key: cord-0843597-xt0tpkae authors: Summer, S.; Schmidt, R.; Herdina, A. N.; Krickl, I.; Madner, J.; Greiner, G.; Mayer, F.; Perkmann-Nagele, N.; Strassl, R. title: High stability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA under minimal storage conditions for detection by Real-Time PCR date: 2020-07-25 journal: nan DOI: 10.1101/2020.07.21.20158154 sha: cfa2cbe3d916c575d069aa319a960d3f0ddf9c03 doc_id: 843597 cord_uid: xt0tpkae Reliable diagnosis, executed by Real-Time PCR (RT-PCR), builds the current basis in SARS-CoV-2 containment. Transport and storage conditions are the main indicators determining the quality of respiratory specimens. According to shortages in commercially available viral transport media, the primary aim of this study was to explore the reliability of minimal transport media including saline and CDC Viral Transport Media (HBSS VTM) composition for SARS-CoV-2 diagnosis by Real-time PCR compared to recommended commercially available standard Universal Transport Media (UTM). This study also implicated the stability of other respiratory viruses, including influenza A, respiratory syncytial virus, adenovirus, rhinovirus and human metapneumovirus, providing further evidence for future recommendations on transport and storage of respiratory viruses. Both viral transport media (self-made HBSS VTM and UTM) and saline (0.9% NaCl) allow adequate detection of SARS-CoV-2 and other respiratory viruses, regardless of an increase in storage temperature (up to 28 {degrees}C) and time (over 28 days). Treatment of SARS-CoV-2 specimens with varying chlorine concentrations, commonly used in swimming pools, resulted in a significant decrease of viral RNA. Detection of respiratory viruses including SARS-CoV-2 depends on the quality of respiratory specimens, predominantly determined by transport and storage conditions. Our study revealed the high resilience of SARS-CoV-2 and other respiratory viruses enabling proper detection in clinical specimens even after longtime storage at high temperatures. This study provides evidence for future recommendations for transport and storage of respiratory viruses, including SARS- Treatment of SARS-CoV-2-positive respiratory specimens with chlorine indicates an early degradation of the virus after the addition of the oxidant, proposing sufficient inactivation of the virus in swimming pool water. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint The rapid spread of the novel coronavirus SARS-CoV-2, probably of zoonotic origin (1) , has led to a continuing COVID-19 pandemic (2) . Extensive laboratory testing for SARS-CoV-2 is currently among the most effective measures to curtail the spread of COVID-19, together with quarantine and social distancing measures (3, 4) . Real-time PCR (RT-PCR), typically performed from upper respiratory specimens, represents the current gold standard for SARS-CoV-2 detection. The World Health Organization (WHO) recommends cooled storage (2-8°C) and the transport of respiratory specimens in viral transport medium (VTM) up to 5 days (3) . Viral transport medium exists in several formulas, all consisting of a buffered salt solution, a complex source of protein and/or amino acids, and antimicrobial agents and can be bought ready-touse or prepared at specific sites (5) . The COVID-19 pandemic has severely challenged worldwide supplies of commercial viral transport media as well as commercial swab kits and reagents for SARS-CoV-2 RT-PCR (5, 6) . Due to the increased demand and the shortage in supply, alternative minimal transport buffers such as 0.9% saline solution have to be used. As the pandemic also affects countries with limited infrastructural facilities and as there is only limited knowledge on the stability of SARS-CoV-2, the primary aim of the present study was to evaluate the stability and detectability of SARS-CoV-2 RNA in clinical samples stored in HBSS VTM (prepared according to CDC) (6) and in 0.9% NaCl at three different temperatures and compare those to commercially available universal transport media (UTM). Additionally, this study aimed to provide evidence on storage and transport conditions of other respiratory viruses, including influenza A, respiratory syncytial virus, adenovirus, rhinovirus and human metapneumovirus, for future recommendations. Finally, the study investigated the effect of defined All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint chlorine concentrations, commonly used in out and indoor swimming pools (7, 8) , on the stability of SARS-CoV-2. After one freeze-thaw cycle 12 anonymized SARS-CoV-2-positive samples and 3 anonymized SARS-CoV-2-negative respiratory swab samples were diluted 50-fold in 700 µl self-made viral transport medium (HBSS VTM, HBSS buffer based recipe after CDC recommendations: 500 ml HANKS Balanced Salt Solution HBSS (Gibco), 2% FBS (Gibco), 100 µg/ml Gentamycin (B. Braun Melsungen AG), 0.5 µg/ml Amphotericin B (Cheplapharm)) (6) and regular saline (0.9% NaCl; B. Braun Melsungen AG), respectively. Following the initial measurement (T0) the samples were stored at different temperatures (4 °C, 21 °C and 28 °C) for up to 28 days. Total All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint 6 RNA was extracted after 4 (T4), 7 (T7), 14 (T14) and 28 (T28) days. pH values were determined, which served as a surrogate parameter for bacterial contamination. For comparison of HBSS VTM and saline, respectively, with UTM, 12 anonymized, undiluted SARS-CoV-2-positive samples, 6 in UTM (Universal Transport Medium, Cepheid, Copan Diagnostics) and 6 in saline, were stored at 21 °C after the initial measurement (T0). RNA was extracted after 7 (T7) and 14 (T14) days. The presence of other respiratory viruses used in this study, Influenza virus A, Adenovirus, respiratory syncytial virus, rhinovirus and human metapneumovirus, were confirmed by TaqMan-based RT-PCR using the LightCycler Multiplex RNA All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. seconds at 40 °C. Primers and probes (9) (10) (11) (12) (13) are listed in Supplementary Table S1 . The threshold was set manually and Cq-values were exported from CFX Manager Dx Software version 3.1. Version 5.0 (GraphPad). In most cases, a two-tailed repeated measurement ANOVA was performed with adjustments for multiple comparisons following the Bonferroni post-hoc test. In one case, when data was not normal, a Wilcoxon signed-rank test was done. Significant differences (p<0.05) were marked by a * above the corresponding curves in the figures. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. To evaluate the stability of the SARS-CoV-2 RNA for diagnostics assessed by RT-PCR paired identical swab specimens of 15 positively-tested SARS-CoV-2 patients were stored in viral transport medium (HBSS VTM, self-made) and saline (0.9% NaCl) over 28 days at different temperatures (4, 21 and 28 °C). Additionally, 12 swab specimens, 6 transported in UTM and 6 in saline, were stored at 21°C for 14 days for comparing self-made HBSS VTM and saline, respectively, to commercial UTM. The specimens. Besides, we also included 3 negative specimens as control. A comparison of the changes in the Cq-values of specimens transported in VTM and saline over 28 days did not show any significant differences between the two All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint transport media ( Figure 1 ). Besides, no significant differences between SARS-CoV-2 specimens stored in self-made HBSS VTM and commercially available UTM at 21 °C were observed either (mean However, the stability of SARS-CoV-2 + samples kept at 4 °C and 28 °C varied significantly from SARS-CoV-2 ++ (* 4 °C p E=0.0008, p S=<0.0001; 28 °C p All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint E=0.0049, p S=0.0001), however, no significant changes in the Cq-values could be observed for the specimens stored at 21 °C (p E=0.73, p S=0.60). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint specimens ( Figure 5 ). Comparable kinetics were observed for samples with high and low viral load ( Figure 5 ). Specimens stored in saline showed a more substantial susceptibility to chlorine. Treatment with 3 ppm chlorine of samples stored in saline leads to an almost complete RNA decay, whereas those stored initially in UTM medium showed a moderate viral RNA reduction. The COVID19 pandemic has re-emphasized the importance of molecular diagnostics for the acquisition of timely results aiding in the containment of the pathogen. In the case of the SARS-CoV-2 virus previous studies have shown that the virus, in contrast to other respiratory viruses like influenza, is not susceptible to heat, supporting the assumption that the prevalence of SARS-CoV-2 will not be seasonal (14, 15) . Future demand for testing in the summer season at higher temperatures required a study determining the stability of the virus under these conditions. High outdoor temperatures combined with a long transport time of respiratory specimens could affect the virus detection capabilities of molecular methods such as RT-PCR. This is especially the case in countries with poor infrastructure where rapid, cooled transport and the availability of commercial transport media for respiratory specimens cannot be guaranteed. In the present study we evaluated the stability of SARS-CoV-2, besides other respiratory viruses, for diagnostic analysis by RT-PCR at temperatures up to 28 °C, considering possible effects mediated by the transport medium. Using SARS-CoV-2 positively tested clinical specimens, we demonstrated the remarkable stability of the viral RNA upon long-time storage (up to 28 days) at high temperatures (21 and 28 °C), independent of the transport medium (Figure 1 and Supplementary Figure S1 ). As previously shown, we were able to confirm this in our study for other respiratory viruses as well (16) . Our observations are in line with previous reports about All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint SARS-CoV-2 stability (17) (18) (19) (20) . Storage in HBSS VTM and saline were equally suitable for adequate detection of the two target genes E (betacoronavirus-specific) and S (SARS-CoV-2-specific) in specimens characterized by a low and high viral load, respectively ( Figure 4) . However, at higher temperatures samples stored in HBSS VTM proved to be more susceptible to bacterial contamination, even when supplemented with antibiotics. Nevertheless, bacterial contamination has neither affected sample preparation nor target detection. The findings that saline as transport medium provides stable long-time storage of the clinical samples lacking significant changes in the detection levels of the target genes, not even in case of higher temperatures, could be advantageous to increase future testing capacities. In regards to the minimal alterations in the detected RNA signal, SARS-CoV-2 is highly resilient to high temperatures. Its detection by RT-PCR is sufficiently robust for the diagnosis of COVID-19, even in seasons or countries characterized by higher outdoor temperatures. Furthermore, the study evaluated the susceptibility of the virus to chlorine. As the shutdown ends for many countries in Europe swimming pools start to open for the summer. Based on a one case report of possible transmission of SARS-CoV-2 in a public bathing house (21) , transmission in rehabilitation pools and therapeutic water environments should be considered as well (22) . Other coronaviruses have been shown to be highly susceptible to chlorination (23) . To our knowledge, this is the first study using clinical samples to test SARS-CoV-2 RNA stability in the presence of chlorine in typical swimming pool concentrations. Expectedly, our data support that SARS-CoV-2 is not stable in water containing 1.5 and 3 ppm chlorine, respectively ( Figure 5) . We determined the detectability of the E and S gene after the treatment of SARS-CoV-2-positive specimens with different chlorine concentrations. We observed All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint 13 that short incubation times are sufficient to decrease the detection of the virus. Even though SARS-CoV-2 has been previously shown and confirmed in this study to be highly resistant against changes in environmental conditions (14, 24) , treatment with chlorine, leading to a change in pH, has a dramatic effect on the virus. SARS-CoV-2 is an enveloped virus with a fragile outer membrane (25, 26) and thus more susceptible to oxidants, such as chlorine, which leads to the breakdown of the viral capsid, hence, viral RNA degradation, as previously also shown for other viruses (27) . Our data support that SARS-CoV-2 is inactivated significantly fast by chlorine treatment. Thus, chlorine could not only be used as a disinfectant against SARS-CoV-2 and could also prevent its transmission in swimming pool water. In summary, we have shown that the survival ability of SARS-CoV-2 in human specimens seems to be considered stable, even compared to other coronaviruses ( Figure 1 and Supplementary Figure S1 ) (28) . Increasing temperatures and long-term storage conditions did not affect its stability. We also demonstrated the efficacy of oxidants, such as chlorine, on virus elimination. Besides, our data confirmed that saline and self-made transport media (HBSS VTM) provide useful alternatives to the commercially available UTM that can be deployed for transporting, even in countries with high outdoor temperatures and long-term preservation of SARS-CoV-2 specimens. Thus, an increased capacity of testing for widespread screening of SARS-CoV-2 and early diagnosis of COVID-19 can be achieved. We thank Karin Mildner for excellent technical assistance in RT-PCR detection of other respiratory viruses. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint A pneumonia outbreak associated with a new coronavirus of probable bat origin An interactive web-based dashboard to track COVID-19 in real time World Health Organization (WHO). 2020. Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases: interim guidance No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted doi: medRxiv preprint 11. Fry AM The Burden of Hospitalized Lower Respiratory Tract Infection Real-time reverse transcription-PCR assay for comprehensive detection of human rhinoviruses Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages Stability of SARS-CoV-2 in different environmental conditions Stability of SARS coronavirus in human specimens and environment and its sensitivity to heating and UV irradiation Detection of influenza by real time RT-PCR is not affected by delays in respiratory specimen processing Stability of SARS-CoV-2 in PBS for Molecular Detection Detection of Severe Acute Respiratory Syndrome Coronavirus 2 The authors have nothing to disclose. No external funding was received for this study.All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 25, 2020. . https://doi.org/10.1101/2020.07.21.20158154 doi: medRxiv preprint