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Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings
(2021)
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID50) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.
Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020
(2020)
Containment strategies and clinical management of coronavirus disease (COVID-19) patients during the current pandemic depend on reliable diagnostic PCR assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we compare 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. While most assays performed well, we identified detection problems in a commonly used assay that may have resulted in false-negative test results during the first weeks of the pandemic.
Due to globally rising numbers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, resources for real-time reverse-transcription polymerase chain reaction (rRT-PCR)-based testing have been exhausted. In order to meet the demands of testing and reduce transmission, SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDTs) are being considered. These tests are fast, inexpensive, and simple to use, but whether they detect potentially infectious cases has not been well studied. We evaluated three lateral flow assays (RIDA®QUICK SARS-CoV-2 Antigen (R-Biopharm), SARS-CoV-2 Rapid Antigen Test (Roche)), and NADAL® COVID-19 Ag Test (Nal von Minden GmbH, Regensburg, Germany) and one microfluidic immunofluorescence assay (SARS-CoV-2 Ag Test (LumiraDx GmbH, Cologne, Germany)) using 100 clinical samples. Diagnostic rRT-PCR and cell culture testing as a marker for infectivity were performed in parallel. The overall Ag-RDT sensitivity for rRT-PCR-positive samples ranged from 24.3% to 50%. However, for samples with a viral load of more than 6 log10 RNA copies/mL (22/100), typically seen in infectious individuals, Ag-RDT positivity was between 81.8% and 100%. Only 51.6% (33/64) of the rRT-PCR-positive samples were infectious in cell culture. In contrast, three Ag-RDTs demonstrated a more significant correlation with cell culture infectivity (61.8–82.4%). Our findings suggest that large-scale SARS-CoV-2 Ag-RDT-based testing can be considered for detecting potentially infective individuals and reducing the virus spread.
Objectives: Regarding reactogenicity and immunogenicity, heterologous COVID-19 vaccination regimens are considered as an alternative to conventional immunization schemes.
Methods: Individuals receiving either heterologous (ChAdOx1-S [AstraZeneca, Cambridge, UK]/BNT162b2 [Pfizer-BioNTech, Mainz, Germany]; n = 306) or homologous (messenger RNA [mRNA]-1273 [Moderna, Cambridge, Massachusetts, USA]; n = 139) vaccination were asked to participate when receiving their second dose. Reactogenicity was assessed after 1 month, immunogenicity after 1, 3, and/or 6 months, including a third dose, through SARS-CoV-2 antispike immunoglobulin G, surrogate virus neutralization test, and a plaque reduction neutralization test against the Delta (B.1.167.2) and Omicron (B.1.1.529; BA.1) variants of concern.
Results: The overall reactogenicity was lower after heterologous vaccination. In both cohorts, SARS-CoV-2 antispike immunoglobulin G concentrations waned over time with the heterologous vaccination demonstrating higher neutralizing activity than homologous mRNA vaccination after 3 months to low neutralizing levels in the Delta plaque reduction neutralization test after 6 months. At this point, 3.2% of the heterologous and 11.4% of the homologous cohort yielded low neutralizing activity against Omicron. After a third dose of an mRNA vaccine, ≥99% of vaccinees demonstrated positive neutralizing activity against Delta. Depending on the vaccination scheme and against Omicron, 60% to 87.5% of vaccinees demonstrated positive neutralizing activity.
Conclusion: ChAdOx1-S/BNT162b2 vaccination demonstrated an acceptable reactogenicity and immunogenicity profile. A third dose of an mRNA vaccine is necessary to maintain neutralizing activity against SARS-CoV-2. However, variants of concern-adapted versions of the vaccines would be desirable.