New insights into Trypanosoma cruzi genetic diversity, and its influence on parasite biology and clinical outcomes.

Chagas disease, caused by Trypanosoma cruzi, remains a serious public health problem worldwide. The parasite was subdivided into six distinct genetic groups, called "discrete typing units" (DTUs), from TcI to TcVI. Several studies have indicated that the heterogeneity of T. cruzi species directly affects the diversity of clinical manifestations of Chagas disease, control, diagnosis performance, and susceptibility to treatment. Thus, this review aims to describe how T. cruzi genetic diversity influences the biology of the parasite and/or clinical parameters in humans. Regarding the geographic dispersion of T. cruzi, evident differences were observed in the distribution of DTUs in distinct areas. For example, TcII is the main DTU detected in Brazilian patients from the central and southeastern regions, where there are also registers of TcVI as a secondary T. cruzi DTU. An important aspect observed in previous studies is that the genetic variability of T. cruzi can impact parasite infectivity, reproduction, and differentiation in the vectors. It has been proposed that T. cruzi DTU influences the host immune response and affects disease progression. Genetic aspects of the parasite play an important role in determining which host tissues will be infected, thus heavily influencing Chagas disease's pathogenesis. Several teams have investigated the correlation between T. cruzi DTU and the reactivation of Chagas disease. In agreement with these data, it is reasonable to suppose that the immunological condition of the patient, whether or not associated with the reactivation of the T. cruzi infection and the parasite strain, may have an important role in the pathogenesis of Chagas disease. In this context, understanding the genetics of T. cruzi and its biological and clinical implications will provide new knowledge that may contribute to additional strategies in the diagnosis and clinical outcome follow-up of patients with Chagas disease, in addition to the reactivation of immunocompromised patients infected with T. cruzi.

Encapsulation of benznidazole in nanostructured lipid carriers and increased trypanocidal activity in a resistant Trypanosoma cruzi strain

Abstract Chagas disease is a neglected parasitic disease caused by Trypanosoma cruzi, whose treatment has remained unsatisfactory for over 50 years, given that it is limited to two drugs. Benznidazole (BZN) is an efficient antichagasic drug used as the first choice, although its poor water-solubility, irregular oral absorption, low efficacy in the chronic phase, and various associated adverse effects are limiting factors for treatment. Incorporating drugs with such characteristics into nanostructured lipid carriers (NLC) is a promising alternative to overcome these limiting obstacles, enhancing drug efficacy and bioavailability while reducing toxicity. Therefore, this study proposed NLC-BZN formulations in different compositions prepared by hot-melt homogenization followed by ultrasound, and the optimized formulation was characterized by FTIR, DRX, DSC, and thermogravimetry. Biological activities included in vitro membrane toxicity (red blood cells), fibroblast cell cytotoxicity, and trypanocidal activity against epimastigotes of the Colombian strain of T. cruzi. The optimized NLC-BZN had a small size (110 nm), negative zeta potential (-18.0 mV), and high encapsulation (1.64% of drug loading), as shown by infrared spectroscopy, X-ray diffraction, and thermal analysis. The NLC-BZN also promoted lower in vitro membrane toxicity (<3% hemolysis), and 50% cytotoxic concentration (CC50) for NLC-BZN in L929 fibroblast cells (110.7 µg/mL) was twice the value as the free BZN (51.3 µg/mL). Our findings showed that the NLC-BZN had higher trypanocidal activity than free BZN against the epimastigotes of the resistant Colombian strain, and this novel NLC-BZN formulation proved to be a promising tool in treating Chagas disease and considered suitable for oral and parenteral administration

Phytochemical profile and antiproliferative activity of human lymphocytes of Gomphrena virgata Mart. (Amaranthaceae).

Gomphrena virgata Mart. popularly known as 'Cangussu-branco', is used in Brazilian folk medicine to treat inflammations and infections. This work aimed to carry out phytochemical analysis and evaluate the anti-inflammatory potential of Gomphrena virgata. In the phytochemical investigation, in addition to the presence of two ecdysteroids, 20 R-dihydroxyecdysone and 20-hydroxyecdysone, identified by HPLC-PDA-MS and NMR, 22 compounds were identified by GC-MS. In the cytotoxicity study, the aqueous extract of the roots of this species did not show in vitro toxicity of PBMCs in the concentrations of 250, 500 and 1000 µg/mL when analyzed by the trypan blue exclusion method. Also, it was effective in reducing lymphocyte proliferation, stimulated with the mitogen PHA, by 26.02%, 48.57% and 50.49% when compared to dexamethasone, respectively. In this work we present information about the phytochemicals of G. virgata, showing that the species is promising in obtaining compounds with medicinal potential mainly anti-inflammatory potential.

Impedimetric immunosensor for rapid and simultaneous detection of chagas and visceral leishmaniasis for point of care diagnosis.

In this work, a dual detection system based on an impedimetric immunosensor was developed for the first time for the simultaneous detection of anti-Trypanosoma cruzi and anti-Leishmania infantum antibodies in human and dog serum samples. The IBMP 8.1 and rLci1A/rLci2B recombinant antigens were immobilized over the surface of dual screen-printed carbon electrodes (W1 and W2) modified with poly (4-hydroxyphenylacetic acid). Under optimized conditions, the immunosensor recognized specific interactions for anti-T. cruzi antibodies up to a dilution of 1:10,240 and for anti-L. infantum up to 1:5120 in canine serum samples. Relative standard deviation (RSD) values of 2.8% for W1 and 3.6% for W2 were obtained for T. cruzi (W1) and L. infantum antigen (W2) samples in three different electrodes for 3 days (n = 9). The immunosensor was stored at 4 °C for 8 weeks, with activity retention of 70.2% in W1 and 78.2% in W2. The results using the recombinant proteins revealed that all antigens discriminated between negative and positive samples (p < 0.0001) in both dog and human groups, as well as no cross-reactivity could be detected among sera with other infections. With this approach, immunosensor-based diagnostic tests achieved 100% accuracy, suggesting that the antigens are eligible to enter Phase-II studies.

Contamination by intestinal parasites in vegetables marketed in an area of Jequitinhonha Valley, Minas Gerais, Brazil / Contaminação por parasitas intestinais em hortaliças comercializadas em uma área do Vale do Jequitinhonha, Minas Gerais, Brasil

ABSTRACT Objective: The present study aimed to evaluate the presence of helminthes and intestinal protozoa in vegetables commercialized in Diamantina, a municipality located at Jequitinhonha Valley, one of the poorest regions of the world. Methods: A total of 108 specimens, including lettuce, green onion and rocket, were monthly collected from the most popular open street market, green grocery and supermarket of the municipality. The samples were processed by a concentration method and evaluated by light microscopy for parasitological identification. Results: The percentage of contamination was 50.9% (55/108), with predominance of nematode larvae (36.5%), cysts of Entamoeba coli (26.0%) and eggs of hookworms/Strongyloides spp. (12.9%). Lettuce showed greater contamination rate (61.1%) and samples from the open street market were more contaminated (77.8%). Information collected at each point of sale pointed the field cultivation as the critical step for such contaminations. Conclusion: Vegetables marketed in Diamantina presents a wide variety of intestinal parasites, which may represent a potential risk to the health of consumers of fresh vegetables.

RESUMO Objetivo: O presente estudo teve como objetivo avaliar a presença de helmintos e protozoários intestinais em hortaliças comercializados em Diamantina, um município localizado no Vale do Jequitinhonha, uma das regiões mais pobres do mundo. Métodos: Cento e oito exemplares, incluindo alface, cebolinha e rúcula, foram mensalmente coletados em uma feira livre, uma quitanda e um supermercado do município. As amostras foram processadas por um método de concentração e avaliadas por microscopia óptica para pesquisa de estruturas parasitárias. Resultados: O percentual global de contaminação foi de 50,9% (55/108), com predominância de larvas de nematódeos (36,5%), cistos de Entamoeba coli e ovos de ancilostomídeos/Strongyloides spp. (12,9%). A alface demonstrou a maior taxa de contaminação (61,1%) e as amostras da feira livre foram as mais contaminadas (77,8%). Informações coletadas em cada ponto de venda apontaram o cultivo em campo como a etapa crítica para a contaminação. Conclusão: Hortaliças comercializadas em Diamantina apresentam uma ampla variedade de parasitas intestinais, o que representa um risco potencial à saúde dos consumidores da área.

Spatial distribution of triatomines in domiciles of an urban area of the Brazilian Southeast Region.

Reports of triatomine infestation in urban areas have increased. We analysed the spatial distribution of infestation by triatomines in the urban area of Diamantina, in the state of Minas Gerais, Brazil. Triatomines were obtained by community-based entomological surveillance. Spatial patterns of infestation were analysed by Ripley's K function and Kernel density estimator. Normalised difference vegetation index (NDVI) and land cover derived from satellite imagery were compared between infested and uninfested areas. A total of 140 adults of four species were captured (100 Triatoma vitticeps, 25 Panstrongylus geniculatus, 8 Panstrongylus megistus, and 7 Triatoma arthurneivai specimens). In total, 87.9% were captured within domiciles. Infection by trypanosomes was observed in 19.6% of 107 examined insects. The spatial distributions ofT. vitticeps, P. geniculatus, T. arthurneivai, and trypanosome-positive triatomines were clustered, occurring mainly in peripheral areas. NDVI values were statistically higher in areas infested by T. vitticeps and P. geniculatus. Buildings infested by these species were located closer to open fields, whereas infestations of P. megistus and T. arthurneivai were closer to bare soil. Human occupation and modification of natural areas may be involved in triatomine invasion, exposing the population to these vectors.

Spatial distribution of triatomines in domiciles of an urban area of the Brazilian Southeast Region

Reports of triatomine infestation in urban areas have increased. We analysed the spatial distribution of infestation by triatomines in the urban area of Diamantina, in the state of Minas Gerais, Brazil. Triatomines were obtained by community-based entomological surveillance. Spatial patterns of infestation were analysed by Ripley’s K function and Kernel density estimator. Normalised difference vegetation index (NDVI) and land cover derived from satellite imagery were compared between infested and uninfested areas. A total of 140 adults of four species were captured (100 Triatoma vitticeps, 25Panstrongylus geniculatus, 8 Panstrongylus megistus, and 7 Triatoma arthurneivai specimens). In total, 87.9% were captured within domiciles. Infection by trypanosomes was observed in 19.6% of 107 examined insects. The spatial distributions ofT. vitticeps, P. geniculatus, T. arthurneivai, and trypanosome-positive triatomines were clustered, occurring mainly in peripheral areas. NDVI values were statistically higher in areas infested by T. vitticeps and P. geniculatus. Buildings infested by these species were located closer to open fields, whereas infestations of P. megistus andT. arthurneivai were closer to bare soil. Human occupation and modification of natural areas may be involved in triatomine invasion, exposing the population to these vectors.

Trypanosoma cruzi Discret Typing Units (TcII and TcVI) in samples of patients from two municipalities of the Jequitinhonha Valley, MG, Brazil, using two molecular typing strategies.

BACKGROUND: Trypanosoma cruzi is classified into six discrete taxonomic units (DTUs). For this classification, different biological markers and classification criteria have been used. The objective was to identify the genetic profile of T. cruzi samples isolated from patients of two municipalities of Jequitinhonha Valley, MG, Brazil. METHODS: Molecular characterization was performed using two different criteria for T. cruzi typing to characterize 63 T. cruzi samples isolated from chronic Chagas disease patients. The characterizations followed two distinct methodologies. Additionally, the RAPD technique was used to evaluate the existence of genetic intragroup variability. RESULTS: The first methodology identified 89% of the samples as TcII, but it was not possible to define the genetic identity of seven isolates. The results obtained with the second methodology corroborated the classification as TcII of the same samples and defined the classification of the other seven as TcVI. RAPD analysis showed lower intra-group variability in TcII. CONCLUSIONS: The results confirmed the preliminary data obtained in other municipalities of the Jequitinhonha Valley, showing a predominance of TcII, similar to that verified in northeast/south axis of Brazil and the first detection of TcVI in the study region. The second protocol was more simple and reliable to identify samples of hybrid character.

Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley, Minas Gerais, Brazil, with Chagas disease.

Trypanosoma cruzi strains from distinct geographic areas show differences in drug resistance and association between parasites genetic and treatment response has been observed. Considering that benznidazole (BZ) can reduce the parasite burden and tissues damage, even in not cured animals and individuals, the goal is to assess the drug response to BZ of T. cruzi II strains isolated from children of the Jequitinhonha Valley, state of Minas Gerais, Brazil, before treatment. Mice infected and treated with BZ in both phases of infection were compared with the untreated and evaluated by fresh blood examination, haemoculture, polymerase chain reaction, conventional (ELISA) and non-conventional (FC-ALTA) serologies. In mice treated in the acute phase, a significant decrease in parasitaemia was observed for all strains. Positive parasitological and/or serological tests in animals treated during the acute and chronic (95.1-100%) phases showed that most of the strains were BZ resistant. However, beneficial effect was demonstrated because significant reduction (p < 0.05%) and/or suppression of parasitaemia was observed in mice infected with all strains (acute phase), associated to reduction/elimination of inflammation and fibrosis for two/eight strains. BZ offered some benefit, even in not cured animals, what suggest that BZ use may be recommended at least for recent chronic infection of the studied region.

Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley, Minas Gerais, Brazil, with Chagas disease

Trypanosoma cruzi strains from distinct geographic areas show differences in drug resistance and association between parasites genetic and treatment response has been observed. Considering that benznidazole (BZ) can reduce the parasite burden and tissues damage, even in not cured animals and individuals, the goal is to assess the drug response to BZ of T. cruzi II strains isolated from children of the Jequitinhonha Valley, state of Minas Gerais, Brazil, before treatment. Mice infected and treated with BZ in both phases of infection were compared with the untreated and evaluated by fresh blood examination, haemoculture, polymerase chain reaction, conventional (ELISA) and non-conventional (FC-ALTA) serologies. In mice treated in the acute phase, a significant decrease in parasitaemia was observed for all strains. Positive parasitological and/or serological tests in animals treated during the acute and chronic (95.1-100%) phases showed that most of the strains were BZ resistant. However, beneficial effect was demonstrated because significant reduction (p < 0.05%) and/or suppression of parasitaemia was observed in mice infected with all strains (acute phase), associated to reduction/elimination of inflammation and fibrosis for two/eight strains. BZ offered some benefit, even in not cured animals, what suggest that BZ use may be recommended at least for recent chronic infection of the studied region.

Molecular and biological characterization of Trypanosoma cruzi strains isolated from children from Jequitinhonha Valley, State of Minas Gerais, Brazil.

INTRODUCTION: The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. METHODS: Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. RESULTS: The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2 ± 0.25 to 13.7 ± 3.08 days), whereas the patent period ranged from 3.3 ± 1.08 to 34.5 ± 3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. CONCLUSIONS: The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis.

Molecular and biological characterization of Trypanosoma cruzi strains isolated from children from Jequitinhonha Valley, State of Minas Gerais, Brazil

Introduction The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. Methods Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. Results The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2±0.25 to 13.7±3.08 days), whereas the patent period ranged from 3.3±1.08 to 34.5±3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. Conclusions The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis. .

Genetic modulation in Be-78 and Y Trypanosoma cruzi strains after long-term infection in Beagle dogs revealed by molecular markers.

The genetic profile of Trypanosoma cruzi was evaluated in parasite populations isolated from Beagle dogs experimentally infected with Be-78 and Y strains that present distinct biological and genetic characteristics. Molecular characterization of the isolates obtained 30days and 2years after infection was carried out. For typing MLEE, sequence polymorphisms of the mitochondrial cytochrome oxidase subunit II gene (COII) and RAPD profiles were used. The profiles of MLEE were the same for the parental Be-78 strains as their respective isolates. However, changes of MLEE profile were observed in two T. cruzi isolates from dogs inoculated with Y strain. Changes in the mitochondrial DNA (COII) and RAPD profiles of the Y strain were also observed. The dendogram constructed by UPGMA with RAPD results indicated two major branches. Global data show that the genetic modulation in polyclonal strains during the long-term infection occurred and was strain-dependent. This study still suggests that each host (here each dog) harbors a determinate T. cruzi population that may change or be modulated throughout long-term infection. This might to hinder the observation of correlation between the genetics of T. cruzi and their biological properties and behavior in different host species due to the complexity of the parasite-host interaction in which probably the genetic background of both should be considered.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity.

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity / Método de ELISA in-house na análise da reatividade de IgG anti-Trypanosoma cruzi para o diagnóstico diferencial e avaliação da morbidade da doença de Chagas

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100 percent sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

INTRODUÇÃO: O objetivo foi desenvolver um método sorológico in-house de alta especificidade e sensibilidade para diagnosticar e monitorar a morbidade da doença de Chagas. MÉTODOS: Para tal, a reatividade sorológica de IgG e subclasses foi testada em soros de pacientes chagásicos de Berilo, Vale do Jequitinhonha/MG/Brasil. A reatividade sorológica foi também avaliada em amostras de pacientes com outras doenças infecto-contagiosas relevantes, incluindo o HIV, vírus da hepatite C (VHC), sífilis (SYP), leishmaniose visceral (LV), leishmaniose tegumentar americana (LTA) e controles não infectados (NI) para verificar o desempenho do método. Outras análises foram feitas para avaliar a aplicabilidade desta metodologia no monitoramento da morbidade da doença de Chagas. Com este propósito os pacientes com doença de Chagas foram anteriormente classificados em três grupos: indeterminados (IND), cardíacos (CARD) e digestivos/mistos (DIG/Mis) conforme seu estado clínico. RESULTADOS: A análise da reatividade sorológica de IgG total na diluição 1:40 mostrou ser uma abordagem importante no diagnóstico da doença de Chagas (100 por cento de sensibilidade e especificidade e ausência de reação cruzada com as demais infecções). A análise das subclasses de IgG mostrou reação cruzada principalmente com NI, LV e LTA em todas as diluições. O grupo IND apresentou a maior reatividade para IgG3 e o grupo DIG/Mis apresentou nível mais elevado de IgG se comparados aos grupos IND e CARD. CONCLUSÕES: Estes achados demonstram que o método de ELISA in-house apresenta uma promissora aplicabilidade no diagnóstico diferencial e na avaliação da morbidade da doença de Chagas.

Trypanosoma cruzi: effect of benznidazole therapy combined with the iron chelator desferrioxamine in infected mice.

Iron chelators have been employed in various studies aimed at evaluating the relationship between the iron status of the host and the development of infection. In the present study, the effects of benznidazole (BZ) therapy in combination with the iron chelator desferrioxamine (DFO) on the development of infection in mice inoculated with Trypanosoma cruzi Y strain have been investigated. Infected mice treated with DFO presented lower levels of parasitemia compared with infected untreated animals. Therapy with BZ for 21 days, with or without DFO, led to decreased parasitemia and reduced mortality, but BZ in combination with DFO treatment for 35 days (BZ/DFO-35) gave 0% mortality. All infected groups presented lower levels of iron in the liver, but serum iron concentrations were greater in DFO-35 and BZ/DFO-35, whereas hemoglobin levels were higher in BZ/DFO-35 and lower in DFO-35 compared with other treated groups. The percentage cure, determined from negative hemoculture and PCR results in animals that had survived for 60 days post-infection, was 18% for BZ and BZ/DFO-35, 42% for BZ combined with DFO for 21 days, and 67% for DFO-35. The results demonstrate that modification in iron stores increases BZ efficacy.

Tratamento com Benzonidazol em camundongos infectados com misturas de populações do Trypanosoma cruzi pertencentes aos genótipos principais / Treatment with Benznidazole in mice infected with mixtures of populations of Trypanosoma cruzi major genotypes to belonging

O objetivo desse trabalho foi avaliar o impacto de infecções mistas por clones de Trypanosoma cruzi pertencentes aos quatro genótipos principais na eficácia terapêutica do Benzonidazol (BZ) em camundongos BALB/c. Para esta proposta, foram empregados dois clones do T. cruzi de cada genótipo com diferentes níveis de virulência e de susceptibilidade ao BZ, que foram combinados aos pares totalizando 24 misturas distintas avaliadas comparativamente às respectivas monoinfecções. O tratamento com BZ reduziu os níveis de parasitemia além de outros parâmetros relacionados na maioria das infecções mistas, exceto nas da combinação dos genótipos 39+32. As taxas de mortalidade dos animais tratados (IT) e seus controles (INT) não diferiram significativamente entre as monoinfecções e as infecções mistas. A cura após tratamento foi definida empregando o critério de cura clássico, proposto por Krettli & Brener (1982) por meio de métodos parasitológicos (exame de sangue a fresco-ESF, hemocultura-Hc e PCR no sangue) e sorológicos (ELISA e pesquisa de anticorpos antitripomastigotas vivos-AATV) e, permitiu classificar os animais em tratados não curados (TNC=72,2%), tratados curados (TC=19,1) e dissociados (DIS=8,7%). Em geral, o ESF apresentou baixa positividade para a detecção da falha terapêutica (18,1%).

A Hc apresentou sensibilidade satisfatória para a maioria das infecções, exceto nos animais infectados com o genótipo 39 e com a combinação 39+32; enquanto a PCR foi o método parasitológico mais sensível, a despeito da diversidade genética do T. cruzi. Os dados demonstraram que a ELISA não deve ser utilizada em amostras coletadas antes de três meses após tratamento, uma vez que pode apresentar resultados falsos negativos. A AATV foi mais eficiente na categorização dos animais em relação à resposta terapêutica e na definição precoce de cura. Os dados do presente estudo elegem a AATV e a PCR em sangue como as técnicas mais adequadas para a definição precoce de cura após tratamento da fase aguda da infecção pelo T. cruzi no modelo murino. Um importante achado deste estudo foi a observação de resultados positivos da PCR em tecidos de animais considerados curados pelo critério clássico, sugerindo infecção residual. Uma importante questão levantada por esses resultados é se a cura parasitológica de fato existe. A avaliação dos perfis de susceptibilidade ao BZ das infecções mistas foi realizada pela comparação entre os índices de cura observado e o esperado e, revelou mudança em direção ao padrão de menor susceptibilidade ao BZ nas combinações 19+39 e 19+32. Entretanto, quando os resultados foram analisados em nível dos clones do T. cruzi, foram observadas mudanças em nove das 24 misturas tendo sido detectados tanto aumento como diminuição na susceptibilidade ao fármaco.

A caracterização molecular da amostra Cuica cl1, até então considerada um clone, demonstrou ser constituída de fato por duas populações geneticamente distintas cuja dinâmica populacional pode mudar dependendo da manutenção laboratorial. Entretanto, essas duas populações não apresentaram diferenças no perfil de susceptibilidade ao BZ. Em geral, a PCR de microssatélites apresentou boa correlação com a análise de isoenzimas na identificação dos clones presentes nas amostras isoladas de animais TNC e INT e confirmou a relação filogenética previamente determinada entre os clones por meio das análises de isoenzimas e RAPD. Apesar da análise de microssatélites apresentar baixa eficiência na identificação dos clones diretamente no sangue e nos tecidos de animais INT e TNC, os resultados obtidos dessas amostras biológicas foram similares aos obtidos para clones caracterizados após o isolamento pela Hc. Foram detectadas misturas em 7,7% dos isolados provenientes de animais INT e TNC obtidos por Hc. Em algumas misturas, resultados interessantes foram detectados revelando a presença do clone mais susceptível nas monoinfecções. Considerando a estrutura clonal do T. cruzi, um resultado inesperado foi a identificação de 15,5% dos isolados apresentando subpopulações do parasito com alelos de microssatélites distintos comparados aos clones originais. Esses achados sugerem que em alguns casos o tratamento com Benzonidazol pode resultar em um desequilíbrio da relação parasito-hospedeiro e levar ao estabelecimento de um curso da infecção distinto. Em conjunto, esses achados sugerem que infecções mistas podem apresentar um importante impacto na eficácia terapêutica. Estudos futuros são necessários para o melhor entendimento dos mecanismos envolvidos nas mudanças genéticas e no perfil de susceptibilidade ao Benzonidazol observado nas infecções mistas e o real benefício do tratamento para o hospedeiro

Tratamento com Benzonidazol em camundongos infectados com misturas de populações do Trypanosoma cruzi pertencentes aos genótipos principais

O objetivo desse trabalho foi avaliar o impacto de infecções mistas por clones de Trypanosoma cruzi pertencentes aos quatro genótipos principais na eficácia terapêutica do Benzonidazol (BZ) em camundongos BALB/c. Para esta proposta, foram empregados dois clones do T. cruzi de cada genótipo com diferentes níveis de virulência e de susceptibilidade ao BZ, que foram combinados aos pares totalizando 24 misturas distintas avaliadas comparativamente às respectivas monoinfecções. O tratamento com BZ reduziu os níveis de parasitemia além de outros parâmetros relacionados na maioria das infecções mistas, exceto nas da combinação dos genótipos 39+32. As taxas de mortalidade dos animais tratados (IT) e seus controles (INT) não diferiram significativamente entre as monoinfecções e as infecções mistas. A cura após tratamento foi definida empregando o critério de cura clássico, proposto por Krettli & Brener (1982) por meio de métodos parasitológicos (exame de sangue a fresco-ESF, hemocultura-Hc e PCR no sangue) e sorológicos (ELISA e pesquisa de anticorpos antitripomastigotas vivos-AATV) e, permitiu classificar os animais em tratados não curados (TNC=72,2%), tratados curados (TC=19,1) e dissociados (DIS=8,7%). Em geral, o ESF apresentou baixa positividade para a detecção da falha terapêutica (18,1%).

A Hc apresentou sensibilidade satisfatória para a maioria das infecções, exceto nos animais infectados com o genótipo 39 e com a combinação 39+32; enquanto a PCR foi o método parasitológico mais sensível, a despeito da diversidade genética do T. cruzi. Os dados demonstraram que a ELISA não deve ser utilizada em amostras coletadas antes de três meses após tratamento, uma vez que pode apresentar resultados falsos negativos. A AATV foi mais eficiente na categorização dos animais em relação à resposta terapêutica e na definição precoce de cura. Os dados do presente estudo elegem a AATV e a PCR em sangue como as técnicas mais adequadas para a definição precoce de cura após tratamento da fase aguda da infecção pelo T. cruzi no modelo murino. Um importante achado deste estudo foi a observação de resultados positivos da PCR em tecidos de animais considerados curados pelo critério clássico, sugerindo infecção residual. Uma importante questão levantada por esses resultados é se a cura parasitológica de fato existe. A avaliação dos perfis de susceptibilidade ao BZ das infecções mistas foi realizada pela comparação entre os índices de cura observado e o esperado e, revelou mudança em direção ao padrão de menor susceptibilidade ao BZ nas combinações 19+39 e 19+32. Entretanto, quando os resultados foram analisados em nível dos clones do T. cruzi, foram observadas mudanças em nove das 24 misturas tendo sido detectados tanto aumento como diminuição na susceptibilidade ao fármaco.

A caracterização molecular da amostra Cuica cl1, até então considerada um clone, demonstrou ser constituída de fato por duas populações geneticamente distintas cuja dinâmica populacional pode mudar dependendo da manutenção laboratorial. Entretanto, essas duas populações não apresentaram diferenças no perfil de susceptibilidade ao BZ. Em geral, a PCR de microssatélites apresentou boa correlação com a análise de isoenzimas na identificação dos clones presentes nas amostras isoladas de animais TNC e INT e confirmou a relação filogenética previamente determinada entre os clones por meio das análises de isoenzimas e RAPD. Apesar da análise de microssatélites apresentar baixa eficiência na identificação dos clones diretamente no sangue e nos tecidos de animais INT e TNC, os resultados obtidos dessas amostras biológicas foram similares aos obtidos para clones caracterizados após o isolamento pela Hc. Foram detectadas misturas em 7,7% dos isolados provenientes de animais INT e TNC obtidos por Hc. Em algumas misturas, resultados interessantes foram detectados revelando a presença do clone mais susceptível nas monoinfecções. Considerando a estrutura clonal do T. cruzi, um resultado inesperado foi a identificação de 15,5% dos isolados apresentando subpopulações do parasito com alelos de microssatélites distintos comparados aos clones originais. Esses achados sugerem que em alguns casos o tratamento com Benzonidazol pode resultar em um desequilíbrio da relação parasito-hospedeiro e levar ao estabelecimento de um curso da infecção distinto. Em conjunto, esses achados sugerem que infecções mistas podem apresentar um importante impacto na eficácia terapêutica. Estudos futuros são necessários para o melhor entendimento dos mecanismos envolvidos nas mudanças genéticas e no perfil de susceptibilidade ao Benzonidazol observado nas infecções mistas e o real benefício do tratamento para o hospedeiro

Trypanosoma cruzi: treatment with the iron chelator desferrioxamine reduces parasitemia and mortality in experimentally infected mice.

The effects of prolonged treatment with iron chelator (desferrioxamine) on the development of infection in mice inoculated with Y Trypanosoma cruzi were determined. Infected/treated mice presented lower levels of parasitemia and reduced mortality rate compared with infected/non-treated animals. The five out of twenty infected/treated mice that survived the acute phase of infection showed negative hemoculture and positive ELISA in the acute and chronic phases and positive PCR in the acute phase: in the chronic phase, three of the animals presented negative PCR. The single surviving infected/non-treated animal exhibited positive hemoculture, PCR and ELISA in both phases of infection. Infected groups presented lower levels of iron in the liver compared with treated/non-infected or non-treated/non-infected animals. The serum iron levels of the infected/non-treated group were higher on the 21st day post-infection in comparison with control and infected/treated groups. These results suggest that decrease of iron in the host leads to T. cruzi infection attenuation.