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Understanding the burden and risk factors of dengue virus (DENV) infection in Puerto Rico is important for the prevention of dengue in local, traveler and military populations. Using sera from the Department of Defense Serum Repository, we estimated the prevalence and predictors of DENV seropositivity in those who had served in Puerto Rico, stratified by birth or prior residence (“birth/residence”) in dengue-endemic versus non-endemic regions. We selected sera collected in early 2015 from 500 U.S. military members, a time-point also permitting detection of early cryptic Zika virus (ZIKV) circulation. 87.2% were born or resided in a DENV-endemic area before their military service in Puerto Rico. A high-throughput, flow-cytometry-based neutralization assay was employed to screen sera for ZIKV and DENV neutralizing antibodies, and confirmatory testing was done by plaque-reduction neutralization test (PRNT). We identified one Puerto Rico resident who seroconverted to ZIKV by June 2015, suggesting cryptic ZIKV circulation in Puerto Rico at least 4 months before the first reported cases. A further six PRNT-positive presumptive ZIKV infections which were resolved as DENV infections only by the use of paired sera. We noted 66.8% of the total study sample was DENV seropositive by early 2015. Logistic regression analysis indicated that birth/residence in a dengue non-endemic region (before military service in Puerto Rico) was associated with a lower odds of DENV exposure by January—June 2015 (aOR = 0.28, p = 0.001). Among those with birth/residence in a non-endemic country, we noted moderate evidence to support increase in odds of DENV exposure for each year of military service in Puerto Rico (aOR = 1.58, p = 0.06), but no association with age. In those with birth/residence in dengue-endemic regions (before military service in Puerto Rico), we noted that age (aOR = 1.04, p = 0.02), rather than duration of Puerto Rico service, was associated with dengue seropositivity, suggesting earlier lifetime DENV exposure. Our findings provide insights into the burden and predictors of DENV infection in local, traveler and military populations in Puerto Rico. Our study also highlights substantial PRNT ZIKV false-positivity when paired sera are not available, even during periods of very low ZIKV prevalence.

In the latest World Health Organization (WHO) recommendation for Dengvaxia implementation, either serological testing or a person’s history of prior dengue illness may be used as supporting evidence to identify dengue virus (DENV)-immune individuals eligible for vaccination, in areas with limited capacity for laboratory confirmation. This analysis aimed to estimate the concordance between self-reported dengue illness histories and seropositivity in a prospective cohort study for dengue virus infection in Kamphaeng Phet province, a dengue-endemic area in northern Thailand. The study enrolled 2,076 subjects from 516 multigenerational families, with a median age of 30.6 years (range 0–90 years). Individual and family member dengue illness histories were obtained by questionnaire. Seropositivity was defined based on hemagglutination inhibition (HAI) assays. Overall seropositivity for DENV was 86.5% among those aged 9–45 years, which increased with age. 18.5% of participants reported a history of dengue illness prior to enrollment; 30.1% reported a previous DENV infection in the family, and 40.1% reported DENV infection in either themselves or a family member. Relative to seropositivity by HAI in the vaccine candidate group, the sensitivity and specificity of individual prior dengue illness history were 18.5% and 81.6%, respectively; sensitivity and specificity of reported dengue illness in a family member were 29.8% and 68.0%, and of either the individual or a family member were 40.1% and 60.5%. Notably, 13.4% of individuals reporting prior dengue illness were seronegative. Given the high occurrence of asymptomatic and mild DENV infection, self-reported dengue illness history is poorly sensitive for prior exposure and may misclassify individuals as ‘exposed’ when they were not. This analysis highlights that a simple, highly sensitive, and highly specific test for determining serostatus prior to Dengvaxia vaccination is urgently needed., Author summary Dengue is common worldwide and more effective counter-measures are needed. The only DENV vaccine to have achieved licensure is more effective in DENV-immune individuals and is contra-indicated in DENV-naïve individuals due to safety concerns. Self-reports of prior dengue illness have been proposed to serve as a proxy for DENV exposure, due to limited resources for serological testing in many DENV-endemic locales. Here, we compare the concordance of self-reported DENV infection histories relative to serological confirmation of prior DENV exposure and report very low sensitivity and limited specificity, perhaps placing as many as one in seven individuals reporting a history of prior dengue illness at risk for an adverse outcome with receipt of Dengvaxia. We propose that reported histories of dengue illness are not sufficiently accurate to replace serological testing prior to vaccination.

Dengue virus (DENV) is transmitted by infectious mosquitoes during blood-feeding via saliva containing biologically-active proteins. Here, we examined the effect of varying DENV infection modality in rhesus macaques in order to improve the DENV nonhuman primate (NHP) challenge model. NHPs were exposed to DENV-1 via subcutaneous or intradermal inoculation of virus only, intradermal inoculation of virus and salivary gland extract, or infectious mosquito feeding. The infectious mosquito feeding group exhibited delayed onset of viremia, greater viral loads, and altered clinical and immune responses compared to other groups. After 15 months, NHPs in the subcutaneous and infectious mosquito feeding groups were re-exposed to either DENV-1 or DENV-2. Viral replication and neutralizing antibody following homologous challenge were suggestive of sterilizing immunity, whereas heterologous challenge resulted in productive, yet reduced, DENV-2 replication and boosted neutralizing antibody. These results show that a more transmission-relevant exposure modality resulted in viral replication closer to that observed in humans., Author summary Dengue virus is transmitted into the skin of humans by mosquitoes as they take a blood meal. In contrast, many animal models are infected in the laboratory using a syringe to inject below the skin. Here, we looked at how different routes and methods of infection altered dengue infection in rhesus macaques. We found that infection via mosquito feeding resulted in a number of changes compared to other routes and methods, including a delay in the time to detection of dengue virus and overall greater quantities of dengue virus in the blood, and changes in the amounts of various components of blood that have been associated with dengue disease in humans. After 15 months, we exposed the macaques again to either the same or a different type of dengue virus. We found that animals exposed to the same type of dengue virus were protected from infection, whereas those animals exposed to a different type were only partially protected. Overall, our results show that dengue virus delivery using the natural transmission vector, mosquitoes, results in infection that is closer to what is observed in humans and may influence the interpretation of future studies of candidate vaccines.

Background Hyperendemic circulation of all four types of dengue virus (DENV-1-4) has expanded globally, fueling concern for increased incidence of severe dengue. While the majority of DENV infections are subclinical, epidemiologic studies suggest that type-cross-reactive immunity can influence disease outcome in subsequent infections. The mechanisms controlling these differential clinical outcomes remain poorly defined. Methodology/Principal findings Blood samples were collected from a cohort of school-aged Thai children who subsequently experienced a subclinical DENV infection or developed dengue illness. PBMC collected prior to infection were stimulated in vitro with DENV and the secretion of 30 cytokines was measured using a multiplexed, bead-based array. Significant differences were found in cytokine production based on both the type of DENV used for stimulation and the occurrence of clinical illness. Secretion of IL-15 and MCP-1 was significantly higher by PBMC of subjects who later developed symptomatic DENV infection. In addition, IL-6 was produced by PBMC from all subjects who subsequently developed symptomatic infection, versus 59% of subjects who had subclinical infection. Secretion of IL-12, IL-2R, MIP-1α, RANTES, GM-CSF, and TNFα was significantly lower by PBMC from subjects with symptomatic infection. Conclusions/Significance These data demonstrate significant differences in pre-existing immune responses to DENV associated with the clinical outcome of subsequent infection. The finding of higher levels of some cytokines in subjects with symptomatic infection and higher levels of other cytokines in subjects with subclinical infection supports the existence of both protective and pathologic immune profiles. Clinical-immunological correlations identified in the context of natural DENV infection may be useful for evaluating immune responses to dengue vaccines., Author summary Dengue is one of the most prevalent mosquito-borne infectious diseases worldwide. It is caused by one of four viruses, types 1–4, and ranges in severity from subclinical (mild or no symptoms) to dengue fever (febrile illness with headache and bone pain), or less frequently, dengue hemorrhagic fever, where patients experience leaky blood vessels, sometimes significant bleeding, and may be severe enough to cause death. While many risk factors have been associated with development of severe dengue, sequential infection with different virus types is a major factor, implying that the immune response generated after an initial infection is partly responsible for making subsequent exposure clinically worse. This study sought to identify profiles of immune markers that correlate with increased or decreased risk of dengue. Using samples from individuals ~5 months prior to dengue virus infection, who later experienced either subclinical infection or overt disease, we modeled virus exposure in vitro and compared the production of various immune proteins between the two outcome groups. Three of the proteins studied were produced at higher levels by subjects who subsequently had dengue, and six of the proteins were produced at higher levels by subjects who subsequently had subclinical infection. These results help define what constitutes beneficial versus potentially harmful immune responses, aiding in the design of effective dengue vaccines.

Zika virus (ZIKV) recently caused a pandemic complicated by Guillain-Barre syndrome (GBS) and birth defects. ZIKV is structurally similar to the dengue viruses (DENV) and in vitro studies suggest antibody dependent enhancement occurs in ZIKV infections preceded by DENV; however, the clinical significance of this remains unclear. We undertook a PRISMA-adherent systematic review of all current human and non-human primate (NHP) data to determine if prior infection with DENV, compared to DENV-naïve hosts, is associated with a greater risk of ZIKV clinical complications or greater ZIKV peak viremia in vivo. We identified 1146 studies in MEDLINE, EMBASE and the grey literature, of which five studies were eligible. One human study indicated no increase in the risk of GBS in ZIKV infections with prior DENV exposure. Two additional human studies showed a small increase in ZIKV viremia in those with prior DENV exposure; however, this was not statistically significant nor was it associated with an increase in clinical severity or adverse pregnancy outcomes. While no meta-analysis was possible using human data, a pooled analysis of the two NHP studies leveraging extended data provided only weak evidence of a 0.39 log10 GE/mL rise in ZIKV viremia in DENV experienced rhesus macaques compared to those with no DENV exposure (p = 0.22). Using a customized quality grading criteria, we further show that no existing published human studies have offered high quality measurement of both acute ZIKV and antecedent DENV infections. In conclusion, limited human and NHP studies indicate a small and non-statistically significant increase in ZIKV viremia in DENV-experienced versus DENV-naïve hosts; however, there is no evidence that even a possible small increase in ZIKV viremia would correlate with a change in ZIKV clinical phenotype. More data derived from larger sample sizes and improved sero-assays are needed to resolve this question, which has major relevance for clinical prognosis and vaccine design., Author summary Zika virus (ZIKV) is a mosquito borne virus that recently caused a large epidemic with some cases complicated by ascending paralysis (Guillain-Barre syndrome) and birth defects. One major concern is that such complications may be more common in those who have had previous infection with the closely related mosquito-borne dengue virus (DENV) which also circulates in the tropics. Here, we undertook a thorough, structured review of all human and high-order animal literature to synthesize the current evidence about whether ZIKV outcomes are worse in those with previous DENV exposure. We further undertook a pooled analysis across the two major non-human primate studies to improve statistical power. We summarize that, in humans and in non-human primates, prior DENV exposure may lead to a small increase in ZIKV viral load during infection. However, we do not show that any possible increase in ZIKV viral replication is associated with a higher rate of Zika complications or Zika clinical severity. We further graded the quality of these published literature and indicate that substantial improvements in the immunological measurement of ZIKV and DENV exposure in humans are needed to answer these and other pertinent questions about these two epidemic pathogens.

Background Zika virus (ZIKV) has extended its known geographic distribution to the New World and is now responsible for severe clinical complications in a subset of patients. While substantial genetic and vector susceptibility data exist for ZIKV, less is known for the closest related flavivirus, Spondweni virus (SPONV). Both ZIKV and SPONV have been known to circulate in Africa since the mid-1900s, but neither has been genetically characterized by gene and compared in parallel. Furthermore, the susceptibility of peridomestic mosquito species incriminated or suspected in the transmission of ZIKV to SPONV was unknown. Methodology/Principal Findings In this study, two geographically distinct strains of SPONV were genetically characterized and compared to nine genetically and geographically distinct ZIKV strains. Additionally, the susceptibility of both SPONV strains was determined in three mosquito species. The open reading frame (ORF) of the SPONV 1952 Nigerian Chuku strain, exhibited a nucleotide and amino acid identity of 97.8% and 99.2%, respectively, when compared to the SPONV 1954 prototype South African SA Ar 94 strain. The ORF of the SPONV Chuku strain exhibited a nucleotide and amino acid identity that ranged from 68.3% to 69.0% and 74.6% to 75.0%, respectively, when compared to nine geographically and genetically distinct strains of ZIKV. The ORF of the nine African and Asian lineage ZIKV strains exhibited limited nucleotide divergence. Aedes aegypti, Ae. albopictus and Culex quinquefasciatus susceptibility and dissemination was low or non-existent following artificial infectious blood feeding of moderate doses of both SPONV strains. Conclusions/Significance SPONV and ZIKV nucleotide and amino acid divergence coupled with differences in geographic distribution, ecology and vector species support previous reports that these viruses are separate species. Furthermore, the low degree of SPONV infection or dissemination in Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus following exposure to two geographically and genetically distinct virus strains suggest a low potential for these species to serve as vectors., Author Summary Spondweni virus (SPONV) is a mosquito-transmitted flavivirus reported in Africa. Human infection with SPONV may result in a febrile illness similar to symptomatic Zika virus (ZIKV) infection, as well as many other tropical infections. Previously, little was known about the genetic relationships between SPONV and ZIKV. Additionally, the ability of SPONV to infect peridomestic mosquito species suspected or incriminated in the transmission of ZIKV was unknown. Both SPONV strains exhibited a high degree of nucleotide and amino acid identity to each other, but considerable nucleotide and amino acid divergence to ZIKV. The open reading frame (ORF) of the nine African and Asian lineage ZIKV strains originally isolated in West Africa, Central Africa, East Africa, Southeast Asia, the Pacific Islands and the New World all exhibited limited nucleotide divergence. Both strains of SPONV exhibited a low degree of infection and/or dissemination in Aedes albopictus, Ae. aegypti and Culex quinquefasciatus mosquitoes suggesting that these species have a low potential to serve as vectors. These results coupled with differences in geographic distribution, ecology and vector species indicate that SPONV and ZIKV are similar but separate species.

Dengue viruses (DENVs) are among the most rapidly and efficiently spreading arboviruses. WHO recently estimated that about half of the world’s population is now at risk for DENV infection. There is no specific treatment or vaccine available to treat or prevent DENV infections. Here, we report the development of a novel dengue nanovaccine (DNV) composed of UV-inactivated DENV-2 (UVI-DENV) and Mycobacterium bovis Bacillus Calmette-Guerin cell wall components (BCG-CWCs) loaded into chitosan nanoparticles (CS-NPs). CS-NPs were prepared by an emulsion polymerization method prior to loading of the BCG-CWCs and UVI-DENV components. Using a scanning electron microscope and a zetasizer, DNV was determined to be of spherical shape with a diameter of 372.0 ± 11.2 nm in average and cationic surface properties. The loading efficacies of BCG-CWCs and UVI-DENV into the CS-NPs and BCG-CS-NPs were up to 97.2 and 98.4%, respectively. THP-1 cellular uptake of UVI-DENV present in the DNV was higher than soluble UVI-DENV alone. DNV stimulation of immature dendritic cells (iDCs) resulted in a significantly higher expression of DCs maturation markers (CD80, CD86 and HLA-DR) and induction of various cytokine and chemokine productions than in UVI-DENV-treated iDCs, suggesting a potential use of BCG- CS-NPs as adjuvant and delivery system for dengue vaccines., Author Summary Among the flavivirus family, dengue virus (DENV) represents the most important public health issue, affecting a large area of the world. The incidence rate of DENV infections is increasing every year. In lieu of the uncontrolled mosquito vector and the lack of specific treatments for DENV disease, an effective dengue vaccine is required. Several dengue vaccines have been developed but none of them are licensed yet. Here, we reported a novel dengue nanovaccine (DNV), composed of UV-inactivated DENV (UVI-DENV) immunogen loaded on BCG adjuvanted chitosan nanoparticle (BCG-CS-NPs). DNV particles were of spherical shape with a diameter of 372 nm and cationic surface properties. The immunogenic properties of DNV have been demonstrated. BCG-CS-NPs could improve the uptake of UVI-DENV by THP1 cells. DNV could also stimulate iDCs maturation, an important step in antigen processing and presentation to T cells and up-regulation of CD80, CD86 and HLA-DR expression. In addition, the DNV was capable of increasing the secretion of various cytokines and chemokines in iDCs. These immunogenic properties point to the potential use of DNV as an effective dengue vaccine.

Internet-based surveillance methods for vector-borne diseases (VBDs) using "big data" sources such as Google, Twitter, and internet newswire scraping have recently been developed, yet reviews on such "digital disease detection" methods have focused on respiratory pathogens, particularly in high-income regions. Here, we present a narrative review of the literature that has examined the performance of internet-based biosurveillance for diseases caused by vector-borne viruses, parasites, and other pathogens, including Zika, dengue, other arthropod-borne viruses, malaria, leishmaniasis, and Lyme disease across a range of settings, including low- and middle-income countries. The fundamental features, advantages, and drawbacks of each internet big data source are presented for those with varying familiarity of "digital epidemiology." We conclude with some of the challenges and future directions in using internet-based biosurveillance for the surveillance and control of VBD.

Background Long-term homologous and temporary heterologous protection from dengue virus (DENV) infection may be mediated by neutralizing antibodies. However, neutralizing antibody titers (NTs) have not been clearly associated with protection from infection. Methodology/Principal Findings Data from two geographic cluster studies conducted in Kamphaeng Phet, Thailand were used for this analysis. In the first study (2004–2007), cluster investigations of 100-meter radius were triggered by DENV-infected index cases from a concurrent prospective cohort. Subjects between 6 months and 15 years old were evaluated for DENV infection at days 0 and 15 by DENV PCR and IgM ELISA. In the second study (2009–2012), clusters of 200-meter radius were triggered by DENV-infected index cases admitted to the provincial hospital. Subjects of any age ≥6 months were evaluated for DENV infection at days 0 and 14. In both studies, subjects who were DENV PCR positive at day 14/15 were considered to have been “susceptible” on day 0. Comparison subjects from houses in which someone had documented DENV infection, but the subject remained DENV negative at days 0 and 14/15, were considered “non-susceptible.” Day 0 samples were presumed to be from just before virus exposure, and underwent plaque reduction neutralization testing (PRNT). Seventeen “susceptible” (six DENV-1, five DENV-2, and six DENV-4), and 32 “non-susceptible” (13 exposed to DENV-1, 10 DENV-2, and 9 DENV-4) subjects were evaluated. Comparing subjects exposed to the same serotype, receiver operating characteristic (ROC) curves identified homotypic PRNT titers of 11, 323 and 16 for DENV-1, -2 and -4, respectively, to differentiate “susceptible” from “non-susceptible” subjects. Conclusions/Significance PRNT titers were associated with protection from infection by DENV-1, -2 and -4. Protective NTs appeared to be serotype-dependent and may be higher for DENV-2 than other serotypes. These findings are relevant for both dengue epidemiology studies and vaccine development efforts., Author Summary Dengue is caused by four different dengue virus serotypes (DENV-1, -2, -3, -4). Infection induces long-term protection against the same serotype, but only short-term protection, and possible enhancement, from different serotypes. DENV neutralizing antibody titers (NTs) are thought to mediate protection or modify disease. Association of NTs with protection from infection has not, however, been clearly demonstrated. We analyzed data from two geographic clusters studies conducted in Kamphaeng Phet, Thailand, in which DENV NTs just before virus exposure were compared between DENV-infected “susceptible” and non-infected “non-susceptible” subjects. NTs appeared to be associated with protection against DENV-1, -2, and -4, but at different NT cutoff levels, with the cutoff for DENV-2 appearing to be the highest. These findings are relevant for ongoing efforts to investigate dengue epidemiology and develop dengue vaccine candidates.

Background Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death. Methodology/Principal Findings Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons. Conclusions/Significance The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE., Author Summary Japanese encephalitis (JE) is one of the most important causes of viral encephalitis worldwide, with no specific antiviral treatment available. Despite some recent successes with widespread vaccination, JE will likely remain an important public health problem; because the virus is mosquito-borne and has natural animal hosts, it will never be eradicated. We have little understanding of what determines the severity and outcome of infection. Data from human post mortem studies is very limited because of cultural constraints on autopsies in areas where JE occurs. Circumstantial evidence suggests that in addition to cytopathology caused directly by infection of neurons, there may be bystander cell death of non-infected neurons, caused by an excessive inflammatory response. Our study used archived brain samples from a prior challenge study in a validated macaque model of JE. We stained for the presence of JEV antigen, apoptosis, and pro-inflammatory markers in affected areas, such as the thalamus and brainstem. We show that bystander neuronal cell death is important, and elucidate the inflammatory and apoptotic mechanisms underlying it. Currently there is no proven efficacious therapy for most viral infections of the central nervous system, including JE. Novel strategies for treating such infections are urgently needed. Our findings suggest new anti-inflammatory and anti-apoptotic therapeutic approaches may be useful in treating this debilitating disease.

Background Accurate determination of neutralization antibody titers supports epidemiological studies of dengue virus transmission and vaccine trials. Neutralization titers measured using the plaque reduction neutralization test (PRNT) are believed to provide a key measure of immunity to dengue viruses, however, the assay's variability is poorly understood, making it difficult to interpret the significance of any assay reading. In addition there is limited standardization of the neutralization evaluation point or statistical model used to estimate titers across laboratories, with little understanding of the optimum approach. Methodology/Principal Findings We used repeated assays on the same two pools of serum using five different viruses (2,319 assays) to characterize the variability in the technique under identical experimental conditions. We also assessed the performance of multiple statistical models to interpolate continuous values of neutralization titer from discrete measurements from serial dilutions. We found that the variance in plaque reductions for individual dilutions was 0.016, equivalent to a 95% confidence interval of 0.45–0.95 for an observed plaque reduction of 0.7. We identified PRNT75 as the optimum evaluation point with a variance of 0.025 (log10 scale), indicating a titer reading of 1∶500 had 95% confidence intervals of 1∶240–1∶1000 (2.70±0.31 on a log10 scale). The choice of statistical model was not important for the calculation of relative titers, however, cloglog regression out-performed alternatives where absolute titers are of interest. Finally, we estimated that only 0.7% of assays would falsely detect a four-fold difference in titers between acute and convalescent sera where no true difference exists. Conclusions Estimating and reporting assay uncertainty will aid the interpretation of individual titers. Laboratories should perform a small number of repeat assays to generate their own variability estimates. These could be used to calculate confidence intervals for all reported titers and allow benchmarking of assay performance., Author Summary Plaque Reduction Neutralization Tests (PRNTs) remain the most popular approach to characterize an individual's ability to neutralize dengue viruses and are widely used in both epidemiological studies and vaccine trials. However, the underlying variability in the assay is poorly understood, hindering the interpretation of PRNT titer estimates. Further, there is little standardization of its use across laboratories limiting our ability to compare results across settings. Here we used many repeated experiments on the same serum under identical laboratory conditions to estimate the variance in titer measurements. We also identified both the optimum PRNT evaluation point and statistical model to calculate titers. By providing an estimate of the variability in the assay, laboratories will be able to provide a confidence bound on individual PRNT readings. In addition by providing recommended statistical approaches that could be used across laboratories, our findings will help the standardization of the assay across settings.

Background Currently, no dengue NS1 detection kit has regulatory approval for the diagnosis of acute dengue fever. Here we report the sensitivity and specificity of the InBios DEN Detect NS1 ELISA using a panel of well characterized human acute fever serum specimens. Methodology/Principal Findings The InBios DENV Detect NS1 ELISA was tested using a panel composed of 334 serum specimens collected from acute febrile patients seeking care in a Bangkok hospital in 2010 and 2011. Of these patients, 314 were found to have acute dengue by either RT-PCR and/or anti-dengue IgM/IgG ELISA. Alongside the InBios NS1 ELISA kit, we compared the performance characteristics of the BioRad Platelia NS1 antigen kit. The InBios NS1 ELISA Ag kit had a higher overall sensitivity (86% vs 72.8%) but equal specificity (100%) compared to the BioRad Platelia kit. The serological status of the patient significantly influenced the outcome. In primary infections, the InBios NS1 kit demonstrated a higher sensitivity (98.8%) than in secondary infections (83.5%). We found significant variation in the sensitivity of the InBios NS1 ELISA kit depending on the serotype of the dengue virus and also found decreasing sensitivity the longer after the onset of illness, showing 100% sensitivity early during illness, but dropping below 50% by Day 7. Conclusion/Significance The InBios NS1 ELISA kit demonstrated high accuracy when compared to the initial clinical diagnosis with greater than 85% agreement when patients were clinically diagnosed with dengue illness. Results presented here suggest the accurate detection of circulating dengue NS1 by the InBios DENV Detect NS1 ELISA can provide clinicians with a useful tool for diagnosis of early dengue infections., Author Summary Infections by the mosquito-transmitted dengue virus continue to increase, threatening the health of people in over a hundred countries worldwide. The lack of effective mosquito control, licensed dengue vaccines or specific therapeutics to treat dengue infections presents challenges to reduce the burden of this disease. Rapid and accurate diagnosis of dengue infections is critical for the reduction of patients' morbidity and mortality. We present data that support the use of the InBios DENV Detect NS1 ELISA for the detection of dengue NS1 in patient serum. The InBios NS1 ELISA kit was tested against sera collected from acute fever patients seeking medical care in a Bangkok, Thailand hospital during 2010 and 2011. The data demonstrate the InBios DENV Detect NS1 ELISA accurately detects circulating dengue NS1 in the tested specimens, demonstrating high sensitivity and specificity. Nonetheless, the sensitivity of the NS1 ELISA kits was found to vary depending on the serological status of the patient (primary versus secondary dengue infection), time of specimen collection and dengue serotype. Further, the performance characteristics of the InBios DENV Detect NS1 ELISA were found to meet and exceed those of the commonly used Platelia Dengue NS1 Ag kit.

Background Dengue viruses are a major cause of morbidity in tropical and subtropical regions of the world. Inapparent dengue is an important component of the overall burden of dengue infection. It provides a source of infection for mosquito transmission during the course of an epidemic, yet by definition is undetected by health care providers. Previous studies of inapparent or subclinical infection have reported varying ratios of symptomatic to inapparent dengue infection. Methodology/Principal Findings In a prospective study of school children in Northern Thailand, we describe the spatial and temporal variation of the symptomatic to inapparent (S:I) dengue illness ratio. Our findings indicate that there is a wide fluctuation in this ratio between and among schools in a given year and within schools over several dengue seasons. The most important determinants of this S:I ratio for a given school were the incidence of dengue infection in a given year and the incidence of infection in the preceding year. We found no association between the S:I ratio and age in our population. Conclusions/Significance Our findings point to an important aspect of virus-host interactions at either a population or individual level possibly due to an effect of heterotypic cross-reactive immunity to reduce dengue disease severity. These findings have important implications for future dengue vaccines., Author Summary Dengue viruses are a major cause of illness and hospitalizations in tropical and subtropical regions of the world. Severe dengue illness can cause prolonged hospitalization and in some cases death in both children and adults. The majority of dengue infections however are inapparent, producing little clinical illness. Little is known about the epidemiology or factors that determine the incidence of inapparent infection. We describe in a study of school children in Northern Thailand the changing nature of symptomatic and inapparent dengue infection. We demonstrate that the proportion of inapparent dengue infection varies widely among schools during a year and within schools during subsequent years. Important factors that determine this variation are the amount of dengue infection in a given and previous year. Our findings provide an important insight in the virus-host interaction that determines dengue severity, how severe a dengue epidemic may be in a given year, and important clues on how a dengue vaccine may be effective.

Background It is unclear whether dengue serotypes differ in their propensity to cause severe disease. We analyzed differences in serotype-specific disease severity in children presenting for medical attention in Bangkok, Thailand. Methodology/Principal Findings Prospective studies were conducted from 1994 to 2006. Univariate and multivariate logistic and multinomial logistic regressions were used to determine if dengue hemorrhagic fever (DHF) and signs of severe clinical disease (pleural effusion, ascites, thrombocytopenia, hemoconcentration) were associated with serotype. Crude and adjusted odds ratios were calculated. There were 162 (36%) cases with DENV-1, 102 (23%) with DENV-2, 123 (27%) with DENV-3, and 64 (14%) with DENV-4. There was no significant difference in the rates of DHF by serotype: DENV-2 (43%), DENV-3 (39%), DENV-1 (34%), DENV-4 (31%). DENV-2 was significantly associated with increased odds of DHF grade I compared to DF (OR 2.9 95% CI 1.1, 8.0), when using DENV-1 as the reference. Though not statistically significant, DENV-2 had an increased odds of total DHF and DHF grades II, III, and IV. Secondary serologic response was significantly associated with DHF (OR 6.2) and increased when considering more severe grades of DHF. DENV-2 (9%) and -4 (3%) were significantly less often associated with primary disease than DENV-1 (28%) and -3 (33%). Restricting analysis to secondary cases, we found DENV-2 and DENV-3 to be twice as likely to result in DHF as DEN-4 (p = 0.05). Comparing study years, we found the rate of DHF to be significantly less in 1999, 2000, 2004, and 2005 than in 1994, the study year with the highest percentage of DHF cases, even when controlling for other variables. Conclusions/Significance As in other studies, we find secondary disease to be strongly associated with DHF and with more severe grades of DHF. DENV-2 appears to be marginally associated with more severe dengue disease as evidenced by a significant association with DHF grade I when compared to DENV-1. In addition, we found non-significant trends with other grades of DHF. Restricting the analysis to secondary disease we found DENV-2 and -3 to be twice as likely to result in DHF as DEN-4. Differences in severity by study year may suggest that other factors besides serotype play a role in disease severity., Author Summary The four dengue viruses (DENV) represent the most common human arbovirus infections in the world and are currently a challenging problem, particularly in the tropical and subtropical regions of Asia and the Americas. Infection with DENV may produce symptoms of varying severity. While access to care, appropriate interventions, host genetic factors, and previous exposure to DENV are all known to affect the outcome of the infection, it is not entirely understood why some individuals develop more severe disease. It has been hypothesized that the four dengue serotypes differ in disease severity and clinical manifestations. This analysis assessed whether there were significant differences in severity of disease caused by the dengue serotypes in a pediatric population in Thailand. We found significant and non-significant correlations between dengue serotype 2 infection and more severe dengue disease. We also found that individual serotypes varied in disease severity between study years, perhaps supporting the hypothesis that the particular sequences of primary and secondary DENV infections influence disease severity.

Revealing the patterns and determinants of the spread of dengue virus (DENV) at local scales is central to understanding the epidemiology and evolution of this major human pathogen. We performed a phylogenetic analysis of the envelope (E) genes of DENV-1, -2, -3, and -4 isolates (involving 97, 23, 5, and 74 newly collected sequences, respectively) sampled from school-based cohort and village-based cluster studies in Kamphaeng Phet, Thailand, between 2004 and 2007. With these data, we sought to describe the spatial and temporal patterns of DENV spread within a rural population where a future vaccine efficacy trial is planned. Our analysis revealed considerable genetic diversity within the study population, with multiple lineages within each serotype circulating for various lengths of time during the study period. These results suggest that DENV is frequently introduced into both semi-urban and rural areas in Kamphaeng Phet from other populations. In contrast, the persistence of viral lineages across sampling years was observed less frequently. Analysis of phylogenetic clustering indicated that DENV transmission was highly spatially and temporally focal, and that it occurred in homes rather than at school. Overall, the strength of temporal clustering suggests that seasonal bottlenecks in local DENV populations facilitate the invasion and establishment of viruses from outside of the study area, in turn reducing the extent of lineage persistence., Author Summary Long-term cohort studies of dengue virus (DENV), the most common vector-borne viral disease of humans, are essential to understand the epidemiology and evolution of this important human pathogen, and may assist in predicting the evolutionary response to vaccination. We utilized DENV gene sequences and information on the locations and timing of infected children within a primary school-based cohort in Kamphaeng Phet, Thailand to investigate the spatial and temporal relationships among viruses isolated from 2004 to 2007. We found that all four DENV serotypes circulated in the region during the study period, with the presence of multiple viral lineages within each serotype. Viruses sampled closely in time and space were generally very closely related. More genetic variation was observed across districts in a given year and within the same district across different years. The high genetic similarity among viruses during each season and the rare persistence of these lineages through multiple seasons suggest that seasonal reductions in the force of infection through changes in mosquito transmission and fluctuations in human population immunity are key factors shaping the genetic diversity of dengue virus diversity in this region. The importation of DENV by human movement from other populations is therefore an important generator of DENV diversity even in hyperendemic areas.

Background Currently dengue viruses (DENV) pose an increasing threat to over 2.5 billion people in over 100 tropical and sub-tropical countries worldwide. International air travel is facilitating rapid global movement of DENV, increasing the risk of severe dengue epidemics by introducing different serotypes. Accurate diagnosis is critical for early initiation of preventive measures. Different reverse transcriptase PCR (RT-PCR) methods are available, which should be evaluated and standardized. Epidemiological and laboratory-based surveillance is required to monitor and guide dengue prevention and control programmes, i.e., by mosquito control or possible vaccination (as soon as an effective and safe vaccine becomes available). Objective The purpose of the external quality assurance (EQA) study described is to assess the efficiency and accuracy of dengue molecular diagnosis methods applied by expert laboratories. Study Design A panel of 12 human plasma samples was distributed and tested for DENV-specific RNA. The panel comprised 9 samples spiked with different DENV serotypes (DENV-1 to DENV-4), including 10-fold dilution series of DENV-1 and DENV-3. Two specificity controls consisted of a sample with a pool of 4 other flaviviruses and a sample with chikungunya virus. A negative control sample was also included. Results Thirty-seven laboratories (from Europe, Middle East Asia, Asia, the Americas/Caribbean, and Africa) participated in this EQA study, and reports including 46 sets of results were returned. Performance among laboratories varied according to methodologies used. Only 5 (10.9%) data sets met all criteria with optimal performance, and 4 (8.7%) with acceptable performance, while 37 (80.4%) reported results showed the need for improvement regarding accomplishment of dengue molecular diagnosis. Failures were mainly due to lack of sensitivity and the presence of false positives. Conclusions The EQA provides information on each laboratory's efficacy of RT-PCR techniques for dengue diagnosis and indicates for most laboratories an urgent need to improve sensitivity and specificity., Author Summary Dengue viruses (DENV) are the most widespread arthropod-borne viruses which have shown an unexpected geographic expansion, as well as an increase in the number and severity of outbreaks in the last decades. In this context, the accurate diagnosis and reliable surveillance of dengue infections are essential. The laboratory diagnosis of dengue relies on the use of several methods detecting markers of DENV infection present in patient serum. Molecular diagnosis methods are usually rapid, sensitive, and simple when correctly standardized. Moreover, PCR-based diagnosis techniques are able to readily detect DENV during the acute phase of the disease and may assume an important role in dengue diagnosis and surveillance. Different reverse transcriptase PCR (RT-PCR) methods have been developed and are currently available and should be standardized in each laboratory to maintain high quality performance. In this work an External quality assessment (EQA) activity has been carried out to evaluate the accuracy and quality of laboratory data for the molecular diagnosis and surveillance of dengue, which involved worldwide dengue reference laboratories. In conclusion, RT-PCR techniques for dengue diagnosis applied by the participating laboratories demonstrated the need of further improvement in most laboratories.