RESUMEN
Trypanosoma cruzi, the protozoan causative of Chagas disease (ChD), exhibits striking genetic and phenotypic intraspecific diversity, along with ecoepidemiological complexity. Human-pathogen interactions lead to distinct clinical presentations of ChD. In 2009, an international consensus classified T. cruzi strains into six discrete typing units (DTUs), TcI to TcVI, later including TcBat, and proposed reproducible genotyping schemes for DTU identification. This article aims to review the impact of classifying T. cruzi strains into DTUs on our understanding of biological, ecoepidemiological, and pathogenic aspects of T. cruzi. We will explore the likely origin of DTUs and the intrinsic characteristics of each group of strains concerning genome organization, genomics, and susceptibility to drugs used in ChD treatment. We will also provide an overview of the association of DTUs with mammalian reservoirs, and summarize the geographic distribution, and the clinical implications, of prevalent specific DTUs in ChD patients. Throughout this review, we will emphasize the crucial roles of both parasite and human genetics in defining ChD pathogenesis and chemotherapy outcome.
RESUMEN
Trypanosoma cruzi, the agent of Chagas disease (ChD), exhibits remarkable biological and genetic diversity, along with eco-epidemiological complexity. In order to facilitate communication among researchers aiming at the characterisation of biological and epidemiological aspects of T. cruzi, parasite isolates and strains were partitioned into seven discrete typing units (DTUs), TcI-TcVI and TcBat, identifiable by reproducible genotyping protocols. Here we present the potential origin of the genetic diversity of T. cruzi and summarise knowledge about eco-epidemiological associations of DTUs with mammalian reservoirs and vectors. Circumstantial evidence of a connection between T. cruzi genotype and ChD manifestations is also discussed emphasising the role of the host's immune response in clinical ChD progression. We describe genomic aspects of DTUs focusing on polymorphisms in multigene families encoding surface antigens that play essential functions for parasite survival both in the insect vector and the mammalian host. Such antigens most probably contributed to the parasite success in establishing infections in different hosts and exploring several niches. Gaps in the current knowledge and challenges for future research are pointed out.
RESUMEN
Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.
Asunto(s)
Animales , Humanos , Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Medicamentos/genética , Nitroimidazoles/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , ADN Protozoario/genética , Genotipo , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Parasitaria , FilogeniaRESUMEN
This opinion piece presents an approach to standardisation of an important aspect of Chagas disease drug discovery and development: selecting Trypanosoma cruzi strains for in vitro screening. We discuss the rationale for strain selection representing T. cruzi diversity and provide recommendations on the preferred parasite stage for drug discovery, T. cruzi discrete typing units to include in the panel of strains and the number of strains/clones for primary screens and lead compounds. We also consider experimental approaches for in vitro drug assays. The Figure illustrates the current Chagas disease drug-discovery and development landscape.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Descubrimiento de Drogas , Tripanocidas/uso terapéutico , Trypanosoma cruzi/clasificación , Biodiversidad , Ensayos Clínicos como Asunto , Enfermedad de Chagas/parasitología , Estadios del Ciclo de Vida/efectos de los fármacos , Enfermedades Desatendidas/tratamiento farmacológico , Enfermedades Desatendidas/parasitología , Especificidad de la Especie , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC50 to BZ between 7.6-32 µM. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC50 of the strains varied from 15.6 ± 3-51.4 ± 1 µM. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.
Asunto(s)
Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de Chagas , Nitroimidazoles , Tripanocidas , Trypanosoma cruzi , Enfermedad de Chagas , Resistencia a Medicamentos , Repeticiones de Microsatélite , Nitroimidazoles , Pruebas de Sensibilidad Parasitaria , Resultado del Tratamiento , Tripanocidas , Trypanosoma cruziRESUMEN
Classes e subclasses de anticorpos apresentam diferentes funçöes, influenciando a resposta imune humoral de um hospedeiro, frente a um agente infeccioso. Na maioria dos sistemas, o alvo principal é representado pelos antígenos de membrana do parasita. Entretanto, a identificaçäo de antígenos de superficie de parasitas, reativos para classe (e subclasse) de imunoglobulinas que näo se ligam a proteína-A implica em imunoprecipitaçöes sucessivas, que levam a perda de antígenos e/ou reaçöes inespecíficas. Visando esse estudo, foi desenvolvida ua técnica denominada "radio-immunoblotting", através da qual a reatividade de imunoglobulinas de diferentes classes para antígenos de membrana (e/ou internos) foi analisada simultaneamente. O método constitui na marcaçäo prévia da superfície dos parasitas por radioiodaçäo, fracionamento dos polipeptídeos por SDS/PAGE, transferência das fraçöes para nitrocelulose, reaçäo com soros e conjugados anti-Igs - peroxidase a autroradiografia dos mesmos, a análise é feita comparando-se os antígenos comuns evidenciados na autoradiografia e nas tiras de nitrocelulose coradas com o substrato da peroxidase. Essa técnica foi utilizada para analisar antígenos de superfície de formas tripomastigotas de T. cruzi reativas para IgG, IgM e IgA provenientes de soros de pacientes com doença de Chagas na fase aguda. Obtiveram-se distintos padröes de reatividade para as diferentes classes de anticorpos provenientes de um mesmo soro humano
Asunto(s)
Humanos , Antígenos de Protozoos/análisis , Antígenos de Superficie/análisis , Trypanosoma cruzi/inmunología , Western Blotting , Enfermedad de Chagas/inmunología , Inmunoglobulina G/análisisRESUMEN
Meeting of the Biotechnology Subcommittee Advisory Committee on Health Research in Buenos Aires, Argentina on April 16-19, 1996
Meeting of the Advisory Committee on Health Research, 31. Pan American Health Organization; Jul. 15-17, 1996
Asunto(s)
Biotecnología , Investigación sobre Servicios de SaludRESUMEN
Meeting of the Biotechnology Subcommittee Advisory Committee on Health Research in Buenos Aires, Argentina on April 16-19, 1996
Reunión del Comité Asesor de Investigaciones en Salud, 31. Organizacion Panamericana de la Salud; Jul. 15-17, 1996
Asunto(s)
Biotecnología , Investigación sobre Servicios de SaludRESUMEN
Immunoglobbulin G and M humoral response to recombinant protein B13 and glycoconjugate LPPG Trypanosoma cruzi defined antigens was evaluated by ELISA in 18 patients in the acute phase of Chagas disease, who were contaminated on the same occasion. LPPG showed 100 por cento positivity detecting both IgM and IgG antibodies, while positivity of 55-65 por cento was observed for B13. An epimastigote alkaline extract (EPI) also showed high sensitivity for acute IgM (100 por cento) and IgG (90 por cento) antibodies. However LPPG had better discriminatory reactivity since with EPI two patients showed negative IgG and several other sera presented OD values for IgG and IgM antibodies very close to the cutoff. Thus, it is suggested that detection of Igm antibodies by LPPG may be used for diagnosis of the acute phase of Chagas disease. An intense decline of IgG and IgM antibodies to three antigens was observed in response to anti-T. cruzi chemoterapy in all acute phase patients. After treatment, six (30 por cento) individuals maintained IgG positivity to EPI, LPPG, and B13 with lower reactivity than that measured at the acute phase. For comparison, serology of a group of 22 patients in the chronic phase of Chagas disease and also submitted to chemotherapy was determined. Positive IgM antibodies to EPI, LPPG abd B13 were detected in only 5-9 por cento cases. In all chronic-phase patients IgG antibodies highly reactive to the three antigens were present and no significant decrease resulted after benznidazole administration. These observations reinforce previous reports that treatment in the acute phase may reduce or eliminate the parasite.
Asunto(s)
Humanos , Animales , Inmunoglobulina G/análisis , Trypanosoma cruzi/inmunología , Anticuerpos Antivirales/análisis , Enfermedad de Chagas/inmunología , Inmunoglobulina M/análisisRESUMEN
Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains
Asunto(s)
Animales , Humanos , Repeticiones de Microsatélite , Trypanosoma cruzi/genética , ADN Protozoario/análisis , ADN Protozoario/genética , Genotipo , Técnicas de Amplificación de Ácido Nucleico , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo Genético/genética , Sensibilidad y EspecificidadRESUMEN
Clone CL Brener is the reference organism used in the Trypanosma cruzi Genome Project. Some biological paramenters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28ºC is 58ñ13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20 per cent Grace's medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37ºC; (c) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24 alpha ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed.
Asunto(s)
Animales , Células Clonales/microbiología , Trypanosoma cruzi/genética , Genoma de ProtozoosRESUMEN
The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/lOJ) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected
Asunto(s)
Ratones , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Enfermedad de Chagas/inmunología , Trypanosoma cruzi/inmunología , Reacciones Antígeno-Anticuerpo , Western Blotting , Susceptibilidad a Enfermedades , Electroforesis en Gel de Poliacrilamida , Sueros Inmunes , Isotipos de Inmunoglobulinas/análisis , Ratones Endogámicos A , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
We evaluated the performance of two defined antigens in the serological diagnosis of Chagas' disease. One of them is a recombinant protein named B13 isolated from a genomic library of Trypanosoma cruzi in the expression vector lambda gtll. We show that the gene corresponding to B13 is conserved in the evolutive stages of the two ®polar® strains of T. cruzi. The protein epitopes cloned in B13 are represented in 140 kDa, 116 kDa and 35 kDa polypeptides of trypomastigotes. The other antigen chosen for serodiagnosis is a lipopeptidophosphoglycan (LPPG). This glycoconjugate is also widely distributed in T. cruzi strains. The use of a rabbit serum to LPPG allowed the demonstration that this molecule bears epitopes in common to LPPG-like components and to 80-90 kDa glycoproteins of trypomastigotes. Both B13 and LPPG were evaluated in serodiagnosis by ELISA and RIA using a panel of normal human, Chagasic and Leishmaniasis sera. It was observed that B13 presents high sensitivity and specificity for Chagasic sera. For LPPG it was also concluded that this reagent discriminates between individuals infected and non-infected with T. cruzi. A heterogeneity in the level of antibodies to LPPG in Chagasic patients was detected. No correlation was found between the clinical form of Chagas' disease and the preferential reactivity to B13 or LPPG. We also report preliminary studies towards the characterization of a 100 bp sequence of the 24S alpha rRNA as a target for DNA-based detection systems for diagnosis. We show that polymerase chain reaction of total DNA of different trypanosomatids lead to the specific amplification of a 100 bp fragment only for T. cruzi. Northern blots confirmed the presence of the target region in the mature 24S alpha rRNA. Titration experiments based on the direct amplification of RNA with Taq DNA polymerase allowed the detection of 50 parasites. Studies are in progress to increase the sensitivity of the proposed system