A comprehensive antigen production and characterization study for easy-to-implement, highly specific and quantitative SARS-CoV-2 antibody assays

Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection. While first-generation antibody tests have primarily provided qualitative results with low specificity, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Here, we describe two quantitative ELISA antibody tests based on the SARS-CoV-2 spike receptor-binding domain and the nucleocapsid protein. Comparative expression in bacterial, insect, mammalian and plant-based platforms enabled the identification of new antigen designs with superior quality and high suitability as diagnostic reagents. Both tests scored excellently in clinical validations with multi-centric specificity and sensitivity cohorts and showed unprecedented correlation with SARS-CoV-2 neutralization titers. Orthogonal testing increased assay specificity to 99.8%, thereby enabling robust serodiagnosis in low-prevalence settings. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19.

Analysis of the specificity of the SD Biosensor Standard Q Ag-Test based on Slovak mass testing data

From 31.10. - 1.11.2020 Slovakia has used the SD Biosensor Standard Q Ag-Test for nationwide tests for SARS-CoV-2, in which 3,625,332 persons from 79 counties were tested. Based on this data, we calculate that the specificity of the test is at least 99.6% (with a 97.5% one-sided lower confidence bound). Our analysis is based on a worst case approach in which all positives are assumed to be false positives. Therefore, the actual specificity is expected to exceed 99.6%.

Increasing both specificity and sensitivity of SARS-CoV-2 antibody tests by using an adaptive orthogonal testing approach

BackgroundSerological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems is often poor, leaving room for false positive and false negative results. However, conventional methods used to increase specificity decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the "Sensitivity Improved Two-Test" or " SIT2" algorithm. MethodsSIT2 involves confirmatory re-testing of samples with results falling in a predefined retesting-zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT2 to single tests and orthogonal testing (OTA) in an Austrian cohort (1,117 negative, 64 post-COVID positive samples) and validated the algorithm in an independent British cohort (976 negatives, 536 positives). ResultsThe specificity of SIT2 was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT2 when compared to single tests or OTA. SIT2 allowed correct identification of infected individuals even when a live virus neutralization assay could not detect antibodies. Compared to single testing or OTA, SIT2 significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. ConclusionFor SARS-CoV-2 serology, SIT2 proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.

Covid-19 serology in nephrology health care workers

BackgroundChronic kidney disease patients show a high mortality in case of a SARS-CoV-2 infection. Thus, to be informed on Nephrology personnels sero-status might be crucial for patient protection. However, limited information exists about the presence of SARS-CoV-2 antibodies in asymptomatic individuals. MethodsWe examined the seroprevalence of SARS-CoV-2 IgG and IgM antibodies among health care workers of a tertiary care kidney center during the peak phase of the Covid-19 crisis in Austria using an orthogonal test strategy and a total of 12 commercial nucleocapsid protein or spike glycoprotein based assays as well as Western blotting and a neutralization assay. ResultsAt baseline 60 of 235 study participants (25.5%, 95% CI: 20.4-31.5) were judged to be borderline positive or positive for IgM or IgG using a high sensitivity/low specificity threshold in one test system. Follow-up analysis after about two weeks revealed IgG positivity in 12 (5.1%, 95% CI: 2.9-8.8) and IgM positivity in six (2.6%, 95% CI: 1.1-5.6) in at least one assay. 2.1% (95% CI: 0.8-5.0) of health care workers showed IgG nucleocapsid antibodies in at least two assays. By contrast, positive controls with proven Covid-19 showed antibody positivity among almost all test systems. Moreover, serum samples obtained from health care workers did not show SARS-CoV-2 neutralizing capacity, in contrast to positive controls. ConclusionsUsing a broad spectrum of antibody tests the present study revealed inconsistent results for SARS-CoV-2 seroprevalence among asymptomatic individuals, while this was not the case among Covid-19 patients. Trial registration numberCONEC, ClinicalTrials.gov number NCT04347694

High stability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA under minimal storage conditions for detection by Real-Time PCR

Reliable diagnosis, executed by Real-Time PCR (RT-PCR), builds the current basis in SARS-CoV-2 containment. Transport and storage conditions are the main indicators determining the quality of respiratory specimens. According to shortages in commercially available viral transport media, the primary aim of this study was to explore the reliability of minimal transport media including saline and CDC Viral Transport Media (HBSS VTM) composition for SARS-CoV-2 diagnosis by Real-time PCR compared to recommended commercially available standard Universal Transport Media (UTM). This study also implicated the stability of other respiratory viruses, including influenza A, respiratory syncytial virus, adenovirus, rhinovirus and human metapneumovirus, providing further evidence for future recommendations on transport and storage of respiratory viruses. Both viral transport media (self-made HBSS VTM and UTM) and saline (0.9% NaCl) allow adequate detection of SARS- CoV-2 and other respiratory viruses, regardless of an increase in storage temperature (up to 28 {degrees}C) and time (over 28 days). Treatment of SARS-CoV-2 specimens with varying chlorine concentrations, commonly used in swimming pools, resulted in a significant decrease of viral RNA. ImportanceDetection of respiratory viruses including SARS-CoV-2 depends on the quality of respiratory specimens, predominantly determined by transport and storage conditions. Our study revealed the high resilience of SARS-CoV-2 and other respiratory viruses enabling proper detection in clinical specimens even after long- time storage at high temperatures. This study provides evidence for future recommendations for transport and storage of respiratory viruses, including SARS- CoV-2. Treatment of SARS-CoV-2-positive respiratory specimens with chlorine indicates an early degradation of the virus after the addition of the oxidant, proposing sufficient inactivation of the virus in swimming pool water.

Side by side comparison of three fully automated SARS-CoV-2 antibody assays with a focus on specificity

Background: In the context of the COVID-19 pandemic, numerous new serological test systems for the detection of anti-SARS-CoV-2 antibodies have become available quickly. However, the clinical performance of many of them is still insufficiently described. Therefore we compared three commercial, CE-marked, SARS-CoV-2 antibody assays side by side. Methods: We included a total of 1,154 specimens from pre-COVID-19 times and 65 samples from COVID-19 patients ([≥]14 days after symptom onset) to evaluate the test performance of SARS-CoV-2 serological assays by Abbott, Roche, and DiaSorin. Results: All three assays presented with high specificities: 99.2% (98.6-99.7) for Abbott, 99.7% (99.2-100.0) for Roche, and 98.3% (97.3-98.9) for DiaSorin. In contrast to the manufacturers specifications, sensitivities only ranged from 83.1% to 89.2%. Although the three methods were in good agreement (Cohens Kappa 0.71-0.87), McNemars test revealed significant differences between results obtained from Roche and DiaSorin. However, at low seroprevalences, the minor differences in specificity resulted in profound discrepancies of positive predictability at 1% seroprevalence: 52.3% (36.2-67.9), 77.6% (52.8-91.5), and 32.6% (23.6-43.1) for Abbott, Roche, and DiaSorin, respectively. Conclusion: We find diagnostically relevant differences in specificities for the anti-SARS-CoV-2 antibody assays by Abbott, Roche, and DiaSorin that have a significant impact on the positive predictability of these tests.