key: cord-0812215-kp5o17yw authors: Thom, Ruth E.; Eastaugh, Lin S.; O’Brien, Lyn M.; Ulaeto, David O.; Findlay, James S.; Smither, Sophie J.; Phelps, Amanda L.; Stapleton, Helen L.; Hamblin, Karleigh A.; Weller, Simon A. title: Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms date: 2021-09-16 journal: Front Cell Infect Microbiol DOI: 10.3389/fcimb.2021.716436 sha: bf4a3e7bb479dbdbcfb9ee1317cffcdcba866bc2 doc_id: 812215 cord_uid: kp5o17yw Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 10(5) tissue culture infectious dose (TCID)(50)/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 10(4) TCID(50)/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 10(3) TCID(50)/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to the Coronaviridae family and is the causative agent of the respiratory illness, coronavirus disease 2019 (COVID-19) (Gorbalenya et al., 2020) . The enveloped positive-sense singlestranded RNA virus was first discovered in early 2020 after a cluster of viral pneumonia cases of unknown cause were reported in the Hubei Province of China (Wu et al., 2020) . The virus is highly contagious in humans, and in March 2020, the WHO declared a global pandemic (Chen, 2020) . Diagnostic testing is critical in the fight against the COVID-19 pandemic (Patel et al., 2020) , not just for patients displaying symptoms but also for asymptomatic carriers and presymptomatic patients (Shental et al., 2020) . SARS-CoV-2 has been classified in the United Kingdom as a Hazard Group (HG) 3 pathogen by the Advisory Committee on Dangerous Pathogens (ACDP), meaning that this virus must be handled under Containment Level (CL) 3 conditions [biosafety level (BSL) 3]. However, guidance from the WHO (World Health Organization 2020) and Public Health England, United Kingdom (Public Health England, 2020), has permitted non-propagative diagnostic testing to be carried out at CL 2 with non-inactivated samples being handled within a Class 1 microbiology safety cabinet. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) is the gold standard test for the detection of SARS-CoV-2 from nasopharyngeal swab samples (Tahamtan and Ardebili, 2020) . Inactivation of viral pathogens prior to PCR is typically carried out at the same time as extraction of viral nucleic acids from samples, with chemical or physical methods employed. Typically buffers provided in nucleic acid extraction kits contain chaotropic salts, solvents, and detergents to lyse the virus. Guanidinium salts, such as guanidinium isothiocyanate (GITC), are chaotropic agents found in many lysis buffers, which in some cases have been demonstrated to inactivate viral pathogens, including alphaviruses, flaviviruses, filoviruses, and a bunyavirus (Blow et al., 2004; Ngo et al., 2017) . Other reports suggest that a combination of a GITC containing extraction buffer (such as Qiagen AVL) and a solvent (such as ethanol) is required for the inactivation of viruses such as Ebola virus (Smither et al., 2015) and Middle East respiratory syndrome coronavirus (MERS-CoV) (Kumar et al., 2015) . Detergents such as Tween, sodium dodecyl sulphate (SDS), and Triton X-100 have also been shown to disrupt viral envelopes and reduce viral titres (Mayo and Beckwith, 2002; van Kampen et al., 2017; Patterson et al., 2020) , with a combination of the GITC-based reagent (Buffer AVL) and Triton X-100 having been reported to inactivate Ebola virus (Burton et al., 2017) . Physical processes such as heat can also be incorporated in the nucleic acid extraction workflow and can have an inactivation effect. Some reports suggest that the application of heat alone can inactivate SARS-CoV, MERS-CoV, and SARS-CoV-2 following a heat regimen of 65°C for at least 15 min (Darnell et al., 2004; Leclercq et al., 2014; Kim et al., 2020) . Since the pandemic was declared, United Kingdom's Defence Science and Technology Laboratory (Dstl) and British military clinicians have set up the Defence COVID Laboratory (DCL), which has been awarded an extension to scope (under ISO17025) for the provision of a SARS-CoV-2 PCR test by the United Kingdom Accreditation Service (UKAS). The DCL analyses samples from UK military units and operates two automated high-throughput RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex). In this study, conducted entirely under CL 3 laboratory conditions (BSL 3), we report the inactivation efficacy of SARS-CoV-2 by buffers from three commercially available kits used on these two platforms. Buffer volumes and ratios were chosen for their suitability for RNA extraction (following manufacturer's instructions) rather than their potential inactivation efficacy; however, in doing so, we have further investigated the inactivation efficacy of combinations of GITC containing buffers, solvents, and/or detergents with and without an additional heat inactivation step. We provide evidence to support protocols for the inactivation of SARS-CoV-2 and the safe use of clinical samples in downstream RT-PCR in highthroughput diagnostic laboratories. All virus manipulations were carried out using the SARS-CoV-2 England 2 strain (GISAID reference EPI_ISL_407073), provided by Public Health England. Virus stock was propagated in Vero C1008 cell, harvested at day 3 and clarified by centrifugation at 350 × g for 15 min (Sigma 3-16K centrifuge). Viral stocks were concentrated by centrifugation at 11,000 × g for 3 h at 4°C to achieve 1 × 10 8 tissue culture infectious dose (TCID) 50 /ml and stored at −80°C. All cell cultures were carried out using confluent monolayers of Vero C1008 cells (European Collection of Authenticated Cell Cultures [ECACC], United Kingdom; catalogue no. 85020206) maintained in Dulbecco's minimal essential medium (DMEM; Sigma, United Kingdom) supplemented with 10% foetal calf serum, 1% L-glutamine, and 1% penicillin-streptomycin (Sigma, United Kingdom) and incubated at 37°C in a 5% CO 2 environment. Prior to virus being added to cell monolayers, 10% DMEM was replaced with Leibovitz's L-15 (to buffer for the lack of CO 2 at CL 3), supplemented as described for DMEM, with the exception of 2% foetal calf serum, and incubated at 37°C. Viral enumeration (for determining starting concentrations and measuring reductions in concentrations post-inactivation) was carried out by an end-point TCID 50 assay (Piercy et al., 2010) . In brief, Vero C1008 cells were prepared in 96-well microtitre plates to achieve confluent monolayers on the day of assay. To all wells of column 1 of the plate, 100 µl of test sample was added. From column 1, 20 µl of sample was transferred sequentially across the plate to achieve a 10-fold serial dilution to column 9. Cells in columns 11 and 12 were left in tissue culture medium (TCM) as controls. Plates were incubated in a humidified atmosphere for 3-4 days at 37°C, after which they were scored for cytopathic effect (CPE) by microscopic observation. The TCID 50 value was calculated by the method of Reed and Muench (1938) . Mean values were calculated as the geometric mean. Buffers and reagents from three different RNA extraction kits were assessed to determine inactivation of SARS-CoV-2 ( Table 1 ). The composition of these initial reagents and their suitability for extraction of SARS-CoV-2 RNA from clinical samples was determined based on manufacturers' protocols and after discussions with each manufacturer. The inactivation efficacy of each lysis buffer was evaluated with and without the inclusion of a heat step. Table 1 summarises the components and volumes used for each lysis buffer preparation. MS2 bacteriophage (10 6 plaque-forming unit (PFU)/ml) was added to each lysis buffer preparation as an internal control in the DCL and was therefore included in these experiments. Test samples for each experiment were set up in triplicate, and each experiment was performed on at least three separate occasions. Viral transport medium (VTM; EO Labs, United Kingdom) was inoculated with SARS-CoV-2 to achieve a starting concentration of 5 × 10 6 TCID 50 /ml for all experiments. To the lysis buffer preparations, 200 µl of virus in VTM was added; the samples were briefly vortexed and incubated for 10 min at room temperature. For heat-treated samples, the tubes were incubated for 25 min in a heat block (Eppendorf ThermoMixer C heat) set at 75°C. Laboratory tests showed that this was the temperature setting required for this individual heat block to heat and maintain the samples at 68°C for 15 min. Heat steps were carried out after the addition of virus to either lysis buffer reagents or an equivalent volume of TCM, to assess the effect of viability following heat in the presence or absence of reagents. Further controls included shaminactivated virus, where appropriate volume of TCM replaced the lysis buffer reagents and negative controls consisting of VTM only were added to lysis buffer reagents to assess the effect of the reagents on cell monolayers. After inactivation (with or without heat treatment), all samples and controls were centrifuged at 6,000 × g for 5 min in a microcentrifuge (Hermle Microlitre Centrifuge Z 160 M), and the supernatant was discarded and replaced with 1 ml of TCM. This Water was used to replace the magnetic beads, as the washing steps described below would not remove the beads, and the beads interfered with the read-out of the TCID 50 assay. High-Throughput SARS-CoV-2 Inactivation step was required to dilute the chemical components that would otherwise cause toxicity in the cell culture-based enumeration assay. Although virus pellets were not visible, this method is known to pellet virus with appropriate efficiency, as demonstrated by virus recovery in positive controls and is similar to methods used successfully in previous studies (Smither et al., 2016) . In experiments (data not shown), this step was shown to be required four times for the Qiagen reagents and two times for the KingFisher reagents in order to remove all traces of the inactivation chemicals from the sample and to avoid toxicity during cell culture. After the final wash, the pellets were re-suspended in 1 ml of TCM. Controls in each experiment were washed the same number of times as required by the reagent being evaluated. To quantify and determine the viability of the virus following inactivation, the samples were enumerated by the TCID 50 endpoint dilution assay described above; and the remaining sample underwent three rounds of serial passage in tissue culture flasks for a secondary confirmation of viral inactivation. In brief, all of the remaining samples (approx. 180 µl) were added to confluent monolayer of Vero C1008 cells in a 12.5-cm 2 tissue culture flask. Flasks were incubated in a humidified atmosphere for 3-4 days after which presence or absence of cytopathic effect was recorded. A total of three passages were performed, and CPE was recorded after each round. To control for cross-contamination, a set of un-infected flasks were also prepared, and supernatant was passaged in parallel to the experimental samples. A 10-fold serial dilution of SARS-CoV-2 was also inoculated into a set of flasks starting from 1.7 × 10 7 TCID 50 /ml and diluted to 1.1 TCID 50 /ml to show the limit of detection (LOD) of the flask passage assay and demonstrate a suitable environment for the passage and propagation of the virus. All data were graphically represented and statistically analysed using GraphPad Prism 8. The Kruskal-Wallis analysis of variance (ANOVA) was performed on data sets with Dunn's multiple comparison post hoc. The inactivation of SARS-CoV-2 was assessed using three different RNA lysis buffers with and without the inclusion of a heat step. The viability of virus was determined quantitatively using the TCID 50 assay and qualitatively by serially passaging samples in flask. These studies used the highest working concentration of SARS-CoV-2 that was available, and this ranged from 5.9 × 10 5 to 3.5 × 10 6 TCID 50 /ml (Figure 1 ). Following the inactivation procedure, residual toxic lysis buffer components were removed by way of multiple wash steps. Residual chemical components would otherwise be toxic to the cell-based assays. To determine if the multiple wash steps by centrifugation resulted in a loss of virus, virus was inoculated into TCM without the addition of lysis reagents (as described in the Methods) and assayed as described. This highlighted that there was approximately a 1-Log 10 drop in titre in each experiment. When virus was added to the Qiagen lysis buffer, there was a statistically significant 5-Log 10 drop (p = 0.002) in virus titre from 3.3 × 10 5 TCID 50 /ml to below the lower limit of quantification (LLoQ) of 32 TCID 50 /ml. Complete inactivation was not achieved however, as virus was detected below the LLoQ, but this was not quantifiable. However, by extrapolation, it was estimated that the titre was 6.2 TCID 50 /ml ( Figure 1A ). Similar results were observed when virus was inactivated using the MagMAX Protocol 2; complete inactivation was not achieved as virus was detected below the LLoQ and was not quantifiable. The starting titre of virus for these experiments, following washing steps, was 5.8 × 10 4 TCID 50 /ml, demonstrating a 4-Log 10 drop in viral titre following inactivation (p < 0.001) ( Figure 1C) . Virus inactivation following the MagMAX Protocol 1 resulted in no detectable virus by TCID 50 assay. The starting concentration of virus, following washing steps, was calculated to be 1.4 × 10 5 TCID 50 /ml, thus demonstrating a 5-Log 10 drop in viral titre with this particular protocol (p < 0.0001) ( Figure 1B ). Heat alone or in combination with lysis buffer was also investigated as a means to inactivate SARS-CoV-2. For each experiment, virus in TCM was heated at 68°C for 15 min and centrifuged to maintain consistency with samples in lysis buffer. Although not statistically significant, at least a 3-Log 10 drop in viral titre was observed following heat treatment alone, though viable virus was observed in replicates across all three heat alone experiments, even when below LLoQ (Figure 1) . When the virus was added to one of the three lysis buffers and subsequently heated, no viable virus was detected following TCID 50 assay and an average drop in viral titre of 5-Log 10 across all experiments (p < 0.0001) ( Figures 1A-C) . To confirm findings by TCID 50 assay, viral samples were propagated in cell culture flasks over a total of three passages to identify potential viral breakthrough. Table 2 shows the results of the presence of CPE after the first passage. The LOD for viral propagation was determined following propagation of serially diluted virus stocks (Table 2 row 1 to 5), and on average, the LOD was 1.3 TCID 50 /ml. When virus was added to TCM, CPE was present in all flasks as expected (Table 2 row 6, positive control). No cell toxicity was observed from negative control samples where TCM only was added to lysis buffer and washed as described previously (Table 2 row 10, negative control). When SARS-CoV-2 was inactivated following the Qiagen protocol, three out of the nine flasks were scored as positive for CPE. Of the flasks where no CPE was observed, no breakthrough of virus was seen as a result of serial passage ( Table 2 row 7) . These data align with the TCID 50 assays, where Qiagen lysis buffer alone did not completely inactivate the virus. Following both MagMAX protocols, zero out of the nine flasks were scored positively for CPE (Table 2 row 7) . For the MagMAX Protocol 1, this confirms the TCID 50 results, where no viable virus was also observed. For the MagMAX Protocol 2, virus was detected but not quantifiable in the TCID 50 assay (below the LLoQ); however, subsequent serial passage did not provide evidence of viability, as all flasks were negative for CPE. When SARS-CoV-2 was added to TCM and heated for 15 min at 68°C, CPE was observed in all but one flask ( Table 2 row 8), confirming the TCID 50 results that the heating protocol described here does not completely inactivate the virus. For all inactivation protocols, when SARS-CoV-2 samples were treated in a two-step manner (lysis buffer and heat), no viable virus was detected in either the quantitative or qualitative assays ( Figure 1 and Table 2 row 9). These data provide strong evidence that the lysis buffers described here in combination with the heat protocol can completely inactivate up to 5-Log 10 TCID 50 /ml SARS-CoV-2. Real-time PCR is the gold standard clinical diagnostic method for the detection of SARS-CoV-2 in patients displaying symptoms of COVID-19. There has been a rapid development in RNA extraction and RT-PCR diagnostic methods in order to help prevent further spread of infection through communities. It is crucial that testing is accurate and efficient, both of which must not compromise safety of those processing the samples (Dhamad 1. SARS-CoV-2 starting titre 3/3 1.7 × 10 7 3/3 5.9 × 10 6 3/3 3.0 × 10 6 2. SARS-CoV-2 10 −4 dilution 3/3 1.7 × 10 3 * 3/3 5.9 × 10 2 * 3/3 3.0 × 10 2 * 3. SARS-CoV-2 10 −5 dilution 3/3 1.7 × 10 2 * 3/3 59.4* 2/3 20.0* 4. SARS-CoV-2 10 −6 dilution 3/3 17* 1/3 2.0* 1/3 0.7* 5. SARS-CoV-2 10 −7 dilution 2/3 1.1* 0/3 ND 0/3 ND 6. SARS-CoV-2 + TCM 9/9 3.3 × 10 5 9/9 1.4 × 10 5 9/9 5.8 × 10 4 7. SARS-CoV-2 + lysis buffer 3/9