RESUMO
The discovery and characterization of novel parasite antigens to improve the diagnosis of Trypanosoma cruzi by serological methods and for accurate and rapid follow-up of treatment efficiency are still needed. TcTASV is a T. cruzi-specific multigene family, whose products are expressed on the parasite stages present in the vertebrate host. In a previous work, a mix of antigens from subfamilies TcTASV-A and TcTASV-C (Mix A + C) was sensitive and specific to identify dogs with active infection of high epidemiological relevance. Here, TcTASV-A and TcTASV-C were assayed separately as well as together (Mix A + C) in an ELISA format on human samples. The Mix A + C presented moderate sensitivity (78%) but high diagnostic accuracy with a 100% of specificity, evaluated on healthy, leishmaniasic, and Strongyloides stercoralis infected patients. Moreover, antibody levels of pediatric patients showed-2 years posttreatment-diminished reactivity against the Mix A + C (P < 0.0001), pointing TcTASV antigens as promising tools for treatment follow-up.
Assuntos
Antígenos de Protozoários/sangue , Antiprotozoários/uso terapêutico , Doença de Chagas/sangue , Doença de Chagas/tratamento farmacológico , Trypanosoma cruzi/imunologia , Anticorpos Antiprotozoários/imunologia , Biomarcadores/sangue , Criança , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Sensibilidade e EspecificidadeRESUMO
Background. Endemic areas of tegumentary leishmaniasis (TL) in Salta, Argentina, present some overlap zones with the geographical distribution of Chagas disease, with mixed infection cases being often detected. Objectives. The purpose of this study was to determine the magnitude of Leishmania sp. infection and potential associated risk factors, the serologic prevalence of T. cruzi, and the presence of T. cruzi-Leishmania sp. mixed infection in a region of the northwest of Argentina. Methods. Cross-sectional studies were conducted to detect TL prevalence and T. cruzi seroprevalence. A case-control study was conducted to examine leishmaniasis risk factors. Results. Prevalence of TL was 0.17%, seroprevalence of T. cruzi infection was 9.73%, and mixed infection proportion-within the leishmaniasic patients group-was 16.67%. The risk factors associated with TL transmission were sex, age, exposure to bites at work, staying outdoors more than 10 hours/day, bathing in the river, and living with people who had lesions or were infected during the study. Discussion. The endemic pattern of TL seems to involve exposure of patients to vectors in wild as well as peridomestic environment. Cases of T. cruzi infection are apparently due to migration. Therefore, a careful epidemiological surveillance is necessary due to the contraindication of antimonial administration to chagasic patients.
Assuntos
Coinfecção/parasitologia , Leishmaniose/epidemiologia , Trypanosoma cruzi , Tripanossomíase/epidemiologia , Adolescente , Adulto , Idoso , Argentina/epidemiologia , Estudos de Casos e Controles , Doença de Chagas/epidemiologia , Criança , Coinfecção/epidemiologia , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Geografia , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Floresta Úmida , Fatores de Risco , Estudos Soroepidemiológicos , Clima Tropical , Adulto JovemRESUMO
In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/isolamento & purificação , Doença de Chagas/diagnóstico , Doença de Chagas/veterinária , Doenças do Cão/diagnóstico , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/imunologia , Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Cães , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Camundongos , Sensibilidade e EspecificidadeRESUMO
The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco.
Assuntos
Doença de Chagas/parasitologia , Doença de Chagas/veterinária , Doenças do Cão/sangue , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Adolescente , Adulto , Animais , Argentina , Doença de Chagas/sangue , Doença de Chagas/genética , Criança , Coinfecção , Estudos Transversais , DNA de Protozoário/genética , Doenças do Cão/parasitologia , Cães , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , População Rural , Trypanosoma cruzi/isolamento & purificação , Adulto JovemRESUMO
Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI.
Assuntos
Doença de Chagas/parasitologia , Evolução Molecular , Variação Genética , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Alelos , Argentina , DNA Intergênico , Loci Gênicos , Genótipo , Humanos , Desequilíbrio de Ligação , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Filogenia , Polimorfismo Genético , RNA Líder para ProcessamentoRESUMO
Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.
Assuntos
Tipagem de Sequências Multilocus/métodos , Parasitologia/métodos , Trypanosoma cruzi/genética , GenótipoRESUMO
The biological behavior of the different Trypanosoma cruzi strains is still unclear and the importance of exploring the relevance of these differences in natural isolates is of great significance. Herein we describe the biological behavior of four T. cruzi isolates circulating sympatrically in a restricted geographic area in Argentina endemic for Chagas Disease. These isolates were characterized as belonging to the Discrete Typing Units (DTUs) TcI, TcIII, TcV and TcVI as shown by Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing. In order to study the natural behavior of the different isolates and to preserve their natural properties, we developed a vector transmission model that allows their maintenance in the laboratory. The model consisted of serial passages of these parasites between insect vectors and mice. Vector-derived parasite forms were then inoculated in C57BL/6J mice and number of parasite in peripheral blood, serological response and histological damage in acute and chronic phases of the infection were measured. Parasites from DTUs TcI, TcIII and TcVI were detected by direct fresh blood examination, while TcV parasites could only be detected by Polimerase Chain Reaction. No significant difference in the anti-T. cruzi antibody response was found during the chronic phase of infection, except for mice infected with TcV parasites where no antibodies could be detected. Histological sections showed that TcI isolate produced more damage in skeletal muscle while TcVI induced more inflammation in the heart. This work shows differential biological behavior among different parasite isolates obtained from the same cycle of transmission, permitting the opportunity to formulate future hypotheses of clinical and epidemiological importance.
Assuntos
Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Doenças Endêmicas , Trypanosoma cruzi/patogenicidade , Animais , Anticorpos Antiprotozoários/sangue , Argentina/epidemiologia , Sangue/parasitologia , Doença de Chagas/imunologia , Doença de Chagas/patologia , Impressões Digitais de DNA , DNA de Protozoário/genética , Modelos Animais de Doenças , Enzimas/análise , Variação Genética , Coração/parasitologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/parasitologia , Músculo Esquelético/patologia , Miocárdio/patologia , Trypanosoma cruzi/classificação , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference.
Assuntos
DNA Intergênico/genética , Variação Genética , Tipagem de Sequências Multilocus/métodos , RNA Líder para Processamento , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Argentina , Teorema de Bayes , Doença de Chagas/parasitologia , Doença de Chagas/transmissão , DNA de Protozoário/genética , Genótipo , Geografia , Haplótipos , Humanos , Repetições de Microssatélites , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Of 139 children who received an orthotopic liver transplant in our center between March 1984 and July 1989, a total of 17 patients (12%) had transplants before their first birthday (mean age 10.3 months; range 8 to 11). The mean weight was 7.3 kg (range 5.2 to 13). Nine retransplantations were performed in five children because of primary nonfunction (three children), hepatic artery thrombosis (four), or rejection (two). A reduced donor liver was used for 11 of 26 transplants. Baseline immunosuppression included cyclosporine, prednisone, and azathioprine with OKT3 or anti-thymocyte globulin for steroid-resistant rejection episodes. Survivors were discharged after a mean hospital stay of 47 days (range 22 to 87), and nonsurvivors died within a mean of 40 days (range 0 to 120). The 1 year actuarial survival rate was 64.7%, in comparison with 75.8% in the whole series. One patient died perioperatively, two died from primary nonfunction, one from adenovirus infection, two from rejection, and one from bone marrow aplasia. Eighteen rejection episodes, of which 11 were steroid resistant, occurred in 11 patients. Our series shows that liver transplantation can be successful in this age group.