RESUMO
BACKGROUND: In humans, Trypanosoma cruzi infection is controlled by a complex immune response. Immunoglobulin G (IgG) is important for opsonizing blood trypomastigotes, activating the classic complement pathway, and reducing parasitemia. The trypanocidal activity of benznidazole is recognized, but its effects on the prevention and progression of Chagas disease is not well understood OBJECTIVE: We aimed to evaluate the levels of total IgG and cross-specific IgG subclasses in patients with chronic Chagas disease of different clinical forms before and after 4 years of benznidazole treatment. METHODS: Eight individuals with the indeterminate form and nine with the cardiac form who completed the treatment protocol were evaluated. The levels of total IgG and IgG1, IgG2, IgG3, and IgG4 isotypes were quantified in the serum of each individual using the fluorescent immunosorbent assay. The results are expressed as relative fluorescence unit. RESULTS: Patients with chronic Chagas disease presented decreased levels of total IgG at 48 months after benznidazole treatment. Increased IgG1 and decreased IgG3 levels were observed in patients with the cardiac form and those with exacerbated clinical forms. In addition, a decrease in the IgG3/IgG1 ratio was observed in individuals with the cardiac form of Chagas disease. CONCLUSIONS: Benznidazole administration in the chronic phase differentially changes IgG subclasses in patients with cardiac and indeterminate forms, and monitoring the IgG3 level may indicate the possible prognosis to the cardiac form or worsening of the already established clinical form.
Assuntos
Doença de Chagas , Nitroimidazóis , Doença de Chagas/tratamento farmacológico , Humanos , Imunoglobulina G , Nitroimidazóis/uso terapêutico , ParasitemiaRESUMO
Trypanosoma rangeli is an avirulent flagellate protozoan that could mislead correct diagnosis of Trypanosoma cruzi infection, the causative agent of Chagas' disease, given their high similarity. Besides, T. rangeli presents two genetic groups, whose differentiation is achieved mainly by molecular approaches. In this context, ribosomal DNA (rDNA) is a useful target for intra and interspecific molecular differentiation. Analyzing the rDNA of T. rangeli and comparison with other trypanosomatid species, two highly divergent regions (Trß1 and Trß2) within the 28Sß gene were found. Those regions were amplified and sequenced in KP1(+) and KP1(-) strains of T. rangeli, revealing group-specific polymorphisms useful for intraspecific distinction through restriction fragment length polymorphism technique. Also, amplification of Trß1 allowed differentiation between T. rangeli and T. cruzi. Trß2 predicted restriction length profile, allowed differentiation between T. rangeli, T. cruzi, Trypanosoma brucei, and Leishmania braziliensis, increasing the use of Trß1 and Trß2 beyond a molecular approach for T. rangeli genotyping, but also as a useful target for trypanosomatid classification.
Assuntos
DNA Ribossômico , Trypanosoma rangeli/classificação , Trypanosoma rangeli/genética , DNA de Protozoário/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Especificidade da Espécie , Trypanosoma/classificação , Trypanosoma/genética , Trypanosoma cruzi/genéticaRESUMO
Chagas Disease is a zoonosis caused by the parasite Trypanosoma cruzi. Several high-resolution markers have subdivided T. cruzi taxon into at least seven lineages or Discrete Typing Units (DTUs) (TcI-TcVI and TcBat). Trypanosoma cruzi I is the most diverse and geographically widespread DTU. Recently a TcI genotype related to domestic cycles was proposed and named as TcIDOM. Herein, we combined traditional markers and housekeeping genes and applied a Multispecies Coalescent method to explore intra-TcI relationships, lineage boundaries and genetic diversity in a random set of isolates and DNA sequences retrieved from Genbank from different countries in the Americas. We found further evidence supporting TcIDOM as an independent and emerging genotype of TcI at least in Colombia and Venezuela. We also found evidence of high phylogenetic incongruence between parasite's gene trees (including introgression) and embedded species trees, and a lack of genetic structure among geography and hosts, illustrating the complex dynamics and epidemiology of TcI across the Americas. These findings provide novel insights into T. cruzi systematics and epidemiology and support the need to assess parasite diversity and lineage boundaries through hypothesis testing using different approaches to those traditionally employed, including the Bayesian Multispecies coalescent method.
Assuntos
Variação Genética , Filogenia , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , América Central , DNA de Protozoário/análise , México , América do SulRESUMO
The major problem with Chagas disease is evolution of the chronic indeterminate form to a progressive cardiac disease. Treatment diminishes parasitemia but not clinical progression, and the immunological features involved are unclear. Here, we studied the clinical course and the immune response in patients with chronic-phase Chagas disease at 48 months after benznidazole treatment. Progression to the cardiac form of Chagas disease or its aggravation was associated with higher in vitro antigen-specific production of interferon gamma (IFN-γ) in patients with cardiac Chagas disease than in patients with the indeterminate form. Predominance of IFN-γ production over interleukin-10 (IL-10) production in antigen-specific cultures was associated with cardiac involvement. Significantly higher numbers of antigen-specific T helper 1 cells (T-Bet+ IFN-γ+) and a significantly higher IFN-γ+/IL-10+ ratio were observed in patients with cardiac Chagas disease than in patients with the indeterminate form. Cardiac damage was associated with higher numbers of T helper cells than cytotoxic T lymphocytes producing IFN-γ. Patients with cardiac Chagas disease had predominant CD25- and CD25low T regulatory (Treg) subpopulations, whereas patients with the indeterminate form manifested a higher relative mean percentage of CD25high Treg subpopulations. These findings suggest that at 48 months after benznidazole treatment, the disease can worsen or progress to the cardiac form. The progression may be related to increased IFN-γ production (mostly from CD4+ T cells) relative to IL-10 production and increased Treg percentages. Patients with the indeterminate form of Chagas disease show a more balanced ratio of proinflammatory and anti-inflammatory cytokines.
Assuntos
Doença de Chagas/tratamento farmacológico , Citocinas/biossíntese , Nitroimidazóis/uso terapêutico , Linfócitos T/imunologia , Idoso , Doença de Chagas/imunologia , Feminino , Humanos , Imunofenotipagem , Interferon gama/biossíntese , Interleucina-10/biossíntese , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/imunologiaRESUMO
Several bat species can be infected by trypanosomes, but there is not much information about which of these parasites infect bats from Triângulo Mineiro and Alto Paranaíba, Minas Gerais state, Brazil, a formerly endemic region for Trypanosoma cruzi, the causative agent of Chagas disease. The aim of this study was to describe, characterize, and identify the presence of trypanosomes in bats. The captured bats (448) belong to four families and to 19 different species. Of those, 37 bats were found to be positive for trypanosomes by microhematocrit, (infection rate 8.3%) and 27 were positive after hemoculture analysis. Initially, the isolates were identified by PCR (18S rDNA, 24Sα rDNA, spliced leader, COII RFLP-PCR) using primers originally designed for T. cruzi. PCRs (18S rDNA, 24Sα rDNA) showed compatible bands for TcI, whereas COII RFLP-PCR showed a similar pattern associated to TcII. However, there was no DNA amplification using spliced leader as a target, revealing a discrepancy between the results. Phylogenetic analysis of Cathepsin L-like and 18S rDNA sequences proved that 15 of the isolates corresponded to Trypanosoma cruzi marinkellei and one to Trypanosoma dionisii. These results revealed that the diversity of trypanosome species in a region considered endemic for Chagas disease is greater than previous descriptions. All this can confirm the necessity of using DNA sequencing approaches in order to determinate trypanosomes species isolated from bats.
Assuntos
Quirópteros/parasitologia , Trypanosoma/isolamento & purificação , Animais , Brasil/epidemiologia , Catepsina L/genética , Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , DNA de Protozoário , DNA Ribossômico/genética , Filogenia , Análise de Sequência de DNA , Trypanosoma/classificação , Trypanosoma/genética , Trypanosoma cruzi/genéticaRESUMO
Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4-110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(-). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(-) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts.
Assuntos
DNA de Protozoário/análise , Trypanosoma cruzi/genética , Trypanosoma rangeli/genética , Animais , Citometria de Fluxo , Genoma de ProtozoárioRESUMO
Leishmaniasis is a complex of zoonotic diseases caused by parasites of the genus Leishmania, which can develop in domestic as well as wild animals and humans throughout the world. Currently, this disease is spreading in rural and urban areas of non-endemic regions in Brazil. Recently, bats have gained epidemiological significance in leishmaniasis due to its close relationship with human settlements. In this study, we investigated the presence of Leishmania spp. DNA in blood samples from 448 bats belonging to four families representing 20 species that were captured in the Triangulo Mineiro and Alto Paranaiba areas of Minas Gerais State (non-endemic areas for leishmaniasis), Brazil. Leishmania spp. DNA was detected in 8·0% of the blood samples, 41·6% of which were Leishmania infantum, 38·9% Leishmania amazonensis and 19·4% Leishmania braziliensis. No positive correlation was found between Leishmania spp. and bat food source. The species with more infection rates were the insectivorous bats Eumops perotis; 22·2% (4/18) of which tested positive for Leishmania DNA. The presence of Leishmania in the bat blood samples, as observed in this study, represents epidemiological importance due to the absence of Leishmaniasis cases in the region.
Assuntos
Quirópteros , Leishmania/fisiologia , Leishmaniose/veterinária , Animais , Brasil/epidemiologia , DNA de Protozoário/análise , Leishmania/genética , Leishmania braziliensis/genética , Leishmania braziliensis/fisiologia , Leishmania infantum/genética , Leishmania infantum/fisiologia , Leishmaniose/epidemiologia , Filogenia , Especificidade da EspécieRESUMO
INTRODUCTION: This work shows that 3% (v/v) human urine (HU) in semisolid Liver Infusion Tryptose (SSL) medium favors the growth of Trypanosoma cruzi and T. rangeli. METHODS: Parasites were plated as individual or mixed strains on SSL medium and on SSL medium with 3% human urine (SSL-HU). Isolate DNA was analyzed using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE). RESULTS: SSL-HU medium improved clone isolation. PCR revealed that T. cruzi strains predominate on mixed-strain plates. PFGE confirmed that isolated parasites share the same molecular karyotype as parental cell lines. CONCLUSIONS: SSL-HU medium constitutes a novel tool for obtaining T. cruzi and T. rangeli clonal lineages.
Assuntos
Meios de Cultura/química , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma rangeli/crescimento & desenvolvimento , Urina/química , Eletroforese em Gel de Campo Pulsado , Humanos , Cariótipo , Compostos Orgânicos/farmacologia , Reação em Cadeia da Polimerase , Trypanosoma cruzi/genética , Trypanosoma rangeli/genéticaRESUMO
In previous studies, we have demonstrated that inoculation with a Trypanosoma cruzi marinkellei (avirulent RM1 strain) was able to reduce parasitemia in mice challenged with T. cruzi, although it was not able to prevent histopathological lesions. Th1 response stimulation by immunization is necessary for T. cruzi infection control, but the resistance is also dependent on immunoregulatory mechanisms, which can be induced by adjuvants. Thus, we evaluated whether inoculation of T. cruzi marinkellei associated with administration of different adjuvants would be capable of inducing different patterns of immune response to maximize the immune response against T. cruzi (virulent Romildo strain) infection. Two hundred eighty nonisogenic mice were divided into 14 groups according to the immunization scheme and the subsequent challenge with virulent Romildo T. cruzi strain. Nonimmunized groups and animals inoculated without adjuvants were also included. Immune protection was not observed with Th2 adjuvants (incomplete Freund's adjuvant [IFA] and Alum) due to high parasitemia. Th1/Th2-polarizing adjuvants also did not induce immune protection because inulin was unable to maintain survival, and immune-stimulating complexes induced intense inflammatory processes. Animals sensitized with RM1 strain without adjuvants were able to reduce parasitemia, increase survival, and protect against severe histological lesions, followed by adequate cytokine stimulation. Finally, our results demonstrate that the early and balanced IFN-γ production becomes critical to promote protection and that Th1 adjuvant elicited a controversial infection control due to increased histopathological damage. Therefore, the host's immunomodulation remains one of the most important challenges in the research for effective protection against T. cruzi infection. Similarly, the identification of protective antigens in the RM1 strain of T. cruzi marinkellei may contribute to further studies on vaccine development against human Chagas disease.
Assuntos
Adjuvantes Imunológicos , Doença de Chagas/prevenção & controle , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Animais , Doença de Chagas/metabolismo , Masculino , Camundongos , Trypanosoma cruzi/classificaçãoRESUMO
An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.