Validation of a SARS-CoV-2 Surrogate Virus Neutralization Test in Recovered and Vaccinated Healthcare Workers.

Vaccination against COVID-19 is the main public health approach to fight against the pandemic. The Spike (S) glycoprotein of SARS-CoV-2 is the principal target of the neutralizing humoral response. We evaluated the analytical and clinical performances of a surrogate virus neutralization test (sVNT) compared to conventional neutralization tests (cVNTs) and anti-S eCLIA assays in recovered and/or vaccinated healthcare workers. Our results indicate that sVNTs displayed high specificity and no cross-reactivity. Both eCLIA and sVNT immunoassays were good at identifying cVNT serum dilutions ≥1:16. The optimal thresholds when identifying cVNT titers ≥1:16, were 74.5 U/mL and 49.4 IU/mL for anti-S eCLIA and sVNT, respectively. Our data show that neutralizing antibody titers (Nab) differ from one individual to another and may diminish over time. Specific assays such as sVNTs could offer a reliable complementary tool to routine anti-S serological assays.

Persistence of Neutralizing Antibodies and Clinical Protection up to 12†Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection in the Elderly.

Background: Coronavirus disease 2019 (COVID-19) has severely affected the elderly, who are expected to display decreased immune responses due to immunosenescence. Methods: This study retrospectively assesses neutralizing antibody (NAb) production up to 12 months after infection in long-term care patients. We used Roche Diagnostics immunoassay to quantify anti-spike (S) antibodies and a competitive immunoassay from YHLO as a surrogate test for NAb. Results: We included 91 patients (mean age, 86 years). There was no significant variation in anti-S titers over time. There was a significant decrease of NAb titers between month 3 and month 6 but no further significant change up to month 12. Overall, 75 of 91 (82%) and 52 of 91 (57%) patients had, at least once, anti-S titers >75 U/mL and NAb titers >50 AU/mL, respectively, corresponding to a significant neutralizing activity in vitro. All 68 patients studied at M12 had detectable anti-S antibodies and 60 (88%) had detectable NAb; 60 of 68 (88%) and 29 of 68 (42.6%) still had anti-S titers >75 U/mL and NAb titers >50 AU/mL. Higher NAb titers were correlated with severe infection, higher levels of C-reactive protein, and lower lymphocyte counts. No patient developed reinfection. Conclusions: Elderly people can display robust and persistent humoral response after severe acute respiratory syndrome coronavirus 2 infection, with NAb lasting up to 12 months.

Clinical Added Value of SARS-CoV-2 Antigen Detection in Blood Samples.

This study evaluated the performances of immunoassays (LFIA and ELISA) designed for SARS-CoV-2 Antigen (Ag)-detection in nasopharyngeal (NP) and serum samples in comparison to RT-PCR. NP samples from patients with respiratory symptoms (183 RT-PCR-positive and 74 RT-PCR-negative samples) were collected from March to April and November to December 2020. Seroconversion and antigen dynamics were assessed by symptom onset and day of RT-PCR diagnosis. Serum samples from 87 COVID-19 patients were used to investigate the added value of Ag quantification, at diagnosis and during follow-up. The sensitivity of COVID-VIRO-LFIA on samples with Ct ≤ 33, considered as the contagious threshold, was 86% on NPs (CI 95%: 79-90.5) and 76% on serum samples (CI 95%: 59.4-88), with a specificity of 100%. Serum N-Ag was detected during active infection as early as day two from symptom onset, with a diagnostic sensitivity of 81.5%. Within one week of symptom onset, diagnostic sensitivity and specificity reached 90.9% (95% CI, 85.1%-94.6%) and 98.3% (95% CI, 91.1%-99.9%), respectively. Serum N-Ag concentration closely correlated with disease severity. Longitudinal analysis revealed the simultaneous increase of antibodies and decrease of N-Ag. Sensitivities of COVID-VIRO-LFIA and COV-QUANTO-ELISA tests on NP and serum samples were close to 80%. They are suitable COVID-19-laboratory diagnostic tests, particularly when blood samples are available, thus reducing the requirement for NP sampling, and subsequent PCR analysis. ELISA titers may help to identify patients at risk of poor outcomes.

SARS-CoV-2 Genomic Characteristics and Clinical Impact of SARS-CoV-2 Viral Diversity in Critically Ill COVID-19 Patients: A Prospective Multicenter Cohort Study.

The SARS-CoV-2 variant of concern, α, spread worldwide at the beginning of 2021. It was suggested that this variant was associated with a higher risk of mortality than other variants. We aimed to characterize the genetic diversity of SARS-CoV-2 variants isolated from patients with severe COVID-19 and unravel the relationships between specific viral mutations/mutational patterns and clinical outcomes. This is a prospective multicenter observational cohort study. Patients aged ≥18 years admitted to 11 intensive care units (ICUs) in hospitals in the Greater Paris area for SARS-CoV-2 infection and acute respiratory failure between 1 October 2020 and 30 May 2021 were included. The primary clinical endpoint was day-28 mortality. Full-length SARS-CoV-2 genomes were sequenced by means of next-generation sequencing (Illumina COVIDSeq). In total, 413 patients were included, 183 (44.3%) were infected with pre-existing variants, 197 (47.7%) were infected with variant α, and 33 (8.0%) were infected with other variants. The patients infected with pre-existing variants were significantly older (64.9 ± 11.9 vs. 60.5 ± 11.8 years; p = 0.0005) and had more frequent COPD (11.5% vs. 4.1%; p = 0.009) and higher SOFA scores (4 [3-8] vs. 3 [2-4]; 0.0002). The day-28 mortality was no different between the patients infected with pre-existing, α, or other variants (31.1% vs. 26.2% vs. 30.3%; p = 0.550). There was no association between day-28 mortality and specific variants or the presence of specific mutations. At ICU admission, the patients infected with pre-existing variants had a different clinical presentation from those infected with variant α, but mortality did not differ between these groups. There was no association between specific variants or SARS-CoV-2 genome mutational pattern and day-28 mortality.

Performance of the QIAprep& Viral RNA UM Kit assay (Qiagen), an automatable method for RT-qPCR detection of SARS-CoV-2 without RNA extraction.

We evaluated the performance of the QIAprep& Viral RNA UM Kit (Qiagen) for SARS-CoV-2 detection. It displayed specificity and sensitivity required for SARS-CoV-2 RNA detection from swab transport media without RNA extraction. This method identifies accurately patients at risk of transmission while saving time and cost of extraction.

Rituximab Impairs B Cell Response But Not T Cell Response to COVID-19 Vaccine in Autoimmune Diseases.

OBJECTIVE: Antibody response to the messenger RNA (mRNA) COVID-19 vaccine has been shown to be diminished in rituximab (RTX)-treated patients. We undertook this study to compare humoral and T cell responses between healthy controls, patients with autoimmune diseases treated with RTX, and those treated with other immunosuppressants, all of whom had been vaccinated with 2 doses of the mRNA COVID-19 vaccine. METHODS: We performed anti-spike IgG and neutralization assays just before and 28 days after the second BNT162b2 (Pfizer-BioNTech) vaccine dose. The specific T cell response was assessed in activated CD4 and CD8 T cells using intracellular flow cytometry staining of cytokines (interferon-γ, tumor necrosis factor, and interleukin-2) after stimulation with SARS-CoV-2 spike peptide pools. RESULTS: A lower proportion of responders with neutralizing antibodies to the vaccine was observed in the RTX group (29%; n = 24) compared to the other immunosuppressants group (80%; n = 35) (P = 0.0001) and the healthy control group (92%; n = 26) (P < 0.0001). No patients treated with RTX in the last 6 months showed a response. Time since last infusion was the main factor influencing humoral response in RTX-treated patients. The functional CD4 and CD8 cellular responses to SARS-CoV-2 peptides for each single cytokine or polyfunctionality were not different in the RTX group compared to the other immunosuppressants group or the control group. In RTX-treated patients, the T cell response was not different between patients with and those without a humoral response. CONCLUSION: RTX induced a diminished antibody response to the mRNA COVID-19 vaccine, but the functional T cell response was not altered compared to healthy controls and autoimmune disease patients treated with other immunosuppressants. Further work is needed to assess the clinical protection granted by a functionally active T cell response in the absence of an anti-spike antibody response.

Emergence of SARS-CoV-2 resistance mutations in a patient who received anti-SARS-COV2 spike protein monoclonal antibodies: a case report.

BACKGROUND: To manage severe or potentially severe cases of CoronaVirus Disease 2019 (COVID-19), therapeutic monoclonal antibodies targeting Spike protein of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) have been designed. It has been noted in vitro that upon exposure to these treatments, mutations could be selected. CASE PRESENTATION: We here report the case of an immunosuppressed patient infected with a B.1.1.7 variant, who received a combination of monoclonal antibodies, and subsequently selected mutations K417N, E484K and Q493R on Spike protein of SARS-CoV-2. CONCLUSIONS: Our case raises the importance of monitoring SARS-CoV-2 mutations in patients receiving monoclonal antibodies and having persistent excretion of the virus, in order to offer optimal management of their infection, and strengthen prevention measures to avoid subsequent transmission of these selected variants.

Response to COVID-19 mRNA vaccination in multiple myeloma is conserved but impaired compared to controls.

Patients with multiple myeloma are at high risk of severe forms of COVID-19. Despite data showing diminished response to vaccine, the era of highly efficient mRNA vaccine might be a gamechanger. We sought to examine response to mRNA vaccine between healthy controls (n = 28) and multiple myeloma (MM) patients (n = 27). Response was analyzed 1 month after the second dose of anti-SARS-CoV-2 BNT162b2 vaccine. Multiple myeloma patients showed diminished levels of Anti-Spike IgG levels compared to controls, but with a high proportion of patients achieving a humoral response (89% vs. 97% in controls). Neutralizing antibodies were present in 74% of patients versus 96% of controls. Patients under current daratumumab treatment had neutralizing activity of anti-SARS-CoV-2 antibodies. Multiple myeloma patients show diminished response to SARS-COV-2 vaccine but with still high response rate. The main potential risk factor of non-response to COVID-19 vaccine was uncontrolled disease under treatment.

Indirect effects of severe acute respiratory syndrome coronavirus 2 on the kidney in coronavirus disease patients.

Among patients hospitalized for novel coronavirus disease (COVID-19), between 10 and 14% develop an acute kidney injury and around half display marked proteinuria and haematuria. Post-mortem analyses of COVID-19 kidney tissue suggest that renal tubular cells and podocytes are affected. Here we report two cases of collapsing glomerulopathy and tubulointerstitial lesions in living COVID-19 patients. Despite our use of sensitive reverse transcription polymerase chain reaction techniques in this study, we failed to detect the virus in blood, urine and kidney tissues. Our observations suggest that these kidney lesions are probably not due to direct infection of the kidney by severe acute respiratory syndrome coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 indirectly damages kidney structures.

BACKGROUND: The objectives were to characterize Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease 2019 (COVID-19) in patients with acute kidney injury (AKI). METHODS: Kidney biopsy samples in two Caucasian patients and one African with COVID-19 AKI were investigated. RESULTS: All patients had a high-level non-selective glomerular proteinuria. SARS-CoV-2 samples by real-time polymerase chain reaction (RT- PCR) assay were all-negative, as well as for virus particles in the kidney by electron microscopy. The three patients and patients with other AKI did not differ significantly with regard to angiotensin-converting enzyme 2 and transmembrane protease serine 2 kidney staining. CONCLUSIONS: The kidney damage particularly in Caucasians in COVID-19 seems to be an AKI, possibly by the systemic inflammatory response.

Quantitative Assessment of SARS-CoV-2 Virus in Nasopharyngeal Swabs Stored in Transport Medium by a Straightforward LC-MS/MS Assay Targeting Nucleocapsid, Membrane, and Spike Proteins.

Alternative methods to RT-PCR for SARS-CoV-2 detection are investigated to provide complementary data on viral proteins, increase the number of tests performed, or identify false positive/negative results. Here, we have developed a simple mass spectrometry assay for SARS-CoV-2 in nasopharyngeal swab samples using common laboratory reagents. The method employs high sensitivity and selectivity targeted mass spectrometry detection, monitoring nine constitutive peptides representative of the three main viral proteins and a straightforward pellet digestion protocol for convenient routine applications. Absolute quantification of N, M, and S proteins was achieved by addition of isotope-labeled versions of best peptides. Limit of detection, recovery, precision, and linearity were thoroughly evaluated in four representative viral transport media (VTM) containing distinct total protein content. The protocol was sensitive in all swab media with limit of detection determined at 2 × 103 pfu/mL, corresponding to as low as 30 pfu injected into the LC-MS/MS system. When tested on VTM-stored nasopharyngeal swab samples from positive and control patients, sensitivity was similar to or better than rapid immunoassay dipsticks, revealing a corresponding RT-PCR detection threshold at Ct ∼ 24. The study represents the first thorough evaluation of sensitivity and robustness of targeted mass spectrometry in nasal swabs, constituting a promising SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 and compatible with the constraints of clinical settings. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01646.

Evaluating 10 Commercially Available SARS-CoV-2 Rapid Serological Tests by Use of the STARD (Standards for Reporting of Diagnostic Accuracy Studies) Method.

Numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid serological tests have been developed, but their accuracy has usually been assessed using very few samples, and rigorous comparisons between these tests are scarce. In this study, we evaluated and compared 10 commercially available SARS-CoV-2 rapid serological tests using the STARD (Standards for Reporting of Diagnostic Accuracy Studies) methodology. Two hundred fifty serum samples from 159 PCR-confirmed SARS-CoV-2 patients (collected 0 to 32 days after the onset of symptoms) were tested with rapid serological tests. Control serum samples (n = 254) were retrieved from pre-coronavirus disease (COVID) periods from patients with other coronavirus infections (n = 11), positivity for rheumatoid factors (n = 3), IgG/IgM hyperglobulinemia (n = 9), malaria (n = 5), or no documented viral infection (n = 226). All samples were tested using rapid lateral flow immunoassays (LFIAs) from 10 manufacturers. Only four tests achieved ≥98% specificity, with the specificities ranging from 75.7% to 99.2%. The sensitivities varied by the day of sample collection after the onset of symptoms, from 31.7% to 55.4% (days 0 to 9), 65.9% to 92.9% (days 10 to 14), and 81.0% to 95.2% (>14 days). Only three of the tests evaluated met French health authorities' thresholds for SARS-CoV-2 serological tests (≥90% sensitivity and ≥98% specificity). Overall, the performances varied greatly between tests, with only one-third meeting acceptable specificity and sensitivity thresholds. Knowledge of the analytical performances of these tests will allow clinicians and, most importantly, laboratorians to use them with more confidence; could help determine the general population's immunological status; and may help diagnose some patients with false-negative real-time reverse transcription-PCR (RT-PCR) results.

Rapid Determination of SARS-CoV-2 antibodies using a bedside, point-of-Care, serological test.

Background: Several serological tests for SARS-CoV-2 have been developed or use, but most have only been validated on few samples, and none provide medical practitioners with an easy-to-use, self-contained, bedside test with high accuracy. Material and methods: Two-hundred fifty-six sera from 101 patients hospitalized with SARS-CoV-2 infection (positive RT-PCR) and 50 control sera were tested for IgM/IgG using the NG-Test IgM-IgG COVID all-in-one assay. The seroconversion dynamic was assessed by symptom onset and day of RT-PCR diagnosis. Results: Among the SARS-CoV-2 infected patients, positive IgG and/or IgM result was observed for 67.3% of patients (68/101), including 17 (16.8%) already positive at the day of RT-PCR, and 51 (50.5%) with observable seroconversion, and 32.7% (33/101) remained negative as subsequent sampling was not possible (patient discharge or death). The sensitivity increased with the delay between onset of symptoms and sampling, going from 29.1%, 78.2% and 86.5% for the time periods of 0-9-, 10-14- and >14-days after the onset of symptoms, respectively. Cumulative sensitivity, specificity, Positive Predictive Value and Negative Predictive Value were 97.0%, 100%, 100% and 96.2%, respectively 15-days after the onset of symptoms. No difference in seroconversion delay was observed regardless of whether patients received ventilation. Conclusions: The NG-test is a bedside serological assay that could serve as a complementary source of diagnostic information to RT-PCR and chest imaging. It may also be useful to monitor immunological status of medical and non-medical workers during the ongoing pandemic, and the general population after social distancing measures have eased.