We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.

Reinfecções pelo Trypanosoma cruzi em pacientes de áreas endêmicas têm sido mencionadas como fator agravante das manifestações cardíacas na doença de Chagas. No presente estudo, a influência da tríplice infecção com cepas de diferentes biodemas, sobre as lesões do miocárdio e de músculo esquelético foi investigada experimentalmente. Cinqüenta e oito camundongos cronicamente infectados com a cepa Colombiana do Trypanosoma cruzi (Biodema Tipo III) foram sucessivamente reinoculadas como a seguir: 1º grupo - reinfectados com a cepa 21 SF (Tipo II) seguido pela cepa Y (Tipo I); 2º grupo - reinfecção com a cepa Y seguida pela cepa 21SF. A análise isoenzimática dos parasitas das hemoculturas obtidas dos animais com tríplice infecção, revelou os padrões dos diferentes zimodemas no mesmo animal. Cada cepa do Trypanosoma cruzi foi re-isolada após quatro passagens em camundongos no 7º, no 14º, ou no 30º dia após a inoculação com o sangue de camundongos com tríplice infecção. Resultados da histopatologia demonstraram uma significante exacerbação das lesões inflamatórias de miocárdio e músculo esquelético, confirmadas pela avaliação morfométrica. Não foi detectada acentuação do parasitismo. A possibilidade de aumento da resposta celular nos animais com tríplice infecção é sugerida.

Reinfections with Trypanosoma cruzi in patients from endemic areas have been claimed to be an aggravation factor of cardiac manifestations in Chagas' disease. In the present study, the influence of triple infections with strains of different biodemes, on cardiac and skeletal muscle lesions was experimentally tested. Fifty eight mice chronically infected with the Colombian strain (Biodeme Type III) were successively reinfected as follows: 1st group - reinfected with 21 SF strain (Type II) followed by Y strain (Type I ); 2nd - group reinfections with Y strain followed by 21SF strain. Isoenzyme analysis of parasites from hemocultures obtained from triple infected mice, revealed the patterns of three distinct zymodemes in the same animal. Each Trypanosoma cruzi strain was reisolated after four passages in mice on either the 7th, 14th or 30th day after inoculation with the blood of triple infected mice. Histopathology results demonstrated a significant exacerbation of cardiac and skeletal muscle inflammatory lesions, confirmed by morphometric evaluation, in mice with triple infection. No aggravation of parasitism was detected. The possibility of an enhancement of cellular response in the triple infected mice is suggested.

A suscetibilidade à quimioterapia com o benzonidazol, de 5 clones isolados da cepa 21SF (biodema Tipo II, T. cruzi II), foi investigada. Camundongos suíços foram infectados com a cepa parental e com cada clone e submetidos à quimioterapia com benzonidazol (100mg/k/dia durante 90 dias). Os índices de cura foram avaliados pelos testes de cura parasitológicos. A sorologia foi avaliada para os animais tratados e (de negativo a 1: 640) e para os controles não tratados( 1:160 a 1:640). Os índices de cura variaram de 30% a 100% para os 5 clones sendo de 25% para a cepa parental. Os resultados sugerem que a variabilidade de resposta ao tratamento das populações clonais das cepas Trypanosoma cruzi II é responsável pela grande variação na resposta à quimioterapia com benzonidazol e nifurtimox das cepas deste biodema.

Susceptibility to chemotherapy with benznidazole was investigated of 5 clones isolated from the 21 SF strain (biodeme Type II, Trypanosoma cruzi II). Swiss mice were infected with the parental strain for each clone and submitted to chemotherapy with benznidazole (100mg/kg/day during 90 days). Treatment determined negativity of the parasitemia. Cure rates were evaluated by parasitological cure tests. Serology was evaluated for treated animals (titers from negative to 1:640) and untreated controls (1:160 to 1:640). Cure rates varied from 30 to 100% for the 5 clones, and were 25% for the parental strain. Results suggested that the variability of response to treatment of the clonal populations of Trypanosoma cruzi II strains is responsible for the high variation in the response to chemotherapy with benznidazole and nifurtimox by strains of this biodeme.

A estrutura clonal da cepa Colombiana do Trypanosoma cruzi, biodema Tipo III e zimodema 1, foi analisada com o objetivo de caracterizar as suas populações e estabelecer a homogeneidade ou heterogeneidade das mesmas. Foram isolados sete clones, os quais apresentaram as características básicas do biodema Tipo III, com o mesmo padrão de curvas parasitêmicas, tropismo tecidual para músculo esquelético e miocárdio, alta patogenicidade, com extensas lesões necrótico-inflamatórias, do 20º ao 30º dia de infecção. A cepa parental e os clones C1, C3, C4 e C6 apresentaram os niveis mais elevados de parasitemia entre 20 e 30 dias pós-infecção e alto indice de mortalidade até 30 dias (79 a 100%); os clones C2, C5 e C7 apresentaram niveis mais baixos de parasitemia com baixa mortalidade até 30 dias (7,6 a 23%). Os padrões isoenzimáticos foram característicos do zimodema 1 (Z1) para a cepa parental e os sete clones. Os achados do presente trabalho indicam uma homogeneidade fenotípica entre os clones isolados da cepa Colombiana e sugerem a predominância de um clone principal, responsável pelo padrão de comportamento biológico da cepa parental e dos clones.

The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%); clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%). Isoenzymic patterns, characteristic of zymodeme 1, (Z1) were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.