Development and Evaluation of a Set of Spike and Receptor Binding Domain-Based Enzyme-Linked Immunosorbent Assays for SARS-CoV-2 Serological Testing.

The implementation and validation of anti-SARS-CoV-2 IgG serological assays are reported in this paper. S1 and RBD proteins were used to coat ELISA plates, and several secondary antibodies served as reporters. The assays were initially validated with 50 RT-PCR positive COVID-19 sera, which showed high IgG titers of mainly IgG1 isotype, followed by IgG3. Low or no IgG2 and IgG4 titers were detected. Then, the RBD/IgG assay was further validated with 887 serum samples from RT-PCR positive COVID-19 individuals collected at different times, including 7, 14, 21, and 40 days after the onset of symptoms. Most of the sera were IgG positive at day 40, with seroconversion happening after 14-21 days. A third party conducted an additional performance test of the RBD/IgG assay with 406 sera, including 149 RT-PCR positive COVID-19 samples, 229 RT-PCR negative COVID-19 individuals, and 28 sera from individuals with other viral infections not related to SARS-CoV-2. The sensitivity of the assay was 99.33%, with a specificity of 97.82%. All the sera collected from individuals with infectious diseases other than COVID-19 were negative. Given the robustness of this RBD/IgG assay, it received approval from the sanitary authority in Mexico (COFEPRIS) for production and commercialization under the name UDISTEST-V2G®.

Poor Survival in COVID-19 Associated with Lymphopenia and Higher Neutrophile-Lymphocyte Ratio.

BACKGROUND: Immune cell counts in blood in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection may be useful prognostic biomarkers of disease severity, mortality, and response to treatment. OBJECTIVES: To analyze sub-populations of lymphocytes at hospital admission in survivors and deceased from severe pneumonia due to coronavirus disease-2019 (COVID-19). METHODS: We conducted a cross-sectional study of healthcare workers confirmed with SARS-CoV-2 in convalescents (control group) and healthy controls (HC) diagnosed with severe COVID-19. Serum samples were taken at hospital admission and after recovery. Serum samples ≥ 25 days after onset of symptoms were analyzed for lymphocyte subpopulations through flow cytometry. Descriptive statistics, Kruskall-Wallis test, receiver operating characteristic curve, calculation of sensitivity, specificity, predictive values, and Kaplan-Meier analysis were performed. RESULTS: We included 337 patients: 120 HC, 127 convalescents, and 90 severe COVID-19 disease patients (50 survivors, 40 deceased). For T cells, total lymphocytes ≥ 800/µL, CD3+ ≥ 400/µL, CD4+ ≥ 180/µL, CD8+ ≥ 150/µL, B cells CD19+ ≥ 80/µL, and NK ≥ 34/µL subsets were associated with survival in severe COVID-19 disease patients. All subtypes of lymphocytes had higher concentrations in survivors than deceased, but similar between HC and convalescents. Leukocytes ≥ 10.150/µL or neutrophils ≥ 10,000/µL were associated with increased mortality. The neutrophil-to-lymphocyte ratio (NLR) ≥ 8.5 increased the probability of death in severe COVID-19 (odds ratio 11.68). CONCLUSIONS: Total lymphocytes; NLR; and levels of CD3+, CD4+, CD8+, and NK cells are useful as biomarkers of survival or mortality in severe COVID-19 disease and commonly reach normal levels in convalescents.

Osteoblast response to zirconia modified-ORMOSILs.

In this study, an in vitro evaluation of the human osteoblasts response to Organically Modified Silicate (ORMOSIL) biomaterials was conducted. These materials were synthetized by sol-gel process being modified with zirconia (ZrO2) and/or Ca2+. The materials were immersed into phosphate buffer solution (PBS) in order to test precipitation of mimetic apatite-like on their surfaces. ORMOSILs were characterized by SEM, FT-IR and X-RD analysis. The response of osteoblast to ORMOSILs was analyzed as a measure of cell adhesion, proliferation and differentiation. The results showed that the addition of Ca2+ ions modifies the surface morphology of ORMOSILs by forming precipitates of mimetic apatite-like with cauliflower and scales morphologies. On the other hand, biological results suggest that the incorporation of zirconia to ORMOSILs increases their ability to support cell adhesion and proliferation. However, the inclusion of both zirconia and Ca2+ in the ORMOSILs decreases their biological compatibility by showing less cell proliferation and lower osteonectin expression, a protein related to osteoblasts. The unfavorable effect of Ca2+ on cell proliferation and cell viability could be due to its ability to induce the formation of mimetic apatite-like with incompatible morphology. The analysis of other proteins related to bone formation on ORMOSIL-Zr and ORMOSIL-Zr-Ca surfaces demonstrated clear expression of osteopontin and osteocalcin in cells growth. In the case of ORMOSIL-Zr, the expression of osteonectin occurred at early stages while the expression of osteopontin and osteocalcin begun at later stages, indicating a switch from an early to a mature stage being stimulated by the biomaterial. Together, these results highlight the important role of zirconia and Ca2+ ions in the composition of materials regulating their biocompatibility when used as scaffolds in bone regeneration.