Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

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.

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009).

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.

Reinfections with strains of Trypanosoma cruzi, of different biodemes as a factor of aggravation of myocarditis and myositis in mice.

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.

Reinfections with strains of Trypanosoma cruzi, of different biodemes as a factor of aggravation of myocarditis and myositis in mice

Reinfeccões pelo Trypanosoma cruzi em pacientes de áreas endêmicas têm sido mencionadas como fator agravante das manifestacões cardíacas na doenca de Chagas. No presente estudo, a influência da tríplice infeccão 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 - reinfeccão com a cepa Y seguida pela cepa 21SF. A análise isoenzimática dos parasitas das hemoculturas obtidas dos animais com tríplice infeccão, 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 inoculacão com o sangue de camundongos com tríplice infeccão. Resultados da histopatologia demonstraram uma significante exacerbacão das lesões inflamatórias de miocárdio e músculo esquelético, confirmadas pela avaliacão morfométrica. Não foi detectada acentuacão do parasitismo. A possibilidade de aumento da resposta celular nos animais com tríplice infeccão é sugerida.

Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined

Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study.

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500 microliters of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500 microliters of normal mice blood. Positive control: 500 microliters of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers: S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500 microliters of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

Biodemes and zymodemes of Trypanosoma cruzi strains: correlations with clinical data and experimental pathology

With the objective of establishing biological and biochemical characteristics of a significant number of Trypanosoma cruzi strains from different geographical areas, 138 strains isolated from naturally infected humans, triatomine or vertebrate hosts were studied; 120 were isolated from different areas of Brazil and 18 from other South and Central American countries. Inocula from triatomine or culture forms were injected into suckling Swiss mice, followed by passages into mice 10 to 12 g. Biological characters and histopathological study permitted the inclusion of the strains into three Types or biodemes: I, II, III. Isoenzymic analysis confirmed a correspondence between the biodemes and zymodemes: Type I and Z2b, Type II and Z2, Type III and Z1. Results showed the ubiquitary distribution of the several types of strains. The predominance of the same Type and zymodeme in one geographical area was confirmed: Type II strains among the human cases from eastern Bahia and east of Goiás; Type III strains from humans of north Brazil and Central America and from silvatic vectors or vertebrates from other geographical areas. The biological types of strains correlate with different histopathological lesions considering cardiac involvement and neuronal lesions. These findings suggest that the biological behavior together with isoenzymes patterns and pathological pictures in the vertebrate host can be an important tool for establishing correlations between strains behavior and clinico-pathological manifestations of Chagas' disease in different geographical areas.

Trypanosoma cruzi strains: behavior after passage into authoctonous or foreign species of treatomine (biological and biochemical patterns)

Foi estudada a influencia da passagem de cepas do T. cruzi de Sao Felipe - BA (19 SF, 21 SF e 22 SF), Tipo II, Zimodema 2, no vetor autoctone (P. megistus) e em vetores nao autoctones (T. infestans e R. prolixus). Para cada cepa, camundongos suicos de 10 a 12 g foram inoculados com I - formas sanguicolas (controles); II - metaciclicos do P. megistus; III - metaciclicos de T. infestans; IV - metaciclicos do R. prolixus. O inoculo para cada grupo foi de 10 a quarta potencia tripomastigotas. A obtencao dos metaciclicos foi feita 60 a 120 dias apos infeccao dos triatomineos...

Investigation on the possibility of spontaneous cure mice infected with diferent strains of Trypanosoma cruzi

Setenta camundongos suicos, cronicamente infectados com diferentes cepas do Trypanosoma cruzi, cuja parasitemia se manteve negativa ao exame de rotina do sangue periferico, foram investigados parasitologicamente com o objetivo de verficar se houve cura espontanea dos mesmos. A duracao da infeccao variava entre 90 e 250 dias quando os camundongos foram inicialmente investigados. Os testes parasitologicos usados foram: pesquisa direta de parasitos no sangue periferico, feita diariamente, durante pelo menos 25 dias, seguida de xenodiagnostico e subinoculacao do sangue em camundongos recem-nascidos. Os camundongos que mostraram resultados negativos foram tratados com uma dose de Ciclosfofamida de 250mg/kg peso corporal foi aplicada aos camundongos cronicamente infectados tiveram testes parasitologicos positivos em diferentes etapas da presente investigacao. Concluimos que neste grupo representativo da fase cronica da infeccao por diferentes cepas do T. cruzi, nao ocorreu cura espontanea.

Estudo do comportamento de cepas de Trypanosoma cruzi após passagem em diferentes espécieis de triatomíneos / Behavior of Trypanosoma cruzi strains after passage in different triatominae species

Foi estudada a influência da passagem em diferentes espécies de triatomíneos de cepas do T. cruzi biologicamente distintas, mantidas em camundongos. Cepas: Peruana (Tipo I), 12 SF (Tipo II), Colombiana (Tipo III), Triatomíneos: Rhodnius prolixus, Panstrongylus megistus e Triatoma ingestans. Ninfas do 4§ estágio foram alimentadas em camundongos infectados com as respectivas cepas e obtidos os inóculos (10**4 formas metacíclicas) para infecçäo de camundongos de 8 a 10g. Grupos experimentais para cada cepa: I - Controles inoculados com formas sanguícolas; II, III, IV - inoculados com metacíclicos de P. megistus, T. infestans ou R. prolixus, respectivamente. Os perfis de parasitemia foram mantidos para cada cepa, com piques mais elevados nas infecçöes com metacíclicos do P. megistus e do R. prolixus, para a cepa Peruana, do P. megistus para a 12 SF e do R. prolixus, para a Colombiana. O histotropismo e o padräo de lesöes foram mantidos para três tipos de cepas. O grau de virulência da cepa Peruana foi idêntico em todos os grupos e variou para as cepas 12 SF e Colombiana de acordo com a espécie de triatomíneo usada, com predominância de formas delgadas, nos inoculados com formas metacíclicas o que foi interpretado como uma fase adaptativa das formas metacíclicas ao hospedeiro vertebrado

Terapêutica da fase crônica da infecçäo experimental pelo Trypanosoma cruzi com o benzonidazol e o nifurtimox / Therapy of the chronic phase of the experimental infection by Trupanosoma cruzi with benzonidazole and nifurtimox

Foram submetidos à quimioterapia com Nifurtimox (Bay 2502) ou com o Benzonidazol (Ro7-1051) cinqüenta e oito camundongos cronicamente infectados com diferentes cepas do Trypanosoma cruzi (Tipos II e III) por períodos de 90 a 100 dias. Vinte e um camundongos cronicamente infectados foram utilizado como controles näo tratados. Os inóculos variaram ente 1 x 10***4 e 5 x 10***4 tripomastigotas sanguícolas. O tratamento foi feito durante 90 dias, nas doses de 200 mg/Kg/dia durante 4 dias, seguidas de doses de 50 mg/Kg/dia, para o Nifurtimox e de 100 mg/Kg/dia, para o Benzonidazol. Os resultados dos testes parasitológicos (xenodiagnóstico, subnoculaçäo do sangue e hemocultura), nos camundongos tratados com Benzonidazol, mostraram 85,3% de negativaçäo nos animais infectados com as cepas de Tipo II e 43% com as cepas de Tipo III. Nos camundongos tratados com o Nifurtimox observou-se 71,4% de cura parasitológica nos infectados com as cepas de Tipo II e 66% nos infectados com as cepas de Tipo III. Os testes de IFI permaneceram positivos em 90% dos animais tratados e curados. Houve regressäo total ou parcial das lesöes miocárdicas e de músculo esquelético nos animais tratados e curados. Em 50% dos animais em que os testes de cura permaneceram positivos, houve persistência de lesöes histopatológicas discretas. Conclui-se que o tratamento na fase crônica pode determinar negativaçäo parasitológica em percentagem elevada de casos, compáravel ao obtido na fase aguda, com as cepas de Tipo II e mais elevadas com as cepas de Tipo III, com persistência da positividade da IFI

Aspectos imunológicos da infecçäo de seis linhagens isogênicas de camundongos por três diferentes cepas do Trypanosoma cruzi / Immunological aspects of infection of 6 inbred strains of mice by 3 different strains of Trypanosoma cruzi

Foram avaliados aspectos da imunidade humoral e celular em seis linhagens de camundongos isogênicos (BALB/c, B-10, C3H, A/J, AKR e DBA) infectados por três cepas do Trypanosoma cuzi, representantes dos três tipos de cepas da classificaçäo de Andrade (cepas Peruana, 21SF e Colombiana). A imunidade celular, avaliada pelo teste de hipersensibilidade cutânea tardia contra antigenos do parasita, estava suprimida. A avaliaçäo dos níveis de imunoglobulinas (imunodifusäo radial), mostrou queda precose de IgG1 e elevaçäo de IgM em praticamente todas as linhagens infectadas por qualquer das cepas estudadas. A elevaçäo de IgG2a e/ou IgG2b foi mais intensa nas linhagens mais resistentes. Os níveis de anticorpos anti-T, cruzi (Imunofluorescência indireta e ELISA) näo se correlacionam com a sobrevida dos animais. Apesar de diferenças entre as linhagens observou-se uma regularidade na resposta do hospedeiro e a manutençäo dos padröes biológicos que caracterizam os tipos de cepa

Comportamento das cepas Y e Peruana do Trypanosoma cruzi no camundongo, após passagem em diferentes meios / Behavior of the Y and Peruvian strains of Trypanasoma cruzi in mice, ofter passage through different media

Com o objetivo de verificar se o comportamento morfobiológico e histopalógico de cerpa do Trypanosoma cruzi é mantido após modificaçöes no seu meio de manutençäo e multiplicaçäo, foram estudados oito grupos de infecçäo experimental em camundongos com as cepas Y e Peruana, após manutençäo nas seguintes condiçöes cultura acelular em meio de Warren, criopreservaçäo em Nitrogênio líquido durante trinta dias, passagem em triatomíneos e passagem em camundongos. Os parâmetros avaliados foram: parasitemia, mortalidade, sobrevida, morfologiaq dos parasitos no sangue periférico, tropismo tíssular e lesöes histopatológicoas. Tanto com a cepa Y como Peruana, cada grupo experimental foi dividido em dois subgrupos de acordo com a dose de inóculo, sendo um inoculado com 10.000 e outro com 50.000 formas tripomastigotas, menos o inoculado com formas de triatomíneos em que o inóculo foi apenas com 10.000 tripomastigotas. Observou-se na infecçäo pela cepa Y, retardo na evoluçäo da parasitemia nos inoculados com formas de cultura e atenuaçäo de virulência com o inóculo de 10.000 formas. Os caracteres básicos da cepa foram mantidos com predominância de macrofagotropismo na fase inicial da infecçäo e miotropismo nas fases tardias,predominância de forma delgadas e elevada patogenicidade, com 100% de mortalidade embora com variaçäo nos periodos de sobrevida. Na infecçäo pela cepa Peruana observou-se também retardo da evoluçäo da parasitemia com o inóculo de 10.000 formas provenientes de triatomíneos, de cultura e de criopreservaçäo. Entretanto, as características morfológicas, o tropismo tissular e a patogenicidade foram mantidos