Interruption of viral interference by anti-SARS-CoV-2 vaccination (preprint)

Epstein-Barr virus (EBV) reactivation may be involved in long-COVID symptoms. Here we evaluated reactivation of parvovirus B19 and several viruses of the herpes family in patients with long-COVID syndrome, how vaccination affected viral interference, and how virus reactivation influenced clinical conditions. Clinical and laboratory data on 252 consecutive patients (97 vaccinated and 155 non-vaccinated) were recorded between April 2021–May 2022 (median 243 days post-COVID-19 infection). Viral IgG and IgM titers were compared between vaccinated or non-vaccinated patients, and age and sex-matched healthy controls. Vaccination was associated with significantly less frequent fatigue and multiorgan symptoms (P < 0.001), significantly less cumulative IgM positivity of the investigated viruses, significantly lower plasma levels of IgG subfractions 2 and 4, and significantly lower quantitative Cytomegalovirus (CMV) IgG, CMV IgM, and EBV IgM titers. These results indicate that anti-SARS-CoV2 vaccination interrupts viral crosstalk in patients with long-COVID syndrome. (ClinicalTrials.gov Identifier: NCT05398952)

The Systemic Renin-Angiotensin System in COVID-19 (preprint)

Introduction: SARS-CoV-2 gains cell entry via angiotensin-converting enzyme (ACE) 2, a membrane-bound enzyme of the “alternative” (alt) renin-angiotensin system (RAS). ACE2 counteracts angiotensin II by converting it to potentially protective angiotensin 1–7.Methods Using mass spectrometry, we assessed key metabolites of the classical RAS (angiotensins I–II) and alt-RAS (angiotensins 1–7 and 1–5) pathways as well as ACE and ACE2 concentrations in 159 patients hospitalized with COVID-19, stratified by disease severity (severe, n = 76; non-severe: n = 83). Plasma renin activity (PRA-S) was calculated as the sum of RAS metabolites. We estimated ACE activity using the angiotensin II:I ratio (ACE-S) and estimated systemic alt-RAS activation using the ratio of alt-RAS axis metabolites to PRA-S (ALT-S). We applied mixed linear models to assess how PRA-S and ACE/ACE2 concentrations affected ALT-S, ACE-S, and angiotensins II and 1–7.Results Median angiotensin I and II levels were higher with severe versus non-severe COVID-19 (both p < 0.05), demonstrating activation of classical RAS. The difference disappeared with analysis limited to patients not taking a RAS inhibitor. ALT-S in severe COVID-19 increased with time (days 1–6: 0.12; days 11–16: 0.22) and correlated with ACE2 concentration (r = 0.831). ACE-S was lower in severe versus non-severe COVID-19 (p < 0.001), but ACE concentrations were similar between groups and weakly correlated with ACE-S (r = 0.232). ACE2 and ACE-S trajectories in severe COVID-19, however, did not differ between survivors and non-survivors. Overall RAS alteration in severe COVID-19 resembled severity of disease-matched patients with influenza. In mixed linear models, renin activity most strongly predicted angiotensin II and 1–7 levels. ACE2 also predicted angiotensin 1–7 levels and ALT-S. No single factor or the combined model, however, could fully explain ACE-S. ACE2 and ACE-S trajectories in severe COVID-19 did not differ between survivors and non-survivors.Conclusions Angiotensin II was elevated in severe COVID-19 but markedly influenced by RAS inhibitors and driven by overall RAS activation. ACE-S was significantly lower with severe COVID-19 and did not correlate with ACE concentrations. A shift to the alt-RAS axis because of increased ACE2 could partially explain the relative reduction in angiotensin II levels.

SARS-CoV-2 Postvaccination Infections Among Staff Members of a Tertiary Care University Hospital – Vienna, January-July 2021; an Exploratory Study on 8 500 Employees with Better Outcome of Vector than m-RNA Vaccine (preprint)

Background: The identification of SARS-CoV-2 antigen or RNA in a respiratory specimen collected from a person ≥14 days after completing all recommended doses of authorized COVID-19 vaccine series is a rare condition and defined as „breakthrough infection“. Data on mildly symptomatic or asymptomatic SARS-CoV-2 vaccine breakthrough infection cases especially with respect to viral characteristics and temporal relation to vaccination date is scarce. The purpose of the present investigation was to investigate the prevalence of postvaccination infections in hospital employees and to compare between m-RNA and vector based SARS-CoV-2 vaccines.Methods: At the General Hospital of Vienna 8 553 actively employed staff members participated at an voluntary in-house vaccination program, which took place between January and May 2021 and comprised of application of two doses of either COMIRNATY (BNT162b2; Pfizer/BioNTech, Inc.) or VAXZEVRIA (AstraZeneca, Inc.). Findings: By the end of July 2021 a total of 78 postvaccination infection cases after administration of a minimum of one dose of vaccination had been identified (median age: 40·5 years (IQR: 19-60 years); 53 women) of whom 53 had been vaccinated with VAXZEVRIA and 25 with COMIRNATY. The majority of infections was related to an insufficient or partial vaccination status; the incidence of postvaccination infection ≥14 days after complete vaccination (i.e. breakthrough infection) was 34·8: 10 000 for COMIRNATY and 8·8: 10 000 for VAXZEVRIA. There was no difference in PCR-CT values between the two vaccine brands (24·67 (SD: 7·41) for VAXZEVRIA vaccinated versus 24·0 (SD: 6·53) for COMIRNATY vaccinated persons). Genotyping of positive PCR specimens revealed 42 variant of concerns: B.1.1.7 (n=34); B.1.351 (n=2), B.1.617.2 (n=6). The prevalence of „real brakthrough infections“ (≥14 days after second vaccination) was higher in COMIRNATY vaccinated subjects than in the VAXZEVRIA vaccinated subjects with a ratio of 14:4 (p<0,01). Interpretation: mRNA and vector vaccines against COVID-19 are both highly effective in protecting against a broad genomic spectrum of SARS-CoV-2 infections, at least with respect to severe illness. In the present investigation VAXZEVRIA was more effective than COMIRNATY with respect to prevent breakthrough infections after full immunization, however, it seems important, that all recommended vaccine doses are applicated. Maintaining distinct precautions and ongoing testing, at least for the immediate period after vaccination, to detect asymptomatic infections is highly recommended.Funding: None to declare. Declaration of Interest: We decline no conflicting interest. Ethical Approval: The study was approved by the ethics committee of the Medical University of Vienna [No.1721/2021] and the medical directorate of the hospital.

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

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 (preprint)

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%.

Detection of SARS-CoV-2 by Real-Time PCR under challenging pre-analytical conditions reveals independence of swab media and cooling chain (preprint)

With global demand for SARS-CoV-2 testing ever rising, shortages in commercially available viral transport media pose a serious problem for laboratories and health care providers. For reliable diagnosis of SARS-CoV-2 and other respiratory viruses, executed by Real-time PCR, the quality of respiratory specimens, predominantly determined by transport and storage conditions, is crucial. Therefore, our aim was to explore the reliability of minimal transport media, comprising saline and the CDC recommended Viral Transport Media (HBSS VTM), for the diagnosis of SARS-CoV-2 and other respiratory viruses (influenza A, respiratory syncytial virus, adenovirus, rhinovirus and human metapneumovirus) compared to commercial products, such as the Universal Transport Media (UTM). We question the assumptions, that the choice of medium and temperature for storage and transport affect the accuracy of viral detection by RT-PCR. Both alternatives to the commercial transport medium (UTM), namely the CDC viral transport media (HBSS VTM) and saline, allow adequate detection of SARS-CoV-2 and other respiratory viruses, regardless of the storage temperature and time. 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, independent of the transport medium’s composition.

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

Background SARS-CoV-2 antibody tests have undergone a remarkable improvement in performance. However, due to the low seroprevalence in several areas, very high demands are made on their specificity. Furthermore, the low antibody-response in some individuals requires high test sensitivity to avoid underestimating true seroprevalence. Optimization of testing has been reported through lowering manufacturer cut-offs to improve SARS-CoV-2 assay sensitivity or by combining two tests to improve specificity at the cost of sensitivity. However, these strategies have thus far been used in isolation of each other. Methods To increase sensitivity, cut-offs of three commercially available SARS-CoV-2 automated assays (Roche, Abbott, and DiaSorin) were reduced according to published values in a pre-pandemic specificity cohort (n=1117) and a SARS-CoV-2 positive cohort (n=64). All three testing systems were combined in an orthogonal approach with a confirmatory test, which was one of the remaining automated assays or one of two commercial ELISAs directed against the spike protein receptor binding-domain (RBD) or the nucleocapsid antigen (NP). Results The modified orthogonal test strategy resulted in an improved specificity of at least 99.8%, often even 100%, in all 12 tested combinations with no significant decline in sensitivity. In our cohort, regardless of whether the assays were used for screening or confirmation, combining Roche and Abbott delivered the best overall performance (+~10% sensitivity compared to the single tests and 100% specificity). Conclusion Here we propose a novel orthogonal assay strategy that approaches 100% specificity while maintaining or even significantly improving the screening test's sensitivity.

Covid-19 serology in nephrology health care workers (preprint)

Background: Chronic kidney disease patients show a high mortality in case of a SARS-CoV-2 infection. Thus, to be informed on Nephrology personnel's sero-status might be crucial for patient protection. However, limited information exists about the presence of SARS-CoV-2 antibodies in asymptomatic individuals. Methods: We 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. Results: At 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. Conclusions: Using 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.

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

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.

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

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 (Cohen's Kappa 0.71-0.87), McNemar's 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.