Impact of dual infections on chemotherapeutic efficacy in BALB/c mice infected with major genotypes of Trypanosoma cruzi.

The aim of this work was to investigate the impact of dual infections with stocks of Trypanosoma cruzi major genotypes on benznidazole (BZ) treatment efficacy. For this purpose, T. cruzi stocks representative of the genetic T. cruzi lineages, displaying different susceptibilities to BZ, belonging to the major T. cruzi genotypes broadly dispersed in North and South America and important in Chagas' disease epidemiology were used. Therapeutic efficacy was observed in 27.8% of the animals treated. Following BZ susceptibility classification, significant differences were observed in dual infections on the major genotype level, demonstrating that combinations of genotypes 19+39 and genotypes 19+32 led to a shift in the expected BZ susceptibility profile toward the resistance pattern. Analysis on the T. cruzi stock level demonstrated that 9 out of 24 dual infections shifted the expected BZ susceptibility profile compared with the respective single infections, including shifts toward lower and higher BZ susceptibilities. Microsatellite identification was able to identify a mixture of T. cruzi stocks in 7.7% of the T. cruzi isolates from infected and untreated mice (6.9%) and infected and treated but not cured mice (9.0%), revealing in some mixtures of BZ-susceptible and -resistant stocks that the T. cruzi stock identified after BZ treatment was previously susceptible in single infections. Considering the clonal structure and evolution of T. cruzi, an unexpected result was the identification of parasite subpopulations with distinct microsatellite alleles in relation to the original stocks observed in 12.2% of the isolates. Taken together, the data suggest that mixed infections, already verified in nature, may have an important impact on the efficacy of chemotherapy.

Trypanosoma cruzi: Impact of dual-clone infections on parasite biological properties in BALB/c mice.

Herein, we have analyzed major biological properties following dual-clone Trypanosoma cruzi infections in BALB/c mice. Eight T. cruzi clonal stocks, two of each principal genotype, including genotype 19 and 20 (T. cruzi I), hybrid genotype 39 (T. cruzi) and 32 (T. cruzi II) were combined into 24 different dual-clone infections. Special attention was given to characterize biological parameters assayed including: prepatent period, patent period, maximum of parasitemia, day of maximum parasitemia, area under the parasitemia curve, infectivity, mortality, and hemoculture positivity. Our findings clearly demonstrated that features resultant of dual-clone infections of T. cruzi clonal stocks did not display either the characteristics of the corresponding monoclonal infections or the theoretical mixture based on the respective monoclonal infections. Significant changes in the expected values were observed in 4.2-79.2% of the mixtures considering the eight biological parameters studied. A lower frequency of significant differences was found for mixtures composed by phylogenetically distant clonal stocks. Altogether, our data support our hypothesis that mixed T. cruzi infections have a great impact on the biological properties of the parasite in the host and re-emphasizes the importance of considering the possible occurrence of natural mixed infections in humans and their consequences on the biological aspects of ongoing Chagas' disease.

Trypanosoma cruzi: sensitivity of the polymerase chain reaction for detecting the parasite in the blood of mice infected with different clonal genotypes.

The polymerase chain reaction showed high sensitivity for detecting Trypanosoma cruzi in the blood of mice, independent of clonal genotype (19, 20-T. cruzi I; 32, 39-T. cruzi II) or phase of the infection (acute or chronic).

Trypanosoma cruzi: two genetic groups in Paraná state, Southern Brazil.

This paper presents the genetic characterization of Trypanosoma cruzi strains isolated from chronic chagasic patients, triatomines, and sylvatic reservoirs from Paraná state, Southern Brazil, using the RAPD and SSR-PCR techniques. It has shown the presence of both phylogenetic groups of T. cruzi (I and II), describing for the first time the existence of T. cruzi II in Paraná state.

Effects of specific treatment on parasitological and histopathological parameters in mice infected with different Trypanosoma cruzi clonal genotypes.

The goal of this study was to verify the effect of specific treatment on parasitological and histopathological parameters in mice experimentally infected with different Trypanosoma cruzi clonal genotypes. Twenty cloned stocks were selected, representative of the whole phylogenetic diversity of the protozoan and belonging to the clonal genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II). The stocks were inoculated in 40 BALB/c mice divided into four groups: (i) treated with benznidazole, (ii) treated with itraconazole and (iii and iv) untreated control groups (NT) for each drug, respectively. Seven parameters related to parasitaemia curves and histopathological lesions were analysed. Four during the acute phase (AP) and three during both the AP and chronic phase (CP) of infection. Statistical comparison between benznidazole-treated and NT groups for the biological parameters showed significant differences for all genotypes. Benznidazole treatment led to lower patent period, maximum of parasitaemia, day of maximum parasitaemia and area under the parasitaemia curve for all genotypes analysed. Percentage of positive haemoculture during AP and CP was lower for genotypes 19 and 32. Tissue parasitism (TP) and inflammatory process (IP) during AP were lower for genotypes 19 and 32, respectively. In general, itraconazole treatment induced a smaller reduction in these same parameters between treated and NT animals in relation to benznidazole treatment. Our results indicate that phylogenetic divergence among T. cruzi clonal genotypes must be taken in account in chemotherapy and studies dealing with all aspects of the parasite and the disease.

Impact of Trypanosoma cruzi clonal evolution on its biological properties in mice.

Twenty Trypanosoma cruzi stocks attributed to the 19, 20, 39, and 32 clonal genotypes were comparatively studied in BALB/c mice during the acute and chronic phases of the infection to test the working hypothesis that T. cruzi clonal structure has a major impact on its biological properties. Fourteen parameters were assayed: (1) infectivity; (2) prepatent period; (3) patent period; (4) maximum of parasitemia; (5) day of maximum of parasitemia; (6) parasitemia; (7) mortality, (8) percentage of positive hemoculture, (9) tissue parasitism; (10) inflammatory process during the acute phase of the infection; (11) mortality, (12) percentage of positive hemoculture; (13) tissue parasitism; and (14) inflammatory process during the chronic phase of the infection. Statistical comparison showed that the results are overall consistent with the working hypothesis that biological differences are proportional to the evolutionary divergence among the genotypes. Thus, closely related genotypes (19 vs 20 and 32 vs 39) show in general fewer differences than distantly related groups (19 or 20 vs 32 or 39) except for the comparison between 19 and 32. The working hypothesis is even more strongly supported by the result of the nonparametric Mantel test, which showed a highly significant correlation (P = 2.3 x 10(-3)) between biological differences and genetic distances among all pairs of stocks. These data taken together emphasize that it is crucial to take into account the phylogenetic diversity of T. cruzi natural clones in all applied studies dealing with diagnosis, drug and vaccine design, epidemiological surveys, and clinical diversity of Chagas' disease. Index Descriptors and Abbreviations: Trypanosoma cruzi; phylogenetic distance; biological properties; clonal theory; multilocus enzyme electrophoresis (MLEE); randomly amplified polymorphic DNA (RAPD); acute phase (AP); chronic phase (CP); days after inoculation (d.a.i.); liver infusion tryptose (LIT); gastrointestinal tract (GIT); genitourinary tract (GUT); percentage of infectivity (%INF); percentage of mortality during the acute phase (%MORT AP); percentage of mortality during the chronic phase (%MORT CP); prepatent period (PPP); patent period (PP); maximum of parasitemia (MP); day of maximum of parasitemia (DMP); parasitemia (PAR); percentage of positive hemoculture during the acute phase (% + HC AP); percentage of positive hemoculture during the chronic acute phase (% + HC CP); tissue parasitism (TP); inflammatory process (IP); tissue parasitism during the acute phase (TP AP); tissue parasitism during chronic phase (TP CP); inflammatory process during acute phase (IP AP); inflammatory process chronic phase (IP CP); Mann-Whitney test (MW); Kruskal-Wallis (KW); Kolmogorow-Smirnov test (KS).

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.

Estudo sobre triatomíneos e reservatórios silvestres de Trypanosoma cruzi no Estado do Paraná, Sul do Brasil: resultados preliminares / Triatomids and forest reservoirs of Trypanosoma cruzi in the state of Paraná, southern Brazil: preliminary results

Em localidades da zona rural de cinco municípios da região noroeste do Paraná adultos e ninfas de Triatoma sordida foram capturados em 21 (41,2%) de 51 unidades domiciliares pesquisadas. Foram capturados 154 exemplares de T. sordida e 2 Panstrongylus megistus. De 135 exemplares examinados 58 (43,0%) apresentavam o Trypanosoma tipo cruzi. Constatou-se também a infecção em 57,1% (4/7) dos gambás (Didelphis sp) examinados. O peridomicílio apresentou-se mais infestado que o intradomicílio, sendo a casa de madeira abandonada a construção mais freqüente (34,7% do total investigado) e com maior taxa de infestação (53,9%). Os dados mostram um elevado índice de infestação da zona rural por espécies secundárias de triatomíneos com altas taxas de infecção por flagelados do tipo T. cruzi, em uma área endêmica para a doença de Chagas já em fase de vigilância epidemiológica.

In the rural area of five municipalities of the Northeast of the State of Paraná, Brazil, 154 adults or nymphs of Triatoma sordida and 2 Panstrongylus megistus were captured in 21 (41.2%) of 51 dwellings at the peridomestic sites. Trypanosoma cruzi-like organisms were found in 58 out of the 135 (43.0%) triatomids. Moreover, it was also found in the blood of 4/7 opossum (Didelphis sp). The triatomine infestation was more prevalent in the peridomicile than inside house. The forsaken wooden house presented the highest level of infestation (53.8%). Although the rural area of the Northeast of the State of Paraná is considered under epidemiologic surveillance it still presents a high level of peridomestic infestation by triatomids highly infected with T. cruzi-like organisms.

[Triatomids and forest reservoirs of Trypanosoma cruzi in the state of Paraná, southern Brazil. Preliminary results]. / Estudo sobre triatomíneos e reservatórios silvestres de Trypanosoma cruzi no Estado do Paraná, Sul do Brasil. Resultados preliminares.

In the rural area of five municipalities of the Northeast of the State of Paraná, Brazil, 154 adults or nymphs of Triatoma sordida and 2 Panstrongylus megistus were captured in 21 (41.2%) of 51 dwellings at the peridomestic sites. Trypanosoma cruzi-like organisms were found in 58 out of the 135 (43.0%) triatomids. Moreover, it was also found in the blood of 4/7 opossum (Didelphis sp). The triatomine infestation was more prevalent in the peridomicile than inside house. The forsaken wooden house presented the highest level of infestation (53.8%). Although the rural area of the Northeast of the State of Paraná is considered under epidemiologic surveillance it still presents a high level of peridomestic infestation by triatomids highly infected with T. cruzi-like organisms.

Trypanosoma cruzi: chemotherapy with benznidazole in mice inoculated with strains from Paraná state and from different endemic areas of Brazil.

Strains of Trypanosoma cruzi from different geographical areas have shown different levels of susceptibility to trypanosomicidal drugs. The susceptibility in vivo to benznidazole was investigated in eighteen strains of T. cruzi. Twelve were isolated from chronic chagasic patients from different Chagas' disease endemic areas. The other six strains were isolated from the northwestern region of Paraná state; two of them from patients, three from triatomines (Triatoma sordida) and one from wild reservoir (Didelphis sp.). To test drug the infected mice were divided into two groups of twenty. One group was treated with benznidazole for twenty consecutive days and the other group was used as untreated control. The treatment began after detection of the infection by direct blood examination or haemoculture. The control of cure was done through haemoculture and indirect immunofluorescence test. The drug eliminated the inflammatory lesions of the skeletal muscle of mice considered cured and from the heart of most of them. Moreover, the inflammatory lesions were reduced in treated but not cured animals. The T. cruzi strains studied showed a gradient of drug susceptibility that varied from 0% to 100%. Ten strains were considered sensitive to the treatment (61 to 100% of cure), one strain was partially sensitive (50% of cure) and seven strains were considered resistant to the treatment (0 to 40% of cure). This variation was observed both in strains of T. cruzi isolated from domestic and sylvatic cycles.