Dengue virus (DENV) infection imposes a major global health burden, with around 100 million symptomatic cases and about 40 000 deaths each year. Despite decades of effort, no direct-acting antivirals are licensed and current vaccines show serotype-dependent and baseline serostatus-dependent efficacy, raising concerns about antibody-dependent enhancement. Early phase therapeutic trials therefore rely mainly on quantitative viraemia as a virological endpoint. However, DENV RNA in blood becomes undetectable soon after presentation, and may be an imperfect surrogate for infectious virus owing to defective interfering particles and continued replication in tissues. The secreted non-structural protein 1 (NS1) correlates with viral replication and disease severity in many studies and remains detectable after virological clearance, making it an attractive complementary biomarker. Here, we review mechanistic and clinical evidence linking NS1 to viral burden, vascular leakage and thrombocytopenia, and summarize data from human challenge studies, natural infection cohorts and pharmacometric modelling. We highlight the extended analytical window and growing availability of quantitative NS1 assays and propose practical analysis frameworks for incorporating NS1 into early phase dengue trials alongside viral RNA. We also discuss limitations, including serotype, immune status and assay-dependent effects and outline priorities for standardization of NS1-based endpoints. This article is part of the Theo Murphy meeting issue 'Evaluating anti-infective drugs'.

Dengue virus (DENV) infection is a major global health problem. While DENV infection rarely results in serious complications, the more severe illness dengue hemorrhagic fever (DHF) has a significant mortality rate due to the associated plasma leakage that may lead to hypovolemic shock. Proper care thus requires identifying patients with DHF among those with suspected dengue so that they can be provided with adequate and prompt fluid replacement. In this study we used seventeen years of pediatric patient data from a prospective cohort study in two hospitals in Thailand to develop models to predict DHF among patients with suspected dengue infection. We produced models for a general hospital setting and for a primary care unit setting lacking lab facilities. The best model using combined data from both hospitals achieved an AUC of 0.90 for the general hospital setting and 0.79 for the primary care unit setting. We then investigated the generalizability of the models by training models with data from one hospital and testing them with data from the other. For some models, we found a significant reduction in performance. Possible sources of this are differences in how attributes are defined or measured and differences in the hematological parameters of the two patient populations. We conclude that while high accuracy prediction of DHF is possible, care must be taken when applying DHF predictive models from one clinical setting to another.

This study introduces a novel approach to dengue diagnostics by leveraging surface-enhanced Raman spectroscopy (SERS) coupled to machine learning. This method addresses the critical need for rapid and accurate identification of dengue virus (DENV) infection and prediction of the disease severity. For the first time, a commercialized SERS substrate is applied to analyze plasma samples from 60 pediatric patients, equally distributed among other febrile illnesses (OFI), dengue fever (DF), and dengue hemorrhagic fever (DHF) cases. This innovative application of SERS technology captures unique molecular signature characteristics of each disease state, offering a new paradigm in viral diagnostics. Our methodology employs various machine learning algorithms, notably linear discriminant analysis (LDA) and logistic regression, to classify the SERS spectral data. The models exhibited exceptional performance in distinguishing dengue from OFI, with both achieving an outstanding area under the curve (AUC) of 0.99. In the more complex task of discriminating between DF, DHF, and OFI, LDA demonstrated remarkable AUC values of 0.81, 0.90, and 0.99, respectively, while logistic regression slightly outperformed with AUC values of 0.82, 0.88, and 0.99. Even in the challenging differentiation of DF from DHF, the models achieved notable AUC values of 0.84 (LDA) and 0.79 (logistic regression). This pioneering SERS-based approach represents a significant advancement over existing dengue diagnostic methods, offering unparalleled speed and accuracy, particularly in resource-limited settings. By providing a new tool for early detection and classification of dengue severity, this innovative technique has the potential to improve patient outcomes and guide targeted therapeutic strategies in dengue management.

Background:Dengue is an immune-mediated disease with severe manifestations often linked to secondary infections. Dengue virus nonstructural protein 1 (DENV NS1) and corresponding antibodies are thought to play a crucial role in disease pathogenesis. Methods:This study investigated the kinetics of anti-NS1 antibody responses in relation to NS1 levels and NS1 immune complexes (ICs) in pediatric dengue patients with varying disease severity following secondary DENV infections with all four serotypes, using retrospective plasma samples collected in Thailand and analyzed by enzyme-linked immunosorbent assays (ELISAs). Plasma samples from pediatric patients with other febrile illnesses (OFI) and healthy adults served as non-dengue control groups. Results:Anti-NS1 IgG (particularly IgG1 and IgG4) and IgA persisted for at least 6 months after illness and were associated with increased dengue severity, whereas the shorter-lived anti-NS1 IgM was higher in patients with milder dengue. Anti-NS1 IgG also persisted notably longer than anti-DENV IgG. Anti-NS1 IgG3 showed a transient surge across all four serotypes, suggesting its potential as a marker of recent infection. NS1 proteins and NS1 ICs were positively correlated, inversely related to anti-NS1 IgG, and strongly associated with severe dengue. Conclusions:Anti-NS1 antibody isotypes and IgG subclasses exhibited distinct associations with dengue severity, influenced by both infecting serotypes and time since illness onset. Correlations among NS1, anti-NS1 antibodies, and NS1 ICs were observed during the acute phase of secondary infection, potentially influencing dengue disease outcomes. These findings enhance our understanding of dengue immunopathogenesis and may inform improved diagnostic and surveillance tools.

ObjectivesEffective, real-time surveillance of dengue may provide early warning of outbreaks and support targeted disease-control intervention but requires widespread accurate diagnosis and timely case reporting. Research directing innovation in diagnostics for dengue surveillance is lacking. This study aimed to describe experience and requirements of relevant prospective users.DesignA qualitative, focus group study was conducted.ParticipantsData were collected from 19 users of diagnostic technology who work across the Thai dengue surveillance system.Data collection and analysisContextual knowledge, experience and needs were explored in focus groups. Discussions were translated, transcribed, analysed thematically and mapped to Consolidated Framework for Implementation Research domains.ResultsParticipants expressed a need for rapid, accurate, serotype-specific tests which can be operated easily by non-expert users without laboratory equipment. They supported integration of diagnostics with surveillance systems and felt this would increase the quantity and speed of case reporting as well as provide healthcare professionals with up-to-date information about the number of cases locally, thereby aiding interpretation of test results. Concerns included those relating to data security and the cost of tests.ConclusionsEngagement to understand prospective user experience and requirements can improve relevance and uptake of new technology, leading to system efficiencies. The present study highlights specific needs for accurate, serotype-specific, remote-connected diagnostics which are integrated with surveillance systems and support dengue case reporting at the point-of-care.

The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.

Natural killer cells (NK cells) are the front line of immune cells to combat pathogens and able to influence the subsequent adaptive immune responses. One of the factors contributing to pathogenesis in dengue hemorrhagic fever (DHF) disease is aberrant immune activation during early phase of infection. This study explored the profile of NK cells in dengue infected pediatric patients with different degrees of disease severity. DHF patients contained higher frequency of activated NK cells but lower ratio of CD56dim:CD56bright NK subsets. Activated NK cells exhibited alterations in several NK receptors. Interestingly, the frequencies of NKp30 expressing activated NK cells were more pronounced in dengue fever (DF) than in DHF pediatric patients. In vitro functional analysis indicated that degranulation of NK cells in responding to dengue infected dendritic cells (DCs) required cell-cell contact and type I IFNs. Meanwhile, Interferon gamma (IFN-γ) production initially required cell-cell contact and type I IFNs followed by Interleukin-12 (IL-12), Interleukin-15 (IL-15) and Interleukin-18 (IL-18) resulting in the amplification of IFN-γ producing NK cells over time. This study highlighted the complexity and the factors influencing NK cells responses to dengue virus. Degree of activation, phenotypes of activated cells and the crosstalk between NK cells and other immune cells, could modulate the outcome of NK cells function in the dengue disease.

This study describes a blockade-of-binding assay for the determination of antibodies that recognize a selected set of serotype-specific or group-reactive epitopes in the envelope of dengue virus. By employing blood samples collected from dengue virus-infected or -immunized macaques, moderate to strong correlations of the epitope-blocking activities with the virus-neutralizing antibody titers were observed with serotype-specific blocking activities for each of the four dengue serotypes.

BACKGROUND:The error-prone replication of dengue virus (DENV) in host results in the highly diverse viral population. Together with the host factor, intra-host diversity may influence the disease severity. Therefore, it is worth investigating whether there is a correlation between intra-host genetic diversity and disease severity.OBJECTIVE:To investigate the genetic diversity in DENV for four serotypes of the dengue population from patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) using next-generation sequencing (NGS) technology.METHODS:Forty RNA samples categorized into eight groups by severity and serotypes were sequenced and analyzed for genetic variation. Analysis on the hot-cold genomic regions, selection pressure and correlation between genotype and disease severity were performed in this study.RESULTS:Comparison between the NGS data of the DF and DHF specimens showed conservation between their major populations with the consensus sequences for DF and DHF sharing 99% similarity. However, the minor populations in DF and DHF were more diverse. Many genes in DF had an #NS/#S ratio higher than in DHF. Only NS4B of DENV1 DF has #NS/#S ratio higher than one. Hot regions of the DF were detected in NS3 of DENV1, DENV2 and Envelope of DENV3, whereas the hot regions of the DHF samples were detected in the small region in 3'UTR of DENV2 and DENV3.CONCLUSIONS:Various explorations of the variations of DF and DHF were performed in this study. However, we have not yet found any specific characteristics of intra-host diversity associated with disease severity.

ABSTRACT Currently licensed dengue vaccines do not induce long-term protection in children without prior dengue virus exposure. A better understanding of the mechanism by which the immune system prevents dengue virus infection is urgently needed to improve vaccine efficacy. In this study, the induction of protective antibody responses against dengue virus infection was tested in a non-human primate model using the heterologous prime-boost vaccination approach. Groups of cynomolgus macaques were immunized with a priming dose of attenuated dengue viruses and followed by two booster doses of virus-like particles in four monovalent arms, or in the tetravalent arm (prM + E)-expressing plasmids. At 1 month post-immunization, all macaques had elevated levels of neutralizing antibodies, and live viral challenges revealed an overall protective efficacy of 91% (40/44 macaques protected) against infection with clinical isolates. Breakthrough infections occurred in macaques with distinctive antibody profiles at the time of challenge: two macaques had the lowest neutralizing antibodies against the respective DENV-1 and -4 challenge strains among the respective groups, whereas two other DENV-4-infected macaques exhibited high levels of neutralizing and virus-binding antibodies. The ratio of antibodies recognizing a DENV-4-specific epitope and those that bound viral particles was at the lowest levels in the latter DENV-4-infected macaques, indicating an underrepresentation of antibodies targeting the serotype-specific epitope. Protection among macaques challenged with DENV-2 or -3 coincided with vigorous EDIII-binding antibody responses induced by booster immunization. A combination of attenuated viruses for priming and non-infectious particle-based antigens for boosting may be a more effective means of preventing dengue. IMPORTANCE Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Dengue virus infection is a major global health problem. While dengue fever rarely results in serious complications, the more severe illness dengue hemorrhagic fever (DHF) has a significant mortality rate due to the associated plasma leakage. Proper care thus requires identifying patients with DHF among those with suspected dengue so that they can be provided with adequate and prompt fluid replacement. In this paper, we use 18 years of pediatric patient data collected prospectively from two hospitals in Thailand to develop models to predict DHF among patients with suspected dengue. The best model using pooled data from both hospitals achieved an AUC of 0.92. We then investigate the generalizability of the models by constructing a model for one hospital and testing it on the other, a question that has not yet been adequately explored in the literature on DHF prediction. For some models, we find significant degradation in performance. We show this is due to differences in attribute values among the two hospital patient populations. Possible sources of this are differences in the definition of attributes and differences in the pathogenesis of the disease among the two sub-populations. We conclude that while high predictive accuracy is possible, care must be taken when seeking to apply DHF predictive models from one clinical setting to another.

Partial cleavage of a dengue virus envelope protein, prM, by furin results in a mixture of extracellular particles with variable levels of maturation and infectivity. Partially mature particles can infect leukocytes via interaction between the prM-anti-prM antibody complex with Fcγ receptors. Known prM epitopes involved in antibody-mediated infection are localized to the pr domain. In this study, a group of murine anti-prM monoclonal antibodies with strong infection-enhancing activity was found to reduce the focus size of subsets of multiple dengue serotypes that they could enhance. By employing sets of overlapping peptides, four antibodies recognizing 2-mercaptoethanol-insensitive epitopes were mapped to a common tetrapeptide located distantly in the b-c loop and furin binding site. Substitution mutations of each, or both, of the tetrapeptides in virus-like particles, however, failed to reduce binding. Further mapping experiments were performed using immature virus-like particles with abolished furin binding site to minimize the differential influence of various pr substitutions on pr-M cleavage. Reduction of antibody binding was detected when single alanine substitutions were introduced into the 'a' strand and 'c' strand of pr domain. These findings suggest that the pr 'a and c' strands region is the major binding site of these unusual focus size-reducing anti-prM antibodies.

Non-structural protein 1 (NS1) is a glycoprotein component of dengue virus (DENV) that is essential for viral replication, infection and immune evasion. Immunization with NS1 has been shown to elicit antibody-mediated immune responses which protect mice against DENV infections. Here, we obtained peripheral blood mononuclear cells from human subjects with secondary dengue infections, which were used to construct a dengue immune phage library displaying single-chain variable fragments. Phage selective for DENV NS1 were obtained by biopanning. Twenty-one monoclonal antibodies (mAbs) against DENV NS1 were generated from the selected phage and characterized in detail. We found most anti-NS1 mAbs used IGHV1 heavy chain antibody genes. The mAbs were classified into strongly and weakly-reactive groups based on their binding to NS1 expressed in dengue virus 2 (DENV2)-infected cells. Antibody binding experiments with recombinant NS1 proteins revealed that the mAbs recognize conformational epitopes on the β-ladder domain (amino acid residues 178-273) of DENV NS1. Epitope mapping studies on alanine-substituted NS1 proteins identified distinct but overlapping epitopes. Protruding amino acids distributed around the spaghetti loop are required for the binding of the strongly-reactive mAbs, whereas the recognition residues of the weakly-reactive mAbs are likely to be located in inaccessible sites facing toward the cell membrane. This information could guide the design of an NS1 epitope-based vaccine that targets cross-reactive conserved epitopes on cell surface-associated DENV NS1.

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device–termed Cygnus–with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-μl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58–0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63–0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction.

Dengue virus (DENV) infection is a significant global health problem. There are no specific therapeutics or widely available vaccines. Early diagnosis is critical for patient management. Viral RNA detection by multiplex RT-PCR using multiple pairs of primers/probes allowing the simultaneous detection of all four DENV serotypes is commonly used. However, increasing the number of primers in the RT-PCR reaction reduces the sensitivity of detection due to the increased possibility of primer dimer formation. Here, a one tube, singleplex real-time RT-PCR specific to DENV 3′-UTR was developed for the detection and quantification of pan-DENV with no cross reactivity to other flaviviruses. The sensitivity of DENV detection was as high as 96.9% in clinical specimens collected at the first day of hospitalization. Our assay provided equivalent PCR efficiency and RNA quantification among each DENV serotype. The assay’s performance was comparable with previously established real-time RT-PCR targeting coding sequences. Using both assays on the same specimens, our results indicate the presence of defective virus particles in the circulation of patients infected with all serotypes. Dual regions targeting RT-PCR enhanced the sensitivity of viral genome detection especially during the late acute phase when viremia rapidly decline and an incomplete viral genome was clinically evident.

The non-structural protein-1 (NS1) of dengue virus (DENV) contributes to several functions related to dengue disease pathogenesis as well as diagnostic applications. Antibodies against DENV NS1 can cross-react with other co-circulating flaviviruses, which may lead to incorrect diagnosis. Herein, five anti-DENV NS1 monoclonal antibodies (mAbs) were investigated. Four of them (1F11, 2E3, 1B2, and 4D2) cross-react with NS1 of all four DENV serotypes (pan-DENV mAbs), whereas the other (2E11) also reacts with NS1 of other flaviviruses (flavi-cross-reactive mAb). The binding epitopes recognized by these mAbs were found to overlap a region located on the disordered loop of the NS1 wing domain (amino acid residues 104 to 123). Fine epitope mapping employing phage display technology and alanine-substituted DENV2 NS1 mutants indicates the critical binding residues W115, K116, and K120 for the 2E11 mAb, which are conserved among flaviviruses. In contrast, the critical binding residues of four pan-DENV mAbs include both flavi-conserved residues (W115 to G119) and DENV-conserved flanking residues (K112, Y113, S114 and A121, K122). Our results highlight DENV-conserved residues in cross-reactive epitopes that distinguish pan-DENV antibodies from the flavi-cross-reactive antibody. These antibodies can be potentially applied to differential diagnosis of DENV from other flavivirus infections.

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.

We present RNA sequencing data sets and their genome sequence assembly for dengue virus that was isolated from a patient with dengue hemorrhagic fever and serially propagated in Vero cells. RNA sequencing data obtained from the first, third, and fifth passages and their corresponding whole-genome sequences are provided in this work.

Background. Dengue is the most significant mosquito-borne viral disease; there are no specific therapeutics. The antiparasitic drug ivermectin efficiently inhibits the replication of all 4 dengue virus serotypes in vitro. Methods. We conducted 2 consecutive randomized, double-blind, placebo-controlled trials in adult dengue patients to evaluate safety and virological and clinical efficacies of ivermectin. After a phase 2 trial with 2 or 3 days of 1 daily dose of 400 mu g/kg ivermectin, we continued with a phase 3, placebo-controlled trial with 3 days of 400 mu g/kg ivermectin. Results. The phase 2 trial showed a trend in reduction of plasma nonstructural protein 1 (NS1) clearance time in the 3-day ivermectin group compared with placebo. Combining phase 2 and 3 trials, 203 patients were included in the intention to treat analysis (100 and 103 patients receiving ivermectin and placebo, respectively). Dengue hemorrhagic fever occurred in 24 (24.0%) of ivermectin-treated patients and 32 (31.1%) patients receiving placebo (P = .260). The median (95% confidence interval [CI]) clearance time of NS1 antigenemia was shorter in the ivermectin group (71.5 [95% CI 59.9-84.0] hours vs 95.8 [95% Cl 83.9-120.0] hours, P = .014). At discharge, 72.0% and 47.6% of patients in the ivermectin and placebo groups, respectively had undetectable plasma NS1 (P = .001). There were no differences in the viremia clearance time and incidence of adverse events between the 2 groups. Conclusions. A 3-day 1 daily dose of 400 mu g/kg oral ivermectin was safe and accelerated NS1 antigenemia clearance in dengue patients. However, clinical efficacy of ivermectin was not observed at this dosage regimen.

Dengue hemorrhagic fever (DHF) is caused by infection with dengue virus (DENV). Four different serotypes (DENV1-4) co-circulate in dengue endemic areas. The viral RNA genome-based reverse-transcription PCR (RT-PCR) is the most widely used method to identify DENV serotypes in patient specimens. However, the non-structural protein 1 (NS1) antigen as a biomarker for DENV serotyping is an emerging alternative method. We modified the serotyping-NS1-enzyme linked immunosorbent assay (stNS1-ELISA) from the originally established assay which had limited sensitivity overall and poor specificity for the DENV2 serotype. Here, four biotinylated serotype-specific antibodies were applied, including an entirely new design for detection of DENV2. Prediction of the infecting serotype of retrospective acute-phase plasma from dengue patients revealed 100% concordance with the standard RT-PCR method for all four serotypes and 78% overall sensitivity (156/200). The sensitivity of DENV1 NS1 detection was greatly improved (from 62% to 90%) by the addition of a DENV1/DENV3 sub-complex antibody pair. Inclusive of five antibody pairs, the stNS1-ELISA (plus) method showed an overall increased sensitivity to 85.5% (171/200). With the same clinical specimens, a commercial NS1 rapid diagnostic test (NS1-RDT) showed 72% sensitivity (147/200), significantly lower than the stNS1-ELISA (plus) performance. In conclusion, the stNS1-ELISA (plus) is an improved method for prediction of DENV serotype and for overall sensitivity. It could be an alternative assay not only for early dengue diagnosis, but also for serotype identification especially in remote resource-limited dengue endemic areas.