troponin T and myosin autoantibodies., Conclusion: These findings indicate that increased production of anti-cardiac troponin T and myosin autoantibodies probably influences the left ventricular ejection fraction and could be related to chagasic cardiomyopathy., (© 2013 John Wiley & Sons Ltd.)

Dogs are the primary reservoir for Leishmania parasites. The immune response induced by Leishmania infantum infection in these animals has not been completely elucidated, and few studies have investigated the relationship between the expression levels of chemokines and chemokine receptors and the clinical status of dogs with canine visceral leishmaniasis (CVL). The aim of this study was to correlate the clinical status of naturally L. infantum-infected dogs (from rural areas of Mossoró city, State of Rio Grande do Norte, Brazil) with the expression levels of chemokines (ccl1, ccl2, ccl3, ccl4, ccl5, ccl17, ccl20, ccl24, ccl26, cxcl9, cxcl10) and chemokine receptors (cxcr3, ccr3, ccr4, ccr5, ccr6, ccr8) in the liver and spleen determined using real-time PCR. Twenty-one dogs were clinically evaluated and classified as asymptomatic (n=11) or symptomatic (n=10). Splenomegaly, weight loss and onychogryphosis were the most pronounced symptoms. In the liver, the mRNA expression levels of ccl1, ccl17, ccl26, ccr3, ccr4, ccr5, ccr6, and ccr8 were lower in symptomatic animals than in asymptomatic animals. Compared with uninfected animals, symptomatic dogs had lower expression levels of almost all molecules analyzed. Moreover, high clinical scores were negatively correlated with ccr5 and ccr6 expression and positively correlated with cxcl10 expression. We conclude that the impairment of the expression of chemokines and chemokine receptors results in deficient leukocyte migration and hampers the immune response, leading to the development of disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The genetic variability of 24 Trypanosoma cruzi isolates from humans (11) and triatomines (13) in northeastern Brazil was analyzed by random amplified polymorphic DNA (RAPD) and compared with taxonomic groups, host, and geographical origin of the parasite. TcI (12.5%), TcII (45.8%), and TcIII (41.6%) showed a similarity coefficient (SC) of 0.74 using the mean of three primers and 0.80, 0.75, and 0.66 for λgt11-F, M13-40F, and L15996 primers, respectively. The samples were clustered according to their phylogenetic origin in two polymorphic and divergent branches: one associated with TcI and the other with two subbranches corresponding to TcII and TcIII. TcI was only identified in humans and correlated with the Id homogenous group (0.80 SC). TcII from humans and Triatoma brasiliensis showed 0.86 SC and was clustered according monoclonal or polyclonal populations with similar RAPD profiles detected among the vector and/or humans in different municipalities. TcIII was isolated exclusively in sylvatic cycles from T. brasiliensis and Panstrongylus lutzi and showed low variability (0.84 SC) and high homology mainly among isolated populations at the same locality. The homology of T. cruzi among different hosts and locations suggests the distribution of principal clones circulating and reveals an overlapping between the sylvatic and domestic cycles in this area, where T. brasiliensis infected with TcII acts as link in both environments. This species is important to maintain TcII and TcIII in wild cycles and deserves particular attention due an emergent risk of these populations being introduced into the domestic cycle; moreover, its clinical and epidemiological implications remain unknown.

The genetic profile of Trypanosoma cruzi was evaluated in parasite populations isolated from Beagle dogs experimentally infected with Be-78 and Y strains that present distinct biological and genetic characteristics. Molecular characterization of the isolates obtained 30days and 2years after infection was carried out. For typing MLEE, sequence polymorphisms of the mitochondrial cytochrome oxidase subunit II gene (COII) and RAPD profiles were used. The profiles of MLEE were the same for the parental Be-78 strains as their respective isolates. However, changes of MLEE profile were observed in two T. cruzi isolates from dogs inoculated with Y strain. Changes in the mitochondrial DNA (COII) and RAPD profiles of the Y strain were also observed. The dendogram constructed by UPGMA with RAPD results indicated two major branches. Global data show that the genetic modulation in polyclonal strains during the long-term infection occurred and was strain-dependent. This study still suggests that each host (here each dog) harbors a determinate T. cruzi population that may change or be modulated throughout long-term infection. This might to hinder the observation of correlation between the genetics of T. cruzi and their biological properties and behavior in different host species due to the complexity of the parasite-host interaction in which probably the genetic background of both should be considered., (Published by Elsevier B.V.)

Introduction: A seroepidemiological survey was carried out to evaluate Trypanosoma cruzi infection in an endemic area of the State of Rio Grande do Norte, Brazil, involving rural residents., Methods: Sixteen municipalities were randomly selected, 15 from the west mesoregion and one from the central, with an estimated population of 83,852 individuals. A total of 1,950 blood samples were collected in the west mesoregion and 390 in Caicó. Anti-T. cruzi antibodies were detected using the Chagatest® ELISA HAI-hemagglutination kits and indirect immunofluorescence. As sera presented indeterminate results, TESAcruzi® western blot was performed to confirm reactivity., Results: An estimated seroprevalence of 6.5% was determined for the west mesoregion and 3.3% for Caicó. Seropositivity rises progressively with the age of individuals, up to 40 years in Caicó and up to 50 years in the west mesoregion. Only educational level and knowledge regarding the triatomine were associated with seropositivity. No seroreactive individuals under 18 years of age were identified., Conclusions: Infection by T. cruzi remains high and is concentrated in municipalities in the central western area of the west mesoregion; however, evidence suggests a decline in vector transmission in this mesoregion and in Caicó. Epidemiological variables appear not to influence seropositivity, with the exception of education and knowledge concerning the triatomine, among seroreactive individuals from the west mesoregion.

To confirm that Beagle dogs are a good experimental model for Chagas disease, we evaluated hematological alterations during the acute and chronic phases in Beagle dogs infected with the Y, Berenice-78 (Be-78) and ABC strains of Trypanosomacruzi, correlating clinical signs with the parasitemia curve. We demonstrate that the acute phase of infection was marked by lethargy and loss of appetite. Simultaneously, we observed anemia, leukocytosis and lymphocytosis. Also,we describe hematological alterations and clinical signs that were positively correlated with the parasitemia during the experimental infection with the three strains of T.cruzi, and demonstrate that experimental infection of Beagle is a trustworthy model for Chagas disease.

Chemokines and chemokine receptors interaction have presented important role in leukocyte migration to specific immune reaction sites. Recently, it has been reported that chemokine receptors CXC (CXCR3) and CC (CCR5) were preferentially expressed on Th1 cells while CCR3 and CCR4 were preferentially expressed on Th2 cells. This study evaluated the mRNA expression of type 1 and type 2 chemokine and chemokine receptors in the cardiac tissue of Beagle dogs infected with distinct genetic groups of Trypanosoma cruzi (Y, Berenice-78 and ABC strains) during acute and chronic phases. To analyze the correlation between chemokine and chemokine receptors expression and the development of heart pathology, the chronic infected animals were divided into groups, according to the parasite strain and based on the degree of heart damage: cardiac and indeterminate form of Chagas disease. Our results indicated that cardiac type1/2 chemokines and their receptors were partially dependent on the genetic diversity of parasites as well as the polarization of clinical forms. Also, dogs presenting cardiac form showed lower heart tissue mRNA expression of CCL24 (type 2) and higher expression of CCL5, CCL4 and CXCR3 (type 1) when compared with those with indeterminate form of disease. Together, these data reinforce a close-relation between T. cruzi genetic population and the host specific type 1 immune response and, for the first time, we show the distribution of type 1/2 chemokines associated with the development of cardiac pathology using dogs, a well similar model to study human Chagas disease., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC₅₀ to BZ between 7.6-32 µM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC₅₀ of the strains varied from 15.6 ± 3-51.4 ± 1 µM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.

We have previously demonstrated selection favoring the JG strain of Trypanosoma cruzi in hearts of BALB/c mice that were chronically infected with an equal mixture of the monoclonal JG strain and a clone of the Colombian strain, Col1.7G2. To evaluate whether cell invasion efficiency drives this selection, we infected primary cultures of BALB/c cardiomyocytes using these same T. cruzi populations. Contrary to expectation, Col1.7G2 parasites invaded heart cell cultures in higher numbers than JG parasites; however, intracellular multiplication of JG parasites was more efficient than that of Col1.7G2 parasites. This phenomenon was only observed for cardiomyocytes and not for cultured Vero cells. Double infections (Col1.7G2 + JG) showed similar results. Even though invasion might influence tissue selection, our data strongly suggest that intracellular development is important to determine parasite tissue tropism.

Trypanosoma cruzi genetic diversity was investigated in 25 isolates (vectors and humans) from the semiarid zone of the State of Rio Grande do Norte, Brazil. Molecular markers (3' region of the 24Salpha rRNA; mitochondrial cytochrome oxidase subunit 2 (COII) gene; spliced leader intergenic region (SL-IR) gene; allelic size microsatellite polymorphism) identified 56% TcIII (100% Panstrongyluslutzi; 50% Triatomabrasiliensis); 40% TcII (91.7% humans; 50% T. brasiliensis) and 4% TcI (human). Microsatellite analysis revealed monoclonal and heterozygous patterns on one or more microsatellite loci in 64% of T. cruzi isolates (92.3% triatomines; 33.3% humans) and 36% putative polyclonal populations (66.7% humans; 7.7% triatomines) by loci SCLE10, SCLE11, TcTAT20, TcAAAT6, all belonging to TcII. Identical T. cruzi polyclonal profiles (88.9%) were detected, mostly from humans. The adaptative natural plasticity of TcII and TcIII and their potential for maintaining human infection in T. brasiliensis were confirmed. Intraspecific and phylogenetic T. cruzi diversity in the sylvatic and domestic transmission cycles in this specific region will provide exclusive control strategies., (2010 Elsevier B.V. All rights reserved.)

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form, IF). Each year, approximately 3% of them develop lesions in the heart or gastrointestinal tract. Cardiomyopathy (CCHD) is the most severe manifestation of Chagas disease. The factors that determine the outcome of the infection are unknown, but certainly depend on complex interactions amongst the genetic make-up of the parasite, the host immunogenetic background and environment. In a previous study we verified that the maxicircle gene NADH dehydrogenase (mitochondrial complex I) subunit 7 (ND7) from IF isolates had a 455 bp deletion compared with the wild type (WT) ND7 gene from CCHD strains. We proposed that ND7 could constitute a valuable target for PCR assays in the differential diagnosis of the infective strain. In the present study we evaluated this hypothesis by examination of ND7 structure in parasites from 75 patients with defined pathologies, from Southeast Brazil. We also analysed the structure of additional mitochondrial genes (ND4/CR4, COIII and COII) since the maxicircle is used for clustering Trypanosoma cruzi strains into three clades/haplogroups. We conclude that maxicircle genes do not discriminate parasite populations which induce IF or CCHD forms. Interestingly, the great majority of the analysed isolates belong to T. cruzi II (discrete typing unit, (DTU) IIb) genotype. This scenario is at variance with the prevalence of hybrid (DTU IId) human isolates in Bolivia, Chile and Argentina. The distribution of WT and deleted ND7 and ND4 genes in T. cruzi strains suggests that mutations in the two genes occurred in different ancestrals in the T. cruzi II cluster, allowing the identification of at least three mitochondrial sub-lineages within this group. The observation that T. cruzi strains accumulate mutations in several genes coding for complex I subunits favours the hypothesis that complex I may have a limited activity in this parasite.

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions.

Our research aimed to characterize the genetic profiles of 102 Trypanosoma cruzi isolates recently obtained from 44 chronic chagasic patients from different regions of the states of Minas Gerais and Goiás in Brazil. At least two isolates were obtained from each patient at different times in order to study the parasite population dynamics during disease progression in the chronic phase. The isolates were characterized molecularly by genotyping the 3' region of the 24S alpha rRNA, the mitochondrial cytochrome oxidase subunit 2 (COII) gene, and the intergenic region of the spliced leader intergenic region (SL-IR) gene. Seventy-seven isolates were analyzed for nine microsatellite loci. The data presented here show a strong correlation between the T. cruzi lineage II (T. cruzi II) and human infection in these regions of Brazil. Interestingly, isolates from two patients were initially characterized (by rRNA genotyping) as T. cruzi I and hybrid strains, but subsequent analyses of the COII and SL-IR genes confirmed that those isolates belonged to T. cruzi III and a hybrid group, respectively. Our results confirm the risk of misclassifying T. cruzi isolates on the basis of analysis of a single molecular marker. The microsatellite profiles showed that different isolates obtained from the same patient were genetically identical and monoclonal. Exceptions were observed for T. cruzi isolates from two patients who presented differences for the SCLE11 locus and also from two other patients who showed amplification of three peaks for a microsatellite locus (TcAAAT6), implying that they were multiclonal. On the basis of the findings of the studies described here, we were not able to establish a correlation between the clinical forms of Chagas' disease and the genetic profiles of the T. cruzi isolates.

The immune response is crucial for protection against disease; however, immunological imbalances can lead to heart and digestive tract lesions in chagasic patients. Several studies have evaluated the cellular and humoral immune responses in chagasic patients in an attempt to correlate immunological findings with clinical forms of Chagas disease. Moreover, immunoglobulins and cytokines are important for parasitic control and are involved in lesion genesis. Here, cytokine and IgG isotype production were studied, using total epimastigote antigen on sera of chagasic patients with indeterminate (IND, n = 27) and cardiac (CARD, n = 16) forms of the disease. Samples from normal,uninfected individuals (NI, n = 30) were use as controls. The results showed that sera from both IND and CARD patients contained higher levels of Trypanosoma cruzi-specific IgG1 (IgG1) antibodies than sera from NI. No difference in IgG2 production levels was observed between NI, IND and CARD patients, nor was a difference in IL-10 and IFN-gamma production detected in the sera of IND, CARD and NI patients. However, IND patients displayed a positive correlation between IL-10 and IFN-gamma levels in serum, while CARD patients showed no such correlation, indicating an uncontrolled inflammatory response in CARD patients. These findings support the hypothesis that a lack of efficient regulation between IFN-gamma and IL-10 productions in CARD patients may lead to cardiac immunopathology.

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

A systematic study following infection by various strains of the protozoan parasite, Trypanosoma cruzi, and the simultaneous monitoring of the humoral immune response together with the elicited cellular response, could add greatly to our understanding of differences between strains of this important human pathogen. In that sense, acute and chronic infections with distinct T. cruzi strains (Y, Berenice-78 and ABC) in Beagle dogs were studied through a longitudinal evaluation of immunoglobulin G (IgG), IgG1 and IgG2 isotypes (by ELISA and flow cytometry (FC)), as well as measurements of peripheral blood mononuclear cell (PBMC) proliferation over a 100-week period, and their correlation with cardiomegaly. Our results show that infected animals presenting cardiomegaly showed lower or absent levels of IgG1 during the chronic phase of the infection, when compared to those that did not show an increase in heart weight. In that manner, our results suggest that IgG1 could be used as a marker for cardiac pathogenicity in Chagas disease.

Trypanosoma cruzi is a hemoflagelate parasite associated with heart dysfunctions causing serious problems in Central and South America. Beagle dogs develop the symptoms of Chagas disease in humans, and could be an important experimental model for better understanding the immunopathogenic mechanisms involved in the chagasic infection. In the present study we investigated the relation among biological factors inherent to the parasite (trypomastigote polymorphism and in vitro infectivity) and immunoglobulin production, inflammation, and fibrosis in the heart of Beagle dogs infected with either T. cruzi Y or Berenice-78 strains. In vitro infectivity of Vero cells as well as the extension of cardiac lesions in infected Beagle was higher for Y strain when compared to Berenice-78 strain. These data suggested that in vitro infectivity assays may correlate with pathogenicity in vivo. In fact, animals infected with Y strain, which shows prevalence of slender forms and high infectivity in vitro, presented cardiomegaly, inflammation, and fibrosis in heart area. Concerning the immunoglobulin production, no statistically significant difference was observed for IgA, IgM or IgG levels among T. cruzi infected animals. However, IgA together IgM levels have shown to be a good marker for the acute phase of Chagas disease.

Neuronal lesions and peripheral denervation in Chagas' disease are related to local inflammation; however, the pathogenic mechanisms of neuronal lesions in the heart and megavisceras are still unclear. We investigated the involvement of nitric oxide (NO) on neuronal lesion in co-cultures of neurons and macrophages. Trypanosoma cruzi-infected and interferon-gamma (IFN-gamma)-activated co-cultures of neurons and wild-type (WT) macrophages showed significant reduction of both neuronal survival and neurite density. These findings correlated with the levels of NO and the expression of inducible nitric oxide synthase (iNOS). Accordingly, neuronal survival rate in the co-cultures was recovered to control levels by treatment of the cultures with the iNOS inhibitor, aminoguanidine. Moreover, neither neuronal survival nor the neurite density was affected in the co-cultures when the macrophages were harvested from iNOS-deficient mice. These results demonstrate that iNOS-derived NO is the major molecule involved in neuronal damage mechanism in our in vitro model of Chagas' disease neuropathology.

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form). Every year 2-3% of these individuals develop severe clinical manifestations (cardiac and digestive forms). In this study a Trypanosoma cruzi DNA microarray was used to compare the transcript profiles of six human isolates: three from asymptomatic and three from cardiac patients. Seven signals were expressed differentially between the two classes of isolates, including tryparedoxin, surface protease GP63, cyclophilin, some hypothetical proteins and the pre-edited maxicircle gene NADH dehydrogenase subunit 7 (ND7). The approximately 30-fold greater signal in cardiac strains for ND7 was the most pronounced of the group, and differential levels of pre-edited ND7 transcript confirmed the microarray analysis. The ND7 gene from asymptomatic isolates showed a deletion of 455bp from nt 222 to nt 677 relative to ND7 of the CL Brener reference strain. The ND7 gene structure correlated with disease manifestation for 20 isolates from clinically characterised, chronic phase patients. The ND7 lesion produces a truncated product that could impair the function of mitochondrial complex I. Possible links between the integrity of the electron transport chain and symptom presentation are discussed. We propose that ND7 and other genes of the pathway constitute valuable targets for PCR assays in the differential diagnosis of the infective T. cruzi strain. While this hypothesis requires validation by the examination of additional recent parasite isolates from patients with defined pathologies, the identification of specific molecular markers represents a promising advance in the association between parasite genetics and disease pathology.

The change in parasitemia profile, measured by sequential blood cultures of 27 benznidazole (Bz) treated patients compared with 13 untreated patients, on the chronic phase of chagas disease, is described. All patients were adults (age limits: 23-88) with positive serology (three tests); 23 of them were females. All patients were submitted to six blood cultures, three before and three after Bz treatment. The parasitemia was classified as nondetected (with three negative blood cultures), medium (one positive culture in three), and high (two or three positive cultures). From the eight patients with nondetected parasitemia before Bz, seven still had the same profile and only one switched to medium; from eight with medium parasitemia, seven shifted to nondetected, and one to high parasitemia. From the 11 patients with high parasitemia before Bz, ten converted to nondetected and only one was positive after Bz. Nineteen of the 27 patients changed the parasitemia profile (70.4%), and the rate of therapeutic failure was 11.1% (3/27) during the first 24 months of follow-up after Bz. The shift to nondetected parasitemia profile was from 8/27 to 24/27 patients after Bz treatment for the first 2 years. Only 46.2% (6/13) of the nontreated individuals changed the parasitemia profile. We conclude that there is a strong trypanocide effect of Bz (88.8%) and a rate of therapeutic failure of 11.1% during the first 2 years after trypanocidal treatment.

The clinical course of Chagas' disease varies widely among different patients and geographic regions. For reasons that are not completely understood but involve host and parasite factors, some patients never develop the disease while others present cardiac and/or gastrointestinal symptoms. Many studies have been conducted in order to correlate the genetic variability of the parasites with the clinical forms of the disease, but no conclusive data have been obtained. Our research aims at characterizing the genetic profiles of Trypanosoma cruzi isolates recently obtained from 70 chagasic patients who either showed pathological lesions with symptoms of various intensities or were asymptomatic. All patients came from an area where Chagas' disease is endemic in southeast Brazil where vectorial transmission has been controlled and different clinical forms of the disease can be found. The molecular characterization of parasites evaluated the polymorphisms of the 3' region of the 24Salpha rRNA gene and the variability of kinetoplast DNA (kDNA) minicircles of T. cruzi populations by low-stringency single specific primer PCR. Data presented here provide a strong correlation between T. cruzi II and human infection in this region. However, a high degree of variability was observed within T. cruzi II, as demonstrated by intense kDNA polymorphism among all clinical forms and also within each of them, irrespective of the intensity of pathological processes.

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

One of the greatest concerns in Chagas' disease is the absence of reliable methods for the evaluation of chemotherapy efficacy in treated patients. The tests available to evaluate cure after the specific treatment are the complement-mediated lysis (CoML) and flow cytometry tests, but they are not feasible for routine clinical use. In this study, we evaluated an enzyme-linked immunosorbent assay (ELISA) based on the recombinant Trypanosoma cruzi complement regulatory protein (rCRP) as a method to determine parasite clearance in comparison to the CoML and other methods such as conventional serology, hemoculture, and PCR in serum samples of 31 patients collected before and after the treatment, monitored for an average of 27.7 months after chemotherapy. The results showed that the percentage of patient samples that were positive by rCRP ELISA was reduced from 100 to 70.3, 62.5, 71.4, and 33.4% in the first, second, third, and fourth years after treatment, respectively, while the samples positive by CoML were reduced to 85.2, 81.2, 71.4, and 33.4% during the same period, demonstrating the same significant tendency in the reduction of positive samples. On the other hand, the conventional serology (CS) tests did not present this reduction. The percentage of samples positive by PCR was initially 77.4% and decreased to 55.5, 68.7, 47.7, and 50.0% at the fourth year after treatment, confirming the drastic clearance of circulating parasites after treatment. Our results strongly suggest that the rCRP ELISA was capable of detecting the early therapeutic efficacy in treated patients and confirmed its superiority over the CS tests and parasitologic methods.

Assessment of cure of Trypanosoma cruzi infection by antibody seroconversion usually involves several years of follow-up. Parasitological negativity is useless for cure assessment, since even untreated patients mostly show negative results; conversely, positive tests are of great value because they indicate treatment failure. Here, PCR was used to assess the rate of specific chemotherapy failure in a well-characterized Brazilian cohort of T. cruzi-seropositive children, who were enrolled in a field trial of benznidazole (Bz) efficacy. Paired blood samples from 111 children were taken at baseline and 36 months after treatment with either Bz (n = 58) or a placebo (n = 53). DNA extraction and PCR amplification were carried out as previously described, and hybridization was performed with all PCR products. At the end of follow-up, PCR was positive for 39.6% of the patients in the Bz group versus 64.2% in the placebo group (P = 0.01). Untreated patients had a 1.6-fold-higher chance of remaining positive by PCR than those in the Bz group (P < 0.05). We conclude that PCR is a useful tool for revealing therapeutic failure of T. cruzi infection on a short-term basis.

In rats, CL-Brener clone caused high mortality, severe acute myocarditis, and myositis that subsided completely in surviving animals. Accordingly, no parasite kDNA could be amplified in several organs after 4 months. The monoclonal JG strain caused null mortality, acute predominantly focal myocarditis, discrete and focal myositis, and a chronic phase with sparse inflammatory foci. Double infection with both Trypanosoma cruzi populations turned mortality very low or null. At the end of the acute phase, the heart exhibited only JG strain kDNA (LSSP-PCR), while skeletal muscles and rectum exhibited only CL-Brener kDNA. Molecular and histopathological findings were accordant. In double infection chronic phase, JG strain remains in heart and appeared in organs previously parasitized by CL-Brener clone. Understanding the virulence and histotropism shifts now described could be important to clarify the variable clinical course and epidemiological peculiarities of Chagas' disease.

The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b and C4b to restrict activation of the complement cascade. Here, we report production of functional, recombinant T. cruzi CRP in mammalian cells and a one-step purification of the recombinant protein. Exchange of the crp DNA sequence encoding the carboxy-terminal GPI signal sequence with the corresponding sequence of decay accelerating factor (DAF) was necessary for recognition, cleavage, and addition of GPI in mammalian cells. CRP production was assessed in two mammalian cell lines with crp-daf gene expression driven by three different transcription control regions: Rous sarcoma virus long terminal repeat, cytomegalovirus (CMV) immediate early gene, and chicken beta-actin promoter/CMV enhancer. We present evidence that CRP produced in transfected Chinese hamster Ovary (CHO) cells was functional and protected the cells from complement-mediated lysis. To facilitate purification of the recombinant protein, a hexahistidyl tag was incorporated at 3(') end of the cDNA upstream of the GPI anchor addition sequence. An additional histidine fusion construct was made that allowed for secretion and recovery of recombinant protein from culture supernatant fluid. Both membrane and secreted forms of the protein were purified in one step by nickel nitrilotriacetic acid. The production and purification of functionally active CRP in a non-infectious expression system will allow for structure and function studies aimed at identifying the active site(s) of this protein.

Changes in tubulin expression are among the biochemical and morphological adaptations that occur during the life cycle of Trypanosomatids. To investigate the mechanism responsible for the differential accumulation of tubulin mRNAs in Trypanosoma cruzi, we determine the sequences of alpha- and beta-tubulin transcripts and analyzed their expression during the life cycle of the parasite. Two beta-tubulin mRNAs of 1.9 and 2.3 kb were found to differ mainly by an additional 369 nucleotides at the end of the 3' untranslated region (UTR). Although their transcription rates are similar in epimastigotes and amastigotes, alpha- and beta-tubulin transcripts are 3- to 6-fold more abundant in epimastigotes than in trypomastigotes and amastigotes. Accordingly, the half-lives of alpha- and beta-tubulin mRNAs are significantly higher in epimastigotes than in amastigotes. Transient transfection experiments indicated that positive regulatory elements occur in the 3' UTR plus downstream intergenic region of the alpha-tubulin gene and that both positive and negative elements occur in the equivalent regions of the beta-tubulin gene.

In the present study, we evaluated for the first time the profile of blood parasitism in untreated, chronic Chagas' disease. The study was conducted on 60 patients and a control group of nine serologically negative individuals. Analysis of three blood samples showed 70% cumulative positivity for blood culture and 86.7% positivity for PCR. The comparison of the two tests revealed that 41.1% (74/180) of the samples presented positive results for both PCR and blood culture, 22.2% (40/180) were positive for PCR alone, and 4.4% (8/180) were positive for blood culture and negative for PCR. The addition of the second sample raised positivity significantly for both blood culture ( P=0.0000) and PCR ( P=0.0369). Addition of the third sample was also statistically significant for blood culture ( P=0.0001) but not for PCR ( P=0.1186). These data point to the importance of studying the parasitemia of Trypanosoma cruzi-infected individuals before specific treatment. They also suggest that at least two blood samples should be collected and that two tests should be used, if possible--a procedure that considerably improves the parasitologic diagnosis of Chagas' disease and the evaluation of therapeutic efficacy.

In the present study, we investigated the role of dogs as experimental models for acute and chronic phases of Chagas' disease, before and after therapeutic treatments. Dogs were infected with Trypanosoma cruzi strains of different susceptibilities to benznidazole (Bz) and treated with the same therapeutic scheme as used for human chagasic. The treatment with Bz was able to prevent death and induced parasitological cure in 62.5% (acute phase) and 38.7% (chronic recent phase) of the tested animals. These results were similar to those reported in clinical trials for treated human patients (cured and uncured) in both phases of the disease. We also showed that parasitologic and serologic tests for monitoring the cure were similar to those obtained for human trials. In addition, Polymerase chain reaction showed the highest sensitivity when compared with hemoculture as an indicator of parasite clearance. In conclusion, the proposed experimental model should be relevant for chemotherapy studies for the control of Chagas' disease.