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Dengue haemorrhagic fever in children in the 1996 Delhi epidemic.

An epidemic of dengue haemorrhagic fever (DHF) occurred in Delhi in 1996. A total of 240 children between the age of 4 months to 13 years of either sex, admitted in one hospital, were evaluated. Two hundred and sixteen (90%) children were from Delhi. A clinical diagnosis of dengue fever (DF) was made in 25 (10%), dengue fever with unusual bleeding (DFB) in 22 (9%), DHF in 80 (33%) and dengue shock syndrome (DSS) in 113 (47%) of the children strictly according to the WHO classification. The age peaked at 8 years. There was no association between various grades of severity of illness and age-groups though girls suffered from more severe illness. No association between severity of malnutrition and severity of illness was observed. Tourniquet test was positive in 40% with DF, 18% with DFB, 62% with DHF and 64% with DSS. In DSS haematemesis was present in 55 (49%), epistaxis in 39 (35%), melaena in 27 (24%) and ecchymosis in 34 (30%) patients. Children diagnosed as DFB had haematemesis and epistaxis in 12 (55%) and 10 (45%) respectively. Intravenous fluid requirement was clearly less in DFB patients than in DHF/DSS patients. Unusual clinical features in the form of jaundice were present in 7 (6%), hepatic encephalopathy in 6 (5%) and dengue encephalopathy in 6 (5%) patients. Dengue 2 virus was isolated from 10 of the 50 patients for whom viral culture was done on C6/36 clone of Aedes albopictus cell line. Eighteen patients suffering from DSS died giving an overall case fatality of 7.5%. The mortality rate in DHF/DSS was 9.3%. It is further suggested that DFB is a distinct entity. Most patients could be classified by the WHO classification if a retrospective packed cell volume was used to assess haemoconcentration. We suggest that development of area-specific criteria for diagnosis and management is desirable.

Antibodies, Viral↗
PubMed1998

Cardiac involvement in Dengue Haemorrhagic Fever.

We studied 17 consecutive patients of Dengue Haemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) to assess cardiac function by radionuclide ventriculography, echocardiography and electrocardiography (ECG) during the epidemic of Dengue virus type-2 (DEN-2) in Delhi, India (1996). Case definitions laid down by the WHO were followed. Fourteen patients were seropositive for Dengue infection. In radionuclide ventriculography study, the mean left-ventricular ejection fraction was 41.69 (5.04% (range 33-49%) and 7 patients had an ejection fraction less than 40%, global hypokinesia was detected in 12 (70.59%) patients. In echocardiography, the mean ejection fraction was 47.06 (3.8%). Eight patients had Dengue Shock Syndrome and the mean ejection fraction was 39.63% (4.97% in radionuclide ventriculography, out of which 5 patients had an ejection fraction below 40%. To find out the nature of myocardial involvement, 99m Tc-pyrophosphate imaging was done in 4 patients and it was discontinued further because no myocardial necrosis was detected in those patients. Five patients had ST and T changes in the electrocardiogram, radionuclide ventriculography and echocardiography revealed no abnormalities after 3 weeks of follow up and the ejection fraction was more than 50% in all cases. Global hypokinesia also improved and ECG changes reverted back to normal within 3 weeks. Acute reversible cardiac insult may be noticed in Dengue Haemorrhagic Fever/Dengue Shock Syndrome and could be responsible for hypotension/shock seen in some of these patients. Further studies are required to establish the pathogenic mechanisms of cardiac dysfunction in patients with Dengue Haemorrhagic Fever/Dengue Shock Syndrome.

Adolescent↗
PubMed1996

Multicentre evaluation of dengue IgM dot enzyme immunoassay.

BACKGROUND: The traditional methods used in the diagnosis of dengue infection do not lend themselves to field application. As such, clinical specimens have to be sent to a central laboratory for processing which invariably leads to delay. This affects patient management and disease control. The development of the dengue IgM dot enzyme immunoassay has opened up the possibility of carrying out the test in peripheral health settings. OBJECTIVES: This multicentre study was conducted to evaluate a new, commercial nitrocellulose membrane based IgM capture enzyme immunoassay. STUDY DESIGN: The sensitivity and specificity of the test were compared with in-house dengue IgM enzyme-linked immunoassays routinely performed by each of the selected centres. Known positive and negative dengue specimens, as well as specimens from non-dengue cases, were included in the evaluation. RESULTS: Based on 402 specimens tested by the six centres, the sensitivity was 92.1% and specificity 88.1%, with an overall agreement of 92.8% when compared with IgM EIA assays performed on microplates. CONCLUSIONS: The results suggest that this commercial kit has a role to play in the diagnosis of dengue infection, especially in peripheral health settings.

Antibodies, Viral↗
PubMed2003

Non-specific alert system for dengue epidemic outbreaks in areas of endemic malaria. A hospital-based evaluation in Cayenne (French Guiana).

The emergence of dengue haemorrhagic fever is a public health problem in Latin America and the Caribbean. This study, carried out in French Guiana where malaria is endemic, evaluated the value and the limitations of a non-specific alert system including all patients admitted to the emergency department of Cayenne Hospital, between 1 January 1996 and June 2001. Four indices were studied on a weekly basis: the emergency malaria negative index (EMN), the EMN thrombocytopenia index (EMNT), the dengue suspected index: EMNT/EMN ratio; and the number of hospitalized patients with dengue fever according to the Department of Medical Information. These indices were retrospectively compared with data from the Arbovirus Reference Centre at the Pasteur Institute in French Guiana. Using the non-specific indices, we were able to identify four clear epidemics, two of which were shown to be linked to dengue. Variations in the incidence of malaria had no marked effect on this alert system. We propose that this simple, cheap, sensitive and reactive alert system be used to improve the serological and virological monitoring of dengue and to facilitate adequate and timely vector control measures. It could be used in all regions at risk of dengue and malaria.

Adult↗
PubMed2006

Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides.

The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.

Dengue Virus↗
PubMed2003

Inhibition of interferon signaling by dengue virus.

Dengue virus is a worldwide-distributed mosquito-borne flavivirus with a positive strand RNA genome. Its transcribed polyprotein is cleaved by host- and virus-encoded peptidases into 10 proteins, some of which are of unknown function. Although dengue virus-infected cells seem to be resistant to the antiviral action of IFN, the viral products that mediate this resistance are unknown. Therefore, we have analyzed the ability of the 10 dengue virus-encoded proteins to antagonize the IFN response. We found that expression in human A549 cells of the dengue virus nonstructural proteins NS2A, NS4A, or NS4B enhances replication of an IFN-sensitive virus. Moreover, expression of NS4B and, to a lesser extent, of NS2A and NS4A proteins results in down-regulation of IFN-beta-stimulated gene expression. Cells expressing NS4B or infected with dengue virus do not exhibit nuclear signal transducer and activator of transcription (STAT) 1 on treatment with IFN-beta or IFN-gamma, indicating that NS4B might be involved in blocking IFN signaling during dengue virus infections. This protein, encoded by a positive strand RNA virus, is implicated as an IFN-signaling inhibitor.

Animals↗
PubMed1977

Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection.

Studies were made on the identity of human and monkey mononuclear leukocytes permissive to antibody-enhanced dengue 2 virus (D2V) infection. In cultures of peripheral blood leukocytes (PBL) inoculated immediately after separation, it was concluded that only mononuclear phagocytes support dengue infection. This is based upon observations that D2V-permissive cells were resistant to 1,200 rads, were both plastic adherent and nonadherent, were removed when passed through nylon wool columns in 10 percent fetal bovine serum or 100 percent autologous serum, and were destroyed by incubation with 100 mug/ml particulate silica. On direct immunofluorescence staining, perinuclear dengue antigen was visualized at 24 h, becoming maximal at 60 h. Antigen-containing cells had ample cytoplasm, ruffled cytoplasmic membrane, and 73 percent were actively phagocytic. As further evidence of the infection of mononuclear phagocytes, antibody-enhanced D2V replication was observed in bone marrow cultures from five of five rhesus monkeys, but not in cell cultures of spleen, thymus, or lymph nodes prepared from the same animals. It is hypothesized that dengue virus complexed with non-neutralizing antibody is internalized by immune phagocytosis in a mononuclear phagocyte with a defective virus-destroying mechanism. Dengue permissiveness may depend upon cellular immaturity since bone marrow leukocytes could be infected even when held for 4 days before infection while PBL held for this time decreased in permissiveness. In vitro antibody-dependent infection of mononuclear phagocytes should prove useful as a model for study of immunopathologic mechanisms in human dengue.

Animals↗
PubMed2001

Plasma free amino acid concentrations in healthy Guatemalan adults and in patients with classic dengue.

BACKGROUND: Plasma free amino acid patterns in health and disease have been reported. However, amino acid concentrations in adult populations in developing countries and in patients with dengue, as a model for an acute infectious viral disease endemic to the tropics, have not been reported. OBJECTIVE: The purpose of this study was to determine the amino acid profile in both healthy Guatemalan adults from different socioeconomic backgrounds and at 3 time points during the course of classic dengue. DESIGN: The study was carried out in Guatemala and included measurement of plasma free amino acids in 22 healthy control subjects (14 low income, 8 middle class) and 17 febrile patients. Measurements of amino acids were repeated within a 48-h interval in 20 of the healthy Guatemalans. In 9 patients with dengue, amino acids were assayed 3 times: on admission to a local hospital in the coastal plain of Guatemala, on hospital discharge, and 7 d after hospital discharge. RESULTS: Branched-chain amino acid concentrations in healthy adults and dengue patients in Guatemala were lower than normal values reported in the literature for healthy Swedish adults. With the exception of increased phenylalanine concentrations and an increased ratio of phenylalanine to tyrosine, all amino acids as well as the Fischer molar ratio were decreased in the acute phase of dengue. CONCLUSIONS: Healthy Guatemalans have different amino acid patterns than do Swedish subjects independent of socioeconomic status. The systemic viral disease dengue is associated with changes in the plasma free amino acid pattern, reflecting infection-related alterations in amino acid metabolism.

Adult↗
PubMed2003

Inferring the rate and time-scale of dengue virus evolution.

Dengue is often referred to as an emerging disease because of the rapid increases in incidence and prevalence that have been observed in recent decades. To understand the rate at which genetic diversification occurs in dengue virus and to infer the time-scale of its evolution, we employed a maximum likelihood method that uses information about times of virus sampling to estimate the rate of molecular evolution in a large number of viral envelope (E) gene sequences and to place bounds around the dates of appearance of all serotypes and specific genotypes. Our analysis reveals that dengue virus generally evolves according to a molecular clock, although some serotype-specific and genotype-specific rate differences were observed, and that its origin is more recent than previously suggested, with the virus appearing approximately 1,000 years ago. Furthermore, we estimate that the zoonotic transfer of dengue from sylvatic (monkey) to sustained human transmission occurred between 125 and 320 years ago, that the current global genetic diversity in the four serotypes of dengue virus only appeared during the past century, and that the recent rise in genetic diversity can be loosely correlated both to human activities such as population growth, urbanization, and mass transport and to the emergence of dengue hemorrhagic fever as a major disease problem.

Animals↗
PubMed2004

Pulmonary hemorrhage syndrome associated with an autochthonous case of dengue hemorrhagic fever.

Dengue fever is a major public health problem worldwide. Dengue hemorrhagic fever, a much rarer form of the disease, occurs when a person previously infected with dengue is re-infected with a different viral serotype. In recent years the infection rates of dengue and both clinical syndromes have increased along the United States-Mexico border. We present the case of a 61-year-old Laotian female who presented with a 1-week history of fever, altered mental status, oral ulceration, and rash. The patient developed diffuse pulmonary hemorrhage and anemia requiring multiple transfusions. She eventually sustained multi-organ system failure and expired. Both the titer data and serologies were consistent with the diagnosis of dengue hemorrhagic fever. We hypothesize that this syndrome was the result of re-infection occurring within the United States. This case is also unusual in that it is the second reported in the literature of pulmonary hemorrhages associated with dengue hemorrhagic fever.

Fatal Outcome↗
PubMed2005

Aggressive management of dengue shock syndrome may decrease mortality rate: a suggested protocol.

OBJECTIVES: Dengue shock syndrome is a leading cause of mortality among Indian children. In January 2000, we instituted a protocol for aggressive management of children with dengue shock syndrome. The objective of this study was to compare outcomes (duration of ventilation, pediatric intensive care unit stay, incidence of acute respiratory distress syndrome, and intensive care unit and hospital mortality) before and after the protocol. DESIGN: Retrospective chart review. SETTING: Pediatric intensive care unit at a tertiary teaching hospital. PATIENTS: One hundred and fourteen patients admitted between July 1997 and December 1999 received standard therapy recommended by the World Health Organization (WHO) and were designated as the WHO guidelines group (W), whereas 96 patients admitted between January 2000 and December 2001 were treated by our protocol and designated as the protocol group (P). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The patients in each group were equally matched in terms of age, Pediatric Risk of Mortality, and number with dengue hemorrhage fever grade IV, although the platelet counts were higher in the W group compared with the P group (geometric mean 42.2, confidence interval 36.9, 48.4 vs. geometric mean 36.7, confidence interval 33.3, 40.5, p < .05). Patients in the W group received less fluids in the first hour compared with the P group (median and interquartile range 20 mL/kg, 15 and 20 vs. 30 mL/kg, 20 and 60). Fluid was actively removed less often in the W group than the P group (6 of 111 vs. 45 of 96, p < .05). There was no difference in the need for ventilation or incidence of acute respiratory distress syndrome between groups, although among dengue hemorrhage fever grade IV patients, the number requiring ventilation (17 of 30 vs. 20 of 23, p < .05) and the incidence of acute respiratory distress syndrome (9 of 30 vs. 17 of 23, p < .05) were significantly greater in the W group compared with the P group. The duration of ventilation (1.5 +/- 1.7 vs. 4.2 +/- 2.9 days, p < .05) and length of intensive care unit stay (3.0 +/- 2.8 vs. 3.4 +/- 2.9 days, p < .05) were significantly less in the W group. The pediatric intensive care unit mortality (16.6% vs. 6.3%, p < .05) was significantly higher in the W group than in the P group. CONCLUSIONS: Patients with dengue shock syndrome are at high risk of mortality due to refractory shock and multiple organ failure. Survival was better for patients in the P group. Aggressive shock management and possibly the use of judicious fluid removal may decrease mortality rates in the severest forms of dengue shock syndrome.

Algorithms↗
PubMed1998

Definition of the region on NS3 which contains multiple epitopes recognized by dengue virus serotype-cross-reactive and flavivirus-cross-reactive, HLA-DPw2-restricted CD4+ T cell clones.

The epitopes recognized by six CD4+ CD8- cytotoxic T lymphocyte (CTL) clones established from a dengue-3 virus-immune donor were defined. (i) Three CTL clones, JK10, JK34 and JK39, were cross-reactive for dengue virus types 1-4. (ii) One clone, JK28, was cross-reactive for dengue virus types 1-4 and West Nile virus. (iii) Two clones, JK26 and JK49, were cross-reactive for dengue virus types 1-4, West Nile virus and yellow fever virus. The clones, except for JK49, recognized the same epitope on NS3 in an HLA-DPw2-restricted fashion. The smallest synthetic peptide recognized by the five CTL clones was a 10 aa peptide which comprises aa 255-264 on dengue virus NS3. JK49 recognized the overlapping epitope which comprises aa 257-266 in an HLA-DPw2-restricted fashion. Analysis of T cell receptor (TCR) usage by these T cell clones revealed that (i) JK10 and JK34 use V alpha11, and JK34 and JK28 use V beta23, and (ii) the amino acid sequences of the V(D)J junctional region of the TCR were different among these five CTL clones. There were, however, single amino acid conservations among TCRs of some of these T cell clones. These results indicate that the region on NS3 which comprises aa 255-266 contains multiple epitopes recognized by dengue serotype-cross-reactive and flavivirus-cross-reactive CD4+ CTL in an HLA-DPw2-restricted fashion and that a single epitope can be recognized by T cells which have heterogeneous virus specificities.

Amino Acid Sequence↗
PubMed2004

Identification of the homotypic interaction domain of the core protein of dengue virus type 2.

Dengue virus causes dengue haemorrhagic fever or dengue shock syndrome with a high mortality rate. The genome of dengue virus is a positive-sense, single-stranded RNA encoding three structural and seven non-structural proteins. The core protein is one of the three structural proteins and is the building block of the nucleocapsid of dengue virus. The core protein of dengue virus type 2 (DEN2) is composed of 100 aa with four alpha-helix domains. An internal hydrophobic domain located at aa 44-60 was identified. The DEN2 core protein was shown to form homodimers. Deletion of aa 1-36 or 73-100 decreased but did not completely abolish the core-to-core homotypic interaction, whereas deletion of a portion (aa 44-60) within aa 37-72 completely abolished the ability of the DEN2 core proteins to interact with each other. A recombinant DEN2 core protein corresponding to aa 37-72 was able to undergo homotypic interaction and bound to a native DEN2 core protein. The results of this study indicated that the homotypic interaction domain of the DEN2 core protein is located at aa 37-72 and that the internal hydrophobic domain located at aa 44-60 plays a pivotal role in core-to-core homotypic interaction.

Active Transport, Cell Nucleus↗
PubMed2004

Evaluation of an algorithm for integrated management of childhood illness in an area of Vietnam with dengue transmission.

OBJECTIVES: To determine whether nurses, using the WHO/UNICEF algorithm for integrated management of childhood illness (IMCI), modified to include dengue infection, satisfactorily classified children in an area endemic for dengue haemorrhagic fever (DHF). METHODS: Nurses assessed and classified, using the modified IMCI algorithm, a systematic sample of 1250 children aged 2 months to 10 years (n = 1250) presenting to a paediatric hospital in Dong Nai Province, Vietnam. Their classification was compared with that of a paediatrician, blind to the result of the nurses' assessment, which could be modified in the light of simple investigations, e.g. dengue serology. RESULTS: In children aged 2-59 months (n = 859), the nurses were able to classify, using the modified chart, the presenting illness in >99% of children and found more than one classification in 70%. For the children with pneumonia, diarrhoea, dengue shock syndrome, severe DHF and severe disease requiring urgent admission, the nurse's classification was >60% sensitive and >85% specific compared with that of the paediatrician. For the nurse's classification of DHF the specificity was 50-55% for the children <5 years and in children with definitive dengue serology. Alterations in the DHF algorithm improved specificity at the expense of sensitivity. CONCLUSION: Using the IMCI chart, nurses classified appropriately many of the major clinical problems in sick children <5 years in southern Vietnam. However, further modifications will be required in the fever section, particularly for dengue. The impact of using the IMCI chart in peripheral health stations remains to be evaluated.

Algorithms↗
PubMed2004

Risk factors and clinical features associated with severe dengue infection in adults and children during the 2001 epidemic in Chonburi, Thailand.

OBJECTIVES: Dengue haemorrhagic fever (DHF) is an important cause of morbidity in South-east Asia and used to occur almost exclusively in young children. In recent years, there has been a progressive shift in age-distribution towards older children and adults. We investigated an outbreak in 2001 in both children and adults, in an endemic area of Thailand. METHODS: Retrospective study of 347 patients with serologically confirmed dengue infection admitted to Chonburi Hospital during an epidemic in 2001. RESULTS: A total of 128 (37%) patients had dengue fever (DF) and 219 (63%) had DHF. Patients with DHF were significantly older than patients with DF (11 years vs. 8 years). Clinical bleeding was noted in 124 individuals, both with DF (n = 24) and DHF (n = 100), and significantly more frequently in adults. Twenty-nine (13.2%) of all DHF cases were caused by primary infection. Secondary dengue infection was associated significantly with the development of DHF in children, OR (95% CI) = 3.63 (1.94-6.82), P < 0.0001, but not in adults, OR (95% CI) = 0.6 (0.02-6.04), P = 1. Unusual clinical manifestations were observed in 23 patients: three presented with encephalopathy and 20 with highly elevated liver-enzymes. In the latter group, four patients were icteric and nine had gastrointestinal bleeding. CONCLUSION: These results indicate that DHF in South-east Asia is common in both children and adults. In dengue-endemic countries, dengue should be considered as a differential diagnosis in patients with clinical gastrointestinal bleeding in association with increased liver enzymes.

Adolescent↗
PubMed2006

Headache features in patients with dengue virus infection.

The aim of this study was to describe the frequency and features of headache among patients with confirmed dengue virus infection and to compare the headache features in patients with dengue fever and dengue haemorrhagic fever, primary and secondary dengue infection, and patients with and without neurological involvement. Patients with classic dengue fever had a more intense headache than those with the more severe form of the disease, dengue haemorrhagic fever.

Adult↗
PubMed1970

Dengue virus plaque development in simian cell systems. I. Factors influencing virus adsorption and variables in the agar overlay medium.

Chemical and physical variables influencing the plaquing of all dengue serotypes in two simian cell systems were studied. Calf serum in the nutrient overlay may be replaced by mouse ascitic fluid or bovine plasma albumin when employing the rhesus monkey kidney LLC-MK(2) cell system for plaquing all dengue serotypes. Doubling the serum concentration in the overlay had little effect in modifying dengue types 1, 2, 3, and 4 plaque titers. Newborn agamma, 4-week-old and 8-week-old calf serum gave comparable titers with all dengue virus serotypes. Dengue virus titers, plaque size, and development time were unaffected by sodium bicarbonate concentrations ranging from 1.1 to 4.4 mg/ml of overlay. A twofold increase (0.00332 g%) in the amount of either autoclaved or filtered-sterilized neutral red reduced the dengue 2 virus titer as much as 2.2 logs. An increased Mg(++) and decreased Ca(++) concentration in the overlay medium increased the efficiency of the plaquing system.

Adsorption↗
PubMed1980

Monoclonal antibodies specific for dengue virus type 3.

Mouse lymphocyte hybridomas were prepared by polyethylene glycol-mediated fusion of cells from a mouse plasmacytoma line with lymphocytes from a mouse hyperimmunized with dengue virus type 3 (dengue-3). Media from 50 hybrid colonies were screened; 46 of them showed antibody activity against dengue-3-infected cells as determined by an indirect immunofluorescent antibody technique. Dengue monoclonal antibody obtained after cloning one of these colonies demonstrated activity in hemagglutination inhibition and indirect immunofluorescent antibody assays with dengue-3 antigen, but not type 1, 2, and 4 antigens. In addition, this antibody activity could be removed from culture media only by absorption with dengue-3 antigen.

Absorption↗