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.

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.

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.