944 resultados para Leishmania chagasi Recombinant antigens


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A doença de Chagas é uma zoonose causada pelo protozoário flagelado Trypanosoma cruzi. Estima-se que 8 milhões de pessoas estão infectadas com o T. cruzi em todo o mundo, principalmente na América Latina. Testes tradicionais de diagnóstico estão sendo gradualmente substituídos por métodos inovadores. A utilização de antígenos recombinantes foi proposta nos anos 90, e várias combinações foram testadas com soros de pacientes com diferentes formas clínicas de diferentes regiões da América Latina. Apesar do ganho em especificidade, estes testes apresentaram menor sensibilidade, frustrando expectativas. Este estudo objetivou analisar a variabilidade genética dos genes KMP11 e 1F8 que codificam antígenos comumente utilizados em diagnóstico experimental. Cepas de T. cruzi pertencentes a diferentes sub-grupos taxonômicos e de diferentes regiões foram analisadas para avaliar o impacto da variação antigênica em testes de diagnóstico. Maximizando a sensibilidade, evitar reatividade cruzada com epítopos de outros agentes patogênicos deve permitir a concepção de melhores testes rápidos. Num primeiro passo, DNA genômico foi extraído das seguintes cepas: Dm28c, Colombiana, Y, 3663, 4167, LL014 e CL Brener e foi realizada a amplificação dos genes 1F8 e KMP11 que codificam antígenos a partir destas cepas. Em seguida foram realizadas a clonagem, sequenciamento, expressão e detecção dos antígenos recombinantes. Na etapa final, análises de estruturas secundárias e terciárias, a última apenas para o antígeno 1F8, visualizaram as diferenças nas sequências de aminoácidos obtidas a partir de sequenciamento de DNA. Os resultados apresentados neste estudo mostram que o antígeno KMP11 de T. cruzi possui uma similaridade na sequência de aminoácidos muito elevada com o T. rangeli, mostrando a necessidade de um mapeamento antigênico desta proteína em todos os tripanosomatídeos que apresentaram alta similaridade com o antígeno de T. cruzi, como o T. rangeli, para verificar a presença de epítopos específicos de T. cruzi. O antígeno 1F8 pode ser uma ferramenta útil no diagnóstico da doença de Chagas e que será necessário aprofundar os conhecimentos sobre os determinantes antigênicos para futuramente elaborar poli-epítopos sintéticos adaptados de um maior número possível de antígenos, obtendo a maior especificidade e sensibilidade nos testes de diagnóstico sorológico e de teste rápido da doença de Chagas. Este estudo representa um passo crucial para a otimização de antígenos recombinantes para o diagnóstico da doença de Chagas.

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Metalworking fluid-associated hypersensitivity pneumonitis (MWF-HP) is a pulmonary disease caused by inhaling microorganisms present in the metalworking fluids used in the industrial sector. Mycobacterium immunogenum is the main etiological agent. Among the clinical, radiological and biological tools used for diagnosis, serological tests are important. The aim of this study was to identify immunogenic proteins in M. immunogenum and to use recombinant antigens for serological diagnosis of MWF-HP. Immunogenic proteins were detected by two-dimensional Western blot and candidate proteins were identified by mass spectrometry. Recombinant antigens were expressed in Escherichia coli and tested by enzyme-linked immunosorbent assay (ELISA) with the sera of 14 subjects with MWF-HP and 12 asymptomatic controls exposed to M. immunogenum. From the 350 spots visualized by two-dimensional gel electrophoresis with M. immunogenum extract, 6 immunogenic proteins were selected to be expressed as recombinant antigens. Acyl-CoA dehydrogenase antigen allowed for the best discrimination of MWF-HP cases against controls with an area under the receiver operating characteristics (ROC) curve of 0.930 (95% CI=0.820-1), a sensitivity of 100% and a specificity of 83% for the optimum threshold. Other recombinant antigens correspond to acyl-CoA dehydrogenase FadE, cytosol aminopeptidase, dihydrolipoyl dehydrogenase, serine hydroxymethyltransferase and superoxide dismutase. This is the first time that recombinant antigens have been used for the serodiagnosis of hypersensitivity pneumonitis. The availability of recombinant antigens makes it possible to develop standardized serological tests which in turn could simplify diagnosis, thus making it less invasive.

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To find the most reliable screening method for Trypanosoma cruzi infection in blood banks. Epidemiological data, lymphoproliferation assay, parasitological, conventional serological tests: immunofluorescence, haemagglutination, ELISA with epimastigote and trypomastigote antigens and reference serological tests: trypomastigote excreted-secreted antigens (TESA) blot and chemiluminescent ELISA assay with mucine from trypomastigote forms were applied to individuals with inconclusive serology, non-chagasic individuals and chronic chagasic patients. TESA blot had the best performance when used as a single test in all the groups. In the inconclusive group 20.5% of individuals were positive for TESA blot, 23.3% for either lymphoproliferation or TESA blot, and 17.8% for lymphoproliferation only. Positive lymphoproliferation without detectable antibodies was observed in 5.47% of all inconclusive serology cases. Analysis of six parameters (three serological assays, at least one parasitological test, one lymphoproliferation assay and epidemiological data) in the inconclusive group showed that diagnosis of Chagas` disease was probable in 15 patients who were positive by two or more serological tests or for whom three of those six parameters were positive. TESA blot is a good confirmatory test for Chagas` disease in the inconclusive group. Although lymphoproliferation suggests the diagnosis of Chagas` disease in the absence of antibodies when associated with a high epidemiological risk of acquiring Chagas` disease, the data from this study and the characteristics of the lymphoproliferation assay (which is both laborious and time-consuming) do not support its use as a confirmatory test in blood-bank screening. However, our findings underscore the need to develop alternative methods that are not based on antibody detection to improve the diagnosis when serological tests are inconclusive.

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BACKGROUND: A major problem in Chagas disease donor screening is the high frequency of samples with inconclusive results. The objective of this study was to describe patterns of serologic results among donors to the three Brazilian REDS-II blood centers and correlate with epidemiologic characteristics. STUDY DESIGN AND METHODS: The centers screened donor samples with one Trypanosoma cruzi lysate enzyme immunoassay (EIA). EIA-reactive samples were tested with a second lysate EIA, a recombinant-antigen based EIA, and an immunfluorescence assay. Based on the serologic results, samples were classified as confirmed positive (CP), probable positive (PP), possible other parasitic infection (POPI), and false positive (FP). RESULTS: In 2007 to 2008, a total of 877 of 615,433 donations were discarded due to Chagas assay reactivity. The prevalences (95% confidence intervals [CIs]) among first-time donors for CP, PP, POPI, and FP patterns were 114 (99-129), 26 (19-34), 10 (5-14), and 96 (82-110) per 100,000 donations, respectively. CP and PP had similar patterns of prevalence when analyzed by age, sex, education, and location, suggesting that PP cases represent true T. cruzi infections; in contrast the demographics of donors with POPI were distinct and likely unrelated to Chagas disease. No CP cases were detected among 218,514 repeat donors followed for a total of 718,187 person-years. CONCLUSION: We have proposed a classification algorithm that may have practical importance for donor counseling and epidemiologic analyses of T. cruzi-seroreactive donors. The absence of incident T. cruzi infections is reassuring with respect to risk of window phase infections within Brazil and travel-related infections in nonendemic countries such as the United States.

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Investigation of the bioactive crude extract from the sponge Plakortis angulospiculatus from Brazil led to the isolation of plakortenone (1) as a new polyketide, along with five known polyketides (2-6) previously isolated from other Plakortis sponges. The known polyketides were tested in antileishmanial, antitrypanosomal, antineuroinflammatory, and cytotoxicity assays. The results show that plakortide P (3) is a potent antiparasitic compound, against both Leishmania chagasi and Trypanosona cruzi, and exhibited antineuroinflammatory activity. The known polyketides 2-6 were tested for cytotoxicity against four human cancer cell lines, but displayed only moderate cytotoxic activity.

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The Visceral Leishmaniose (LV) disease is endemic in some places in Brazil. It is caused by the protozoa Leishmania chagasi, being transmitted for vector, the phlebotomies, Lutzomyia longipalpis. In virtue of the expansion of the illness in Rio Grande do Norte, it is necessary to evaluate the determinative ambient factors in the proliferation of the vector for better control of the illness. The variable rainfall and the social variables had been analyzed using space regression with two models and the ambient variable of ZANE and the variables analyzed in 205 houses in the cities of Natal, Extremoz, Nísia Floresta, São Gonçalo do Amarante, São Jose do Mipibu, Parnamirim and Macaíba the Person and ML Chi-square were used . The analyses had shown that high rainfall, plain relief, the forest, the humid tropical climate the activities of production culture of sugar cane and fruit culture and the presence of bovines increase the risk of the LV. The work showed that it has space aggregation and that ambient factors influence in the LV in the State

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Leishmaniasis are endemic diseases wild spread in the New and Old World, caused by the flagelated protozoan Leishmania. In the New World, the distribution of different forms of leishmaniasis is mostly in tropical regions. In the State of Rio Grande do Norte, Northeast Brazil, 85% of the captured sand flies fauna is Lutzomyia longipalpis. The distribution of the sand fly vector in the state overlaps with the disease distribution, where the presence of sand flies is associated with presence of animals shelters. The aim of this study was to analyse the blood meal preference of sand flies vector from the genus Lutzomyia spp. in laboratory conditions, to verify the vector life cicle at different temperatures sets and to identify the main blood meal source in endemic areas for visceral leishmaniasis (VL) at peri-urban regions of Natal. Sand flies samples were collected from the municipalities of São Gonçalo do Amarante and Nísia Floresta where female sand flies were grouped for the colony maintenance in the laboratory and for the analysis of the preferred source of sand fly blood meal in natural environment. The prevalence of blood meal preference and oviposition for the females sand flies was 97% for Cavia porcellus with oviposition of 19 eggs/female; 97% for Eqqus caballus with 19 eggs/female; 98% for human blood with 14 eggs/female; 71.3% for Didelphis albiventris with 8.4 eggs/female; 73% for Gallus gallus with 14 eggs/female; 86% for Canis familiaris with 10.3 eggs/female; 81.4% for Galea spixii with 26 eggs/female; 36% for Callithrix jachus with 15 eggs/female; 42.8% for Monodelphis domestica with 0% of oviposition. Female sand flies did not take a blood meal from Felis catus. Sand flies life cycle ranged from 32-40 days, with 21-50 oviposition rates approximately. This study also showed that at 32°C the life cycle had 31 days, at 28° C it had 50 days and at 22°C it increased to 79 days. Adjusting the temperature to 35°C the eggs did not hatch, thus blocking the life cycle. A total of 1540 sand flies were captured, among them, 1.310 were male and 230 were female. Whereas 86% of the sand flies captured were Lu. longipalpis as compared to 10.5% for Lu. evandroi and, 3.2% for L. lenti and 0.3% for Lu whitmani. The ratio between female and male sandfly was approximately 6 males to 1 female. In Nísia Floresta, 50.7% of the collected females took their blood meal from armadillo, 12.8% from human. Among the female sand flies captured in São Gonçalo do Amarante, 80 of them were tested for the Leishmania KDNA infectivity where 5% of them were infected with Leishmania chagasi. Female Lutzomyia spp. showed to have an opportunistic blood meal characteristic. The behavioral parameters seem to have a higher influence in the oviposition when compared to the level of total proteins detected in the host s bloodstream. A higher Lu. longipalpis life cycle viability was observed at 28°C. The increase of temperature dropped the life cycle time, which means that the life cycle is modified by temperature range, source of blood meal and humidity. Lu longipalpis was the most specie found in the inner and peridomiciliar environment. In Nísia Floresta, armadillos were the main source of blood meal for Lutzomyia spp. At São Gonçalo do Amarante, humans were the main source of blood meal due to CDC nets placed inside their houses

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Lectin obtained from the marine sponge Tedania ignis was purified and characterized by extraction of soluble proteins (crude extract) in 50mM Borax, pH 7.5. The purification procedure was carried out by crude extract precipitation with ammonium sulfate 30% (FI). The precipitated was resuspended in the same buffer and fractionated with acetone 1.0 volume (F1.0). A lectin was purified from this specific fraction by using an affinity chromatography Sepharose 6B. This lectin preferentially agglutinated human erythrocytes from B type previously treated with papain enzyme. The hemagglutinating activity lectin was dependent of divalent Mn2+ cation and was inhibited by the carbohydrates galactose, xylose and fructose. SDS-PAGE analysis indicated a molecular mass of the lectin around 45 kDa. This protein showed stability until 40°C for 1 h. Further, it showed activity between pH 2.5 and 11.5, with an enhanced activity at pH 7.5. Leishmania chagasi promastigotes stained with Coomassie brilliant blue R-250 were agglutinated by F1,0 and in the presence of galactose this interaction was abolished. These results show that this lectin could be implicated in defense procedures and it will can be used as biological tools in studies with this protozoon

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Visceral leishmaniasis (VL) has undergone changes in terms of clinical and epidemiological presentation worldwide. Urbanization has been described in different regions of Brazil and the world, as well as in the state of Rio Grande do Norte. These changes have impacted in the clinical outcome of Leishmania infection. A new clinical entity called co-infection of HIV/Leishmania has been described as a consequence of overlapping areas of occurrence of VL and HIV / AIDS in different countries including Brazil. The aim of this study was to define the process of periurbanization of the LV and describe a case series of co-infection HIV / Leishmania in Rio Grande do Norte. A new demographic pattern of VL was detected, with an increase in the number VL adult male subjects. Analysis of spatial distribution of VL in the state of Rio Grande do Norte showed that in the past 20 years VL tends to occur in larger cities and therefore the highest risk disease is greater in the eastern and western regions. The first region included Natal, the state capital, where the process of suburbanization began in 1990, and more recently the city of Mossoró, the second largest state, where periurbanization began in the last five years. In 1990, the emergence of co-infection HIV/Leishmania in the state was observed. Case-control study revealed that the new clinical entity affects adult males, who acquired HIV through sexual intercourse, 40% of those with a preivous history of leishmania infection Relapse and death from LV is increased in HIV positive compared with HIV-negative patients matched by sex and age. This pattern is similar to the observed in Europe, except of the route of transmission, where in Europe occured concomitantly, by parenteral route in drug users. Analysis of spatial distribution identified overlapping new areas of occurrence of HIV / AIDS and LV potentially signaling to increased risk of this new clinical entity as described above. Therefore, epidemiological surveillance for co-infection HIV / Leishmania should be adopted in all areas of risk of VL. At the same time, it is necessary to evaluate drug resistance currently used in the treatment of VL, as well as parenteral transmission of L infantum/ chagasi in areas where drug dependence is a risk factor for HIV acquisition

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO(R) vector which contains thioredoxin and polyhistidine tags at the C-and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 mu g/mL growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

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The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety Vitória de Verão genetically modified.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Leishmaniasis is a parasitic zoonosis caused by protozoans of the genus Leishmania transmitted by insects known as phlebotomines, which are found in wild or urban environments. It affects domestic and wild animals and transmission to man happens by accident. The disease occurs in tropical and sub-tropical areas, mainly in Asia, Europe, Africa, and the Americas. There are two forms that affect man: American cutaneous leishmaniasis (ACL) and American visceral leishmaniasis (AVL). The latter is caused by three species of Leishmania: Leishmania (Leishmania) donovani, Leishmania (Leishmania) infantum, and Leishmania (Leishmania) chagasi, which are grouped in the Leishmania (Leishmania) donovani complex. Wild reservoir hosts of L. chagasi known so far are foxes and marsupials. In domestic environment, dogs are the most important reservoir hosts and sources of infection to the vectors Lutzomyia longipalpis. Leishmaniasis is difficult to control, causing epidemic outbreaks, thus being an important public health problem. Due to lesions caused by the mucocutaneous type and the severity of those caused by the visceral type in humans, visceral leishmaniasis is one of the main public health concerns. This paper is part of the monograph presented at the end of the residency program in the field of Zoonosis and Public Health at the School of Veterinary Sciences and Animal Husbandry, São Paulo State University, UNESP, Botucatu, São Paulo State, Brazil, in 2005.

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Background: Leishmaniasis is one of the most important vector-borne diseases of humans. This parasitic disease can be caused by many species of Leishmania. In humans, different species of the parasite are associated with different forms of the disease, cutaneous and visceral. Among domesticated animals, dogs are the most important species in the epidemiology of this disease. Leishmania chagasi, an important zoonosis, is well established as the agent of visceral leishmaniasis in Brazil. The disease is endemic in north, northeast, midwest and southeast, and is transmitted to mammals by hematophagous insects such as the Lutzomyia longipalpis. In 2008, our research group has diagnosed a case of canine leishmaniasis in the municipality of Uruguaiana and subsequently there were several cases in the city and the neighbor municipality of Sao Borja. Most Brazilian states are endemic for leishmaniasis, with the exception of Rio Grande do Sul. In southern Brazil, the reports of humans and dogs infected by Leishmania spp. are the source of endemic area in the country. Therefore, the aim of this study is register the first clinical case of canine visceral leishmaniasis in the municipality of Santa Maria, RS.Case: In october 2010, a veterinary clinic of Santa Maria received a canine, female, Doberman, with two years of age. The animal had severe skin lesions on the head and limbs, pale mucous membranes, and enlarged lymph nodes. According to the owner, the animal showed progressive weight loss and anorexia for more than five days. During the clinical examination the blood was collected for hemogram and cytology of lymph nodes was performed by puncture aspiration with a fine needle. In the erythrogram, it was observed a decrease in the total number of erythrocytes (2.8 x 10(6)/mu L), hematocrit (21%), hemoglobin (6.8 g/dL) and platelets (98 x 10(3)/mu L). In the leucogram, any alteration was observed. The cytology of lymph nodes showed amastigotes forms, suggestive of the Leishmania spp. Based on this finding; we performed the blood collection for PCR, to confirm parasitism and to determine the species of Leishmania. At the molecular test was used PCR-specific for L. chagasi, and the result was positive.Discussion: This is the first autochthonous clinical case in the central region of the RS, non-endemic area for leishmaniasis. In serological studies of visceral leishmaniasis it was diagnosed in five asymptomatic dogs in the municipalities of Santa Maria, Julio de Castilhos and Itaara, however not confirmed by molecular analysis. In the municipalities of Cruz Alta and Uruguaiana cases of L. chagasi have been reported in dogs which previously resided in Leishmania sp. endemic areas. The municipality of Sao Borja had the first record of L. longipalpis in the RS during the leishmaniasis outbreak in 2008-2009. In the central region of the RS vector has not been found, but because in this first autochthonous case dog in Santa Maria believe that the parasite is present and/or doing other insect transmission of leishmaniasis. Clinical signs associated with hematologic and coagulation disorders observed in the canine are commonly described in symptomatic dogs in endemic regions. This case of autochthonous leishmaniasis reinforces the idea of the vector presence in Santa Maria, center of the RS. We believe that canine leishmaniasis is an emerging disease in the southern region of Brazil.