Differential expression of proteins in genetically distinct Trypanosoma cruzi samples (TcI and TcII DTUs) isolated from chronic Chagas disease cardiac patients.

Background Trypanosoma cruzi, a hemoflagellate protozoan parasite and the etiological agent of Chagas disease (CD), exhibits great genetic and biological diversity. Infected individuals may present clinical manifestations with different levels of severity. Several hypotheses have been proposed to attempt to correlate the diversity of clinical signs and symptoms to the genetic variability of T. cruzi. This work aimed to investigate the differential expression of proteins from two distinct genetic groups of T. cruzi (discrete typing units TcI and TcII), isolated from chronically infected individuals displaying the cardiac form of CD. For this purpose, epimastigote forms of the two isolates were cultured in vitro and the cells recovered for protein extraction. Comparative two-dimensional (2D) gel electrophoreses were performed and differentially expressed spots selected for identification by mass spectrometry, followed by database searching and protein categorization. Results The 2D electrophoretic profiles revealed the complex composition of the T. cruzi extracted proteome. Protein spots were distributed along the entire pH and molecular mass ranges attesting for the integrity of the protein preparations. In total, 46 differentially expressed proteins were identified present in 40 distinct spots found in the comparative gel analyses. Of these, 16 displayed upregulation in the gel from TcI-typed parasites and 24 appeared overexpressed in the gel from TcII-typed parasites. Functional characterization of differentially expressed proteins revealed major alterations associated with stress response, lipid and amino acid metabolism in parasites of the TcII isolate, whilst those proteins upregulated in the TcI sample were primarily linked to central metabolic pathways. Conclusions The comparative 2D-gel electrophoresis allowed detection of major differences in protein expression between two T. cruzi isolates, belonging to the TcI and TcII genotypes. Our findings suggest that patients displaying the cardiac form of the disease harbor parasites capable of exhibiting distinct proteomic profiles. This should be of relevance to disease prognosis and treatment

Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley, Minas Gerais, Brazil, with Chagas disease.

Trypanosoma cruzi strains from distinct geographic areas show differences in drug resistance and association between parasites genetic and treatment response has been observed. Considering that benznidazole (BZ) can reduce the parasite burden and tissues damage, even in not cured animals and individuals, the goal is to assess the drug response to BZ of T. cruzi II strains isolated from children of the Jequitinhonha Valley, state of Minas Gerais, Brazil, before treatment. Mice infected and treated with BZ in both phases of infection were compared with the untreated and evaluated by fresh blood examination, haemoculture, polymerase chain reaction, conventional (ELISA) and non-conventional (FC-ALTA) serologies. In mice treated in the acute phase, a significant decrease in parasitaemia was observed for all strains. Positive parasitological and/or serological tests in animals treated during the acute and chronic (95.1-100%) phases showed that most of the strains were BZ resistant. However, beneficial effect was demonstrated because significant reduction (p < 0.05%) and/or suppression of parasitaemia was observed in mice infected with all strains (acute phase), associated to reduction/elimination of inflammation and fibrosis for two/eight strains. BZ offered some benefit, even in not cured animals, what suggest that BZ use may be recommended at least for recent chronic infection of the studied region

Diretriz da Sociedade Brasileira de Cardiologia sobre Diagnóstico e Tratamento de Pacientes com Cardiomiopatia da Doença de Chagas

This guideline aimed to update the concepts and formulate the standards of conduct and scientific evidence that support them, regarding the diagnosis and treatment of the Cardiomyopathy of Chagas disease, with special emphasis on the rationality base that supported it.&nbsp; Chagas disease in the 21st century maintains an epidemiological pattern of endemicity in 21 Latin American countries. Researchers and managers from endemic and non-endemic countries point to the need to adopt comprehensive public health policies to effectively control the interhuman transmission of T. cruzi infection, and to obtain an optimized level of care for already infected individuals, focusing on diagnostic and therapeutic opportunistic opportunities. &nbsp; Pathogenic and pathophysiological mechanisms of the Cardiomyopathy of Chagas disease were revisited after in-depth updating and the notion that necrosis and fibrosis are stimulated by tissue parasitic persistence and adverse immune reaction, as fundamental mechanisms, assisted by autonomic and microvascular disorders, was well established. Some of them have recently formed potential targets of therapies.&nbsp; The natural history of the acute and chronic phases was reviewed, with enhancement for oral transmission, indeterminate form and chronic syndromes. Recent meta-analyses of observational studies have estimated the risk of evolution from acute and indeterminate forms and mortality after chronic cardiomyopathy. Therapeutic approaches applicable to individuals with Indeterminate form of Chagas disease were specifically addressed. All methods to detect structural and/or functional alterations with various cardiac imaging techniques were also reviewed, with recommendations for use in various clinical scenarios. Mortality risk stratification based on the Rassi score, with recent studies of its application, was complemented by methods that detect myocardial fibrosis.&nbsp; The current methodology for etiological diagnosis and the consequent implications of trypanonomic treatment deserved a comprehensive and in-depth approach. Also the treatment of patients at risk or with heart failure, arrhythmias and thromboembolic events, based on pharmacological and complementary resources, received special attention. Additional chapters supported the conducts applicable to several special contexts, including t. cruzi/HIV co-infection, risk during surgeries, in pregnant women, in the reactivation of infection after heart transplantation, and others.&nbsp; &nbsp;&nbsp; Finally, two chapters of great social significance, addressing the structuring of specialized services to care for individuals with the Cardiomyopathy of Chagas disease, and reviewing the concepts of severe heart disease and its medical-labor implications completed this guideline.Esta diretriz teve como objetivo principal atualizar os conceitos e formular as normas de conduta e evidências científicas que as suportam, quanto ao diagnóstico e tratamento da CDC, com especial ênfase na base de racionalidade que a embasou. A DC no século XXI mantém padrão epidemiológico de endemicidade em 21 países da América Latina. Investigadores e gestores de países endêmicos e não endêmicos indigitam a necessidade de se adotarem políticas abrangentes, de saúde pública, para controle eficaz da transmissão inter-humanos da infecção pelo T. cruzi, e obter-se nível otimizado de atendimento aos indivíduos já infectados, com foco em oportunização diagnóstica e terapêutica. Mecanismos patogênicos e fisiopatológicos da CDC foram revisitados após atualização aprofundada e ficou bem consolidada a noção de que necrose e fibrose sejam estimuladas pela persistência parasitária tissular e reação imune adversa, como mecanismos fundamentais, coadjuvados por distúrbios autonômicos e microvasculares. Alguns deles recentemente constituíram alvos potenciais de terapêuticas. A história natural das fases aguda e crônica foi revista, com realce para a transmissão oral, a forma indeterminada e as síndromes crônicas. Metanálises recentes de estudos observacionais estimaram o risco de evolução a partir das formas aguda e indeterminada e de mortalidade após instalação da cardiomiopatia crônica. Condutas terapêuticas aplicáveis aos indivíduos com a FIDC foram abordadas especificamente. Todos os métodos para detectar alterações estruturais e/ou funcionais com variadas técnicas de imageamento cardíaco também foram revisados, com recomendações de uso nos vários cenários clínicos. Estratificação de risco de mortalidade fundamentada no escore de Rassi, com estudos recentes de sua aplicação, foi complementada por métodos que detectam fibrose miocárdica. A metodologia atual para diagnóstico etiológico e as consequentes implicações do tratamento tripanossomicida mereceram enfoque abrangente e aprofundado. Também o tratamento de pacientes em risco ou com insuficiência cardíaca, arritmias e eventos tromboembólicos, baseado em recursos farmacológicos e complementares, recebeu especial atenção. Capítulos suplementares subsidiaram as condutas aplicáveis a diversos contextos especiais, entre eles o da co-infecção por T. cruzi/HIV, risco durante cirurgias, em grávidas, na reativação da infecção após transplante cardíacos, e outros.&nbsp;&nbsp;&nbsp; Por fim, dois capítulos de grande significado social, abordando a estruturação de serviços especializados para atendimento aos indivíduos com a CDC, e revisando os conceitos de cardiopatia grave e suas implicações médico-trabalhistas completaram esta diretriz.&nbsp

Genotipagem de amostras de Trypanosoma cruzi isoladas de pacientes chagásicos de dois municípios da região do Vale do Jequitinhonha, MG, Brasil.

A espécie do Trypanosoma cruzi possui uma estrutura genética que permite a subdivisão intraespecífica em seis grupos genéticos distintos, denominados TcI, TcII, TcIII, TcIV, TcV e TcVI (Zingales et al., 2009) que tem apresentado diferenças frente a distribuição geográfica, propriedades biológicas e susceptibilidade a droga. É conhecido o predomínio das DTU’s TcII e TcVI em diversas regiões Brasileiras, associados tanto ao ciclo doméstico como ao silvestre da Doença e Chagas, e estando intimamente relacionados com as manifestações clínicas graves em pacientes chagásicos crônicos. Conhecendo o predomínio dessas DTU’s no estado de Minas Gerais, trabalhos anteriores do grupo realizando a genotipagem de um número limitado de amostras destas mesmas localidades (municípios de Berilo e José Gonçalves de Minas, Vale do Jequitinhonha, MG) mostrou a presença somente de amostras de T. cruzi II, como ocorre em outras regiões do Brasil. Desse modo, o objetivo principal do estudo foi caracterizar molecularmente amostras de T. cruzi isoladas de pacientes chagásicos crônicos dos municípios de Berilo e José Gonçalves de Minas, Vale do Jequitinhonha, MG, a fim de determinar o perfil genético do parasito circulante nesta região e comparar esses dados com o observado no Brasil e em outras regiões do Cone Sul. Para isto, os parasitos foram isolados dos pacientes através de hemocultura e mantidos em crescimento em meio LIT para obtenção das massas úmidas e posterior extração de DNA e caracterização por cinco diferentes marcadores moleculares. A identificação inicial dos grupos genéticos do T. cruzi foi realizada seguindo o tríplice ensaio proposto por Lewis et al.(2009). Essa metodologia explora a análise em conjunto dos perfis de bandas gerados após a amplificação do domínio D7 do 24Sα rDNA, do perfil de corte gerado após a digestão dos produtos amplificados de dois genes com suas respectivas enzimas de restrição (HSP60/ECORV e GPI/HhaI) via RFLP-PCR. Nesta primeira etapa de caracterização foram identificadas 43 amostras, sendo todas pertencentes ao grupo TcII, segundo a nova classificação consensual proposta por Zingales et al. (2009). Entretanto, oito isolados não puderam ter sua identificação definida baseada nessa metodologia e foram submetidas à análise do polimorfismo do gene da citocromo oxidase subunidade II (CoII), 24Sα rDNA e do espaçador intergênico do mini-exon (SL-IR), que permitiram em conjunto, classificá-las como TcVI. A seguir, a técnica de RAPD foi empregada na avaliação da variabilidade intra-específica dos isolados do T. cruzi empregando 10 iniciadores. Os dados obtidos pela análise dos dez iniciadores, foram submetidos à análise de UPGMA para obtenção do fenograma e os resultados obtidos corroboram os dos outros marcadores na identificação da maioria dos isolados (43) como TcII e dos oito restantes como TcVI, revelando ainda pouca variabilidade no interior desses grupos. Os resultados desse trabalho confirmam dados preliminares obtidos na região em estudo, demonstrando a predominância dos isolados de T. cruzi pertencentes ao grupo TcII no município de Berilo, semelhante ao demonstrado por outros estudos que avaliaram isolados obtidos de pacientes em outras regiões no eixo nordeste-sul do Brasil. Adicionalmente, foram detectados ainda alguns isolados de TcVI cuja ocorrência é mais rara no Brasil, diferentemente de outros países da América do Sul.Currently Trypanosoma cruzi is classified into six discrete taxonomic units (DTUs) TcI, TcII, TcIII, TcIV, TcV and TcVI that present significant differences concerned the geographic distribution and biological properties. This study was conducted to verify the genetic profile of this parasite isolated of patients from Berilo and José Gonçalves de Minas municipalities, Jequitinhonha Valley, MG, an important endemic area of Chagas disease in Brazil. Molecular characterization was performed through of five different markers in fifty T. cruzi stocks isolated by hemoculture from patients in chronic phase of Chagas disease. DNA extraction was performed using the DNA Purification KIT (Promega, USA). The first identification of the six T. cruzi genetic groups followed the methodology of Lewis et al. (2009). This methodology explores the set of profiles of bands originated after the amplified products of dominium D7 of 24Sα rDNA, profile of bands originated after the digestion of products amplified of two genes with their respective restriction enzymes (HSP60/ECORV e GPI/HhaI) via RFLP-PCR. In this first phase of characterization 43 samples were typed as T. cruzi of TcII group according to the new consensual classification of Zingales et al. (2009). However, eight isolates did not have the genetic identity defined based on this methodology, being submitted to polymorphic analysis of the subunit II (CoII) of citochrome oxidase and of the spliced leader intergenic region of mini-exon (SL-IR) which when associated classified these isolates as TcVI. After, the RAPD technique was employed in the evaluation of intra-especific variability of the T. cruzi isolates using ten primers. Data obtained were submitted to the UPGMA analysis and the results corroborate the originated from the other markers identifying the majority of the isolates as TcII and the other eight isolates as TcVI, showing yet some intra-groups variability with two groups defined into them. The results of this work confirm the preliminary data obtained of the studied region, showing a predominance of T. cruzi isolates of TcII in Berilo, similar to the verified in other studies that evaluated isolates of patients in other regions of Brazil. Additionally, although less frequently, some isolates of TcVI were also detected. These data confirmed the distinct profile observed in Brazil compared to other countries of South America

Propriedades biol?gicas fundamentais de diferentes grupos gen?ticos do Trypanosoma cruzi, e avalia??o inicial da investiga??o da express?o diferencial de prote?nas associadas aos grupos gen?ticos TcI e TcII.

Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.A Doen?a de Chagas (DCh) tem como agente etiol?gico o protozo?rio hemoflagelado Trypanosoma cruzi que apresenta grande diversidade gen?tica, que por sua vez apresenta-se relacionada ? diversidade biol?gica da esp?cie. Diversas estudo foram conduzidos ao longo do tempo, na tentativa de relacionar a variabilidade gen?tica do T. cruzi com seus par?metros biol?gicos resist?ncia ao tratamento e as diferentes manifesta??es cl?nicas da DCh, mas at? o presente momento sem sucesso. Sabendo-se da import?ncia e necessidade de busca de novos alvos que possam responder a essas quest?es, a proposta desse projeto foi avaliar os par?metros biol?gicos principais de seis amostras do T. cruzi, pertencentes a grupos gen?ticos distintos (TcI, TcII e TcVI), isoladas de pacientes chag?sicos cr?nicos, apresentando formas cl?nicas distintas da DCh. Foram utilizadas ferramentas prote?micas para determinar o proteoma diferencial de duas amostras do T. cruzi (TcI e TcII) para verificar se h? correla??o entre o perfil proteico com os par?metros biol?gicos, evolu??o da infec??o em modelo murino e as formas cl?nicas da doen?a humana. Este trabalho avaliou ainda a biologia de todas as seis amostras em meio acelular (LIT), em c?lulas ?Vero? e camundongos Swiss. Neste modelo animal foi tamb?m avaliada a resposta ao tratamento com o Nifurtimox nas fases aguda e cr?nica da infec??o. O conjunto de dados biol?gicos tais como crescimento em meio acelular, infec??o em meio celular e desenvolvimento da infec??o em modelo murino, obtidos de cada amostra de T. cruzi revelou diferen?as significativas entre os gen?tipos. Em rela??o ? resposta ao tratamento etiol?gico, o crit?rio de cura cl?ssico (ELISA negativa), demonstrou cura em apenas 6,25% dos animais infectados e tratados na fase aguda (ITFA). Em contrapartida, quando foi adotado um crit?rio mais recente (CF-AATV negativa), o ?ndice de cura foi 10 vezes maior (62.5%), e os mesmos animais considerados curados por este segundo crit?rio foram tamb?m negativos na qPCR em tecido card?aco. Na fase cr?nica, nenhuma amostra foi curada considerando ambos os crit?rios de cura, e a qPCR no cora??o tamb?m foi positiva em todos os animais, confirmando a conhecida dificuldade de curar infec??es cr?nicas. Os dados obtidos da prote?mica das duas cepas (TcI e TcII) a partir das formas epimastigotas, demonstraram diferen?as significativas na express?o diferencial de prote?nas, principalmente em rela??o ao grupo proteico com fun??o de resposta ao estresse. Dessa forma, pode-se cloncuir com esse trabalho, que a hip?tese de correla??o entre gen?tica do T. cruzi com a biologia das amostras foi confirmada, e que ? importante adotar metodologias mais atuais que antecipam a demonstra??o da cura parasitol?gica da DCh. Esse trabalho demonstrou ainda a import?ncia de busca de alvos diferencialmente expressos no parasita que sirvam como marcadores de progn?stico da infec??o experimental e da DCh humana.Chagas disease (CD) has as etiologic agent the hemoflagellate protozoan Trypanosoma cruzi that presents great genetic diversity, which in turn is related to the biological diversity of the species. Several studies have been conducted over time as an attempt to correlate the genetic variability of T. cruzi with the main biological parameters, resistance to treatment and the different clinical manifestations of CD, but so far without success. Considering the importance and need of to search new targets that may answer these questions, the purpose of this project was to evaluate the main biological parameters of six samples of T. cruzi belonging to different genetic groups (TcI, TcII and TcVI), isolated from chronic chagasic patients with different clinical forms of the disease. Proteomics tools were used to determine the proteome of two different T. cruzi samples (TcI and TcII) to check for correlation between the protein profile with the biological parameters, resistance to treatment, course of the infection in mice and the clinical forms of human disease. This study also evaluated the biology of all six samples in acellular medium (LIT), "Vero" cells and Swiss mice. In this animal model it was also evaluated the response to treatment with Nifurtimox in the acute and chronic phases of the infection. The set of biological data, such as, growth in acellular medium, infection in "Vero"cells and evolution of the infection in mice obtained from each T. cruzi sample showed significant differences between the genotypes. Regarding the response to specific treatment, the classical criteria of cure (ELISA negative) showed cure in only 6.25% of the infected animals and treated in the acute phase (ITAP). In contrast, when a more recent criterion (FC-ALTA negative) was adopted, the cure rate was 10 times higher (62.5%). Interestingly, the same cured animals were also negative in qPCR in the heart. In the chronic phase it was not observed cure when both cure criteria were considered, and the heart was also qPCR negative in all animals, what confirms the known difficulty of to cure chronic infections. Data of proteomics obtained from epimastigotes of two samples (TcI and TcII) showed significant differences in the differential expression of proteins, especially in relation to the protein group with function of response to stress. Thus, it was possible to verify with this work that the hypothesis of correlation between T. cruzi genetics and the biology of the samples was confirmed, and that is important to use more current methodologies that anticipate the demonstration of parasitological cure of CD. This work also demonstrated the importance of to search targets differentially expressed in T. cruzi that work as prognostic markers of the experimental infection and human disease

Differential expression of proteins in genetically distinct Trypanosoma cruzi samples (TcI and TcII DTUs) isolated from chronic Chagas disease cardiac patients

Abstract Background Trypanosoma cruzi, a hemoflagellate protozoan parasite and the etiological agent of Chagas disease (CD), exhibits great genetic and biological diversity. Infected individuals may present clinical manifestations with different levels of severity. Several hypotheses have been proposed to attempt to correlate the diversity of clinical signs and symptoms to the genetic variability of T. cruzi. This work aimed to investigate the differential expression of proteins from two distinct genetic groups of T. cruzi (discrete typing units TcI and TcII), isolated from chronically infected individuals displaying the cardiac form of CD. For this purpose, epimastigote forms of the two isolates were cultured in vitro and the cells recovered for protein extraction. Comparative two-dimensional (2D) gel electrophoreses were performed and differentially expressed spots selected for identification by mass spectrometry, followed by database searching and protein categorization. Results The 2D electrophoretic profiles revealed the complex composition of the T. cruzi extracted proteome. Protein spots were distributed along the entire pH and molecular mass ranges attesting for the integrity of the protein preparations. In total, 46 differentially expressed proteins were identified present in 40 distinct spots found in the comparative gel analyses. Of these, 16 displayed upregulation in the gel from TcI-typed parasites and 24 appeared overexpressed in the gel from TcII-typed parasites. Functional characterization of differentially expressed proteins revealed major alterations associated with stress response, lipid and amino acid metabolism in parasites of the TcII isolate, whilst those proteins upregulated in the TcI sample were primarily linked to central metabolic pathways. Conclusions The comparative 2D-gel electrophoresis allowed detection of major differences in protein expression between two T. cruzi isolates, belonging to the TcI and TcII genotypes. Our findings suggest that patients displaying the cardiac form of the disease harbor parasites capable of exhibiting distinct proteomic profiles. This should be of relevance to disease prognosis and treatment

Evaluation of parasite and host genetics in two generations of a family with Chagas disease.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered to be a multifactorial disease associated with host and parasite genetics, which influence clinical aspects of the disease and other host conditions. In order to understand better the evolution of the disease, this study intended to evaluation of parasite and host genetics in two generations of a family with Chagas disease from the Alto Paranaiba region, Minas Gerais, Brazil, comprising a mother and her five daughters. Several features were evaluated, including the characterization of T. cruzi directly from the blood of patients, host polymorphisms of genes related to cardiomyopathy (TNF, WISP1, CCR5, and TGF-?1) and clinical aspects of the patients. To verify the intraspecific variability of the parasite, the characterization was done directly from human blood using the PCR-LSSP technique and analyzed based on Dice coefficient and unweighted pair group analysis (UPGMA). The host polymorphism was evaluated by PCR-RFLP. The global results showed low variability of the parasites characterized from blood of patients, through Shannon index (0.492) and mean heterozygosity value per locus (0.322). All six patients presented the same genetic polymorphism profile for TNF, WISP1, and TGF-?1, and only one patient was homozygous to CCR5, which suggests that there is no association between the clinical aspects of the patients and their genetic profiles. In conclusion, the findings confirm that the understanding of the clinical evolution of Chagas disease goes beyond the genetic aspects of the parasite and the host

Molecular and biological characterization of Trypanosoma cruzi strains isolated from children from Jequitinhonha Valley, State of Minas Gerais, Brazil

Introduction The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. Methods Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. Results The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2±0.25 to 13.7±3.08 days), whereas the patent period ranged from 3.3±1.08 to 34.5±3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. Conclusions The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis