IgG Subclass Analysis in Patients with Chagas Disease 4 Years After Benznidazole Treatment.

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.

Trypanosoma rangeli 28Sß Ribosomal Gene Allows Intra and Interspecific Molecular Differentiation.

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.

T-Cell Immunophenotyping and Cytokine Production Analysis in Patients with Chagas Disease 4 Years after Benznidazole Treatment.

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.

Evaluation of the multispecies coalescent method to explore intra-Trypanosoma cruzi I relationships and genetic diversity.

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.

Identification of bat trypanosomes from Minas Gerais state, Brazil, based on 18S rDNA and Cathepsin-L-like targets.

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.

DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli.

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.

Leishmania infection in bats from a non-endemic region of Leishmaniasis in Brazil.

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.

Semisolid liver infusion tryptose supplemented with human urine allows growth and isolation of Trypanosoma cruzi and Trypanosoma rangeli clonal lineages.

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.

The Driving of Immune Response by Th1 Adjuvants in Immunization of Mice with Trypanosoma cruzi marinkellei Elicits a Controversial Infection Control.

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.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

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.

Trypanosoma cruzi DTU TcII presents higher blood parasitism than DTU TcI in an experimental model of mixed infection.

Trypanosoma cruzi (Tc), the causative agent of Chagas disease, affects millions of people worldwide. One of the major characteristics of T. cruzi is related to its heterogeneity due to the variability of its biological properties, parasite growth rates, infectivity, tissue tropism, morbidity and virulence among different isolates observed during experimental or human infection. Moreover, presence of mixed infections in the same host in endemic areas is a matter of study due to its impact on clinical manifestations and disease progression. In this study, we evaluated the biological behavior of two Tc I strains AQ1-7 (AQ) and MUTUM (MT) and one Tc II strain (JG) during the acute phase of infection, in unique and mixed infections. A patent blood parasitism was detected only in mice inoculated with JG strain . In addition blood parasitism parameters (peak and average blood parasitism) were positively associated when JG and AQ strains were combined. In contrast, a negative association was observed in the JG+MUTUM group. The predominance of TcII strain over TcI strains was highlighted using the LSSP-PCR technique, which was performed in samples from hemoculture. Thus, this study showed important biological differences between different T. cruzi strains and discrete typing units (DTUs) in acute phase. Finally, we observed that blood parasitism during early period of infection seems to be more related to DTU than to a specific strain.

Immunomodulation by Trypanosoma cruzi: toward understanding the association of dendritic cells with infecting TcI and TcII populations.

Dendritic cells (DCs) are major immune components, and depending on how these cells are modulated, the protective host immune response changes drastically. Trypanosoma cruzi is a parasite with high genetic variability and modulates DCs by interfering with their capacity for antigen recognition, migration, and maturation. Despite recent efforts, the association between DCs and T. cruzi I (TcI) and TcII populations is unknown. Herein, it was demonstrated that AQ1.7 and MUTUM TcI strains present low rates of invasion of bone marrow-derived DCs, whereas the 1849 and 2369 TcII strains present higher rates. Whereas the four strains similarly induced the expression of PD-L1, the production and expression of IL-10 and TLR-2, respectively, in DCs were differentially increased. The production of TNF-α, IL-12, IL-6, and CCL2 and the expression of CD40, CD80, MHC-II, CCR5, and CCR7 changed depending on the strain. The 2369 strain yielded the most remarkable results because greater invasion correlated with an increase in the levels of anti-inflammatory molecules IL-10 and PD-L1 but not with a change in the levels of TNF-α, MHC-II, or CD40 molecules. These results suggest that T. cruzi strains belonging to different populations have evolved specific evasion strategies that subvert DCs and consequently the host response.

High similarity of Trypanosoma cruzi kDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil.

INTRODUCTION: Determining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease. METHODS: An epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture). RESULTS: T. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected. CONCLUSIONS: LSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.

High similarity of Trypanosoma cruzi kDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil

Introduction Determining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease. Methods An epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture). Results T. cruzi I (TcI) was isolated from one child and TcII was found in the remaining (97.1%) subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) showed high heterogeneity among TcII populations (46% of shared bands); however, high similarities (80-100%) among pairs of mothers/children, siblings, or cousins were detected. Conclusions LSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease. .

Effects of cholinergic stimulation with pyridostigmine bromide on chronic chagasic cardiomyopathic mice.

The aim of the present study was to assess the effects of an anticholinesterase agent, pyridostigmine bromide (Pyrido), on experimental chronic Chagas heart disease in mice. To this end, male C57BL/6J mice noninfected (control:Con) or chronically infected (5 months) with Trypanosoma cruzi (chagasic:Chg) were treated or not (NT) with Pyrido for one month. At the end of this period, electrocardiogram (ECG); cardiac autonomic function; heart histopathology; serum cytokines; and the presence of blood and tissue parasites by means of immunohistochemistry and PCR were assessed. In NT-Chg mice, significant changes in the electrocardiographic, autonomic, and cardiac histopathological profiles were observed confirming a chronic inflammatory response. Treatment with Pyrido in Chagasic mice caused a significant reduction of myocardial inflammatory infiltration, fibrosis, and hypertrophy, which was accompanied by a decrease in serum levels of IFNγ with no change in IL-10 levels, suggesting a shift of immune response toward an anti-inflammatory profile. Lower nondifferent numbers of parasite DNA copies were observed in both treated and nontreated chagasic mice. In conclusion, our findings confirm the marked neuroimmunomodulatory role played by the parasympathetic autonomic nervous system in the evolution of the inflammatory-immune response to T. cruzi during experimental chronic Chagas heart disease in mice.

Species-specific markers for the differential diagnosis of Trypanosoma cruzi and Trypanosoma rangeli and polymorphisms detection in Trypanosoma rangeli.

Trypanosoma cruzi and Trypanosoma rangeli are kinetoplastid parasites which are able to infect humans in Central and South America. Misdiagnosis between these trypanosomes can be avoided by targeting barcoding sequences or genes of each organism. This work aims to analyze the feasibility of using species-specific markers for identification of intraspecific polymorphisms and as target for diagnostic methods by PCR. Accordingly, primers which are able to specifically detect T. cruzi or T. rangeli genomic DNA were characterized. The use of intergenic regions, generally divergent in the trypanosomatids, and the serine carboxypeptidase gene were successful. Using T. rangeli genomic sequences for the identification of group-specific polymorphisms and a polymorphic AT(n) dinucleotide repeat permitted the classification of the strains into two groups, which are entirely coincident with T. rangeli main lineages, KP1 (+) and KP1 (-), previously determined by kinetoplast DNA (kDNA) characterization. The sequences analyzed totalize 622 bp (382 bp represent a hypothetical protein sequence, and 240 bp represent an anonymous sequence), and of these, 581 (93.3%) are conserved sites and 41 bp (6.7%) are polymorphic, with 9 transitions (21.9%), 2 transversions (4.9%), and 30 (73.2%) insertion/deletion events. Taken together, the species-specific markers analyzed may be useful for the development of new strategies for the accurate diagnosis of infections. Furthermore, the identification of T. rangeli polymorphisms has a direct impact in the understanding of the population structure of this parasite.

A DTU-dependent blood parasitism and a DTU-independent tissue parasitism during mixed infection of Trypanosoma cruzi in immunosuppressed mice.

Trypanosoma cruzi (Tc) diversity is determined by different biological, genetic, and biochemical markers and has been grouped into six discrete typing units (DTUs) or taxonomic groups (TcI-TcVI). This variability, coupled with natural reinfection or the hosts' immunosuppression, may play an important role in the pathogenesis of Chagas disease. Therefore, we evaluated the blood and tissue parasitism and genetic profile of mice coinfected with the TcII (JG) strain and TcI AQ1-7 (AQ) or MUTUM (MT) strains during the acute and chronic phases of the disease and during immunosuppression. T. cruzi blood populations in mixed infections were clearly associated with the TcII strain during acute and chronic phases or during immunosuppression. However, in tissues, the parasite populations were distributed according to the strain and the stage of infection. TcII populations overlapped TcI strains during the acute phase; in contrast, during chronic phase, both TcI strains were more prevalent than the TcII strain. The immunosuppression induced selective exacerbation of parasite populations, leading to reactivation of the TcII strain when associated with the AQ, but not with MT strain. Thus, a differential distribution of T. cruzi populations in blood and tissues with overlapping according to the stage of infection and strain used was observed. Blood parasitism was associated with the DTU TcII and tissue parasitism with a specific parasite strain and not with DTUs. Finally, to our knowledge, this is the first study to analyze subpatent blood parasitism and to simultaneously identify different T. cruzi populations in tissues and blood.

First evidence of genetic intraspecific variability and occurrence of Entamoeba gingivalis in HIV(+)/AIDS.

Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4(+) lymphocyte count (≤200 cells/mm(3) (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(-) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4(+) lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis isolated from HIV patients.

Gene identification and comparative molecular modeling of a Trypanosoma rangeli major surface protease.

Trypanosoma rangeli is a hemoflagellate parasite which is able to infect humans. Distinct from Trypanosoma cruzi, the causative agent of Chagas disease, T. rangeli is non-pathogenic to the vertebrate host. The manner by which the T. rangeli interacts with the host is still unknown, but it certainly depends on the surface molecules. Major surface proteins (MSP) are GPI-anchored, zinc-dependent metalloproteases present in the surface of all trypanosomatids studied so far, which are implicated as virulence factors in pathogenic trypanosomatids, such as Leishmania spp and T. cruzi. The aims of this work were to generate the complete sequence of a T. rangeli MSP (TrMSP) gene and to determine the 3D-structure of the predicted protein by homology modeling. The plasmid bearing a complete copy of a TrMSP gene was completely sequenced and the predicted protein was modeled using Modeller software. Results indicate that TrMSP open reading frame (ORF) codes for a predicted 588 amino acid protein and shows all elements required for its posttranslational processing. Multiple sequence alignment of TrMSP with other trypanosomatids' MSPs showed an extensive conservation of the N-terminal and central regions and a more divergent C-terminal region. Leishmania major MSP (LmMSP), which had its crystal structure previously determined, has an overall 35% identity with TrMSP. This identity allowed the comparative molecular modeling of TrMSP, which demonstrated a high degree of structural conservation between MSPs from other trypanosomatids (TrypMSPs). All modeled MSPs have a conserved folding pattern, apart from structural divergences in the C-domain and discrete differences of charge and topology in the catalytic cleft, and present the same geometry of the canonical HEXXH zinc-binding motif. The determination of surface charges of the molecules revealed that TrMSP is a predominantly positive protein, whereas LmMSP and Trypanosoma cruzi MSP (TcMSP) are negative proteins, suggesting that substrates recognized by TcMSP and LmMSP could not interact with TrMSP. Moreover, the comparison between TrMSP and TcMSP protein sequences has revealed 45 non-neutral amino acid substitutions, which can be further assessed through protein engineering. The characteristics of TrMSP could explain, at least in part, the lack of pathogenicity of T. rangeli to humans and point to the necessity of identifying the biological targets of this enzyme.

Homogeneity of Trypanosoma cruzi I, II, and III populations and the overlap of wild and domestic transmission cycles by Triatoma brasiliensis in northeastern Brazil.

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.