A avidez de anticorpos específicos anti-toxoplasma da classe IgG e sua utilização na diferenciação entre toxoplasmose recente e crônica em caprinos

Foi avaliada a avidez de anticorpos IgG como marcador sorológico de infecção recente e crônica pelo Toxoplasma gondii, através da dissociação do complexo antígeno-anticorpo com uréia. A avaliação foi realizada medindo-se a percentagem de queda (%Q) da absorbância, pelo ELISA, após a lavagem do complexo Ag - Ac formado com solução de uréia. Ficou determinado que a concentração dc uréia 9 Molar (M) foi a que melhor diferenciou infecções recentes e crônicas de cabras experimentalmente infectadas com Toxoplasma gondii, e que a %Q da absorbância decresce com o tempo de infecção do animal, tornando-se estável em torno do 100º. dia após a inoculação. Em um grupo de 116 amostras de soro, coletadas de caprinos naturalmente infectados, foram demonstrados perfis característicos de toxoplasmose recente, crônica ou em fase de transição. Os perfis foram previamente determinados pela avaliação da evolução dos níveis de anticorpos IgG pela reação de imunofluorescência indireta. Os animais caracterizados como portadores de toxoplasmose crônica apresentaram uma %Q da absorbância de 26,32 ± 10,84. Para toxoplasmose recente a %Q da absorbância observada foi de 77,61 ± 13,89 e para o perfil de transição, 46,22 ± 11,94

Toxoplasma - specific IgG antibodies avidity ELISA and its role in the differentiation between recent and chronic toxoplasmosis in goats

For serological characterization of recent Toxoplasma infection in goats low avidity IgG specific antibodies were studied. Avidity was evaluated as the decrease of absorbance in ELISA test after treating plates with 3, 6 and 9M urea, as a dissociating solution of low avidity antigen-antibody complexes. A concentration of 9M urea best discriminated between recent and old infections in goats experimentally infected with Toxoplasma gondii. Absorbance after treating plates with 9M urea decreased in relation to the duration of infection, levelling off about the 100th day after inoculation. In a group of 116 serum samples from naturally infected goats patterns of recent, transitional or chronic toxoplasmosis were demonstrated. Serological patterns were previously determined by immunofluorescent assay. For chronic toxoplasmosis infection, the observed decrease of absorbance was 26.32 ± 10.84. For recent toxoplasmosis it was 77.61 ± 13.89, and for transitional pattern, 46.22 ± 11.94.Foi avaliada a avidez de anticorpos IgG como marcador sorológico de infecção recente e crônica pelo Toxoplasma gondii, através da dissociação do complexo antígeno-anticorpo com uréia. A avaliação foi realizada medindo-se a percentagem de queda (%Q) da absorbância, pelo ELISA, após a lavagem do complexo Ag - Ac formado com solução de uréia. Ficou determinado que a concentração dc uréia 9 Molar (M) foi a que melhor diferenciou infecções recentes e crônicas de cabras experimentalmente infectadas com Toxoplasma gondii, e que a %Q da absorbância decresce com o tempo de infecção do animal, tornando-se estável em torno do 100º. dia após a inoculação. Em um grupo de 116 amostras de soro, coletadas de caprinos naturalmente infectados, foram demonstrados perfis característicos de toxoplasmose recente, crônica ou em fase de transição. Os perfis foram previamente determinados pela avaliação da evolução dos níveis de anticorpos IgG pela reação de imunofluorescência indireta. Os animais caracterizados como portadores de toxoplasmose crônica apresentaram uma %Q da absorbância de 26,32 ± 10,84. Para toxoplasmose recente a %Q da absorbância observada foi de 77,61 ± 13,89 e para o perfil de transição, 46,22 ± 11,94

Profile of interstitial cells of Cajal in a murine model of chagasic megacolon

Abstract Disorders of gastrointestinal motility are the major physiologic problem in chagasic megacolon. The contraction mechanism is complex and controlled by different cell types such as enteric neurons, smooth muscle, telocytes, and an important pacemaker of the intestine, the interstitial cells of Cajal (ICCs). The role of ICCs in the progression of acute and chronic Chagas disease remains unclear. In the present work, we investigate the aspects of ICCs in a long-term model of Chagas disease that mimics the pathological aspects of human megacolon. Different subsets of ICCs isolated from Auerbach’s myenteric plexuses and muscle layers of control and Trypanosoma cruzi infected animals were determined by analysis of CD117, CD44, and CD34 expression by flow cytometer. Compared with the respective controls, the results showed a reduced frequency of mature ICCs in the acute phase and three months after infection. These results demonstrate for the first time the phenotypic distribution of ICCs associated with functional dysfunction in a murine model of chagasic megacolon. This murine model proved valuable for studying the profile of ICCs as an integrative system in the gut and as a platform for understanding the mechanism of chagasic megacolon development

Therapeutic responses to different anti-Trypanosoma cruzi drugs in experimental infection by benznidazole-resistant parasite stock

This study describes the role of parasite clearance time induced by benznidazole, fexinidazole and posaconazole treatments upon mice infection with a benznidazole-resistant Trypanosoma cruzi strain in the pathological outcomes. Trypanosoma cruzi-infected mice were treated with different drugs and parasite clearance time was detected by blood and tissue qPCR, to determine the dynamic relationship between the efficacy of the treatments and the intensity of heart lesion/serum inflammatory mediators. Our results indicate that anti-T. cruzi treatments were able to reduce parasite replication and consequently induce immunomodulatory effects, where the degree of the immunopathology prevention was related to the time of parasite clearance induced by different treatments. Nevertheless, in benznidazole and posaconazole treatments, parasite rebounding was detected with parasitism reaching levels similar to infected and non-treated mice; the time for parasitic rebound being earlier among benznidazole-treated mice. In parallel, an increase of cardiac lesions and plasma chemokine levels was also detected and was more accentuated in benznidazole-treated animals. Interestingly, in the presence of parasitological cure (fexinidazole treatment), basal levels of these inflammatory mediators were evidenced as well as an absence of cardiac inflammation or fibrosis. Overall, our data indicate that all treatments have positive effects on the clinical evolution of T. cruzi infection, with success in preventing cardiac alterations being drug-dependen

Use of dried blood sample for serological diagnosis of toxoplasmosis in goats

Eluates of 183 dried caprine blood samples on filter paper were tested for anti-Toxoplasma gondii antibodies, using IFA, ELISA and dot-ELISA, and compared with results obtained by direct observations on comparable sera, usin the same tests. The results has showed that papers with dried blood can be preserved for up to 45 days at room temperature and for six months at 4°C, provided they are protected against humid conditions by using desiccating agents such as silica-gel. Comparison between serum and eluate reactions revelated 97-100% correspondance. The antibody titers remained constant throughout the study period.Foi realizada pesquisa de anticorpos anti-Toxoplasma gondii em 183 amostras de sangue dessecado em papel de filtro utilizando as reações de imunofluorescência indireta ELISA e dot-ELISA, tomando como referência os resultados obtidos nos soros. A análise dos resultados demonstrou que papéis com sangue dessecado podem ser conservados por um período mínimo de 45 dias à temperatura ambiente e por seis meses a 4°C, desde que mantidos livres de umidade pela utilização de agentes dessecantes como a sílica-gel. A reprodutibilidade das reações, avaliada por meio da curva dos títulos de anticorpos no decorrer do tempo após a coleta do sangue em papéis de filtro, demonstrou uma concordância de 97 a 100% entre os resultados obtidos nos soros e eluatos. Os títulos de anticorpos permaneceram estáveis durante o período observado. Os resultados obtidos com eluato de sangue dessecado foram semelhantes na RIFI, ELISA e dot-ELISA, indicando que qualquer uma das três reações pode ser utilizada em eluatos de sangue dessecado para o diagnóstico da toxoplasmose caprina

Trypanosoma cruzi I and IV Stocks from Brazilian Amazon Are Divergent in Terms of Biological and Medical Properties in Mice

Background: In the Brazilian Amazon, clinical and epidemiological frameworks of Chagas disease are very dissimilar in relation to the endemic classical areas of transmission, possibly due to genetic and biological characteristics of the circulating Trypanosoma cruzi stocks. Twenty six T. cruzi stocks from Western Amazon Region attributed to the TcI and TcIV DTUs were comparatively studied in Swiss mice to test the hypothesis that T. cruzi clonal structure has a major impact on its biological and medical properties. Methodology/Principal Findings: Seventeen parameters were assayed in mice infected with 14 T. cruzi strains belonging to DTU TcI and 11 strains typed as TcIV. In comparison with TcI, TcIV stocks promoted a significantly shorter pre-patent period (p<0.001), a longer patent period (p<0.001), higher values of mean daily parasitemia (p = 0.009) and maximum of parasitemia (p = 0.015), earlier days of maximum parasitemia (p<0.001) and mortality (p = 0.018), higher mortality rates in the acute phase (p = 0.047), higher infectivity rates (p = 0.002), higher positivity in the fresh blood examination (p<0.001), higher positivity in the ELISA at the early chronic phase (p = 0.022), and a higher positivity in the ELISA at the late chronic phase (p = 0.003). On the other hand TcI showed higher values of mortality rates in the early chronic phase (p = 0.014), higher frequency of mice with inflammatory process in any organ (p = 0.005), higher frequency of mice with tissue parasitism in any organ (p = 0.027) and a higher susceptibility to benznidazole (p = 0.002) than TcIV. Survival analysis showing the time elapsed from the day of inoculation to the beginning of the patent period was significantly shorter for TcIV strains and the death episodes triggered following the infection with TcI occurred significantly later in relation to TcIV. The notable exceptions come from positivity in the hemocultures and PCR, for which the results were similar. Conclusion/Significance: T. cruzi stocks belonging to TcI and TcIV DTUs from Brazilian Amazon are divergent in terms of biological and medical properties in mice.publishersversionpublishe

Soroprevalência da doença de Chagas em escolares de dois municípios do Vale do Jequitinhonha, Minas Gerais, Brasil, seis anos após a implantação da vigilância epidemiológica

Six years after the beginning of the epidemiological surveillance of Chagas disease in Berilo and José Gonçalves de Minas, Jequitinhonha Valley, MG, Brazil, a serological inquiry was performed to observe whether the transmission of this endemy was occurring in this area. A randomized sample of 1,412 children seven to 14 years old, was screened. Six asymptomatic children were found to be positive, leading to 0.4% of prevalence. Hemoculture confirmed infection in five out of the six positive cases. Additional epidemiological investigation revealed important antecedents, such as disease reports in relatives and predisposing ecological and housing conditions. Our results demonstrated similar seroprevalence (0.4%) in schoolchildren, ranging from seven to 14 years old, and that were observed six years ago (0.2%) for children 0-9 year-old. Thus, considering the constant presence of Panstrogylus megistus in the peridomicile these findings emphasize the need of continuous improved epidemiological surveillance of Chagas disease in this region.Seis anos após o início da vigilância epidemiológica para doença de Chagas em Berilo e José Gonçalves de Minas, Vale do Jequitinhonha, MG, Brasil, foi realizado um inquérito sorológico para verificar se a transmissão desta endemia estava ocorrendo naquela área. Uma amostra aleatória de 1.412 crianças, de 7 a 14 anos, foi avaliada. Foram encontradas seis crianças positivas assintomáticas, totalizando uma prevalência de 0,4%. Hemocultura confirmou a infecção em cinco dos seis casos positivos. Uma investigação epidemiológica adicional, revelou importantes antecedentes, tais como: casos da doença em parentes e condições de moradia e ecológicas predisponentes. Nossos resultados demonstram uma soroprevalência similar (0,4%) em escolares de 7 a 14 anos àquela observada há seis anos (0,2%) em crianças de 0-9 anos. Dessa forma, considerando a presença constante de Panstrogylus megistus estes achados reforçam a necessidade de uma vigilância epidemiológica contínua e aperfeiçoada para Doença de Chagas naquela região

Ravuconazole self-emulsifying delivery system : in vitro activity against Trypanosoma cruzi amastigotes and in vivo toxicity.

Self-emulsifying drug delivery systems (SEDDSs) are lipid-based anhydrous formulations composed of an isotropic mixture of oil, surfactant, and cosurfactants usually presented in gelatin capsules. Ravuconazole (Biopharmaceutics Classification System [BCS] Class II) is a poorly water-soluble drug, and a SEDDS type IIIA was designed to deliver it in a predissolved state, improving dissolution in gastrointestinal fluids. After emulsification, the droplets had mean hydrodynamic diameters ,250 nm, zeta potential values in the range of -45 mV to -57 mV, and showed no signs of ravuconazole precipitation. Asymmetric flow field-flow fractionation with dynamic and multiangle laser light scattering was used to characterize these formulations in terms of size distribution and homogeneity. The fractograms obtained at 37?C showed a polydisperse profile for all blank and ravuconazole?SEDDS formulations but no large aggregates. SEDDS increased ravuconazole in vitro dissolution extent and rate (20%) compared to free drug (3%) in 6 h. The in vivo toxicity of blank SEDDS comprising Labrasol? surfactant in different concentrations and preliminary safety tests in repeated-dose oral administration (20 days) showed a dose-dependent Labrasol toxicity in healthy mice. Ravuconazole?SEDDS at low surfactant content (10%, v/v) in Trypanosoma cruzi-infected mice was safe during the 20-day treatment. The anti-T. cruzi activity of free ravuconazole, ravuconazole?SEDDS and each excipient were evaluated in vitro at equivalent ravuconazole concentrations needed to inhibit 50% or 90% (IC50 and IC90), respectively of the intracellular amastigote form of the parasite in a cardiomyocyte cell line. The results showed a clear improvement of the ravuconazole anti-T. cruzi activity when associated with SEDDS. Based on our results, the repurposing of ravuconazole in SEDDS dosage form is a strategy that deserves further in vivo investigation in preclinical studies for the treatment of human T. cruzi infections

Hematological alterations during experimental canine infection by Trypanosoma cruzi

To confirm that Beagle dogs are a good experimental model for Chagas disease, we evaluated hematological alterations during the acute and chronic phases in Beagle dogs infected with the Y, Berenice-78 (Be-78) and ABC strains of Trypanosoma cruzi, correlating clinical signs with the parasitemia curve. We demonstrate that the acute phase of infection was marked by lethargy and loss of appetite. Simultaneously, we observed anemia, leukocytosis and lymphocytosis. Also,we describe hematological alterations and clinical signs that were positively correlated with the parasitemia during the experimental infection with the three strains of T cruzi, and demonstrate that experimental infection of Beagle is a trustworthy model for Chagas disease.Research Support Foundation of the State of Minas Gerais (FAPEMIG)Research Support Foundation of the State of Minas Gerais (FAPEMIG)National Research Council for Scientific and Technological Development (CNPq)National Research Council for Scientific and Technological Development (CNPq)Coordination Office for Advancement of Universitylevel Personnel (CAPES)Coordination Office for Advancement of University-level Personnel (CAPES

IgG isotype profile is correlated with cardiomegaly in Beagle dogs infected with distinct Trypanosoma cruzi strains.

A systematic study following infection by various strains of the protozoan parasite, Trypanosoma cruzi, and the simultaneous monitoring of the humoral immune response together with the elicited cellular response, could add greatly to our understanding of differences between strains of this important human pathogen. In that sense, acute and chronic infections with distinct T. cruzi strains (Y, Berenice-78 and ABC) in Beagle dogs were studied through a longitudinal evaluation of immunoglobulin G (IgG), IgG1 and IgG2 isotypes (by ELISA and flow cytometry (FC)), as well as measurements of peripheral blood mononuclear cell (PBMC) proliferation over a 100-week period, and their correlation with cardiomegaly. Our results show that infected animals presenting cardiomegaly showed lower or absent levels of IgG1 during the chronic phase of the infection, when compared to those that did not show an increase in heart weight. In that manner, our results suggest that IgG1 could be used as a marker for cardiac pathogenicity in Chagas disease