Detection and differentiation of dengue virus serotypes by one-step multiplex reverse transcription PCR assays / Detecção e diferenciação de sorotipos do vírus da dengue por ensaios de PCR com transcrição reversa multiplexada em uma etapa

Background: Dengue infections are a severe public health problem in Brazil. The Ministry of Health recommends an immunosorbent assay (ELISA) for the capture of IgM (MAC-ELISA) to diagnose dengue. However, it detects antibodies that cross-react with other flaviviruses and requires confirmation in reference laboratories. Methods: One-step multiplex RT-PCR assay was used to amplify RNA of 197 serum from patients with clinical suspicion of dengue infection. The samples had been screened with the IgM ELISA kit in the Central Public Health Laboratory of the State of Maranhão. Results: Of the 197 samples evaluated by IgM ELISA, 135 were positive; of these, 96 (71.1%) were from patients in the acute phase of the infection. The one-step multiplex RT-PCR detected viral RNA in 88 (91.7%) of this serum. Among the 62-negative serum by ELISA, 29 samples (46.8%) were amplified using the molecular method. Conclusions: One-step multiplex RT-PCR was sensitive in the detection of viral particles from the first day until the sixth day after the onset of the feverish period. Moreover, it was specific and 100% reproducible. Based on these results, we recommend the use of this molecular assay to diagnose and differentiate the DENV serotypes in the acute phase of the disease.Background: Dengue infections are a severe public health problem in Brazil. The Ministry of Health recommends an immunosorbent assay (ELISA) for the capture of IgM (MAC-ELISA) to diagnose dengue. However, it detects antibodies that cross-react with other flaviviruses and requires confirmation in reference laboratories. Methods: One-step multiplex RT-PCR assay was used to amplify RNA of 197 serum from patients with clinical suspicion of dengue infection. The samples had been screened with the IgM ELISA kit in the Central Public Health Laboratory of the State of Maranhão. Results: Of the 197 samples evaluated by IgM ELISA, 135 were positive; of these, 96 (71.1%) were from patients in the acute phase of the infection. The one-step multiplex RT-PCR detected viral RNA in 88 (91.7%) of this serum. Among the 62-negative serum by ELISA, 29 samples (46.8%) were amplified using the molecular method. Conclusions: One-step multiplex RT-PCR was sensitive in the detection of viral particles from the first day until the sixth day after the onset of the feverish period. Moreover, it was specific and 100% reproducible. Based on these results, we recommend the use of this molecular assay to diagnose and differentiate the DENV serotypes in the acute phase of the disease

Regulatory T Cells Phenotype in Different Clinical Forms of Chagas' Disease

CD25High CD4+ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3+ CD25HighCD4+ cells that was also IL-10+ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3+ CD25High CD4+ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans – anteaters, sloths, and armadillos – have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with 24 domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, ten anteaters, and six sloths. Our dataset includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data-paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the south of the USA, Mexico, and Caribbean countries at the northern portion of the Neotropics, to its austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n=5,941), and Cyclopes sp. has the fewest (n=240). The armadillo species with the most data is Dasypus novemcinctus (n=11,588), and the least recorded for Calyptophractus retusus (n=33). With regards to sloth species, Bradypus variegatus has the most records (n=962), and Bradypus pygmaeus has the fewest (n=12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other datasets of Neotropical Series which will become available very soon (i.e. Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans dataset

Amazonia Camtrap: a data set of mammal, bird, and reptile species recorded with camera traps in the Amazon forest.

Abstract : The Amazon forest has the highest biodiversity on Earth. However, information on Amazonian vertebrate diversity is still deficient and scatteredacross the published, peer-reviewed, and gray literature and in unpublishedraw data. Camera traps are an effective non-invasive method of surveying vertebrates, applicable to different scales of time and space. In this study, we organized and standardized camera trap records from different Amazonregions to compile the most extensive data set of inventories of mammal,bird, and reptile species ever assembled for the area. The complete data setcomprises 154,123 records of 317 species (185 birds, 119 mammals, and13 reptiles) gathered from surveys from the Amazonian portion of eightcountries (Brazil, Bolivia, Colombia, Ecuador, French Guiana, Peru,Suriname, and Venezuela). The most frequently recorded species per taxawere: mammals:Cuniculus paca (11,907 records); birds: Pauxi tuberosa (3713 records); and reptiles:Tupinambis teguixin(716 records). The infor-mation detailed in this data paper opens up opportunities for new ecological studies at different spatial and temporal scales, allowing for a moreaccurate evaluation of the effects of habitat loss, fragmentation, climatechange, and other human-mediated defaunation processes in one of themost important and threatened tropical environments in the world. The data set is not copyright restricted; please cite this data paper when usingits data in publications and we also request that researchers and educator sinform us of how they are using these data

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Purificação de albumina humana por cromatografia de troca ionica para estudos imunoquimicos

Orientador : H. A. RangelDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Experiências foram realizadas com o sentido de verificar a possibilidade de obtenção de albumina humana imunoquimicamente pura, utilizando-se uma metodologia disponível em laboratório medianamente equipados. Foram utilizados soros humanos obtidos a partir de sangue rejeitado para serviços de transfusão por ser impróprio para uso humano por apresentar reações sorológicas positivas para sífilis ou Doença de Chagas. Estes soros foram fracionados por ¿salting-out¿ e por cromatografia e a pureza das frações foi testada por eletroforese em acetato de celulose, imunoeletroforese e imunoeletroforese cruzada. Os resultados obtidos mostraram que: 1. O fracionamento com sulfato de amoneo permitiu a obtenção de uma fração enriquecida em albumina (80%) com um rendimento médio de 70%. 2. Por cromatografia em CM-celulose em condições controladas pode-se obter uma fração que de acordo com os resultados dos testes de pureza utilizados, contem apenas albumina com um rendimento global da ordem de aproximadamente 30%. 3. Experimentos de digestão da albumina obtida com a catepsina D de coelho, mostraram resultados similares aos obtidos com albuminas humanas purificadas, obtidas comercialmenteAbstract: Not informed.MestradoMestre em Ciências Biológica

Aspectos de imunodiagnóstico, imunopatologia e proteinases ácidas na doença de Chagas

Made available in DSpace on 2017-10-30T15:42:10Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) rosa_pinho_ioc_dout_2001.pdf: 4508486 bytes, checksum: 51694e4206fa1f971462d585bddaf66c (MD5)Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Neste trabalho, nós estudamos aspectos da doença de Chagas relacionados ao imunodiagnóstico desenvolvendo um novo teste de diagnóstico utilizando saliva, estudamos aspectos relacionados à imunopatologia e isolamos proteinases ácidas do Trypanosoma cruzi. Em nosso primeiro estudo desenvolvemos um teste para o diagnóstico de doença de Chagas. Para isto, utilizamos saliva de pacientes na fase crônica da doença e detectamos, por ELISA, anticorpos específicos da classe IgG em 103 dos 114 testados com 95 e 90 % de especificidade e sensibilidade, respectivamente. Embora, a utilização de soro comparado à da saliva forneça melhores resultados, concluímos que este pode ser um teste alternativo para imunodiagnóstico da doença de Chagas. No segundo estudo caracterizamos os antígenos liberados de T. cruzi que se ligam em células não infectadas de mamíferos. Assim, detectamos através de marcação com metionina (35S), que antígenos liberados por tripomastigotas do T. cruzi, adsorveram-se às células não-infectadas e estes foram identificados com massa molecular de 85-110 e 160-170 kDa. Foi também demonstrado o efeito destes antígenos nestas células por imuno-citoquímica e detectamos aumento de expressão de componentes da matriz extracelular (ECM) como colágeno, laminina e fibronectina. Assim, concluímos, que estes antígenos podem ser importantes na inflamação secundária à infecção pelo T. cruzi No terceiro estudo isolamos, através de coluna de afinidade agarosepepstatina A, duas proteinases (CZP-I e CZP-II) de formas epimastigotas de T. cruzi. A atividade enzimática destas foi observada na região de 56-48 kDa em pH 3,5. O conteúdo de a- hélice, estruturas b, e estruturas não definidas foi de 9, 62 e 17% respectivamente. Ambas as enzimas foram inibidas pelo inibidor clássico pepstatina A (\2A7E68%) e pelo marcador de sitio aspártico protease ativo, 1,2-epoxy-3-(phenylnitrophenoxy) propane (\2A7E80%). Usando-se soros imunes produzidos em coelhos contra estas duas enzimas detectou-se a presença de ambas em todas as formas do parasito (Anexo II). Os resultados obtidos nos permitem concluir que estas proteinases têm características da classe das aspártico poteinases. Ainda, avaliamos a imunogenicidade da proteinase (CZP-II) em camundongos e obtivemos resposta proliferativa proporcional à quantidade de enzima adicionada. Foram também avaliadas, através de imunofenotipagem, as subpopulações das células T presentes ao final da linfoproliferação e verificou-se um predomínio de células CD8+ em relação às células CD4+. Foi ainda observada produção de IFN-g enquanto a detecção de producão de IL-4 foi baixa. Estes resultados sugerem que esta enzima possa ter papel na imuno regulação na infecção pelo T. cruzi no modelo murino.We have studied some aspects of immunodiagnosis and immunopatology in Chagas\2019disease. A new diagnostic test using saliva was developed and aspartic proteinases from Trypanosoma cruzi were isolated. On the first study, we developed a new test for Chagas\2019 disease. Enzyme linked immunosorbent assay (ELISA) was applied to saliva from chronic infected patients to detect infection by Trypanosoma cruzi. Specific IgG antibodies were detected in saliva from 103 of 114 samples of infected patients and it showed 90,4% of sensitivity and 95% of specificity. This test could be used as an alternative diagnostic method for Chagas\2019 disease. On the second study, we characterized T. cruzi antigens that bound to noninfected mammalian cells. These released T. cruzi trypomastigotes antigens were labeled with (35S) methionine and were able to adsorb to non-infected cells, they showed molecular mass of 85-110 and 160-170 kDa. The adsorption of these antigens to mammalian cells induced an increase expression of extracellular matrix components (ECM) as colagen, laminin and fibronectin as detected by immunohistochemistry. We concluded that these antigens could be important in inflammation secondary to the infection by T. cruzi On the third study, two proteinases (CZP-I and CZP-II) from T. cruzi epimastigotes were isolated by affinity chromatography column. The enzymatic activity of these proteinases was observed at 56-48 kDa region in a pH 3.5. The CZPI and CZP-II content of a-helix, b-structures and random coil were calculated to be approximately 9, 62 and 29%, respectively. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (\2A7E68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (\2A7E80%). Hyperimmune rabbit sera against the enzymes were able to detect both enzymes in all parasite stages (attachment II). These results suggested that these proteinases have characteristics of aspartic proteinases. We also studied the immunogenicity of proteinase CZP-II in mice and it showed proliferative response that had direct relation to the amount of enzyme used. Immunophenotyping of the T cells subsets showed predominance of CD8 over CD4 cells and g-IFN production was observed in contrast to lower levels of IL-4. These results suggest that these enzymes could play some role in the immunoregulation of T. cruzi infection in the mouse model