Interleukin-6 as a Biomarker of Early-Onset Neonatal Sepsis.

OBJECTIVE: The aim of this study is to determine the utility of C reactive protein (CRP) and interleukin (IL)-6 in the diagnosis of neonatal sepsis (NS) in a neonatal intensive care unit (NICU) in the south of Colombia. STUDY DESIGN: A nonmatched case-control study was conducted. Convenience sampling was performed. Data were obtained from clinical records. IL-6 levels were determined using enzyme-linked immunosorbent assay. Receiver operator characteristic (ROC) curve analysis and Youden's index were used to determine the optimal cutoffs for CRP and IL-6 levels in diagnosing NS, early-onset NS (EONS), and late-onset NS (LONS). RESULTS: Data from 31 cases and 62 controls were included. History of chorioamnionitis (infinite odds ratio [OR] [3.07-infinity]), and the presence of meconium-stained amniotic fluid during birth (OR: 9.04 [1.35-112]) were identified as risk factors for NS. Differences in CRP (p < 0.0001) and IL-6 (p < 0.0485) levels were also found, more significantly for LONS and EONS patients, respectively. In the diagnosis of LONS using CRP levels, the area under the ROC curve (AUC) was 0.8371 (p < 0.0001). The optimal cutoff was 0.53 mg/dL. For EONS diagnosis using IL-6, the AUC was 0.6869 (p = 0.0315) and the optimal cutoff was 17.75 pg/mL. CONCLUSION: Differences between CRP and IL-6 levels were found between control and NS groups. Furthermore, CRP showed greater potential diagnostic utility in the LONS group, whereas IL-6 showed greater potential utility in the EONS group. KEY POINTS: · NS is a major morbimortality cause worldwide. · CRP and IL-6 levels may be useful NS biomarkers. · No biomarker alone is enough for the diagnosis of NS.

Locally-secreted interleukin-6 is related with radiological severity in smear-negative pulmonary tuberculosis.

Pulmonary tuberculosis (PTB) has been identified as a substantial public health threat and diagnostic challenge. A large proportion of patients exhibit negative smear tests despite active infection. The role of cytokines in the pathophysiology and clinical severity of PTB remains a controversial question. We evaluated the pattern of cytokines presents locally in patients with smear-negative PTB. Levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, and IL-17 in bronchoalveolar lavage fluid (BALf) from patients with smear-negative PTB, as well as in those with other pulmonary diseases and controls, were performed by flow cytometry. ROC curve and a radiological severity scale were used to establish the potential diagnosis use and the relationship of the cytokine levels with disease severity, respectively. The levels of IL-6 were higher in the PTB (P = 0.0249) and pneumonia (P = 0.0047) groups compared to controls. Low to undetectable levels of TNF-α, IFN-γ, IL-2, IL-4, IL-10, and IL-17 were found in BALf, even after sample concentration using filtration columns and centrifugation. IL-6 levels measured in BALf could distinguish PTB patients or pneumonia patients from controls (AUC: 0.91, P = 0.002 and AUC: 0.86, P = 0.001, respectively), but not patients with PTB from those with pneumonia (AUC: 0.51, P = 0.86). IL-6 levels were related with the severity of PTB, as levels were higher in patients with higher radiological severity. These results confirm the importance of IL-6 in the immunopathology of smear-negative PTB.

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.

First Analysis of SERPING1 Gene in Patients with Hereditary Angioedema in Colombia Reveals Two Genotypic Variants in a Highly Symptomatic Individual.

Hereditary angioedema (HAE) is a heterogeneous genetic disease caused by a deficit in C1 inhibitor (C1-INH) and clinically characterized by sudden events of edema, swelling, and pruritus. Here, we describe the first SERPING1 genotyping in 22 subjects from 4 non-related families, all from southern Colombia. The previously reported heterozygous gene mutations, c.1081C>T (p.Gln361*), c.1396C>G (p.Arg466Gly), c.1029+84G>A, or c.106_107del (p.Ser36Phefs*21), were found in 12 patients. Of note, a single patient clinically characterized as severe HAE type 2 expressed mutations in exon 8 and intron 6, whereas all the others have type 1 HAE and expressed one pathogenic variant. One of the subjects, a 5-year-old girl was discovered to have a pathogenic variant, and she is still asymptomatic. This is the first report focused on HAE genetic analysis in a Colombian population.

Human rotavirus-specific IgM Memory B cells have differential cloning efficiencies and switch capacities and play a role in antiviral immunity in vivo.

Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM(+) mBc, we found that 62% and 21% of total (non-antigen-specific) IgM(+) and RV-IgM(+) mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM(+) (17%) and RV-IgM(+) (7%) mBc were lower than those of the corresponding switched (IgG(+) IgA(+)) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM(+) RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2Rγ(null)) immunodeficient mice were adoptively transferred highly purified human IgM(+) mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM(+) mBc are phenotypically diverse and significantly enriched in the IgM(hi) IgD(low) subset. Thus, RV-IgM(+) mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.

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.

Clinical and Laboratory Characteristics of Hemophagocytic Lymphohistiocytosis in Children With Severe Dengue During the 2019-2020 Outbreak in Southern Colombia.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled activation of inflammatory cells and an exaggerated release of cytokines. It can be triggered by different factors, including viruses, such as dengue. The objective of this study was to characterize the clinical and laboratory profiles of children with severe dengue and HLH, and to identify the risk factors for this clinical complication. METHODS: An analytical study was conducted in children with severe dengue who were treated in an intensive care unit between January 2019 and March 2020. Clinical and laboratory factors were compared between patients with and without HLH. RESULTS: HLH represented 13.4% (15/112) of children with severe dengue. Patients with HLH had a long-lasting fever (10.1 vs. 5.8 days; P = 0.012), low hemoglobin levels (7.6 vs. 10.8 g/dL; P = 0.000) and high aspartate aminotransferase values (4443 vs. 1061 U/L; P = 0.002), alanine transaminase (1433 vs. 487 U/L; P = 0.004), partial thromboplastin time (80.6 vs. 51.8 seconds; P = 0.010), prothrombin time (23.5 vs. 19.6 seconds; P = 0.024), triglycerides (333.7 vs. 223.2 mg/dL; P = 0.005), lactate dehydrogenase (4209 vs. 1947 U/L; P = 0.006), soluble CD25 (3488 vs. 1026 pg/mL; P = 0.014), and presented with higher frequency of myocarditis (66.7% vs. 38.3%; P = 0.048), hepatitis (5.3% vs. 1.3%; P = 0.014), bacterial coinfection (73.3% vs. 26.7%; P = 0.010) and fatal outcome (26% vs. 5%; P = 0.037). CONCLUSIONS: HLH is a serious life-threatening clinical complication of dengue virus infection that must be considered, particularly during outbreaks.

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.

A Specific Pattern and Dynamics of Circulating Cytokines Are Associated with the Extension of Lung Injury and Mortality in Colombian Adults with Coronavirus Disease-19.

Increased systemic levels of inflammatory cytokines have been associated with the development of pathophysiologic events during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To further explore differences in the pattern and dynamics of plasma cytokines in individuals with coronavirus disease-19 (COVID-19), and the relationship with disease mortality, here we evaluated the plasma levels of proinflammatory and regulatory cytokines in Colombian patient survivors and nonsurvivors of SARS-CoV-2 infection. Individuals with confirmed COVID-19, with other respiratory diseases requiring hospitalization, and healthy controls, were included. Plasma levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon-γ, IL-10, soluble tumor necrosis factor receptor I (sTNFRI), and transforming growth factor-ß1 were measured by a bead-based assay or enzyme-linked immunosorbent assay and clinical, laboratory, and tomographic parameters were registered during hospitalization. The levels of most of the evaluated cytokines were increased in COVID-19 individuals relative to healthy controls. The levels of IL-6, IL-10, and sTNFRI were directly associated with the development of respiratory failure, immune dysregulation, and coagulopathy, as well as with COVID-19 mortality. Particularly, the early, robust, and persistent increase of circulating IL-6 characterized COVID-19 nonsurvivors, while survivors were able to counteract the inflammatory cytokine response. In addition, IL-6 systemic levels positively correlated with the tomographic extension of lung damage in individuals with COVID-19. Thus, an exacerbated inflammatory cytokine response, particularly mediated by IL-6 added to the inefficiency of regulatory cytokines, distinguishes COVID-19-associated tissue disturbances, severity, and mortality in Colombian adults.

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.

Frequency, Markers and Costs of Secondary Bacterial Infection in Pediatric Dengue.

BACKGROUND: Secondary bacterial infection (SBI) occurs in a proportion of individuals with dengue and results in longer hospitalization, higher mortality, and increased health-related costs. However, the frequency, risk factors and predictive biomarkers of this comorbidity in pediatric dengue is partially known. METHODS: We conducted a retrospective multicenter study in a dengue hyperendemic region of Colombia, analyzing 1597 children from two pediatric cohorts. We included children with confirmed dengue (mild to severe disease) and evaluated the rate of SBI, their clinical characteristics, diagnostic predictors and attention costs. We also assessed the diagnostic performance of plasma interleukin (IL)-6 for detecting SBI in pediatric dengue. RESULTS: The frequency of SBI in children with dengue with warning signs in cohorts 1 and 2 was 2.4% and 7.3%, respectively, and this rate reached 30.7% and 38.2% in children with severe disease. Staphylococcus aureus and Escherichia coli were the more frequent infectious agents. Increased total leukocytes and C-reactive protein levels, as well as high IL-6 at hospital admission, in children <48 months of age were early indications of SBI in dengue. Higher rates of organ dysfunction, the requirement of a longer hospitalization and a 2.3-fold increase in attention costs were observed in SBI. CONCLUSIONS: An important proportion of children with dengue course with SBI and exhibit higher morbidity. Elevated leukocytes, C-reactive protein and IL-6 in young children are early markers of SBI. Physicians should identify children with dengue and risk factors for SBI, microbiologically confirm the bacterial infection, and rationally and timely provide antimicrobial therapy.

Human myeloid dendritic cells treated with supernatants of rotavirus infected Caco-2 cells induce a poor Th1 response.

We have previously shown that human myeloid dendritic cells treated with purified rotavirus induce an allogenic Th1 response. To determine if rotavirus in the context of an intestinal microenvironment modulates the function of dendritic cells, we treated these cells with supernatants from non-infected or infected Caco-2 cells and studied their capacity to promote Th1 or Th2 responses. Dendritic cells treated with supernatants from rotavirus-infected Caco-2 cells promoted a significantly lower Th1 response, in comparison with those treated with purified rotavirus. We wanted to establish if TGF-ß1, induced, or TSLP, not induced, during rotavirus infection, could mediate this effect. Neutralization of TGF-ß but not TSLP in the supernatant prior to treatment of dendritic cells increased their capacity to promote a Th1 response. The results suggest that the TGF-ß1 induced by rotavirus could be an immune evasion mechanism, and may partially explain the poor rotavirus-specific T cell response we have previously evidenced.

Dengue Virus and Platelets: From the Biology to the Clinic.

Dengue is one of the most important vector-borne viral illnesses found in tropical and subtropical regions. Colombia has one of the highest rates of dengue cases in the Americas. Severe dengue virus (DENV) infection presents with capillary leakage, hemorrhage, and organ compromise, eventually leading to death. Over the years, there have been many efforts to develop a vaccine that guarantees protective immunity, but they have been partially successful, as such immunity would need to guarantee protection against four distinct viral serotypes. Absolute platelet count is a laboratory parameter used to monitor the clinical progression of DENV, as infection is often accompanied by thrombocytopenia. Although this finding is well described with respect to the natural history of the disease, there are various hypotheses as to the cause of this rapid decrease, and several in vivo and ex vivo models have been used to explain the effect of DENV infection on platelets and their precursors. DENV infects and activates platelets, facilitating their elimination through recognition by phagocytic cells and peripheral margination. However, infection also affects the precursors in the bone marrow by modulating megakaryopoiesis. The objective of this article is to explore various proposed mechanisms of DENV-induced thrombocytopenia to better understand the pathophysiology and clinical presentations of this highly relevant viral infection.

Combination of the Focus-Forming Assay and Digital Automated Imaging Analysis for the Detection of Dengue and Zika Viral Loads in Cultures and Acute Disease.

Optimized methods for the detection of flavivirus infections in hyperendemic areas are still needed, especially for working with patient serum as a starting material. The focus-forming assay (FFA) reveals critical aspects of virus-host interactions, as it is a quantitative assay to determine viral loads. Automated image analysis provides evaluations of relative amounts of intracellular viral protein at the single-cell level. Here, we developed an optimized FFA for the detection of infectious Zika virus (ZIKV) and dengue virus (DENV) viral particles in cell cultures and clinical serum samples, respectively. Vero-76 cells were infected with DENV-2 (16681) or ZIKV (PRVA BC59). Using a panel of anti-DENV and anti-ZIKV NS1-specific monoclonal antibodies (mAbs), the primary mAbs, concentration, and the optimal time of infection were determined. To determine whether intracellular accumulation of NS1 improved the efficiency of the FFA, brefeldin A was added to the cultures. Focus formation was identified by conventional optical microscopy combined with CellProfiler™ automated image analysis software. The FFA was used with spike assays for ZIKV and clinical specimens from natural infection by DENV-1 and DENV-2. mAb 7744-644 for ZIKV and mAb 724-323 for DENV used at a concentration of 1 µg/ml and a time of 24 hours postinfection produced the best detection of foci when combining conventional counting and automated digital analysis. Brefeldin A did not improve the assessment of FFUs or their digitally assessed intensity at single-cell level. The FFA showed 95% ZIKV recovery and achieved the detection of circulating DENV-1 and DENV-2 in the plasma of acutely ill patients. The combination of the two techniques optimized the FFA, allowing the study of DENV and ZIKV in culture supernatants and clinical specimens from natural infection in hyperendemic areas.

Dengue and Zika virus differential infection of human megakaryoblast MEG-01 reveals unique cellular markers.

Platelet count is widely used for the diagnosis and follow-up of patients with dengue. Despite its close viral structural and symptomatic homology, ZIKV infection does not typically induce significant thrombocytopenia. To determine the effect of DENV-2 and ZIKV infection on human platelet precursors we utilized MEG-01 cell line to evaluate the viral infection, viability, innate gene expression and release of platelet-like particles (PLPs). DENV-2 induced a higher proportion of cell death at 48-72 h post-infection than ZIKV. The median range of intracellular NS1+/E+ cells was 11.2% (3.3%-25%) and 5% (3%-8.1%) for DENV-2 and ZIKV, respectively (p = 0.03). MEG-01 cells infected with DENV-2 quickly expressed higher levels of IFN-ß, indolamine 2,3-dioxygenase and CXCL10 mRNA compared to ZIKV infected cells and DENV-2 but not ZIKV infection reduced the number PLPs from stimulated MEG-01 cells. The results shed light into mechanisms including thrombocytopenia present in patients with DENV but absent in ZIKV infections.

Rotavirus differentially infects and polyclonally stimulates human B cells depending on their differentiation state and tissue of origin.

We have shown previously that rotavirus (RV) can infect murine intestinal B220(+) cells in vivo (M. Fenaux, M. A. Cuadras, N. Feng, M. Jaimes, and H. B. Greenberg, J. Virol. 80:5219-5232, 2006) and human blood B cells in vitro (M. C. Mesa, L. S. Rodriguez, M. A. Franco, and J. Angel, Virology 366:174-184, 2007). However, the effect of RV on B cells, especially those present in the human intestine, the primary site of RV infection, is unknown. Here, we compared the effects of the in vitro RV infection of human circulating (CBC) and intestinal B cells (IBC). RV infected four times more IBC than CBC, and in both types of B cells the viral replication was highly restricted to the memory subset. RV induced cell death in 30 and 3% of infected CBC and IBC, respectively. Moreover, RV induced activation and differentiation into antibody-secreting cells (ASC) of CBC but not IBC when the B cells were present with other mononuclear cells. However, RV did not induce these effects in purified CBC or IBC, suggesting the participation of other cells in activating and differentiating CBC. RV infection was associated with enhanced interleukin-6 (IL-6) production by CBC independent of viral replication. The infection of the anti-B-cell receptor, lipopolysaccharide, or CpG-stimulated CBC reduced the secretion of IL-6 and IL-8 and decreased the number of ASC. These inhibitory effects were associated with an increase in viral replication and cell death and were observed in polyclonally stimulated CBC but not in IBC. Thus, RV differentially interacts with primary human B cells depending on their tissue of origin and differentiation stage, and it affects their capacity to modulate the local and systemic immune responses.

A Case Series of Severe Dengue with Neurological Presentation in Children from a Colombian Hyperendemic Area.

Dengue transmission is sustained in Colombia with increasing prevalence mainly in children. This work aimed to describe a case series of children diagnosed with dengue presenting neurological disease in Huila Province of Colombia. Eleven pediatric febrile patients confirmed for dengue disease and presenting neurological signs were studied in the University Hospital of Neiva, Huila Province. Clinical and laboratory findings, CSF cytochemical analysis, neurology images, and serology and molecular studies were performed. Viral RNA was detected in all patients' sera by RT-PCR. Nine out of 11 were primary infections. Tonic-clonic seizures (73%), consciousness alterations (27%), irritability (27%), and ataxia (18%) were the most frequent neurological signs. None of the patients had plasma leakage, hypovolemic shock, or liver disease, confirming the encephalitis diagnosis. Diagnostic images did not show abnormal findings, but neither bacterial nor fungal infections were detected in CSF analysis. All patients survived without sequelae except for one patient that presented ataxia for months. In conclusion, we described a group of children with neurological signs during severe dengue disease as the main finding, indicating the importance to including dengue as a differential diagnosis in neurological patients from endemic areas.

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.

Serotype-specific detection of dengue viruses in a nonstructural protein 1-based enzyme-linked immunosorbent assay validated with a multi-national cohort.

BACKGROUND: Dengue virus (DENV) infections pose one of the largest global barriers to human health. The four serotypes (DENV 1-4) present different symptoms and influence immune response to subsequent DENV infections, rendering surveillance, risk assessments, and disease control particularly challenging. Early diagnosis and appropriate clinical management is critical and can be achieved by detecting DENV nonstructural protein 1 (NS1) in serum during the acute phase. However, few NS1-based tests have been developed that are capable of differentiating DENV serotypes and none are currently commercially available. METHODOLOGY/PRINCIPLE FINDINGS: We developed an enzyme-linked immunosorbent assay (ELISA) to distinguish DENV-1-4 NS1 using serotype-specific pairs of monoclonal antibodies. A total of 1,046 antibodies were harvested from DENV-immunized mice and screened for antigen binding affinity. ELISA clinical performance was evaluated using 408 polymerase chain reaction-confirmed dengue samples obtained from patients in Brazil, Honduras, and India. The overall sensitivity of the test for pan-DENV was 79.66% (325/408), and the sensitivities for DENV-1-4 serotyping were 79.1% (38/48), 80.41% (78/97), 100% (45/45), and 79.6% (98/123), respectively. Specificity reached 94.07-100%. SIGNIFICANCE: Our study demonstrates a robust antibody screening strategy that enabled the development of a serotype NS1-based ELISA with maximized specific and sensitive antigen binding. This sensitive and specific assay also utilized the most expansive cohort to date, and of which about half are from Latin America, a geographic region severely underrepresented in previous similar studies. This ELISA test offers potential enhanced diagnostics during the acute phase of infection to help guide patient care and disease control. These results indicate that this ELISA is a promising aid in early DENV-1-4 diagnosis and surveillance in regions of endemicity in addition to offer convenient monitoring for future vaccine interventions.