Navegando por Autor "Freitas, Jorge Marcelo de"
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Item Further genetic characterization of the two Trypanosoma cruzi Berenice strains (Be-62 and Be-78) isolated from the first human case of Chagas disease (Chagas, 1909).(2006) Cruz, Ruth Elizabeth; Macedo, Andréa Mara; Barnabé, Christian; Freitas, Jorge Marcelo de; Chiari, Egler; Veloso, Vanja Maria; Carneiro, Cláudia Martins; Bahia, Maria Terezinha; Tafuri, Washington Luiz; Lana, Marta deWe describe here an extension of a previous genetic characterization of Trypanosoma cruzi strains (Be-62 and Be-78) isolated from the patient Berenice, the first human case of Chagas disease [Chagas, C., 1909. Nova Tripanom´ıase humana. Estudos sobre morfologia e o ciclo evolutivo do Schizotrypanum cruzi, n. gen., n. sp., agente etiol´ojico da nova entidade morbida do homem. Mem. Inst. Oswaldo Cruz 1, 159–218]. We wanted to verify the composition of T. cruzi populations originated from these two isolates. In the present work, 22 enzymatic loci (MLEE), nine RAPD primers and 7 microsatellite loci were analyzed. Clones from both strains were also characterized to verify whether these strains are mono or polyclonal. Be-62 and Be-78 strains were different in 3 out of 22 enzymatic systems, in 3 out of 9 RAPD primers tested and in all microsatellite loci investigated. However, our data suggests that both strains are phylogenetically closely related, belonging to genetic group 32 from Tibayrenc and Ayala [Tibayrenc, M., Ayala, F.J., 1988. Isoenzime variability in Trypanosoma cruzi, the agent of Chagas’ disease: genetical, taxonomical, and epidemiological significance. Evolution 42, 277–292], equivalent to zymodeme 2 and T. cruzi II major lineage which, in Brazil, comprises parasites from the domestic cycle of the disease. Microsatellite analyses showed differences betweenthe parental strains but suggested that both populations are monoclonal since each strain and their respective clones showed the same amplification products.Item Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites.(2008) Pimenta, Juliana Ramos; Freitas, Jorge Marcelo de; Duffy, Tomás; Bartholomeu, Daniella Castanheira; Oliveira, Riva de Paula; Chiari, Egler; Moreira, Maria da Consolação Vieira; Brasileiro Filho, Geraldo; Schijman, Alejandro Gabriel; Franco, Glória Regina; Machado, Carlos Renato; Pena, Sérgio Danilo Junho; Macedo, Andréa MaraThe investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci – six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.