A dengue é uma doença que afeta milhões de pessoas e possui um número significativo de mortes. Não há nenhum tratamento vacinal legalizado para uso. As estratégias vacinais contra a dengue baseadas em proteínas não estruturais têm demonstrado serem mais seguras do que as baseadas em proteínas estruturais. A proteína não estrutural 5 (NS5) do vírus dengue é a proteína mais conservada entre os quatro sorotipos e desempenha um papel crucial na replicação viral. Neste estudo, foi gerada uma forma recombinante da NS5 expressa em E. coli com propriedades antigênicas preservados. As condições de cultura foram optimizadas, o que permitiu a expressão dessa proteína na forma solúvel. A imunização de camundongos Balb/c com a NS5 sozinha ou em combinação com um adjuvante (poli (I:C)) promoveu o aumento da sobrevida de camundongos após desafio letal com DENV2. A combinação da NS5 com poli (I:C) emulsionado em Montanide 720 levou a expansão de linfócitos T CD8+ específicos. Os resultados indicam que a proteína NS5 obtida preserva determinantes antigênicos da proteína nativa e pode ser uma ferramenta útil para estudos sobre a biologia do DENV, busca de drogas antivirais e desenvolvimento de vacinas. / Dengue fever is a disease affecting millions of people worldwide and causing a significant number of deaths. There are no effective treatments or vaccine approaches capable of preventing such infection. Anti-DENV vaccine strategies based on nonstructural proteins as antigens have been shown to be safer than those based on structural proteins. The DENV nonstructural protein 5 (NS5), plays a crucial role in viral replication. In this study, we generated a recombinant form of DENV2 NS5 expressed in E. coli in high amounts and with preserved antigenic properties with regard to the native protein. Culture conditions were optimized in order to allow expression of NS5 as a soluble protein. The immunization of Balb/c mice using this protein alone or in combination with poly (I:C) led to increased survival after intracranial challenge with the DENV2 JHA1 strain. The combination of the protein with poly (I:C) emulsified in Montanide 720 led to the activation of NS5-specific CD8+ T lymphocytes. Altogether, the results indicate that the recombinant NS5 protein preserves antigenic determinants of the native protein and may be a useful tool for studies dealing with the DENV\'s biology, search for anti-viral drugs and vaccine development.

O desenvolvimento de uma vacina para o controle da dengue é uma prioridade em todo o mundo. O domínio helicase da proteína NS3 (NS3H) viral alberga epítopos reconhecidos por linfócitos T citotóxicos, os quais tem papel importante na eliminação de células infectadas. Esse trabalho propôs a obtenção de uma forma recombinante, produzida em linhagens de Escherichia coli, da NS3H do DENV2 com características similares à proteína nativa e sua utilização como um potencial antígeno vacinal. A NS3H foi obtida na forma solúvel, foi reconhecida por anticorpos de camundongos e de humanos infectados e foi capaz de interagir com o RNA viral. Camundongos imunizados com NS3H coadministrada com diferentes adjuvantes desenvolveram respostas imunológicas específicas mas não foram protegidos após desafio. Em conjunto, os resultados indicam que a proteína NS3H recombinante preserva conformação e determinantes antigênicos da proteína viral nativa e pode ser útil em estudos sobre a biologia viral e na busca de estratégias anti-virais voltadas para o controle da dengue. / The development of a dengue vaccine is a worldwide priority. The helicase domain of viral NS3 protein (NS3H) preserves epitopes recognized by cytotoxic T lymphocytes, which plays an important role in the elimination of infected cells. This study aimed the generation of a recombinant NS3H form of a type 2 dengue virus (DENV2) lineage, in Escherichia coli strains, with properties similar to the native protein and its use as a potential vaccine antigen. The NS3H was obtained in soluble form, was recognized by antibodies from mice and human subjects and was able to interact with the viral RNA. Mice immunized with NS3H combined with different adjuvants developed specific immune responses but did not confer protection to a lethal challenge. Altogether, the results indicate that the recombinant NS3H protein preserves conformational and antigenic determinants of the native protein and may be a useful tool for studies dealing with the DENV biology and the search for anti-virus approaches.

La dengue est une préoccupation mondiale majeure en santé publique, touchant principalement les jeunes enfants en Asie du sud est. Les vaccins contre la dengue sont actuellement en développement. Des études coût-efficacité seront nécessaires en cas d'introduction d'un nouveau vaccin. Pour cela, il est indispensable d’obtenir des informations sur le coût économique lié à la dengue (qui sont relativement rares) et des données précises sur l'incidence de la maladie, ceci afin de fournir des estimations solides quant à l'importance de la dengue dans les régions endémiques.Ce travail de thèse propose une approche méthodologique par laquelle le Ministère de la Santé devrait envisager lors de l'évaluation de la pertinence et de la faisabilité de l'adoption d’un vaccin contre la dengue par les programmes de nationaux de vaccination, particulièrement dans les pays en voie de développement ; le Cambodge par exemple. Nous avons mis en place des études prospectives d'incidence de la dengue sur plusieurs années, réalisées dans la population générale, incluant un large éventail de groupes d'âge. Les résultats de ces études ont été comparés avec les données de surveillance nationale réunis dans la même région en calculant des facteurs de multiplication afin d’estimer la sous-déclaration. D'autres études de coûts de la maladie ont été menées pour estimer le poids financier la dengue sur plusieurs années et d'identifier les groupes vulnérables. Cette approche a également permis de mieux comprendre l'épidémiologie de la dengue dans le pays, dont certaines caractéristiques sont nécessaires pour évaluer la faisabilité de l'introduction d’un nouveau vaccin contre la dengue. Les résultats rapportés dans cette thèse vont permettre (1) d’initier à partir des données cambodgiennes des simulations de coût-efficacité à partir des caractéristiques attendues du vaccin et (2) de développer un modèle dynamique afin de permettre aux autorités nationales d'analyser les études de coût-efficacité en tenant compte des spécificités immunologiques et épidémiologiques des pays concernés. Si un vaccin efficace et fiable est disponible à un prix abordable, son adoption par le programme national de vaccination serait probablement rentable pour le Cambodge mais répondrait surtout à un problème d'inéquité sociale. / Dengue is a major public health concern worldwide, particularly in young children in Southeast Asia. Dengue vaccines are currently in development and policymakers need appropriate economic studies to determine their potential financial and public health impact. Alongside economic assessment, accurate disease incidence data are required to provide robust estimates of disease burden across the regions where dengue is endemic. This thesis proposes a methodological approach by which the Ministry of Health should consider when assessing feasibility of adoption dengue vaccines, with specific reference to developing country settings, e.g. Cambodia. We implemented prospective dengue incidence studies over several years, performed in a general population and include a wide range of ages. The results of these studies were compared with national surveillance data gathered in the same region by calculating multiplication factors to estimate underreporting on several years. Additional prospective cost of illness study was conducted to generate accurate economic burden of disease estimates over several years and identify vulnerable groups. This strategy also allowed better understanding of the epidemiology of dengue in Cambodia, of which some characteristics are needed to evaluate the feasibility of introducing dengue vaccine. Designing cost-effectiveness studies before a vaccine has been fully evaluated requires assumptions about variables such as efficacy and effectiveness, dosage and costs. Our next step will be to determine the threshold price – sensitivity analyses - for a vaccine to be cost-effective rather than assigning a specific cost, because it is difficult to predict cost for a vaccine that has not been marketed. Should this vaccine be safe and affordable enough, the adoption of the new vaccine into a National Immunization Programme would probably be cost-effective but, above all, a matter of social equity.

Predictive models based in symptomatology of suspected dengue patients seeking medical care in Honduras. The models based on logistic regression models predicted the outcomes of dengue fever/ severe dengue. Sensitivity and specificity are discussed. It also describe the level of agreement between Honduran classification of severe dengue and the ones based on World Health Organization guidelines of 1997 and 2009. / Introduction: Dengue is a major public health problem in tropical and subtropical countries but its clinical presentation may be similar to many febrile illnesses. Since in endemic countries laboratory confirmation is frequently delayed, the majority of dengue cases are diagnosed based on patient’s symptomatology. This can often lead to misdiagnosis and potential serious health complications. The objective of this study was to identify clinical, hematological and demographical parameters that could be used as predictors of dengue fever among patients with febrile illness. Methods: We conducted a retrospective cohort study of 548 patients presenting with febrile syndrome to the largest public hospitals in Honduras. Patients’ clinical, laboratory, and demographical data as well as dengue laboratory confirmation by either serology or viral isolation were used to build a predictive statistical model to identify dengue cases. Results: Of 548 patients, 390 were confirmed with dengue infection while 158 had negative results. Univariable analysis revealed seven variables associated with dengue: male sex, petechiae, skin rash, myalgia, retro-ocular pain, positive tourniquet test, and bleeding gums. In multivariable logistic regression analysis, retro-ocular pain petechiae and bleeding gums were associated with increased risk, while epistaxis and paleness of skin were associated with reduced risk of dengue. Using a value of 0.6 (i.e., 60% probability for a case to be positive based on the equation values), our model had a sensitivity of 86.2%, a specificity of 27.2%, and an overall accuracy of 69.2%; allowing for the diagnosis of dengue to be ruled out and for other febrile conditions to be investigated. Conclusions: The application of predictive models can be valuable when laboratory confirmation is delayed. Among Honduran patients presenting with febrile illness, our data reveal key symptoms associated with dengue fever, however the overall accuracy of our model is still low and specificity remains a concern. Our model requires validation in other populations with similar pattern of dengue transmission. Key Words: Dengue, fever, Predictive model, symptoms, Honduras / Thesis / Doctor of Philosophy (PhD) / Predictive models based in symptomatology of suspected dengue patients seeking medical care in Honduras. The models based on logistic regression models predicted the outcomes of dengue fever/ severe dengue. Sensitivity and specificity are discussed. It also describe the level of agreement between Honduran classification of severe dengue and the ones based on World Health Organization guidelines of 1997 and 2009.

No description available.

O dengue e o dengue hemorrágico são considerados como as arboviroses mais importantes do ponto de vista da saúde pública além de serem também as mais disseminadas. O greening ou huanglongbing (HLB), por sua vez, é uma doença de difícil controle e rápida disseminação que afeta seriamente a produção de citros no mundo todo. É considerada a pior doença de citros da atualidade pois não tem cura e leva ao declínio e morte das árvores em alguns anos. O DMDS é um composto sulfúrico volátil derivado de plantas e que tem despertado um crescente interesse devido a sua comprovada atividade repelente e inseticida além de ação nematicida e disinfectante do solo. Visando oferecer uma alternativa para o controle de ambas as doenças, este trabalho teve como objetivo desenvolver um método de microencapsulação por spray drying e spray congealing para o dimetil dissulfeto (DMDS), visando a redução da sua volatilidade através de uma liberação controlada para o ambiente. Dessa forma, tornar-se-ia viável sua utilização como repelente, larvicida e inseticida no combate ao vetor do dengue além da sua utilização como repelente no controle do greening nas lavouras de citrus. Tentou-se a microencapsulação através de spray congealing utilizando enxofre como microencapsulante mas não se obteve sucesso devido as características térmicas do enxofre. Foram obtidas micropartículas de DMDS microencapsulado em goma arábica através da técnica de spray drying. As micropartículas e o processo de secagem foram caracterizados com relação ao rendimento de secagem, rendimento de microencapsulação, morfologia, teor de água residual, atividade de água, densidades aparente e compactada e propriedades de fluxo. Confirmou-se a possibilidade de microencapsulação de DMDS por spray drying e eficiência da goma arábica na retenção do mesmo. As partículas obtidas apresentaram boas propriedades de atividade de água, teor de água residual e densidade mas propriedades de fluxo que requerem melhoria. Foi feito também um estudo simplificado da viabilidade técnico-econômica da implantação de uma unidade de produção de DMDS microencapsulado. Esse estudo foi baseado no processo de microencapsulação estudado. Foram analisados diversos parâmetros econômicos e através destas análises verificou-se que a produção de DMDS microencapsulado pelo método utilizado seria economicamente viável. / Dengue fever and dengue hemorrhagic fever are not only considered the most important arboviruses from public health standpoint but also the most disseminated ones. Greening or huanglongbing (HLB) is a citrus disease of difficult control and fast dissemination that affects citrus production all over the world. It is considered the worst citrus disease nowadays since it does not have a cure and it causes the decline and death of the trees in a few years. Dimethyl disulfide is a volatile sulphur compound derived from plants and that has aroused growing interest due to its proved repellence and insecticidal activity, soil disinfection and nematocidal properties. Therefore, this work aimed at developing a DMDS microencapsulation method via spray drying and spray congealing in order to decrease DMDS\' volatility through a controlled release to the environment and offer a control alternative to both diseases. This would make DMDS use as a repellent, larvicide and insecticide of dengue fever\'s vector and also its use in greening control as a repellent viable. We tried to microencapsulate the DMDS through spray congealing using sulfur as a microenpsulating agent but it was unsuccessful due to the thermal characteristics of sulfur. DMDS was microencapsulated in Arabic gum through spray drying. Drying yield, microencapsulation yield, morphology of the particles, residual water percentage, water activity, bulk and tapped density and flow properties were used to characterize the microparticles and the drying process. Feasibility of DMDS microencapsulation and Arabic gum efficiency in DMDS retention were confirmed. The microparticles presented good bulk and tapped density, residual water percentage and water activity properties though its flow properties requiring further improvement. A simplified technical-economical evaluation of a DMDS microencapsulation factory was also done. This evaluation was based on the studied microencapsulation process. Many economical parameters were analysed and those analyses showed that DMDS microparticles production using the studied method would be economically viable.

Orientadora: Janete Apparecida Desidério Sena / Banca: Manoel Victor Franco Lemos / Banca: Cristina Lacerda Soares Petrarolha Silva / Banca: Gislayne Fernandes Lemes Trindade Vilas-Bôas / Banca: Odair Aparecido Fernandes / Resumo: Aedes aegypti (Linnaeus), principal vetor da dengue no Brasil, tem sido combatido com o uso maciço de produtos químicos, contribuindo com o desenvolvimento de resistência e inviabilizando o controle do inseto. Bioinseticidas à base de Bacillus thuringiensis vêm apresentando resultados satisfatórios no controle de dípteros, devido à produção de proteínas bioinseticidas denominadas Cry (cristal), Cyt (citolíticas) e Chi (quitinase) e os efeitos sinergísticos existentes entre elas. O presente trabalho objetivou a seleção de isolados de B. thuringiensis, portadores de genes cry, cyt e chi com alta eficiência no controle de A. aegypti. Uma coleção de 1073 isolados de B. thuringiensis, provenientes de diversas regiões brasileiras, foi submetida à Reação em Cadeia da Polimerase (PCR), com base nos iniciadores específicos para os genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. Os isolados dípteros-específico foram avaliados quanto à estruturação genética e através de bioensaios seletivos e quantitativos, para determinação da CL50 e CL90. Apenas 45 (4,19%) apresentaram amplificação para os genes cry e cyt e destes, 25 (54,34%) eram quitinolíticos. Foram definidos 21 haplótipos entre os 45 isolados de B. thuringiensis, os quais foram submetidos aos bioensaios seletivos, indicando 13 isolados que causaram 100% de mortalidade às larvas de A. aegypti. Os bioensaios quantitativos e análise de agrupamento permitiram selecionar quatro isolados altamente tóxicos às larvas de A. aegypti. A identificação dos genes cry, cyt e chi de B. thuringiensis e análise dos efeitos sinergísticos entre as toxinas, associadas à análise da toxicidade a insetos vetores permitiram a seleção de isolados que poderão ser utilizados em formulações de novos bioinseticidas brasileiros, podendo contornar possíveis problemas de resistência. / Abstract: Aedes aegypti (Linnaeus), the main vector of dengue fever in Brazil, has been controlled with massive use of chemical products wich, contributed to the development of resistance decreasing the insect control efficacy. Bioinsecticides based on Bacillus thuringiensis is currently presenting satisfactory results on controlling dipterans, due to the production of Cry proteins (crystal proteins), Cyt (citolitic) and Chi (chitinase) with synergistic effects between them. The present work aimed to select B. thuringiensis isolates with cry, cyt and chi genes exhibiting high efficiency on A. aegypti larvae. A bacterial collection containing 1073 isolates of B. thuringiensis, obtained from different Brazilian locations, had their DNA isolated and submitted to PCR amplifications using specific primers for the genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. The dipterans-especific isolates were then evaluated for the genetic structure and through selective and latter to quantitative bioassays for the determination of LC50 and LC90. Forty five out of 1073 isolates (4.19%) presented amplicons for the cry and cyt genes and from these 25 (54.34%) were detected as chitinolitic. Twenty one haplotypes were identified among the 45 isolates of B. thuringiensis, which were submitted to the selective bioassays, indicating 13 isolates that caused 100% mortality of A. aegypti larvae. The quantitative bioassays and the statistical analysis have pointed four toxic isolates to A. aegypti larvae. The identification of the cry, cyt and chi genes of B. thuringiensis and the analysis of the synergistic effects among the toxins, along with the toxicity analysis towards the vector insects allowed the selection of a set of isolates that can be used for the formulation of new bioinsecticides from Brazilian origin, that would avoid or at least delay the appearance of resistance problems. / Doutor

Dengue fever is a debilitating, viral, mosquito-borne disease occurring in tropical and subtropical areas in the world. The majority of dengue cases in the United States were acquired in endemic areas by travelers or immigrants. However, in recent years, autochthonous (locally acquired) dengue cases have been diagnosed in Florida. The purpose of this study was to find an association between potential risk factors and the expansion of dengue fever in the United States. Guided by the eco-bio-social framework, which offers a broad assessment of risk factors for the illness, a retrospective design was used with archival data to correlate changes in climatic variables and imported dengue cases with autochthonous dengue cases in Southeast Florida from 1980 to 2013. A Spearman correlation indicated weak correlations between temperature and autochthonous dengue cases (rs = .999, p = 000) and imported dengue cases with autochthonous dengue cases (rs = .162, p = 000). A negative binomial multivariate regression was used to analyze the expansion of dengue to each monthly unit of temperature, rainfall, and imported dengue cases over 34 years. The results indicated that temperature (IRR = 2.198; 95% CI [1.903, 2.538]) and precipitation (IRR = .991; 95% CI [.988, .994]) were predictors for the geographic expansion of dengue fever in Southeast Florida. The positive social changes include the use of the results to develop an understanding of how climatic variables and migration may influence the expansion of dengue fever to nonendemic regions. The results can be used by public health authorities to address risk factors and to formulate evidence-based decisions in regard to prevention and education concerning dengue fever.

Dengue fever is a debilitating, viral, mosquito-borne disease occurring in tropical and subtropical areas in the world. The majority of dengue cases in the United States were acquired in endemic areas by travelers or immigrants. However, in recent years, autochthonous (locally acquired) dengue cases have been diagnosed in Florida. The purpose of this study was to find an association between potential risk factors and the expansion of dengue fever in the United States. Guided by the eco-bio-social framework, which offers a broad assessment of risk factors for the illness, a retrospective design was used with archival data to correlate changes in climatic variables and imported dengue cases with autochthonous dengue cases in Southeast Florida from 1980 to 2013. A Spearman correlation indicated weak correlations between temperature and autochthonous dengue cases (rs = .999, p = 000) and imported dengue cases with autochthonous dengue cases (rs = .162, p = 000). A negative binomial multivariate regression was used to analyze the expansion of dengue to each monthly unit of temperature, rainfall, and imported dengue cases over 34 years. The results indicated that temperature (IRR = 2.198; 95% CI [1.903, 2.538]) and precipitation (IRR = .991; 95% CI [.988, .994]) were predictors for the geographic expansion of dengue fever in Southeast Florida. The positive social changes include the use of the results to develop an understanding of how climatic variables and migration may influence the expansion of dengue fever to nonendemic regions. The results can be used by public health authorities to address risk factors and to formulate evidence-based decisions in regard to prevention and education concerning dengue fever.

Made available in DSpace on 2016-12-12T20:22:52Z (GMT). No. of bitstreams: 1 Diego Buchinger.pdf: 6508385 bytes, checksum: 43c3902c34434ea92c276412dcd6d710 (MD5) Previous issue date: 2014-11-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The dengue fever disease is a public health problem in many countries. Whilst efforts on creating a vaccine have been expended, the adopted practice for controlling this disease is raising the population awareness about this problem. Teaching to youngsters however, has proven a challenging task. In this sense, using Serious Games is a way for increasing motivation, and the use of collaborative and competitive interactions altogether can provide more learning. Thus, a new collaborative-competitive Serious Game about dengue was designed and developed. As no design methodology to this type of game was found, motivational fundaments for games were adopted in order to guide the game design. The game was promoted and used by various audiences, from K-12 to post-graduates, in joint efforts for dengue fever awareness. In order to assess learning, 71 joint effort participants answered a questionnaire of dengue fever knowledge and confidence before and after the game usage. Based on the data gathered, an increase of 17.35% in the participants mean knowledge about dengue fever and an increase of 51.23% in the answers confidence were observed. Also, there was an increase of 74.07% participants that have obtained a grade equal to or greater than seven, on a scale of zero to ten. The results are strong evidence that Sherlock Dengue 8 can promote learning and confidence on the knowledge regarding dengue fever. / A doença da dengue tem se mostrado um problema de saúde pública em muitos países. Enquanto esforços estão sendo despendidos para a criação de uma vacina, a prática adotada para o controle desta doença é a conscientização da população sobre este problema. Ensinar os jovens entretanto, tem se mostrado uma tarefa desafiadora. Neste sentido, a proposta do uso de Jogos Sérios é um meio de aumentar a motivação e, o uso de interações colaborativas e competitivas, juntas, pode trazer maior aprendizado. Assim, um Jogo Sério Colaborativo-Competitivo sobre dengue foi projetado e desenvolvido. Como não se encontrou uma metodologia de design para este tipo de jogo, adotaram-se os fundamentos da área motivacional de jogos para orientar o design. O jogo foi promovido e utilizado por diversos públicos, do ensino fundamental à pós-graduação em mutirões de conscientização sobre a dengue. A fim de avaliar a aprendizagem com a utilização do jogo, 71 participantes responderam a um questionário de conhecimento e confiança antes e após o uso do jogo. Com base nos dados obtidos foi verificado um aumento médio de 17,35% no conhecimento sobre dengue, um aumento médio de 51,23% na confiança nas respostas e um aumento de 74,07% no número de participantes que obtiveram nota igual ou superior a sete, numa escala de zero a dez. Os resultados são fortes indícios de que o Sherlock Dengue 8 pode promover aprendizado e confiança no conhecimento sobre a dengue.