key: cord-0822562-nrwlc491
authors: Pryce, Todd M.; Boan, Peter A.; Kay, Ian D.; Flexman, James P.
title: Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing
date: 2021-08-24
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2021.115519
sha: 47580121f251de6a3f7af948bfa72d533a2dfbb2
doc_id: 822562
cord_uid: nrwlc491

To improve laboratory safety we thermally treated naso-oropharyngeal samples before testing with the cobas SARS-CoV-2 assay. This study aimed to determine if thermal treatment significantly affects the qualitative detection of SARS-CoV-2 and the quantitative measurement of cobas SARS-CoV-2 ORF1a and E-gene target copy number using an in-house quantitative method. A collection of positive (n=238) and negative samples (n=196) was tested in parallel comparing thermal treatment (75°C for 15 minutes) to room-temperature. There were no significant differences in the final qualitative outcomes for thermal treatment versus room-temperature (99.8% agreement) despite a statistically significant reduction (p<0.05) in target copy number following thermal treatment. The median ORF1a and E-gene reduction in target copy number was ­0.07 (1.6%) and ­0.22 (4.2%) log(10) copies/mL respectively. The standard curves for both ORF1a and E-gene targets were highly linear (r(2)=0.99). Good correlation was observed for ORF1a (r(2)=0.96) and E-gene (r(2)=0.98) comparing thermal treatment to room-temperature control.

Since the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have seen shortages of laboratory reagents, consumables and personal protective equipment (PPE) [1, 2] . Our initial testing protocols included guanidine hydrochloride (GuHCl) inactivation of SARS-CoV-2 to improve laboratory safety and avoid the use of additional PPE (eye protection, N95 mask, disposable gown) [2, 3] . When we transitioned SARS-CoV-2 testing to the cobas 6800 system (Roche Diagnostics, Basel, Switzerland) we were concerned about transferring un-capped samples to the cobas 6800 instrument so returned to using additional PPE as a precaution. We were also concerned about the pipetting workload associated with the addition of GuHCl to samples before cobas testing, potential loss of assay sensitivity from sample dilution and assay nonspecific interference. The effects of temperature on the viability of SARS-CoV-2 and other coronaviruses have been reported [4] [5] [6] [7] . Our initial investigations suggested thermal treatment of nasopharyngeal samples resulted in statistically significant higher cycle threshold (C t ) values for E-gene (p 0.040), suggesting a reduction in detectable virus RNA [8] . Despite marginally higher cobas C t values for thermally treated samples, conflicting findings were observed for the qualitative detection of SARS-CoV-2, specifically detection of ORF1a and E-gene targets at the limit of detection. The chief aim of this study was to determine if thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing affects the qualitative detection of SARS-CoV-2 and the quantitative detection of RNA target copy number. In addition, we developed and present here a quantitative method for cobas SARS-CoV-2 ORF1a and E-gene targets, using a commercially available SARS-CoV-2 standard to assess the effects of thermal 4 treatment on RNA target copy number. We also investigated quantitative and qualitative results of storage at room-temperature for up to 48 hours for thermally treated and nonthermally treated samples in case we encounter testing delays.

A combined nasopharyngeal and oropharyngeal swab from each patient was inoculated into 3 mL of either Copan UTM-RT media (Brescia, Italy), CITOSWAB (Citotest Scientific Jiangsu, People's Republic of China) or Virus Transport Media (VTM) prepared by PathWest Media [9] . All samples were initially tested as part of routine testing using the cobas SARS-CoV-2 assay (Roche Diagnostics, Basel, Switzerland). This test is performed on either the cobas 6800 or cobas 8800 instrument (Roche) and is a walkaway sample-to-result assay. The cobas SARS-CoV-2 assay targets ORF1a (a nonstructural region that is unique to SARS-CoV-2) and E-gene (a structural protein envelope gene for pan-sarbecovirus detection). According to the manufacturer's instructions, a sample is SARS-CoV-2 positive if ORF1a is detected with or without Egene detection. In the case of positivity with E-gene alone, the result should be reported as SARS-CoV-2 presumptive positive.

All SARS-CoV-2 positive samples were stored as aliquots at -80C. To prepare positive samples for this study (n=238; positive sample group) a 0.2 mL aliquot from each sample was diluted with 1.8 mL of a nasopharyngeal/throat matrix (1:10 dilution). The matrix consisted of pooled cobas SARS-CoV-2 negative patient samples (oro-nasopharyngeal swabs) in VTM. The pooled matrix tested negative with cobas SARS-CoV-2 and Xpert 5 Xpress SARS-CoV-2 (Cepheid, Sunnyvale, California, USA). All dilutions were prepared in cobas omni secondary tubes (Ref. 06438776001). Cobas SARS-CoV-2 negative samples were stored at 4C and were not diluted (n=196; negative sample group). All samples were tested following protocols issued by the manufacturer (roomtemperature control). Un-capped samples in cobas omni secondary tubes were transferred to the cobas 6800 system by laboratory personnel wearing additional PPE (eye protection, N95 mask, disposable gown). Samples were retrieved from the cobas 6800 following sample aspiration with additional PPE, capped and thermally treated for 75C for 15 minutes in a QBD4 dry block heater (Grant Instruments, Cambridge, United Kingdom), then retested within 2 hours (thermal treatment) without the use of additional PPE. The qualitative results and C t values were recorded for ORF1a, E-gene and Internal Control (IC).

Quantitative standards were prepared from a commercially available SARS-CoV-2 standard (Exact Diagnostics, Fort Worth, Texas). Exact Diagnostics SARS-CoV-2 standard contains E-gene and ORF1ab synthetic RNA transcripts quantitated to 200,000 copies/mL using BioRad Digital Droplet PCR. We pooled multiple vials and 10-fold serially diluted in molecular grade water (G-Biosciences, St. Louis, Missouri, USA) to prepare six standards over the range of 0.30 to 5.30 log 10 copies/mL. Each standard was tested with cobas SARS-CoV-2 in duplicate (no thermal treatment) on a single run using cobas SARS-CoV-2 kit lot G18524. The mean C t value at each concentration was used to calculate ORF1a and E-gene standard curves and regression. Both replicates at each 6 dilution were required to be positive to be included in the standard curve and regression analysis. The regression formulas were used to calculate the ORF1a and E-gene copy number for all positive samples and controls over three consecutive runs. The testing for all positive samples and the quantitative standards was performed using the same cobas SARS-CoV-2 kit lot (G18524). As part of the QConnect programme an external control (EQC) was also performed routinely to monitor reproducibility (Optitrol NAT SARS-CoV-2; DiaMex, Heidelberg, Germany) [10] . A single lot number of EQC (DM20119) was tested over 19 runs.

A low-titre positive patient sample in VTM (≈3.00 log 10 copies/mL) was used to study effects over time. Two replicates were thermally treated then tested at 2, 4, 6, 10, 24 and 48-hour intervals (kept at room temperature). Another two replicates remained at roomtemperature and were tested in parallel with the thermally treated samples. The C t values for ORF1a, E-gene and IC was recorded for each time point and the ORF1a and E-gene copy number was calculated using the standard curves.

All results of ORF1a and E-gene (C t values and log 10 copies/mL) for thermal treatment and room-temperature were compared using the Wilcoxon Signed-Ranks test (p < 0.05).

The same statistical approach was applied for all IC C t values. The median for each group was calculated and used to determine the percentage reduction or gain in C t value or log 10 target copy number/mL. Differences in the mean and standard deviation were also 7 calculated for comparison. Correlation between comparing thermal treatment to room temperature for ORF1a-positive samples (n=180) and E-gene-positive samples (n=201) was also performed. The mean and standard deviation was calculated for the EQC. All statistical analyses were performed by Excel (Microsoft, Redmond, WA, USA) and

MedCalc v15.4 (New York, NY, USA). 

The qualitative results comparing thermal treatment to room-temperature for the positive sample group (n=238) and negative sample group (n=196) are shown in the Supplementary Material and are summarised in Table 1 

The C t values for each target concentration and standard curves for ORF1a and E-gene are shown in the Supplementary Material. The results for both targets were highly linear.

ORF1a demonstrated R-squared value of 0.9988 over the range of 1.30 to 5.30 log 10 copies/mL (5 standards). ORF1a detection at 0.30 log 10 copies/mL was not reproducible and was omitted from the standard curve. E-gene demonstrated R-squared value of 0.9994 over the range of 2.30 to 5.30 log 10 copies/mL (4 standards). E-gene detection at 1.30 log 10 copies/mL was not reproducible and was omitted from the standard curve. Both replicates were negative at 0.30 log 10 copies/mL for E-gene.

All C t values for ORF1a, E-gene and internal control for all samples are shown in the Supplementary Material. The quantitative measurement of ORF1a and E-gene targets in copies/mL and log 10 copies/mL are also shown, including the ORF1a and E-gene targets 9 above and below the calculated standard curve range; the standard curve for each target was assumed to be linear above and below the calculated standard curve range to simplify statistical analysis. A summary of the Wilcoxon Signed-Ranks statistical analysis for

ORF1a, E-gene and IC are summarised in Table 2 . The mean differences and standard deviation differences are also shown for comparison. For thermal treatment compared to room-temperature, we found a significant difference (p<0.05) in median C t and/or quantitative values for ORF1a, E-gene and IC for all samples. The median ORF1a and Egene reduction in target copy number was -0.07 (1.6%) and -0.22 (4.2%) log 10 copies/mL respectively. Good correlation was observed for ORF1a (r 2 =0.96) and E-gene (r 2 =0.98) comparing thermal treatment to room-temperature control (Fig. 1) .

demonstrated a mean of 3.95 ±0.20 log 10 copies/mL and E-gene 4.77 ±0.23 log 10 copies/mL across 12 reagent lot numbers. ORF1a demonstrated a mean of 3.88 ±0.12 log 10 copies/mL and E-gene 4.63 ±0.21 log 10 copies/mL for the positive sample group tested with the quantitative standards (lot number G18524).

The 

We implemented thermal treatment of patient samples to improve laboratory safety and reduce additional PPE use [8] . Preliminary results with a limited number of positive samples (n=34) showed increased C t values for thermal treatment compared to roomtemperature despite a potential improvement in the qualitative detection of SARS-CoV-2 for thermally treated samples. Our aim was to provide clarity using a greater number of positive samples (n=238) and include more negative control samples to verify that thermal treatment does not cause nonspecific results. We sought to improve this assessment by measuring ORF1a and E-gene target copy number with a commercial quantitative standard. We also assessed thermal treatment compared to room temperature over time to obtain a better understanding of the stability of the ORF1a and E-gene targets with storage at ambient temperature.

A direct comparison of thermal treatment and room-temperature using undiluted original patient material would have been ideal. However, following initial routine testing and our SARS-CoV-2 surveillance testing, the residual sample volume was limited to conduct parallel re-testing with sufficient sample remaining for future research. To overcome this we diluted all positive samples in our collection with a pooled oro-nasopharyngeal matrix derived from SARS-CoV-2-negative patient samples to maximise the number of positive samples from different patients in this study. The closest representation to an original sample was maintained with this approach.

We found no significant differences in the qualitative outcomes (detected, presumptive or negative) for thermally treated samples compared to room-temperature samples in this 11 study. In the previous study [8] we observed additional positive results (n=3) and presumptive results (n=3) for thermal treatment compared to room-temperature (17.6%; 6/34 samples in the positive group), compared to only one additional positive result for thermal treatment (0.4%; 1/238 samples in the positive group) in this study. In the previous study we performed 10-fold serial dilutions (n=34) for each patient sample (n=8) compared to a single dilution for each patient this study (n=238). Although assay sensitivity is best shown by testing replicates at the lower limit of detection, we were limited due to costs and reagents to perform similar 10-fold serial dilutions for all positive samples. Nevertheless, the current study included sufficient samples of low concentration where the qualitative results were not different overall. We conclude with this larger study that there are no significant qualitative differences between thermally treated and room-temperature samples. Although not validated by the manufacturer, there was no evidence that thermal treatment leads to non-specificity.

Other investigators using a digital droplet PCR method have demonstrated a median drop in SARS-CoV-2 copy number of 50% to 66% after heating samples (n=63) at different SARS-CoV-2 concentrations for 80C for 20 minutes [11] . Whilst digital droplet PCR methods are useful for the sensitive detection and quantification of SARS-CoV-2 [12] , they are not practical for routine diagnostic SARS-CoV-2 detection as digital droplet PCR machines lack the necessary throughput required for front-line testing [13] . The cobas 6800/8800 instrument is a sample-to-result platform widely used for molecular diagnostics and SARS-CoV-2 was added in response to the pandemic with recent reports of strong correlation with other assays and utility emerging [14] . The exceptional test performance of quantitative cobas 6800/8800 assays (using an internal quantitation 12 standard) for blood-borne virus testing is well known [15] [16] [17] . We sought to utilise the platform as a quantitative assay using an external standard as a reference. To our knowledge a quantitative cobas SARS-COV-2 method has not been published. To assess the loss of target copy number following thermal treatment we developed standard curves for the quantitation of ORF1a and E-gene copy number for cobas SARS-CoV-2 test. The

ORF1a and E-gene standard curves were linear (r 2 >0.99) and the cobas SARS-CoV-2 test was shown to be a reproducible assay in our laboratory using a commercially available quality control. We demonstrated statistically significant reduction in the median target copy number for ORF1a (p 0.03) and E-gene (p<0.01) after heating samples for 75C for 15 minutes. However in contrast, the median and mean difference was quantitatively small for both targets (< 5%) and is unlikely to be clinically significant. We conclude the negligible loss in ORF1a and E-gene target copy number following thermal treatment is increase of three C t values when treated at 80C for 30 minutes [19] . In comparison, we demonstrated mean C t value increases of +0.13 for ORF1a and +0.31 for E-gene 13 following thermal treatment at 75C for 15 minutes when tested with a commercial SARS-CoV-2 assay, using a large number of positive samples (key point of difference).

We also assess the effects of thermal treatment of negative controls (nonspecific results were not observed).

We routinely thermally treat samples and leave the aliquots overnight at 4°C to avoid delays in processing the next day and to maximise throughput. Investigations past 48 hours were not performed as our laboratory has an expected test-turnaround-time of <24 hours from collection. Initially we speculated that thermal treatment may inactivate nucleases and preserve the sample over extended periods of time. To investigate we performed quantitative measurement of ORF1a and E-gene targets comparing thermal treatment and room temperature over time intervals. No reduction in ORF1a or E-gene copy number was observed over a 48-hour duration. This is an important finding for remote collection where refrigeration may not be immediately available and transport to the testing laboratory may be delayed. Delays in testing may also occur due to overwhelming workload. We conclude that SARS-CoV-2 RNA targets remain stable in VTM over the 6-12 hour time delays that may be encountered due to workload.

Investigations with other media and manufacturers are ongoing with similar results (data not shown). We recommend other laboratories conduct their own in-house evaluation of the media locally available for cobas or other SARS-CoV-2 testing.

Due to safety concerns at the time and suboptimal recovery of SARS-CoV-2 from culture, our laboratory did not confirm the inactivation efficacy of 75C for 15 minutes.

However, we implemented a thermal treatment method that exceeds the temperature and time duration from a previously published method of 70C for 5 min for complete SARS- 14 CoV-2 inactivation in virus transport medium [4] . Subsequently, a report from the Institut Pasteur (France) has shown that SARS-CoV-2 is relatively sensitive to heat inactivation using a dry heating block and can be inactivated in less than 30 minutes, 15 minutes and 3 minutes at 56°C, 65°C and 95°C respectively [18] . In our laboratory, thermal treatment before cobas SARS-CoV-2 testing is a simple precautionary method to improve safety when transferring un-capped samples to the cobas 6800 instrument, which does not affect the qualitative detection SARS-CoV-2 with this assay. The use of additional PPE has also been reduced. Writing -Reviewing and Editing. Fig. 1 Correlation between log 10 copies/mL obtained by cobas SARS-CoV-2 following thermal treatment for ORF1a (target 1) and E-gene (target 2) compared to cobas SARS-CoV-2 room-temperature control. Linear regression was performed using samples positive for SARS-CoV-2 for both thermal treatment and room-temperature control for ORF1a (n=180) and E-gene (n=201). The r 2 correlation is indicated. R² cobas SARS-CoV-2 ORF1a correlation Fig. 1 Correlation in log 10 copies/mL obtained by cobas SARS-CoV-2 following thermal treatment for ORF1a (target 1) and E-gene (target 2) compared to cobas SARS-CoV-2 room-temperature control. Linear regression was performed using samples positive for SARS-CoV-2 for both thermal treatment and roomtemperature control for ORF1a (n=180) and E-gene (n=201). The r 2 correlation is indicated. cobas SARS-CoV-2 E-gene correlation

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The authors are grateful to all laboratory personnel that work in the Department of Clinical Microbiology, for their dedication, diligence and perseverance.

The authors have provided raw data in the supplementary file submitted.

All authors have no conflict of interest.

This study did not receive funding support.

The authors declare that they have no competing interests. 15