Isoenzyme profiles of Trypanosoma cruzi stocks from different areas of Paraguay

Twenty one Trypanosoma cruzi stocks from humans, domiciliary triatomines and one sylvatic animal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were studied. MN cl2 (clonets 39) and SO34 cl4 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard´s distances matrix. Among the Paraguayan stocks 14 zymodemes were identified (Par1 to Par14), Par 5 being the most frequent. Polymorphism rate and clonal diversity were 0.73 and 0.93, respectively. Average number of alleles per polymorphic locus was 2.5 (range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stocks belong to the same lineage, as MN cl2 reference strain (T. cruzi II). Moreover three distinct subgroups were identified and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonality for T. cruzi populations. However sample size was not adequate to detect genetic recombination in any single locality.

Morphometry of submucous and myenteric esophagic plexus of dogs experimentally reinfected with Trypanosoma cruzi

We carried out a morphometric study of the esophagus of cross-bred dogs experimentally infected or consecutively reinfected with Trypanosoma cruzi 147 and SC-1 strains, in order to verify denervation and/or neuronal hypertrophy in the intramural plexus. The animals were sacrificed in the chronic stage, 38 months after the initial infection. Neither nests of amastigotes, nor myositis or ganglionitis, were observed in all third inferior portions of esophageal rings analyzed. No nerve cell was identified in the submucous of this organ. There was no significant difference (p>0.05) between the number, maximum diameter, perimeter, or area and volume of the nerve cells of the myenteric plexus of infected and/or reinfected dogs and of the non-infected ones. In view of these results we may conclude that the 147 and SC-1 strains have little neurotropism and do not determine denervation and/or hypertrophy in the intramural esophageal plexuses in the animals studied, independent of the reinfections.

Trypanosoma evansi control and horse mortality in the Brazilian Pantanal

The impact of three treatment strategies for Trypanosoma evansi control on horse mortality in the Brazilian Pantanal based on four size categories of cattle ranches is explored. The region's 49,000 horses are indispensable to traditional extensive cattle ranching and T. evansi kills horses. About 13% of these horses would be lost, annually, due to T. evansi if no control were undertaken. One preventive and two curative treatment strategies are financially justifiable in the Pantanal. The best available technology for the treatment of T. evansi from a horse mortality perspective is the preventive strategy, which spares 6,462 horses, annually. The year-round cure spares 5,783 horses, and the seasonal cure saves 5,204 horses on a regional basis relative to no control strategy. Regardless of the strategy adopted, 39% of the costs or benefits fall to the largest ranches, while 18% fall to the smallest ranches.

Diagnostic performance characteristics of rapid dipstick test for Plasmodium vivax malaria

We compared the diagnostic performance characteristics of newly developed method, the rapid dipstick test, which provides colorimetric determination by developing antibody to the lactate dehydrogenase enzyme of parasites, with conventional standard thick-blood film examination. For the rapid test, OptiMAL commercial kits were used. The results were also evaluated with clinical findings from patients. The parasites were determined by microscopic examination of thick-blood films from 81 patients with vivax malaria from southeastern Anatolia, Turkey. The OptiMAL test results were found to be negative in five patients who were diagnosed clinically and through thick-film testing as having vivax malaria. There was no false positivity observed with the OptiMAL test. We concluded that this rapid malaria test has a lower level of sensitivity than the classical thick-blood-film test for malaria, but that these methods have equal specificity.

A α-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes

α-glycerophosphate dehydrogenase (α-GPDH-EC.1.1.1.8) has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance.

Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates

Phenotypic diversity has been described in the central repeated region of the circumsporozoite protein (CSP) from Plasmodium vivax. Two sequences VK210 (common) and VK247 (variant) have been found widely distributed in P. vivax isolates from several malaria endemic areas around the world. A third protein variant called P. vivax-like showing a sequence similar to the simian parasite P. simio-ovale has also been described. Here, using an immunofluorescent test and specific monoclonal antibodies, we assessed the presence of two of these protein variants (VK210 and VK247) in laboratory produced sporozoite. Both sequences were found in parasite isolates coming from different geographic regions of Colombia. Interestingly, sporozoites carrying the VK247 sequence were more frequently produced in Anopheles albimanus than sporozoites with the VK210 sequence. This difference in sporozoites production was statistically significant (p <0.05, Kruskal-Wallis); not correlation was found with parameters as the total number of parasites or gametocytes in blood from human donors used to feed mosquitoes. Previous studies in the same region have shown a higher prevalence of anti-VK210 antibodies which in theory may suggest their role in blocking the development of sporozoites carrying the CSP VK210 sequence.

Experimental transmission of Trypanosoma cruzi through the genitalia of albino mice

Trypanosoma cruzi is usually transmitted by contact with the excreta of infected Triatominae; among non-vectorial infections, direct transmission through coitus has been proposed. We investigated this possibility by instilling, through the external meatus of the vagina and the penis of previously anesthetized NMRI albino mice, blood of mice infected with strains isolated from Didelphis marsupialis (opossum, strain CO57), Rattus rattus (rat, strain CO22) and human (strain EP). Some animals were allowed to copulate the same day of the instillation. In other experiments, the strains were inoculated in the scrotum. To determine the effect of immunosuppression, some mice were treated with cyclophosphamide 30 days post-instillation. Controls were instilled orally and ocularly. Vaginal instillation with strain CO22 produced systemic infection with tropism to the heart, skeletal muscle, skin, duodenum, pancreas, ovary and sternum. Scrotal inoculation with strain EP likewise invaded liver, spleen, lung, lymph nodes and urogenital organs; while strain CO57 invaded skeletal and cardiac muscle, pancreas, testis, and vas deferens. Penile infection with strain CO22 was detected by xenodiagnosis. Immunosuppression did not increase parasitemia of vaginally infected mice or controls. Mating did not produce infection. Our results show that contact of blood trypomastigotes of T. cruzi with genital mucosa can produce blood and tissue infections. These results are discussed in relation to reports of frequent experimental tropism of T. cruzi toward urogenital organs.

Trypanosoma evansi isolated from capybara (Hidrochaeris hidrochaeris)

A study was conducted to determine the morphological and biometric characteristics of Trypanosoma isolated from 50 capybaras animals, raised in captivity in the Peruvian Amazon. Trypanosoma was found in 14 blood samples using the microhaematocrit, wide drop, and Giemsa-stain methods and T. evansi was identified through morphological details in all 14 positive samples (the subterminal kinetoplast, the developed undulating membrane, and a long free flagellum were used for the identification of the agent).

Specific antibody levels and antigenic recognition of Wistar rats inoculated with distinct isolates of Trypanosoma evansi

"Mal de Cadeiras", an enzootic disease caused by Trypanosoma evansi, is one of the most important trypanosomiases in the Brazilian Pantanal region. The disease affects mainly horses, which are widely used in extensive cattle production, an activity of greatest economical significance for the region. The parasite also infects sylvan (coatis and capybaras) and domestic (dogs) animals, respectively considered wild and domestic reservoirs of T. evansi. For a better understanding of the interaction of T. evansi with its rodent host, we evaluated the differences in the specific antibody level patterns and in the parasitic peptides recognition patterns of experimentally infected Wistar rats. The rats experimentally infected with T. evansi isolates obtained from coatis, dogs and horses were submitted to indirect immunofluorescence test (IgM e IgG) and Western blotting. The serological titers for IgM and IgG ranged between 1:40 and 1:160. The most recognized polypeptide profiles were in a range of 17 and 74 kDa. Our data suggest that the humoral immune response in Wistar rats is not sufficient for granting an effective control of T. evansi infections.

Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains

In this work, the susceptibility to benznidazole of two parental Trypanosoma cruzi strains, Colombian and Berenice-78, was compared to isolates obtained from dogs infected with these strains for several years. In order to evaluate the susceptibility to benznidazole two groups of mice were infected with one of five distinct populations isolated from dogs as well as the two parental strains of T. cruzi. The first group was treated with benznidazole during the acute phase and the second remained untreated controls. The animals were considered cured when parasitological and serological tests remained persistently negative. Mice infected with the Colombian strain and its isolates Colombian (A and B) did not cure after treatment. On the other hand, all animals infected with Berenice-78 were cured by benznidazole treatment. However, 100%, 50% and 70% of cure rates were observed in animals infected with the isolates Berenice-78 B, C and D, respectively. No significant differences were observed in serological profile of infected control groups, with all animals presenting high antibody levels. However, the ELISA test showed differences in serological patterns between mice inoculated with the different T. cruzi isolates and treated with benznidazole. This variability was dependent on the T. cruzi population used and seemed to be associated with the level of resistance to benznidazole.

Triatomines Involved in Domestic and Wild Trypanosoma cruzi Transmission in Concepción, Corrientes, Argentina

An entomological and serological survey was performed in three localities of the Department of Concepción, Province of Corrientes, Argentina in 1998 and 1999, to identify triatomines species involved in domestic and wild transmission of Chagas disease. Triatomines were collected by man/hour capture in 32 houses randomly selected and 44 nearby outdoor ecotopes. Trypanosoma cruzi infection in triatomines was assessed by direct microscopic observation (400x) of feces and polymerase chain reaction. Serological techniques used for people were Indirect Hemagglutination Test and Indirect Fluorescent Test. Triatomines were collected in 28.1% of the houses and 31.8% of the wild biotopes. Triatoma infestans (Klug 1834) was exclusively found indoors and T. cruzi infected 60% of them. Triatoma sordida (Stål 1859) was mainly found in extradomestic ecotopes where trypanosome infection rate reached 12.7%. Serological study of 98 local people showed that 29.6% were seroreactive; most of their houses were closed to wild biotopes colonized by T. sordida. Results indicate that there is an active T. infestans mediated transmission of Chagas disease in this zone that yields important human prevalence and that the populations of T. sordida in wild biotopes not only sustain the wild T. cruzi cycle but also represent an actual risk for people living in the area.

In Vitro and in Vivo Assays of 3,5-Disubstituted-Tetrahydro-2H-1,3,5-Thiadiazin-2-Thione Derivatives against Trypanosoma cruzi

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 µg/ml for macrophages and a few of them maintained this cytotoxicity even at 10 µg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1µg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.

Integrate Study of a Bolivian Population Infected by Trypanosoma cruzi, the Agent of Chagas Disease

A cross section of a human population (501 individuals) selected at random, and living in a Bolivian community, highly endemic for Chagas disease, was investigated combining together clinical, parasitological and molecular approaches. Conventional serology and polymerase chain reaction (PCR) indicated an active transmission of the infection, a high seroprevalence (43.3%) ranging from around 12% in < 5 years to 94.7% in > 45 years, and a high sensitivity (83.8%) and specificity of PCR. Abnormal ECG tracing was predominant in chagasic patients and was already present among individuals younger than 13 years. SAPA (shed acute phase antigen) recombinant protein and the synthetic peptide R-13 were used as antigens in ELISA tests. The reactivity of SAPA was strongly associated to Trypanosoma cruzi infection and independent of the age of the patients but was not suitable neither for universal serodiagnosis nor for discrimination of specific phases of Chagas infection. Anti-R-13 response was observed in 27.5% only in chagasic patients. Moreover, anti-R13 reactivity was associated with early infection and not to cardiac pathology. This result questioned previous studies, which considered the anti-R-13 response as a marker of chronic Chagas heart disease. The major clonets 20 and 39 (belonging to Trypanosoma cruzi I and T. cruzi II respectively) which circulate in equal proportions in vectors of the studied area, were identified in patients' blood by PCR. Clonet 39 was selected over clonet 20 in the circulation whatever the age of the patient. The only factor related to strain detected in patients' blood, was the anti-R-13 reactivity: 37% of the patients infected by clonet 39 (94 cases) had anti-R13 antibodies contrasting with only 6% of the patients without clonet 39 (16 cases).

Prevalence of Trypanosoma lewisi in Rattus norvegicus from Belo Horizonte, State of Minas Gerais, Brazil

From April 1984 to March 1985, a Trypanosoma lewisi prevalence of 21.7% was found in 429 Rattus norvegicus trapped in Belo Horizonte, State of Minas Gerais, Brazil. The infection rates were higher in male and young rats and could be attributed to ecological and behavioral factors. T. lewisi was observed in rats measuring between 60 and 250 mm. Data about monthly T. lewisi infections throughout the year are presented for the first time in Brazil, with the highest prevalences observed in the warm-rainy season (October to March).

In Vitro and in Vivo Anti-Trypanosoma cruzi Activity of a Novel Nitro-derivative

Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at concentrations in which it was no longer cytotoxic, thus it was assayed against intracellular amastigotes. Its anti-amastigote activity was similar to that of nifurtimox. Afterwards in vivo toxicity and anti-chagasic activity were determined. A reduction in parasitemia was observed.