Plasma cell-free DNA: a potential biomarker for early prediction of severe dengue.

BACKGROUND: Considerable progress has been made in dengue management, however the lack of appropriate predictors of severity has led to huge number of unwanted admissions mostly decided on the grounds of warning signs. Apoptosis related mediators, among others, are known to correlate with severe dengue (SD) although no predictive validity is established. The objective of this study was to investigate the association of plasma cell-free DNA (cfDNA) with SD, and evaluate its prognostic value in SD prediction at acute phase. METHODS: This was a hospital-based prospective cohort study conducted in Vietnam. All the recruited patients were required to be admitted to the hospital and were strictly monitored for various laboratory and clinical parameters (including progression to SD) until discharged. Plasma samples collected during acute phase (6-48 h before defervescence) were used to estimate the level of cfDNA. RESULTS: Of the 61 dengue patients, SD patients (n = 8) developed shock syndrome in 4.8 days (95% CI 3.7-5.4) after the fever onset. Plasma cfDNA levels before the defervescence of SD patients were significantly higher than the non-SD group (p = 0.0493). From the receiver operating characteristic (ROC) curve analysis, a cut-off of > 36.9 ng/mL was able to predict SD with a good sensitivity (87.5%), specificity (54.7%), and area under the curve (AUC) (0.72, 95% CI 0.55-0.88; p = 0.0493). CONCLUSIONS: Taken together, these findings suggest that cfDNA could serve as a potential prognostic biomarker of SD. Studies with cfDNA kinetics and its combination with other biomarkers and clinical parameters would further improve the diagnostic ability for SD.

Association between dengue severity and plasma levels of dengue-specific IgE and chymase.

There is no definitive predictor of dengue severity, and this has led to a very large number of unnecessary hospitalizations worldwide. Although mast cell mediators are believed to a play role in dengue severity, the lack of precise kinetic data demands further research on early predictors. We enrolled 111 patients with confirmed dengue and 85 with "other febrile illness" (OFI) in a hospital-based prospective study in Vietnam. Dengue patients were classified as level 1, 2, or 3 based on the clinical intervention received. Blood samples were collected from each patient every day (pre- and post-defervescence) and after discharge. Plasma chymase, total IgE, and dengue-specific IgE were measured. Dengue-specific IgE levels showed an increasing trend during the course of illness and remained high even at post-discharge, although no significant difference was observed among severity levels. Total IgE showed no such trend. The specific IgE/total IgE ratio (S/T ratio) remained constantly higher in level 3 patients compared to other levels, with a significant difference at some time points. The S/T ratio of acute phase samples (before defervescence) tended to increase with increasing severity (level 1 < 2 < 3), and was significantly higher in level 3 patients than in level 1 and OFI patients. As an early predictor of severity allowing level 3 patients to be distinguished from other dengue patients, the S/T ratio achieved a sensitivity of 75% and specificity of 68%. We describe the kinetic profiles of IgEs, their ratio, and chymase levels at different severity levels. The S/T ratio was found to be associated with dengue severity, suggesting that it could potentially be used as an early predictor of severity.

Evolution and Spatiotemporal Dynamics of Enterovirus A71 Subgenogroups in Vietnam.

Background: Enterovirus A71 (EV-A71) is the major cause of severe hand, foot, and mouth disease and viral encephalitis in children across the Asia-Pacific region, including in Vietnam, which has experienced a high burden of disease in recent years. Multiple subgenogroups (C1, C4, C5, and B5) concurrently circulate in the region with a large variation in epidemic severity. The relative differences in their evolution and epidemiology were examined within Vietnam and globally. Methods: A total of 752 VP1 gene sequences were analyzed (413 generated in this study combined with 339 obtained from GenBank), collected from patients in 36 provinces in Vietnam during 2003-2013, along with epidemiological metadata. Globally representative VP1 gene datasets of subgenogroups were used to coestimate time-resolved phylogenies and relative genetic diversity to infer virus origins and regional transmission network. Results: Despite frequent virus migration between countries, the highest genetic diversity of individual subgenogroups was maintained independently for several years in specific Asian countries representing genogroup-specific sources of EV-A71 diversity. Conclusion: This study highlights a persistent transmission network of EV-A71, with specific Asian countries seeding other countries in the region and beyond, emphasizing the need for improved EV-A71 surveillance and detailed genetic and antigenic characterization.

Development of clinical decision rules to predict recurrent shock in dengue.

INTRODUCTION: Mortality from dengue infection is mostly due to shock. Among dengue patients with shock, approximately 30% have recurrent shock that requires a treatment change. Here, we report development of a clinical rule for use during a patient's first shock episode to predict a recurrent shock episode. METHODS: The study was conducted in Center for Preventive Medicine in Vinh Long province and the Children's Hospital No. 2 in Ho Chi Minh City, Vietnam. We included 444 dengue patients with shock, 126 of whom had recurrent shock (28%). Univariate and multivariate analyses and a preprocessing method were used to evaluate and select 14 clinical and laboratory signs recorded at shock onset. Five variables (admission day, purpura/ecchymosis, ascites/pleural effusion, blood platelet count and pulse pressure) were finally trained and validated by a 10-fold validation strategy with 10 times of repetition, using a logistic regression model. RESULTS: The results showed that shorter admission day (fewer days prior to admission), purpura/ecchymosis, ascites/pleural effusion, low platelet count and narrow pulse pressure were independently associated with recurrent shock. Our logistic prediction model was capable of predicting recurrent shock when compared to the null method (P < 0.05) and was not outperformed by other prediction models. Our final scoring rule provided relatively good accuracy (AUC, 0.73; sensitivity and specificity, 68%). Score points derived from the logistic prediction model revealed identical accuracy with AUCs at 0.73. Using a cutoff value greater than -154.5, our simple scoring rule showed a sensitivity of 68.3% and a specificity of 68.2%. CONCLUSIONS: Our simple clinical rule is not to replace clinical judgment, but to help clinicians predict recurrent shock during a patient's first dengue shock episode.

Kinetics of CD4+ T Helper and CD8+ Effector T Cell Responses in Acute Dengue Patients.

Background: The protective or pathogenic role of T lymphocytes during the acute phase of dengue virus (DENV) infection has not been fully understood despite its importance in immunity and vaccine development. Objectives: This study aimed to clarify the kinetics of T lymphocyte subsets during the clinical course of acute dengue patients. Study design: In this hospital-based cohort study, 59 eligible Vietnamese dengue patients were recruited and admitted. They were investigated and monitored for T cell subsets and a panel of clinical and laboratory parameters every day until discharged and at post-discharge from the hospital. Results: We described for the first time the kinetics of T cell response during the clinical course of DENV infection. Severe cases showed significantly lower levels of effector CD8+ T cells compared to mild cases at day -1 (p = 0.017) and day 0 (p = 0.033) of defervescence. After defervescence, these cell counts in severe cases increased rapidly to equalize with the levels of mild cases. Our results also showed a decline in total CD4+ T, Th1, Th1/17 cells during febrile phase of dengue patients compared to normal controls or convalescent phase. On the other hand, Th2 cells increased during DENV infection until convalescent phase. Cytokines such as interferon-γ, IL-12p70, IL-5, IL-23, IL-17A showed tendency to decrease on day 0 and 1 compared with convalescence and only IL-5 showed significance indicating the production during acute phase was not systemic. Conclusion: With a rigorous study design, we uncovered the kinetics of T cells in natural DENV infection. Decreased number of effector CD8+ T cells in the early phase of infection and subsequent increment after defervescence day probably associated with the T cell migration in DENV infection.

Isolation and characterization of two phenotypically distinct dengue type-2 virus isolates from the same dengue hemorrhagic Fever patient.

Dengue is the one of the most prevalent arthropod-borne viral diseases. Dengue virus circulates between humans and mosquitoes, and causes a wide range of disease in humans. To elucidate the link between the cell tropism of dengue virus and its pathogenesis, peripheral blood cells of infected patients were analyzed by flow cytometry. The dengue virus antigen was detected in peripheral CD19+ cells (B cells) in one dengue hemorrhagic fever patient. Two dengue type-2 virus isolates were recovered from this patient using mosquito cell line C6/36 and human hematopoietic cell line K562, and designated VNHCM18-C/02 and VNHCM18-K/02, respectively. VNHCM18-K/02 exhibited strong binding ability and high infectivity to a B-lymphocyte cell line (RPMI8226) but showed poor growth in C6/36 cells, while VNHCM18-C/02 more efficiently and dominantly grew in C6/36 cells but did not efficiently bind to nor infect the B-cell line. Three amino acid differences were detected; one in an envelope protein (E-62) and two in nonstructural proteins. The distinct cell-binding to RPMI8226 was attributed to the difference between the two isolates in envelope protein E-62. Thus, we isolated two dengue type-2 virus variants with different cell-tropisms from the same patient, suggesting possible co-circulation in the patient.

Fetal Zika Virus Infection in Vietnam.

As of 13 July 2016, 13 countries have reported fetal Zika virus (ZIKV) infection. Here we report a case of fetal ZIKV infection that resulted from an infection originating in Vietnam.

Post-dengue acute disseminated encephalomyelitis: A case report and meta-analysis.

BACKGROUND: Dengue is one of the most common infectious diseases. The aim of this study was to systematically review acute disseminated encephalomyelitis (ADEM) and to represent a new case. METHODOLOGY/PRINCIPAL FINDINGS: We searched for articles in nine databases for case reports, series or previous reviews reporting ADEM cases in human. We used Fisher's exact and Mann-Whitney U tests. Classification trees were used to find the predictors of the disease outcomes. We combined findings using fixed- and random-effects models. A 13-year-old girl was admitted to the hospital due to fever. She has a urinary retention. The neurological examinations revealed that she became lethargic and quadriplegic. She had upper limbs weakness and lower limbs complete paraplegia. Her status gradually improved after the treatment. She was nearly intact with the proximal part of her legs had a mild weakness in discharge. The prevalence of ADEM among dengue patients was 0.4% [95% confidence intervals (95% CI) 0.1-2.5%], all neurological disorders among dengue was 2.6% [95% CI 1.8-3.8%], and ADEM among neurological disorders was 6.8% [95% CI 3.4-13%]. The most frequent manifestation of ADEM was altered sensorium/consciousness (58%), seizures and urination problems (35%), vision problems (31%), slurred speech (23%), walk problems (15%) then ataxia (12%). There was a significant difference between cases having complete recovery or bad outcomes in the onset day of neurological manifestations being earlier and in temperature being higher in cases having bad outcomes (p-value < 0.05). This was confirmed by classification trees which included these two variables. CONCLUSIONS/SIGNIFICANCE: The prevalence of ADEM among dengue and other dengue-related neurological disorders is not too rare. The high fever of ADEM cases at admission and earlier onset day of neurological manifestations are associated with the bad outcomes.

Criteria of "persistent vomiting" in the WHO 2009 warning signs for dengue case classification.

INTRODUCTION: Dengue is a viral disease that spreads rapidly in the tropic and subtropic regions of the world and causes 22,000 deaths annually. In 2009, the World Health Organization (WHO) released a new classification of dengue infections, which divided them into three categories: dengue without warning sign (D), dengue with warning sign (DWS), and severe dengue (SD). However, researchers have been using different criteria to define persistent vomiting; therefore, we aimed to evaluate the ability of the number of vomiting times in early prediction of SD development among D/DWS patients. METHOD: A hospital-based cohort study was conducted in Ben Tre-south of Vietnam. We enrolled confirmed dengue patients with D and DWS at admission. The final classification was determined on the discharged day for every patient based on the classification of WHO 2009 without using vomiting symptom, using the receiver operating characteristic (ROC) curve to evaluate the ability of the number of vomiting times in early prediction of SD development among D/DWS patients. RESULT: The prevalence of vomiting symptom was higher in SD group than D/DWS group (92 versus 46 %, p = 0.006), and the median of the number of vomiting times was higher in SD group than D/DWS group (2.5 versus 0, p = 0.001). To distinguish SD from D/DWS, the ROC curve of the number of vomiting episodes showed that the area under the curve was 0.77; with the cut point of two, the sensitivity and specificity were 92 and 52 %, respectively. DISCUSSION: The number of vomiting times could be a good clinical sign which can early predict SD from the group of D/DWS. We suggest the definition of persistent vomiting should be vomiting two times or more per day.

A Proteomic Approach Identifies Candidate Early Biomarkers to Predict Severe Dengue in Children.

BACKGROUND: Severe dengue with severe plasma leakage (SD-SPL) is the most frequent of dengue severe form. Plasma biomarkers for early predictive diagnosis of SD-SPL are required in the primary clinics for the prevention of dengue death. METHODOLOGY: Among 63 confirmed dengue pediatric patients recruited, hospital based longitudinal study detected six SD-SPL and ten dengue with warning sign (DWS). To identify the specific proteins increased or decreased in the SD-SPL plasma obtained 6-48 hours before the shock compared with the DWS, the isobaric tags for relative and absolute quantification (iTRAQ) technology was performed using four patients each group. Validation was undertaken in 6 SD-SPL and 10 DWS patients. PRINCIPAL FINDINGS: Nineteen plasma proteins exhibited significantly different relative concentrations (p<0.05), with five over-expressed and fourteen under-expressed in SD-SPL compared with DWS. The individual protein was classified to either blood coagulation, vascular regulation, cellular transport-related processes or immune response. The immunoblot quantification showed angiotensinogen and antithrombin III significantly increased in SD-SPL whole plasma of early stage compared with DWS subjects. Even using this small number of samples, antithrombin III predicted SD-SPL before shock occurrence with accuracy. CONCLUSION: Proteins identified here may serve as candidate predictive markers to diagnose SD-SPL for timely clinical management. Since the number of subjects are small, so further studies are needed to confirm all these biomarkers.

Alpha tryptase allele of Tryptase 1 (TPSAB1) gene associated with Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) in Vietnam and Philippines.

We previously reported, significantly higher levels of Chymase and Tryptase in early stage plasma of DSS patients prior to the occurrence of shock suggesting a possible role of mast cells in dengue pathogenesis. To further investigate, we analyzed CMA1 promoter SNP (rs1800875) and TPSAB1 gene alleles, which encode the Human Chymase and α- and ß- tryptase 1 enzymes respectively, for susceptibility to Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) in patients from hospitals in Vietnam (Ho Chi Minh City and Vinh Long) and the Philippines. While the CMA1 promoter SNP (rs1800875) was not associated with DHF/DSS, the homozygous form of α-tryptase allele was associated with DSS patients in Vinh Long and the Philippines (OR=3.52, p<0.0001; OR=3.37, p<0.0001, respectively) and with DHF in Ho Chi Minh City (OR=2.54, p=0.0084). Also, a statistically significant association was observed when DHF and DSS were combined in Vinh Long (OR=1.5, p=0.034) and the Philippines (OR=2.36, p=0.0004); in Ho Chi Minh City when DHF and DSS were combine an association was observed, but it was not statistically significant (OR=1.5, p=0.0505). Therefore, the α-tryptase might have a possible effect on the susceptibility to severe form of Dengue infection.

Kinetics of dengue non-structural protein 1 antigen and IgM and IgA antibodies in capillary blood samples from confirmed dengue patients.

Large-scale epidemiological surveillance of dengue in the field and dengue patient management require simple methods for sample collection, storage, and transportation as well as effective diagnostic tools. We evaluated the kinetics of three biological markers of dengue infection-non-structural protein 1 (NS1) antigen, immunoglobulin M (IgM), and IgA-in sequential capillary blood samples collected from fingertips of confirmed dengue patients. The overall sensitivities and specificities of the tests were 96% and 100%, respectively, for NS1, 58.1% and 100%, respectively, for IgM, and 33% and 100%, respectively, for IgA. During the acute phase of the disease, NS1 was the best marker of dengue infection, with a sensitivity of 98.7%, whereas from day 5, all three markers exhibited relevant levels of sensitivity. This first descriptive study of the kinetics of biological markers of dengue in capillary blood samples confirms the usefulness of this biological compartment for dengue diagnosis and argues for its exploitation in community-level and remote settings.

The association of cytokines with severe dengue in children.

BACKGROUND: Dengue virus infection is a major public health problem. A hypothesis put forward for severe dengue is the cytokine storm, a sudden increase in cytokines that induces vascular permeability. Previous studies and our recent meta-analysis showed that IL-6, IL-8, IFNγ, TNFα, VEGF-A and VCAM-1 are associated with dengue shock syndrome. Therefore, in this study we aim to validate the association of these cytokines with severe dengue. METHODS & FINDINGS: In a hospital based-case control study in Vietnam, children with dengue fever, other febrile illness and healthy controls were recruited. Dengue virus infection was confirmed by several diagnostic tests. Multiplex immunoassay using Luminex technology was used to measure cytokines simultaneously. A positive association with dengue shock syndrome was found for VCAM-1, whereas a negative association was found for IFNγ. Furthermore, multivariate logistic analysis also showed that VCAM-1 and IFNγ were independently correlated with dengue shock syndrome. CONCLUSION: IFNγ and VCAM-1 were associated with dengue shock syndrome, although their role in the severe dengue pathogenesis remains unclear. Additional studies are required to shed further light on the function of these cytokines in severe dengue.

Studies on hantavirus infection in small mammals captured in southern and central highland area of Vietnam.

To investigate the distribution of hantaviruses among animals in Southern and Central Highland area of Vietnam, a total of 1311 serum samples were obtained from rats and Asian house shrews (Suncus murinus) captured at 11 locations between 2006 and 2009. A total of 1066 serum samples from rats were examined for IgG antibodies against Hantaan virus, and there were 30 antibody-positive serum samples from rats that had been captured mainly in a port area and urban area in Ho Chi Minh City (HCMC) (2.8%). All of the antibody-positive rats were Rattus norvegicus, and they had Seoul virus (SEOV) genome in their lungs. SEOV sequences detected from rats captured in Southern Vietnam belonged to the same lineage as those from rats captured at Haiphong Port and a market area in Hanoi City. SEOV strain CSG5 was isolated from a rat captured at Saigon Harbor. Strain CSG5 showed a cross-neutralization pattern almost the same as that of a representative strain of SEOV. A total of 245 Asian house shrews were captured in the Central Highland area and near HCMC. Sera were examined for IgG antibodies against Thottapalayam virus (TPMV), and 32 (13.1%) of the antibody-positive shrews were mainly from the Central Highland area and showed a neutralizing antibody against TPMV. These results indicated that SEOV is distributed among R. norvegicus inhabiting harbor and urban areas of Southern Vietnam and that TPMV or an antigenically related virus is distributed among Asian house shrews in Central Highland area.

Association of mast cell-derived VEGF and proteases in Dengue shock syndrome.

BACKGROUND: Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9. CONCLUSIONS: As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

Community-based control of Aedes aegypti by using Mesocyclops in southern Vietnam.

We previously reported a new community-based mosquito control strategy that resulted in elimination of Aedes aegypti (Linn.) in 40 of 46 communes in northern and central Vietnam, and with annual recurrent total costs (direct and indirect) of only $0.28-$0.89 international dollars per person. This control strategy was extended to four provinces in southern Vietnam in Long An and Hau Giang (2004-2007) and to Long An, Ben Tre, and Vinh Long (2005-2010). In a total of 14 communes with 124,743 residents, the mean ± SD of adult female Ae. aegypti was reduced from 0.93 ± 0.62 to 0.06 ± 0.09, and the reduction of immature Ae. aegypti averaged 98.8%. By the final survey, no adults could be collected in 6 of 14 communes, and one commune, Binh Thanh, also had no immature forms. Although the community-based programs also involved community education and clean-up campaigns, the prevalence of Mesocyclops in large water storage containers > 50 liters increased from 12.77 ± 8.39 to 75.69 ± 9.17% over periods of 15-45 months. At the conclusion of the study, no confirmed dengue cases were detected in four of the five communes for which diagnostic serologic analysis was performed. The rate of progress was faster in communes that were added in stages to the program but the reason for this finding was unclear. At the completion of the formal project, sustainability funds were set up to provide each commune with the financial means to ensure that community-based dengue control activities continued.

Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection.

BACKGROUND: Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue. METHODS AND FINDINGS: A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome. CONCLUSIONS: Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

A prospective cohort study of dengue infection in schoolchildren in Long Xuyen, Viet Nam.

A dynamic school-based cohort of 2-15 year-olds was established in Long Xuyen, Viet Nam to provide epidemiological data for a dengue vaccine efficacy trial. Active surveillance of febrile episodes identified clinically-suspected dengue and acute and convalescent sera were collected. IgG seroconversion between annual seroprevalence surveys identified sub-clinical infections. In 2004, 2190 children were enrolled with 3239, 3146, and 3081 present each year from 2005 to 2007 consecutively. In all, 627 children had a total of 690 clinically-suspected dengue episodes (394 hospitalisations, 296 outpatients) with 284-310 (41.2-45.0%) laboratory-confirmed depending on testing. Dengue serotype 2 was predominant in 2004 and 2005, and serotype 1 in 2006 and 2007. The acute dengue disease incidence rate per 1000 person-years ranged from 16.9 in 2005 to 40.4 in 2007. The average annual incidence of primary dengue infection (IgG seroconversion in previously naïve children) was 11.4% and the symptomatic to asymptomatic primary infection ratio ranged from 1:3-1:6. Study withdrawal rate, a feasibility indicator for conducting efficacy trials, was low: 4.2% per year when excluding children who changed schools. Our 2004-2007 results confirm the high transmission of dengue in children in Long Xuyen and demonstrate the suitability of this study site for a large scale efficacy trial.

Early clinical and biological features of severe clinical manifestations of dengue in Vietnamese adults.

BACKGROUND: Dengue is a major public health problem in both children and adults in Vietnam, but dengue severity in adults has been poorly investigated. OBJECTIVES: To describe severe manifestations of dengue in Vietnamese adults and to identify early indicators of these manifestations. STUDY DESIGN: A prospective longitudinal study was carried out from July to October, 2007, in People's Hospital 115, Ho Chi Minh City, Vietnam. RESULTS: One hundred ninety-five clinically suspected dengue patients were enrolled. 151 of these were laboratory-confirmed using serum samples on day 3 after onset of fever for RT-PCR and/or virus isolation. Dengue was associated with plasma leakage in 51 patients (33.8%), gallbladder thickening in 30 patients (20%), spontaneous bleeding in 127 patients (84.1%), and internal bleeding in 37 patients (24.5%). Several early indicators were associated with severe manifestations of dengue. Frequent vomiting (> or =3 times a day), marked lymphopenia, thrombocytopenia, and elevated liver enzymes on day 3 after onset of fever were significantly associated with plasma leakage and gallbladder thickening. Increased alanine aminotransferase level in plasma on day 3 was significantly associated with internal bleeding. Gallbladder thickening and internal bleeding were more common with specific serotypes. CONCLUSION: Several severe manifestations of dengue were observed in Vietnamese adults. These manifestations were associated with early clinical and biological indicators. This information may be useful for clinicians to better monitor adult dengue patients, particularly in tropical areas where health resources may be limited.