Ketoconazole promotes parasitological cure of mice infected with Trypanosoma cruzi

Administration of 120 mg/kg/day of ketoconazole for 9 weeks resulted in parasitological cure of at least 78.5% of mice infected with 10⁵ blood trypanosomes of Trypanosoma cruzi, strain Y. These results and the fact that ketoconazole is widely used in humans for prolonged therapy of fungal infections without significant side effects strongly suggest that further evaluation of ketoconazole for treatment of Chagas disease is highly desirable.     

Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a major public health problem in Latin America. This parasite has a complex population structure comprised by six or seven major evolutionary lineages (discrete typing units or DTUs) TcI-TcVI and TcBat, some of which have apparently resulted from ancient hybridization events. Because of the existence of significant biological differences between these lineages, strain characterization methods have been essential to study T. cruzi in its different vectors and hosts. However, available methods can be laborious and costly, limited in resolution or sensitivity. In this study, a new genotyping strategy by real-time PCR to identify each of the six DTUs in clinical blood samples have been developed and evaluated.Two nuclear (SL-IR and 18S rDNA) and two mitochondrial genes (COII and ND1) were selected to develop original primers. The method was evaluated with eight genomic DNA of T. cruzi populations belonging to the six DTUs, one genomic DNA of Trypanosoma rangeli, and 53 blood samples from individuals with chronic Chagas disease. The assays had an analytical sensitivity of 1–25 fg of DNA per reaction tube depending on the DTU analyzed. The selectivity of trials with 20 fg/μL of genomic DNA identified each DTU, excluding non-targets DTUs in every test. The method was able to characterize 67.9% of the chronically infected clinical samples with high detection of TcII followed by TcI.With the proposed original genotyping methodology, each DTU was established with high sensitivity after a single real-time PCR assay. This novel protocol reduces carryover contamination, enables detection of each DTU independently and in the future, the quantification of each DTU in clinical blood samples.     

Vaccination with Trypanosoma rangeli reduces the infectiousness of dogs experimentally infected with Trypanosoma cruzi

The goal of this work was to test the efficacy of the vaccination with Trypanosoma rangeli in dogs. Mongrel dogs received three subcutaneous injections of fixed T. rangeli epimastigotes at 6-week intervals. Such immunisation induced antibodies against Trypanosoma cruzi. While both control and immunised dogs developed detectable parasitemia, this was lower and shorter in vaccinated animals. Interestingly, feeding of Triatoma infestans nymphs on vaccinated and chronically infected dogs led to a sharp reduction in the rate of bug infection. These results suggest that it might be possible to reduce the vectorial parasitemia through vaccination of dogs. As dogs are known to play a major role in the domestic cycle of T. cruzi, this might represent a strategy to reduce parasite transmission to humans.     

Immune precipitation and immunoblotting for the detection of Trypanosoma cruzi antigens

The efficiency of immunoprecipitation and immunoblotting for detecting Trypanosoma cruzi antigens using rabbit and mouse antisera has been examined. Comparison of the starting material for each technique showed that the extraction methods resulted in a similar range of polypeptides when assessed by apparent Mr. Reaction with either hyperimmune rabbit sera or a sequential series of sera from infected mice, showed a significant disparity in the range of antigens revealed by each technique. Epitopes on polypeptides greater than 50 kDa were poorly preserved by immunoblotting compared to immunoprecipitation; however, below this threshold both techniques were equally efficient. Under the conditions of assay, it is probable that immunoprecipitation allows effective detection of both sequence and conformational determinants, whereas immunoblotting favours the detection of sequence determinants alone.     

Evaluation of chemotherapy with benznidazole and nifurtimox in mice infected with Trypanosoma cruzi strains of different types

A test was made of the susceptibility of 30 strains of Trypanosoma cruzi to chemotherapy with nifurtimox (Bay 2502) and benznidazole (Ro 7-1051). The strains had previously been classified as type I, II, or III according to their morphobiological and isoenzymic characteristics. Three type I strains, 14 type II strains, and 13 type III strains were studied. Mice were infected with 2 × 10⁵ blood forms of these parasites and treated for 90 days with benznidazole or nifurtimox. All the surviving mice were submitted to parasitological tests (direct parasitaemia, xenodiagnosis, inoculation in new-born mice, and haemoculture) and serological tests (indirect immunofluorescence). As the latter remained positive in about 80% of the parasitologically negative animals, the cure rates were based on the more reliable parasitological tests. Type I strains displayed high susceptibility, type II strains showed medium to high susceptibility, and type III strains were highly resistant to both drugs. The fact that a particular strain type, with its own level of susceptibility, usually predominates in a given geographical area may explain the contradictory results after chemotherapy from different endemic areas.     

Experimental chemotherapy of Trypanosoma cruzi infection: persistence of parasite antigens and positive serology in parasitologically cured mice

Mice infected with Trypanosoma cruzi, but parasitologically cured after specific chemotherapy, continued to exhibit positive indirect immunofluorescence serological tests 3-6 months after the therapy. Treatment of trypanosome antigens with monospecific antisera produced in rabbits, and examination by immunoelectron-microscopy following peroxidase labelling disclosed the presence of membrane deposits in cell processes in the spleens of the mice. Similar deposits were observed in the external membranes of T. cruzi amastigotes in the spleens of acutely infected mice, but not in normal control mice. No reaction occurred in tissues not previously treated with the monospecific anti-T. cruzi serum. Positive cells in treated and cured mice, as well as in the not cured or untreated control mice, were located in germinal centres of the splenic white pulp and presented long and branching cytoplasmic processes, which are indicative of dendritic cells of the lymphoid follicles of the spleen.     

Cross reactivity between Trypanosoma cruzi and Leishmania antigens in the lymphocyte blastogenesis assay

The in vitro blastogenesis response of lymphocytes from 19 patients, with either visceral or mucocutaneous leishmaniasis or Chagas disease, to antigens of the Leishmania-Trypanosoma complex was studied. Cells from all patients responded to both homologous and heterologous antigens and the magnitude of the responses did not differ for any of the patient groups or antigens.     

Effects of clomipramine on Trypanosoma cruzi infection in mice.

Trypanosoma cruzi, widely distributed in Latin American countries, provokes Chagas disease, characterized by cardiomyopathy and mega-viscera. The drugs used currently for treatment of acute Chagas disease are highly toxic; the side-effects are undesirable and patients may abandon treatment. We have previously demonstrated that clomipramine (CLO) exerts trypanocidal effects upon epimastigotes and trypomastigotes in vitro with anticalmodulin activity. The present study analyses the effectiveness of CLO treatment in mice infected with a low number of T. cruzi, an animal model that reproduces acute, indeterminate and chronic phases of this trypanosomiasis. In this work, our results demonstrated that CLO 5 mg/kg daily for 30 days, or 2 doses of CLO 40 mg/kg given intraperitoneally at 1 h and 7 days after infection, was not toxic for the host, but was effective against the parasite in that parasitaemias became negative and only mild heart structural and electrocardiographic alterations were detected in the chronic phase in the group treated with CLO 5 mg/kg. In mice treated with CLO 40 mg/kg, none of these alterations was detected. Cardiac beta receptor density and affinity returned to normal in the chronic stage in both experimental groups. T. cruzi enzymes such as calmodulin and trypanothione reductase represent potential drug targets. It has been reported that both can be inhibited by CLO, a tricyclic drug used in clinical therapeutics. We have shown that CLO strongly decreased the mortality rate and electrocardiographic alterations; in addition cardiac beta receptor density and heart histology returned to, or close to, normality 135 days post infection. These results clearly demonstrated that CLO treatment modified significantly the natural evolution of T. cruzi infection.     

The influence of the strain of Trypanosoma cruzi in placental infections in mice

Pregnant female mice were infected with Trypanosoma cruzi strains which differed according to several parameters and were classified as three different types. Mice were killed during either the acute or the chronic phase of infection. Animals' tissues and foetuses together with the placentas, were studied histopathologically. Clear cut differences were noted in the incidence of placental parasitism and in the localization of amastigotes in the vascular sinus of the placenta amongst the animals in the acute phase of the infection with different strains. No parasitism of the foetal tissues was seen. The incidence of placental parasitism reached 98% for the Colombian strain, 18·4% for the Peruvian strain, 17% for the Y strain and 13·2% for the Honorina strain (isolated from a woman that transmitted the infection to twins). The presence of parasites in the vascular part of the placenta was prominent with the Colombian strain and rare with the others. These experimental data seem to show that parasite strain plays a role in congenital T. cruzi infection.