Classification of "Near-patient" and "Point-of-Care" SARS-CoV-2 Nucleic Acid Amplification Test Systems and a first approach to evaluate their analytical independence of operator activities.

BACKGROUND: European legislation defines as "near-patient testing" (NPT) what is popularly and in other legislations specified as "point-of-care testing" (POCT). Systems intended for NPT/POCT use must be characterized by independence from operator activities during the analytic procedure. However, tools for evaluating this are lacking. We hypothesized that the variability of measurement results obtained from identical samples with a larger number of identical devices by different operators, expressed as the method-specific reproducibility of measurement results reported in External Quality Assessment (EQA) schemes, is an indicator for this characteristic. MATERIALS AND METHODS: Legal frameworks in the EU, the USA and Australia were evaluated about their requirements for NPT/POCT. EQA reproducibility of seven SARS-CoV-2-NAAT systems, all but one designated as "POCT", was calculated from variabilities in Ct values obtained from the respective device types in three different EQA schemes for virus genome detection. RESULTS: A matrix for characterizing test systems based on their technical complexity and the required operator competence was derived from requirements of the European In Vitro Diagnostic Regulation (IVDR) 2017/746. Good EQA reproducibility of the measurement results of the test systems investigated implies that different users in different locations have no recognizable influence on their measurement results. CONCLUSION: The fundamental suitability of test systems for NPT/POCT use according to IVDR can be easily verified using the evaluation matrix presented. EQA reproducibility is a specific characteristic indicating independence from operator activities of NPT/POCT assays. EQA reproducibility of other systems than those investigated here remains to be determined.

Design of external quality assessment schemes and definition of the roles of their providers in future epidemics.

During an epidemic, individual test results form the basis of epidemiological indicators such as case numbers or incidence. Therefore, the accuracy of measures derived from these indicators depends on the reliability of individual results. In the COVID-19 pandemic, monitoring and evaluating the performance of the unprecedented number of testing facilities in operation, and novel testing systems in use, was urgently needed. External quality assessment (EQA) schemes are unique sources of data reporting on testing performance, and their providers are recognised contacts and support for test facilities (for technical-analytical topics) and health authorities (for planning the monitoring of infection diagnostics). To identify information provided by SARS-CoV-2 genome detection EQA schemes that is relevant for public health microbiology, we reviewed the current literature published in PubMed between January, 2020, and July, 2022. We derived recommendations for EQA providers and their schemes for best practices to monitor pathogen-detection performance in future epidemics. We also showed laboratories, test facilities, and health authorities the information and benefits they can derive from EQA data, and from the non-EQA services of their providers.

Immunity of Heterologously and Homologously Boosted or Convalescent Individuals Against Omicron BA.1, BA.2, and BA.4/5 Variants.

BACKGROUND: The emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants BA.1, BA.2, and BA.4/5 demonstrate higher transmission and infection rates than previous variants of concern. To evaluate effectiveness of heterologous and homologous booster vaccination, we directly compared cellular and humoral immune responses as well as neutralizing capacity against replication-competent SARS-CoV-2 wild type, Delta, and Omicron variants BA.1, BA.2, and BA.4/5. METHODS: Peripheral blood mononuclear cells and serum samples from 137 participants were investigated, in 3 major groups. Individuals in the first group were vaccinated twice with ChAdOx1 and boosted with a messenger RNA (mRNA) vaccine (BNT162b2 or mRNA-1273); the second group included triple mRNA--vaccinated participants, and the third group, twice-vaccinated and convalescent individuals. RESULTS: Vaccination and convalescence resulted in the highest SARS-CoV-2-specific antibody levels, stronger T-cell responses, and best neutralization against wild type, Delta Omicron BA.2, and BA.4/5, while a combination of ChAdOx1 and BNT162b2 vaccination elevated neutralizing capacity against Omicron BA.1. In addition, heterologous booster regimens, compared with homologous regimens, showed higher efficacy against Omicron BA.2 as well as BA.4/5. CONCLUSIONS: We showed that twice-vaccinated and convalescent individuals demonstrated the strongest immunity against Omicron BA.2 and BA.4/5 variant, followed by those receiving heterologous and homologous booster vaccine regimens.

Three rounds of a national external quality assessment reveal a link between disharmonic anti-SARS-CoV-2 antibody quantifications and the infection stage.

OBJECTIVES: The WHO's standardized measuring unit, "binding antibody units per milliliter (BAU/mL)," should allow the harmonization of quantitative results by different commercial Anti-SARS-CoV-2 immunoassays. However, multiple studies demonstrate inter-assay discrepancies. The antigenic changes of the Omicron variant affect the performance of Spike-specific immunoassays. This study evaluated the variation of quantitative Anti-SARS-CoV-2-Spike antibody measurements among 46, 50, and 44 laboratories in three rounds of a national external quality assessment (EQA) prior to and after the emergence of the Omicron variant in a diagnostic near-to-real-life setting. METHODS: We analyzed results reported by the EQA participant laboratories from single and sequential samples from SARS-CoV-2 convalescent, acutely infected, and vaccinated individuals, including samples obtained after primary and breakthrough infections with the Omicron variant. RESULTS: The three immunoassays most commonly used by the participants displayed a low intra-assay and inter-laboratory variation with excellent reproducibility using identical samples sent to the participants in duplicates. In contrast, the inter-assay variation was very high with all samples. Notably, the ratios of BAU/mL levels quantified by different immunoassays were not equal among all samples but differed between vaccination, past, and acute infection, including primary infection with the Omicron variant. The antibody kinetics measured in vaccinated individuals strongly depended on the applied immunoassay. CONCLUSIONS: Measured BAU/mL levels are only inter-changeable among different laboratories when the same assay was used for their assessment. Highly variable ratios of BAU/mL quantifications among different immunoassays and infection stages argue against the usage of universal inter-assay conversion factors.

Converting to an international unit system improves harmonization of results for SARS-CoV-2 quantification: Results from multiple external quality assessments.

BACKGROUND: The detection of SARS-CoV-2 vRNA in clinical samples has relied almost exclusively on RT-qPCR as the gold standard test. Published results from various external quality assessments ("ring trials") worldwide have shown that there is still a large variability in results reported for the same samples. As reference standards of SARS-CoV-2 RNA are available, we tested whether using standard curves to convert Ct values into copies/mL (cp/mL) improved harmonization. METHODS: Nine laboratories using 23 test systems (15 of which were unique) prepared standard dilution curves to convert Ct values of 13 SARS-CoV-2 positive samples to cp/mL (hereafter IU/mL). The samples were provided in three rounds of a virus genome detection external quality assessment (EQA) scheme. We tested the precision and accuracy of results reported in IU/mL, and attempted to identify the sources of variability. RESULTS: Reporting results as IU/mL improved the precision of the estimated concentrations of all samples compared to reporting Ct values, although some inaccuracy remained. Variance analysis showed that nearly all variability in data was explained by individual test systems within individual laboratories. When controlling for this effect, there was no significant difference between all other factors tested (test systems, EQA rounds, sample material). CONCLUSIONS: Converting results to copies/mL improved precision across laboratory test systems. However, it seems the results are still very specific to test systems within laboratories. Further efforts could be made to improve accuracy and achieve full harmonization across diagnostic laboratories.

One-Year Follow-Up of COVID-19 Patients Indicates Substantial Assay-Dependent Differences in the Kinetics of SARS-CoV-2 Antibodies.

Determination of antibody levels against the nucleocapsid (N) and spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are used to estimate the humoral immune response after SARS-CoV-2 infection or vaccination. Differences in the design and specification of antibody assays challenge the interpretation of test results, and comparative studies are often limited to single time points per patient. We determined the longitudinal kinetics of antibody levels of 145 unvaccinated coronavirus disease 2019 (COVID-19) patients at four visits over 1 year upon convalescence using 8 commercial SARS-CoV-2 antibody assays (from Abbott, DiaSorin, Roche, Siemens, and Technoclone), as well as a virus neutralization test (VNT). A linear regression model was used to investigate whether antibody results obtained in the first 6 months after disease onset could predict the VNT results at 12 months. Spike protein-specific antibody tests showed good correlation to the VNT at individual time points (rS, 0.74 to 0.92). While longitudinal assay comparison with the Roche Elecsys anti-SARS-CoV-2 S test showed almost constant antibody concentrations over 12 months, the VNT and all other tests indicated a decline in serum antibody levels (median decrease to 14% to 36% of baseline). The antibody level at 3 months was the best predictor of the VNT results at 12 months after disease onset. The current standardization to a WHO calibrator for normalization to binding antibody units (BAU) is not sufficient for the harmonization of SARS-CoV-2 antibody tests. Assay-specific differences in absolute values and trends over time need to be considered when interpreting the course of antibody levels in patients. IMPORTANCE Determination of antibodies against SARS-CoV-2 will play an important role in detecting a sufficient immune response. Although all the manufacturers expressed antibody levels in binding antibody units per milliliter, thus suggesting comparable results, we found discrepant behavior between the eight investigated assays when we followed the antibody levels in a cohort of 145 convalescent patients over 1 year. While one assay yielded constant antibody levels, the others showed decreasing antibody levels to a varying extent. Therefore, the comparability of the assays must be improved regarding the long-term kinetics of antibody levels. This is a prerequisite for establishing reliable antibody level cutoffs for sufficient individual protection against SARS-CoV-2.

Results of a SARS-CoV-2 virus genome detection external quality assessment round focusing on sensitivity of assays and pooling of samples.

OBJECTIVES: Results of earlier external quality assessment (EQA) rounds suggested remarkable differences in the sensitivity of SARS-CoV PCR assays. Although the test systems are intended to detect SARS-CoV-2 in individual samples, screening is often applied to sample pools to increase efficiency and decrease costs. However, it is unknown to what extent these tests actually meet the manufacturer's specifications for sensitivity and how they perform when testing sample pools. METHODS: The sensitivity of assays in routine use was evaluated with a panel of positive samples in a round of a SARS-CoV-2 virus genome detection EQA scheme. The panel consisted of samples at or near the lower limit of detection ("weakly positive"). Laboratories that routinely test sample pools were asked to also analyze the pooled EQA samples according to their usual pool size and dilution method. RESULTS: All participants could detect a highly positive patient-derived sample (>106 copies/mL). Most (96%) of the test systems could detect at least 1,000 copies/mL, meeting the minimum acceptable benchmark, and many (94%) detected the vRNA in a sample with lower concentration (500 copies/mL). The false negative ratio increased to 16 and 26% for samples with 100 and 50 copies/mL, respectively. CONCLUSIONS: The performance of most assays met or exceeded their specification on sensitivity. If assays are to be used to analyze sample pools, the sensitivity of the assay and the number of pooled samples must be balanced.

Borderline and weakly positive antibody levels against the S-protein of SARS-CoV-2 exhibit limited agreement with virus neutralization titres.

The presence of neutralizing antibodies against SARS-CoV-2 in a large number of people is - besides cellular immunity - important to overcome the SARS-CoV-2 pandemic. While determination of neutralizing antibodies via virus neutralization tests are laborious, assays to determine the antibody levels serologically are fully automated and widely available. Correlations between these methodologies were recently given by the manufacturers, however performance in samples close to the cut off value have not yet been fully validated. Thus, we analysed 22 borderline and low positive (<100 BAU/ml) samples and 9 high positive (≥ 100 BAU/ml) from infected and/or vaccinated individuals and compared the SARS-CoV-2 IgG II Quant assay (Abbott), LIAISON SARS-CoV-2 TrimericS IgG (Diasorin), Elecsys Anti-SARS-CoV-2 S (Roche), and SARS-CoV-2 IgG (Siemens) with results obtained from a virus neutralization test. Based on the cut off values given by Abbott, Diasorin, Roche, and Siemens, the positive serologic results were concordant with the virus neutralization test in 100%, 76%, 88%, and 71%, respectively, while in turn, negative ones were in agreement in 29%, 79%, 93%, and 86%, respectively. In conclusion, weakly positive, serologic results are challenging to correctly predict the presence of neutralizing antibodies. Our study suggests, that different cut off values (for positivity vs. presence of neutralizing antibodies) could improve the test's performance, but determination thereof requires more samples to be analysed.

Inadequate design of mutation detection panels prevents interpretation of variants of concern: results of an external quality assessment for SARS-CoV-2 variant detection.

OBJECTIVES: Mutation-specific PCR assays have quickly found their way into laboratory diagnostics due to their capacity to be a fast, easy to implement and high-throughput method for the detection of known SARS-CoV-2 variants of concern (VoCs). However, little is known about the performance of such assays in routine laboratory analysis. METHODS: The results reported in a recent round of an external quality assessment (EQA) scheme for SARS-CoV-2 mutation-specific PCR were retrospectively analyzed. For the determination of individual variant-specific sequences as well as for the interpretation results for certain virus variants, correct, incorrect, and unreported results were evaluated, and their possible causes were investigated. RESULTS: A total of 34 laboratories participated in this study. For five samples containing the VoC Alpha + E484K, Beta, Gamma, Delta, or B.1.1.318 (as a variant of interest), 848 results for SARS-2-CoV mutation detection were reported, 824 (97.2%, range per sample 88-100%) of which were correct. Melting curve assays gave 99% correct results, real-time RT-qPCR 94%, microarray-based assays 100%, and MALDI-TOF MS 96%. A total of 122/167 (73%) reported results for SARS-CoV-2 variant determination were correct. Of the 45 inconclusive or incorrect results, 33 (73%) were due to inadequate selection of targets that did not allow identification of contemporary VoC, 11 (24%) were due to incorrect results, and one (3%) was due to correct results of mutation-specific PCR. CONCLUSIONS: Careful and up-to-date selection of the targets used in mutation-specific PCR is essential for successful detection of current SARS-CoV-2 variants.

RT-PCR based SARS-CoV-2 variant screening assays require careful quality control.

BACKGROUND: Distinctive genotypes of SARS-CoV-2 have emerged that are or may be associated with increased transmission, pathogenicity, and/or antibody escape. In many countries, clinical and diagnostic laboratories are under mandate to identify and report these so-called variants of concern (VOC). OBJECTIVES: We used an external quality assessment scheme to determine the scope, accuracy, and reliability of laboratories using various molecular diagnostic assays to identify current VOC (03 March 2021). STUDY DESIGN: Participant laboratories were sent the same five patient-derived samples and were asked to provide their variant detection methods, variant detection results and interpretation of results. RESULTS: Twenty-five laboratories reported a range of RT-qPCR-based assays to identify specific variations in the SARS-CoV-2 spike protein that are characteristic of three VOC lineages. Laboratories that detected VOC-associated nucleotide mutations at four specific sites had the highest ratio of correct classification. Low template copy number and additional variation in target regions resulted in loss of confidence and accuracy in sample classification. CONCLUSIONS: Melting-curve-based assays to identify genomic variants are less time-consuming and require less bioinformatic analysis compared to partial or whole genome sequencing. However, our results suggest that correct classification of a given genotype/lineage (e.g., a VOC) relies on the ability to detect more than one variant site, adequate template in the sample (i.e., relatively high viral load/copy number) and results may be unclear in certain samples with additional genetic variations. These initial results suggest that some diagnostic laboratories may require additional training to interpret and report complex genetic information about a dynamic emerging virus.

Testosterone Deficiency Is a Risk Factor for Severe COVID-19.

Background: Male sex is related to increased COVID-19 severity and fatality although confirmed infections are similarly distributed between men and women. The aim of this retrospective analysis was to investigate the impact of sex hormones on disease progression and immune activation in men with COVID-19. Patients and Methods: We studied for effects of sex hormones on disease severity and immune activation in 377 patients (230 men, 147 women) with PCR-confirmed SARS-CoV-2 infections hospitalized at the Innsbruck University Hospital between February and December 2020. Results: Men had more severe COVID-19 with concomitant higher immune system activation upon hospital admission when compared to women. Men with a severe course of infection had lower serum total testosterone (tT) levels whereas luteinizing hormone (LH) and estradiol (E2) levels were within the normal range. tT deficiency was associated with elevated CRP (rs = - 0.567, p < 0.001), IL-6 levels (rs = - 0.563, p < 0.001), lower cholesterol levels (rs = 0.407, p < 0.001) and an increased morbidity and mortality. Men with tT levels < 100 ng/dL had a more than eighteen-fold higher in-hospital mortality risk (OR 18.243 [95%CI 2.301 - 144.639], p = 0.006) compared to men with tT levels > 230 ng/dL. Moreover, while morbidity and mortality showed a positive correlation with E2 levels at admission, we detected a negative correlation with the tT/E2 ratio upon hospital admission. Conclusion: Hospitalized men with COVID-19 present with rather low testosterone levels linked to more advanced immune activation, severe clinical manifestations translating into an increased risk for ICU admission or death. The underlying mechanisms remain elusive but may include infection driven hypogonadism as well as inflammation mediated cholesterol reduction causing gonadotropin suppression and impaired androgen formation. Finally, in elderly late onset hypogonadism might also contribute to lower testosterone levels.

Potent SARS-CoV-2-Specific T Cell Immunity and Low Anaphylatoxin Levels Correlate With Mild Disease Progression in COVID-19 Patients.

T cells play a fundamental role in the early control and clearance of many viral infections of the respiratory system. In SARS-CoV-2-infected individuals, lymphopenia with drastically reduced CD4+ and CD8+ T cells correlates with Coronavirus disease 2019 (COVID-19)-associated disease severity and mortality. In this study, we characterized cellular and humoral immune responses induced in patients with mild, severe and critical COVID-19. Peripheral blood mononuclear cells of 37 patients with mild, severe and critical COVID-19 and 10 healthy individuals were analyzed by IFNγ ELISpot and multi-color flow cytometry upon stimulation with peptide pools covering complete immunodominant SARS-CoV-2 matrix, nucleocapsid and spike proteins. In addition SARS-CoV-2 antibody levels, neutralization abilities and anaphylatoxin levels were evaluated by various commercially available ELISA platforms. Our data clearly demonstrates a significantly stronger induction of SARS-CoV-2 specific CD8+ T lymphocytes and higher IFNγ production in patients with mild compared to patients with severe or critical COVID-19. In all patients SARS-CoV-2-specific antibodies with similar neutralizing activity were detected, but highest titers of total IgGs were observed in critical patients. Finally, elevated anaphylatoxin C3a and C5a levels were identified in severe and critical COVID-19 patients probably caused by aberrant immune complex formation due to elevated antibody titers in these patients. Crucially, we provide a full picture of cellular and humoral immune responses of COVID-19 patients and prove that robust polyfunctional CD8+ T cell responses concomitant with low anaphylatoxin levels correlate with mild infections. In addition, our data indicates that high SARS-CoV-2 antibody titers are associated with severe disease progression.

The versatility of external quality assessment for the surveillance of laboratory and in vitro diagnostic performance: SARS-CoV-2 viral genome detection in Austria.

OBJECTIVES: External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. METHODS: Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. RESULTS: A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (Ct ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of Ct values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. CONCLUSIONS: Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.