Predictive factors of dengue severity in hospitalized children and adolescents in Rio de Janeiro, Brazil

Abstract INTRODUCTION: Dengue is one of the most important mosquito-borne infections. Severe cases are more frequently observed in adults. However, in 2008, the State of Rio de Janeiro, Brazil, experienced a severe dengue epidemic that primarily affected children and caused many cases of dengue hemorrhagic fever (DHF) and death. METHODS: A cross-sectional analytical study was conducted to examine laboratory diagnosis and clinical epidemiologic factors for confirmed dengue cases in patients aged less than 16 years, from January to June 2008, at a municipal hospital in the City of Rio de Janeiro, Brazil. Variables associated with severe outcomes and P values less than .05 were evaluated by means of a logistic regression model. RESULTS: Of the 419 dengue cases studied, 296 were classified as DHF and 123 as classical dengue. Six patients who had DHF died. In multivariate analysis, some laboratory and clinical variables were independently associated with DHF: age 5 years or older (odds ratio [OR], 4.94; 95% confidence interval [CI], 1.30-18.71), abdominal pain (OR, 8.59; 95% CI, 3.17-23.27), hepatomegaly (OR, 15.87; 95% CI, 5.38-46.85), and positive tourniquet test (OR, 10.84; 95% CI, 3.96-29.71). Hypoalbuminemia occurred more frequently than hemoconcentration in DHF cases, and high aminotransferase levels were associated with severity. CONCLUSIONS: Age greater than 5 years, abdominal pain, painful hepatomegaly, and positive tourniquet test were predictors of DHF. The high frequency of hepatic impairment suggests that acetaminophen should be avoided in severe cases of dengue.

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7

ABSTRACT BACKGROUND Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN CONCLUSIONS The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Cissampelos sympodialis has anti-viral effect inhibiting dengue non-structural viral protein-1 and pro-inflammatory mediators

ABSTRACT Dengue is the most important viral infection transmitted among humans by arthropod-borne. There are currently no vaccines or specific therapeutical treatment. Therefore, immunomodulatory compounds from plants have been widely examined for their antiviral effects. Cissampelos sympodialis Eichler, Menispermaceae, has scientifically proven to present immunomodulatory activities. Here we assessed the antiviral activity of leaf hydroalcoholic extract, warifteine or methylwarifteine from C. sympodialis in an in vitro dengue virus infection model. The results demonstrated that leaf hydroalcoholic extract or warifteine/methylwarifteine treatment did not reduce dengue virus-Ag+ hepatocyte (Huh-7 cell) rates in present experimental conditions. However, we assessed the potential antiviral effect of leaf hydroalcoholic extract or warifteine/methylwarifteine on dengue virus-infection by the production of inflammatory molecules, TNF-α, MIF, IL-8 and PGE2. Dengue virus infection enhanced TNF-α, MIF, IL-8 and PGE2 production in infected Huh-7 cells and leaf hydroalcoholic extract but not warifteine/methylwarifteine treatments, significantly reduced these molecules in infected cells. In dengue virus-infected Huh-7 cells, non-structural protein-1 is produced and leaf hydroalcoholic extract significantly inhibited it independently of alkaloids. Our findings imply that leaf hydroalcoholic extract may attenuate dengue virus infection in Huh-7 cells by inhibiting the enhanced of pro-inflammatory mediators and non-structural protein-1 production induce by dengue virus independently of warifteine/methywarifteine its major compound.

From primary care to hospitalization: clinical warning signs of severe dengue fever in children and adolescents during an outbreak in Rio de Janeiro, Brazil / Da atenção primária à hospitalização: sinais clínicos de alarme para dengue grave em crianças e adolescentes durante uma epidemia no Rio de Janeiro, Brasil / De la atención primaria a la hospitalización: señales clínicas de alarma del dengue grave en niños y adolescentes durante una epidemia en Río de Janeiro, Brasil

We analyzed factors associated with severe cases of dengue in children and adolescents hospitalized during the 2007/2008 epidemic in Rio de Janeiro, Brazil. This is a retrospective case-control study that covers 88 cases of severe dengue in patients admitted to four tertiary care children's hospitals. Controls consisted of 22 children with non-severe dengue living in the same neighborhood as the patients with severe dengue. Differences in prevalence of the clinical signs - abdominal pain, breathing difficulty, drowsiness or irritability - emerged on the third day after the onset of symptoms, in the febrile stage. Cases and controls received first medical care at the same clinical stage of disease. However, hospital admission of severe cases occurred later, on average between the third and fourth day after the onset of the disease. Early discharge of patients with fever whose condition could have progressed to severe dengue may have been a consequence of the type of medical assistance provided by primary care units, suggesting deficiencies both in the use of the risk classification protocol and patient triage.

Foram avaliados fatores associados à ocorrência de casos graves de dengue em crianças/adolescentes hospitalizados durante a epidemia de 2007/2008 no Rio de Janeiro, Brasil. Trata-se de estudo caso-controle retrospectivo com 88 casos graves de dengue, admitidos em quatro hospitais de atenção terciária infantil. Os controles foram 22 crianças com dengue não grave residentes na vizinhança dos casos. Foram observadas diferenças na prevalência de sinais clínicos - dor abdominal, dificuldade respiratória, sonolência/irritabilidade - a partir do terceiro dia do início dos sintomas, ainda na presença da febre. Casos e controles receberam o primeiro atendimento médico no mesmo estágio clínico da doença. Contudo, as hospitalizações dos casos graves ocorreram mais tardiamente, em média entre o terceiro e quarto dia da doença. A liberação precoce de pacientes com quadro febril e potencialmente graves pode ter sido consequência do atendimento médico prestado nas unidades de atenção primária, sugerindo deficiências na aplicação do protocolo de classificação de risco de dengue e triagem de pacientes.

Fueron evaluados factores asociados a la ocurrencia de casos graves de dengue en niños/adolescentes hospitalizados, durante la epidemia de 2007/2008 en Río de Janeiro, Brasil. Se trata de un estudio caso-control retrospectivo con 88 casos graves de dengue, admitidos en cuatro hospitales de atención terciaria infantil. Los controles se efectuaron con 22 niños con dengue no grave, residentes en los alrededores de los casos. Se observaron diferencias en la prevalencia de señales clínicas -dolor abdominal, dificultad respiratoria, somnolencia/irritabilidad- a partir del tercer día del inicio de los síntomas, todavía con presencia de fiebre. Casos y controles recibieron la primera atención médica en la misma fase clínica de la enfermedad. No obstante, las hospitalizaciones de los casos graves se produjeron más tardíamente, en media entre el tercer y cuarto día de la enfermedad. El alta precoz de pacientes con cuadro febril, y potencialmente graves, puede haber sido consecuencia de la atención médica prestada en las unidades de atención primaria, sugiriendo deficiencias en la aplicación del protocolo de clasificación de riesgo de dengue y selección de pacientes.

Clinical and hepatic evaluation in adult dengue patients: a prospective two-month cohort study / Avaliação das alterações clínicas e hepáticas em pacientes adultos com dengue: estudo prospectivo em um período de dois meses

INTRODUCTION: To analyze the liver dysfunction and evolution of signs and symptoms in adult dengue patients during a two-month follow-up period. METHODS: A prospective cohort study was conducted in Campos dos Goytacazes, Rio de Janeiro, Brazil, from January to July, 2008. The evolution of laboratory and clinical manifestations of 90 adult dengue patients was evaluated in five scheduled visits within a two-month follow-up period. Twenty controls were enrolled for the analysis of liver function. Patients with hepatitis B, hepatitis C, those known to be human immunodeficiency virus (HIV) seropositive and pregnant women were excluded from the study. RESULTS: At the end of the second month following diagnosis, we observed that symptoms persisted in 33.3% (30/90) of dengue patients. We also observed that, 57.7% (15/26) of the symptoms persisted at the end of the second month. The most persistent symptoms were arthralgia, fatigue, weakness, adynamia, anorexia, taste alteration, and hair loss. Prior dengue virus (DENV) infection did not predispose patients to a longer duration of symptoms. Among hepatic functions, transaminases had the most remarkable elevation and in some cases remained elevated up to the second month after the disease onset. Alanine aminotransferase (ALT) levels overcame aspartate aminotransferase (AST) during the convalescent period. Male patients were more severely affected than females. CONCLUSIONS: Dengue fever may present a wide number of symptoms and elevated liver transaminases at the end of the second month.

INTRODUÇÃO: Analisar prospectivamente a disfunção hepática e a evolução dos sinais e sintomas em pacientes adultos com dengue durante um período de dois meses. MÉTODOS: Realizamos um estudo prospectivo em Campos dos Goytacazes, Rio de Janeiro, Brasil, de janeiro a julho de 2008. Foi avaliada a evolução das manifestações clínicas e laboratoriais em 90 pacientes adultos com dengue, em um período de dois meses. Vinte controles foram arrolados para análise da função hepática. Em ambos os grupos foram realizadas coletas de dados e sangue nos primeiros cinco dias da doença, e aos 8, 15, 30 e 60 dias após o início da doença. Foram excluídos pacientes com hepatite B, hepatite C, gestantes e aqueles sabidamente soropositivos para HIV. RESULTADOS: No final do segundo mês do início da dengue, 33,3% (30/90) dos pacientes apresentaram persistência de pelo menos um sinal ou sintoma. Estavam presentes no final do segundo mês 57,7% (15/26) dos sinais ou sintomas. Os maiores percentuais de persistência foram: artralgia, adinamia, fraqueza, fadiga, anorexia, alteração do paladar e queda de cabelo. A infecção prévia pelo vírus da dengue (DENV) não predispôs a uma maior duração dos sintomas. Da função hepática, observamos alterações relevantes somente nos níveis das transaminases, que em alguns casos permaneceram elevados até o final do segundo mês. Os níveis de ALT ultrapassaram os de AST na convalescença. Homens apresentaram níveis mais elevados de transaminases quando comparados aos de mulheres. CONCLUSÕES: Dengue apresenta grande número de sintomas e transaminases elevadas no final do segundo mês de doença.

Profile of circulating levels of IL-1Ra, CXCL10/IP-10, CCL4/MIP-1β and CCL2/MCP-1 in dengue fever and parvovirosis

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1β, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

Dengue-2 infection and the induction of apoptosis in human primary monocytes

Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-á and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Detection of circulant tumor necrosis factor-alpha, soluble tumor necrosis factor p75 and interferon-gamma in Brazilian patients with dengue fever and dengue hemorrhagic fever

Pro-inflammatory cytokines are believed to play an important role in the pathogenesis of dengue infection. This study reports cytokine levels in a total of 54 patients examined in Recife, State of Pernambuco, Brazil. Five out of eight patients who had hemorrhagic manifestations presented tumor necrosis factor-alpha (TNF-alpha) levels in sera which were statistically higher than those recorded for controls. In contrast, only one out of 16 patients with mild manifestations had elevated TNF-alpha levels. The levels of interleukin-6 (IL), IL-1beta tested in 24 samples and IL-12 in 30 samples were not significantly increased. Interferon-g was present in 10 out of 30 patients with dengue. The data support the concept that the increased level of TNF-alpha is related to the severity of the disease. Soluble TNF receptor p75 was found in most patients but it is unlikely to be related to severity since it was found with an equivalent frequency and levels in 15 patients with dengue fever and another 15 with dengue hemorrhagic fever

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Increased pro-inflammatory cytokines (TNF-[alfa] and IL-6) and anti-inflammatory compounds (sTNFRp55 and sTNFRp75) in brazilian patients during exanthematic dengue fever

Pro-inflammatory cytokines, tumor necrosis factor (TNF-[alfa]), interleukin-6(IL-6) and interleukin-1ß (IL-1ß) as well as anti-inflammatory compounds, soluble TNF-Receptor p55 (sTNFRp55), sTNFRp75 and IL-1 receptor antagonist (sIL-1Ra), were investigated in 34 Brazilian cases of dengue fever (DF) originated from a study of exanthematic virosis. The presence of pro-inflammatory cytokines was detected in sera from these patients by ELISA. TNF-[alfa] and IL-6 levels were significantly higher than control subjects in 32 (por cento) and 52(por cento) patients, respectively. To our knowledge this was the first time a receptor antagonist and soluble receptors for cytokines were detected in sera obtained during exanthematic DF without hemorrhagic manifestations. Both sTNFRp55 and sTNFRp75 were consistently elevated in 42 (por cento) and 84 (por cento) patients, respectively. Most patients had IL-1ß levels not different from those of normal subjects, except for one case. Only 16 (por cento) patients had altered levels of IL-Ra. Previous studies in dengue hemorrhagic fever patients demonstrated production of these soluble factors; here we observed that they are found in absence of hemorrhagic manifestations. The possible role of these anti-inflammatory compounds in immune cell activation and in regulating cytokine-mediated pathogenesis during dengue infection is discussed.

Ultrastructural study on experimental infection of rotavirus in a murine heterologous model

Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11). Samples taken from duodenum, jejunun and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

Characterization of CD4 and CD8 bearing cells in intestines of rotavirus susceptible (suckling) and resistant (adult/weanling) mice by flow cytometry

Suckling mice are susceptible to several virus infections and develop diarrhea after rotavirus inoculation whereas 3 week-old and older mice are resistant. Since young mice have a n immature immune system, we investigated the status of CD4 and CD8 bearing T-lymphocytes in intestines of 1, 3-4 and 8-10 week-old mice. Unicellular suspensions of the total small intestine were prepared. Cells were stained with monoclonal antibodies reactive to CD4 and CD8 molecules and were analyzed by flow cystometry. Percentages of CD8+ and CD4+CD8+ cells were markedly increased in intestines of suckling mice when compared to adults. CD4+ cells were apparently not altered. Rotavirus SA-11 infected diarrheic suckling mice presented a decrease of all three studied lymphocyte subpopulations, whereas no changes were observed in virus inoculated weanling mice. We suggest that higher proportions of CD4+CD8+ and CD8+ cells in intestines of suckling mice may play a role in the susceptibility to rotavirus, which would disable the animals to develop a rapid and efficient immune response resulting in resistance.