Early resuscitation of dengue shock syndrome in children with hyperosmolar sodium-lactate: a randomized single-blind clinical trial of efficacy and safety.

INTRODUCTION: Dengue shock syndrome (DSS) fluid resuscitation by following the World Health Organization (WHO) guideline usually required large volumes of Ringer lactate (RL) that might induce secondary fluid overload. Our objective was to compare the effectiveness of the recommended volume of RL versus a smaller volume of a hypertonic sodium lactate solution (HSL) in children with DSS. The primary end point was to evaluate the effect of HSL on endothelial cell inflammation, assessed by soluble vascular cell adhesion molecule-1 (sVCAM-1) measurements. Secondarily, we considered the effectiveness of HSL in restoring hemodynamic fluid balance, acid-base status, and sodium and chloride balances, as well as in-hospital survival. METHODS: A prospective randomized single-blind clinical trial including 50 DSS children was conducted in the Pediatrics Department of Hasan Sadikin Hospital, Bandung, Indonesia. Only pediatric patients (2 to 14 years old) fulfilling the WHO criteria for DSS and new to resuscitation treatments were eligible. Patients were resuscitated with either HSL (5 ml/kg/BW in 15 minutes followed by 1 ml/kg/BW/h for 12 hours), or RL (20 ml/kg/BW in 15 minutes followed by decreasing doses of 10, 7, 5, and 3 ml/kg BW/h for 12 hours). RESULTS: In total, 50 patients were randomized and included in outcome and adverse-event analysis; 46 patients (8.2 ± 0.5 years; 24.9 ± 1.9 kg; mean ± SEM) completed the protocol and were fully analyzed (24 and 22 subjects in the HSL and RL groups, respectively). Baseline (prebolus) data were similar in both groups. Hemodynamic recovery, plasma expansion, clinical outcome, and survival rate were not significantly different in the two groups, whereas fluid accumulation was one third lower in the HSL than in the RL group. Moreover, HSL was responsible for a partial recovery from endothelial dysfunction, as indicated by the significant decrease in sVCAM-1. CONCLUSION: Similar hemodynamic shock recovery and plasma expansion were achieved in both groups despite much lower fluid intake and fluid accumulation in the HSL group. TRIAL REGISTRATION: ClinicalTrials.gov NCT00966628. Registered 26 August 2009.

Increase of plasminogen activator inhibitor-1 and decrease of transforming growth factor-b1 in children with dengue haemorrhagic fever in Indonesia.

Mortality in children with severe dengue haemorrhagic fever (DHF) in Indonesia is high. The origin of the elevated plasminogen activator inhibitor-1 (PAI-1) levels in these children is unclear. We measured PAI-1, transforming growth factor-ß1 (TGF-ß1), platelet counts, plasma leakage and liver function in 71 children with DHF (3-15 years old) and in 30 healthy children. We found that PAI-1 concentrations in children with DHF were significantly higher on admission than on Day 2. Circulating TGF-ß1 concentrations on admission were significantly lower in DHF than in controls, but on Day 2 increased towards levels in controls. TGF-ß1 and PAI-1 concentrations were not correlated on either day. PAI-1 was correlated with platelet count and serum albumin on admission, and with degree of pleural effusion. Liver function tests were mildly elevated but not correlated with PAI-1. In conclusion, elevated PAI-1 concentrations in DHF were associated with platelet counts and plasma leakage.

RNA secondary structures in the proximal 3'UTR of Indonesian Dengue 1 virus strains.

The characteristics of DENV-1 viruses, isolated during the 2001-2002 outbreak in Indonesia were studied. The secondary structure of the 3'UTR of different DENV-1 strains derived from Indonesian patients was compared with the 3'UTR of previously described DENV-1 sequences. The complete 3'UTR of DENV-1 was sequenced from 13 patients suffering from the severe form of dengue virus infection (dengue hemorrhagic fever). Prediction of RNA secondary structure of the 3'UTR revealed some previously unidentified conserved structures in the proximal region of the 3'UTR, the role of which in viral replication is still unknown. In addition our data suggest that some structural elements previously described in the distal part of the 3'UTR are partly dependent on the proximal part of the UTR. Our data support the existence of previously unidentified conserved secondary structures in the proximal part of the 3'UTR and their roles need to be further investigated.

Dengue disease severity in Indonesian children: an evaluation of the World Health Organization classification system.

BACKGROUND: Dengue disease severity is usually classified using criteria set up by the World Health Organization (WHO). We aimed to assess the diagnostic accuracy of the WHO classification system and modifications to this system, and evaluated their potential practical usefulness. METHODS: Patients, admitted consecutively to the hospital with severe dengue, were classified using the WHO classification system and modifications to this system. Treating physicians were asked to classify patients immediately after discharge. We calculated the sensitivity of the various classification systems for the detection of shock and the agreement between the various classification systems and the treating physician's classification. RESULTS: Of 152 patients with confirmed dengue, sixty-six (43%) had evidence of circulatory failure. The WHO classification system had a sensitivity of 86% (95%CI 76-94) for the detection of patients with shock. All modifications to the WHO classification system had a higher sensitivity than the WHO classification system (sensitivity ranging from 88% to 99%). The WHO classification system was in only modest agreement with the intuitive classification by treating physicians whereas several modified classification systems were in good agreement. CONCLUSION: The use of the WHO classification system to classify dengue disease severity is to be questioned, because it is not accurate in correctly classifying dengue disease severity and it lacks sufficient agreement with clinical practice.

The role of cytokines in activation of coagulation and fibrinolysis in dengue shock syndrome.

In a prospective clinical study of 50 patients with Dengue Shock Syndrome (DSS), we investigated the association of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), and IL-6 with activation markers of coagulation (F1+2 and TATc) and fibrinolysis (t-PA, PAPc, and D-dimer). We found that TNF-alpha, IL-1beta and Il-1Ra, but not IL-6, concentrations were elevated in the circulation during the early stage of infection and at discharge from hospital. TNF-alpha was significantly associated with D-dimer, an activation marker of fibrinolysis (p < 0.003), but not with activation markers of coagulation. IL-1beta was significantly associated with t-PA (p < 0.03). IL-1Ra was significantly associated with F1+2, TATc (p < 0.04 and p < 0.02, respectively), whereas IL-6 was significantly associated with both, activation markers of coagulation (F1+2; p < 0.03) and fibrinolysis (PAPc; p = 0.002). Our data are in line with studies in bacterial sepsis. In severe dengue virus infection the same cytokines are involved in the onset and regulation of hemostasis.

Cytokine patterns during dengue shock syndrome.

OBJECTIVE: To investigate the patterns of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, interferon-gamma (IFN-gamma) and interleukin-1 receptor antagonist (IL-1Ra) during the course of dengue shock syndrome. DESIGN: Prospective clinical study. SETTING: Pediatric Intensive Care Unit, Dr. Kariadi Hospital, the university hospital of Diponegoro University, Semarang, Indonesia. PATIENTS: Fifty children with dengue shock syndrome. MEASUREMENTS: The plasma concentration and the ex vivo production, with and without lipopolysaccharide (LPS), of TNF-alpha, IL-1beta and IL-1Ra were measured in duplicate by nonequilibrium radioimmunoassay (RIA); IFN-gamma and IL-6 were measured by ELISA. RESULTS: During the acute phase, the plasma concentrations and the ex vivo production without LPS of IL-1Ra were considerably elevated and returned to normal on recovery. However, the ex vivo LPS-stimulated production of the proinflammatory cytokines TNF-alpha and IL-1beta were considerably depressed. Also, these concentrations returned towards normal on recovery. In non-survivors, the plasma concentrations of IL-6 and IL-1Ra were significantly higher than in survivors (p = 0.00001 and p = 0.0005, respectively). In addition, the ex vivo production of IL-1Ra in non-survivors was significantly higher than in survivors, both without LPS stimulation (p = 0.0008) and with LPS (p < 0.004). IL-1Ra was significantly associated with mortality (p = 0.007). CONCLUSION: Since IL-1Ra was significantly associated with mortality, this measurement may be used as an index of disease severity in dengue shock syndrome.

Plasma levels of inter-alpha inhibitor proteins in children with acute Dengue virus infection.

BACKGROUND: Inter-alpha inhibitor proteins (IaIp) belong to a family of protease inhibitors that are involved in the haemostatic and the vascular system. Dengue viruses (DENV) infections are characterized by coagulopathy and increased vascular permeability. In this study we measured the concentration of IaIp during DENV infections and evaluated its potential as a biomarker. METHODS AND FINDINGS: Concentrations of IaIp were measured in patients with acute DENV infections using a quantitative, competitive enzyme linked immunoassay. Concentrations of IaIp measured in pediatric patients suffering from severe DENV infections were significantly lower than in healthy controls. CONCLUSIONS: This is the first report to demonstrate changes in concentration of IaIp during viral infections. The data also highlight the potential of IaIp as a biological marker for severity of DENV infections.

Differential gene expression changes in children with severe dengue virus infections.

BACKGROUND: The host response to dengue virus infection is characterized by the production of numerous cytokines, but the overall picture appears to be complex. It has been suggested that a balance may be involved between protective and pathologic immune responses. This study aimed to define differential immune responses in association with clinical outcomes by gene expression profiling of a selected panel of inflammatory genes in whole blood samples from children with severe dengue infections. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood mRNA from 56 Indonesian children with severe dengue virus infections was analyzed during early admission and at day -1, 0, 1, and 5-8 after defervescence. Levels were related to baseline levels collected at a 1-month follow-up visit. Processing of mRNA was performed in a single reaction by multiplex ligation-dependent probe amplification, measuring mRNA levels from genes encoding 36 inflammatory proteins and 14 Toll-like receptor (TLR)-associated molecules. The inflammatory gene profiles showed up-regulation during infection of eight genes, including IFNG and IL12A, which indicated an antiviral response. On the contrary, genes associated with the nuclear factor (NF)-kappaB pathway were down-regulated, including NFKB1, NFKB2, TNFR1, IL1B, IL8, and TNFA. Many of these NF-kappaB pathway-related genes, but not IFNG or IL12A, correlated with adverse clinical events such as development of pleural effusion and hemorrhagic manifestations. The TLR profile showed that TLRs were differentially activated during severe dengue infections: increased expression of TLR7 and TLR4R3 was found together with a decreased expression of TLR1, TLR2, TLR4R4, and TLR4 co-factor CD14. CONCLUSIONS/SIGNIFICANCE: These data show that different immunological pathways are differently expressed and associated with different clinical outcomes in children with severe dengue infections.

Elevated plasma levels of the long pentraxin, pentraxin 3, in severe dengue virus infections.

C-reactive protein is one of the most widely used indicators of the response of acute-phase proteins. The measurement of C-reactive protein in dengue, however, is clinically not useful, because of marginally elevated levels and absent association with disease severity. The prototypic long pentraxin, pentraxin 3, is an acute phase protein that is structurally related but distinct from C-reactive protein which has proven to correlate with the severity of bacterial infection in critically ill patients. The potential involvement of pentraxin 3 in dengue and its aptitude to predict more severe disease or poor clinical outcome has not been studied previously. We therefore measured pentraxin 3 plasma levels in 44 dengue virus infected patients. Pentraxin 3 levels were strikingly higher when compared to C-reactive protein levels, with highest pentraxin 3 values observed in the first 7 days after the onset of symptoms. Median pentraxin 3 levels at admission and peak levels during follow up were higher in patients suffering from dengue shock syndrome (at admission: 119.3 ng/ml [interquartile range 61.8--188.7], peak values during follow up: 147.9 ng/ml [interquartile range 85.7--204.3]) compared to levels found in patients with dengue fever and dengue hemorrhagic fever (at admission: 59.0 ng/ml [interquartile range 28.6--100.3], P=0.040; peak values during follow up: 80.8 ng/ml [interquartile range 36.1--168.1], P=0.020). Our results indicate that pentraxin 3 seems to be a marker of infection better than C-reactive protein in dengue. The role of pentraxin 3 in the pathogenesis of dengue and its potential as an early prognostic indicator of disease severity needs further assessment.

Elevation of soluble VCAM-1 plasma levels in children with acute dengue virus infection of varying severity.

Approximately 1,000 million infections with dengue viruses are estimated to occur annually. The majority of the cases develop mild disease, whereas only small proportion of the infected individuals develop severe hemorrhagic manifestations at the end of the acute phase of illness. In this study, the value of plasma levels of vascular cell adhesion molecule 1 (VCAM-1) in the pathogenesis and prognosis of dengue illness was investigated in children with dengue infections of varying severity. The plasma levels of soluble VCAM-1 (sVCAM-1) were measured in serial plasma samples obtained from 168 children aged between 7 months and 14 years with confirmed dengue infection. Of those children, 71 were suffering from dengue fever, 30 were suffering from dengue hemorrhagic fever, and 67 were suffering from dengue shock syndrome. Plasma samples obtained from 21 patients with febrile illness other than dengue served as controls. A commercially available kit (R&D Systems, Oxon, UK) was used to measure the levels of sVCAM-1 in plasma samples. sVCAM-1 was elevated during acute dengue infection, and significantly elevated among dengue shock syndrome patients as compared to dengue fever or dengue hemorrhagic fever patients (P < 0.05). Statistical analysis revealed that sVCAM-1 was associated with dengue disease severity and the time post infection (acute vs. convalescent phase) and not with age, sex, or previous exposure of the patients to dengue infection. A significant difference was found in the plasma levels of sVCAM-1 between dengue shock syndrome and dengue fever patients, however, the prognostic value of this marker in the acute stage of dengue illness proved to be limited. These data also favor to study the further elucidation of the role of sVCAM-1 in the pathogenesis of dengue infections.

Detection of immune-complex-dissociated nonstructural-1 antigen in patients with acute dengue virus infections.

Accurate and timely diagnosis of dengue virus (DEN) infections is essential for the differential diagnosis of patients with febrile illness and hemorrhagic fever. In the present study, the diagnostic value of a newly developed immune-complex dissociated nonstructural-1 (NS-1) antigen dot blot immunoassay (DBI) was compared to a commercially available DEN antigen detection kit (denKEY Blue kit; Globio Co., Beverly, Mass.) and a reverse transcription-PCR (RT-PCR) kit. Serial serum or plasma samples (n = 181) obtained from 55 acute DEN-infected patients were used. In samples obtained from 32 of these 55 DEN-infected patients, viral RNA could be detected by RT-PCR. DEN antigen was detected in only 10 of these 55 patient samples by using the denKEY kit. When these samples were treated with acid to release the immune-complex-associated NS-1 antigen for detection by DBI, 43 of these 55 patients were found to be positive for DEN NS-1 antigen. In nondissociated samples, 22 of these patients were found to be positive by the DBI. In the presence of DEN-specific immunoglobulin M antibodies, both viral RNA and DEN (NS-1) antigen could be detected. The number of positive samples identified by RT-PCR and DBI from these patients with primary DEN infections varied between 28 and 78%. In secondary DEN infections, the number of samples that tested positive by the DBI after immune-complex dissociation (DIS-DBI) was 25% higher than the number of samples that tested positive by RT-PCR and was 35% higher than that determined by nondissociated antigen (NDIS-DBI) detection. We conclude that the denKEY kit has limited diagnostic value for acute DEN infections compared to the RT-PCR and the NDIS-DBI and DIS-DBI methods. We clearly demonstrate that in secondary DEN infections the dissociation of NS-1 immune complexes is essential for early diagnosis of DEN infections.

Elevated levels of total and dengue virus-specific immunoglobulin E in patients with varying disease severity.

The kinetics of total and dengue virus-specific immunoglobulin E (IgE) were studied in serial serum samples obtained from 168 patients, 41 of whom suffered from primary dengue virus infection and 127 suffered from secondary dengue virus infection. Seventy-one patients were classified as dengue fever, 30 as dengue hemorrhagic fever, and 67 as dengue shock syndrome. A control group included single serum samples from patients with a herpes virus infection (n = 14), non-dengue febrile patients (n = 10), and healthy blood donors (n = 10). Patients with dengue virus infection had higher levels of total and dengue virus-specific IgE than non-dengue patients (P < 0.05). Patients with secondary dengue virus infections had not significantly increased levels of both total and dengue virus-specific IgE in the acute phase of disease compared to patients with primary dengue virus infections. Dengue virus-specific IgE was significantly higher in dengue hemorrhagic fever and/or dengue shock syndrome patients compared to dengue fever and non-dengue patients (P < 0.05). In conclusion, this study showed elevated total and dengue virus-specific IgE serum antibody levels in the acute stage of disease. Therefore, measurement of both total and dengue virus-specific IgE serum antibodies can be used as an additional prognostic marker in the development of severe complications in dengue virus infections. In addition, the presence and increase of dengue virus-specific IgE serum antibodies in patients with dengue virus infections is suggestive of the pathogenetic role that IgE may play in the hemostatic disorders observed in dengue hemorrhagic fever and dengue shock syndrome.

Impaired fibrinolysis in the pathogenesis of dengue hemorrhagic fever.

The mechanisms contributing to bleeding complications in dengue hemorrhagic fever were studied by investigating the pattern of activation of the coagulation and fibrinolytic systems in 50 children with severe dengue hemorrhagic fever. Thirteen patients (26%) died, and activation of coagulation was most pronounced in the deceased group. Fibrinolysis was also activated, but this activation was relatively weak compared with that of coagulation as a result of persistently high plasminogen activator inhibitor levels. Plasminogen activator inhibitor also prevented a switch from the procoagulant to the profibrinolytic state in lethal dengue hemorrhagic fever, which was further enhanced by an acquired protein C deficiency. The present study is the first to demonstrate such a mechanism in a viral infection. This imbalance between coagulation and fibrinolysis may be used as a prognostic marker, but it may also be a target for future therapeutic intervention.