Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have seen shortages of diagnostic reagents, consumables and personal protective equipment [1,2]. Initially we inactivated samples with guanidine hydrochloride (GuHCl) before SARS-CoV-2 testing [2,3]. Following implementation of cobas SARS-CoV-2 testing (Roche, Basel, Switzerland), we encountered shortages of GuHCl and personal protective equipment. To overcome these issues, we investigated a number of rapid heat treatment steps before cobas testing. Temperatures and durations investigated were based on reports demonstrating the effects of temperature on the viability of SARS-CoV-2 (70°C for 5 minutes) [4] and other coronaviruses [[5], [6], [7]].
To test the effects of thermal treatment on cobas assay performance, we tenfold serially diluted cobas-positive clinical samples collected in Copan UTMRT media (Brescia, Italy) from different patients (n = 8) using cobas-negative nasopharyngeal matrix and thermally treated incrementally at 60°C, 65°C and 75°C (UTM internal temperature) for a total time of 15 minutes, 30 minutes and 60 minutes at each temperature point. Aliquots were prepared in cobas omni secondary tubes (ref. 06438776001) and were thermally treated in a Dri-Bath. An aliquot of each dilution remained untreated (room-temperature control), and cobas testing was performed in parallel for all samples (n = 34). We also prospectively tested 40 consecutive patient samples comparing 75°C for 15 minutes to room temperature (Table 1 and Supplementary Material).
Table 1.
Summary of cobas SARS-CoV-2 results in 74 samples
| Result outcome | No. of samples | cobas SARS-CoV-2 result for: |
|
|---|---|---|---|
| Heat treatment | Room-temperature control | ||
| 1 | 22 | Detected | Detected |
| 2 | 3 | Detected | Negative |
| 3 | 3 | Presumptive positive | Negative |
| 4 | 1 | Presumptive positive | Presumptive positive |
| 5 | 45 | Negative | Negative |
Detected indicates ORF1a positive, E-gene positive; presumptive positive, ORF1a negative, E-gene positive.
SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
All samples were heated immediately before extraction and were loaded without delay (<5 minutes). We recorded the cycle threshold (C t) for ORF1a, E-gene and internal control. All C t values are shown in the Supplementary Material, and the qualitative results are shown in Table 1. Positive C t values in both ORF1a and E-gene for thermally treated and room-temperature control were compared by the two-tailed paired t test (p < 0.05). The same statistical approach was applied for all internal control C t values. All C t values were normally distributed (D'Agostino-Pearson test). For thermal treated compared to room-temperature control, we found no significant difference in C t values for ORF1a (22 samples compared, mean difference +0.13 ± 0.89 SD, p 0.502). However, a significant difference was observed for E-gene (21 samples compared, mean difference +0.55 ± 1.15 SD, p 0.040) and internal control (all 74 samples compared, −0.27 ± 0.40 SD, p 0.00001). In summary, the mean ORF1a and E-gene C t values were 0.13 and 0.55 C t higher for heat treatment than control respectively.
Higher C t values for thermally treated samples may suggest a reduction in detectable virus RNA. A recent study using a commercial qualitative method (BioGerm Medical Biotechnology, Shanghai, China) and quantitative digital PCR (TargetingOne, Beijing, China) demonstrated a drop in SARS-CoV-2 copy number by 50% to 66% after heating at 80°C for 20 minutes [8]. However, internal control test performance was not evaluated (or not included as part of the assay), and correlation with other commercial in vitro diagnostic assays or cobas is not known. Despite marginally higher cobas C t values for thermally treated samples in our study, 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 (Table 1). Although we observed C t shifts in the cobas E-gene target, the cobas assay is dual target, and therefore the delay in one target may not be critical to the qualitative detection of SARS-CoV-2. Of 34 dilutions prepared, SARS-CoV-2 was detected in three thermally treated samples (ORF1a ± E-gene) and three were presumptively positive for SARS-CoV-2 (E-gene only), all of which were negative for the room-temperature control.
On the basis of these results, heat treatment may improve the qualitative detection of SARS-CoV-2. To confirm our qualitative findings, many replicates at the lower limit of detection combined with probit analysis are required. As a result of safety concerns and suboptimal recovery of SARS-CoV-2 from culture, our laboratory did not confirm the inactivation efficacy of thermal treatment. However, using standard biosafety level 2 (BSL2) laboratory safety procedures, we continue to use the highest temperature assessed with a time that suits the work flow (75°C for 15 minutes), thereby exceeding a previously published temperature and duration of 70°C for 5 minutes for complete SARS-CoV-2 inactivation in virus transport medium [4].
We acknowledge that thermal inactivation of SARS-CoV-2 may not be 100% efficient [[4], [5], [6], [7]]. However, we consider the risk to staff in a well-equipped BSL2 laboratory is greatly reduced with thermal pretreatment. Moreover, thermal treatment negates the need for GuHCl and enables the redirection of personal protective equipment to frontline personnel.
Transparency declaration
Funding was provided by PathWest. All authors report no conflicts of interest relevant to this article.
Acknowledgements
The authors are grateful to Adrian Bautista (Acting Chief Executive of PathWest at the commencement of COVID-19 testing) for provisioning the cobas kits.
Editor: F. Allerberger
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.cmi.2020.07.042.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
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