Serum SARS-CoV-2 Antigens for the Determination of COVID-19 Severity.

The diagnostic of SARS-CoV-2 infection relies on reverse transcriptase polymerase chain reactions (RT-PCRs) performed on nasopharyngeal (NP) swabs. Nevertheless, false-negative results can be obtained with inadequate sampling procedures, making the use of other biological matrices worthy of investigation. This study aims to evaluate the kinetics of serum N antigens in severe and non-severe patients and compare the clinical performance of serum antigenic assays with NP RT-PCR. Ninety patients were included in the study and monitored for several days. Disease severity was determined according to the WHO clinical progression scale. Serum N antigen levels were measured with a chemiluminescent assay (CLIA) and the Single Molecular Array (Simoa) assay. Viremia thresholds for severity were determined and proposed. In severe patients, the peak antigen response was observed 7 days after the onset of symptoms, followed by a decline. No real peak response was observed in non-severe patients. Severity thresholds for the Simoa and the CLIA provided positive likelihood ratios of 30.0 and 10.9 for the timeframe between day 2 and day 14, respectively. Sensitive detection of N antigens in serum may thus provide a valuable new marker for COVID-19 diagnosis and evaluation of disease severity. When assessed during the first 2 weeks since the onset of symptoms, it may help in identifying patients at risk of developing severe COVID-19 to optimize better intensive care utilization.

Waning of IgG, Total and Neutralizing Antibodies 6 Months Post-Vaccination with BNT162b2 in Healthcare Workers.

Data about the long-term duration of antibodies after SARS-CoV-2 vaccination are still scarce and are important to design vaccination strategies. In this study, 231 healthcare professionals received the two-dose regimen of BNT162b2. Of these, 158 were seronegative and 73 were seropositive at baseline. Samples were collected at several time points. The neutralizing antibodies (NAbs) and antibodies against the nucleocapsid and the spike protein of SARS-CoV-2 were measured. At day 180, a significant antibody decline was observed in seronegative (-55.4% with total antibody assay; -89.6% with IgG assay) and seropositive individuals (-74.8% with total antibody assay; -79.4% with IgG assay). The estimated half-life of IgG from the peak humoral response was 21 days (95% CI: 13-65) in seronegative and 53 days (95% CI: 40-79) in seropositive individuals. The estimated half-life of total antibodies was longer and ranged from 68 days (95% CI: 54-90) to 114 days (95% CI: 87-167) in seropositive and seronegative individuals, respectively. The decline of NAbs was more pronounced (-98.6%) and around 45% of the subjects tested were negative at day 180. Whether this decrease correlates with an equivalent drop in the clinical effectiveness against the virus would require appropriate clinical studies.

Persistence of Anti-SARS-CoV-2 Antibodies Depends on the Analytical Kit: A Report for Up to 10 Months after Infection.

Several studies have described the long-term kinetics of anti-SARS-CoV-2 antibodies but long-term follow-up data, i.e., >6 months, are still sparse. Additionally, the literature is inconsistent regarding the waning effect of the serological response. The aim of this study was to explore the temporal dynamic changes of the immune response after SARS-CoV-2 infection in hospitalized and non-hospitalized symptomatic patients over a period of 10 months. Six different analytical kits for SARS-CoV-2 antibody detection were used. Positivity rates, inter-assay agreement and kinetic models were determined. A high inter-individual and an inter-methodology variability was observed. Assays targeting total antibodies presented higher positivity rates and reached the highest positivity rates sooner compared with assays directed against IgG. The inter-assay agreement was also higher between these assays. The stratification by disease severity showed a much-elevated serological response in hospitalized versus non-hospitalized patients in all assays. In this 10-month follow-up study, serological assays showed a clinically significant difference to detect past SARS-CoV-2 infection with total antibody assays presenting the highest positivity rates. The waning effect reported in several studies should be interpreted with caution because it could depend on the assay considered.

Antibody titres decline 3-month post-vaccination with BNT162b2.

Several studies reported on the humoral response in subjects having received the BNT162b2 mRNA COVID-19 vaccine. However, data on the kinetics of antibodies 3 months post-vaccination are currently lacking and are important to drive the future vaccination strategy. The CRO-VAX HCP study is an ongoing multicentre, prospective and interventional study designed to assess the antibody response in a population of healthcare professionals who had received two doses of the BNT162b2 mRNA COVID-19 vaccine. Two hundred individuals underwent a blood drawn within 2 days before the first vaccine dose. One-hundred and forty-two persons (71%) were categorized as seronegative at baseline while 58 (29%) were seropositive. Samples were then collected after 14, 28, 42, 56, and 90 days. Antibodies against the SARS-CoV-2 nucleocapsid and the receptor binding domain of the S1 subunit of the spike protein were measured in all individuals at different time points. Using a one-compartment kinetics model, the time to maximum concentration was estimated at 36 ± 3 days after the first dose and the estimated half-life of antibodies was 55 days (95% CI: 37-107 days) in seronegative participants. In seropositive participants, the time to maximum concentration was estimated at 24 ± 4 days and the estimated half-life was 80 days (95% CI: 46-303 days). The antibody response was higher in seropositive compared to seronegative participants. In both seropositive and seronegative subjects, a significant antibody decline was observed at 3 months compared to the peak response. Nevertheless, the humoral response remained robust in all participants.

Neutralizing Antibodies in COVID-19 Patients and Vaccine Recipients after Two Doses of BNT162b2.

The evaluation of the neutralizing capacity of anti-SARS-CoV-2 antibodies is important because they represent real protective immunity. In this study we aimed to measure and compare the neutralizing antibodies (NAbs) in COVID-19 patients and in vaccinated individuals. One-hundred and fifty long-term samples from 75 COVID-19 patients were analyzed with a surrogate virus neutralization test (sVNT) and compared to six different SARS-CoV-2 serology assays. The agreement between the sVNT and pseudovirus VNT (pVNT) results was found to be excellent (i.e., 97.2%). The NAb response was also assessed in 90 individuals who had received the complete dose regimen of BNT162b2. In COVID-19 patients, a stronger response was observed in moderate-severe versus mild patients (p-value = 0.0006). A slow decay in NAbs was noted in samples for up to 300 days after diagnosis, especially in moderate-severe patients (r = -0.35, p-value = 0.03). In the vaccinated population, 83.3% of COVID-19-naive individuals had positive NAbs 14 days after the first dose and all were positive 7 days after the second dose, i.e., at day 28. In previously infected individuals, all were already positive for NAbs at day 14. At each time point, a stronger response was observed for previously infected individuals (p-value < 0.05). The NAb response remained stable for up to 56 days in all participants. Vaccinated participants had significantly higher NAb titers compared to COVID patients. In previously infected vaccine recipients, one dose might be sufficient to generate sufficient neutralizing antibodies.

Confounding Factors Influencing the Kinetics and Magnitude of Serological Response Following Administration of BNT162b2.

BACKGROUND: Little is known about potential confounding factors influencing the humoral response in individuals having received the BNT162b2 vaccine. METHODS: Blood samples from 231 subjects were collected before and 14, 28, and 42 days following coronavirus disease 2019 (COVID-19) vaccination with BNT162b2. Anti-spike receptor-binding-domain protein (anti-Spike/RBD) immunoglobulin G (IgG) antibodies were measured at each time-point. Impact of age, sex, childbearing age status, hormonal therapy, blood group, body mass index and past-history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were assessed by multivariable analyses. RESULTS AND CONCLUSIONS: In naïve subjects, the level of anti-Spike/RBD antibodies gradually increased following administration of the first dose to reach the maximal response at day 28 and then plateauing at day 42. In vaccinated subjects with previous SARS-CoV-2 infection, the plateau was reached sooner (i.e., at day 14). In the naïve population, age had a significant negative impact on anti-Spike/RBD titers at days 14 and 28 while lower levels were observed for males at day 42, when corrected for other confounding factors. Body mass index (BMI) as well as B and AB blood groups had a significant impact in various subgroups on the early response at day 14 but no longer after. No significant confounding factors were highlighted in the previously infected group.

Clinical performance of three fully automated anti-SARS-CoV-2 immunoassays targeting the nucleocapsid or spike proteins.

This study assesses the clinical performance of three anti-SARS-CoV-2 assays, namely EUROIMMUN anti-SARS-CoV-2 nucleocapsid (IgG) ELISA, Elecsys anti-SARS-CoV-2 nucleocapsid (total antibodies) assay, and LIAISON anti-SARS-CoV-2 spike proteins S1 and S2 (IgG) assay. One hundred and thirty-seven coronavirus disease 2019 (COVID-19) samples from 96 reverse-transcription polymerase chain reaction confirmed patients were chosen to perform the sensitivity analysis. Non-SARS-CoV-2 sera (n = 141) with a potential cross-reaction to SARS-CoV-2 immunoassays were included in the specificity analysis. None of these tests demonstrated a sufficiently high clinical sensitivity to diagnose acute infection. Fourteen days since symptom onset, we did not find any significant difference between the three techniques in terms of sensitivities. However, Elecsys performed better in terms of specificity. All three anti-SARS-CoV-2 assays had equivalent sensitivities 14 days from symptom onset to diagnose past-COVID-19 infection. We also confirmed that anti-SARS-CoV-2 determination before Day 14 is of less clinical interest.

Clinical performance of the Panbio assay for the detection of SARS-CoV-2 IgM and IgG in COVID-19 patients.

Following the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, numerous serological tests have been developed, including rapid diagnostic tests. This study aims at assessing the clinical performance of the Panbio immunoglobulin G (IgG)/IgM coronavirus disease 2019 (COVID-19) test (Abbott), a rapid lateral flow assay for the qualitative detection of IgG and IgM against SARS-CoV-2. One hundred and thirty-eight samples from 95 COVID-19 patients with a positive SARS-CoV-2 reverse-transcriptase polymerase chain reaction were analyzed to assess the clinical sensitivity. Seventy-six pre-COVID-19 samples were used to evaluate the clinical specificity. Two independent and blinded raters determined visually the presence or absence of the IgG, IgM, and control lines for each test after 10 and 20 min. The sensitivity obtained from collected samples more than 14 days after the onset of symptoms was 95.2% for IgG. IgM was less frequently detected (highest sensitivity of 20.5%). The specificities obtained were 98.7% and 100% for IgG and IgM, respectively. In addition, the sensitivity of the assay was better when the reading was performed at 20 min than at 10 min, whereas the specificity was unchanged. The Panbio COVID-19 IgG/IgM rapid test detects IgG with high sensitivity 14 days since symptom onset but presents a low sensitivity for IgM. The specificity was excellent for both IgG and IgM.

Head-to-Head Comparison of Rapid and Automated Antigen Detection Tests for the Diagnosis of SARS-CoV-2 Infection.

(1) Background: The detection of SARS-CoV-2 RNA in nasopharyngeal samples through real-time reverse transcription-polymerase chain reaction (RT-PCR) is considered the standard gold method for the diagnosis of SARS-CoV-2 infection. Antigen detection (AD) tests are more rapid, less laborious, and less expensive alternatives but still require clinical validation. (2) Methods: This study compared the clinical performance of five AD tests, including four rapid AD (RAD) tests (biotical, Panbio, Healgen, and Roche) and one automated AD test (VITROS). For that purpose, 118 (62.8%) symptomatic patients and 70 (37.2%) asymptomatic subjects were tested, and results were compared to RT-PCR. (3) Results: The performance of the RAD tests was modest and allowed us to identify RT-PCR positive patients with higher viral loads. For Ct values ≤25, the sensitivity ranged from 93.1% (95% CI: 83.3-98.1%) to 96.6% (95% CI: 88.1-99.6%), meaning that some samples with high viral loads were missed. Considering the Ct value proposed by the CDC for contagiousness (i.e., Ct values ≤33) sensitivities ranged from 76.2% (95% CI: 65.4-85.1%) to 88.8% (95% CI: 79.7-94.7%) while the specificity ranged from 96.3% (95% CI: 90.8-99.0%) to 99.1% (95% CI: 95.0-100%). The VITROS automated assay showed a 100% (95% CI: 95.5-100%) sensitivity for Ct values ≤33, and had a specificity of 100% (95% CI: 96.6-100%); (4) Conclusions: Compared to RAD tests, the VITROS assay fully aligned with RT-PCR for Ct values up to 33, which might allow a faster, easier and cheaper identification of SARS-CoV-2 contagious patients.

An Original ELISA-Based Multiplex Method for the Simultaneous Detection of 5 SARS-CoV-2 IgG Antibodies Directed against Different Antigens.

Strategies to detect SARS-CoV-2 are increasingly being developed. Among them, serological methods have been developed. Nevertheless, although these may present an interesting clinical performance, they are often directed against only one antigen. This study aims at evaluating the clinical performance of an innovative multiplex immunoassay (i.e., CoViDiag assay) detecting simultaneously the presence of antibodies directed against N, S1, S2, RBD and NTD antigens. Sensitivity was evaluated in 135 samples obtained from 94 rRT-PCR confirmed coronavirus disease 2019 (COVID-19) patients. Non-SARS-CoV-2 sera (n = 132) collected before the COVID-19 pandemic with potential cross-reactions to the SARS-CoV-2 immunoassay were included in the specificity analysis. The antibody signature was also studied in hospitalized and non-hospitalized patients. The specificity of the CoViDiag assay was excellent for all antibodies (99.2 to 100%) using adapted cut-offs. None of the false positive samples were positive for more than one antibody. The sensitivity obtained from samples collected 14 days since symptom onset varied from 92.0 to 100.0% depending on the antibody considered. Among samples collected more than 14 days after symptom onset, 12.8, 66.3, 3.5, 9.3, 5.8 and 2.3% were positive for 5, 4, 3, 2, 1 or 0 antibodies, respectively. A trend toward higher antibody titers was observed in hospitalized patient in the early days since symptom onset. However, no significant difference was observed compared to non-hospitalized patients after 14 days since symptom onset. The clinical performance of the CoViDiag 5 IgG assay is sufficient to recommend its use for the detection and the characterization of the antibody signature following SARS-CoV-2 infection. The combination of several antigens in the same test improves the overall specificity and sensitivity of the test. Further research is needed to investigate whether this strategy may be of interest to identify severe disease outcome in patients with SARS-CoV-2 infection.

High clinical performance and quantitative assessment of antibody kinetics using a dual recognition assay for the detection of SARS-CoV-2 IgM and IgG antibodies.

OBJECTIVES: Several serological SARS-CoV-2 immunoassays have been developed recently but require external validation before widespread use. This study aims at assessing the analytical and clinical performance of the iFlash® anti-SARS-CoV-2 chemiluminescence assay for the detection of both IgM and IgG antibodies. The kinetics of the antibody response was also evaluated. DESIGN & METHODS: The precision, carry-over, linearity, limit of blank, detection and quantification were assessed. Sensitivity analysis was performed by using 178 sera collected from 154 RT-PCR confirmed COVID-19 patients. The specificity analysis was performed from 75 selected non-SARS-CoV-2 sera with a potential cross-reaction to the SARS-CoV-2 immunoassay. RESULTS: This iFlash® SARS-CoV-2 assay showed excellent analytical performance. After 2 weeks since symptom onset, the sensitivities for IgM and IgG were 62.2% (95% CI: 52.3-71.2%) and 92.9%% (95% CI: 85.7-96.7%), respectively by using the cut-off provided by the manufacturer. After cut-off optimization (i.e. >2.81 for IgM and >4.86 for IgG), the sensitivity for IgM and IgG were 81.6 (95% CI: 72.7-88.1%) and 95.9% (95% CI: 89.4-98.7%), respectively. Optimized cut-off for IgG improved the sensitivity to reach 100% (95%CI: 87.6-100) from 28 days since symptom onset. CONCLUSIONS: This study shows that the iFlash® SARS-CoV-2 assay from YHLO biotechnology, has satisfactory analytical performance. Nevertheless, the sensitivity of the IgM is limited for a proper clinical use compared to IgG. The determination of anti-SARS-CoV-2 IgG antibodies from 28 days since symptom onset was associated with high sensitivity, especially using optimized cut-offs (i.e. 100%).