Heterologous Vector-mRNA Based SARS-CoV-2 Vaccination Strategy Appears Superior to a Homologous Vector-Based Vaccination Scheme in German Healthcare Workers Regarding Humoral SARS-CoV-2 Response Indicating a High Boosting Effect by mRNA Vaccines.

BACKGROUND: Longitudinal humoral SARS-CoV-2 (severe acute respiratory syndrome coronavirus type 2) immunity for up to 15 months due to vaccination, the efficacy of vaccination strategies (homologous, vector-vector versus heterologous, vector-mRNA), the influence of vaccination side effects, and the infection rate in German healthcare workers need to be investigated. METHODS: In this study, 103 individuals vaccinated against SARS-CoV-2 were enrolled to examine their anti-SARS-CoV-2 anti-N- and anti-RBD/S1-Ig levels. A total of 415 blood samples in lithium heparin tubes were prospectively obtained, and a structured survey regarding medical history, type of vaccine, and vaccination reactions was conducted. RESULTS: All participants demonstrated a humoral immune response, among whom no values decreased below the positivity cutoff. Five to six months after the third vaccination, three participants showed anti-RBD/S1 antibodies of less than 1000 U/mL. We observed higher levels for heterologous mRNA-/vector-based combinations compared to pure vector-based vaccination after the second vaccination, which is harmonized after a third vaccination with the mRNA-vaccine only in both cohorts. The incidence of vaccine breakthrough in a highly exposed cohort was 60.3%. CONCLUSION: Sustained long-term humoral immunity was observed, indicating the superiority of a heterologous mRNA-/vector-based combination compared to pure vector-based vaccination. There was longevity of anti-RBD/S1 antibodies of at least 4 and up to 7 months without external stimulus. Regarding vaccination reactogenity, the occurrence of local symptoms as pain at the injection site was increased after the first mRNA application compared to the vector-vector cohort with a general decrease in adverse events at later vaccination time points. Overall, a correlation between the humoral vaccination response and vaccination side effects was not observed. Despite the high prevalence of vaccine breakthroughs, these only occurred in the later course of the study when more infectious variants, which are, however, associated with milder courses, were present. These results provide insights into vaccine-related serologic responses, and the study should be expanded using additional vaccine doses and novel variants in the future.

Humoral SARS-CoV-2 Immune Response in COVID-19 Recovered Vaccinated and Unvaccinated Individuals Related to Post-COVID-Syndrome.

BACKGROUND: The duration of anti-SARS-CoV-2-antibody detectability up to 12 months was examined in individuals after either single convalescence or convalescence and vaccination. Moreover, variables that might influence an anti-RBD/S1 antibody decline and the existence of a post-COVID-syndrome (PCS) were addressed. METHODS: Forty-nine SARS-CoV-2-qRT-PCR-confirmed participants completed a 12-month examination of anti-SARS-CoV-2-antibody levels and PCS-associated long-term sequelae. Overall, 324 samples were collected. Cell-free DNA (cfDNA) was isolated and quantified from EDTA-plasma. As cfDNA is released into the bloodstream from dying cells, it might provide information on organ damage in the late recovery of COIVD-19. Therefore, we evaluated cfDNA concentrations as a biomarker for a PCS. In the context of antibody dynamics, a random forest-based logistic regression with antibody decline as the target was performed and internally validated. RESULTS: The mean percentage dynamic related to the maximum measured value was 96 (±38)% for anti-RBD/S1 antibodies and 30 (±26)% for anti-N antibodies. Anti-RBD/S1 antibodies decreased in 37%, whereas anti-SARS-CoV-2-anti-N antibodies decreased in 86% of the subjects. Clinical anti-RBD/S1 antibody decline prediction models, including vascular and other diseases, were cross-validated (highest AUC 0.74). Long-term follow-up revealed no significant reduction in PCS prevalence but an increase in cognitive impairment, with no indication for cfDNA as a marker for a PCS. CONCLUSION: Long-term anti-RBD/S1-antibody positivity was confirmed, and clinical parameters associated with declining titers were presented. A fulminant decrease in anti-SARS-CoV-2-anti-N antibodies was observed (mean change to maximum value 30 (±26)%). Anti-RBD/S1 antibody titers of SARS-CoV-2 recovered subjects boosted with a vaccine exceeded the maximum values measured after single infection by 235 ± 382-fold, with no influence on preexisting PCS. PCS long-term prevalence was 38.6%, with an increase in cognitive impairment compromising the quality of life. Quantified cfDNA measured in the early post-COVID-19 phase might not be an effective marker for PCS identification.

Results of a European-Wide External Quality Assessment (EQA) Scheme for Serological Detection of Anti-SARS-CoV-2 (CoVimm)-Pitfalls of Routine Application.

BACKGROUND: During the last two years, a variety of assays for the serological detection of antibodies to the new SARS-CoV-2 virus have been launched and used as part of standard care in many laboratories. The pace with which these tests have been introduced into routine care emphasizes the importance of quality measures for analytical methods, particularly with regard to the implications of results for clinical and epidemiologic decisions. Accuracy, reliability and comparability of analytical test results are thus essential, and here external quality assessment (EQA) is the most important quality assurance tool. It allows us to achieve harmonization of test methods as a prerequisite for a high standard of performance for laboratory and analytical techniques and their interpretation. METHODS: This EQA scheme consisted of pre-characterized clinical biospecimens dedicated to the analysis of anti-SARS-CoV-2 IgG total antibodies and differentiation into spike protein-specific IgG antibodies against SARS-CoV-2 (anti-S-SARS-CoV-2) and nucleocapsid-specific IgG antibodies against SARS-CoV-2 (anti-N-SARS-CoV-2). RESULTS: A total of 239 laboratories across Europe participated in this scheme, called CoVimm. In detail, 536 results for anti-SARS-CoV-2 IgG, 431 results for anti-S-SARS-CoV-2 IgG, and 200 results for anti-N-SARS-CoV-2 IgG were reported. Based on the pre-defined thresholds, the success rates for the determination of anti-S-SARS-CoV-2 IgG and anti-N-SARS-CoV-2 IgG were 96% and 90%, respectively. Interestingly, only 64% of the participating laboratories successfully passed the EQA scheme for the determination of total anti-SARS-CoV-2 IgG. CONCLUSIONS: This EQA revealed serious concerns regarding the reliability and appropriate use of anti-SARS-CoV-2 antibody assays in routine care. In addition to the wide heterogeneity of different assays used by participating laboratories, a lack of standardization and harmonization is also evident. This is of particular importance for reliable and clinically meaningful interpretation of test results.

Anosmia Testing as Early Detection of SARS-CoV-2 Positivity; A Prospective Study under Screening Conditions.

Sudden onset of anosmia is a phenomenon often associated with developing COVID-19 disease and has even been described as an initial isolated symptom in individual cases. In this case-control study, we investigated the feasibility of this condition as a suitable screening test in a population at risk. We performed a prospective study with a total of 313 subjects with suspected SARS-CoV-2 infection. In parallel to routine PCR analysis, a modified commercial scent test was performed to objectify the presence of potential anosmia as a predictor of SARS-CoV-2 positivity. Furthermore, a structured interview assessment of the participants was conducted. A total of 12.1% of the study participants had molecular genetic detection of SARS-CoV-2 infection in the nasopharyngeal swab. It could be demonstrated that these subjects had a significantly weaker olfactory identification performance of the scents. Further analysis of the collected data from the scent test and medical history via random forest (Boruta) algorithm showed that no improvement of the prediction power was achieved by this design. The assay investigated in this study may be suitable for screening general olfactory function. For the screening of COVID-19, it seems to be affected by too many external and internal biases and requires too elaborate and selective pre-test screening.

Liquid profiling of circulating tumor DNA in colorectal cancer: steps needed to achieve its full clinical value as standard care

The analysis of circulating tumor DNA (ctDNA) is at the threshold of implementation into standard care for colorectal cancer (CRC) patients. However, data about the clinical utility of liquid profiling (LP), its acceptance by clinicians, and its integration into clinical workflows in real‐world settings remain limited. Here, LP tests requested as part of routine care since 2016 were retrospectively evaluated. Results show restrained request behavior that improved moderately over time, as well as reliable diagnostic performance comparable to translational studies, with an overall agreement of 91.7%. Extremely low ctDNA levels at < 0.1% in over 20% of cases, a high frequency of concomitant driver mutations (in up to 14% of cases), and ctDNA levels reflecting the clinical course of disease were revealed. However, certain limitations hampering successful translation of ctDNA into clinical practice were uncovered, including the lack of clinically relevant ctDNA thresholds, appropriate time points of LP requests, and integrative evaluation of ctDNA, imaging, and clinical findings. In conclusion, these results highlight the potential clinical value of LP for CRC patient management and demonstrate issues that need to be addressed for successful long‐term implementation in clinical workflows.

Recruitment of highly cytotoxic CD8+ T cell receptors in mild SARS-CoV-2 infection.

T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8+ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8+ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8+ TCRs-classic features of protective immunity-are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8+ T cell immunity.

SARS-CoV-2 Antibody Testing - How to Increase the Positive Predictive Value.

BACKGROUND: Despite increasing COVID-19 infection rates, low overall prevalence resulting in a poor positive predictive value (PPV) of serological tests requires strategies to increase specificity. We therefore investigated a dual diagnostic strategy and evaluated the correlation between the severity of a SARS-CoV-2 infection and the detectable immune-response. METHODS: Participants were systematically categorized into positive and control cohorts and a probability score of COVID-19 was calculated based on clinical symptoms. Six hundred eighty-two serum samples were analyzed using a highly specific high-throughput system. Combining the serological test result and probability score was performed as a dual diagnostic strategy. RESULTS: Specificity of 99.61% and sensitivity of 86.0% were the basis of our approach. A dual diagnostic strategy led to increased pre-test probability and thus to a test specificity of 100%. In a flu-like symptomatic population, we estimated a COVID-prevalence of 4.79%. Moreover, we detected significantly higher antibody values in patients with fever than without fever. CONCLUSIONS: Based on sensitivity and specificity results of our study being in line with previous findings, we demonstrated a dual assessment strategy including a symptom-based probability score and serological testing to increase the PPV. Moreover, the presence of fever seems to trigger a stronger immune-response.

Assessing the Quality of Serological Testing in the COVID-19 Pandemic: Results of a European External Quality Assessment (EQA) Scheme for Anti-SARS-CoV-2 Antibody Detection.

External quality assessment (EQA) is a key instrument for achieving harmonization, and thus a high quality, of diagnostic procedures. As reliable test results are crucial for accurate assessment of SARS-CoV-2 infection prevalence, vaccine response, and immunity, and thus for successful management of the ongoing COVID-19 pandemic, the Reference Institute for Bioanalytics (RfB) was the first EQA provider to offer an open scheme for anti-SARS-CoV-2 antibody detection. The main objectives of this EQA were (i) to gain insights into the current diagnostic landscape and the performance of serological tests in Europe and (ii) to provide recommendations for diagnostic improvements. Within the EQA, a blinded panel of precharacterized human serum samples with variable anti-SARS-CoV-2 antibody titers was provided for detection of anti-SARS-CoV-2 IgG, IgA, and IgM antibodies. Across the three distribution rounds in 2020, 284 laboratories from 22 countries reported a total of 3,744 results for anti-SARS-CoV-2 antibody detection using more than 24 different assays for IgG. Overall, 97/3,004 results were false for anti-SARS-CoV-2 IgG, 88/248 for IgA, and 34/124 for IgM. Regarding diagnostic sensitivity and specificity, substantial differences were found between the different assays used, as well as between certified and noncertified tests. For cutoff samples, a drop in the diagnostic sensitivity to 46.3% and high interlaboratory variability were observed. In general, this EQA highlights the current variability of anti-SARS-CoV-2 antibody detection, technical limitations with respect to cutoff samples, and the lack of harmonization of testing procedures. Recommendations are provided to help laboratories and manufacturers further improve the quality of anti-SARS-CoV-2 serological diagnostics.

Longitudinal assessment of anti-SARS-CoV-2 antibody dynamics and clinical features following convalescence from a COVID-19 infection.

INTRODUCTION: The longevity of antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the duration of immunity are current topics of major scientific interest. Antibody kinetics during the acute phase are well studied, whereas the long-term kinetics are yet to be determined, with contradictory results from the studies to date. Here, we present a longitudinal analysis of the serological responses to a SARS-CoV-2 infection following convalescence and the association with post-COVID syndrome (PCS). MATERIALS AND METHODS: A total of 237 serum samples were prospectively collected from 61 participants who had had a SARS-CoV-2 infection, which was confirmed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). For each participant, anti-nucleocapsid (N) and anti-spike subunit 1 receptor binding domain (RBD/S1) immunoglobulin (Ig) levels were regularly determined over a period of 8 months. COVID-19-associated symptoms were assessed using a standardized questionnaire at study entry and again after 6 months. RESULTS: Antibodies were detectable in 56 of the 61 participants. No substantial decline in anti-SARS-CoV-2 pan-Ig levels was observed for the duration of the follow-up period. Antibody levels correlated positively with the disease severity, body mass index, fever, and smoking status. It was found that 46.8% of the participants suffered from PCS, with olfactory and gustatory dysfunctions being the most commonly reported symptoms. CONCLUSION: The results demonstrate stable anti-SARS-CoV-2 antibody titers and thus may indicate a long-lasting immunity. The results are in line with recently published data and provide further insight concerning asymptomatic to mildly-affected patients, the association with clinical features, and the frequency of PCS.

Specificity testing by point prevalence as a simple assessment strategy using the Roche Elecsys® anti-SARS-CoV-2 immunoassay.

BACKGROUND: The detection of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mandatory for the diagnosis, retrospective assessment of disease progression, and correct evaluation of the current infection situation in the population. Many such assays have been launched by various manufacturers. Unfortunately, the new US Food and Drug Administration emergency use regulations have resulted in a situation where laboratories have to perform their own validation studies but many of these laboratories do not have the biobank needed to conduct the studies. METHODS: We introduce a method that allows institutions to quickly perform a verification study in a low-prevalence infection situation. As proof of concept, we used the Roche Elecsys® anti-SARS-CoV-2 electrochemiluminescence immunoassay and an SAP-based hospital information system. The Shenzhen YHLO Biotech IgM and IgG assay targeting other surface patterns was used as a confirmatory test. RESULTS: The Roche assay demonstrated a limit of detection of 0.069 cutoff index and successfully passed the performance validation according to Clinical and Laboratory Standards Institute EP15-A3. The study population of 627 inpatients has a median age of 64 years, and approximately 13% of the group were under intensive care at the respective time point. All patients included tested negative for SARS-CoV-2 infection by quantitative reverse transcription polymerase chain reaction (cobas® 6800, Roche, Mannheim, Germany). Only one false-positive result was obtained, resulting in a specificity for the Roche Elecsys anti-SARS-CoV-2 test of 99.84% and a negative predictive value of 99.98%. CONCLUSIONS: The anonymized use of residual material enables quick evaluation of anti-SARS-CoV-2 immunoassays, as shown in this work with the Roche Elecsys assay. Comparison of the control population with economic data makes it possible to validate the sampling set and therefore to determine diagnostic specificity. By use of the approach chosen, it was shown that the Roche test achieved very good results in terms of diagnostic specificity, reproducibility, and limit of detection.

Clinical evaluation of commercial automated SARS-CoV-2 immunoassays.

OBJECTIVE: Numerous immunoassays for detecting antibodies directed against SARS-CoV-2 have been rapidly developed and released. Validations of these have been performed with a limited number of samples. The lack of standardisation might lead to significantly different results. This study compared ten automated assays from six vendors in terms of sensitivity, specificity and reproducibility. METHODS: This study compared ten fully automated immunoassays from the following vendors: Diasorin, Epitope Diagnostics, Euroimmun, Roche, YHLO, and Snibe. The retrospective part of the study included patients with a laboratory-confirmed COVID-19 infection, and controls comprised patients with a suspected infection, in whom the disease was excluded. Furthermore, biobanked sera were taken as negative controls (n = 97). The retrospective part involved four groups: (1) laboratory-confirmed COVID-19 infection (n = 183); (1B) suspected COVID-19 infection (n = 167) without a qRT-PCR result but positive serological results from at least two different assays, and suspected COVID-19 infection due to a positive serological result from the Roche assay (n = 295); (2) biobanked sera obtained from patients before the emergence of SARS-CoV-2 (n = 97) as negative controls; and (2A) probably COVID-19-negative sera with negative serological results from at least two different assays (n = 152). RESULTS: Overall diagnostic sensitivities were: Euroimmun (IgA) 87%; Epitope Diagnostics (IgG) 83%; YHLO (IgG) 77%; Roche (IgM/IgG) 77%; Euroimmun (IgG) 75%; Diasorin (IgG) 53%; Epitope Diagnostics (IgM) 52%; Snibe (IgG) 47%; YHLO (IgM) 35%; and Snibe (IgM) 26%. Diagnostic specificities were: YHLO (IgG) 100%; Roche, 100%; Snibe (IgM/IgG) 100%; Diasorin (IgG) 97%; Euroimmun (IgG) 94%; YHLO (IgM) 94%; Euroimmun (IgA) 83%. CONCLUSION: Assays from different vendors substantially varied in terms of their performance. These findings might facilitate selection of appropriate serological assays.

Results of the first pilot external quality assessment (EQA) scheme for anti-SARS-CoV2-antibody testing.

Objectives Assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevalence and immunity is cornerstones in the fight against COVID-19 pandemic. For pandemic control, reliable assays for the detection of anti-SARS-CoV-2 antibodies are required. This pilot external quality assessment (EQA) scheme aimed to independently assess the participants' clinical performance of anti-SARS-CoV-2 testing, to identify shortcomings in clinical practice and to evaluate the suitability of the scheme format. Methods The EQA scheme consisted of eight serum samples with variable reactivity against SARS-CoV-2 intended for the analysis of anti-SARS-CoV-2 immunoglobulin (Ig)G, IgA, and IgM antibodies. Laboratories reported: (1) results for each sample and the respective method, (2) raw data from replicate testing of each sample. Results The 16 selected pilot EQA participants reported 294 interpreted results and 796 raw data results from replicate testing. The overall error rate for the anti-SARS-CoV-2 IgG, IgA, and IgM tests was 2.7, 6.9, and 16.7%, respectively. While the overall diagnostic specificity was rated as very high, sensitivity rates between 67 and 98% indicate considerable quality differences between the manufacturers, especially for IgA and IgM. Conclusions Even the results reported by the small number of participants indicate a very heterogeneous landscape of anti-SARS-CoV-2 serological testing. Differences of available tests and the individual performance of laboratories result in a success rate of 57.1% with one laboratory succeeding for all three antibody-classes. These results are an incentive for laboratories to participate in upcoming open EQA schemes that are needed to achieve a harmonization of test results and to improve serological testing.

Comparison of test performance of commercial anti-SARS-CoV-2 immunoassays in serum and plasma samples.

BACKGROUND: For epidemiologic, social and economic reasons, assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevalence and immunity are important to adapt decisions to current demands. Hence, immunoassays for detection of anti-SARS-CoV-2 antibodies are introduced rapidly without requiring FDA emergency use authorization approval. Thus, evaluation of test performance predominantly relies on laboratories. This study aimed to evaluate the test performance of recently launched commercial immunoassays in serum and plasma samples. METHODS: 51 serum samples from 26 patients with confirmed SARS-CoV-2 infection after end of quarantine and 25 control patients were analyzed using anti-SARS-CoV-2 IgG immunoassays from Roche, Euroimmun and Epitope to assess diagnostic sensitivity and specificity. 20 matching pairs of serum and plasma samples were included to analyze comparability between different specimens. RESULTS: Overall, a diagnostic sensitivity of 92.3%, 96.2-100% and 100% with a respective diagnostic specificity of 100%, 100% and 84-86% for the immunoassays from Roche, Euroimmun and Epitope were determined. In total, 84-96% of samples were correctly classified as negative and 92.3-95.2% as positive. The level of concordance between plasma- and serum-based testing diverged between the assays (Epitope r2 = 0.97; Euroimmun r2 = 0.91; Roche r2 = 0.76). CONCLUSIONS: The immunoassays from Euroimmun and Roche revealed a higher specificity than the Epitope assay without a substantial drop of diagnostic sensitivity. Significant differences between plasma- and serum-based testing highlights the need for determination of appropriate cut-offs per specimen type. Hence, there is an urgent need for test harmonization and establishment of quality standards for an appropriate use of COVID-19 serological tests.

SARS-CoV-2 antibody testing-questions to be asked.

Severe acute respiratory syndrome coronavirus 2 infection and development of coronavirus disease 2019 presents a major health care challenge of global dimensions. Laboratory diagnostics of infected patients, and the assessment of immunity against severe acute respiratory syndrome coronavirus 2, presents a major cornerstone in handling the pandemic. Currently, there is an increase in demand for antibody testing and a large number of tests are already marketed or are in the late stage of development. However, the interpretation of test results depends on many variables and factors, including sensitivity, specificity, potential cross-reactivity and cross-protectivity, the diagnostic value of antibodies of different isotypes, and the use of antibody testing in identification of acutely ill patients or in epidemiological settings. In this article, the recently established COVID-19 Task Force of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL) addresses these issues on the basis of currently available data sets in this rapidly moving field.