Identification and Characterization at the Single-Cell Level of Cytokine-Producing Circulating Cells in Children With Dengue.

In this study, we identified, at the single-cell level, naturally induced cytokine-producing circulating cells (CPCCs) in children with dengue virus (DENV) infection ranging clinically from mild to severe disease. Tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) CPCCs were detected in children with primary or secondary acute dengue virus (DENV) infection, and the pattern of these cytokines was similar to that seen in the supernatant of cultured peripheral blood mononuclear cells and partially comparable to that found in plasma. Monocytes, B cells, and myeloid dendritic cells (mDCs) were the primary CPCCs detected, and the frequency of mDCs was significantly higher in severe disease. B cells isolated from children with dengue spontaneously secreted TNF-α, IL-6, and interleukin 10, and supernatants from cultures of purified B cells induced activation of allogeneic T cells, supporting an antibody-independent function of these cells during DENV infection. Thus, CPCCs could be a new immune parameter with potential use to evaluate pathogenesis in this infection.

High plasma levels of soluble ST2 but not its ligand IL-33 is associated with severe forms of pediatric dengue.

Identification of early determinants of dengue disease progression, which could potentially enable individualized patient care are needed at present times. Soluble ST2 (sST2) has been recently reported to be elevated in the serum of children older than 2 years old and adults with dengue infection and it was correlated with secondary infections as well as with severe presentations of the disease. The mechanism by which secreted ST2 is linked to severe dengue and plasma leakage remains unclear. One possibility is that IL-33 ligand may be elevated, contributing to membrane bound ST2 as part of the immune activation in dengue infection. We determined plasma levels of sST2 and the ligand IL-33 in 66 children with acute secondary dengue infections clinically classified using the guidelines of the World Health Organization, 2009. Dengue infection showed significant increases in cytokines IL-12p70, IL-10, IL-8, IL-6, IL-1ß and TNFα measured by flow cytometry based assay compared to uninfected individuals. In contrast, IL-33 levels remained unchanged between infected and uninfected individuals. The levels of sST2 positively correlated with values of IL-6 and IL-8 and inversely correlated with number of median value of platelet levels. In addition to circulating cytokine positive correlations we found that sST2 and isoenzyme creatine kinase-MB (CK-MB), a marker of myocardial muscle damage present in severe dengue cases were associated. Our pediatric study concluded that in dengue infections sST2 elevation does not involve concomitant changes of IL-33 ligand. We propose a study to assess its value as a predictor factor of disease severity.

Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas.

The isolation of nucleic acids is a critical and limiting step for molecular assays, which prompted the arrival in Colombia of automated purification instruments during the SARS-CoV-2 pandemic. The local application of this technology in the study of tropical diseases, such as dengue and zika, is beginning to be tested. We evaluated the efficiency of the automated extraction of viral RNA for studies of pediatric dengue and zika. Clinical samples of children with dengue that were well characterized through RNA isolation by silica columns and serotype-specific nested RT-PCR (DENV-1 n = 7, DENV-2 n = 5, and negatives n = 8) in addition to 40 pediatric plasma samples spiked with ZIKV (strain PRVA BC59) and 209 from negative pre-epidemic children were analyzed. RNA from patients was extracted by two automated standard and high-throughput protocols on the KingFisher™ Flex instrument. The isolated RNA was evaluated for concentration and purity by spectrophotometry, for structural and functional integrity by electrophoresis and expression of the RNase P gene, and usefulness in serotype-specific DENV detection by conventional and real-time RT-PCR. For the evaluation of ZIKV RNA, the commercial TaqMan Triplex® assay was used, along with a well-tested in-house RT-qPCR assay. The concentration of RNA (5.2 vs. 7.5 ng/µL, P=0.03) and the number of integral bands (9 vs. 11) were higher with the high-throughput protocol. However, the number of specimens serotyped for DENV by RT-qPCR was comparable for both protocols. The cycle thresholds of the TaqMan Triplex® commercial kit and the in-house assay for the detection of plasma ZIKV RNA isolated with the standard protocol showed a strong association (r = 0.93, P < 0.0001) and a Cohen Kappa index of 0.98 when all 249 samples were analyzed. These preliminary results suggest that automated instruments could be used in studies of cocirculating flaviviruses that have represented a public health problem in recent decades in Colombia. They boast advantages such as efficiency, precision, time savings, and lower risk of cross-contamination.

NS1-Specific Antibody Response Facilitates the Identification of Children With Dengue and Zika in Hyperendemic Areas.

BACKGROUND: Infections by dengue virus (DENV) and Zika virus (ZIKV) have some similar symptoms and a cross-reactive immune response, although with different risk populations and outcomes. Here, we evaluated the virological characteristics and the nonstructural protein 1 (NS1)-specific antibody responses to DENV and ZIKV in children suspected of dengue in different epidemiological moments in Colombia. METHODS: Viral RNA, circulating NS1 and IgM/IgG specific for DENV and ZIKV were performed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) in 301 children suspected of dengue enrolled in a hospital setting during the ZIKV epidemic and a primary healthcare setting during a DENV epidemic. For the detection of DENV and ZIKV-specific IgM, an NS1-based ELISA was validated using characterized pediatric samples. Clinical and laboratory parameters were also evaluated. RESULTS: DENV RNA or NS1 antigen was detected in the plasma of 62% of children, and in none, the ZIKV RNA was found. NS1-based ELISA for DENV and ZIKV IgM showed a sensitivity/specificity of 90/84% and 73/98%, respectively. Of 114 children without detectable viremia or antigenemia, 30.7%, 17.5%, 22% and 30% were IgM-DENV+, IgM-ZIKV+, IgM-DENV+ZIKV+ and IgM-DENV-ZIKV-, respectively. The ZIKV/DENV IgM-NS1 ratio allows the identification of the infecting ortho flavivirus in 88% of the children with IgM-DENV+ZIKV+, confirming a high predominance of DENV infections in the 2 pediatric settings. CONCLUSION: Overall, 88% of the children with clinical suspicion of dengue had an identifiable ortho flaviviral infection, with 80% caused by DENV, 7% by ZIKV and 0.7% classified as recent infections or coinfection, demonstrating active viral cocirculation in the pediatric population of southern Colombia. The IgM-NS1 detection improved the identification of ortho flaviviral infections in children without viremia or antigenemia, suggesting it is a helpful complementary tool for medical personnel in tropical regions with high viral cocirculation and different clinical scenes.

Unique Immune Blood Markers Between Severe Dengue and Sepsis in Children.

BACKGROUND: Pediatric dengue and sepsis share clinical and pathophysiologic aspects. Multiple inflammatory and regulatory cytokines, decoy receptors and vascular permeability factors have been implicated in the pathogenesis of both diseases. The differential pattern and dynamic of these soluble factors, and the relationship with clinical severity between pediatric dengue and sepsis could offer new diagnosis and therapeutic strategies. METHODS: We evaluated the concentration levels of 11 soluble factors with proinflammatory, regulatory and vascular permeability involvement, in plasma from children with dengue or sepsis, both clinically ranging from mild to severe, in the early, late and convalescence phases of the disease. RESULTS: During early acute infection, children with sepsis exhibited specific higher concentration levels of IL-6, vascular endothelial growth factor (VEGF), and its soluble decoy receptor II (sVEGFR2) and lower concentration levels of IL-10 and the soluble tumor necrosis factor receptor 2 (sTNFR2), in comparison with children with severe dengue. In addition, the circulating amounts of soluble ST2, and VEGF/sVEGFR2 were widely associated with clinical and laboratory indicators of dengue severity, whereas secondary dengue virus infections were characterized by an enhanced cytokine response, relative to primary infections. In severe forms of dengue, or sepsis, the kinetics and the cytokines response during the late and convalescence phases of the disease also differentiate. CONCLUSIONS: Dengue virus infection and septic processes in children are characterized by cytokine responses of a specific magnitude, pattern and kinetics, which are implicated in the pathophysiology and clinical outcome of these diseases.

Levels of soluble ST2 in serum associated with severity of dengue due to tumour necrosis factor alpha stimulation.

The interleukin-1 receptor-like-1 protein (IL1RL1), also known as ST2, has been shown previously to regulate T-cell function and is produced by T cells and endothelial cells. It was reported recently to be elevated in mild dengue patients during acute disease. The ST2 gene encodes several splice products: L (long), V (short) and s (soluble). A cohort of 38 patients with dengue haemorrhagic fever (DHF) and mild dengue fever (DF) were evaluated using a secreted soluble ST2 (sST2) ELISA. The RNA expression of ST2 was evaluated by real-time quantitative RT-PCR using patients' peripheral blood mononuclear cells (PBMCs) and in vitro using human umbilical vein endothelial cells (HUVECs) exposed to sera from dengue patients. DHF patients had higher levels of serum sST2, tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8 and IL-10 compared with DF patients and normal healthy control individuals. However, viraemia was indistinguishable between mild and severe cases. No changes in ST2 mRNA expression were found in PBMCs from these two groups of dengue patients. In vitro, sST2 was elevated in HUVECs treated with patient sera. Neutralization of TNF-alpha in patient sera by pre-treatment with a TNF-alpha antibody inhibited the upregulation of sST2 expression in HUVECs. These results implicate serum TNF-alpha in the modulation of expression of sST2 in an in vitro system, and indicate that sST2 could be associated with the severity of disease. Further studies to determine whether sST2 levels are predictive of the severe form of the disease and the role of sST2 in immune regulation are warranted.

Clinical, laboratory and immune aspects of Zika virus-associated encephalitis in children.

OBJECTIVE: To evaluate the clinical, laboratory, and immune characteristics of Zika virus (ZIKV)-associated encephalitis in pediatric patients after the epidemic in Huila, southern Colombia. METHODS: A pediatric neuro-surveillance hospital study was conducted in a referral health center in southern Colombia, from October 2016 to October 2017. Cases of encephalitis were confirmed by nucleic acid amplification tests and serological methods in cerebrospinal fluid (CSF), plasma, and/or urine. Levels of six cytokines were evaluated by flow cytometry. Patients underwent daily clinical and laboratory follow-up. RESULTS: Twenty children with probable encephalitis were included for further studies and 16 of them were confirmed. Four cases of bacterial meningoencephalitis (Streptococcus pneumoniae, group B Streptococcus, Staphylococcus epidermidis, and Escherichia coli) and 12 cases of viral encephalitis were identified, six of them associated with ZIKV infection. Other viral encephalitis cases were caused by herpes viruses (n=3), enterovirus (n=2), and dengue virus type 2 (DENV-2; n=1) infections. ZIKV-associated encephalitis symptoms subsided faster than those of patients with encephalitis caused by other agents. CSF analysis revealed lymphocytic pleocytosis. Compared to healthy controls, children with ZIKV-associated encephalitis presented modest plasma interleukin (IL)-10 but not IL-2, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor alpha (TNF-α). Cytokine expression was differentially regulated, as dramatically elevated IL-6, IL-10, and IFN-γ levels were observed in CSF but not in paired plasma samples in one of the patients with ZIKV detectable in CSF. CONCLUSIONS: This study provides evidence that ZIKV is responsible for pediatric encephalitis in endemic areas, and the local presence of the virus may induce cephalic but not systemic expression of cytokines.

Association of IL4R-rs1805016 and IL6R-rs8192284 polymorphisms with clinical dengue in children from Colombian populations.

BACKGROUND: Etiologic studies provide evidence that IL-4R and IL-6R receptors may play important roles in the regulatory mechanisms of the development of clinical dengue, especially in children which is a segment of the population with high severe dengue risk. Moreover, the allele frequencies and genetic associations may be influenced by the populational genetic background. Therefore, we performed a case-control study to evaluate possible associations between SNPs in IL4R and IL6R genes and clinical dengue in children from two Colombian populations. METHODS: We genotyped the rs1805016 (IL4R) and rs8192284 (IL6R) by PCR-RFLP method, in 298 symptomatic children and 648 asymptomatic controls. Three individual genetic ancestral proportions (APs) (European, Amerindian, African) were inferred by genotyping 29 AIMs (Ancestry informative markers). The variables gender, APs, and the population of origin were used like confusion variables. RESULTS: We found IL4R-rs1805016 GG genotype and G-allele carriers and IL6R-rs8192284 AA genotype associated with clinical dengue in the pooled and Huila samples. Nevertheless, we found no association of these polymorphisms in the sample of Antioquia. CONCLUSIONS: For the first time, we report SNPs in IL4R and IL6R genes associated with clinical dengue, which contributes to understanding the genetic susceptibility to dengue disease. Moreover, these results may be influenced by genetic background and must be evaluated through functional analysis.

Evaluation of variants in IL6R, TLR3, and DC-SIGN genes associated with dengue in sampled Colombian population. / Evaluación de variantes en los genes IL6R, TLR3 y DC-SIGN asociadas con dengue en una población colombiana muestreada

INTRODUCTION: Host genetics is recognized as an influential factor for the development of dengue disease. OBJECTIVE: This study evaluated the association of dengue with the polymorphisms rs8192284 for gene IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN. MATERIALS AND METHODS: Of the 292 surveyed subjects, 191 were confirmed for dengue fever and the remaining 101 were included as controls. The genotypes were resolved using polymerase chain reaction and restriction fragment length polymorphism (PCRRFLP). In an attempt to determine the risk (Odds Ratio) of suffering dengue fever, data were analyzed using chi-square for alleles and logistic regression for both genotypes and allelic combinations. Confidence intervals were set to 95% for all tests regardless of the adjustment by either self-identification or ancestry. RESULTS: For Afro-Colombians, the allele rs8192284 C offered protection against dengue [OR=0.425,(0.204-0.887), p=0.020]. The alleles rs7248637 A and rs3775290 A posed, respectively, an increased risk of dengue for Afro-Colombians [OR=2.389, (1.170-4.879), p=0.015] and Mestizos [OR=2.329, (1.283-4.226), p=0.005]. The reproducibility for rs8192284 C/C [OR=2.45, (1.05-5.76), p=0.013] remained after adjustment by Amerindian ancestry [OR=2.52, (1.04-6.09), p=0.013]. The reproducibility for rs3775290 A/A [OR=2.48, (1.09-5.65), p=0.033] remained after adjustment by European [OR=2.34, (1.02-5.35), p=0.048], Amerindian [OR=2.49, (1.09-5.66), p=0.035], and African ancestry [OR=2.37, (1.04-5.41), p=0.046]. Finally, the association of dengue fever with the allelic combination CAG [OR=2.07, (1.06-4.05), p=0.033] remained after adjustment by Amerindian ancestry [OR=2.16, (1.09-4.28), p=0.028]. CONCLUSIONS: Polymorphisms rs8192284 for IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN were associated with the susceptibility to suffer dengue fever in the sampled Colombian population.

Introducción. La genética del huésped se reconoce como un factor que influye en el desarrollo del dengue. Objetivo. Este estudio evaluó la asociación del dengue con los polimorfismos rs8192284 del gen IL6R, rs3775290 del TLR3 y rs7248637 del DC-SIGN. Materiales y métodos. De los 292 sujetos encuestados, en 191 se confirmó la presencia de fiebre por dengue y los restantes 101 se incluyeron como controles. Los genotipos se resolvieron mediante reacción en cadena de la polimerasa y polimorfismos en la longitud de los fragmentos de restricción (PCR-RFLP). En un intento por determinar el riesgo de sufrir dengue, los datos se analizaron mediante la prueba de ji al cuadrado para los alelos y la regresión logística para los genotipos y las combinaciones alélicas. Los intervalos de confianza se calcularon a 95 % para todas las pruebas independientemente ajustadas por autoidentificación o componente genético ancestral. Resultados. En los afrocolombianos, el alelo C rs8192284 ofreció protección contra el dengue (OR=0,425; 0,204-0,887, p=0,020). Los alelos A rs7248637 y A rs3775290 plantearon un mayor riesgo de dengue para los afrocolombianos (OR=2,389; 1,170-4,879; p=0,015) y los mestizos (OR=2,329; 1,283-4,226: p=0,005), respectivamente. La reproducibilidad para rs8192284 C/C (OR=2,45; 1,05-5,76; p=0,013) permaneció después del ajuste por el componente genético ancestral amerindio (OR=2,52; 1,04-6,09; p=0,013). La reproducibilidad del rs3775290 A/A (OR=2,48; 1,09-5,65; p=0,033) permaneció después del ajuste por el componente europeo (OR=2,34; 1,02-5,35; p=0,048), el amerindio (OR=2,49; 1,09- 5,66; p=0,035), y el africano (OR=2,37; 1,04-5,41; p=0,046). Por último, la asociación del dengue con la combinación alélica CAG (OR=2,07; 1,06-4,05; p=0,033) permaneció después del ajuste por el componente genético amerindio (OR=2,16; 1,09-4,28; p=0,028). Conclusión. Los polimorfismos rs8192284 en IL6R, rs3775290 en TLR3 y rs7248637 en DC-SIGN, se asociaron con la propensión a sufrir dengue en una muestra de población colombiana.

[Clinical and epidemiological characterisation of dengue haemorrhagic fever in Neiva, Colombia, 2004]. / Caracterización Clínica y Epidemiológica de Dengue Hemorrágico en Neiva, Colombia, 2004.

OBJECTIVE: Neiva (a southern Colombia city) is endemic for dengue fever and dengue haemorrhagic fever. Neiva has suffered outbreaks of dengue, that in 2004 being the latest one. This study was designed to characterise epidemiological and clinical data from that outbreak of dengue. MATERIAL AND METHODS: This was a descriptive, retrospective study of children aged less than 13 years who were admitted to the University Hospital and fulfilled the WHO's clinical and laboratory criteria for dengue or dengue haemorrhagic fever. Demographic, clinical and paraclinical data were recorded and analysed using Chi square (chi(2)) bivariate tabular test. RESULTS: 105 children were diagnosed as suffering from either dengue haemorrhagic fever (87.6%) or dengue fever (12.4%); 67% of them were aged less than 5 years. Girls were more frequently affected by severe clinical manifestations. 83% of the children were admitted during the first six days of the disease; dengue shock syndrome was diagnosed in 20% and 76% presented clinical complications (chi(2) 29.53, gl 6, p=0.0000). Aminotransferases were 3 to 5 times above normal levels. There was a statistical correlation between low platelet count (less than 20 000 per mm(3)) and shock during admission (chi(2) 20.65, gl 4, p=0.0004). Complications arose during clinical evolution in 32% of the cases (13% myocarditis, 19% hepatitis or encephalitis and 2% sepsis). CONCLUSION: The clinical and epidemiological characteristics observed in this cohort evidenced differences in age, gender and organs affected compared to data described in the literature; there was a high incidence of myocarditis.

Selective dysfunction of subsets of peripheral blood mononuclear cells during pediatric dengue and its relationship with clinical outcome.

During dengue virus (DENV) infection, a blockage of secretion of cytokines such as tumor necrosis factor (TNF)-α and members of the interferon (IFN) family has been described in vitro. We evaluated the functionality of monocytes as well as dendritic, B and T cells isolated from children with mild and severe dengue. Compared with those of healthy children, stimulated monocytes, CD4+ T cells and dendritic cells from children with dengue had lower production of proinflammatory cytokines. The interferon axis was dramatically modulated by infection as plasmacytoid dendritic cells (pDCs) and CD4+ T cells had low production of IFN-α and IFN-γ, respectively; plasma levels of IFN-α and IFN-γ were lower in severely ill children, suggesting a protective role. Patients with antigenemia had the highest levels of IFN-α in plasma but the lowest frequency of IFN-α-producing pDCs, suggesting that DENV infection stimulates a systemic type I IFN response but affects the pDCs function.

Magnitude of viremia, antigenemia and infection of circulating monocytes in children with mild and severe dengue.

Dengue is a major public health problem in tropical regions around the world. Viral and immune host factors determine the clinical courses of the infection. We analyzed the dynamics of viremia (by real-time polymerase chain reactions), antigenemia (through detection of the viral non-structural protein [NS]-1 by enzyme-linked immunosorbent assays) and the frequency of virus-infected peripheral blood mononuclear cells (PBMCs) (by multiparametric flow cytometry) in children with primary or secondary dengue virus (DENV) infection in mild to severe cases. Additionally, we evaluated the association of these factors with clinical severity and laboratory parameters. The levels of viremia and antigenemia peaked during the early days of illness and these viral parameters were correlated (rho=0.37, P=0.003). Circulating monocytes were the most naturally infected subset within the PBMCs population, with kinetics similar to those of viremia and antigenemia. The levels of viremia and antigenemia were higher in children with primary infections than in those with secondary infections (P≤0.04). Although there were no associations between the three evaluated factors and clinical severity, the levels of plasma NS1 and the frequency of dengue virus-infected monocytes correlated with prolonged coagulation times. In short, the viremia, antigenemia and infected monocytes were detected early and were not related to clinical severity. The magnitude of antigenemia and infected circulating monocytes was associated with coagulation disorders.

Levels of Circulating Tumor Necrosis Factor-α in Children with Symptomatic Dengue Evaluated by ELISA and Bead-Based Assays.

Tumor necrosis factor (TNF)-α is a key cytokine in the pathogenesis of dengue virus infection, and its accurate detection in several types of human samples is critical. The enzyme-linked immunosorbent assay (ELISA) is the gold standard for the detection of TNF-α, but multiplexed bead-based assays such as cytometric bead array (CBA) are now frequently used. Here, using ELISA and two CBAs commercially available, we measured TNF-α concentrations in plasma and serum from children with acute dengue virus infection and healthy controls. To evaluate the detection efficiency and factors affecting it, spiked recovery and immune complex dissociation assays were also performed. The levels of TNF-α evaluated by ELISA in paired serum and plasma samples from children with dengue positively correlated (rho = 0.99, p < 0.0001). Children with dengue had higher levels of plasma TNF-α than those of healthy children (p = 0.004). The ELISA detected TNF-α in a higher number of plasma samples than the CBA (p < 0.0001), and both methods only correlated when TNF-α was evaluated in buffer-based solutions but not in plasma, indicating the presence of a factor interfering with the detection of TNF-α in plasma. The recovery of several types of human recombinant TNF-α was dramatically decreased in plasma but not in tissue culture media (p ≤ 0.01), and this effect was similar in the plasma obtained from the children with dengue or the healthy controls. The dissociation of immune complexes did not improve TNF-α recovery. Dilution of the plasma samples increased the recovery of TNF-α, but at high concentrations of the cytokine. In short, plasma affects the efficiency of TNF-α detection, and this effect should be considered in the measurement of this cytokine.

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum.

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-µl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-µl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.

B cells naturally induced during dengue virus infection release soluble CD27, the plasma level of which is associated with severe forms of pediatric dengue.

The CD27 and CD38 antigens are highly expressed on the plasmablast surface, and a massive plasmablast response has been described for dengue virus infection. Soluble CD27 and CD38 forms (sCD27 and sCD38, respectively) increase after immune activation. Here, we show increased sCD27 release in cultures of purified polyclonally stimulated B cells. T and B cells isolated from children with dengue spontaneously produced higher levels of sCD27 but not sCD38, compared with healthy children (P=0.03), and sCD27 levels positively correlated with plasmablast frequency in the cultures (rho=0.58, P=0.01). Children with dengue had higher plasma levels of sCD27 and sCD38 than healthy children, which decreased during convalescence. Plasma sCD27 was higher in severe than with mild dengue, but the opposite was observed for sCD38. These findings support a potential new role for B cells in dengue pathogenesis, and sCD27 and sCD38 are novel biomarkers associated with clinical outcome during dengue virus infection.

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.

Total and Envelope Protein-Specific Antibody-Secreting Cell Response in Pediatric Dengue Is Highly Modulated by Age and Subsequent Infections.

The response of antibody-secreting cells (ASC) induced by dengue has only recently started to be characterized. We propose that young age and previous infections could be simple factors that affect this response. Here, we evaluated the primary and secondary responses of circulating ASC in infants (6-12 months old) and children (1-14 years old) infected with dengue showing different degrees of clinical severity. The ASC response was delayed and of lower magnitude in infants, compared with older children. In primary infection (PI), the total and envelope (E) protein-specific IgM ASC were dominant in infants but not in children, and a negative correlation was found between age and the number of IgM ASC (rho = -0.59, P = 0.03). However, infants with plasma dengue-specific IgG detectable in the acute phase developed an intense ASC response largely dominated by IgG and comparable to that of children with secondary infection (SI). IgM and IgG produced by ASC circulating in PI or SI were highly cross-reactive among the four serotypes. Dengue infection caused the disturbance of B cell subsets, particularly a decrease in the relative frequency of naïve B cells. Higher frequencies of total and E protein-specific IgM ASC in the infants and IgG in the children were associated with clinically severe forms of infection. Therefore, the ASC response induced by dengue is highly influenced by the age at which infection occurs and previous immune status, and its magnitude is a relevant element in the clinical outcome. These results are important in the search for correlates of protection and for determining the ideal age for vaccinating against dengue.

Congenital and perinatal complications of chikungunya fever: a Latin American experience.

BACKGROUND: During the years 2014 and 2015, the Region of the Americas underwent a devastating epidemic of chikungunya virus (CHIKV) of the Asian genotype, resulting in millions of affected individuals. However, epidemiological and clinical information on this experience is scarce. Prior knowledge of congenital and neonatal illness caused by CHIKV is limited and almost exclusively based on data obtained from a single outbreak of the East/Central/South African (ECSA) genotype. The effect of chikungunya fever (CHIKF) on pregnancy outcomes and its consequences for infants born to infected mothers at the peak of the epidemic wave in Latin America are reviewed herein. Epidemiological and clinical data on maternal and neonatal infections were collected prospectively and analyzed. METHODS: One hundred sixty-nine symptomatic newborns with CHIKF seen at four large regional maternity hospitals in three different Central and South American countries were evaluated prospectively. The outcomes of pregnancies in symptomatic infected mothers at two of these clinical centers were also analyzed. RESULTS: The observed vertical transmission rate ranged between 27.7% and 48.29%. The incidence of congenital disease was unrelated to the use of cesarean section or natural delivery. The case fatality rate (CFR) at the only center that reported deaths was 5.3%. The most common clinical manifestations included fever, irritability, rash, hyperalgesia syndrome, diffuse limb edema, meningoencephalitis, and bullous dermatitis. Severe complications included meningoencephalitis, myocarditis, seizures, and acute respiratory failure. Leukocytosis with neutrophilia and normal or increased platelets was a common finding, and in those with signs of meningeal involvement, moderate lymphocytic pleocytosis with normal glucose and protein levels was typical. CONCLUSIONS: This study presents the largest number of symptomatic neonates with CHIKF analyzed so far in any region and is the first involving infection with the Asian genotype of CHIKV. Although the clinical manifestations found were similar to those reported previously, the percentage of neurological complications was lower. The CFR was comparatively high. Chikungunya represented a substantial risk for neonates born to symptomatic parturients during the chikungunya outbreak in the Americas Region, with important clinical and public health implications.

Evaluation of variants in IL6R, TLR3, and DC-SIGN genes associated with dengue in a sampled Colombian population / Evaluación de las variantes en los genes IL6R, TLR3 y DC-SIGN asociadas con dengue en una muestra de población colombiana

Abstract Introduction: Host genetics is recognized as an influential factor for the development of dengue disease. Objective: This study evaluated the association of dengue with the polymorphisms rs8192284 for gene IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN. Materials and methods: Of the 292 surveyed subjects, 191 were confirmed for dengue fever and the remaining 101 were included as controls. The genotypes were resolved using polymerase chain reaction and restriction fragment length polymorphism (PCR- RFLP). In an attempt to determine the risk (Odds Ratio) of suffering dengue fever, data were analyzed using chi-square for alleles and logistic regression for both genotypes and allelic combinations. Confidence intervals were set to 95% for all tests regardless of the adjustment by either self-identification or ancestry. Results: For Afro-Colombians, the allele rs8192284 C offered protection against dengue [OR=0.425,(0.204-0.887), p=0.020]. The alleles rs7248637 A and rs3775290 A posed, respectively, an increased risk of dengue for Afro-Colombians [OR=2.389, (1.170-4.879), p=0.015] and Mestizos [OR=2.329, (1.283-4.226), p=0.005]. The reproducibility for rs8192284 C/C [OR=2.45, (1.05-5.76), p=0.013] remained after adjustment by Amerindian ancestry [OR=2.52, (1.04-6.09), p=0.013]. The reproducibility for rs3775290 A/A [OR=2.48, (1.09-5.65), p=0.033] remained after adjustment by European [OR=2.34, (1.02-5.35), p=0.048], Amerindian [OR=2.49, (1.09-5.66), p=0.035], and African ancestry [OR=2.37, (1.04-5.41), p=0.046]. Finally, the association of dengue fever with the allelic combination CAG [OR=2.07, (1.06-4.05), p=0.033] remained after adjustment by Amerindian ancestry [OR=2.16, (1.09-4.28), p=0.028]. Conclusions: Polymorphisms rs8192284 for IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN were associated with the susceptibility to suffer dengue fever in the sampled Colombian population.

Resumen Introducción. La genética del huésped se reconoce como un factor que influye en el desarrollo del dengue. Objetivo. Este estudio evaluó la asociación del dengue con los polimorfismos rs8192284 del gen IL6R, rs3775290 del TLR3 y rs7248637 del DC-SIGN. Materiales y métodos. De los 292 sujetos encuestados, en 191 se confirmó la presencia de fiebre por dengue y los restantes 101 se incluyeron como controles. Los genotipos se resolvieron mediante reacción en cadena de la polimerasa y polimorfismos en la longitud de los fragmentos de restricción (PCR-RFLP). En un intento por determinar el riesgo de sufrir dengue, los datos se analizaron mediante la prueba de ji al cuadrado para los alelos y la regresión logística para los genotipos y las combinaciones alélicas. Los intervalos de confianza se calcularon a 95 % para todas las pruebas independientemente ajustadas por autoidentificación o componente genético ancestral. Resultados. En los afrocolombianos, el alelo C rs8192284 ofreció protección contra el dengue (OR=0,425; 0,204-0,887, p=0,020). Los alelos A rs7248637 y Ars3775290 plantearon un mayor riesgo de dengue para los afrocolombianos (OR=2,389; 1,170- 4,879; p=0,015) y los mestizos (OR=2,329; 1,283-4,226: p=0,005), respectivamente. La reproducibilidad para rs8192284 C/C (OR=2,45; 1,05-5,76; p=0,013) permaneció después del ajuste por el componente genético ancestral amerindio (OR=2,52; 1,04- 6,09; p=0,013). La reproducibilidad del rs3775290 A/A (OR=2,48; 1,09-5,65; p=0,033) permaneció después del ajuste por el componente europeo (OR=2,34; 1,02-5,35; p=0,048), el amerindio (OR=2,49; 1,09- 5,66; p=0,035), y el africano (OR=2,37; 1,04- 5,41; p=0,046). Por último, la asociación del dengue con la combinación alélica CAG (OR=2,07; 1,06-4,05; p=0,033) permaneció después del ajuste por el componente genético amerindio (OR=2,16; 1,09-4,28;p=0,028). Conclusión. Los polimorfismos rs8192284 en IL6R, rs3775290 en TLR3 y rs7248637 en DC-SIGN, se asociaron con la propensión a sufrir dengue en una muestra de población colombiana.

[Dengue-related hepatic compromise in children from the Huila department of Colombia]. / Compromiso hepático por Dengue en niños del Huila, Colombia.

OBJECTIVE: Dengue is the most important arthropod-borne viral disease in the world; it can be life-threatening because of liver involvement. Aim Determining liver involvement frequency and severity in dengue-infected children. METHODS: This was a descriptive case series study which involved studying 108 dengue-infected children aged less than 13 years old whose infection had been confirmed by the detection of dengue-specific IgM and NS1 in plasma. Clinical and biochemical parameters were used for evaluating liver involvement, including transaminases and albumin. Hepatitis A and leptospira infection were also evaluated by using ELISA to detect pathogen-specific IgM in plasma during acute and convalescence phases. The study was carried out at a teaching hospital in Neiva from June 2009 to May 2010. RESULTS: Ninety-eight of the aforementioned cases were clinically classified as dengue with warning signs (DWS) and 10 as severe dengue (SD). Two out of three DWS patients and all SD patients had some degree of liver involvement, shown clinically and biochemically. Regardless of the clinical classification, hepatomegaly was the main clinical sign of liver involvement and was present in 85% of all the children in the study. It is worth noting that 5 patients had probable dengue and leptospirosis co-infection, this being the first instance of this in Colombia. None of the cases analyzed here had acute hepatitis A. CONCLUSIONS: Liver compromise should be considered in confirmed cases of dengue as shown in this series of children. Leptospirosis must be considered as differential diagnosis and also as causing co-infection in a febrile child.