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Background:  Dengue virus infection typically causes mild dengue fever, but, in severe cases, life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) occur. The pathophysiological hallmark of DHF and DSS is plasma leakage that leads to enhanced vascular permeability, likely due to a cytokine storm., Methods:  Ninety patients with dengue during 2010-2012 in Singapore were prospectively recruited and stratified according to their disease phase, primary and secondary infection status, and disease severity, measured by plasma leakage. Clinical parameters were recorded throughout the disease progression. The levels of various immune mediators were quantified using comprehensive multiplex microbead-based immunoassays for 46 immune mediators., Results:  Associations between clinical parameters and immune mediators were analyzed using various statistical methods. Potential immune markers, including interleukin 1 receptor antagonist, interferon γ-inducible protein 10, hepatocyte growth factor, soluble p75 tumor necrosis factor α receptor, vascular cell adhesion molecule 1, and matrix metalloproteinase 2, were significantly associated with significant plasma leakage. Secondary dengue virus infections were also shown to influence disease outcome in terms of disease severity., Conclusions:  This study identified several key markers for exacerbated dengue pathogenesis, notably plasma leakage. This will allow a better understanding of the molecular mechanisms of DHF and DSS in patients with dengue., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Globally, dengue virus (DENV) is one of the most widespread vector-borne viruses. Dengue disease affects populations in endemic areas and, increasingly, tourists who travel to these countries, but there is currently no approved vaccine for dengue. A phase 3 efficacy trial with Sanofi-Pasteur's recombinant, live-attenuated, tetravalent dengue vaccine (CYD-TDV) conducted in South East Asia showed an overall efficacy of 56% against virologically confirmed dengue infections of any severity and any of the 4 serotypes, but the long-term protection of the vaccine has yet to be demonstrated. To address longevity of antibody titers and B cell memory, we recalled study participants from an earlier CYD immunogenicity study (Phase 2) conducted in Singapore that enrolled healthy volunteers in the year 2009. Depending on the age group, 57-84% of the participants initially generated a neutralizing antibody titer ≥ 10 to all 4 DENV serotypes 28 d after the third and final dose. We observed very low antibody titers in blood samples collected from 23 vaccinees 5 y after the first dose, particularly titers of antibodies binding to virus particles compared with those binding to recombinant E protein. The in vivo efficacy of plasma antibodies against DENV-2 challenge was also tested in a mouse model, which found that only 2 out of 23 samples were able to reduce viremia. Although the sample size is too small for general conclusions, dengue immune memory after vaccination with CYD-TDV appears relatively low.

Studies on serotype-specific features of dengue and disease severity on adults are limited. We prospectively recruited adult febrile patients without alternate diagnosis to dengue from April 2005 to December 2011. Outcomes were defined using both the World Health Organization (WHO) 1997 and 2009 criteria; Dengue hemorrhagic fever (DHF) and severe dengue (SD). Infecting serotype was identified in 469 dengue-confirmed patients comprising 22.0% dengue virus serotype 1 (DENV-1), 57.1% DENV-2, 17.1% DENV-3, and 3.8% DENV-4. Cases infected with DENV-1 were more likely to present with red eyes whereas presence of joint pain and lower platelet count was associated with DENV-2 cases. After adjusting for potential confounders, DENV-1 was associated with both DHF (adjusted Relative Risk [aRR] = 1.74) and SD (aRR = 2.1) whereas DENV-2 had a lower risk of DHF (aRR = 0.5). DENV-1 genotype 1 and DENV-2 cosmopolitan were the predominant genotypes identified. Infecting dengue serotype and possibly genotype may play an important role in disease severity among adult dengue patients in Singapore., (© The American Society of Tropical Medicine and Hygiene.)

Dengue, which is the most prevalent mosquito-borne viral disease afflicting human populations, causes a spectrum of clinical symptoms that include fever, muscle and joint pain, maculopapular skin rash, and hemorrhagic manifestations. Patients infected with dengue develop a broad antigen-specific T lymphocyte response, but the phenotype and functional properties of these cells are only partially understood. We show that natural infection induces dengue-specific CD8(+) T lymphocytes that are highly activated and proliferating, exhibit antiviral effector functions, and express CXCR3, CCR5, and the skin-homing marker cutaneous lymphocyte-associated antigen (CLA). In the same patients, bystander human cytomegalovirus -specific CD8(+) T cells are also activated during acute dengue infection but do not express the same tissue-homing phenotype. We show that CLA expression by circulating dengue-specific CD4(+) and CD8(+) T cells correlates with their in vivo ability to traffic to the skin during dengue infection. The juxtaposition of dengue-specific T cells with virus-permissive cell types at sites of possible dengue exposure represents a previously uncharacterized form of immune surveillance for this virus. These findings suggest that vaccination strategies may need to induce dengue-specific T cells with similar homing properties to provide durable protection against dengue viruses., (Copyright © 2015, American Association for the Advancement of Science.)

WHO recommendations for dengue diagnosis require laboratory facilities. Antibody-based rapid diagnostic tests (RDTs) have performed poorly, and clinical diagnosis remains the mainstay in dengue-endemic countries. We evaluated a combination antigen-antibody RDT for point-of-care testing in a high-prevalence setting. In this prospective cohort study, adults were enrolled from a tertiary infectious disease centre for evaluation of undifferentiated febrile illness from October 2011 to May 2012. SD Bioline Dengue Duo was evaluated at point-of-care against a WHO-based reference standard of viral isolation, RT-PCR, NS1-, IgM-, and IgG-ELISA. 246 adults were enrolled (median age 34 years, range 18-69), of which 197 could be confirmed definitively as either dengue or non-dengue. DENV-2 was the predominant serotype (79.5%) and the ratio of primary to secondary cases was 1∶1.1. There were no test failures and minimal interobserver variation with a Fleiss' kappa of 0.983 (95% CI 0.827-1.00). Overall sensitivity and specificity were 93.9% (95% CI 88.8-96.8%) and 92.0% (95% CI 81.2-96.9%) respectively. Using WHO clinical criteria alone for diagnosis had similar sensitivities (95.9%, 95% CI 91.4-98.1%) and lower specificities (20.0%, 95% CI 11.2-33.0%). No significant difference in performance was found when testing early versus late presenters, primary versus secondary cases, or DENV-1 versus DENV-2 infections. The use of a combination RDT fulfills WHO ASSURED criteria for point-of-care testing and can enhance dengue diagnosis in an endemic setting. This has the potential to markedly improve clinical management of dengue in the field.

The authors report two cases of complicated dengue viral infection with acute myocarditis involving young male adults, of which one was fatal. The first case presented with typical signs of myocardial disease: chest pain and diaphoresis with myocardial depression in the electrocardiograph. The second case deteriorated rapidly and demised within the first day of admission. Histology of the heart muscles showed multiple small foci of myocyte necrosis surrounded by lymphocytes, in keeping with viral myocarditis. Both cases fulfilled the World Health Organization (WHO) diagnosis of probable dengue: the first case had positive dengue serology, both IgM and IgG at day six of illness, and the second case was polymerase chain reaction (PCR) positive for dengue and identified as serotype 2. Despite the severe outcome, both cases did not completely fulfil the criteria for dengue haemorrhagic fever (DHF). Although severe cardiac impairment is not commonly reported in dengue infection, it can be life threatening.

Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4(+) and CD8(+) T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4(+) and 21 are CD8(+) T cell epitopes. We observe that whereas CD8(+) T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4(+) epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4(+) T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4(+) and CD8(+) T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.