Secondary Dengue Infection Research Articles

Risk factors influencing severity in pediatric dengue infection

Background: Dengue disease encompasses a wide spectrum of clinical presentations. While the severity of dengue disease can vary from patient to patient, it is, however, still unclear as to what factors determine dengue disease severity. Objective: To analyze the potential risk factors such as: sex, age, nutritional status, dengue serotypes, D-dimer positivity, and serologic responses, and study their correlation with the severity of dengue virus infection. Methods: Dengue patients at King Chulalongkorn Memorial Hospital were enrolled for the study. Dengue serotype was determined by PCR and primary or secondary infection was determined based on serological criteria. D-dimer levels were also tested using a rapid semiquantitation system. Clinical data such as age, body weight, sex, and nutritional status were also collected. Results: Forty-six patients were confirmed for dengue fever (DF), and 52 patients were confirmed for Dengue hemorrhagic fever (DHF). 77.5% of patients had secondary dengue infection, 11.3% had primary infection, and 11.2% were of inconclusive status. Gender and age of patients did not correlate with dengue disease severity (p > 0.05). It was inconclusive as to whether nutritional status was a risk factor. Positivity of the D-dimer test was statistically significant for DHF (p=0.001). Early increasing D-dimer in the febrile stage could predict severity of dengue infection during the initial stage of the disease (positive predictive value=68.4 %). Conclusion: D-dimer is significantly correlated with dengue severity and it may be one of the prognostic factors for dengue infection in children.

Secondary heterologous dengue infection risk: Disequilibrium between immune regulation and inflammation?

Increased serum levels of cytokines released by cells of the immune response have been detected in patients suffering from dengue disease. Likewise, secondary infections by a different dengue virus serotype result in a highest risk of development of the severe dengue disease. Both findings suggest that the memory immune response is one of the key players in the pathogenesis of this disease. Here we take advantage of the particular Cuban epidemiological situation in dengue to analyze a broad spectrum of cell-mediated immune response mediators at mRNA and protein level. Evidences for a regulatory immune pattern in homologous (TGF-β, IL-10) vs. pro-inflammatory pattern (IFN-γ, TNF-α) in heterologous dengue virus re-challenge were found, suggesting a possible association with the higher incidence of severe dengue cases in the latter case.

The benign nature of acute dengue infection in hospitalized older adults in Singapore

Objectives In Singapore, dengue primarily affects adults. This study aimed to determine if older dengue patients in Singapore have greater morbidity and mortality. Methods All laboratory diagnosed dengue patients admitted to Tan Tock Seng Hospital in 2004 were retrospectively reviewed. Cases were re-classified into dengue fever and dengue hemorrhagic fever based on World Health Organization criteria. Demographic, clinical, laboratory, and outcome data of patients aged ≥60 years and <60 years were collected. Results Of 1971 laboratory confirmed dengue cases, 66 were aged ≥60 years. Older patients were significantly less likely to be male (44% vs. 64%), and more likely to have diabetes (17% vs. 2%), hypertension (48% vs. 4%), ischemic heart disease (6% vs. 0.1%), hyperlipidemia (18% vs. 1%), and secondary dengue infections (64% vs. 34%). Clinical features were similar except older patients were significantly less likely to report fever (92% vs. 99%), or have leukopenia (32% vs. 51%) or hemoconcentration (0 vs. 5%) on admission. Older patients had similar dengue hemorrhagic fever, bleeding, hypotension, severe thrombocytopenia, and elevated transaminase rates. Length of hospital stay, risk of intensive care unit admission, and outcome of death were not statistically different. Conclusions Despite greater co-morbidity and secondary dengue infection, older dengue patients in Singapore did not have greater morbidity or mortality.

Foreword

Dengue is a major global health concern. Its increasing incidence and continued geographical spread make the development of an effective vaccine an international health priority. Dengue is currently present in more than one hundred countries, while up to 3 billion people are estimated to be at risk of infection. In addition, dengue is associated with a substantial economic burden that is particularly severe at the individual family level. Establishing the full human cost and economic impact of dengue remains urgent. The approaches being investigated for vaccine development include the use of live, vectored and killed, and recombinant preparations. Vaccine candidates must provide broad and robust immunity to all four dengue serotypes simultaneously as secondary dengue infections may lead to enhanced disease severity. The design, implementation, and surveillance measures associated with dengue vaccine trials need to be particularly rigorous due to the complexity of the disease and its epidemiology. Therefore, eligible trial sites must satisfy several criteria including documented hyper-endemicity and a known epidemiological history of the circulating serotypes. Epidemiological data from Ratchaburi province in Thailand strongly support this location's suitability for a proof-of-concept efficacy trial of the sanofi pasteur tetravalent dengue vaccine. Accurate disease surveillance and carefully monitored clinical trials will provide essential evidence concerning the efficacy of candidate dengue vaccines, which will hopefully herald a new era in dengue disease prevention.

Antibodies Determine Virulence in Dengue

Four dengue viruses (DENV) cause syndromes that are self-limited or severe. The severe syndrome, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), characterized by sudden vascular permeability, is not caused by inherently virulent DENV. This syndrome has consistently been observed to accompany dengue infections in individuals circulating heterotypic dengue antibodies at enhancing concentrations. In humans, dengue infections target monocytes/macrophages where, absent neutralization, heterotypic antibodies, perhaps directed at domain I-II of the envelope protein form immune complexes, attach to Fc receptors, suppress innate immunity, and increase productive infection. Diverse clinical responses to primary infections with different DENV strains do occur and occasionally result in subclinical vascular permeability observed as hypovolemia. The fact that American (AM) genotype DENV 2 produce only mild disease during secondary dengue infections is best explained by the downregulation of disease by DENV 1 antibodies directed at a unique antigenic structure expressed on AM DENV 2.

A travel medicine view of dengue and dengue hemorrhagic fever

Dengue fever is the most prevalent arboviral infection worldwide, with up to 40% of the world population living in endemic regions. Among travelers to tropical countries, dengue infection is increasingly reported, and it is now a leading cause of post-travel fever. Outbreaks of dengue-like illness were already described since the 18th century, but it is only in the last half century that a severe form of the disease - dengue hemorrhagic fever (DHF) has been described. Although the cause of DHF is not established, the prevailing theory attributes the disease to antibody-dependent enhancement of viral replication, in the presence of a secondary dengue infection. Comparative studies of primary vs. secondary infection are difficult to perform in endemic countries because of the rarity of primary infection except during early infancy. Travelers on the other hand are usually diagnosed with primary infection and are therefore a better study population. Data on dengue and DHF among travelers appears to suggest that severe dengue and DHF occur in similar rates among cases with primary and with secondary infections. Epidemiological and physiological data from travelers suggest that the prevailing theory on the causes of DHF needs to be reconsidered.

Reliable Classifier to Differentiate Primary and Secondary Acute Dengue Infection Based on IgG ELISA

BackgroundDengue virus infection causes a wide spectrum of illness, ranging from sub-clinical to severe disease. Severe dengue is associated with sequential viral infections. A strict definition of primary versus secondary dengue infections requires a combination of several tests performed at different stages of the disease, which is not practical.Methods and FindingsWe developed a simple method to classify dengue infections as primary or secondary based on the levels of dengue-specific IgG. A group of 109 dengue infection patients were classified as having primary or secondary dengue infection on the basis of a strict combination of results from assays of antigen-specific IgM and IgG, isolation of virus and detection of the viral genome by PCR tests performed on multiple samples, collected from each patient over a period of 30 days. The dengue-specific IgG levels of all samples from 59 of the patients were analyzed by linear discriminant analysis (LDA), and one- and two-dimensional classifiers were designed. The one-dimensional classifier was estimated by bolstered resubstitution error estimation to have 75.1% sensitivity and 92.5% specificity. The two-dimensional classifier was designed by taking also into consideration the number of days after the onset of symptoms, with an estimated sensitivity and specificity of 91.64% and 92.46%. The performance of the two-dimensional classifier was validated using an independent test set of standard samples from the remaining 50 patients. The classifications of the independent set of samples determined by the two-dimensional classifiers were further validated by comparing with two other dengue classification methods: hemagglutination inhibition (HI) assay and an in-house anti-dengue IgG-capture ELISA method. The decisions made with the two-dimensional classifier were in 100% accordance with the HI assay and 96% with the in-house ELISA.ConclusionsOnce acute dengue infection has been determined, a 2-D classifier based on common dengue virus IgG kits can reliably distinguish primary and secondary dengue infections. Software for calculation and validation of the 2-D classifier is made available for download.

New dengue antibody assay with unique differential detection of IgG and IgM antibodies

Objectives Most of the serological methods, traditional and recent, that have been developed to assess laboratory confirmation of dengue infection, suffer from the need to use separate tests for IgM and for IgG and the need for paired serum specimens in order to establish a clinical interpretation. Moreover, most of the IgG tests ignore early convalescence as well as past exposure. The objectives of this study is to demonstrate the advantages of the ImmunoComb Dengue BiSpot, a new dengue antibody assay, that detects simultaneously both dengue-specific IgM and IgG, in determining type and status of infection in a single test. Designs and methods Single and paired positive and negative specimens obtained from various dengue-high prevalent regions and from dengue-free areas were used. The specimens were comprised of admission samples of suspected patients and secondary dengue infection individuals, a group of hospital patients, asymptomatic to dengue who originated from dengue high prevalence areas and dengue-negative volunteers. All samples were tested by the ImmunoComb Dengue BiSpot kit along with the gold standard assays of Hemagglutination-inhibition (HAI) and MAC ELISA. Results Testing 365 gold standard positive specimens from different regions, the ImmunoComb Dengue BiSpot kit revealed sensitivity for IgM of 97.5% and 99.1% for IgG. The IgG was segregated into 2 groups: 86.6% for high titer and 12.5% for low titer of IgG. The overall sensitivity was 98.9%. The overall specificity of the kit, testing 657 samples, mostly from dengue-free area was 97.9%. The higher diagnostic resolution of the Dengue BiSpot test was demonstrated when the performance of the kit on the various groups was compared to the gold standard tests and the dengue status was determined in paired specimens by the new assay. Conclusions The results of the present studies demonstrate the advantages of ImmunoComb Dengue BiSpot assay as an alternate strategy for the determination of dengue infection status. The combined use of IgM and IgG to discriminate between primary and secondary infections, on one hand, and providing high resolution in distinction of convalescence stage and past infection, on the other hand, endow the kit with improved capacity in assessing the clinical status of a tested individual in a single test.

Dengue vaccine development and dengue viral neutralization and enhancement assays

Dengue fever is a major tropical infectious disease that affects 50-100 million people each year. Its complications, namely dengue haemorrhagic fever and dengue shock syndrome, disproportionately afflict children and young adults. The primary goal of several vaccines now in development is to elicit protective neutralizing antibody responses; however, the exact definition of such responses remain unclear. Here, we review briefly the historical aspects of dengue vaccine development and current candidate dengue vaccines, and discuss various laboratory assays for gauging the neutralizing antibody responses to infection or vaccination, or both. We conclude that modification of current neutralization assays is required to improve the correlation between neutralization end point determinations and protection against secondary heterotypic dengue infections.

Influence of the Dengue Serotype, Previous Dengue Infection, and Plasma Viral Load on Clinical Presentation and Outcome During a Dengue-2 and Dengue-4 Co-Epidemic

Martinique experienced a dengue outbreak with co-circulation of DENV-2 and DENV-4. In an emergency department-based study, we analyzed whether the clinical presentation and outcome of adult patients were related to serotype, immune status, or plasma viral load. Of the 146 adult patients who had confirmed dengue infection, 91 (62.3%) were classified as having classic dengue fever, 11 (7.5%) fulfilled World Health Organization criteria for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), 21 other patients (14.4%) presented with at least one typical feature of DHF/DSS [i.e., internal hemorrhage, plasma leakage, marked thrombocytopenia (platelet count < or = 50,000 platelets/mm(3)) and/or shock], and 23 further patients (15.8%) had unusual manifestations. Four patients died. Severe illness was more frequent in patients with secondary dengue infection (odds ratio, 7.18; 95% confidence interval, 3.1-16.7; P < 0.001). Multivariate regression analysis showed that gastrointestinal symptoms and other unusual manifestations were independently associated with DENV-2 infection, whereas cough and DHF/DSS features were independently associated with secondary immune response. A high plasma viral load was associated with DENV-2 infection, increased serum liver enzymes, and with DHF/DSS features in patients presenting after the third day of illness. The most severe cases of dengue resulted from the combined effects of DENV-2 and secondary infection.

Polymorphisms of the TAP 1 and 2 Gene May Influence Clinical Outcome of Primary Dengue Viral Infection

Antigen peptides are actively transported across the endoplasmic reticulum by the transporters associated with antigen presentation (TAP). TAP genes polymorphism could influence the selection process that determines which antigen peptides play a role in the pathogenesis of dengue infection. The aim of this study was to investigate the association of TAP genes polymorphism in diverse pathogenesis of dengue infection. This study included 197 dengue-infected patients who were further categorized into 64, 23 and 11 primary dengue fever (DF), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) cases, respectively and 26, 52, and 21 secondary DF, DHF and DSS cases, respectively as per WHO grading system. TAP1 and 2 gene polymorphisms were performed by the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Analysis of TAP1 gene polymorphism demonstrated decreased frequency of Ile/Ile genotype at TAP1(333) in primary DHF cases (39.1%) when compared with primary DF (64.1%, P < 0.034, OR = 0.611). The genotype frequency of Val/Val at TAP2(379) locus was significantly decreased among primary DHF (43.5%) in comparison to primary DF (71.9%, P = 0.015, OR = 0.605). Significant low proportion of primary DSS were found to have TAP1(637) Asp/Asp genotypes (54.5%) when compared with primary DF (70.3%, P = 0.043). Asp/Asp genotype at TAP1(637) was found to reduce the risk by 0.643 times for primary DSS. There was no significant difference in the genotypes studied between primary and secondary infection and also within secondary dengue infection in all three clinical groups. This report on TAP gene polymorphisms in dengue suggested that among the primary-infected individuals, homozygous patterns for Ile at TAP1(333) Val at TAP2(379) loci and Asp at TAP1(637) were found to be a protective factor against development of DHF and DSS, respectively.

Elevated levels of soluble ST2 protein in dengue virus infected patients

Levels of the soluble form of the interleukin-1 receptor-like 1 protein (IL-1RL-1/ST2) are elevated in the serum of patients with diseases characterized by an inflammatory response. The objective of this study was to determine the concentration of soluble ST2 (sST2) in dengue infected patients during the course of the disease. Twenty-four patients with confirmed dengue infection, classified as dengue fever, and 11 patients with other febrile illness (OFI) were evaluated. Levels of sST2 in serum and laboratory variables usually altered during dengue infections were measured. Dengue infected patients had higher serum sST2 levels than OFI at the end of the febrile stage and at defervescence ( p = 0.0088 and p = 0.0004, respectively). Patients with secondary dengue infections had higher serum sST2 levels compared with patients with primary dengue infections ( p = 0.047 at last day of fever and p = 0.030 at defervescence). Furthermore, in dengue infected patients, we found a significant negative correlation of sST2 with platelet and WBC counts, and positive correlation with thrombin time and transaminases activity. We suggest that sST2 could be a potential marker of dengue infection, could be associated with severity or could play a role in the immune response in secondary dengue virus infection.

Anti-Platelet and Anti-Endothelial Cell Autoantibodies in Vietnamese Infants and Children with Dengue Hemorrhagic Fever

Dengue hemorrhagic fever (DHF) is a serious public health problem. Increased vascular permeability and thrombocytopenia are the hallmarks of DHF. The mechanisms involved in DHF/Dengue shock syndrome (DSS) pathogenesis is not fully understood. This study gives evidence of the presence of antibodies which cross-reacted with platelets, and endothelial cells in the sera of Vietnamese infants and children with DHF/DSS. The anti-platelet, anti-endothelial cell IgM levels were higher in the sera of DHF/DSS infants and children, compared with controls. However, the levels of these autoantibodies were not correlated with the severity of DHF (non-shock DHF vs DSS). The antiplatelet, and anti-endothelial cell autoantibodies may play a role in the pathogenesis of DHF/DSS in infants and children with predominantly primary, and secondary dengue infections, respectively. The epitopes shared by surface molecules of platelets and endothelial cells and dengue virus antigens need to be identified and avoided in designing the safe candidate vaccines.

High Pro-Inflammatory Cytokine Secretion and Loss of High Avidity Cross-Reactive Cytotoxic T-Cells during the Course of Secondary Dengue Virus Infection

BackgroundDengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing – over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype.Methods and FindingsVirus-specific cytotoxic T lymphocytes (CTL) showing high level cross reactivity between dengue serotypes could be expanded from blood samples taken during the acute phase of secondary dengue infection. These could not be detected in convalescence when only CTL populations demonstrating significant serotype specificity were identified. Dengue cross-reactive CTL clones derived from these patients were of higher avidity than serotype-specific clones and produced much higher levels of both type 1 and certain type 2 cytokines, many previously implicated in dengue pathogenesis.ConclusionDengue serotype cross-reactive CTL clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. That such cells cannot be expanded from convalescent samples suggests that they may be depleted, perhaps as a consequence of activation-induced cell death. Such high avidity cross-reactive memory CTL may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. These cells appear to be subsequently deleted leaving a more serotype-specific memory CTL pool. Further studies are needed to relate these cellular observations to disease phenotype in a large group of patients. If confirmed they have significant implications for understanding the role of virus-specific CTL in pathogenesis of dengue disease.

Lack of Efficacy of High-Dose Intravenous Immunoglobulin Treatment of Severe Thrombocytopenia in Patients with Secondary Dengue Virus Infection

Because most cases of secondary dengue virus infection are associated with an increased level of platelet-associated IgG, a high dose of intravenous immunoglobulin (IVIG) may have an effect on the development of severe thrombocytopenia in this disease. A randomized, controlled study was conducted with two treatment groups consisting of a treatment (IVIG) group (n = 15) and a non-treatment (non-IVIG) group (n = 16) to determine whether a high dose of IVIG is effective in hastening the recovery from thrombocytopenia in patients with secondary dengue virus infection. No significant difference was found in the baseline demographic data between the two groups. No adverse effect of IVIG was observed, but no effect in hastening the recovery of platelet counts was found in patients with secondary dengue infections. The lack of efficacy of IVIG suggests that platelet clearance by macrophages through Fc gamma receptors is not a primary mechanism in this disease.

Characterization of a Dengue Patient Cohort in Recife, Brazil

From 2004 to 2006, 658 patients with suspected dengue virus infections were enrolled in a clinical dengue cohort established in Recife, Pernambuco, located at the northeastern region of Brazil. A total of 2,364 blood samples were collected, and serum, plasma, and cells were cryopreserved. Among the suspected cases, 354 (54%) were confirmed as acute DENV-3 infection based on reverse transcription-polymerase chain reaction, virus isolation, and ELISA-IgM. According to WHO criteria, 29.4% of the positive acute cases were classified as dengue fever (DF) and 8.2% of the cases as dengue hemorrhagic fever (DHF), grade 1 or 2. The DHF cases represent 100% of those confirmed in Recife during the period of the study. The dengue cases that did not fulfill the definition of either DHF or DF were classified as DF complicated and accounted for 44.0% of the cases. All the acute cases were classified as either primary or secondary acute dengue virus infections. Secondary infection was predominant in patients with DF; however, there was no predominance of either primary or secondary infections in patients with DHF.

Involvement of the central nervous system in patients with dengue virus infection

The findings of a neurological evaluation in 85 patients with confirmed, acute, dengue virus infection are described. Signs of central nervous system involvement were present in 18 patients (21.2%). The most frequent neurological symptom was mental confusion. The frequency of neurological involvement did not differ between patients with primary and secondary dengue infection, and the prevalence of central nervous system involvement in dengue fever and dengue hemorrhagic fever also did not differ significantly. The presence of CNS involvement did not influence the prognosis of dengue infection. Dengue viral CSF RNA was found in 7 of 13 patients submitted to a spinal tap, the CSF viral load being less than 1000 copies/ml. PCR was negative in serum samples obtained from three patients on the same day as the CSF samples, suggesting that the dengue virus actively enters the CNS and that the presence of the virus in the CNS does not result from passive crossing of the blood-brain barrier.

Different clinical and laboratory manifestations between dengue haemorrhagic fever and dengue fever with bleeding tendency

The incidence of dengue fever (DF) is estimated to have increased 30-fold in the past 50 years. The incidence of dengue haemorrhagic fever (DHF), a life-threatening complication of DF, is also increasing. The need for better classification of the severity of dengue infections has been proposed in order to clarify different entities of dengue infections. We defined a class of patients with DF with bleeding tendency (DF w/B) to differentiate further the varying pathogenesis among DF, DF w/B and DHF. In a hospital-based study in Taiwan, we compared clinical features, biochemistry and immune mediators among patients with DHF, DF w/B and DF. Results showed that DF w/B patients, similar to DHF patients, had a higher rate of secondary dengue infection (P<0.001) as well as higher IL-10 (P=0.023) and lower IFNgamma (P=0.009) levels than DF patients. In contrast, DHF patients had significantly higher soluble vascular cell adhesion molecule 1 levels than DF w/B patients (P=0.038) and DF patients (P<0.001). This study provides new insight into the different immune mechanisms of DF, DF w/B and DHF. DF involves a Th1 reaction and DF w/B involves an altered Th2 reaction, whereas DHF involves an altered Th2 reaction and augmented vascular insult.

Fulminant liver failure secondary to haemorrhagic dengue in an international traveller

Dengue infections are caused by a single-stranded RNA virus, which has four serotypes (DEN 1-4); mosquitoes of the genus Aedes serve as vectors of transmission. Risk factors for dengue infection are related to both the host and virus. Age, gender, immune status, and genetic background of the host all contribute to the severity of dengue infection. Recently, international travel to endemic areas has also been identified as a major risk factor for both primary and secondary dengue infection. Dengue remains a diagnostic challenge, given its protean nature, ranging from mild febrile illness to profound shock. The most severe manifestation of dengue infection is dengue shock syndrome, which has an estimated mortality rate close to 50%. Dengue shock syndrome typically presents with increased anion gap metabolic acidosis, disseminated intravascular coagulation, severe hypotension, and jaundice. Liver involvement appears to occur more frequently when infections involve DEN-3 and DEN-4 serotypes. While hepatocellular damage has been reported previously in dengue infection, acute liver failure is an extremely rare occurrence in adults. We report a patient with dengue shock syndrome who presented with acute liver failure and hepatic encephalopathy after recent travel to an endemic area.

Sensitivity and specificity of three ELISA-based assays for discriminating primary from secondary acute dengue virus infection

Background Discrimination between primary and secondary dengue virus infection traditionally has been performed using the hemagglutination inhibition (HI) test. However, this test has practical limitations and disadvantages. Objective To evaluate the ability of three ELISA-based methods (IgG avidity test, IgM/IgG ratio and IgG titer) to discriminate primary from secondary dengue infection. Study design Serum samples from convalescent-phase patients with confirmed acute, primary ( n = 46) or secondary ( n = 33) dengue virus infection were tested using three ELISA-based methods. A ROC curve was employed to establish the cut-off points and to evaluate the ability of the three methods to distinguish between acute, primary and secondary dengue virus infection. Results All three assays exhibited sensitivity and negative predictive values of 100% for defining secondary infection. The specificity and positive predictive values were respectively 97.8% and 93.7% for the IgG avidity test, 95.7% and 88.2% for the IgM/IgG ratio assays, and 97.8% and 93.7% for the IgG titer assay. Conclusion All three ELISA-based assays proved reliable tools for discriminating between acute, primary and secondary dengue virus infection when using serum samples from convalescent-phase patients.