Juracy B. Magalhães

Specific chemotherapy of Chagas disease: a comparison between the response in patients and experimental animals inoculated with the same strains

Eleven strains of Trypanosoma cruzi were isolated from patients with Chagas disease in central Brazil by xenodiagnosis and inoculation into newborn mice. Biological characterization and isoenzyme analysis showed that 6 strains were type II (zymodeme 2) and 5 were type III (zymodeme 1). Patients were treated with benznidazole or benznidazole plus nifurtimox. Mice infected with each isolated strain were treated for comparison with the results obtained in the respective patient. Evaluation of cure of the patients was based on the indirect immunofluorescence test, complement fixation reaction and xenodiagnosis. For the mice, haemoculture, indirect immunofluorescence testing, xenodiagnosis and inoculation of blood into newborn mice were used. Tests were performed 3-6 months after the end of treatment. The cure rate was 66-100% in mice infected with type II strains and 0-9% in those infected with type III strains. The correlation between treatment results in patients and mice was 81.8% (9 of 11 cases). Type II strains were more susceptible to treatment, in contrast to type III strains which yielded the majority of therapeutic failures.

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.

Terapêutica da fase crônica da infecção experimental pelo Trypanosoma cruzi com o Benzonidazol e o Nifurtimox

Fifty-eight mice, chronically infected with different T. cruzi strains (Types II and III) were submitted to chemotherapy either with Nifurtimox (Bay 2502) or Benznidazole (Ro 7-1051). Twenty one mice were not treated and were used as infected controls. The duration of infection was from 90 to 400 days. Inocula varied from 1 x 10(4) to 5 x 10(4) blood forms. Treatment lasted for 90 days, doses being 200mg/kg/day during 4 days, followed by 50mg/kg/day for Nifurtimox and 100mg/kg/day for Benznidazole. Parasitological tests (xenodiagnosis, inoculations into baby mice and hemoculture) showed 85.3% negativation for Type II strains and 43% for Type III in animals treated with Benznidazole. As for Nifurtimox, there were 71.4% of parasitological negativation for the animals infected with Type II strains and 66% for those infected with Type III. IFA tests remained positive in 90% of treated and cured animals. Disappearance or marked regression of myocardial and skeletal muscle lesions was seen in the treated and parasitologically negative animals. The conclusion is that the treatment in the chronic phase of T. cruzi infection can result in parasitological cure in a high percentage of cases with regression of histopathological lesions, although with persistence of positivity of the IFA tests.

Immunological response of Swiss mice to infection with three different strains of Trypanosoma cruzi.

The immunological response of Swiss mice to infection with three strains of Trypanosoma cruzi which differ in their morphobiological, antigenic and isoenzymic characters [Peruvian, 12 SF (São Felipe) and Colombian strains] was investigated. The three strains stimulated an elevation of the immunoglobulin fractions IgG2a, IgG2b and IgM during acute infection, as measured by radial immunodiffusion, and an early drop of IgG1 levels. There were low levels of specific antibodies and a negative cutaneous delayed hypersensitivity test to T. cruzi antigens. Cellular reaction of the spleen was evident, with proliferation of lymphocytes and the presence of blastic lymphoid cells in the red and white pulp, and hyperplasia of germinal centres of the lymphoid follicles. Those aspects were consistent with a depletion of the T-cell zone (periarteriolar lymphocyte sheath). Despite these common features, there were clear differences in the onset, intensity and evolution of the splenic cellular reaction and IgG serum levels and in the relationship between these levels and parasitaemia in the mice infected with the three strains of T. cruzi. A positive correlation was seen between high IgG levels and mortality, corresponding to intense exudative tissue lesions, showing that a raised immunoglobulin level was not associated with protection. It is worth observing that the 12 SF strain, which showed the lowest parasitaemic profile and mortality rate, stimulated the greatest elevation of IgG2b during acute infection; and also that IgG2a and IgG2b were the immunoglobulins which showed the greatest increases following infection by all three strains of T. cruzi.

Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III): biological, isoenzymic and histopathological analysis of seven isolated 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.

Comportamento das cepas Y e Peruana do Trypanosoma cruzi no camundongo, após passagem em diferentes meios

The behavior of two strains of Trypanosoma cruzi (Y and Peruvian strains) in experimental mouse infection, after being passed through different conditions of maintainance and cultivation was studied. The conditions were: Warrens acellular culture medium, cryopreservation in liquid Nitrogen, passage through the insect vector and direct blood passage from mice to mice. The parameters considered for comparative study were as follows: parasitemia, mortality rate, maximum survival time, morphology of parasites in peripheral blood, tissue tropism and histopathological lesions. Each experimental group consisted of two sub-groups according to the inocula: 10.000 or 50.000 trypomastigotes. The basic characteristics of the strains remained unchanged. These were macrophagotropism, predominance of slender forms of the parasite in early infection and 100 per cent mortality rate in the acute phase of the infection. However, decrease in the virulence was observed when the culture forms were used or when the infection with low inoculum was used (10.00 forms). Therefore the main biological characteristics of the strains tended to remain the same, regardless of the conditions used for maintainance and cultivation.The behavior of two strains of Trypanosoma cruzi (Y and Peruvian strains) in experimental mouse infection, after being passed through different conditions of maintainance and cultivation was studied. The conditions were: Warrens acellular culture medium, cryopreservation in liquid Nitrogen, passage through the insect vector and direct blood passage from mice to mice. The parameters considered for comparative study were as follows: parasitemia, mortality rate, maximum survival time, morphology of parasites in peripheral blood, tissue tropism and histopathological lesions. Each experimental group consisted of two sub-groups according to the inocula: 10.000 or 50.000 trypomastigotes. The basic characteristics of the strains remained unchanged. These were macrophagotropism, predominance of slender forms of the parasite in early infection and 100 per cent mortality rate in the acute phase of the infection. However, decrease in the virulence was observed when the culture forms were used or when the infection with low inoculum was used (10.00 forms). Therefore the main biological characteristics of the strains tended to remain the same, regardless of the conditions used for maintainance and cultivation.

Aspectos imunológicos da infecção de seis linhagens isogênicas de camundongos por três diferentes cepas do Trypanosoma cruzi

We evaluated humoral and cellular immune responses in 6 inbred mouse strains (BALB/c, B-10, C3H, A/J, AKR and DBA) infected with 3 Trypanosoma cruzi strains (Peruvian, 21 SF and Colombian), which are the standards for the 3 strains Types of Andrades classification. Negative delayed-type hipersensitivity reactions to parasite antigens were evidence of suppressed cell-mediated immunity. An early drop of IgG1 and rise of IgM levels were observed in almost all mouse strains infected by any T. cruzi strain. Elevation of IgG2a and/or IgG2b levels was higher in resistant mouse strains. Anti-T. cruzi antibody levels (Indirect immunofluorescence and ELISA) did not correlate with survival. Despite some differences among mouse strains there was a definition of an overall pattern of host response and the maintenance of biological standards which characterize the basic types of T. cruzi strains.

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

Seventy Swiss mice chronically infected with different strains of Trypanosoma cruzi, with persistently negative parasitemia on routine blood examination were parasitologically investigated to find out whether spontaneous cure occurred. Duration of infection varied from 90 to 250 days in the initial phase of this investigation. Parasitological tests consisted of daily direct blood examination performed during at least 25 days, followed by xenodiagnosis and subinoculation of blood into newborn mice. Mice that persisted negative were treated with Cyclophosphamide with one dose of 250 mg/kg of body weight and then investigated by direct blood examination, xenodiagnosis and subinoculation. A second dose of 250 mg/kg b. w. was given to the persistently negative mice. With one single exception, all mice showed positive parasitological tests in the different stages of the present investigation and we conclude that spontaneous cure did not occur in this group, which is representative of the chronic infection with different strains of T. cruzi.

Estudo do comportamento de cepas de Trypanosoma cruzi após passagem em diferentes espécies de triatomíneos

To study the injluence ofthe intermediate host stage on the course of mouse infection, Trypanosoma cruzi belonging to the Peruvian (Type I), 12 SF (Type II) and Colombian (Type 111) strains were passaged through either Rhodnius prolixus, Panstrongylus megistus or Triatoma infestans. T. cruzimetacyclicforms (dose 10*) from the different strains were obtained from each bug and inoculated into 8-10 gm mice. Comparison was made in mice inoculated with bloodforms. Parasitaemia curves were plotted in the peripheral blood for each strain, reaching more elevated peaks with Peruvian strain parasites from P. megistus and R. prolixus, 12 SF strain from P. megistus and Colombian strain from R. prolixus. Tissue tropism and histopathologiealpatterns were those usually seen in mice infected with each respective strain type. Peruvian virulence was the same for all groups. Slender forms predominate among mice inoculated with metacy clic forms of Colombian and 12 SF strains, probably an adaptative parasite change related to the intermediate host.

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.