Performance of 20 rapid antigen detection tests to detect SARS-CoV-2 B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants using a clinical specimen panel from January 2022, Berlin, Germany.

BackgroundThere are conflicting reports on the performance of rapid antigen detection tests (RDT) in the detection of the SARS-CoV-2 Omicron (B.1.1.529) variant; however, these tests continue to be used frequently to detect potentially contagious individuals with high viral loads.AimThe aim of this study was to investigate comparative detection of the Delta (B.1.617.2) and Omicron variants by using a selection of 20 RDT and a limited panel of pooled combined oro- and nasopharyngeal clinical Delta and Omicron specimens.MethodsWe tested 20 CE-marked RDT for their performance to detect SARS-CoV-2 Delta and Omicron by using a panel of pooled clinical specimens collected in January 2022 in Berlin, Germany.ResultsWe observed equivalent detection performance for Delta and Omicron for most RDT, and sensitivity was widely in line with our previous pre-Delta/Omicron evaluation. Some variation for individual RDT was observed either for Delta vs Omicron detection, or when compared with the previous evaluation, which may be explained both by different panel sizes resulting in different data robustness and potential limitation of batch-to-batch consistency. Additional experiments with three RDT using non-pooled routine clinical samples confirmed comparable performance to detect Delta vs Omicron. Overall, RDT that were previously positively evaluated retained good performance also for Delta and Omicron variants.ConclusionOur findings suggest that currently available RDT are sufficient for the detection of SARS-CoV-2 Delta and Omicron variants.

Monitoring the SARS-CoV-2 Pandemic: Prevalence of Antibodies in a Large, Repetitive Cross-Sectional Study of Blood Donors in Germany-Results from the SeBluCo Study 2020-2022.

SARS-CoV-2 serosurveillance is important to adapt infection control measures and estimate the degree of underreporting. Blood donor samples can be used as a proxy for the healthy adult population. In a repeated cross-sectional study from April 2020 to April 2021, September 2021, and April/May 2022, 13 blood establishments collected 134,510 anonymised specimens from blood donors in 28 study regions across Germany. These were tested for antibodies against the SARS-CoV-2 spike protein and nucleocapsid, including neutralising capacity. Seroprevalence was adjusted for test performance and sampling and weighted for demographic differences between the sample and the general population. Seroprevalence estimates were compared to notified COVID-19 cases. The overall adjusted SARS-CoV-2 seroprevalence remained below 2% until December 2020 and increased to 18.1% in April 2021, 89.4% in September 2021, and to 100% in April/May 2022. Neutralising capacity was found in 74% of all positive specimens until April 2021 and in 98% in April/May 2022. Our serosurveillance allowed for repeated estimations of underreporting from the early stage of the pandemic onwards. Underreporting ranged between factors 5.1 and 1.1 in the first two waves of the pandemic and remained well below 2 afterwards, indicating an adequate test strategy and notification system in Germany.

German federal-state-wide seroprevalence study of 1st SARS-CoV-2 pandemic wave shows importance of long-term antibody test performance.

Background: Reliable data on the adult SARS-CoV-2 infection fatality rate in Germany are still scarce. We performed a federal state-wide cross-sectional seroprevalence study named SaarCoPS, that is representative for the adult population including elderly individuals and nursing home residents in the Saarland. Methods: Serum was collected from 2940 adults via stationary or mobile teams during the 1st pandemic wave steady state period. We selected an antibody test system with maximal specificity, also excluding seroreversion effects due to a high longitudinal test performance. For the calculations of infection and fatality rates, we accounted for the delays of seroconversion and death after infection. Results: Using a highly specific total antibody test detecting anti-SARS-CoV-2 responses over more than 180 days, we estimate an adult infection rate of 1.02% (95% CI: [0.64; 1.44]), an underreporting rate of 2.68-fold (95% CI: [1.68; 3.79]) and infection fatality rates of 2.09% (95% CI: (1.48; 3.32]) or 0.36% (95% CI: [0.25; 0.59]) in all adults including elderly individuals, or adults younger than 70 years, respectively. Conclusion: The study highlights the importance of study design and test performance for seroprevalence studies, particularly when seroprevalences are low. Our results provide a valuable baseline for evaluation of future pandemic dynamics and impact of public health measures on virus spread and human health in comparison to neighbouring countries such as Luxembourg or France.

[Safety of blood and blood products: test methods for the detection of hepatitis B, C, and E virus]. / Sicherheit von Blut und Blutprodukten: Testmethoden zum Nachweis der Hepatitisviren B, C und E.

Infections with hepatitis B, C, and E virus (HBV, HCV, and HEV) can be transmitted via blood and cause severe acute or chronic liver infections. To ensure the safety of blood donations and protect recipients from virus transmissions, blood donations in Germany are tested for viral genomes using nucleic acid amplification techniques (NATs) as well as for viral antigens and antibodies by serological testing. This article describes the relevant regulations on the safety of blood and blood products in Germany and the various screening methods. The safety of blood products is assessed.Currently used NAT methods for detection of hepatitis viruses are based either on polymerase chain reaction (PCR) or isothermal methods such as transcription-mediated amplification (TMA), which enable a highly sensitive detection of viral infections and thereby contribute to the reduction of the diagnostic window. Antigen tests for the detection of viral surface protein of hepatitis B virus in blood donations were introduced in the 1970s in order to prevent potential transmissions. Since the introduction of mandatory testing for HCV-specific antibodies in 1992, HCV NAT testing in 1999, anti-HBc antibody testing in 2006, and the non-mandatory HBV NAT, which is voluntarily performed by most of the blood establishments, blood safety has increased tremendously. Only a few isolated cases of transfusion-transmitted infections in the early window period have been reported since. The success of the recent introduction of mandatory HEV NAT testing in 2020 will have to be assessed in the upcoming years. Besides blood donor screening, the system for blood safety in Germany is supplemented by additional measures for donor selection and pathogen inactivation.

Antibody response to SARS-CoV-2 for more than one year - kinetics and persistence of detection are predominantly determined by avidity progression and test design.

BACKGROUND: Antibody detection of SARS-CoV-2 requires an understanding of its variation, course, and duration. METHODS: Antibody response to SARS-CoV-2 was evaluated over 5-430 days on 828 samples across COVID-19 severity levels, for total antibody (TAb), IgG, IgA, IgM, neutralizing antibody (NAb), antibody avidity, and for receptor-binding-domain (RBD), spike (S), or nucleoprotein (N). Specificity was determined on 676 pre-pandemic samples. RESULTS: Sensitivity at 30-60 days post symptom onset (pso) for TAb-S/RBD, TAb-N, IgG-S, IgG-N, IgA-S, IgM-RBD, and NAb was 96.6%, 99.5%, 89.7%, 94.3%, 80.9%, 76.9% and 92.8%, respectively. Follow-up 430 days pso revealed: TAb-S/RBD increased slightly (100.0%); TAb-N decreased slightly (97.1%); IgG-S and IgA-S decreased moderately (81.4%, 65.7%); NAb remained positive (94.3%), slightly decreasing in activity after 300 days; there was correlation with IgG-S (Rs = 0.88) and IgA-S (Rs = 0.71); IgG-N decreased significantly from day 120 (15.7%); IgM-RBD dropped after 30-60 days (22.9%). High antibody avidity developed against S/RBD steadily with time in 94.3% of patients after 430 days. This correlated with persistent antibody detection depending on antibody-binding efficiency of the test design. Severe COVID-19 correlated with earlier and higher antibody response, mild COVID-19 was heterogeneous with a wide range of antibody reactivities. Specificity of the tests was ≥99%, except for IgA (96%). CONCLUSION: Sensitivity of anti-SARS-CoV-2 assays was determined by test design, target antigen, antibody avidity, and COVID-19 severity. Sustained antibody detection was mainly determined by avidity progression for RBD and S. Testing by TAb and for S/RBD provided the highest sensitivity and longest detection duration of 14 months so far.

Comparative sensitivity evaluation for 122 CE-marked rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021.

IntroductionNumerous CE-marked SARS-CoV-2 antigen rapid diagnostic tests (Ag RDT) are offered in Europe, several of them with unconfirmed quality claims.AimWe performed an independent head-to-head evaluation of the sensitivity of SARS-CoV-2 Ag RDT offered in Germany.MethodsWe addressed the sensitivity of 122 Ag RDT in direct comparison using a common evaluation panel comprised of 50 specimens. Minimum sensitivity of 75% for panel specimens with a PCR quantification cycle (Cq) ≤ 25 was used to identify Ag RDT eligible for reimbursement in the German healthcare system.ResultsThe sensitivity of different SARS-CoV-2 Ag RDT varied over a wide range. The sensitivity limit of 75% for panel members with Cq ≤ 25 was met by 96 of the 122 tests evaluated; 26 tests exhibited lower sensitivity, few of which failed completely. Some RDT exhibited high sensitivity, e.g. 97.5 % for Cq < 30.ConclusionsThis comparative evaluation succeeded in distinguishing less sensitive from better performing Ag RDT. Most of the evaluated Ag RDT appeared to be suitable for fast identification of acute infections associated with high viral loads. Market access of SARS-CoV-2 Ag RDT should be based on minimal requirements for sensitivity and specificity.

Establishment of a specimen panel for the decentralised technical evaluation of the sensitivity of 31 rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021.

IntroductionThe detection of SARS-CoV-2 with rapid diagnostic tests (RDT) has become an important tool to identify infected people and break infection chains. These RDT are usually based on antigen detection in a lateral flow approach.AimWe aimed to establish a comprehensive specimen panel for the decentralised technical evaluation of SARS-CoV-2 antigen rapid diagnostic tests.MethodsWhile for PCR diagnostics the validation of a PCR assay is well established, there is no common validation strategy for antigen tests, including RDT. In this proof-of-principle study we present the establishment of a panel of 50 pooled clinical specimens that cover a SARS-CoV-2 concentration range from 1.1 × 109 to 420 genome copies per mL of specimen. The panel was used to evaluate 31 RDT in up to six laboratories.ResultsOur results show that there is considerable variation in the detection limits and the clinical sensitivity of different RDT. We show that the best RDT can be applied to reliably identify infectious individuals who present with SARS-CoV-2 loads down to 106 genome copies per mL of specimen. For the identification of infected individuals with SARS-CoV-2 loads corresponding to less than 106 genome copies per mL, only three RDT showed a clinical sensitivity of more than 60%.ConclusionsSensitive RDT can be applied to identify infectious individuals with high viral loads but not to identify all infected individuals.

Analysis of Humoral Immune Responses in Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused a pandemic with tens of millions of cases and hundreds of thousands of deaths. The infection causes coronavirus disease 2019 (COVID-19), a disease of the respiratory system of divergent severity. In the current study, humoral immune responses were characterized in a cohort of 143 patients with COVID-19 from the University Hospital Frankfurt am Main, Germany. METHODS: SARS-CoV-2-specific-antibodies were detected by enzyme-linked immunosorbent assay (ELISA). SARS-CoV-2 and human coronavirus NL63 neutralization activity was analyzed with pseudotyped lentiviral vectors. RESULTS: The severity of COVID-19 increased with age, and male patients encountered more serious symptoms than female patients. Disease severity was correlated with the amount of SARS-CoV-2-specific immunoglobulin (Ig) G and IgA and the neutralization activity of the antibodies. The amount of SARS-CoV-2-specific IgG antibodies decreased with time after polymerase chain reaction conformation of the infection, and antibodies directed against the nucleoprotein waned faster than spike protein-directed antibodies. In contrast, for the common flu coronavirus NL63, COVID-19 disease severity seemed to be correlated with low NL63-neutralizing activities, suggesting the possibility of cross-reactive protection. CONCLUSION: The results describe the humoral immune responses against SARS-CoV-2 and might aid the identification of correlates of protection needed for vaccine development.

Detection of hepatitis B virus infection in German blood donors 2008-2015.

BACKGROUND AND OBJECTIVES: Assessment of HBV-NAT testing compared to HBsAg and anti-HBc screening in German blood establishments for the period 2008-2015. MATERIALS AND METHODS: Blood donations screened for HBsAg and anti-HBc along with HBV-NAT were evaluated. Sensitivity of HBsAg and HBV-NAT tests was compared in 30 HBV seroconversion panels and with the viral load of the NAT-only cases. Residual risk for HBV in the WP was modelled. RESULTS: A total of 45 270 111 donations were evaluated. There were 29 NAT-only cases in the HBsAg-negative HBV-WP, one by ID-NAT and 28 by MP-NAT. MP-NAT, on average, showed higher sensitivity than HBsAg testing: MP-NAT-LoD of 146 IU/ml vs. 362 IU/ml HBV DNA for positive HBsAg detection (range 135-1502 IU/ml), resulting in 3·1 days (range 2·0-4·8 days) earlier HBV detection. Viral loads of the NAT-only cases confirmed the sensitivity of the HBV tests in the seroconversion study. One HBsAg-negative case was due to a new HBsAg mutant combination. There was one HBsAg-reactive only case. In addition, HBV incidence in the HBV-WP included 41 HBsAg-/HBV-NAT-positives and three HBV transmission cases. The residual risk for HBsAg was estimated to be 1:1 619 419-1 268 474 compared to 1:2 793 365-2 134 702 for MP-NAT. Within chronic HBV (HBsAg-/anti-HBc-positive and MP-NAT-negative) 70% were ID-NAT positive at low viral load (median 20 IU/ml). Among anti-HBc-only, supplementary ID-NAT detected 23 occult HBV infections. CONCLUSIONS: In the HBV-WP, MP-NAT provided a higher sensitivity than HBsAg testing, obtained a considerably higher yield and reduced the risk for HBV transmission. In later HBV stages, anti-HBc screening and HBV-ID-NAT intercepted potentially infectious donations.

Effectiveness of blood donor screening by HIV, HCV, HBV-NAT assays, as well as HBsAg and anti-HBc immunoassays in Germany (2008-2015).

BACKGROUND AND OBJECTIVES: In Germany, in addition to standard blood donor screening, further mandatory tests were introduced for HCV-RNA, HIV-1-RNA and for anti-HBc. Screening for HBV-DNA is optional. This study investigates the benefits of these additional tests for the detection of HIV, HCV, and HBV infections among German blood donors. MATERIALS AND METHODS: From 2008 to 2015 we collected data on blood donations exclusively testing NAT positive (NAT yield) or reactive in only one of the screening assays. Assuming a Poisson distribution, we calculated NAT yield/reactive only rates on a per donation basis (number of yield/reactive only cases divided by the number of donations tested in the period under review) with 95% confidence intervals. RESULTS: Responding establishments covered 95% of the donations. We identified 20 HIV-1-NAT, 61 HCV-NAT and 29 HBV-NAT yield cases among approximately 46 million blood donations tested corresponding to 0·43 HIV-1 NAT, 1·32 HCV-NAT, and 0·64 HBV-NAT yield cases per million blood donations tested. For one HBsAg reactive only case and 23 anti-HBc reactive only cases in repeat donors, infection was confirmed by ID-NAT which translates into 0·02 and 0·55 cases per million donations tested. During the 8-year-observation period, one HIV-1, no HCV and four HBV transmissions associated with donations in the viremic pre-seroconversion window period were reported. CONCLUSION: Annually, NAT screening alone detected 2·5 HIV-1, 7·6 HCV, and 3·6 HBV infectious donations; anti-HBc screening alone identified 2·9 infectious donations of repeat donors with occult HBV infection. Overall, the survey results support that the currently practiced donor HIV/HCV/HBV screening strategy in Germany does ensure a high standard of blood safety.

Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes.

For human hepatitis B virus eight distinct and two candidate genotypes are described. These genotypes differ with respect to geographic distribution, molecular virology and virus-associated pathogenesis. Comparative analysis of HBV genotypes revealed, with exception of HBV/G that shows impaired HBsAg release, that no fundamental disparities between genotypes exist regarding glycosylation, subcellular distribution, release of HBsAg and formation of subviral particles. However, there are distinctions regarding the proportion of L to M to S HBs proteins detected intra- and extracellularly for different genotypes. 2D electrophoresis revealed different posttranslational modification patterns for LHBs. In light of the relevance of HBsAg as diagnostic marker, detectability of purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated, showing similar sensitivities for genotypes included in this analysis. These data indicate that recombinant HBsAg reproducibly purified following a defined protocol might be used as an alternative to reference materials currently established.

International collaborative study on the 3rd WHO International Standard for hepatitis B surface antigen.

BACKGROUND: The WHO International Standard (IS) for hepatitis B surface antigen (HBsAg) is used to standardize HBsAg assays. Stocks of the 2nd IS for HBsAg are depleted. The proposal to establish its replacement was endorsed by WHO in 2012. OBJECTIVE: Preparation of a freeze-dried candidate 3rd IS (NIBSC 12/226); evaluation of its suitability in a WHO international collaborative study; calibration of its potency in International Units (IU). STUDY DESIGN: The 3rd IS is based on plasma-derived, purified, inactivated HBsAg from Vietnam. Qualitative and quantitative HBsAg assays were used to evaluate 12/226 alongside the 2nd IS and 1st IS. Blinded study samples included a duplicate of 12/226, a negative control and two diluted plasma samples representing hepatitis B virus (HBV) genotypes A and B. RESULTS: Twelve laboratories from 9 countries returned 22 data sets from 15 methods. The overall geometric mean potency of 12/226 is 47.3IU/mL (±13% CV) when compared to the 2nd IS with HBV subgenotype A2. The 3rd IS has HBV subgenotype B4 with a heterogeneous HBsAg subtype population of ayw1 and adw2. Some genotype-dependent effects on the inter-laboratory variability were observed but overall mean potencies were virtually identical irrespective of the IS used for calibration. Stability studies indicate that the candidate is stable for long-term use. CONCLUSIONS: 12/226 was established in October 2014 by the WHO Expert Committee on Biological Standardization as the 3rd IS for HBsAg with a potency of 47.3IU per ampoule maintaining the continuity in the standardization of HBsAg assays.

Hepatitis B virus genotype G monoinfection and its transmission by blood components.

An acute hepatitis B virus (HBV) infection was diagnosed in a regular apheresis (plasma/platelet) donor by the hepatitis B surface antigen (HBsAg) assay and minipool nucleic acid amplification technology (NAT). The acute infection was confirmed by detection of anti-HBc (IgM) and anti-HBs 2 weeks later. The donor showed no clinical symptoms and had normal alanine aminotransferase levels. He had a history of weekly apheresis plasma or platelet donations. Archived material from the donor and the respective recipients was investigated by sensitive HBV NATs as part of a look-back procedure. HBV DNA was detectable in previous donations as well as in two recipients transfused with platelet concentrates. The rare HBV genotype G was identified in all HBV-DNA-positive samples. Strong evidence of genotype G monoinfection was obtained by clonal sequencing, HBV genotype line probe assay, genotype-specific NATs, and restriction pattern analysis. In contrast to previously described genotype G infections, which all appeared as coinfections with genotype A, neither the hepatitis B e antigen (HBeAg) nor anti-HBe was detectable in any of the samples. This shows that HBeAg is dispensable for viral replication. The delay in detecting HBsAg in both the donor and recipient samples may be explained by either decreased genotype G-specific synthesis of incomplete viral forms in early HBV infection or the lower sensitivity to genotype G of the current HBsAg assays. In conclusion, this reported case of an HBV infection was caused exclusively by genotype G.

Evaluation of the performance of 44 assays used in countries with limited resources for the detection of antibodies to hepatitis C virus.

BACKGROUND: This study was conducted by the International Consortium for Blood Safety (ICBS) and its Collaborating Center, the Paul Ehrlich Institute, to identify high-quality, affordable assays for the detection of hepatitis C virus (HCV) antibodies and make available information on their performance for the benefit of developing countries. STUDY DESIGN AND METHODS: Forty-four assays were evaluated for their sensitivity and specificity. The assays' sensitivity was evaluated on a characterized panel of 200 anti-HCV-positive samples comprising major HCV genotypes 1 through 6. Three seroconversion panels were used to estimate sensitivity in the early infectious phase. Specificity was evaluated with a characterized ICBS-negative panel of 181 verified negative samples. RESULTS: Sensitivity was 100 percent for 15 assays, 99.5 percent for 11 assays, 99.0 percent for 6 assays, and less than 99.0 percent for 12 assays. The false-negative results found were not linked to the genotype. Anti-HCV detection in the early infectious phase was, on average, 16.7 days later than for tests licensed in the European Union. Specificity in 25 tests was 100 percent, whereas 11 assays showed 1 false-positive result (99.45%) and the other assays were nonspecific in 2 or more samples. Two assays were not supplied in sufficient quantity to test for specificity. CONCLUSIONS: On applying criteria for highest sensitivity (100%) and high specificity (> or =99.5%), 11 tests met the criteria. An additional 19 tests reached a performance comparable to WHO's criteria for human immunodeficiency virus antibody assays. The genotype diversity of HCV was found not to influence sensitivity of the assays.