key: cord-1036128-k98apm7u
authors: Dzung, Andreas; Cheng, Phil; Stoffel, Corinne; Tastanova, Aizhan; Turko, Patrick; Levesque, Mitchell P.; Bosshard, Philipp P.
title: Prolonged Unfrozen Storage and Repeated Freeze-Thawing of SARS-CoV-2 Patient Samples Have Minor Effects on SARS-CoV-2 Detectability by RT-PCR
date: 2021-03-26
journal: J Mol Diagn
DOI: 10.1016/j.jmoldx.2021.03.003
sha: a9d4531c2b60523a853f345fadba80e99c98c37e
doc_id: 1036128
cord_uid: k98apm7u

Reliable transportation of SARS-CoV-2 patient samples from a swabbing station to a diagnostics facility is essential for the generation of accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, the impact on SARS-CoV-2 detectability by reverse transcriptase polymerase chain reaction (RT-PCR) was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2 positive patient swabs stored in viral transport medium (VTM) were exposed to different temperatures (4°C, 25°C and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by RT-PCR after 21 days of storage in VTM, even when the samples had been stored at 35°C. The change of Ct-value per day was 0.023-0.046 (±0.018-0.019). Additionally, viral RNA was still detected after 15 freeze-thaw cycles with a change of Ct value of 0.106-0.197 (±0.009-0.061) per freeze-thaw cycle. However, compared to storage at 4°C, RNA was significantly less detectable when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels, as measured by RT-PCR assays, are significantly, but not substantially, altered by prolonged unrefrigerated storage of up to 21 days at temperatures ranging up to 35°C or after repeated freeze-thaw cycles.

SARS-CoV-2 detectability by reverse transcriptase polymerase chain reaction (RT-PCR) was 26 assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. 27 SARS-CoV-2 positive patient swabs stored in viral transport medium (VTM) were exposed to 28 different temperatures (4°C, 25°C and 35°C) and to repetitive freeze-thawing, to assess the effect 29 of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by 30 RT-PCR after 21 days of storage in VTM, even when the samples had been stored at 35°C. The 31 change of Ct-value per day was 0.023-0.046 (±0.018-0.019). Additionally, viral RNA was still 32 detected after 15 freeze-thaw cycles with a change of Ct value of 0.106-0.197 (±0.009-0.061) per 33 freeze-thaw cycle. However, compared to storage at 4°C, RNA was significantly less detectable 34 when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate 35 that viral RNA levels, as measured by RT-PCR assays, are significantly, but not substantially, 36 altered by prolonged unrefrigerated storage of up to 21 days at temperatures ranging up to 35°C 37 or after repeated freeze-thaw cycles. 38

In late 2019 a novel Coronavirus, SARS-CoV-2, emerged which caused a pandemic in 2020. 40

With a reproduction number (R0) ranging from 1.4 to 3.9 (depending on country-specific disease 41 prevention measures) 1 , its infection rate was quickly determined to be higher than the seasonal 42 influenza with an R0 of 1.27. 2 To identify infected patients, control and monitor the spread of the 43 virus, massive up-scaling of diagnostics capacities took place, for the detection of SARS-CoV-2 44 viral RNA by reverse-transcriptase polymerase chain reaction (RT-PCR Five SARS-CoV-2 positive samples from the routine diagnostics lab that were stored <30d at -71 20°C were thawed, diluted to 8 mL, and 210 µL aliquoted into cryotubes. The aliquots were then 72 incubated at 4°C, 25°C, or 35°C. Daily for the first 7d and after 10d, 14d, and 21d, respectively, 73 one tube each was collected and frozen at -80°C until further analysis. The transport of nasopharyngeal/oropharyngeal swabs to testing laboratories relies on local 151

infrastructures and thus optimal sample logistics may not always be available. While the CDC 152 official guidelines recommend transport on ice, some of the specimens might be transported 153 without cooling and may therefore be exposed to environmental temperatures. In our hospital for 154 example, specimen transport is accomplished at ambient temperatures and some specimens took 155 several hours until they arrived in our laboratory. We were wondering whether we still would 156 reliably detect SARS-CoV-2 in these cases and to what degree temperature differences 157

influenced Ct values. To answer this question, the stability of the SARS-CoV-2 virus in VTM 158 was tested over time at different temperatures and after different numbers of freeze-thaw cycles. 159

Our data show that the virus can still be reliably detected when patient swabs in VTM are 160 exposed to up to 35°C for as long as 21 days, with minor but significant changes of the Ct-values 161 per day. The VTM that was used here contained antibiotics and antimycotics. No contamination 162

i.e., overgrowth of bacteria or fungi was observed even after prolonged exposure at 35°C. 163

However, it might still be important to store samples cooled, to prevent contamination, which 164 could interfere with RNA extraction and RT-PCR. The samples used in this experiment had a 165 moderately high viral load, i.e. the Ct-values were between 20-30 cycles. We believe that the 166 high stability also accounts for samples with lower viral loads, e.g. Ct values between 30-35 167 cycles. However, it cannot be fully excluded that low positive samples with initial Ct-values 168 between 35-40 cycles might appear negative after a longer storage period at higher temperatures. J o u r n a l P r e -p r o o f 

Assessment of the SARS-CoV-2 basic reproduction number

R0, based on the early phase of COVID-19 outbreak in Italy

Transport of viral specimens

Media, and Specimen Transport Conditions for Optimal Detection of Viruses by PCR

Integrated DNA and RNA 221 extraction using magnetic beads from viral pathogens causing acute respiratory infections

Comparison of RNA extraction methods for the detection of SARS-Cov-2 by 225

A comparative study of real-time RT-PCR based 228

SARS-CoV-2 detection methods and its application to human derived and surface swabbed 229 material

Fitting Linear Mixed-Effects Models Using 231 lme4

Population Marginal Means in the Linear Model

An Alternative to Least Squares Means

False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-236