Dengue viruses (DV) are re-emerging mosquito-borne pathogens for which four distinct lineages, grouped based on serology and referred to as serotypes 1-4 (DIV-D4V), have been described. Epidemiological data imply that re-infection with a "heterologous" serotype, i.e, one other than that to which the individual was originally exposed, enhances the risk for development of severe disease, dengue hemorrhagic fever (DHF). The hallmark of DHF is a transient capillary leakage syndrome of rapid onset, temporally associated with the resolution of fever and viremia. In its most grave form, the vascular permeability phenomenon in DHF may progress to dengue shock syndrome (DSS), which is often fatal in the absence of appropriate medical care. Despite the fulminant nature of vascular leakage during DHF/DSS, this phenomenon does not appear to be due to direct cytopathic effects of DV. Rather, inappropriate reactivation and/or regulation of dengue-specific memory are the prevailing theorized (immunopathological) etiologies. Traditional vaccine development techniques have proven insufficient for DV, since any vaccine must offer complete protection against all four serotypes to avoid enhanced pathology on natural viral challenge. Understanding the underlying mechanisms that contribute to dengue disease, particularly the development of dengue-specific memory, is therefore of critical importance. Dengue immunopathology and the specific aspects of immunological memory that determine disease severity are heatedly debated. Previous research in our lab has suggested that T cell responses contribute to the severity of dengue illness. Clinical data indicate enhanced immune activation in more grave cases of DV infection, and serotype cross-reactive T cells from multiple individuals are present after both primary and secondary dengue infections. However, little is known about the conditions under which T cells are primed and dengue-specific memory is generated. Dendritic cells (DCs) are bone marrow-derived cells that play a central role in directing activity within the immune system. DCs shape quantitative and qualitative aspects of adaptive immunity, and therefore the intrinsic characteristics of host memory to a pathogen. DCs are essential in generating primary immune responses, due to their particular effectiveness in stimulating naïve T cells. DCs also play important roles in the reactivation of memory to an infectious agent, and as reservoirs for the dissemination of invading microorganisms. Exposure to pathogens or their products initiates a series of phenotypic and functional changes in DCs, termed maturation. DC maturation involves a coordinated response of immunomodulatory surface molecule elaboration and cytokine production, culminating in antigen presentation to, and co-stimulation of, T cells specific for the invading agent. The DC response is ostensibly tailored to facilitate effective elimination by regulating effective downstream interactions of the DC with T cells. A number of viruses have evolved to infect DCs and alter their functional behavior, facilitating their own survival within the host, and the herd. DV readily infects DCs both in primary cell cultures and in vivo. However, reports on the effects of DV infection on DC maturation vary both with regard to some of the cytokines produced, and the phenotypes of infected versus bystander cells. Although DCs appear to be activated following DV exposure, responses on the single-cell level appear to depend on the infection state of the cell, hypothetically driven by intracellular virus-mediated effects. Therefore, downstream responses to these divergent populations - i.e., actively infected cells versus uninfected bystander cells - are likely to be the consequence of at least two modes of DC behavior. Because DCs play a pivotal role in adaptive immune development, and because the resulting memory response appears to be critical in affecting disease pathology after heterologous DV re-infection, I sought to explore the phenomena of DC maturation in response to dengue exposure, and to begin to answer the question of how active infection alters the functional capabilities of DCs. Notably, primary dengue infection is generally well-controlled with minimal pathology. Therefore, this thesis addresses the hypothesis that DV infection of DCs results in cellular activation and stimulation of antiviral immunity, despite virus-mediated alteration of DC maturation. In order to address this hypothesis, I examined both DV infection-dependent and independent effects on DC functional responses including surface molecule regulation secretory activity, and CD4 T cell allostimulatory priming. DCs derived from human peripheral blood monocytes were readily infected with multiple strains of DV. DV infection of DCs derived from separate donors was dose-dependent, with substantial variability in DC susceptibility to infection. Exposure to live DV activated surface molecule expression in DCs, similar to the effects of defined maturation stimuli including a combination of TNF-α and IFN-α, or LPS. In addition, UV-inactivated DV induced expression of cell surface molecules, albeit to a lesser extent than did live virus demonstrating inherent stimulatory properties of DV particles. Using intracellular staining for DV envelope (E) protein, I detected increased surface molecule expression on both infected DCs and uninfected bystander DCs from the same culture, as compared to mock-infected DCs. These data indicate that activation was not prevented in cells undergoing active viral replication. However, the degree of surface molecule induction depended on the infection state of the cell. Infected DCs had enhanced PD-L2 and MHC II expression relative to uninfected bystander cells, while PD-L1, CD80, CD86, and MHC I expression were suppressed with active infection. Therefore, intracellular DV replication altered the process of cell surface molecule regulation within these cells. DV infection of DCs also resulted in the secretion of a broad array of cytokines and chernokines. These included the antiviral cytokine IFN-α, inflammatory cytokines TNF-α, IL-6, and IL-1α, and inflammatory chemokines IP10, MCP-1, MIP-1α, and RANTES. DV infection did not induce DC production of the IL-12 p70 heterodimer, and secretion of the immunosuppressive cytokine IL-10 was low in most experiments. Similar to the results seen with surface molecule induction, UV inactivation of DV reduced, but did not eliminate, cytokine and chemokine responses. At the single-cell level, TNF-α and IP10 production profiles of infected DCs and uninfected bystander DCs were distinct. DV infection in DCs reduced production of IP10, but stimulated TNF-α as compared to uninfected bystander cells in the same culture. Blocking experiments demonstrated that IFN-α/β produced by DCs in response to infection actively inhibited viral protein expression and drove IP10, but not TNF-α, production. DV infection of DCs did not consistently suppress DC stimulation of allogeneic CD4 T cell proliferation. In cases where infection enhanced DC stimulatory function, T cell proliferation was less pronounced than that induced by DCs activated with exogenous TNF-α plus IFN-α. Increasing multiplicity of infection (MOI) of DCs with DV resulted in increasing DC infection rates, but a statistically significant trend at the highest MOIs for decreased T cell alloproliferation, suggesting that direct infection of DCs reduces their CD4 T cell priming function. MOI-dependent reduction in DC stimulatory function depended on replication-competent virus. Increased MOIs during DV infection of DCs did not cause an elevation in detectable IL-10 in supernatants derived from T-DC co-cultures. In addition, increased DV MOI of DCs was not associated with increased levels of either IL-13 or IFN-γ in supernatants from T-DC co-culture, suggesting that actively infected DC do not skew CD4 T cells towards a specific Th phenotype. These data demonstrate that DV infection induces functional maturation of DCs that is modified by the presence of virus through both IFN-dependent and independent mechanisms. However, the allostimulatory phenotype of DCs was not universally enhanced, nor was it skewed towards antiviral (Th1)-type responses. These data suggest a model whereby dengue infection during primary illness results in controlled immune stimulation through activation of bystander DCs, and the generation of mixed Th-type responses. Direct DV infection of DCs appears to attenuate activation of, and potentially clearance by, antiviral mechanisms. During secondary infection, reduced IP10 production and enhanced TNF-α secretion by infected cells coupled with MHC I downregulation and enhanced PD-L2 expression, would subvert both Th1 CD4 T cell recruitment and result in CD8 T cell suppression and death. Furthermore, DV-specific effects on DCs would allow for continued viral replication in the absence of effective clearance. These DV-mediated effects would modify T cell memory responses to infected DC, and potentially facilitate the expansion of pathologic T cell subsets. Contributing to this pathological cascade, antibody-dependent enhancement of infection in monocytic cells and macrophages would shift antigen presentation and cytokine production paradigms, increasing the risk of DHF.

This study proposes to directly test the hypothesis that antibody-dependent enhancement (ADE) is the critical factor in the development of dengue hemorrhagic fever (DHF) in infants. DHF occurs in two distinct clinical settings: a) in children and adults with secondary DENV infection, and b) in infants with primary DENV infection born to mothers with prior DENV infection. The ADE hypothesis proposes that pre-existing serotype-cross-reactive non-neutralizing anti-DENV antibodies bind the heterotypic DENV during secondary infection and enhance its uptake into immune cells, leading to increased viral load and DHF. This model suggests that DHF in DENV-infected infants is caused by the enhancing effect of waning maternal anti-DENV antibodies, thus causing a “physiologic secondary infection” during an infant’s primary infection and thereby increasing the infant’s risk for DHF. The effect of maternal immunity on DHF in infants has been studied exclusively in Southeast Asia. However, the maternal DENV seroprevalence approaches 100% in this part of the world. As a consequence, the ADE model of infant DHF cannot truly be tested in Southeast Asia, because all infants possess anti-DENV antibody at birth. In the Western Hemisphere, by contrast, women may have experienced either a single DENV infection, more than one DENV infection, or no DENV infection at all. The ability to include DENV-seronegative mothers as controls allows for the ADE hypothesis to be directly tested in a clinical study. To our knowledge, no such study has been previously conducted. This thesis presents a case-control study designed to evaluate the influence of positive maternal dengue seroprevalence on the risk of DHF in infants. As the MSCI program provides instruction in study design, this thesis does not present findings. The clinical trial described herein began in May 2010 and enrollment is expected to continue through May 2012 (see Table 4).

Made available in DSpace on 2014-07-29T15:30:35Z (GMT). No. of bitstreams: 1 Dissertacao de Benigno Alberto Moraes Rocha.pdf: 429604 bytes, checksum: e7166c606b960cf25df199a5ddc928fd (MD5) Previous issue date: 2008-02-29 / In Brazil, dengue is present in almost all states and territories, with a circulation of three different serotypes (DEN 1, DEN 2 and DEN 3), resulting in about 500 000 cases reported in 2007 (PAHO 2008; MS / SVS 2008a). This dissertation consists of a literature review, with emphasis on the epidemiology of dengue in Brazil and two articles that are designed to analyze the clinical and epidemiological characteristics of this condition, focusing on children under 15 years old, in Goiânia. The first article provides a profile analysis 59. 157 reported cases of dengue (SINAN), in Goiania, from 2001 to 2006. It also presents a reliability evaluation of the clinical classification of dengue, as recorded in the SINAN, compared with a revised classification proposed by the authors. There was an increase of dengue fever with complications or Dengue hemorrhagic fever in all age groups (p <0.05), children showed more hemorrhagic manifestations, however, no difference in the proportion of severe cases among adults and children. The results showed a low capacity of the surveillance system for classifying cases potentially serious in children under 15 years old (Kappa = 0.22; 95% CI 0.20 to 0.24). The second article examines 162 children under 15 years with acute febrile illness suggestive of dengue fever, attended from 2005 to 2006. We collected blood samples for detection of IgM anti-dengue (MAC-ELISA), molecular biology test (RT-PCR) and virus isolation. Half the cases were laboratory confirmed by serology and 84 and five by RT-PCR and / or virus isolation and identification of DEN 3 in all. Myalgia, arthralgia, rash, thrombocytopenia and leukopenia were more frequent among confirmed cases (p <0.05). PPV and NPV for suspected dengue cases, by clinical criteria, were respectively 54.8% and 40.0%. Recommended clinical criteria for diagnosis of dengue were reluctant for this specific age group, even in a region of high endemicity. / No Brasil, a dengue esta presente em quase todos os estados e territórios, com circulação de três sorotipos diferentes (DEN 1, DEN 2 e DEN 3), resultando em cerca de 500 mil casos notificados, em 2007 (PAHO 2008; MS/SVS 2008a). A presente dissertação de mestrado é composta por uma revisão da literatura, com ênfase na epidemiologia da dengue no Brasil e por dois artigos que se propõem analisar as características clínicas e epidemiológicas desse agravo, com enfoque em menores de 15 anos de idade, em Goiânia. O primeiro artigo traz uma análise do perfil de 59. 157 casos notificados de dengue (SINAN), em Goiânia, de 2001 a 2006. Apresenta, também, uma avaliação da confiabilidade da classificação clínica de dengue, conforme registrada no SINAN, em comparação com uma classificação revisada, propostas pelos autores. Houve aumento de dengue com complicação ou dengue hemorrágica, em todas as faixas etárias (p<0,05), Crianças apresentaram mais manifestações hemorrágicas, entretanto, não houve diferença na proporção de formas graves entre adultos e crianças. Foi evidenciada uma baixa capacidade do sistema de vigilância para classificar casos potencialmente graves, em menores de 15 anos de idade (Kappa = 0,22; IC95% 0,20-0,24). O segundo artigo analisa 162 menores de 15 anos, com quadro febril agudo, sugestivo de dengue, atendidas entre 2005 a 2006. Foram colhidas amostras de sangue para detecção de IgM anti-dengue (MAC-ELISA); teste de biologia molecular (RT-PCR) e isolamento viral. Metade dos casos foram confirmados laboratorialmente, sendo 84 por sorologia e cinco por RT-PCR e/ou isolamento viral, com identificação de DEN 3 em todos eles. Mialgia,artralgia, exantema, plaquetopenia e leucopenia foram mais freqüentes entre os casos confirmados (p< 0,05). VPP e VPN para casos suspeitos de dengue, pelos critérios clínicos, foram, respectivamente, de 54,8% e 40,0%. Critérios clínicos preconizados para o diagnóstico de dengue mostraram-se pouco específicos para esta faixa etária, mesmo em região de alta endemicidade.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are emerging infectious diseases which are endemic in many regions of the globe, many of which are resource-poor areas. DHF and DF impose a severe economic health burden in tropical and subtropical areas. Dengue virus causes an acute febrile illness that can be a self-limited febrile illness, as seen in most cases of DF, or a life-threatening illness with plasma leakage and shock, as seen in cases of DHF. A systematic review of the literature revealed gaps in the knowledge base of clinical laboratory findings of dengue illness with regards to longitudinal dynamics and classification and predictive modeling of disease severity. The objective of this thesis was to investigate the utility of clinical laboratory variables for classification and prediction of disease outcomes. The data used in this investigation was derived from a prospective study of Thai children presenting to either of two study hospitals within 72 hours of onset of an acute febrile illness. Systematic data collection, including clinical laboratory parameters, and routine clinical management continued each day until 24 hours after the fever had subsided. A final diagnosis of DHF, DF, or other febrile illness (OFI) was assigned by an expert physician after chart review. The first research objective of this study was to describe the temporal dynamics of clinical laboratory parameters among subjects with DHF, DF, or OFI. Data were analyzed using lowess curves and population-average models. Quadratic functions of clinical variables over time were established and demonstrated significantly divergent patterns between the various diagnostic groups. The second research objective was to establish and validate tools for classification of illness severity using easily obtained clinical laboratory measures. Bivariate logistic regression models were established using data from one hospital in an urban area of Thailand as a training data set and validated with a second data set from a hospital in a rural area of Thailand. The validated models maintained a high sensitivity and specificity in distinguishing severe dengue illnesses without using the hallmark indicators of plasma leakage. The third research objective used classification and regression tree (CART) analysis to established diagnostic decisions trees using data obtained on the day of study enrollment, within the first 3 days of acute illness. Decision trees with high sensitivity were established for severe dengue defined either as: 1) DHF with evidence of shock (dengue shock syndrome, DSS); or 2) DSS or dengue with significant pleural effusion. This study expands existing knowledge of the potential utility of clinical laboratory variables during different phases of dengue illness. The application of the results of these studies should lead to promising opportunities in the fields of epidemiological research and disease surveillance to reduce the health burden, and improve the clinical management, of dengue illness. Future directions involve application of these algorithms to different study populations and age groups. Additionally, other analytical techniques, such as those involving CART analysis, can be explored with these data.

Made available in DSpace on 2016-12-23T13:47:02Z (GMT). No. of bitstreams: 1 Creuza Rachel Vicente.pdf: 3384838 bytes, checksum: c3eca90bf4886725c29d5e5904662f2b (MD5) Previous issue date: 2012-03-15 / Introdução: A ocorrência da dengue sofre influência do comportamento, estrutura social e distribuição da população e sua transmissão pode variar de acordo com as áreas do município. Seu prognóstico depende do diagnóstico precoce e da imediata instituição do tratamento. Este estudo avalia fatores associados à ocorrência da dengue, enfatizando a distribuição territorial e os relacionados à dengue grave. Métodos: Foram realizados dois estudos, sendo um transversal e outro retrospectivo, sobre a totalidade dos casos de dengue que ocorreram em Vitória no ano de 2011, com base nos dados do Sistema de Informações de Agravos de Notificação. Resultados: Entre os casos confirmados, 53,4% ocorreram em mulheres, 74,7% em maiores de 15 anos e 6,3% evoluíram para gravidade. Os territórios de saúde de Jardim Camburi, Maruípe, Ilha das Caieiras, Santa Martha e Santo André responderam por 41,6% das notificações. Quase metade dos casos foram concluídos por critério laboratorial e, destes, 80% realizaram sorologia. Em todos os territórios, mais de 20% dos notificados realizaram sorologia e, na maioria, mais de 51% tiveram resultado positivo. Nas regiões de São Pedro, Maruípe e Santo Antônio, os afetados eram principalmente jovens, enquanto nas regiões Continental e Forte São João, eram pessoas mais velhas. Dos 371 casos de dengue grave, 78,7% foram de dengue com complicações e 21,3% de febre hemorrágica da dengue. Sessenta e sete por cento dos casos ocorreram em pessoas com idade superior a 15 anos. As Regiões de Saúde de Maruípe e São Pedro foram responsáveis por mais da metade dos casos de dengue grave (56,35%). Houve associação estatisticamente significante entre ocorrência de febre hemorrágica da dengue com idades mais jovens (menores de 15 anos) e maior tempo decorrido na procura por atendimento. Também houve associação estatisticamente significante entre maior tempo decorrido na procura pelo atendimento e idade menor que 15 anos. Os casos de dengue grave estavam concentrados em faixas etárias mais jovens na região de São Pedro. Conclusão: A distribuição territorial não foi uniforme, e pode ser determinada pela alta densidade populacional e pelas condições socioeconômicas. As diferenças de idade entre as regiões podem estar relacionadas à incidência da doença nestes locais. A grande proporção de sorologias positivas e o número de exames realizados possibilitaram uma boa detecção e acompanhamento dos casos de dengue. Os resultados corroboram os de outras pesquisas que apontam uma mudança no perfil da febre hemorrágica da dengue nas Américas e no Brasil, com crescente acometimento de jovens, e apontam a demora no tempo de procura por atendimento, baixa qualidade urbana e alta endemicidade como possíveis fatores de risco. / Introduction: The incidence of dengue, influenced by human behavior, social structure and population distribution, may vary with respect to the geographical areas of a given city. Its prognosis depends on early diagnosis and prompt initiation of treatment. This study evaluates factors related to the occurrence of dengue, emphasizing the territorial distribution and risk factors for severe dengue. Methods: A cross-sectional and a retrospective study on all cases were conducted with dengue cases of Vitória in 2011, based on data from the Information System of Notifiable Diseases (SINAN). Results: 53.4% of confirmed cases occurred in women, 74.7% were 15 years old or older, and 6.3% were severe forms. The health territories of Jardim Camburi, Maruípe, Ilha das Caieiras, Santa Martha and Santo André accounted for 41.6% of reported cases. Almost half of the cases had final classification based on laboratory tests, and of these, 80% were submitted to serological tests. Of all territories, more than 20% of those reported cases had been submitted to serological tests, and more than 51% of them had a positive result. In the regions of São Pedro, Santo Antônio e Maruípe, the affected individuals were younger, while in Continental Region and Forte São João Region, they were older. Of the 371 cases of severe dengue, 78.7% were classified as dengue with complications and 21.3% were classified as dengue hemorrhagic fever, whose age distribution disclosed a frequency of 67.1% in individuals 15 years old or older. Regions of Maruípe and São Pedro were responsible for over half of cases of severe dengue (56.3%). There was a statistically significant association between the occurrence of dengue hemorrhagic fever and younger ages (under 15 years) and longer time interval between the beginning of symptoms and seeking for care. People younger than 15 years old take longer to seek for care. The frequencies of cases of severe dengue were concentrated in younger age groups in the region of São Pedro. Conclusion: The geographical distribution was not uniform, and can be influenced by the high population density and socioeconomic conditions. The age differences between regions may be related to disease incidence in these locations. A large proportion of positive tests and a great number of tests performed allowed a good detection and monitoring of dengue cases. The results corroborate those of other studies that indicate a change in the profile of dengue hemorrhagic fever in the Americas and Brazil, with growing involvement of young people, and indicate the time delay in seeking treatment, urban poor quality and high endemic scenario as possible risk factors.