Suitability of Different Diagnostic Platforms for Virological Testing of Blood Samples from Cornea Donors.

Background: To minimize the risk of disease transmission in cornea transplantation, donor screening for blood-derived viral infections is mandatory. Ideally, pre-mortem blood samples are used, but based on availability, cadaveric blood samples of cornea donors may also be used. However, serological and nucleic acid amplification tests (NATs) need to be validated for the use of cadaveric specimens. Methods: Hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV) 1/2, and Treponema pallidum (syphilis)-specific serological and/or NAT assays were validated on different platforms (Abbott Alinity i, Alinity m, Roche Cobas 6800, and Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM)) using (un)spiked paired pre- and post-mortem cornea donor blood samples from the same individual (up to 23.83 h after death) of 28 individuals in accordance with the specifications of the German Federal Institute for Vaccines and Biomedicines (Paul-Ehrlich-Institut [PEI]). In addition, routinely HBV-, HCV- and HIV-PCR-negative tested post-mortem blood samples of 24 individuals were used to assess NAT specificity. Results: For the majority of serological parameters on the Abbott Alinity i (HBsAg, anti-HBc, anti-HBs, anti-HCV, anti-HIV, anti-HTLV 1/2, and anti-Treponema pallidum), ratios of generated test results of (un)spiked paired pre- and post-mortem blood samples differed ≤25%, with an agreement of qualitative pre- and post-mortem test results ranging from 91.2 to 100%. For NAT parameters (HBV, HCV, and HIV) on the Cobas 6800, Alinity m, and CAP/CTM, no significant deviation in virus concentrations (factor >5) of spiked pre- and post-mortem blood samples could be observed. Ct-values of corresponding internal controls did also not differ significantly (>1.5 Ct-values). In addition, no false-positive test results were generated when specificity was assessed. Conclusion: Overall, fluctuations of test results for serological and NAT parameters in pre- and post-mortem blood samples examined in this study, were only limited and within the range of what is also observed when routinely testing fresh patient specimens. We conclude that all examined assays are eligible for the screening of blood samples taken up to about 24 h after the occurrence of death.

Clinical performance of different SARS-CoV-2 IgG antibody tests.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays are urgently needed for rapid diagnosis, contact tracing, and for epidemiological studies. So far, there is limited data on how commercially available tests perform with real patient samples, and if positive tested samples show neutralizing abilities. Focusing on IgG antibodies, we demonstrate the performance of two enzyme-linked immunosorbent assay (ELISA) assays (Euroimmun SARS-CoV-2 IgG and Vircell COVID-19 ELISA IgG) in comparison to one lateral flow assay (FaStep COVID-19 IgG/IgM Rapid Test Device) and two in-house developed assays (immunofluorescence assay [IFA] and plaque reduction neutralization test [PRNT]). We tested follow up serum/plasma samples of individuals polymerase chain reaction-diagnosed with COVID-19. Most of the SARS-CoV-2 samples were from individuals with moderate to the severe clinical course, who required an in-patient hospital stay. For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5-9) and from 93.8% to 100% for the later period (days 10-18).

Utility of Different Surrogate Enzyme-Linked Immunosorbent Assays (sELISAs) for Detection of SARS-CoV-2 Neutralizing Antibodies.

The plaque reduction neutralization test (PRNT) is a preferred method for the detection of functional, SARS-CoV-2 specific neutralizing antibodies from serum samples. Alternatively, surrogate enzyme-linked immunosorbent assays (ELISAs) using ACE2 as the target structure for the detection of neutralization-competent antibodies have been developed. They are capable of high throughput, have a short turnaround time, and can be performed under standard laboratory safety conditions. However, there are very limited data on their clinical performance and how they compare to the PRNT. We evaluated three surrogate immunoassays (GenScript SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript Biotech, Piscataway Township, NJ, USA), the TECO® SARS-CoV-2 Neutralization Antibody Assay (TECOmedical AG, Sissach, Switzerland), and the Leinco COVID-19 ImmunoRank™ Neutralization MICRO-ELISA (Leinco Technologies, Fenton, MO, USA)) and one automated quantitative SARS-CoV-2 Spike protein-based IgG antibody assay (Abbott GmbH, Wiesbaden, Germany) by testing 78 clinical samples, including several follow-up samples of six BNT162b2 (BioNTech/Pfizer, Mainz, Germany/New York, NY, USA) vaccinated individuals. Using the PRNT as a reference method, the overall sensitivity of the examined assays ranged from 93.8 to 100% and specificity ranged from 73.9 to 91.3%. Weighted kappa demonstrated a substantial to almost perfect agreement. The findings of our study allow these assays to be considered when a PRNT is not available. However, the latter still should be the preferred choice. For optimal clinical performance, the cut-off value of the TECO assay should be individually adapted.

Brief clinical evaluation of six high-throughput SARS-CoV-2 IgG antibody assays.

Serological SARS-CoV-2 assays are urgently needed for diagnosis, contact tracing and for epidemiological studies. So far, there is limited data on how recently commercially available, high-throughput immunoassays, using different recombinant SARS-CoV-2 antigens, perform with clinical samples. Focusing on IgG and total antibodies, we demonstrate the performance of four automated immunoassays (Abbott Architect™ i2000 (N protein-based)), Roche cobas™ e 411 analyzer (N protein-based, not differentiating between IgA, IgM or IgG antibodies), LIAISON®XL platform (S1 and S2 protein-based), VIRCLIA® automation system (S1 and N protein-based) in comparison to two ELISA assays (Euroimmun SARS-CoV-2 IgG (S1 protein-based) and Virotech SARS-CoV-2 IgG ELISA (N protein-based)) and an in-house developed plaque reduction neutralization test (PRNT). We tested follow up serum/plasma samples of individuals PCR-diagnosed with COVID-19. When calculating the overall sensitivity, in a time frame of 49 days after first PCR-positivity, the PRNT as gold standard, showed the highest sensitivity with 93.3% followed by the dual-target assay for the VIRCLIA® automation system with 89%. The overall sensitivity in the group of N protein-based assays ranged from 66.7 to 77.8% and in the S protein-based-assays from 71.1 to 75.6%. Five follow-up samples of three individuals were only detected in either an S and/or N protein-based assay, indicating an individual different immune response to SARS-CoV-2 and the influence of the used assay in the detection of IgG antibodies. This should be further analysed. The specificity of the examined assays was ≥ 97%. However, because of the low or unknown prevalence of SARS-CoV-2, the examined assays in this study are currently primarily eligible for epidemiological investigations, as they have limited information in individual testing.