Challenges in Dengue Fever in the Elderly: Atypical Presentation and Risk of Severe Dengue and Hospita-Acquired Infection

Background/methods

To better understand dengue fever in the elderly, we compared clinical features, World Health Organization (WHO) dengue classification and outcomes between adult (<60) and elderly (≥60) dengue patients. We explored the impact of co-morbidity and hospital-acquired infection (HAI) on clinical outcomes in the elderly. All patients managed at the Communicable Disease Centre, Singapore, between 2005 and 2008 with positive dengue polymerase chain reaction (PCR) or who fulfilled WHO 1997 or 2009 probable dengue criteria with positive dengue IgM were included.

Results

Of the 6989 cases, 295 (4.4%) were elderly. PCR was positive in 29%. The elderly suffered more severe disease with more dengue haemorrhagic fever (DHF) (29.2% vs. 21.4%) and severe dengue (SD) (20.3% vs. 14.6%) (p<0.05). Classic dengue symptoms were more common in the adult group. The elderly were less likely to fulfill WHO 1997 (93.6% vs. 96.4%) (p = 0.014), but not WHO 2009 probable dengue (75.3% vs. 71.5%). Time to dengue diagnosis was similar. There was no significant difference in the frequency of warning signs between the two groups, but the elderly were more likely to have hepatomegaly (p = 0.006) and malaise/lethargy (p = 0.033) while the adults had significantly more mucosal bleeding (p<0.001). Intensive care admission occurred in 15 and death in three, with no age difference. Notably, the elderly stayed in hospital longer (median 5 vs. 4 days), and suffered more pneumonia (3.8% vs. 0.7%) and urinary infection (1.9% vs. 0.3%) (p = 0.003). Predictors of excess length of stay were age (adjusted odds ratio [aOR] 2.01, 95% confidence interval [CI] 1.37–2.88), critical illness (aOR 5.13, 95%CI 2.59–9.75), HAI (aOR 12.06, 95%CI 7.39–19.9), Charlson score (aOR 6.9, 95%CI 2.02–22.56) and severe dengue (DHF/dengue shock syndrome/SD) (aOR 2.24, 95%CI 1.83–2.74).

Conclusion

Elderly dengue patients present atypically and are at higher risk of DHF, SD and HAI. Aside from dengue severity, age, co-morbidity and HAI were associated with longer hospital stay.     

Long-Term Arthralgia after Mayaro Virus Infection Correlates with Sustained Pro-inflammatory Cytokine Response

Mayaro virus (MAYV), an alphavirus similar to chikungunya virus (CHIKV), causes an acute debilitating disease which results in the development of long-term arthralgia in more than 50% of infected individuals. Currently, the immune response and its role in the development of MAYV-induced persistent arthralgia remain unknown. In this study, we evaluated the immune response of individuals with confirmed MAYV infection in a one-year longitudinal study carried out in Loreto, Peru. We report that MAYV infection elicits robust immune responses that result in the development of a strong neutralizing antibody response and the secretion of pro-inflammatory immune mediators. The composition of these inflammatory mediators, in some cases, differed to those previously observed for CHIKV. Key mediators such as IL-13, IL-7 and VEGF were strongly induced following MAYV infection and were significantly increased in subjects that eventually developed persistent arthralgia. Although a strong neutralizing antibody response was observed in all subjects, it was not sufficient to prevent the long-term outcomes of MAYV infection. This study provides initial immunologic insight that may eventually contribute to prognostic tools and therapeutic treatments against this emerging pathogen.     

A Non Mouse-Adapted Dengue Virus Strain as a New Model of Severe Dengue Infection in AG129 Mice

The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-α/β and -γ receptors) upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing.     

Suppressor of Cytokine Signaling 4 (SOCS4) Protects against Severe Cytokine Storm and Enhances Viral Clearance during Influenza Infection

Suppressor of cytokine signaling (SOCS) proteins are key regulators of innate and adaptive immunity. There is no described biological role for SOCS4, despite broad expression in the hematopoietic system. We demonstrate that mice lacking functional SOCS4 protein rapidly succumb to infection with a pathogenic H1N1 influenza virus (PR8) and are hypersusceptible to infection with the less virulent H3N2 (X31) strain. In SOCS4-deficient animals, this led to substantially greater weight loss, dysregulated pro-inflammatory cytokine and chemokine production in the lungs and delayed viral clearance. This was associated with impaired trafficking of influenza-specific CD8 T cells to the site of infection and linked to defects in T cell receptor activation. These results demonstrate that SOCS4 is a critical regulator of anti-viral immunity.     

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.     

Targeted Delivery of Small Interfering RNA to Human Dendritic Cells To Suppress Dengue Virus Infection and Associated Proinflammatory Cytokine Production

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-d-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34⁺ hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE^D) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-α), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rγ^−/− mouse model engrafted with human CD34⁺ hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-α effectively suppressed poly(I:C)-induced TNF-α production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.Dengue is a mosquito-borne flavivirus infection that has emerged as a serious public health problem worldwide. Four serotypes of dengue virus (DEN-1 to DEN-4) are capable of causing human disease varying in severity from acute self-limiting febrile illness to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The plasma leakage, hemorrhagic manifestations, and shock that characterize DHF/DSS are considered to have an immunological basis, as they are more common during secondary infection with a heterologous dengue virus strain (15, 28, 33). However, severe clinical manifestations can also occur during primary dengue infection, pointing to a contributory role of viral virulence factors. The WHO estimates that more than 20,000 people worldwide, mainly children, die each year from serious complications of dengue. No specific antiviral therapies are currently available for treating the infection, and efforts to develop a safe prophylactic vaccine have been hindered by the complex role of the immune system in disease pathogenesis (39, 52, 57). Thus, novel treatment strategies that block viral replication and/or to attenuate the exaggerated cytokine response associated with DHF/DSS complications are urgently needed.Potent and specific gene silencing mediated by RNA interference (RNAi) has generated a great deal of interest in development of RNAi as a therapeutic strategy against viral infections (50, 54). Many studies have demonstrated the effectiveness of the RNAi approach to suppress flavivirus infection, including dengue virus replication in experimental cell lines (3, 23, 26, 42, 60). In addition, the versatility of RNAi could also be exploited to block important host mediators that contribute to dengue pathogenesis. However, the existence of four distinct dengue virus serotypes and the ability of viruses to develop resistance to RNAi by mutating their sequences will have to be taken into account before clinical use can be contemplated. A more serious hurdle for RNAi therapeutics is the specific delivery of small interfering RNA (siRNA) to relevant cell types.Even though dengue virus antigens have been detected in many tissues, including liver, spleen, lymph node, and skin of patients with DHF/DSS, macrophages and dendritic cells (DCs) are considered the predominant infected cell types (9, 36, 59). Following the bite of an infected Aedes mosquito, the initial local viral replication is believed to take place in the skin DCs, including myeloid DCs and Langerhans cells (31, 53, 59). Dengue-infected DCs play a key role in the immunopathogenesis of DHF/DSS, as, along with macrophages, they release proinflammatory cytokines and soluble factors that mediate plasma leakage, thrombocytopenia, and hypovolemic shock associated with severe dengue infection (14, 15, 29, 38). Therefore, development of a method to introduce siRNA into DCs would be an important step toward using RNAi therapeutically to suppress viral replication and/or to attenuate the vigorous host cytokine responses in dengue infection (7, 19).To target DCs, we used a previously characterized 12-amino-acid peptide identified from a phage display peptide library that specifically binds to a ligand expressed on DCs (10). In an earlier study, we demonstrated that fusing nucleic acid-binding nine d-arginine residues to a neuronal cell-targeting peptide enabled siRNA delivery to neuronal cells (27). Here, in a similar approach, we synthesized a chimeric peptide consisting of the DC-targeting peptide fused to nona-D-arginines (9dR) to target siRNA selectively to DCs. We investigated whether the DC3-9dR peptide could deliver siRNA targeting a dengue virus envelope sequence to reduce the viral load in DCs. As tumor necrosis factor alpha (TNF-α) is one of the acute-phase cytokines with a major role in inducing plasma leakage in dengue infection (8, 12, 17, 20), we also explored the possibility of reducing TNF-α expression in DC in vitro and in vivo. Our findings demonstrate the potential of a targeted RNAi-based approach for simultaneously decreasing viral load and reducing aberrant cytokine responses in DCs.     

Acute Respiratory Failure and Active Bleeding Are the Important Fatality Predictive Factors for Severe Dengue Viral Infection

Objective

To determine the outcome of severe dengue viral infection (DVI) and the main dengue fatality risk factors.

Study design

The medical records of patients aged <15 years admitted to Songklanagarind Hospital in southern Thailand during 1989–2011 were reviewed. Patients who had dengue hemorrhagic fever (DHF) grades III–IV, organ failure (cardiovascular, respiratory, liver, renal or hematologic), impaired consciousness, or aspartate aminotransferase more than 1,000 units/L, were classified as having severe DVI. To determine the fatality risk factors of severe DVI, the classification trees were constructed based on manual recursive partitioning.

Results

Of the 238 children with severe DVI, 30 (12.6%) died. Compared to the non-fatal DVI cases, the fatal cases had higher rates of DHF grade IV (96.7% vs 24.5%), repeated shock (93.3% vs 27.9%), acute respiratory failure (ARF) (100% vs 6.7%), acute liver failure (ALF) (96.6% vs 6.3%), acute kidney injury (AKI) (79.3% vs 4.5%), and active bleeding requiring blood transfusion (93.3% vs 5.4%), all p<0.01. The combined risk factors of ARF and active bleeding considered together predicted fatal outcome with sensitivity, specificity, and negative and positive predictive values of 0.93 (0.78–0.99), 0.97 (0.93–0.99), 0.99 (0.97–1.00), and 0.82 (0.65–0.93), respectively. The likelihood ratios for a fatal outcome in the patients who had and did not have this risk combination were 32.4 (14.6–71.7) and 0.07 (0.02–0.26), respectively.

Conclusion

Severe DVI patients who have ARF and active bleeding are at a high risk of death, while patients without these things together should survive.     

Cytokine,Antibody and Proliferative Cellular Responses Elicited by Taenia solium Calreticulin upon Experimental Infection in Hamsters

Taenia solium causes two diseases in humans, cysticercosis and taeniosis. Tapeworm carriers are the main risk factor for neurocysticercosis. Limited information is available about the immune response elicited by the adult parasite, particularly the induction of Th2 responses, frequently associated to helminth infections. Calreticulin is a ubiquitous, multifunctional protein involved in cellular calcium homeostasis, which has been suggested to play a role in the regulation of immune responses. In this work, we assessed the effect of recombinant T. solium calreticulin (rTsCRT) on the cytokine, humoral and cellular responses upon experimental infection in Syrian Golden hamsters (Mesocricetus auratus). Animals were infected with T. solium cysticerci and euthanized at different times after infection. Specific serum antibodies, proliferative responses in mesenteric lymph nodes and spleen cells, as well as cytokines messenger RNA (mRNA) were analyzed. The results showed that one third of the infected animals elicited anti-rTsCRT IgG antibodies. Interestingly, mesenteric lymph node (MLN) cells from either infected or non-infected animals did not proliferate upon in vitro stimulation with rTsCRT. Additionally, stimulation with a tapeworm crude extract resulted in increased expression of IL-4 and IL-5 mRNA. Upon stimulation, rTsCRT increased the expression levels of IL-10 in spleen and MLN cells from uninfected and infected hamsters. The results showed that rTsCRT favors a Th2-biased immune response characterized by the induction of IL-10 in mucosal and systemic lymphoid organs. Here we provide the first data on the cytokine, antibody and cellular responses to rTsCRT upon in vitro stimulation during taeniasis.     

Immunodiagnosis of Dengue Virus Infection Using Saliva

Keeping in view the complications and the case fatality associated with dengue virus, several serologic tests have been developed. However, the major drawback of these serologic tests is the need for a venous blood sample obtained by invasive venipuncture. As a noninvasive alternative, saliva provides a body fluid that contains antibodies of diagnostic importance. Hence, the detection of DEN-specific IgM and IgG antibodies in serum and saliva from 80 patients was compared. Salivary IgM antibodies were detected in 100% of the serum IgM-positive samples and in 30% of the serum samples that were negative for IgM antibodies. Salivary IgG antibodies were detected in 93.3% of the serum samples that were positive for anti-dengue IgG antibodies and in none of the serum IgG-negative cases. None of the specimens from the healthy controls showed the presence of IgM or IgG antibodies. The detection of both IgG and IgM antibodies in saliva correlated well with the serum IgG and IgM detection by the ELISA test (r = 0.6322 and r = 0.4227). Detection of salivary IgM antibodies by ELISA showed 100% sensitivity, 70% specificity, 90.9% positive predictive value, and 100% negative predictive value. The detection of IgG in saliva proved to be a promising tool as the sensitivity, specificity, positive predictive value, and negative predictive value were found out to be 93.3%, 100%, 100%, and 83.3%, respectively. Thus, from this study we conclude that the detection of DEN-specific salivary IgG and IgM antibodies are useful markers for dengue infection.     

Cytokine Expression Profile of Dengue Patients at Different Phases of Illness

Background

Dengue is an important medical problem, with symptoms ranging from mild dengue fever to severe forms of the disease, where vascular leakage leads to hypovolemic shock. Cytokines have been implicated to play a role in the progression of severe dengue disease; however, their profile in dengue patients and the synergy that leads to continued plasma leakage is not clearly understood. Herein, we investigated the cytokine kinetics and profiles of dengue patients at different phases of illness to further understand the role of cytokines in dengue disease.

Methods and Findings

Circulating levels of 29 different types of cytokines were assessed by bead-based ELISA method in dengue patients at the 3 different phases of illness. The association between significant changes in the levels of cytokines and clinical parameters were analyzed. At the febrile phase, IP-10 was significant in dengue patients with and without warning signs. However, MIP-1β was found to be significant in only patients with warning signs at this phase. IP-10 was also significant in both with and without warning signs patients during defervescence. At this phase, MIP-1β and G-CSF were significant in patients without warning signs, whereas MCP-1 was noted to be elevated significantly in patients with warning signs. Significant correlations between the levels of VEGF, RANTES, IL-7, IL-12, PDGF and IL-5 with platelets; VEGF with lymphocytes and neutrophils; G-CSF and IP-10 with atypical lymphocytes and various other cytokines with the liver enzymes were observed in this study.

Conclusions

The cytokine profile patterns discovered between the different phases of illness indicate an essential role in dengue pathogenesis and with further studies may serve as predictive markers for progression to dengue with warning signs.     

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.     

登革病毒对人血管内皮细胞感染性的研究

用登革病毒Ⅱ型（DV2）感染体外培养和传代的人脐静脉内皮细胞（HUVEC）,研究发现,HUVEC是登革病毒的允许性细胞。病毒感染后12h即可在培养上清中用微量蚀斑法测出病毒,病毒滴度48h达高峰,以后迅速下降。并发现在一定范围内病毒产量随病毒感染复数（MOI）的增加而增高。间接免疫荧光法证明感染的HUVEC胞浆及胞膜上携带DV2抗原。电镜和光镜下,感染细胞未见明显的形态和结构改变。     

登革病毒感染的检测技术研究进展

登革病毒(dengue virus,DENV)是引起登革热(dengue fever,DF)的病原体,经蚊叮咬而传播疾病.DF主要流行于热带和亚热带地区.目前,DF已经发展成一个日益严重的全球性公共卫生问题.2012年,DF被列为全球最重要的蚊虫传播性疾病.快速而准确的针对DENV感染的检测技术能够有效地控制DF的爆发及减少死亡病例.目前DENV感染的检测技术主要有病毒分离培养、分子生物学诊断及血清学诊断三类,三类检测技术各有优缺点,应该根据具体情况选用适当的检测手段.     

Peridomestic Infection as a Determining Factor of Dengue Transmission

Background

The study of endemic dengue transmission is essential for proposing alternatives to impact its burden. The traditional paradigm establishes that transmission starts around cases, but there are few studies that determine the risk.

Methods

To assess the association between the peridomestic dengue infection and the exposure to a dengue index case (IC), a cohort was carried out in two Mexican endemic communities. People cohabitating with IC or living within a 50-meter radius (exposed cohort) and subjects of areas with no ICs in a 200-meter radius (unexposed cohort) were included.

Results

Exposure was associated with DENV infection in cohabitants (PRa 3.55; 95%CI 2.37–5.31) or neighbors (PRa 1.82; 95%CI 1.29–2.58). Age, location, toilets with no direct water discharge, families with children younger than 5 and the House Index, were associated with infection. Families with older than 13 were associated with a decreased frequency. After a month since the IC fever onset, the infection incidence was not influenced by exposure to an IC or vector density; it was influenced by the local seasonal behavior of dengue and the age. Additionally, we found asymptomatic infections accounted for 60% and a greater age was a protective factor for the presence of symptoms (RR 0.98; 95%CI 0.97–0.99).

Conclusion

The evidence suggests that dengue endemic transmission in these locations is initially peridomestic, around an infected subject who may be asymptomatic due to demographic structure and endemicity, and it is influenced by other characteristics of the individual, the neighborhood and the location. Once the transmission chain has been established, dengue spreads in the community probably by the adults who, despite being the group with lower infection frequency, mostly suffer asymptomatic infections and have higher mobility. This scenario complicates the opportunity and the effectiveness of control programs and highlights the need to apply multiple measures for dengue control.