Sensitive and specific serological ELISA for the detection of SARS-CoV-2 infections.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. METHODS: We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. RESULTS: The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. CONCLUSIONS: We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

Evaluation of S- and M-Proteins Expressed in Escherichia coli and HEK Cells for Serological Detection of Antibodies in Response to SARS-CoV-2 Infections and mRNA-Based Vaccinations.

This study investigated the IgG and IgA antibody response against recombinant S1 and receptor binding domains (RBD) of the spike (S-) protein and the membrane (M-) protein using a set of 115 serum samples collected from patients infected with SARS-CoV-2 in Germany before April 2021 using protein and peptide ELISA. As S1- and RBD-proteins expressed in Escherichia coli provided poor sensitivities in ELISA, they were replaced by proteins expressed in HEK cells. The RBD-ELISA provided a sensitivity of 90.6% (N = 85) for samples collected from patients with confirmed SARS-CoV-2 infections more than 14 days after symptom onset or a positive PCR test. In population-based controls, the specificity was 97.9% (N = 94). In contrast, the sensitivities were only 41.2% and 72.6% for M- and N-proteins, respectively, while the specificities were 88.5% and 100%, respectively. Considering also 20 samples collected during the first two weeks of symptom onset or PCR confirmation, the sensitivity of RBD- and N-protein ELISA decreased to 82.6% and 72.6%, respectively. The combination of two data sets, i.e., N- and RBD-, N- and M-, or RBD- and M-proteins increased the sensitivity to 85.8%, 77.9%, and 87.8%, respectively. Peptide mapping mostly confirmed epitopes previously reported for S1- and M-proteins, but they were only recognized by a few samples already tested positive in the corresponding protein ELISA indicating that peptide-based assays will not improve the diagnostic sensitivity.

Evaluation of the systemic and mucosal immune response induced by COVID-19 and the BNT162b2 mRNA vaccine for SARS-CoV-2.

BACKGROUND: The currently used SARS-CoV-2 mRNA vaccines have proven to induce a strong and protective immune response. However, functional relevance of vaccine-generated antibodies and their temporal progression are still poorly understood. Thus, the central aim of this study is to gain a better understanding of systemic and mucosal humoral immune response after mRNA vaccination with BNT162b2. METHODS: We compared antibody production against the S1 subunit and the RBD of the SARS-CoV-2 spike protein in sera of BNT162b2 vaccinees, heterologous ChAdOx1-S/BNT162b2 vaccinees and COVID-19 patients. We monitored the neutralizing humoral response against SARS-CoV-2 wildtype strain and three VOCs over a period of up to eight months after second and after a subsequent third vaccination. RESULTS: In comparison to COVID-19 patients, vaccinees showed higher or similar amounts of S1- and RBD-binding antibodies but similar or lower virus neutralizing titers. Antibodies peaked two weeks after the second dose, followed by a decrease three and eight months later. Neutralizing antibodies (nAbs) poorly correlated with S1-IgG levels but strongly with RBD-IgGAM titers. After second vaccination we observed a reduced vaccine-induced neutralizing capacity against VOCs, especially against the Omicron variant. Compared to the nAb levels after the second vaccination, the neutralizing capacity against wildtype strain and VOCs was significantly enhanced after third vaccination. In saliva samples, relevant levels of RBD antibodies were detected in convalescent samples but not in vaccinees. CONCLUSIONS: Our data demonstrate that BNT162b2 vaccinated individuals generate relevant nAbs titers, which begin to decrease within three months after immunization and show lower neutralizing potential against VOCs as compared to the wildtype strain. Large proportion of vaccine-induced S1-IgG might be non-neutralizing whereas RBD-IgGAM appears to be a good surrogate marker to estimate nAb levels. A third vaccination increases the nAb response. Furthermore, the systemic vaccine does not seem to elicit readily detectable mucosal immunity.

Evaluation of Simple Lateral Flow Immunoassays for Detection of SARS-CoV-2 Neutralizing Antibodies.

Immunization for the generation of protective antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged to be highly effective in preventing hospital admission, need for intensive care treatment and high mortality in the current SARS-CoV-2 pandemic. Lateral flow immune assays (LFIAs) offer a simple and competitive option to monitor antibody production after vaccination. Here, we compared the diagnostic performance of three different lateral flow assays in detecting nucleocapsid protein (NP), S1 subunit (S1) and receptor binding domain (pseudo)-neutralizing antibodies (nRBD) in sera of 107 health care workers prior (V1), two weeks (V2) after first vaccination with BNT162b2 as well as three weeks (V3) and eight months later (V4). In sera at V1, overall specificity was >99%. At V3, LFIAs showed sensitivities between 98.1 and 100%. The comparison of S1 and nRBD LFIA with S1 ELISA and a focus reduction neutralization assay (FRNT) revealed high concordance at V3. Thus, the use of lateral flow immunoassays appears to have reasonable application in the short-term follow-up after vaccination for SARS-CoV-2.

Bacterial contamination of the smartphones of healthcare workers in a German tertiary-care hospital before and during the COVID-19 pandemic.

BACKGROUND: Assuming that hygiene measures have improved significantly due to COVID-19, we aimed to investigate bacterial colonization on smartphones (SPs) owned by healthcare workers (HCWs) before and during the pandemic. METHODS: Employing a before-and-after study design, randomly selected HCWs were included. Devices underwent sampling under real-life conditions, without prior manipulation. Swabs were collected in 2012 (pre-pandemic) and 2021 to determine microbial colonization. Isolates were identified by MALDI-TOF mass spectrometry and underwent microbiological susceptibility testing. RESULTS: The final analysis included 295 HCWs (67% female, mean age 34 years) from 26 wards. Bacterial contamination was present on 293 of 295 SP screens (99.3%). The proportion of clinically relevant bacterial pathogens (eg Staphylococcus aureus, enterococci, Enterobacterales, non-fermenting bacteria) ranged from 21.2% in 2012 to 39.8% in 2021. Resistance profiles revealed a proportion of multidrug-resistant bacteria such as MRSA and VRE of less than 2%. The comparison of before-and-after sampling showed a significant increase in smartphone use during work from 2012 to 2021 with a simultaneous increase in cleaning intensity, probably as a result of the COVID-19 pandemic. CONCLUSIONS: Bacterial contamination of SPs within the hospital is of concern and can serve as a source of cross-contamination. Hence, in addition to excellent hand hygiene, SPs must be carefully disinfected after handling in healthcare. Behavioral changes related to the COVID-19 pandemic could have a significant impact if implemented sustainably in everyday clinical practice.

Comprehensive investigation of an in-hospital transmission cluster of a symptomatic SARS-CoV-2-positive physician among patients and healthcare workers in Germany.

We investigated potential transmissions of a symptomatic SARS-CoV-2-positive physician in a tertiary-care hospital who worked for 15 cumulative hours without wearing a face mask. No in-hospital transmissions occurred, despite 254 contacts among patients and healthcare workers. In conclusion, exposed hospital staff continued work, accompanied by close clinical and virologic monitoring.

CD99 and polymeric immunoglobulin receptor peptides deregulation in critical COVID-19: A potential link to molecular pathophysiology?

Identification of significant changes in urinary peptides may enable improved understanding of molecular disease mechanisms. We aimed towards identifying urinary peptides associated with critical course of COVID-19 to yield hypotheses on molecular pathophysiological mechanisms in disease development. In this multicentre prospective study urine samples of PCR-confirmed COVID-19 patients were collected in different centres across Europe. The urinary peptidome of 53 patients at WHO stages 6-8 and 66 at WHO stages 1-3 COVID-19 disease was analysed using capillary electrophoresis coupled to mass spectrometry. 593 peptides were identified significantly affected by disease severity. These peptides were compared with changes associated with kidney disease or heart failure. Similarities with kidney disease were observed, indicating comparable molecular mechanisms. In contrast, convincing similarity to heart failure could not be detected. The data for the first time showed deregulation of CD99 and polymeric immunoglobulin receptor peptides and of known peptides associated with kidney disease, including collagen and alpha-1-antitrypsin. Peptidomic findings were in line with the pathophysiology of COVID-19. The clinical corollary is that COVID-19 induces specific inflammation of numerous tissues including endothelial lining. Restoring these changes, especially in CD99, PIGR and alpha-1-antitripsin, may represent a valid and effective therapeutic approach in COVID-19, targeting improvement of endothelial integrity.

Comparison of pre-analytical characteristics for molecular and serological diagnostics of COVID-19.

Background: The diagnosis of SARS-CoV-2 infection relies on RT-PCR from nasopharyngeal swabs. The pre-analytical value of different methods of material harvesting for SARS-CoV-2 are unknown. Methods: We conducted a comprehensive investigation of the pre-analytical performance for different pharyngeal sampling procedures in hospitalized patients with confirmed SARS-CoV-2 infection. In addition to swabs taken simultaneously from different locations, saliva and pharyngeal lavages were also analyzed using RT-PCR. Results: In 10 COVID-19 patients, standard nasopharyngeal swabs detected 8 out of 10 positive patients, whereas swabs taken from the palatoglossal arch resulted in 9 correct-positive results. Brushing the posterior pharynx wall with swabs resulted in detection of 9 out of 10 positive patients with no difference using either dry swabs or liquid Amies medium. A strong correlation between Ct values of both swab materials was observed. Pharyngeal lavages yielded 6 out of 10 positive results in concordance with 85% of nasopharyngeal swabs in late-stage COVID-19 patients. Investigating 23 patients with early SARS-CoV-2 infection, pharyngeal lavages showed a concordance rate of 100% compared to nasopharyngeal swabs. Conclusions: The diagnostic performance of swabs taken from the palatoglossal arch in detecting SARS-CoV-2 infection is similar to that of specimens taken from the nasopharyngeal region. However, the former sampling method is associated with less discomfort and much easier to perform. Pharyngeal lavages may replace swabs for mass screening in early stages of SARS-CoV-2 infection. The predictive values are comparable, and the procedure is performed without exposing healthcare workers to transmission risks.

Differences of SARS-CoV-2 serological test performance between hospitalized and outpatient COVID-19 cases.

BACKGROUND: Serological severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays differ in the target antigen specificity, e.g. of antibodies directed against the viral spike or the nucleocapsid protein, and in the spectrum of detected immunoglobulins. The aim of the study was to evaluate the performance of two different routinely used immunoassays in hospitalized and outpatient COVID-19 cases. METHODS: The test characteristics of commercially available spike1 protein-based serological assays (Euroimmun, EI-assays), determining IgA or IgG and nucleocapsid-based assays (Virotech, VT-assays) determining IgA, IgM or IgG were compared in 139 controls and 116 hospitalized and outpatient COVID-19 cases. RESULTS: Hospitalized COVID-19 patients (n = 51; 115 samples) showed significantly higher concentrations of antibodies against SARS-CoV-2 and differed from outpatient cases (n = 65) by higher age, higher disease severity scores and earlier follow up blood sampling. Sensitivity of the two IgG assays was comparable in hospitalized patients tested ≥ 14 days (EI-assay: 88%, CI95% 67.6-99.9; VT-assay: 96%, CI95% 77.7-99.8). In outpatient COVID-19 cases sensitivity was significantly lower in the VT-assay (86.2%, CI95% 74.8-93.1) compared with the EI-assay (98.5%, CI95% 90.6-99.9). Assays for IgA and IgM demonstrated a lack of specificity or sensitivity. CONCLUSIONS: Our results indicate that SARS-CoV-2 serological assays may need to be optimized to produce reliable results in outpatient COVID-19 cases who are low or even asymptomatic. Assays for IgA and IgM have limited diagnostic performance and do not prove an additional value for population-based screening approaches.