Serum Proteome and Cytokine Analysis in a Longitudinal Cohort of Adults with Primary Dengue Infection Reveals Predictive Markers of DHF

Background

Infections caused by dengue virus are a major cause of morbidity and mortality in tropical and subtropical regions of the world. Factors that control transition from mild forms of disease such as dengue fever (DF) to more life-threatening forms such as dengue hemorrhagic fever (DHF) are poorly understood. Consequently, there are no reliable methods currently available for early triage of DHF patients resulting in significant over-hospitalization.

Methodology/Principal Findings

We have systematically examined the proteome, cytokines and inflammatory markers in sera from 62 adult dengue patients (44 DF; 18 DHF) with primary DENV infection, at three different times of infection representing the early febrile, defervescence and convalescent stages. Using fluorescent bioplex assays, we measured 27 cytokines in these serum samples. Additionally, we used multiple mass spectrometry methods for iTRAQ-based comparative analysis of serum proteome as well as measurements of protein adducts- 3-nitrotyrosine and 3-chlorotyrosine as surrogate measures of free radical activity. Using multiple methods such as OPLS, MRMR and MSVM-RFE for multivariate feature selection and classification, we report molecular markers that allow prediction of primary DHF with sensitivity and specificity of >80%.

Conclusions/Significance

This report constitutes a comprehensive analysis of molecular signatures of dengue disease progression and will help unravel mechanisms of dengue disease progression. Our analysis resulted in the identification of markers that may be useful for early prediction of DHF during the febrile phase. The combination of highly sensitive analytical methods and novel statistical approaches described here forms a robust platform for biomarker discovery.     

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.     

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.     

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.     

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.     

An outbreak of dengue fever in St. Croix (US Virgin Islands), 2005

Background

Periodic outbreaks of dengue fever occur in the United States Virgin Islands. In June 2005, an outbreak of dengue virus (DENV) serotype-2 with cases of dengue hemorrhagic fever (DHF) was detected in St. Croix, US Virgin Islands. The objective of this report is to describe this outbreak of DENV-2 and the findings of a case-control study examining risk factors for DHF.

Methodology/Principal Findings

This is the largest dengue outbreak ever recorded in St. Croix, with 331 suspected dengue cases reported island-wide during 2005 (62.2 cases/10,000 population); 54% were hospitalized, 21% had at least one hemorrhagic manifestation, 28% had thrombocytopenia, 5% had DHF and 1 patient died. Eighty-nine laboratory-positive hospitalized patients were identified. Of these, there were 15 (17%) who met the WHO criteria for DHF (cases) and 74 (83%) who did not (controls). The only variable significantly associated with DHF on bivariate or multivariable analysis was age, with an adjusted odds ratio (95% confidence interval) of 1.033 (1.003,1.064).

Conclusions/Significance

During this outbreak of DENV-2, a high proportion of cases developed DHF and increasing age was significantly associated with DHF.     

Arterial Hypertension and Skin Allergy Are Risk Factors for Progression from Dengue to Dengue Hemorrhagic Fever: A Case Control Study

Background

Currently, knowledge does not allow early prediction of which cases of dengue fever (DF) will progress to dengue hemorrhagic fever (DHF), to allow early intervention to prevent progression or to limit severity. The objective of this study is to investigate the hypothesis that some specific comorbidities increase the likelihood of a DF case progressing to DHF.

Methods

A concurrent case-control study, conducted during dengue epidemics, from 2009 to 2012. Cases were patients with dengue fever that progressed to DHF, and controls were patients of dengue fever who did not progress to DHF. Logistic regression was used to estimate the association between DHF and comorbidities.

Results

There were 490 cases of DHF and 1,316 controls. Among adults, progression to DHF was associated with self-reported hypertension (OR = 1.6; 95% CI 1.1-2.1) and skin allergy (OR = 1.8; 95% CI 1.1-3.2) with DHF after adjusting for ethnicity and socio-economic variables. There was no statistically significant association between any chronic disease and progression to DHF in those younger than 15 years.

Conclusions

Physicians attending patients with dengue fever should keep those with hypertension or skin allergies in health units to monitor progression for early intervention. This would reduce mortality by dengue.     

Predictive Value of Proteinuria in Adult Dengue Severity

Background

Dengue is an important viral infection with different presentations. Predicting disease severity is important in triaging patients requiring hospital care. We aim to study the value of proteinuria in predicting the development of dengue hemorrhagic fever (DHF), utility of urine dipstick test as a rapid prognostic tool.

Methodology and principal findings

Adult patients with undifferentiated fever (n = 293) were prospectively enrolled at the Infectious Disease Research Clinic at Tan Tock Seng Hospital, Singapore from January to August 2012. Dengue infection was confirmed in 168 (57%) by dengue RT-PCR or NS1 antigen detection. Dengue cases had median fever duration of 6 days at enrolment. DHF was diagnosed in 34 cases according to the WHO 1997 guideline. Dengue fever (DF) patients were predominantly younger and were mostly seen in the outpatient setting with higher platelet level. Compared to DF, DHF cases had significantly higher peak urine protein creatinine ratio (UPCR) during clinical course (26 vs. 40 mg/mmol; p<0.001). We obtained a UPCR cut-off value of 29 mg/mmol based on maximum AUC in ROC curves of peak UPCR for DF versus DHF, corresponding to 76% sensitivity and 60% specificity. Multivariate analysis with other readily available clinical and laboratory variables increased the AUC to 0.91 with 92% sensitivity and 80% specificity. Neither urine dipstick at initial presentation nor peak urine dipstick value during the entire illness was able to discriminate between DF and DHF.

Conclusions

Proteinuria measured by a laboratory-based UPCR test may be sensitive and specific in prognosticating adult dengue patients.     

Differential Expression of Toll-like Receptors in Dendritic Cells of Patients with Dengue during Early and Late Acute Phases of the Disease

Background

Dengue hemorrhagic fever (DHF) is observed in individuals that have pre-existing heterotypic dengue antibodies and is associated with increased viral load and high levels of pro-inflammatory cytokines early in infection. Interestingly, a recent study showed that dengue virus infection in the presence of antibodies resulted in poor stimulation of Toll-like receptors (TLRs), thereby facilitating virus particle production, and also suggesting that TLRs may contribute to disease pathogenesis.

Methodology/Principal Findings

We evaluated the expression levels of TLR2, 3, 4 and 9 and the co-stimulatory molecules CD80 and CD86 by flow cytometry. This was evaluated in monocytes, in myeloid and plasmacytoid dendritic cells (mDCs and pDCs) from 30 dengue patients with different clinical outcomes and in 20 healthy controls. Increased expression of TLR3 and TLR9 in DCs of patients with dengue fever (DF) early in infection was detected. In DCs from patients with severe manifestations, poor stimulation of TLR3 and TLR9 was observed. In addition, we found a lower expression of TLR2 in patients with DF compared to DHF. Expression levels of TLR4 were not affected. Furthermore, the expression of CD80 and CD86 was altered in mDCs and CD86 in pDCs of severe dengue cases. We show that interferon alpha production decreased in the presence of dengue virus after stimulation of PBMCs with the TLR9 agonist (CpG A). This suggests that the virus can affect the interferon response through this signaling pathway.

Conclusions/Significance

These results show that during dengue disease progression, the expression profile of TLRs changes depending on the severity of the disease. Changes in TLRs expression could play a central role in DC activation, thereby influencing the innate immune response.     

Metabolomics-Based Discovery of Small Molecule Biomarkers in Serum Associated with Dengue Virus Infections and Disease Outcomes

Background

Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and clinical care of dengue patients throughout the tropical and subtropical world. The ability to predict severe dengue disease outcomes (DHF/DSS) using acute phase clinical specimens would be of enormous value to physicians and health care workers for appropriate triaging of patients for clinical management. Advances in the field of metabolomics and analytic software provide new opportunities to identify host small molecule biomarkers (SMBs) in acute phase clinical specimens that differentiate dengue disease outcomes.

Methodology/Principal Findings

Exploratory metabolomic studies were conducted to characterize the serum metabolome of patients who experienced different dengue disease outcomes. Serum samples from dengue patients from Nicaragua and Mexico were retrospectively obtained, and hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) identified small molecule metabolites that were associated with and statistically differentiated DHF/DSS, DF, and non-dengue (ND) diagnosis groups. In the Nicaraguan samples, 191 metabolites differentiated DF from ND outcomes and 83 differentiated DHF/DSS and DF outcomes. In the Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes. The structural identities of 13 metabolites were confirmed using tandem mass spectrometry (MS/MS). Metabolomic analysis of serum samples from patients diagnosed as DF who progressed to DHF/DSS identified 65 metabolites that predicted dengue disease outcomes. Differential perturbation of the serum metabolome was demonstrated following infection with different DENV serotypes and following primary and secondary DENV infections.

Conclusions/Significance

These results provide proof-of-concept that a metabolomics approach can be used to identify metabolites or SMBs in serum specimens that are associated with distinct DENV infections and disease outcomes. The differentiating metabolites also provide insights into metabolic pathways and pathogenic and immunologic mechanisms associated with dengue disease severity.     

HLA class I and class II associations in dengue viral infections in a Sri Lankan population

Background

HLA class I and class II alleles have been shown to be associated with the development of dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) in different populations. However, the majority of studies have been based on limited numbers of patients. In this study we aimed to investigate the HLA-class I and class II alleles that are positively and negatively associated with the development of DSS in a cohort of patients with DHF and also the alleles associated with development of DHF during primary dengue infections in a Sri Lankan population.

Methodology/Principal Findings

The allele frequencies of HLA class I and class II alleles were compared in 110 patients with DHF and 119 individuals from the population who had never reported a symptomatic dengue infection at the time of recruitment. We found that HLA-A*31 (corrected P = 0.01) and DRB1*08 (corrected P = 0.009) were associated with susceptibility to DSS when infected with the dengue virus, during secondary dengue infection. The frequency of DRB1*08 allele was 28.7 times higher than in the normal population in patients with DSS. HLA-A*31 allele was increased 16.6 fold in DHF who developed shock when compared to those who did not develop shock. A*24 (corrected P = 0.03) and DRB1*12 (corrected P = 0.041) were strongly associated with the development of DHF during primary dengue infection.

Conclusions/Significance

These data suggest that certain HLA alleles confer susceptibility/protection to severe dengue infections. As T cell epitope recognition depend on the HLA type of an individual, it would be now important to investigate how epitope specific T cells associate with primary and secondary dengue infections and in severe dengue infections.     

Classification of Dengue Fever Patients Based on Gene Expression Data Using Support Vector Machines

Background

Symptomatic infection by dengue virus (DENV) can range from dengue fever (DF) to dengue haemorrhagic fever (DHF), however, the determinants of DF or DHF progression are not completely understood. It is hypothesised that host innate immune response factors are involved in modulating the disease outcome and the expression levels of genes involved in this response could be used as early prognostic markers for disease severity.

Methodology/Principal Findings

mRNA expression levels of genes involved in DENV innate immune responses were measured using quantitative real time PCR (qPCR). Here, we present a novel application of the support vector machines (SVM) algorithm to analyze the expression pattern of 12 genes in peripheral blood mononuclear cells (PBMCs) of 28 dengue patients (13 DHF and 15 DF) during acute viral infection. The SVM model was trained using gene expression data of these genes and achieved the highest accuracy of ∼85% with leave-one-out cross-validation. Through selective removal of gene expression data from the SVM model, we have identified seven genes (MYD88, TLR7, TLR3, MDA5, IRF3, IFN-α and CLEC5A) that may be central in differentiating DF patients from DHF, with MYD88 and TLR7 observed to be the most important. Though the individual removal of expression data of five other genes had no impact on the overall accuracy, a significant combined role was observed when the SVM model of the two main genes (MYD88 and TLR7) was re-trained to include the five genes, increasing the overall accuracy to ∼96%.

Conclusions/Significance

Here, we present a novel use of the SVM algorithm to classify DF and DHF patients, as well as to elucidate the significance of the various genes involved. It was observed that seven genes are critical in classifying DF and DHF patients: TLR3, MDA5, IRF3, IFN-α, CLEC5A, and the two most important MYD88 and TLR7. While these preliminary results are promising, further experimental investigation is necessary to validate their specific roles in dengue disease.     

Clinical relevance and discriminatory value of elevated liver aminotransferase levels for dengue severity

Background

Elevation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is prominent in acute dengue illness. The World Health Organization (WHO) 2009 dengue guidelines defined AST or ALT≥1000 units/liter (U/L) as a criterion for severe dengue. We aimed to assess the clinical relevance and discriminatory value of AST or ALT for dengue hemorrhagic fever (DHF) and severe dengue.

Methodology/Principal Findings

We retrospectively studied and classified polymerase chain reaction positive dengue patients from 2006 to 2008 treated at Tan Tock Seng Hospital, Singapore according to WHO 1997 and 2009 criteria for dengue severity. Of 690 dengue patients, 31% had DHF and 24% severe dengue. Elevated AST and ALT occurred in 86% and 46%, respectively. Seven had AST or ALT≥1000 U/L. None had acute liver failure but one patient died. Median AST and ALT values were significantly higher with increasing dengue severity by both WHO 1997 and 2009 criteria. However, they were poorly discriminatory between non-severe and severe dengue (e.g., AST area under the receiver operating characteristic [ROC] curve = 0.62; 95% confidence interval [CI]: 0.57–0.67) and between dengue fever (DF) and DHF (AST area under the ROC curve = 0.56; 95% CI: 0.52–0.61). There was significant overlap in AST and ALT values among patients with dengue with or without warning signs and severe dengue, and between those with DF and DHF.

Conclusions

Although aminotransferase levels increased in conjunction with dengue severity, AST or ALT values did not discriminate between DF and DHF or non-severe and severe dengue.     

DC-SIGN (CD209) Promoter -336 A/G polymorphism is associated with dengue hemorrhagic fever and correlated to DC-SIGN expression and immune augmentation

Background

The C-type lectin DC-SIGN (CD209) is known to be the major dengue receptor on human dendritic cells, and a single nucleotide polymorphism (SNP) in the promoter region of CD209 (−336 A/G; rs4804803) is susceptible to many infectious diseases. We reason that variations in the DC-SIGN gene might have a broad influence on viral replication and host immune responses.

Methods and Findings

We studied whether the rs4804803 SNP was associated with a susceptibility to dengue fever (DF) and/or dengue hemorrhagic fever (DHF) through genotyping analysis in a Taiwanese cohort. We generated monocyte-derived dendritic cells (MDDCs) from individuals with AA or AG genotype of rs4804803 to study the viral replication and immune responses for functional validation. A total of 574 DNA samples were genotyped, including 176 DF, 135 DHF, 143 other non-dengue febrile illnesses (OFI) and 120 population controls. A strong association between GG/AG genotypes of rs4804803 and risk of DHF was found when compared among DF, OFI and controls (p = 0.004, 3×10⁻⁵ and 0.001, respectively). The AA genotype was associated with protection against dengue infection compared with OFI and controls (p = 0.002 and 0.020, respectively). Moreover, MDDCs from individuals with AG genotype with a higher cell surface DC-SIGN expression had a significantly higher TNFα, IL-12p40, and IP-10 production than those with AA genotype in response to dengue infection. However, the viral replication in MDDCs with AG genotype was significantly lower than those with AA genotype. With both genotypes, MDDCs revealed an increase in viral replication following the addition of anti-IP-10 neutralizing antibody.

Conclusions/Significance

The rs4804803 SNP in the CD209 promoter contributed to susceptibility to dengue infection and complication of DHF. This SNP with AG genotype affects the cell surface DC-SIGN expression related to immune augmentation and less viral replication.     

Serum Metabolome and Lipidome Changes in Adult Patients with Primary Dengue Infection

Background

Dengue virus (DENV) is the most widespread arbovirus with an estimated 100 million infections occurring every year. Endemic in the tropical and subtropical areas of the world, dengue fever/dengue hemorrhagic fever (DF/DHF) is emerging as a major public health concern. The complex array of concurrent host physiologic changes has hampered a complete understanding of underlying molecular mechanisms of dengue pathogenesis.

Methodology/Principle Findings

Systems level characterization of serum metabolome and lipidome of adult DF patients at early febrile, defervescence, and convalescent stages of DENV infection was performed using liquid chromatography- and gas chromatography-mass spectrometry. The tractability of following metabolite and lipid changes in a relatively large sample size (n = 44) across three prominent infection stages allowed the identification of critical physiologic changes that coincided with the different stages. Sixty differential metabolites were identified in our metabolomics analysis and the main metabolite classes were free fatty acids, acylcarnitines, phospholipids, and amino acids. Major perturbed metabolic pathways included fatty acid biosynthesis and β-oxidation, phospholipid catabolism, steroid hormone pathway, etc., suggesting the multifactorial nature of human host responses. Analysis of phospholipids and sphingolipids verified the temporal trends and revealed association with lymphocytes and platelets numbers. These metabolites were significantly perturbed during the early stages, and normalized to control levels at convalescent stage, suggesting their potential utility as prognostic markers.

Conclusions/Significance

DENV infection causes temporally distinct serum metabolome and lipidome changes, and many of the differential metabolites are involved in acute inflammatory responses. Our global analyses revealed early anti-inflammatory responses working in concert to modulate early pro-inflammatory processes, thus preventing the host from development of pathologies by excessive or prolonged inflammation. This study is the first example of how an omic- approach can divulge the extensive, concurrent, and dynamic host responses elicited by DENV and offers plausible physiological insights to why DF is self limiting.     

Decision tree algorithms predict the diagnosis and outcome of dengue fever in the early phase of illness

Background

Dengue is re-emerging throughout the tropical world, causing frequent recurrent epidemics. The initial clinical manifestation of dengue often is confused with other febrile states confounding both clinical management and disease surveillance. Evidence-based triage strategies that identify individuals likely to be in the early stages of dengue illness can direct patient stratification for clinical investigations, management, and virological surveillance. Here we report the identification of algorithms that differentiate dengue from other febrile illnesses in the primary care setting and predict severe disease in adults.

Methods and Findings

A total of 1,200 patients presenting in the first 72 hours of acute febrile illness were recruited and followed up for up to a 4-week period prospectively; 1,012 of these were recruited from Singapore and 188 from Vietnam. Of these, 364 were dengue RT-PCR positive; 173 had dengue fever, 171 had dengue hemorrhagic fever, and 20 had dengue shock syndrome as final diagnosis. Using a C4.5 decision tree classifier for analysis of all clinical, haematological, and virological data, we obtained a diagnostic algorithm that differentiates dengue from non-dengue febrile illness with an accuracy of 84.7%. The algorithm can be used differently in different disease prevalence to yield clinically useful positive and negative predictive values. Furthermore, an algorithm using platelet count, crossover threshold value of a real-time RT-PCR for dengue viral RNA, and presence of pre-existing anti-dengue IgG antibodies in sequential order identified cases with sensitivity and specificity of 78.2% and 80.2%, respectively, that eventually developed thrombocytopenia of 50,000 platelet/mm³ or less, a level previously shown to be associated with haemorrhage and shock in adults with dengue fever.

Conclusion

This study shows a proof-of-concept that decision algorithms using simple clinical and haematological parameters can predict diagnosis and prognosis of dengue disease, a finding that could prove useful in disease management and surveillance.     

Sphingosine 1-Phosphate in Acute Dengue Infection

Background

Vascular leak is the hallmark of severe dengue infections and leads to complications such as shock and multi-organ failure. Although many mediators have been implicated in the vascular leak in dengue, the role of sphingosine 1-phosphate (S1P) has not been investigated.

Metholodology/Principal findings

As S1P has been shown to be important in barrier integrity, we assessed the S1P levels in 28 patients with acute dengue and 12 healthy individuals. The S1P levels were significantly lower in patients with acute dengue (p = 0.002) and the levels in patients with grade IV dengue haemorrhagic fever (DHF) were significantly lower than those with dengue fever (p = 0.005). We then investigated the kinetics of S1P levels throughout the course of the illness in another 32 patients in serum samples obtained twice a day. We found that S1P levels were low throughout the course of illness and S1P levels were <0.5 µM in 12/23 patients with DHF when compared to 1/9 with DF.

Conclusions/Significance

As S1P has shown to be important in the endothelial barrier integrity and increases transendothelial resistance, low levels of S1P in acute dengue infection are likely to contribute to increased vascular permeability.     

Host gene expression profiling of dengue virus infection in cell lines and patients

Background

Despite the seriousness of dengue-related disease, with an estimated 50–100 million cases of dengue fever and 250,000–500,000 cases of dengue hemorrhagic fever/dengue shock syndrome each year, a clear understanding of dengue pathogenesis remains elusive. Because of the lack of a disease model in animals and the complex immune interaction in dengue infection, the study of host response and immunopathogenesis is difficult. The development of genomics technology, microarray and high throughput quantitative PCR have allowed researchers to study gene expression changes on a much broader scale. We therefore used this approach to investigate the host response in dengue virus-infected cell lines and in patients developing dengue fever.

Methodology/Principal Findings

Using microarray and high throughput quantitative PCR method to monitor the host response to dengue viral replication in cell line infection models and in dengue patient blood samples, we identified differentially expressed genes along three major pathways; NF-κB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway. Among the most highly upregulated genes were the chemokines IP-10 and I-TAC, both ligands of the CXCR3 receptor. Increased expression of IP-10 and I-TAC in the peripheral blood of ten patients at the early onset of fever was confirmed by ELISA. A highly upregulated gene in the IFN pathway, viperin, was overexpressed in A549 cells resulting in a significant reduction in viral replication. The upregulation of genes in the ubiquitin-proteasome pathway prompted the testing of proteasome inhibitors MG-132 and ALLN, both of which reduced viral replication.

Conclusion/Significance

Unbiased gene expression analysis has identified new host genes associated with dengue infection, which we have validated in functional studies. We showed that some parts of the host response can be used as potential biomarkers for the disease while others can be used to control dengue viral replication, thus representing viable targets for drug therapy.