SARS-CoV-2 infections in "less visible" hospital staff: The roles and safety of environmental services and allied health professionals.

BACKGROUND: During the early SARS-CoV-2 pandemic, all healthcare workers had specific and essential functions. However, environmental services (e.g., cleaning staff) and allied health professionals (e.g., physiotherapists) are often less recognised inpatient care. The aim of our study was to evaluate SARS-CoV-2-infection rates and describe risk factors relevant to workplace transmission and occupational safety amongst healthcare workers in COVID-19 hospitals before the introduction of SARS-CoV-2-specific vaccines. METHODS: This cross-sectional study (from May 2020 to March 2021, standardised WHO early-investigation protocol) is evaluating workplace or health-related data, COVID-19-patient proximity, personal protective equipment (PPE) use, and adherence to infection prevention and control (IPC) measures, anti-SARS-CoV-2-antibody status, and transmission pathways. RESULTS: Out of n = 221 HCW (n = 189 cleaning/service staff; n = 32 allied health professionals), n = 17 (7.7 %) were seropositive. While even SARS-CoV-2-naïve HCW reported SARS-CoV-2-related symptoms, airway symptoms, loss of smell or taste, and appetite were the most specific for a SARS-CoV-2-infection. Adherence to IPC (98.6 %) and recommended PPE use (98.2 %) were high and not associated with seropositivity. In 70.6 %, transmission occurred in private settings; in 23.5 %, at the workplace (by interaction with SARS-CoV-2-positive colleagues [17.6 %] or patient contact [5.9 %]), or remained unclear (one case). CONCLUSIONS: Infection rates were higher in all assessed 'less visible' healthcare-worker groups compared to the general population. Our data indicates that, while IPC measures and PPE may have contributed to the prevention of patient-to-healthcare-worker transmissions, infections were commonly acquired outside of work and transmitted between healthcare workers within the hospital. This finding emphasises the importance of ongoing education on transmission prevention and regular infection screenings at work.

MULTI-PARAMETRIC PREDICTION MODELS FOR COVID-19 VACCINE SELECTION: RESULTS OF A COMPARATIVE POPULATION-BASED COHORT STUDY.

BACKGROUND: Understanding vaccine-dependent effects on protective and sustained humoral immune response are crucial to plan further vaccination strategies against COVID-19. Population-based data comparing different vaccination strategies are lacking. METHODS: This multicenter, population-based cohort study included 4,601 individuals ≥18 years of age after primary vaccination against COVID-19 at least four months ago (full immunization). We compared factors associated with residual antibody levels against SARS-CoV-2 receptor binding domain (RBD) across different vaccination strategies (BNT162b2, mRNA-1273, or ChAdOx1 nCoV-19 [ChAdOx1]). RESULTS: Our main model including 3,787 individuals (2xBNT162b2 n = 2,271, 2xmRNA-1273 n = 251, 2xChAdOx1 n = 1,265) predicted significantly lower levels of anti-RBD-antibodies after 6 months in individuals vaccinated with ChAdOx1 (392.7 BAU/ml) compared to those vaccinated with BNT162b2 (1179.5 BAU/ml) or mRNA-1273 (2098.2 BAU/ml). Vaccine-dependent association of antibody levels was found for age with a significant predicted difference in BAU/ml per year for BNT162b2 (-21.5 [95%CI -24.7 to -18.3]) and no significant association for mRNA-1273 (-4.0 [95%CI -20.0 to 12.1]) or ChAdOx1 (1.7 [95%CI 0.2 to 3.1]). The predicted decrease over time since full immunization was highest in mRNA-1273 (-23.4 [95%CI -31.4 to -15.4]) compared to BNT162b2 -5.9 [95%CI -7 to -4.8]). Higher antibody levels were observed for individuals with systemic adverse events upon vaccination and current smoking (BNT162b2), for days between first and second vaccination (BNT162b2 and ChAdOx1) and for absence of comorbidities (all). CONCLUSION: Our study revealed population-based evidence of vaccine-dependent effects of age and time since full immunization on humoral immune response. Findings underline the importance of an individualized vaccine selection, especially in elderly individuals.

Comparison of five Anti-SARS-CoV-2 antibody assays across three doses of BNT162b2 reveals insufficient standardization of SARS-CoV-2 serology.

OBJECTIVES: To investigate the comparability of WHO standard referenced commercial SARS-CoV-2 antibody tests over three doses of BNT162b2 vaccine and up to 14 months. METHODS: 114 subjects (without previous SARS-CoV-2 infection or immunosuppressive medication) vaccinated with three doses of BNT162b2 were included in this study. Antibody levels were quantified 3 weeks after the first dose, 5-6 weeks and 7 months after the second dose, and 4-5 weeks and 4 months after the third dose using the Roche Elecsys SARS-CoV-2 S, the Abbott SARS-CoV-2 IgG II Quant, the DiaSorin LIAISON SARS-CoV-2 TrimericS IgG, the GenScript cPASS sVNT and the TECO sVNT assays. RESULTS: For each time point analyzed, systematic differences are evident between the results in BAU/mL of the three antibody binding assays. The assay ratios change in a time-dependent manner even beyond administering the third dose (Roche measuring 9 and 3 times higher than Abbott and DiaSorin, respectively). However, changes decrease in magnitude with increasing time intervals from the first dose. IgG-based assays show better agreement across them than with Roche (overall correlations: Abbott x DiaSorin: ρ = 0.94 vs. Abbott x Roche: ρ=0.89, p < 0.0001; DiaSorin x Roche: ρ = 0.87, p < 0.0001), but results are not interchangeable. The sVNTs suggest an underestimation of antibody levels by Roche and slight overestimation by both IgG assays after the first vaccine dose. CONCLUSIONS: Standardization of SARS-CoV-2 antibody binding assays still needs to be improved to allow reliable use of variable assay systems for longitudinal analyses.

Preanalytical stability of SARS-CoV-2 anti-nucleocapsid antibodies.

OBJECTIVES: Anti-nucleocapsid (NC) antibodies are produced in response to SARS-CoV-2 infection. Therefore, they are well suited for the detection of a previous infection. Especially in the case of seroprevalence studies or during the evaluation of a novel in-vitro diagnostic test, samples have been stored at <-70 °C (short- and long-term) or 2-10 °C (short-term) before analysis. This study aimed to assess the impact of different storage conditions relevant to routine biobanking on anti-NC antibodies. METHODS: The preanalytical impact of short-term storage (84 [58-98] days) on <-70 °C and for 14 days at 2-10 °C was evaluated using samples from 111 donors of the MedUni Vienna Biobank. Long-term effects (443 [409-468] days) were assessed using 208 samples from Biobank Graz and 49 samples from Biobank Vienna. Anti-Nucleocapsid antibodies were measured employing electrochemiluminescence assays (Roche Anti-SARS-CoV-2). RESULTS: After short-term storage, the observed changes did not exceed the extent that could be explained by analytical variability. In contrast, results after long-term storage were approximately 20% higher and seemed to increase with storage duration. This effect was independent of the biobank from which the samples were obtained. Accordingly, the sensitivity increased from 92.6 to 95.3% (p=0.008). However, comparisons with data from Anti-Spike protein assays, where these deviations were not apparent, suggest that this deviation could also be explained by the analytical variability of the qualitative Anti-NC assay. CONCLUSIONS: Results from anti-NC antibodies are stable during short-term storage at <-70 °C and 2-10 °C. After long-term storage, a slight increase in sensitivity could not be ruled out.

Increasing test specificity without impairing sensitivity: lessons learned from SARS-CoV-2 serology.

BACKGROUND: Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and 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. METHODS: SIT² involves confirmatory retesting 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 (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives). RESULTS: The specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with 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. CONCLUSION: For 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.

Reduced Sensitivity of Commercial Spike-Specific Antibody Assays after Primary Infection with the SARS-CoV-2 Omicron Variant.

The SARS-CoV-2 Omicron variant is characterized by substantial changes in the antigenic structure of the Spike (S) protein. Therefore, antibodies induced by primary Omicron infection lack neutralizing activity against earlier variants. In this study, we analyzed whether these antigenic changes impact the sensitivity of commercial anti-SARS-CoV-2 antibody assays. Sera from 37 unvaccinated, convalescent individuals after putative primary Omicron infection were tested with a panel of 20 commercial anti-SARS-CoV-2 immunoassays. As controls, we used samples from 43 individuals after primary infection with the SARS-CoV-2 ancestral wild-type strain. In addition, variant-specific live-virus neutralization assays were used as a reference for the presence of SARS-CoV-2-specific antibodies in the samples. Notably, in Omicron convalescents, there was a statistically significant reduction in the sensitivity of all antibody assays containing S or its receptor-binding-domain (RBD) as antigens. Furthermore, antibody levels quantified by these assays displayed a weaker correlation with Omicron-specific neutralizing antibody titers than with those against the wild type. In contrast, the sensitivity of nucleocapsid-protein-specific immunoassays was similar in wild-type and Omicron-infected subjects. In summary, the antigenic changes in the Omicron S lead to reduced immunoreactivity in the current commercial S- and RBD-specific antibody assays, impairing their diagnostic performance. IMPORTANCE This study demonstrates that the antigenic changes of the SARS-CoV-2 Omicron variant affect test results from commercial Spike- and RBD-specific antibody assays, significantly diminishing their sensitivities and diagnostic abilities to assess neutralizing antibodies.

The Comparability of Anti-Spike SARS-CoV-2 Antibody Tests is Time-Dependent: a Prospective Observational Study.

Various commercial anti-Spike SARS-CoV-2 antibody tests are used for studies and in clinical settings after vaccination. An international standard for SARS-CoV-2 antibodies has been established to achieve comparability of such tests, allowing conversions to BAU/mL. This study aimed to investigate the comparability of antibody tests regarding the timing of blood collection after vaccination. For this prospective observational study, antibody levels of 50 participants with homologous AZD1222 vaccination were evaluated at 3 and 11 weeks after the first dose and 3 weeks after the second dose using two commercial anti-Spike binding antibody assays (Roche and Abbott) and a surrogate neutralization assay. The correlation between Roche and Abbott changed significantly depending on the time point studied. Although Abbott provided values three times higher than Roche 3 weeks after the first dose, the values for Roche were twice as high as for Abbott 11 weeks after the first dose and 5 to 6 times higher at 3 weeks after the second dose. The comparability of quantitative anti-Spike SARS-CoV-2 antibody tests was highly dependent on the timing of blood collection after vaccination. Therefore, standardization of the timing of blood collection might be necessary for the comparability of different quantitative SARS-COV-2 antibody assays. IMPORTANCE This work showed that the comparability of apparently standardized SARS-CoV-2 antibody assays (Roche, Abbott; both given in BAU/mL) after vaccination depends on the time of blood withdrawal. Initially (3 weeks after the first dose AZD1222), there were 3 times higher values in the Abbott assay, but this relationship inversed before boosting (11 weeks after the first dose) with Roche 2 times greater than Abbott. After the booster, Roche quantified ca. 5 times higher levels than Abbott. This must be considered by clinicians when interpreting SARS-CoV-2 antibody levels.

Serum antibody response to BNT162b2 after natural SARS-CoV-2 infection.

BACKGROUND: There is preliminary evidence that individuals with previous SARS-CoV-2 infections exhibit a more pronounced antibody response. However, these assumptions have not yet been supported by data obtained through various CE-marked tests. This study aimed to close this gap. METHODS: Sixty-nine seronegatives and 12 individuals post-SARS-CoV-2 infection (tested by CE-labelled Roche NC immunoassay or PCR-confirmed assay) were included 21 ± 1 days after receiving the first dose of the Pfizer/BioNTech BNT162b2 vaccine. Antibody response to viral spike protein (S) was assessed by CE-labelled Roche S and DiaSorin S1/S2 assays and by a surrogate virus neutralization test (sVNT). RESULTS: After a single dose of BNT162b2, individuals after natural SARS-CoV-2 infection presented with markedly higher anti-S levels than naïve individuals (Roche S: 9078.5 BAU/mL [5267.0-24 298.5] vs 79.6 [24.7-142.3]; and DiaSorin S1/S2: 1465.0 AU/mL [631.0-5365.0] vs 63.7 [47.8-87.5]) and showed all the maximum observed inhibition activity in the sVNT (98%), without overlaps between groups. There was a trend for higher responses in those with a more distant infection, although not statistically significant. The relative antibody increase after dose 2 was significantly higher among naïve individuals (25-fold), but antibody levels remained below that of seropositives. CONCLUSIONS: Compared with naïve individuals, seropositives after natural SARS-CoV-2 infection presented with a substantially higher antibody response already after dose 1 of BNT162b2, as measured by two CE-marked in vitro diagnostic tests and a sVNT. These results should stimulate discussion and research on whether individuals after previous SARS-CoV-2 infection would benefit from a two-part vaccination schedule or whether these currently much-needed second doses could be saved.

Initial SARS-CoV-2 vaccination response can predict booster response for BNT162b2 but not for AZD1222.

OBJECTIVE: To determine whether severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibody levels after the first dose of vaccine can predict the final antibody response, and whether this is dependent on the vaccine type. METHODS: Sixty-nine recipients of BNT162b2 (Pfizer/BioNTech) and 55 recipients of AZD1222 (AstraZeneca), without previous infection or immunosuppressive medication, were included in this study. Antibody levels were quantified 3 weeks after the first dose [directly before boostering in the case of AZD1222 (11 weeks after the first dose)] and 3 weeks after the second dose using the Roche Elecsys SARS-CoV-2 S total antibody assay. RESULTS: Median pre-booster {BNT162b2: 80.6 [interquartile range (IQR) 25.5-167.0] binding antibody units (BAU)/mL; AZD1222: 56.4 (IQR 36.4-104.8) BAU/mL; not significant} and post-booster [BNT162b2: 2092.0 (IQR 1216.3-4431.8) BAU/mL; AZD1222: 957.0 (IQR 684.5-1684.8) BAU/mL; P<0.0001] levels correlated well in the recipients of BNT162b2 (ρ=0.53) but not in the recipients of AZD1222. Moreover, antibody levels after the first dose of BNT162b2 correlated inversely with age (ρ=-0.33, P=0.013), whereas a positive correlation with age was observed after the second dose in recipients of AZD1222 (ρ=0.26, P=0.030). CONCLUSIONS: The results of this study suggest that antibody levels quantified by the Roche Elecsys SARS-CoV-2 S assay before the booster shot could infer post-booster responses to BNT162b2, but not to AZ1222. In addition, this study found a vaccine-dependent effect on antibody responses, where age seems to play an ambivalent role.

Anti-Spike Protein Assays to Determine SARS-CoV-2 Antibody Levels: a Head-to-Head Comparison of Five Quantitative Assays.

Reliable quantification of the antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly relevant, e.g., for identifying possible vaccine failure and estimating the time of protection. Therefore, we evaluated five different anti-SARS-CoV-2 antibody assays regarding the quantification of anti-spike (S) antibodies. Sera from 69 SARS-CoV-2-naive individuals 21 ± 1 days after vaccination with a single dose of BNT162b2 (Pfizer/BioNTech) were tested using the following quantitative assays: Roche S total antibody, DiaSorin trimeric spike IgG, DiaSorin S1/S2 IgG, Abbott II IgG, and Serion/Virion IgG. Results were further compared to the percent inhibition calculated from a surrogate virus neutralization test (sVNT). Individual values were distributed over several orders of magnitude for all assays. Although the assays were in good overall agreement (ρ = 0.80 to 0.94), Passing-Bablok regression revealed systematic constant and proportional differences, which could not be eliminated by converting the results to binding antibody units (BAU) per milliliter, as suggested by the manufacturers. Seven (10%) individuals had negative sVNT results (i.e., <30% inhibition). These samples were identified by most assays and yielded significantly lower binding antibody levels. Although all assays showed good correlation, they were not interchangeable, even when converted to BAU per milliliter using the WHO international standard for SARS-CoV-2 immunoglobulin. This highlights the need for further standardization of SARS-CoV-2 serology. IMPORTANCE Reliable quantification of the antibody response to SARS-CoV-2 is highly relevant, e.g., for identifying possible vaccine failure and estimating the time of protection. We compared the performance of five CE marked tests that quantify antibodies against the viral spike protein. Our findings suggest that, although all assays showed good correlation, their results were not interchangeable, even when converted to BAU per milliliter using the WHO international standard for SARS-CoV-2 immunoglobulin. This highlights the need for further standardization of SARS-CoV-2 serology.

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 rapidly have become available. However, the clinical performance of many of these is still insufficiently described. Therefore, we compared 3 commercial CE-marked, SARS-CoV-2 antibody assays side by side. METHODS: We included a total of 1154 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 3 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 3 methods were in good agreement (Cohen's Kappa 0.71-0.87), McNemar tests 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 predictive values 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 found 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 predictive values of these tests.