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.

We have previously demonstrated selection favoring the JG strain of Trypanosoma cruziin hearts of BALB/c mice that were chronically infected with an equal mixture of the monoclonal JG strain and a clone of the Colombian strain, Col1.7G2. To evaluate whether cell invasion efficiency drives this selection, we infected primary cultures of BALB/c cardiomyocytes using these same T. cruzi populations. Contrary to expectation, Col1.7G2 parasites invaded heart cell cultures in higher numbers than JG parasites; however, intracellular multiplication of JG parasites was more efficient than that of Col1.7G2 parasites. This phenomenon was only observed for cardiomyocytes and not for cultured Vero cells. Double infections (Col1.7G2 + JG) showed similar results. Even though invasion might influence tissue selection, our data strongly suggest that intracellular development is important to determine parasite tissue tropism.

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.

Altas temperaturas podem afetar a sobrevivência, estabelecimento e as propriedades simbióticas em estirpes de Rhizobium. As estirpes capazes de nodular o feijoeiro têm sido consideradas particularmente sensíveis, porque nessas estirpes é comum a ocorrência de recombinações e/ou deleções genômicas comprometendo, muitas vezes, a sua utilização como inoculantes. Neste trabalho, procurou-se avaliar a capacidade de crescimento e sobrevivência em temperaturas elevadas de estirpes de Rhizobium efetivas na fixação de nitrogênio no feijoeiro isoladas dos cerrados, bem como avaliar suas características fenotípicas e genotípicas após choque térmico. A capacidade de sobrevivência à temperaturas elevadas, avaliada após choques térmicos sucessivos (45ºC por 4 horas) mostrou ser uma característica própria de cada estirpe, independente de sua termotolerância, que aparentemente foi mais acentuada nas estirpes de R. tropici. Algumas estirpes de R. leguminosarum bv. phaseoli mostraram alterações significativas (Duncan 5% de probabilidade) nas suas características fenotípicas (produção de matéria seca) após choques térmicos e nos seus padrões genômicos evidenciados pela técnica de AP-PCR. As estirpes de R. tropici foram aparentemente mais estáveis não sendo detectadas alterações fenotípicas significativas e com exceção da estirpe FJ2.21, após choque térmico e inoculação na planta hospedeira, mantiveram o padrão genômico original.

High temperatures can affect the survival, establishment and symbiotic properties of Rhizobium strains. Bean nodulating Rhizobium strains are considered particularly sensitive because on this strains genetic recombinations and/or deletions occur frequently, thus compromising the use of these bacteria as inoculants. In this study R. tropici and R. leguminosarum bv. phaseoli strains isolated from Cerrado soils were exposed to thermal stress and the strains’ growth, survival and symbiotic relationships as well as alterations in their genotypic and phenotypic characteristics were analyzed. After successive thermal shocks at 45ºC for four hours, survival capacity appeared to be strain-specific, independent of thermo-tolerance and was more apparent in R. tropici strains. Certain R. leguminosarum bv. phaseoli strains had significant alterations in plant dry weight and DNA patterns obtained by AP-PCR method. R. tropici strains (with the exception of FJ2.21) were more stable than R. leguminosarum bv. phaseoli strains because no significant phenotypic alterations were observed following thermal treatments and they maintained their original genotypic pattern after inoculation in plants.