Kinetics of severe dengue virus infection and development of gut pathology in mice.

IMPORTANCE: Dengue virus, an arbovirus, causes an estimated 100 million symptomatic infections annually and is an increasing threat as the mosquito range expands with climate change. Dengue epidemics are a substantial strain on local economies and health infrastructure, and an understanding of what drives severe disease may enable treatments to help reduce hospitalizations. Factors exacerbating dengue disease are debated, but gut-related symptoms are much more frequent in severe than mild cases. Using mouse models of dengue infection, we have shown that inflammation and damage are earlier and more severe in the gut than in other tissues. Additionally, we observed impairment of the gut mucus layer and propose that breakdown of the barrier function exacerbates inflammation and promotes severe dengue disease. This idea is supported by recent data from human patients showing elevated bacteria-derived molecules in dengue patient serum. Therapies aiming to maintain gut integrity may help to abrogate severe dengue disease.

Lost in post-translational modification-Dengue virus writes its own sequel.

Elevated frequency of afucosylated IgG1 antibodies during dengue virus infection is associated with prior infection and predicts severe disease.

Clinical signs and symptoms associated with WHO severe dengue classification: a systematic review and meta-analysis.

ABSTRACTThe World Health Organization (WHO) introduced the new dengue classification in 2009. We aimed to assess the association of clinical signs and symptoms with WHO severe dengue classification in clinical practice. A systematic literature search was performed using the databases of PubMed, Embase, and Scopus between 2009 and 2018 according to PRISMA guideline. Meta-analysis was performed with the RevMan software. A random or fixed-effect model was applied to pool odds ratios and 95% confidence intervals of important signs and symptoms across studies. Thirty nine articles from 1790 records were included in this review. In our meta-analysis, signs and symptoms associated with higher risk of severe dengue were comorbidity, vomiting, persistent vomiting, abdominal pain or tenderness, pleural effusion, ascites, epistaxis, gum bleeding, GI bleeding, skin bleeding, lethargy or restlessness, hepatomegaly (>2 cm), increased HCT with decreased platelets, shock, dyspnea, impaired consciousness, thrombocytopenia, elevated AST and ALT, gall bladder wall thickening and secondary infection. This review shows new factors comorbidity, epistaxis, GI and skin bleeding, dyspnea, gall bladder wall thickening and secondary infection may be useful to refine the 2009 classification to triage severe dengue patients.

Exposure to Dengue Envelope Protein Domain III Induces Nlrp3 Inflammasome-Dependent Endothelial Dysfunction and Hemorrhage in Mice.

Typically occurring during secondary dengue virus (DENV) infections, dengue hemorrhagic fever (DHF) causes abnormal immune responses, as well as endothelial vascular dysfunction, for which the responsible viral factor remains unclear. During peak viremia, the plasma levels of virion-associated envelope protein domain III (EIII) increases to a point at which cell death is sufficiently induced in megakaryocytes in vitro. Thus, EIII may constitute a virulence factor for endothelial damage. In this study, we examined endothelial cell death induced by treatment with DENV and EIII in vitro. Notably, pyroptosis, the major type of endothelial cell death observed, was attenuated through treatment with Nlrp3 inflammasome inhibitors. EIII injection effectively induced endothelial abnormalities, and sequential injection of EIII and DENV-NS1 autoantibodies induced further vascular damage, liver dysfunction, thrombocytopenia, and hemorrhage, which are typical manifestations in DHF. Under the same treatments, pathophysiological changes in the Nlrp3 inflammasome-deficient mice were notably reduced compared with those in the wild-type mice. These results suggest that the Nlrp3 inflammasome constitutes a potential therapeutic target for treating DENV-induced hemorrhage in DHF.

Effects of chloroquine or hydroxychloroquine treatment on non-SARS-CoV2 viral infections: A systematic review of clinical studies.

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used as antiviral agents for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. We performed a systematic review to examine whether prior clinical studies that compared the effects of CQ and HCQ to a control for the treatment of non-SARS-CoV2 infection supported the use of these agents in the present SARS-CoV2 outbreak. PubMed, EMBASE, Scopus and Web of Science (PROSPERO CRD42020183429) were searched from inception through 2 April 2020 without language restrictions. Of 1766 retrieved reports, 18 studies met our inclusion criteria, including 17 prospective controlled studies and one retrospective study. CQ or HCQ were compared to control for the treatment of infectious mononucleosis (EBV, n = 4), warts (human papillomavirus, n = 2), chronic HIV infection (n = 6), acute chikungunya infection (n = 1), acute dengue virus infection (n = 2), chronic HCV (n = 2), and as preventive measures for influenza infection (n = 1). Survival was not evaluated in any study. For HIV, the virus that was most investigated, while two early studies suggested HCQ reduced viral levels, four subsequent ones did not, and in two of these CQ or HCQ increased viral levels and reduced CD4 counts. Overall, three studies concluded CQ or HCQ were effective; four concluded further research was needed to assess the treatments' effectiveness; and 11 concluded that treatment was ineffective or potentially harmful. Prior controlled clinical trials with CQ and HCQ for non-SARS-CoV2 viral infections do not support these agents' use for the SARS-CoV2 outbreak.

Dengue epidemic in a non-endemic zone of Bangladesh: Clinical and laboratory profiles of patients.

BACKGROUNDS: Approximately, half of the population in the world including tropical and sub-tropical climates region is at risk of dengue. Being an endemic country, Bangladesh has experienced the largest dengue epidemic in 2019. The present study aimed at evaluating the clinical and laboratory profile of dengue patients in northern Bangladesh during the epidemic. METHODS: This cross-sectional study included 319 serologically confirmed dengue patients admitted in Shaheed Ziaur Rahman Medical College Hospital in Bogra district. It is one of the main tertiary care hospitals in northern Bangladesh. Data were collected from July to September 2019. Patients' clinical and laboratory data were extracted from clinical records. Patients were classified into two classes according to the WHO 2009 dengue classification such as (i) non-severe dengue and (ii) severe dengue. Chi-square test and independent t-test were used in this study. RESULTS: Of the 319 patients, 94.1% had non-severe dengue and the remaining 5.9% had severe dengue (severe plasma leakage 68.4%, severe organ involvement 68.4%, and severe clinical bleeding 10.5%). Most of the patients were suffering from primary dengue infection. The most common clinical presentation was fever followed by headache and myalgia. Vomiting and abdominal pain were the most prevalent warning signs. The common hematological findings on admission were leukopenia (63.3%), thrombocytopenia (30.4%) and increased hematocrit (26.6%). Raised serum ALT or AST was observed in 14.1% cases whereas raised serum creatinine was observed in 6.6% cases. Signs of plasma leakage (pleural effusion, respiratory distress, and ascites, rise of hematocrit >20% during hospital stay) and hepatic or renal involvement (serum ALT >42UI/L or serum creatinine >1.2 mg/dL) on admission were mostly associated with severe dengue. CONCLUSION: The study provides clinical evidence on presentation as well as hematological and biochemical profile of dengue patients in northern Bangladesh that should be implicated in effective patient management.

Understanding immunopathology of severe dengue: lessons learnt from sepsis.

Endothelial dysfunction leading to vascular permeability and plasma leakage are characteristic features of severe dengue and sepsis. However, the mechanisms underlying these immune-pathologies remain unclear. The risk of severe dengue and sepsis development depend on patient-related and pathogen-related factors. Additionally, comorbidities increase the risk of severe disease and their incidence hampers correct diagnosis and treatments. To date, there is no efficient therapy to combat severe dengue and sepsis. Here, we discuss the differences and similarities between the pathogenesis of severe dengue and that of bacterial sepsis. We identify gaps in knowledge that need to be better understood in order to move towards the rational development and/or usage of therapeutic strategies to ameliorate severe dengue disease.

Comparison of dengue case classification schemes and evaluation of biological changes in different dengue clinical patterns in a longitudinal follow-up of hospitalized children in Cambodia.

BACKGROUND: The World Health Organization (WHO) proposed guidelines on dengue clinical classification in 1997 and more recently in 2009 for the clinical management of patients. The WHO 1997 classification defines three categories of dengue infection according to severity: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Alternative WHO 2009 guidelines provide a cross-sectional classification aiming to discriminate dengue fever from dengue with warning signs (DWWS) and severe dengue (SD). The primary objective of this study was to perform a comparison of two dengue classifications. The secondary objective was to describe the changes of hematological and biochemical parameters occurring in patients presenting with different degrees of severity during the course of the disease, since progression to more severe clinical forms is unpredictable. METHODOLOGY/PRINCIPAL FINDINGS: We performed a prospective, monocentric, cross-sectional study of hospitalized children in Cambodia, aged from 2 to 15 years old with severe and non-severe dengue. We enrolled 243 patients with acute dengue-like illness: 71.2% were dengue infections confirmed using quantitative reverse transcription PCR or NS1 antigen capture ELISA, of which 87.2% and 9.0% of DF cases were respectively classified DWWS and SD, and 35.9% of DHF were designated SD using an adapted WHO 2009 classification for SD case definition. Systematic use of ultrasound at patient admission was crucial for detecting plasma leakage. No difference was observed in the concentration of secreted NS1 protein between different dengue severity groups. Lipid profiles were different between DWWS and SD at admission, characterized by a decrease in total cholesterol, HDL cholesterol, and LDL cholesterol, in SD. CONCLUSIONS/SIGNIFICANCE: Our results show discrepancies between the two classifications, including misclassification of severe dengue cases as mild cases by the WHO 1997 classification. Using an adapted WHO 2009 classification, SD more precisely defines the group of patients requiring careful clinical care at a given time during hospitalization.

Correlation of host inflammatory cytokines and immune-related metabolites, but not viral NS1 protein, with disease severity of dengue virus infection.

Severe dengue can be lethal caused by manifestations such as severe bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from patients with different disease severity. Cytokines and metabolites profiles were assessed using a multiplex cytokine assay and liquid chromatography mass spectrometry respectively. The findings showed that NS1 was able to induce the loss of barrier function in microvascular endothelium in a dose dependent manner, however, the level of NS1 in serum samples did not correlate with the extent of vascular leakage induced. Further assessment of host factors revealed that cytokines such as CCL2, CCL5, CCL20 and CXCL1, as well as adhesion molecule ICAM-1, that are involved in leukocytes infiltration were expressed higher in dengue patients in comparison to healthy individuals. In addition, metabolomics study revealed the presence of deregulated metabolites involved in the phospholipid metabolism pathway in patients with severe manifestations. In conclusion, disease severity in dengue virus infection did not correlate directly with NS1 level, but instead with host factors that are involved in the regulation of junctional integrity and phospholipid metabolism. However, as the studied population was relatively small in this study, these exploratory findings should be confirmed by expanding the sample size using an independent cohort to further establish the significance of this study.

Serum chymase levels correlate with severe dengue warning signs and clinical fluid accumulation in hospitalized pediatric patients.

Dengue induces a spectrum of severity in humans from the milder dengue fever to severe disease, or dengue hemorrhagic fever (DHF). Chymase is a candidate biomarker that may aid dengue prognosis. This prospective study aimed to identify whether warning signs of severe dengue, including hypovolemia and fluid accumulation, were associated with elevated chymase. Serum chymase levels were quantified prospectively and longitudinally in hospitalized pediatric dengue patients in Sri Lanka. Warning signs were determined based on daily clinical assessments, laboratory tests and ultrasound findings. Chymase was significantly elevated during the acute phase of disease in DHF or Severe dengue, defined by either the 1997 or 2009 WHO diagnosis guidelines, and persisted longer in the most severe patients. Chymase levels were higher in patients with narrow pulse pressure and clinical warning signs such as severe leakage, fluid accumulation, pleural effusion, gall-bladder wall thickening and rapid haematocrit rise concurrent with thrombocytopenia. No association between chymase and liver enlargement was observed. This study confirms that serum chymase levels are associated with DHF/Severe dengue disease in hospitalized pediatric patients. Chymase levels correlate with warning signs of vascular dysfunction highlighting the possible functional role of chymase in vascular leakage during dengue.

Cytokine Expression in Dengue Fever and Dengue Hemorrhagic Fever Patients with Bleeding and Severe Hepatitis.

Dengue is the most common mosquito-borne flaviviral infection in the world today. Several factors contribute and act synergistically to cause severe infection. One of these is dysregulated host immunological mediators that cause transient pathophysiology during infection. These mediators act on the endothelium to increase vascular permeability, which leads to plasma leakage compromising hemodynamics and coagulopathy. We conducted a prospective study to explore the expression of pro- and anti-inflammatory cytokines and how they relate to clinical dengue manifestations, by assessing their dynamics through acute dengue infection in adults admitted to the Hospital for Tropical Diseases, Bangkok, Thailand. We performed cytokine analysis at three phases of infection for 96 hospitalized adults together with serotyping of confirmed dengue infection during the outbreaks of 2015 and 2016. The serum concentrations of seven cytokines (interleukin [IL]-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha, and interferon gamma) were measured in duplicate using a commercial kit (Bio-Plex Human Cytokine Assay). In this study, the cytokine profile was suggestive of a T-helper 2 response. Most patients had secondary infection, and the levels of viremia were higher in patients with plasma leakage than those without plasma leakage. In addition, we observed that bleeding and hepatitis were associated with significantly higher levels of IL-8 during the early phases of infection. Furthermore, IL-6 levels in the early phase of infection were also elevated in bleeding patients with plasma leakage. These results suggest that IL-6 and IL-8 may act in synergy to cause bleeding in patients with plasma leakage.

Severe dengue categories as research endpoints-Results from a prospective observational study in hospitalised dengue patients.

Severe dengue was perceived as one clinical disease entity until the WHO 2009 classification stratified it into severe vascular leakage, severe bleeding, and severe organ dysfunction. The objectives of this study were to investigate the potential use of severe dengue categories as endpoints for intervention research. 271 patients with severe dengue among 1734 confirmed dengue patients were followed prospectively in this hospital-based observational study in Latin America and Asia. We compared the distribution of severe dengue categories according to gender and age (below/above 15y), and determined the relative frequency and the overlap of severe dengue categories in the same patients. In a next step, we extended the analysis to candidate moderate severity categories, based on recently suggested definitions which were adapted for our purposes. Severe vascular leakage occurred in 244 (90%), severe bleeding in 39 (14%), and severe organ dysfunction in 28 (10%) of 271 severe dengue patients. A higher frequency of severe leakage was seen in children or adolescents (<15y) compared to adults. More than 80% of the severe leakage cases, and 30-50% of the cases with severe bleeding or severe organ dysfunction, were defined as severe on the basis of that feature alone. In 136 out of 213 patients with severe leakage alone, neither moderate bleeding manifestation nor hepatic involvement was recorded. On the other hand, moderate leakage manifestations were detected in 4 out of 12 cases that were classified as severe based on bleeding alone. A major proportion of severe dengue patients exhibited clinical manifestations of severe vascular leakage only, which may constitute a useful endpoint for intervention research or pathophysiology studies. Severe bleeding and severe organ manifestation were recorded less frequently and exhibited a higher degree of overlap with severe leakage. Severe bleeding without leakage may be associated with individual predisposition or the presence of comorbidities. More detailed assessments are needed to explore this hypothesis. Candidate moderate disease endpoints were investigated and need to be further validated.

Molecular Signatures of Dengue Virus-Specific IL-10/IFN-γ Co-producing CD4 T Cells and Their Association with Dengue Disease.

Dengue virus (DENV) can cause diseases ranging from dengue fever (DF) to more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Whether antiviral T cells contribute to the protection against or pathogenesis of severe disease is not well defined. Here, we identified antigen-specific IL-10+IFN-γ+ double-positive (DP) CD4 T cells during acute DENV infection. While the transcriptomic signatures of DP cells partially overlapped with those of cytotoxic and type 1 regulatory CD4 T cells, the majority of them were non-cytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1. Although we observed a higher frequency of DP cells in DHF, the transcriptomic profile of DP cells was similar in DF and DHF, suggesting that DHF is not associated with the altered phenotypic or functional attributes of DP cells. Overall, this study revealed a DENV-specific DP cell subset in patients with acute dengue disease and argues against altered DP cells as a determinant of DHF.

Dynamic changes of soluble ST2 levels predicted fatality and were involved in coagulopathy in dengue fever in the elderly.

BACKGROUND: Severe dengue virus (DENV) infection involves plasma leakage and vascular collapse, and leads to significant morbidity and death. Serum soluble ST2 (sST2 [interleukin (IL)-1 receptor like-1 protein: IL-1-RL-1]) levels are high in pediatric cases of DENV infection, and the disease progresses. However, the correlation between serum sST2 levels and the outcomes of DENV infection in the elderly (≥65 years) is unclear. We thus explored the mechanisms of serial sST2 level changes involved in the coagulopathy and bloodstream infections of elderly patients in Taiwan's 2015 DENV outbreak. METHODS: This retrospective study was done in a tertiary medical center in southern Taiwan during the outbreak. All DENV-infected patients who, between July 1, 2015, and December 31, 2015, provided a written informed consent for at least two blood sample analyses were enrolled and reviewed. The serum levels of sST2 were quantified. ΔsST2 is defined as the "changes of sST2 levels in serially paired samples". Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) analyses were used to evaluate the prognostic ability of ΔsST2. RESULTS: Forty-three patients with DENV infection were enrolled. Mean patient age was 75.0 ± 12.2 years and the case fatality rate was 44.2% (19/43). Significantly more non-survivors than survivors had increased ST2 level (78.9% vs. 12.5%, p < 0.001). The AUC value for serum ΔsST2 level was 0.857 for predicting DENV fatality. Moreover, patients given frozen fresh plasma (FFP) transfusions were significantly (p = 0.025) more likely to have higher serum ST2 level changes than were those who had not. DENV-infected patients with early bloodstream infections (BSIs) seemed to have higher ST2 levels than those who did not have BSIs. CONCLUSIONS: Serum ST2 levels increased in the elderly (≥ 65 years of age) with DENV infection. The changes in serum sST2 levels might be a critical indicator of DENV infection severity for the elderly; sST2 is an important modulator of coagulopathy in severe DENV infections.

Clinical relevance of gallbladder wall thickening for dengue severity: A cross-sectional study.

Dengue fever is the most important arthropod-borne viral infection worldwide. Secondary prevention to reduce mortality through improved clinical case management has substantially lowered the mortality rate for severe dengue during the past two decades. Gallbladder wall thickening (GBWT) is a nonspecific finding often associated with more severe cases of dengue infection. This study had the aim to describe the ultrasonographic findings in hospitalized patients with dengue infection from Manaus (in the Western Brazilian Amazon) and to correlate the GBWT with dengue severity, symptoms and laboratorial analysis. Patients from 13-84 years admitted to the emergency department at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD) were enrolled in this study. Patients' selection occurred during the most recent and huge dengue outbreak within the first semester of 2011. All enrolled subjects were systematically tested in order to rule out other possible etiologies for gallbladder inflammation. Abdominal ultrasound was performed by a single physician through bedside portable equipment and all other clinical and laboratorial information were retrieved from patients' electronic files. 54 subjects were considered for analysis, with confirmed dengue infection by NS1 and/or RT-PCR positivity. From all enrolled patients, 50 (42.4%) presented GBWT. GBWT was significantly and independently related to: age under 31 years, pregnancy, presence of bleeding, presence of any cavitary effusion, DHF classification and severe dengue classifications. During dengue outbreaks, the GBWT identification through a non-invasive and bedside procedure is a confident marker for prompt recognition of potential severe cases.

High titres of IgM-bound circulating immune complexes and erythrocytic oxidative damage are indicators of dengue severity.

Global incidence of dengue has drastically increased in the last few years. Despite the global morbidity and mortality associated with dengue infection, mechanisms of immune control and viral pathogenesis are poorly explored. Pancytopenias, along with increased oxidative stress, are salient clinical findings in severe dengue patients. Previously, we demonstrated significant differences of circulating immune complexes (CICs) among severe and non-severe dengue patients. Accordingly, here we sought to determine the contributory role of affinity-purified antibody-bound CICs in dengue severity. To characterize intracellular oxidative stress induced by antibody-bound CICs, 5-(and-6)-chloromethyl-2'-7'-dichlorodihydrofluorescein diacetate (DCFDA) was measured by flow cytometry. At the same time, CICs sensitized healthy red blood cells (RBC) and patients' RBC morphology was determined by scanning electron microscopy and flow cytometry analysis. Erythrophagocytosis and ferritin levels were further determined in severe and non-severe dengue patients. Our results showed that the severe patients had high titres of immunoglobulin (Ig)M-bound CICs (P < 0·0001) in their sera, increased intracellular oxidative stress (P < 0·0001), high ferritin levels (P < 0·0001), altered morphology of RBC and finally enhanced erythrophagocytosis. This study shows for the first time that RBC morphology is altered in severe dengue patients. Taken together, this study suggests that the enhanced IgM-bound CICs could contribute to the increased oxidative stress and act directly on RBC destruction of severe dengue patients, and is an important pathophysiological determinant. Hence, IgM-bound CICs can serve as an important laboratory parameter to monitor dengue infection progression.

Predictive value of persistent NS1 antigen positivity beyond 3rd day for dengue haemorrhagic fever in Sri Lankan children.

OBJECTIVE: Dengue haemorrhagic fever (DHF) is a major public health concern responsible for significant morbidity in both adult and paediatric populations in Sri Lanka. This study examined if persistent non structural protein 1 (NS1) antigen positivity beyond day 3 was predictive of the occurrence of dengue haemorrhagic fever. The patients were followed up during their in-hospital stay and the severity of the illness was classified according to the WHO classification. The NS1 antigen test was repeated after day 3 of the onset of illness, at least 2 days after the initial test. RESULTS: One hundred and fifty-seven patients were enrolled. Persistent NS1 antigen test positivity after day 3 of the illness was not predictive of subsequent development of DHF. Out of multiple other demographic and illness related factors assessed, only having a secondary dengue infection was associated with a high risk of DHF (relative risk = 3.077, 95% CI 1.361, 6.954). Persistent NS1 positivity on day 3 may not be indicative of disease severity. However results need to be confirmed by a larger study with quantitative NS1 testing.

Association of tumour necrosis factor-α (TNF-α) gene polymorphisms (-308 G>A and -238 G>A) and the risk of severe dengue: A meta-analysis and trial sequential analysis.

Individual studies have assessed the association between TNF-α-308G>A and TNF-α-238 G>A polymorphisms and severity of dengue infection. However, the results are inconclusive and most studies had small sample sizes. The objective of this study was to summarize the evidence of association between TNF-α-308 G>A and TNF-α-238 G>A and severity of dengue infection. This study follows the preferred reporting items for systematic reviews and meta- analyses of genetic association studies, recommended by PLOS One. We calculated pooled odds ratio and its 95% confidence interval (CI) to estimate the association between TNF-α-308 G>A or TNF-α-238 G>A and the risk of severe dengue infections. To determine the information size required for this meta-analysis study, a trial sequential analysis (TSA) was done. Eight studies (640 cases and 1275 controls), which assessed the association of TNF-α-308 G>A or TNF-α-238 G>A and the risk of DHF were included. Overall, we found no significant association between TNF-α-308 G>A and the DHF risk in the allelic model (OR, 0.91; 95% CI, 0.51-1.63), the recessive model (OR,1.32;95%CI,0.73-2.37), the dominant model (OR,0.93;95%CI:0.59-1.47) or the additive model (OR,1.43,95;95%CI:0.79-2.59). There was also no significant association between TNF-α-238 G>A and DHF risk under the allele contrast model (OR:1.51;95%CI:0.88-2.58), the recessive model (OR,1.48,95% CI:0.33-6.58), the dominant model (OR,1.48;95%CI:0.56-3.92), or the additive model (OR:1.5;95%CI:0.34-6.69). On subgroup analysis, neither the Asian population nor the non-Asian population showed significant association between TNF-α-308 G>A/TNF-α-238 G>A and the DHF risk under any genetic models. Leave-one-out meta-analysis showed stability of the results. TSA plots suggested that the sample size in this meta-analysis study was below the required information size. The findings suggest an inclusive evidence of the association between TNF-α-308/ TNF-α-238 G>A and the risk of developing severe dengue infection. Large studies with evidence of Hardy-Weinberg equilibrium, assessing gene-gene interactions are recommended.