Dengue Viral RNA Levels in Peripheral Blood Mononuclear Cells Are Associated with Disease Severity and Preexisting Dengue Immune Status

Background

Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.

Method

The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.

Results

Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.

Conclusions

B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.     

Suppression of Virus Specific Immune Responses by IL-10 in Acute Dengue Infection

Background

Elevated IL-10 has been shown to be associated with severe dengue infection (DI). We proceeded to investigate the role of IL-10 in the pathogenesis of acute DI.

Materials and methods

Ex vivo and cultured IFNγ ELISpot assays for dengue virus (DENV) NS3 protein and non dengue viral proteins were carried out in 26 patients with acute DI (16 with dengue haemorrhagic fever) and 12 healthy dengue seropositive individuals from Sri Lanka. DENV serotype specific (SS) responses were determined by using a panel of SS peptides.

Results

Serum IL-10 level were significantly higher (p = 0.02) in those who did not have in vitro responses to DENV-SS peptides (mean 144.2 pg/ml) when compared to those who responded (mean 75.7 pg/ml). DENV-NS3 specific ex vivo IFNγ ELISpot responses were also significantly lower (p = 0.0001) in those who did not respond to DENV-SS peptides (mean 42 SFU/million PBMCs) when compared to those who responded to DENV-SS peptides (mean 1024 SFU/million PBMCs). Serum IL-10 levels correlated significantly (p = 0.03) and inversely (Spearmans R = −0.45) with ex vivo DENV-NS3 specific responses but not with ex vivo non DENV specific responses (Spearmans R = −014, p = 0.52). Blockage of IL-10 in vitro significantly increased (p = 0.04) the ex vivo IFNγ ELISpot DENV-NS3 specific responses but had no effect on responses to non DENV proteins.

Conclusion

IL-10 appears to contribute to the pathogenesis of acute dengue infections by inhibiting DENV-specific T cell responses, which can be restored by blocking IL-10.     

Correlation of serotype-specific dengue virus infection with clinical manifestations

Background

Disease caused by the dengue virus (DENV) is a significant cause of morbidity throughout the world. Although prior research has focused on the association of specific DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) with the development of severe outcomes such as dengue hemorrhagic fever and dengue shock syndrome, relatively little work has correlated other clinical manifestations with a particular DENV serotype. The goal of this study was to estimate and compare the prevalence of non-hemorrhagic clinical manifestations of DENV infection by serotype.

Methodology and Principal Findings

Between the years 2005–2010, individuals with febrile disease from Peru, Bolivia, Ecuador, and Paraguay were enrolled in an outpatient passive surveillance study. Detailed information regarding clinical signs and symptoms, as well as demographic information, was collected. DENV infection was confirmed in patient sera with polyclonal antibodies in a culture-based immunofluorescence assay, and the infecting serotype was determined by serotype-specific monoclonal antibodies. Differences in the prevalence of individual and organ-system manifestations were compared across DENV serotypes. One thousand seven hundred and sixteen individuals were identified as being infected with DENV-1 (39.8%), DENV-2 (4.3%), DENV-3 (41.5%), or DENV-4 (14.4%). When all four DENV serotypes were compared with each other, individuals infected with DENV-3 had a higher prevalence of musculoskeletal and gastrointestinal manifestations, and individuals infected with DENV-4 had a higher prevalence of respiratory and cutaneous manifestations.

Conclusions/Significance

Specific clinical manifestations, as well as groups of clinical manifestations, are often overrepresented by an individual DENV serotype.     

Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using FcγR-expressing cells

Background

Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo.

Methods and Findings

We evaluated serum samples from 80 residents of a dengue endemic country, Malaysia, for neutralizing activity, and infection-enhancing activity at 1∶10 serum dilution by using FcγR-negative BHK cells and FcγR-expressing BHK cells. The serum samples consisted of a panel of patients with acute DENV infection (31%, 25/80) and a panel of donors without acute DENV infection (69%, 55/80). A high proportion of the tested serum samples (75%, 60/80) demonstrated DENV neutralizing activity (PRNT₅₀≥10) and infection-enhancing activity. Eleven of 18 serum samples from patients with acute secondary DENV infection demonstrated neutralizing activity to the infecting serotype determined by using FcγR-negative BHK cells (PRNT₅₀≥10), but not when determined by using FcγR-expressing cells.

Conclusion

Human serum samples with low neutralizing activity determined by using FcγR-negative cells showed DENV infection-enhancing activity using FcγR-expressing cells, whereas those with high neutralizing activity determined by using FcγR-negative cells demonstrate low or no infection-enhancing activity using FcγR-expressing cells. The results suggest an inverse relationship between neutralizing antibody titer and infection-enhancing activity, and that neutralizing activity determined by using FcγR-expressing cells, and not the activity determined by using FcγR-negative cells, may better reflect protection to DENV infection in vivo.     

A Prospective Nested Case-Control Study of Dengue in Infants: Rethinking and Refining the Antibody-Dependent Enhancement Dengue Hemorrhagic Fever Model

Background

Dengue hemorrhagic fever (DHF) is the severe and life-threatening syndrome that can develop after infection with any one of the four dengue virus (DENV) serotypes. DHF occurs almost exclusively in individuals with secondary heterologous DENV infections and infants with primary DENV infections born to dengue immune mothers. The widely accepted explanation for the pathogenesis of DHF in these settings, particularly during infancy, is antibody-dependent enhancement (ADE) of DENV infection.

Methods and Findings

We conducted a prospective nested case-control study of DENV infections during infancy. Clinical data and blood samples were collected from 4,441 mothers and infants in up to two pre-illness study visits, and surveillance was performed for symptomatic and inapparent DENV infections. Pre-illness plasma samples were used to measure the associations between maternally derived anti-DENV3 antibody-neutralizing and -enhancing capacities at the time of DENV3 infection and development of infant DHF.The study captured 60 infants with DENV infections across a wide spectrum of disease severity. DENV3 was the predominant serotype among the infants with symptomatic (35/40) and inapparent (15/20) DENV infections, and 59/60 infants had a primary DENV infection. The estimated in vitro anti-DENV3 neutralizing capacity at birth positively correlated with the age of symptomatic primary DENV3 illness in infants. At the time of symptomatic DENV3 infection, essentially all infants had low anti-DENV3 neutralizing activity (50% plaque reduction neutralizing titers [PRNT₅₀] ≤50) and measurable DENV3 ADE activity. The infants who developed DHF did not have significantly higher frequencies or levels of DENV3 ADE activity compared to symptomatic infants without DHF. A higher weight-for-age in the first 3 mo of life and at illness presentation was associated with a greater risk for DHF from a primary DENV infection during infancy.

Conclusions

This prospective nested case-control study of primarily DENV3 infections during infancy has shown that infants exhibit a full range of disease severity after primary DENV infections. The results support an initial in vivo protective role for maternally derived antibody, and suggest that a DENV3 PRNT₅₀ >50 is associated with protection from symptomatic DENV3 illness. We did not find a significant association between DENV3 ADE activity at illness onset and the development of DHF compared with less severe symptomatic illness. The results of this study should encourage rethinking or refinement of the current ADE pathogenesis model for infant DHF and stimulate new directions of research into mechanisms responsible for the development of DHF during infancy.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00377754","term_id":"NCT00377754"}}NCT00377754 Please see later in the article for the Editors'' Summary     

A Randomized Controlled Trial of Chloroquine for the Treatment of Dengue in Vietnamese Adults

Background

There is currently no licensed antiviral drug for treatment of dengue. Chloroquine (CQ) inhibits the replication of dengue virus (DENV) in vitro.

Methods and Findings

A double-blind, randomized, placebo-controlled trial of CQ in 307 adults hospitalized for suspected DENV infection was conducted at the Hospital for Tropical Diseases (Ho Chi Minh City, Vietnam) between May 2007 and July 2008. Patients with illness histories of 72 hours or less were randomized to a 3-day course of CQ (n = 153) or placebo (n = 154). Laboratory-confirmation of DENV infection was made in 257 (84%) patients. The primary endpoints were time to resolution of DENV viraemia and time to resolution of DENV NS1 antigenaemia. In patients treated with CQ there was a trend toward a longer duration of DENV viraemia (hazard ratio (HR) = 0.80, 95% CI 0.62–1.05), but we did not find any difference for the time to resolution of NS1 antigenaemia (HR = 1.07, 95% CI 0.76–1.51). Interestingly, CQ was associated with a significant reduction in fever clearance time in the intention-to-treat population (HR = 1.37, 95% CI 1.08–1.74) but not in the per-protocol population. There was also a trend towards a lower incidence of dengue hemorrhagic fever (odds ratio = 0.60, PP 95% CI 0.34–1.04) in patients treated with CQ. Differences in levels of T cell activation or pro- or anti-inflammatory plasma cytokine concentrations between CQ- and placebo-treated patients did not explain the trend towards less dengue hemorrhagic fever in the CQ arm. CQ was associated with significantly more adverse events, primarily vomiting.

Conclusions

CQ does not reduce the durations of viraemia and NS1 antigenaemia in dengue patients. Further trials, with appropriate endpoints, would be required to determine if CQ treatment has any clinical benefit in dengue.

Trial Registration

Current Controlled Trials number ISRCTN38002730.     

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

Background

The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.

Methodology/Principal Findings

We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor γ-chain knockout (NOD-scid IL2rγ^null) mice engrafted with human hematopoietic stem cells. Human CD45⁺ cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rγ^null mice with HLA-A2⁺ human cord blood hematopoietic stem cells, were able to secrete IFN-γ, IL-2 and TNF-α in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353_(111–119), NS4b 2423_(181–189), and NS4a 2148_(56–64).

Conclusions/Significance

This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections.     

Dengue infection in children in ratchaburi, Thailand: a cohort study. I. Epidemiology of symptomatic acute dengue infection in children, 2006-2009

Background

There is an urgent need to field test dengue vaccines to determine their role in the control of the disease. Our aims were to study dengue epidemiology and prepare the site for a dengue vaccine efficacy trial.

Methods and Findings

We performed a prospective cohort study of children in primary schools in central Thailand from 2006 through 2009. We assessed the epidemiology of dengue by active fever surveillance for acute febrile illness as detected by school absenteeism and telephone contact of parents, and dengue diagnostic testing. Dengue accounted for 394 (6.74%) of the 5,842 febrile cases identified in 2882, 3104, 2717 and 2312 student person-years over the four years, respectively. Dengue incidence was 1.77% in 2006, 3.58% in 2007, 5.74% in 2008 and 3.29% in 2009. Mean dengue incidence over the 4 years was 3.6%. Dengue virus (DENV) types were determined in 333 (84.5%) of positive specimens; DENV serotype 1 (DENV-1) was the most common (43%), followed by DENV-2 (29%), DENV-3 (20%) and DENV-4 (8%). Disease severity ranged from dengue hemorrhagic fever (DHF) in 42 (10.5%) cases, dengue fever (DF) in 142 (35.5%) cases and undifferentiated fever (UF) in 210 (52.5%) cases. All four DENV serotypes were involved in all disease severity. A majority of cases had secondary DENV infection, 95% in DHF, 88.7% in DF and 81.9% in UF. Two DHF (0.5%) cases had primary DENV-3 infection.

Conclusion

The results illustrate the high incidence of dengue with all four DENV serotypes in primary school children, with approximately 50% of disease manifesting as mild clinical symptoms of UF, not meeting the 1997 WHO criteria for dengue. Severe disease (DHF) occurred in one tenth of cases. Data of this type are required for clinical trials to evaluate the efficacy of dengue vaccines in large scale clinical trials.     

Limited Dengue Virus Replication in Field-Collected Aedes aegypti Mosquitoes Infected with Wolbachia

Introduction

Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus.

Methodology/Principal Findings

Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes.

Conclusions/Significance

Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.     

Autophagy Facilitates Antibody-Enhanced Dengue Virus Infection in Human Pre-Basophil/Mast Cells

Background

Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.

Methodology/Principal Findings

We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4B^C74A mutant.

Conclusions/Significance

Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.     

Invariant NKT Cell Response to Dengue Virus Infection in Human

Background

Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection.

Methods

Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured.

Results

iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated.

Conclusion

iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future.     

Seroepidemiology of Dengue virus in Mayotte, Indian Ocean, 2006

Background

Although Dengue virus (DENV) circulation had been documented in neighbouring South-western Indian Ocean Islands, its presence in Mayotte is poorly characterised. To address this issue, we aimed to assess the seroprevalence of dengue IgG antibodies (DENV-IgG Ab) among the population and to investigate potential associations with individual and household characteristics.

Methods/Principal Findings

In November–December 2006 we conducted a cross-sectional serologic survey in Mayotte among 1,154 inhabitants aged ≥2 years by using a multistage cluster random sampling method. The overall prevalence of DENV-specific IgG antibodies (ELISA) was 22.73% (95% CI, 18.16–27.31). The age-specific seroprevalence increased with age (χ² for trend = 11.86, P<0.0006), and was linked with previous known outbreaks in this region. In multivariate analysis, older age, being born in the Comoros and living in a household with a low socioeconomic index were positively associated with DENV IgG antibody positivity.

Conclusions

These findings document substantial prior exposure of the population of Mayotte to DENV and highlight the risk of severe illness due to the possibility of sequential DENV infections. Further investigations characterizing current DENV circulation patterns and associated serotypes are needed.     

Decreased dengue replication and an increased anti-viral humoral response with the use of combined Toll-like receptor 3 and 7/8 agonists in macaques

Background

Pathogenic versus protective outcomes to Dengue virus (DENV) infection are associated with innate immune function. This study aimed to determine the role of increased TLR3- and TLR7/8-mediated innate signaling after Dengue infection of rhesus macaques in vivo to evaluate its impact on disease and anti-DENV immune responses.

Methodology/Principal Findings

TLR3 and TLR7/8 agonists (emulsified in Montanide) were administered subcutaneously to rhesus macaques at 48 hours and 7 days after DENV infection. The Frequency and activation of myeloid dendritic cells, plasmacytoid dendritic cells, and B cells were measured by flow cytometry while the serum levels of 14 different cytokines and chemokines were quantified. Adaptive immune responses were measured by DENV-specific antibody subtype measurements. Results showed that the combined TLR agonists reduced viral replication and induced the development of a proinflammatory reaction, otherwise absent in Dengue infection alone, without any clear signs of exacerbated disease. Specifically, the TLR-induced response was characterized by activation changes in mDC subsets concurrent with higher serum levels of CXCL-10 and IL-1Ra. TLR stimulation also induced higher titers of anti-DENV antibodies and acted to increase the IgG2/IgG1 ratio of anti-DENV to favor the subtype associated with DENV control. We also observed an effect of DENV-mediated suppression of mDC activation consistent with prior in vitro studies.

Conclusions/Significance

These data show that concurrent TLR3/7/8 activation of the innate immune response after DENV infection in vivo acts to increase antiviral mechanisms via increased inflammatory and humoral responses in rhesus macaques, resulting in decreased viremia and melioration of the infection. These findings underscore an in vivo protective rather than a pathogenic role for combined TLR3/7/8-mediated activation in Dengue infection of rhesus macaques. Our study provides definitive proof-of-concept into the mechanism by which DENV evades immune recognition and activation in vivo.     

Age-specificity of clinical dengue during primary and secondary infections

Background

This study aims to estimate the age-specific risks of clinical dengue attack (i.e., the risk of symptomatic dengue among the total number of dengue virus (DENV) infections) during primary and secondary infections.

Methods

We analyzed two pieces of epidemiological information in Binh Thuan province, southern Vietnam, i.e., age-specific seroprevalence and a community-wide longitudinal study of clinical dengue attack. The latter data set stratified febrile patients with DENV infection by age as well as infection parity. A simple modeling approach was employed to estimate the age-specific risks of clinical dengue attack during primary and secondary infections.

Results

Using the seroprevalence data, the force of infection was estimated to be 11.7% (95% confidence intervals (CI): 10.8–12.7) per year. Median age (and the 25–75 percentiles) of dengue fever patients during primary and secondary infections were 12 (9–20) and 20 (14–31) years, respectively. The estimated age-specific risk of clinical dengue increases as a function of age for both primary and secondary infections; the estimated proportion of symptomatic patients among the total number of infected individuals was estimated to be <7% for those aged <10 years for both primary and secondary infections, but increased as patients become older, reaching to 8–11% by the age of 20 years.

Conclusions/Significance

For both primary and secondary infections, higher age at DENV infection was shown to result in higher risk of clinical attack. Age as an important modulator of clinical dengue explains recent increase in dengue notifications in ageing countries in Southeast Asia, and moreover, poses a paradoxical problem of an increase in adult patients resulting from a decline in the force of infection, which may be caused by various factors including time-dependent variations in epidemiological, ecological and demographic dynamics.     

Dengue infection in children in Ratchaburi, Thailand: a cohort study. II. Clinical manifestations

Background

Dengue infection is one of the most important mosquito-borne diseases. More data regarding the disease burden and the prevalence of each clinical spectrum among symptomatic infections and the clinical manifestations are needed. This study aims to describe the incidence and clinical manifestations of symptomatic dengue infection in Thai children during 2006 through 2008.

Study Design

This study is a school-based prospective open cohort study with a 9,448 person-year follow-up in children aged 3–14 years. Active surveillance for febrile illnesses was done in the studied subjects. Subjects who had febrile illness were asked to visit the study hospital for clinical and laboratory evaluation, treatment, and serological tests for dengue infection. The clinical data from medical records, diary cards, and data collection forms were collected and analyzed.

Results

Dengue infections were the causes of 12.1% of febrile illnesses attending the hospital, including undifferentiated fever (UF) (49.8%), dengue fever (DF) (39.3%) and dengue hemorrhagic fever (DHF) (10.9%). Headache, anorexia, nausea/vomiting and myalgia were common symptoms occurring in more than half of the patients. The more severe dengue spectrum (i.e., DHF) had higher temperature, higher prevalence of nausea/vomiting, abdominal pain, rash, diarrhea, petechiae, hepatomegaly and lower platelet count. DHF cases also had significantly higher prevalence of anorexia, nausea/vomiting and abdominal pain during day 3–6 and diarrhea during day 4–6 of illness. The absence of nausea/vomiting, abdominal pain, diarrhea, petechiae, hepatomegaly and positive tourniquet test may predict non-DHF.

Conclusion

Among symptomatic dengue infection, UF is most common followed by DF and DHF. Some clinical manifestations may be useful to predict the more severe disease (i.e., DHF). This study presents additional information in the clinical spectra of symptomatic dengue infection.     

Evaluation of a Dengue NS1 Antigen Detection Assay Sensitivity and Specificity for the Diagnosis of Acute Dengue Virus Infection

Background

Currently, no dengue NS1 detection kit has regulatory approval for the diagnosis of acute dengue fever. Here we report the sensitivity and specificity of the InBios DEN Detect NS1 ELISA using a panel of well characterized human acute fever serum specimens.

Methodology/Principal Findings

The InBios DENV Detect NS1 ELISA was tested using a panel composed of 334 serum specimens collected from acute febrile patients seeking care in a Bangkok hospital in 2010 and 2011. Of these patients, 314 were found to have acute dengue by either RT-PCR and/or anti-dengue IgM/IgG ELISA. Alongside the InBios NS1 ELISA kit, we compared the performance characteristics of the BioRad Platelia NS1 antigen kit. The InBios NS1 ELISA Ag kit had a higher overall sensitivity (86% vs 72.8%) but equal specificity (100%) compared to the BioRad Platelia kit. The serological status of the patient significantly influenced the outcome. In primary infections, the InBios NS1 kit demonstrated a higher sensitivity (98.8%) than in secondary infections (83.5%). We found significant variation in the sensitivity of the InBios NS1 ELISA kit depending on the serotype of the dengue virus and also found decreasing sensitivity the longer after the onset of illness, showing 100% sensitivity early during illness, but dropping below 50% by Day 7.

Conclusion/Significance

The InBios NS1 ELISA kit demonstrated high accuracy when compared to the initial clinical diagnosis with greater than 85% agreement when patients were clinically diagnosed with dengue illness. Results presented here suggest the accurate detection of circulating dengue NS1 by the InBios DENV Detect NS1 ELISA can provide clinicians with a useful tool for diagnosis of early dengue infections.