Biodemas de cepas do Trypanosoma cruzi isoladas de humanos de três áreas endêmicas de Minas Gerais

Com o objetivo de estudar o comportamento biológico de cepas do T. cruzi e determinar a possível relação entre o biodema da cepa e as formas clínicas da doença de Chagas, 14 cepas do protozoário isoladas de humanos, procedentes de Pains, Iguatama e Berilo, foram avaliadas. Para estudar o comportamento biológico das cepas, grupos de camundongos albinos suíços, machos, pesando entre 10-15 gramas foram infectados com 1x10(4) tripomastigotas sangüíneos para avaliação da infectividade, parasitemia, morfologia dos tripomastigotas sangüíneos, virulência e distribuição do protozoário nos tecidos. Nove cepas apresentaram comportamento similar ao da cepa São Felipe do biodema II e cinco se caracterizaram como pertencentes ao biodema III. Não foi possível estabelecer uma relação entre o biodema da cepa e gravidade da doença nos pacientes.

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.

Caracterização biológica, histopatológica e análise de ácido nucléico de uma cepa Trypanosoma cruzi da região de Marília, SP

Estudou-se o comportamento biológico e histopatológico de uma cepa genuínamente mariliense de Trypanosoma cruzi, isolada em 1997 através de xenodiagnóstico artificial. Vinte e cinco camundongos swiss foram infectados intraperitonealmente, sendo 11 utilizados para a realização da curva parasitêmica e observação da morfologia dos tripomastigotas e 14 foram sacrificados após o 17, 23, 30, 60 e 180 dias pós-infecção e coletados coração, esôfago, fígado, cólon, e músculo esquelético (fragmento da coxa direita) para análise histopatológica. Cultura em meio LIT foi realizada para análise de DNA. Os resultados mostraram predomínio de formas largas, baixa parasitemia com picos médios de 860 tripomastigotas/5mil de sangue ao redor do 20º dia de infecção. Nenhum camundongo morreu na fase aguda da infecção. Exame histopatológico mostrou poucos ninhos de amastigotas em coração, raros em músculo esquelético e cólon com discreto processo inflamatório. Comparada com a cepa Y, que foi isolada de uma paciente da mesma região, notamos diferentes características biológicas e comportamentais, porém a análise de DNA as coloca no mesmo grupo, demonstrando a proximidade dessas cepas.

New evidence of spontaneous cure in human Chagas' disease

A new case of spontaneous cure of human Chagas' disease is described in Uruguay. An 87-year-old man who had a typical acute phase of Trypanosoma cruzi infection in 1947 and never received specific treatment against the disease, when examined in 1998 revealed several completely negative parasitological and serological tests, including traditional serology, PCR and flow cytometry. As a whole, such findings fulfill the current criteria to define the cure of Chagas' disease. Clinical data suggest the possibility of a benign evolution of Chagas' disease in this case, but the basic findings (slight cardiac and esophageal impairment) could also be due to the advanced age of the patient.

Trypanosoma cruzi: susceptibility to chemotherapy with benznidazole of clones isolated from the highly resistant Colombian strain

The present investigation was performed to evaluate the susceptibility of seven clones isolated from the highly resistant Colombian strains, prototype of Biodeme Type III. Seven clones previously obtained, showed a phenotypic homogeneity and high similarity with the parental strain. Eight groups of 30 mice were inoculated with one of seven clones or the parental strain; 20 were treated with benznidazole (100mg/kg/day) and 10 were untreated controls. Cure evaluations were done by parasitological and serological tests and PCR. Cure rates varied from 0% (null) to 16.7%. Correlation between positivity of parasitological and serological tests with positive PCR reached 37%. The results demonstrated the high resistance of the clones, suggesting the predominance of a highly resistant principal clone in this strain. The findings apparently indicate that the possibility of cure is minimal for patients infected with this biodeme; a fact that could affect the control of Chagas' disease through treatment of chronically infected people.

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.

Evaluation of the immune response to CRA and FRA recombinant antigens of Trypanosoma cruzi in C57BL/6 mice

Humoral and cellular immune responses were evaluated in 44 C57BL/6 mice immunized with the Trypanosoma cruzi recombinant antigens CRA and FRA. Both antigens induced cutaneous immediate-type hypersensitivity response. The levels of IgG1, IgG2a, IgG2b and IgG3 were high in CRA immunized mice. IgG3 was the predominant isotype. Although no difference in antibody levels was observed in FRA-immunized mice when compared to control mice, both antigens were able to induce lymphoproliferation in immunized mice. Significant differences were observed between incorporation of [³H]- thymidine by spleen cell stimulated in vitro with CRA or FRA and the control group. These results suggest that CRA and FRA could be involved in mechanisms of resistance to Trypanosoma cruzi infection.

Treatment with benznidazole in association with immunosuppressive drugs in mice chronically infected with Trypanosoma cruzi: investigation into the possible development of neoplasias

Benznidazole is recommended in Brazil for the treatment of Trypanosoma cruzi infection in acute and early chronic phases of Chagas' disease. Observations by others have indicated a higher incidence of neoplasias in immunosuppressed patients, presenting Chagas' disease reactivation, submitted to treatment with benznidazole. In the present study, we investigated whether there is a potentiation in the generation of lymphomas in chronically infected mice, treated with immunosuppressive drugs and benznidazole. For this, 142 Swiss mice chronically infected with the 21 SF strain of T. cruzi and 72 normal Swiss mice were used. Both infected and normal mice were divided into experimental groups and submitted to one of the following treatment regimens: benznidazole alone; immunosuppressive drugs (azathioprine, betamethasone and cyclosporin); a combination of immunosuppressive drugs and benznidazole; and untreated controls. In the infected group treated with benznidazole, one mouse developed a non-Hodgkin's lymphoma. This finding has been interpreted as a spontaneous tumor of mice. The study of the chronically infected mice treated with the combination of immunosuppressive drugs and benznidazole demonstrated an absence of lymphomas or other neoplasias. These findings support the indication of benznidazole, as the drug of choice, for immunosuppressed patients that develop a reactivation of Chagas' disease.

Alta parasitemia pelo Trypanosoma cruzi em paciente com lupus eritematoso sistêmico

É descrito um caso de doença de Chagas com alta parasitemia pelo Trypanosoma cruzi em paciente com lupus eritematoso sistêmico. O xenodiagnóstico foi útil na identificação da parasitemia e o benznidazol foi capaz de reduzir a alta e incomum parasitemia. Em indivíduos com doenças auto-imunes e immunossuprimidos, o benznidazol pode ser uma alternativa no controle da alta parasitemia por Trypanosoma cruzi.

Avaliação hematológica e histopatológica de camundongos BALB/c e C57BL/6 expostos aos antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi

Os antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi foram inoculados em camundongos BALB/c e C57BL/6 e o seu efeito avaliado a nível hematológico e histopatológico. Os resultados mostraram que o padrão histológico normal dos órgãos e o perfil hematológico dos camundongos não foram modificados sugerindo que esses antígenos não parecem causar dano ao animal.

Immunoblotting analyses using two-dimensional gel electrophoresis of Trypanosoma cruzi excreted-secreted antigens

Trypanosoma cruzi trypomastigotes excrete-secrete a complex mixture of antigenic molecules. This antigenic mixture denominated trypomastigote excreted-secreted antigens contains a 150-160 kDa band that shows excellent performance in Chagas' disease diagnosis by immunoblotting. The present study partially characterized by two-dimensional gel electrophoresis the immunoreactivity against the 150-160kDa protein using sera samples from chagasic patients in different phases of the disease. Trypomastigote excreted-secreted antigen preparations were subjected to high-resolution two-dimensional (2D) gel electrophoresis followed by immunoblotting with sera from chagasic and non-chagasic patients. The 150-160kDa protein presented four isoforms with isoelectric focusing ranging from 6.2 to 6.7. The four isoforms were recognized by IgM from acute phase and IgG from chronic phase sera of chagasic patients. The 150-160kDa isoform with IF of approximately 6.4 became the immunodominant spot with the progression of the disease. No cross-reactivity was observed with non-chagasic or patients infected with Leishmania sp. In this study we provide basic knowledge that supports the validation of trypomastigote excreted-secreted antigens for serological diagnosis of Chagas' disease.

Response to chemotherapy with benznidazole of clones isolated from the 21SF strain of Trypanosoma cruzi (biodeme Type II, Trypanosoma cruzi II)

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.

Cytokine serum levels in patients infected by human immunodeficiency virus with and without Trypanosoma cruzi coinfection

This study assessed the number of CD4 T lymphocytes, the parasitemia and serum levels of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-4 and IL-10 of patients infected by human immunodeficiency virus (HIV) and human immunodeficiency virus/Chagas' disease coinfection. CD4 T lymphocytes were low in the two groups of patients, although significantly lower in patients without Chagas' disease. Serum levels of IFN-gamma, IL-4 and TNF-alpha were significantly higher in patients with HIV/Chagas' disease. IL-4/IFN-gamma ratios were higher in patients with HIV/Chagas' disease, which showed a clear balance in favor of Th2-like cytokines in this group of patients. This Th2 balance was higher in patients with detectable parasitemia. We conclude that, although immunosuppression was observed, with CD4 T lymphocytes bellow 200/µm³, these patients did not display reactivation of T. cruzi infection and that a balance favorable to Th2 was associated with the presence of parasitemia.