Utility of the WHO dengue guidelines in pediatric immunological studies.

Dengue is a significant health problem due to the high burden of critical infections during outbreaks. In 1997, the World Health Organization (WHO) classified dengue as dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). It was revised in 2009 (updated in 2015), and the new guidelines recommended classifying patients as dengue without warning signs (DNS), dengue with warning signs (DWS), and severe dengue (SD). Although the utility of the revised 2009 classification for clinical studies is accepted, for immunological studies it needs to be clarified. We determined the usefulness of the 2009 classification for pediatric studies that analyze the circulating interleukin (IL)-6 and IL-8, two inflammatory cytokines. Plasma levels of IL-6 and IL-8 were evaluated in the acute and convalescent phases by flow cytometry in children with dengue classified using the 1997 and 2009 WHO guidelines. The plasma levels of IL-6 and IL-8 were elevated during the acute and decreased during convalescence, and both cytokines served as a good marker of acute dengue illness compared to convalescence. There were no differences in the plasma level of the evaluated cytokines among children with different clinical severity with any classification, except for the IL-8, which was higher in DWS than DNS. Based on the levels of IL-8, the 2009 classification identified DWS plus SD (hospital-treated children) compared to the DNS group [area under the curve (AUC): 0.7, p = 0.028]. These results support the utility of the revised 2009 (updated in 2015) classification in studies of immune markers in pediatric dengue.

Cytokine profiles in dengue fever and dengue hemorrhagic fever: A study from Indonesia.

Recent studies have demonstrated that cytokine dysregulation has a critical role in the pathogenesis of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The aim of this study was to investigate the association between tumor necrosis factor (TNF- α), interleukin 6 (IL-6), interleukin 10 (IL-10), and interleukin 17 (IL-17) with infection status, and severity of dengue. A prospective cross-sectional study was conducted at three hospitals in Gianyar regency and Denpasar municipality, Bali, Indonesia, from June to December 2022. Sixty-four dengue infected patients were involved. Patients' serum was tested for dengue infection using NS1 antigen rapid test, dengue virus immunoglobulin M (IgM) and immunoglobulin G (IgG) test, and reverse transcription polymerase chain reaction (RT-PCR). Cytokine levels (TNF-α, IL-6, IL-10, and IL-17) were measured using enzyme-linked immunosorbent assay (ELISA). Infection status was determined by combining serological and RT-PCR results, categorizing patients into primary and secondary infections. The present study found that DF patients had lower TNF-α, IL-6, and IL-17 but higher IL-10 levels compared to DHF patients (p<0.001). Elevated TNF-α, IL-6, and IL-17 levels were higher in secondary infection, while IL-10 level was higher in primary infection (p<0.001). In conclusion, cytokines play a crucial role in the interplay between cytokine dysregulation and dengue infection dynamics.

Comportamiento clínico y de laboratorio del choque por dengue en pacientes pediátricos / Clinical and laboratory behavior of dengue shock among pediatric patients

Introducción: El dengue es la enfermedad viral transmitida por artrópodos que más morbilidad y mortalidad ocasiona mundialmente. En el mundo actual, esta arbovirosis se considera la décima causa de muerte sobre todo en edades pediátricas. Objetivo: Caracterizar el comportamiento clínico y de laboratorio del choque por dengue en niños a partir de un año de edad. Métodos: Se realizó un estudio de corte transversal. Se estudiaron 19 pacientes con diagnóstico de choque por dengue. Para el análisis estadístico se utilizaron medidas de resumen como frecuencias, porcentaje, rango, mediana y moda. Resultados: Los signos de choque por dengue predominaron en los pacientes mayores de 5 años, femeninos y blancos, normopesos con antecedentes de salud. La mayoría ingresó al cuarto día; la fiebre fue el principal motivo de ingreso. El aumento progresivo del hematocrito fue el principal signo de alarma, y la hipotensión sin otra manifestación de choque constituyó la manifestación clínica más frecuente. Las soluciones cristaloides fueron las más utilizadas con muy buena respuesta clínica. Conclusiones: Todos los pacientes evolucionaron satisfactoriamente; no hubo ningún fallecimiento por dengue a pesar de que la mayoría fueron hospitalizados durante la fase crítica de la enfermedad, existiendo una identificación adecuada de los signos de alarma, y un adecuado control y tratamiento de las formas clínicas de choque por dengue(AU)

Introduction: Dengue is the arthropod-borne disease causing the highest morbidity and mortality worldwide. This condition is currently considered the tenth leading cause of death in the world, mainly in pediatric ages. Objective: Characterize the clinical and laboratory behavior of dengue shock in children aged one year and over. Methods: A cross-sectional study was conducted of 19 patients diagnosed with dengue shock. Statistical analysis was based on the summary measurements frequency, percentage, range, median and mode. Results: Dengue shock signs prevailed in white female patients aged over five years, of normal weight and with a history of good health. Most were admitted on the fourth day; fever was the main reason for admission. Gradual hematocrit increase was the main warning sign, whereas hypotension without any other shock symptom was the most common clinical manifestation. Crystalloid solutions were the most frequently used, with a very good clinical response. Conclusions: All the patients evolved satisfactorily; no death occurred due to dengue, despite the fact that many patients were admitted during the critical stage of the disease; warning signs were appropriately identified and clinical manifestations of dengue shock were controlled and treated(AU)

Effects of chloroquine or hydroxychloroquine treatment on non-SARS-CoV2 viral infections: A systematic review of clinical studies.

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used as antiviral agents for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. We performed a systematic review to examine whether prior clinical studies that compared the effects of CQ and HCQ to a control for the treatment of non-SARS-CoV2 infection supported the use of these agents in the present SARS-CoV2 outbreak. PubMed, EMBASE, Scopus and Web of Science (PROSPERO CRD42020183429) were searched from inception through 2 April 2020 without language restrictions. Of 1766 retrieved reports, 18 studies met our inclusion criteria, including 17 prospective controlled studies and one retrospective study. CQ or HCQ were compared to control for the treatment of infectious mononucleosis (EBV, n = 4), warts (human papillomavirus, n = 2), chronic HIV infection (n = 6), acute chikungunya infection (n = 1), acute dengue virus infection (n = 2), chronic HCV (n = 2), and as preventive measures for influenza infection (n = 1). Survival was not evaluated in any study. For HIV, the virus that was most investigated, while two early studies suggested HCQ reduced viral levels, four subsequent ones did not, and in two of these CQ or HCQ increased viral levels and reduced CD4 counts. Overall, three studies concluded CQ or HCQ were effective; four concluded further research was needed to assess the treatments' effectiveness; and 11 concluded that treatment was ineffective or potentially harmful. Prior controlled clinical trials with CQ and HCQ for non-SARS-CoV2 viral infections do not support these agents' use for the SARS-CoV2 outbreak.

Suppressed humoral immunity is associated with dengue nonstructural protein NS1-elicited anti-death receptor antibody fractions in mice.

Dengue virus (DENV) infections may cause life-threatening dengue hemorrhagic fever (DHF). Suppressed protective immunity was shown in these patients. Although several hypotheses have been formulated, the mechanism of DENV-induced immunosuppression remains unclear. Previously, we found that cross-reactive antibodies against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 1 (death receptor 4 [DR4]) were elicited in DHF patients, and that anti-DR4 autoantibody fractions were elicited by nonstructural protein 1 (NS1) immunizations in experimental mice. In this study, we found that anti-DR4 antibodies could suppress B lymphocyte function in vitro and in vivo. Treatment with the anti-DR4 immunoglobulin (Ig) induced caspase-dependent cell death in immortalized B lymphocyte Raji cells in vitro. Anti-DR4 Igs elicited by NS1 and DR4 immunizations markedly suppressed mouse spleen transitional T2 B (IgM+IgD+), bone marrow pre-pro-B (B220+CD43+), pre-B (B220+CD43-), and mature B cell (B220+IgD+) subsets in mice. Furthermore, functional analysis revealed that the pre-elicitation of anti-NS1 and anti-DR4 Ig titers suppressed subsequently neutralizing antibody production by immunization with DENV envelop protein. Our data suggest that the elicitation of anti-DR4 titers through DENV NS1 immunization plays a suppressive role in humoral immunity in mice.

Molecular Signatures of Dengue Virus-Specific IL-10/IFN-γ Co-producing CD4 T Cells and Their Association with Dengue Disease.

Dengue virus (DENV) can cause diseases ranging from dengue fever (DF) to more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Whether antiviral T cells contribute to the protection against or pathogenesis of severe disease is not well defined. Here, we identified antigen-specific IL-10+IFN-γ+ double-positive (DP) CD4 T cells during acute DENV infection. While the transcriptomic signatures of DP cells partially overlapped with those of cytotoxic and type 1 regulatory CD4 T cells, the majority of them were non-cytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1. Although we observed a higher frequency of DP cells in DHF, the transcriptomic profile of DP cells was similar in DF and DHF, suggesting that DHF is not associated with the altered phenotypic or functional attributes of DP cells. Overall, this study revealed a DENV-specific DP cell subset in patients with acute dengue disease and argues against altered DP cells as a determinant of DHF.

Significance of RANTES-CCR5 axis and linked downstream immunomodulation in Dengue pathogenesis: A study from Guwahati, India.

We aimed to evaluate the significance of the RANTES-CCR5 axis and resulting immunomodulatory status in Dengue pathogenesis involving a Guwahati, India based population where Dengue cases have increased alarmingly. An increased CC-chemokine receptor type 5 (CCR5) messenger RNA expression and CCR5 positive cell count profile was observed in Dengue cases, the highest being in severe cases. CCR5 ligand RANTES expression was significantly decreased in Dengue cases and inversely correlated with Dengue viremia fold change in severe cases. Monocytes are involved in Dengue virus homing and replication. Its levels and activation profile were higher in Dengue cases. A hyper Th1-biased immunomodulatory profile with upregulated tumor necrosis factor-α levels, and downregulated expression of antiviral cytokine interferon-γ and key regulatory Th2 anti-inflammatory cytokine interleukin 10 was observed in severe Dengue cases compared with mild Dengue cases and controls. The results, therefore, suggest the significance of RANTES-CCR5 axis deregulation and resulting altered immunomodulation in Dengue pathogenesis, and holds prognostic and therapeutic significance.

Role of NS1 antibodies in the pathogenesis of acute secondary dengue infection.

The role of NS1-specific antibodies in the pathogenesis of dengue virus infection is poorly understood. Here we investigate the immunoglobulin responses of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) to NS1. Antibody responses to recombinant-NS1 are assessed in serum samples throughout illness of patients with acute secondary DENV1 and DENV2 infection by ELISA. NS1 antibody titres are significantly higher in patients with DHF compared to those with DF for both serotypes, during the critical phase of illness. Furthermore, during both acute secondary DENV1 and DENV2 infection, the antibody repertoire of DF and DHF patients is directed towards distinct regions of the NS1 protein. In addition, healthy individuals, with past non-severe dengue infection have a similar antibody repertoire as those with mild acute infection (DF). Therefore, antibodies that target specific NS1 epitopes could predict disease severity and be of potential benefit in aiding vaccine and treatment design.

Host Genetic Polymorphisms Influencing Susceptibility to Dengue.

Dengue is a pandemic-prone viral disease which is endemic in more than 100 countries and which puts half of the world's population at risk. While the disease presents as subclinical infection or mild fever in the majority of cases, approximately a quarter of the infected individuals experience severe forms of disease like dengue hemorrhagic fever/dengue shock syndrome that cause significant rates of mortality and morbidity. The pathogenesis of this differential outcome of infection is determined by a complex interplay of factors associated with the virus, vector, and host; much of which is not completely understood. In this review, we present an update on the various host genetic polymorphisms that have been reported to influence the susceptibility to dengue. For the convenience of discussion, we have categorized the genetic factors according to the different arms of the immune system with which the corresponding immune determinants are associated.

Analyzing the Human Serum Antibody Responses to a Live Attenuated Tetravalent Dengue Vaccine Candidate.

Background: Dengue virus serotypes 1-4 (DENV-1-4) are the most common vector-borne viral pathogens of humans and the etiological agents of dengue fever and dengue hemorrhagic syndrome. A live-attenuated tetravalent dengue vaccine (TDV) developed by Takeda Vaccines has recently progressed to phase 3 safety and efficacy evaluation. Methods: We analyzed the qualitative features of the neutralizing antibody (nAb) response induced in naive and DENV-immune individuals after TDV administration. Using DENV-specific human monoclonal antibodies (mAbs) and recombinant DENV displaying different serotype-specific Ab epitopes, we mapped the specificity of TDV-induced nAbs against DENV-1-3. Results: Nearly all subjects had high levels of DENV-2-specific nAbs directed to epitopes centered on domain III of the envelope protein. In some individuals, the vaccine induced nAbs that tracked with a DENV-1-specific neutralizing epitope centered on domain I of the envelope protein. The vaccine induced binding Abs directed to a DENV-3 type-specific neutralizing epitope, but findings of mapping of DENV-3 type-specific nAbs were inconclusive. Conclusion: Here we provide qualitative measures of the magnitude and epitope specificity of the nAb responses to TDV. This information will be useful for understanding the performance of TDV in clinical trials and for identifying correlates of protective immunity.

Dengue virus specific IgY provides protection following lethal dengue virus challenge and is neutralizing in the absence of inducing antibody dependent enhancement.

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are severe disease manifestations that can occur following sequential infection with different dengue virus serotypes (DENV1-4). At present, there are no licensed therapies to treat DENV-induced disease. DHF and DSS are thought to be mediated by serotype cross-reactive antibodies that facilitate antibody-dependent enhancement (ADE) by binding to viral antigens and then Fcγ receptors (FcγR) on target myeloid cells. Using genetically engineered DENV-specific antibodies, it has been shown that the interaction between the Fc portion of serotype cross-reactive antibodies and FcγR is required to induce ADE. Additionally, it was demonstrated that these antibodies were as neutralizing as their non-modified variants, were incapable of inducing ADE, and were therapeutic following a lethal, antibody-enhanced infection. Therefore, we hypothesized that avian IgY, which do not interact with mammalian FcγR, would provide a novel therapy for DENV-induced disease. We demonstrate here that goose-derived anti-DENV2 IgY neutralized DENV2 and did not induce ADE in vitro. Anti-DENV2 IgY was also protective in vivo when administered 24 hours following a lethal DENV2 infection. We were also able to demonstrate via epitope mapping that both full-length and alternatively spliced anti-DENV2 IgY recognized different epitopes, including epitopes that have not been previously identified. These observations provide evidence for the potential therapeutic applications of goose-derived anti-DENV2 IgY.

Genetic Polymorphisms of Tumor Necrosis Factor Alpha and Susceptibility to Dengue Virus Infection in a Mexican Population.

To evaluate the association of the -308 and -238 tumor necrosis factor alpha (TNF-α) gene polymorphisms with clinical manifestations of dengue and TNF-α serum levels in a northwestern Mexican population. The study populations included dengue fever (DF) and dengue hemorrhagic fever (DHF) patients, and a group of healthy controls (HCs) without history of dengue. Polymerase chain reaction-restriction fragment length polymorphism and Enzyme-Linked Immunosorbent Assay were performed to determine genotypes and serum concentration of TNF-α, respectively. There were no significant differences in alleles, genotypes, and haplotype frequencies between patients and HCs. However, when patients were separated into DF and DHF, there was an increased prevalence of the -308 GA genotype in HCs compared to DHF (odds ratio [OR] = 0.129, 95% confidence interval [CI] = 0.018-0.945, p = 0.025), as well as the GG haplotype (OR = 0.49, 95% CI = 0.273-0.880, p = 0.01757) in DF. The genotypes of both polymorphisms were not associated with hematologic manifestations. Serum TNF-α levels were significantly higher in patients than in HCs (p = 0.004). Our results suggest a minimal effect of the -308 and -238 TNF-α gene polymorphisms in dengue patients and that their increased serum levels of TNF-α are independent of genotypes.

Severity of Plasma Leakage Is Associated With High Levels of Interferon γ-Inducible Protein 10, Hepatocyte Growth Factor, Matrix Metalloproteinase 2 (MMP-2), and MMP-9 During Dengue Virus Infection.

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

Preventive and therapeutic challenges in combating Zika virus infection: are we getting any closer?

The neuroteratogenic nature of Zika Virus (ZIKV) infection has converted what would have been a tropical disease into a global threat. Zika is transmitted vertically via infected placental cells especially in the first and second trimesters. In the developing central nervous system (CNS), ZIKV can infect and induce apoptosis of neural progenitor cells subsequently causing microcephaly as well as other neuronal complications in infants. Its ability to infect multiple cell types (placental, dermal, and neural) and increased environmental stability as compared to other flaviviruses (FVs) has broadened the transmission routes for ZIKV infection from vector-mediated to transmitted via body fluids. To further complicate the matters, it is genetically similar (about 40%) with the four serotypes of dengue virus (DENV), so much so that it can almost be called a fifth DENV serotype. This homology poses the risk of causing cross-reactive immune responses and subsequent antibody-dependent enhancement (ADE) of infection in case of secondary infections or for immunized individuals. All of these factors complicate the development of a single preventive vaccine candidate or a pharmacological intervention that will completely eliminate or cure ZIKV infection. We discuss all of these factors in detail in this review and conclude that a combinatorial approach including immunization and treatment might prove to be the winning strategy.

Substitution of the precursor peptide prevents anti-prM antibody-mediated antibody-dependent enhancement of dengue virus infection.

Antibody-dependent enhancement (ADE) is currently considered as the mechanism underlying the pathogenesis of severe dengue disease. Many studies have shown that precursor (pr) peptide-specific antibodies do not efficiently neutralize infection but potently promote ADE of dengue virus (DENV) infection. To explore the effect of pr peptide substitution on neutralization and ADE of DENV infection, the rabbit anti-prM polyclonal antibodies (pAbs) and anti-JEVpr/DENV-M pAbs were prepared, and the neutralization and ADE of these two pAbs were further compared. Here, we report that both anti-JEVpr/DENV-M and anti-prM pAbs exhibited broad cross-reactivity and only partial neutralization with four DENV serotypes and immature DENV. Rabbit anti-prM pAbs showed a significant enhancement in a broad range of serum dilutions. However, there was no statistically significant difference in the enhancing activity of rabbit anti-JEVpr/DENV-M pAbs at various levels of dilution. These results demonstrate that anti-prM antibody-mediated ADE can be prevented by JEV pr peptide replacement. The present study contribute further to research on the pathogenesis of DENV infection.

Slow resolution of inflammation in severe adult dengue patients.

BACKGROUND: The pathogenesis of severe dengue has not been fully elucidated. The inflammatory response plays a critical role in the outcome of dengue disease. METHODS: In this study, we investigated the levels of 17 important inflammation mediators in plasma collected from mild or severe adult dengue patients at different time points to understand the contribution of inflammation to disease severity and to seek experimental evidence to optimize the existing clinical treatment strategies. Patients were simply classified as mild and severe dengue according to the 2009 WHO classification. Plasma was collected on day 3-5, 6-7, 8-10 and 14-17 of illness. Levels of 17 inflammation mediators including TNF-α, IL-1α, IFN-γ, IL-6, IFN-α, MIF, IL-10, IL-1RA, IL-8, IP-10, MCP-1, RANTES, GRO, eotaxin-1, sICAM-1 and sVCAM-1 were determined by a multiplex Luminex® system. Different trends of inflammation mediators throughout the disease were compared between mild and severe patients. RESULTS: Inflammation mediators including IL-1α, IFN-γ, IL-10, IL-8, IP-10, MCP-1 and sVCAM-1 displayed significant differences on day 8-10 of illness between mild and severe dengue patients. Their concentrations were higher in severe patients than mild ones at the same time points. Moreover, those cytokines decreased gradually in mild patients but not in severe patients. CONCLUSION: Our results revealed the coexistence of excessive inflammatory response and slow resolution of inflammation in severe adult dengue patients. Hence suppression and/or pro-resolution of inflammation could be a potential therapeutic approach for treatment of severe dengue.

Primary dengue haemorrhagic fever in patients from northeast of Brazil is associated with high levels of interferon-β during acute phase

Dengue is an acute febrile disease caused by the mosquito-borne dengue virus (DENV) that according to clinical manifestations can be classified as asymptomatic, mild or severe dengue. Severe dengue cases have been associated with an unbalanced immune response characterised by an over secretion of inflammatory cytokines. In the present study we measured type I interferon (IFN-I) transcript and circulating levels in primary and secondary DENV infected patients. We observed that dengue fever (DF) and dengue haemorrhagic fever (DHF) patients express IFN-I differently. While DF and DHF patients express interferon-α similarly (52,71 ± 7,40 and 49,05 ± 7,70, respectively), IFN- β were associated with primary DHF patients. On the other hand, secondary DHF patients were not able to secrete large amounts of IFN- β which in turn may have influenced the high-level of viraemia. Our results suggest that, in patients from our cohort, infection by DENV serotype 3 elicits an innate response characterised by higher levels of IFN- β in the DHF patients with primary infection, which could contribute to control infection evidenced by the low-level of viraemia in these patients. The present findings may contribute to shed light in the role of innate immune response in dengue pathogenesis.

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue.

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3+ CD8+ amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3+ cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = -0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs.

Primary dengue virus infections induce differential cytokine production in Mexican patients.

Severe dengue pathogenesis is not fully understood, but high levels of proinflammatory cytokines have been associated with dengue disease severity. In this study, the cytokine levels in 171 sera from Mexican patients with primary dengue fever (DF) and dengue haemorrhagic fever (DHF) from dengue virus (DENV) 1 (n = 116) or 2 (n = 55) were compared. DF and DHF were defined according to the patient's clinical condition, the primary infections as indicated by IgG enzymatic immunoassay negative results, and the infecting serotype as assessed by real-time reverse transcription-polymerase chain reaction. Samples were analysed for circulating levels of interleukin (IL)-12p70, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6, and IL-8 using a commercial cytometric bead array. Significantly higher IFN-γ levels were found in patients with DHF than those with DF. However, significantly higher IL-12p70, TNF-α, and IL-6 levels were associated with DHF only in patients who were infected with DENV2 but not with DENV1. Moreover, patients with DF who were infected with DENV1 showed higher levels of IL-12p70, TNF-α, and IL-6 than patients with DHF early after-fever onset. The IL-8 levels were similar in all cases regardless of the clinical condition or infection serotype. These results suggest that the association between high proinflammatory cytokine levels and dengue disease severity does not always stand, and it once again highlights the complex nature of DHF pathogenesis.

Macrophage Activation Syndrome-Associated Markers in Severe Dengue.

Hemophagocytosis, a phenomenon of which activated macrophages phagocytosed hematopoietic elements was reportedly observed in severe dengue patients. In the present study, we investigated whether markers of macrophage activation syndrome (MAS) can be used as differential diagnostic markers of severe dengue. Two hundred and eight confirmed dengue patients were recruited for the study. Sandwich ELISA was used to determine serum ferritin, soluble CD163 (sCD163), and soluble CD25 (sCD25) levels. The population of circulating CD163 (mCD163) monocytes was determined using flow cytometry. Receiver operating characteristic (ROC) analysis was plotted to determine the predictive validity of the biomarkers. Serum ferritin and sCD163 were found significantly increased in severe dengue patients compared to dengue fever patients (P = 0.003). A fair area under ROC curves (AUC) at 0.72 with a significant P value of 0.004 was observed for sCD163. sCD25 and mCD163 levels were not significantly different between severe dengue and dengue fever patients. Our findings suggest that in addition to serum ferritin, sCD163 can differentiate severe dengue from that of dengue fever patients. Hence, sCD163 level can be considered for use as a predictive marker for impending severe dengue.