key: cord-0682765-y33yhvfr authors: Gehrke, S. G.; Foerderer, C.; Stremmel, W. title: SARS-CoV-2 Airborne Surveillance Using Non-Powered Cold Traps date: 2021-01-26 journal: nan DOI: 10.1101/2021.01.19.21250064 sha: b28df58efa76dade34b48c4e96536feaa0f9e8d6 doc_id: 682765 cord_uid: y33yhvfr Background: COVID-19 pandemic is a worldwide challenge requiring efficient containment strategies. High-throughput SARS-CoV-2 testing and legal restrictions are not effective in order to get the current outbreak under control. Emerging SARS-CoV-2 variants with a higher transmissibility require efficient strategies for early detection and surveillance. Methods: SARS-CoV-2 RNA levels were determined by quantitative RT-PCR in aerosols collected by non-powered cold traps. SARS-CoV-2 spreading kinetics and indoor hotspots could be identified in isolation units and at public places within a high-endemic area. These included an outpatient endoscopy facility, a concert hall, and a shopping mall. Results: Indoor COVID-19 hotspots were found in non-ventilated areas and in zones that are predisposed to a buoyancy (chimney) effect. SARS-CoV-2 RNA in those aerosols reached concentrations of 10^5 copies/mL. Extensive outdoor air ventilation reliably eliminated SARS-CoV-2 aerosol contamination. Conclusions: The method presented herein could predict SARS-CoV-2 indoor hotspots and may help to characterize SARS-CoV-2 spreading kinetics. Moreover, it can be used for the surveillance of emerging SARS-CoV-2 variants. Due to low costs and easy handling, the procedure might enable efficient algorithms for COVID-19 prevention and screening. is spreading around the world for more than 12 months. Since Spanish flu 100 years ago, no pandemic lead to a comparable medical and economic disaster. Only 2 weeks after the first report, a new coronavirus SARS-CoV-2 had been identified being responsible for the outbreak of first COVID-19 cases suffering from severe atypical pneumonias in Wuhan, China. 1 From that on, several diagnostic procedures came onto the market including highly sensitive quantitative RT-PCR methods for detecting SARS-CoV-2 out of oral and nasopharyngeal swabs as well as sputum or bronchoalveolar lavage (BAL). 2 Approximately 80% of infected individuals are completely asymptomatic 4 including cases with high viral load, also designated as "Superspreaders". 5 But also in COVID-19 cases with clinical manifestations, SARS-CoV-2 can be transmitted two days before first symptoms occur. 6 These circumstances clearly demonstrate why early and reliable diagnosis of COVID-19 remains a major challenge. There is growing evidence for a transmission of SARS-CoV-2 via aerosols. [7] [8] [9] However, only a few studies have been published to date. Liu et al. reported SARS-CoV-2 RNA concentrations in different areas of two hospitals in Wuhan. 10 Samples were collected on styrene filter cassettes followed by a two-step RT-PCR protocol using digital droplet PCR (ddPCR). Other studies demonstrated viable SARS-CoV-2 in single hospital rooms of infected patients 11, 12 and in isolation units 13 collected by . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 26, 2021. 14 were able to detect SARS-CoV-2 RNA in central ventilation systems distant from patient indicating that airborne SARS-CoV-2 can be transported over long distances. Therefore, long-range airborne transmission should be taken into consideration as a possible route of infection, even in emerging SARS-CoV-2 variants with increased infectivity. The current outbreak of the SARS-CoV-2 B.1.1.7 lineage in the UK 15 underlines the necessity of efficient strategies for airborne surveillance. In addition, early detection of emerging variants will be of major importance as first SARS-CoV-2 variants with an escape from neutralizing antibodies have been recently detected 16 . Positive selections among immunized individuals might drive the evolution of SARS-CoV-2 toward lineages with a partial or full resistence to current vaccination strategies. Detection of viable SARS-Cov-2 in aerosols requires both, an effective method for sample collection, and a high-sensitive amplification procedure. Herein, we describe a simple and reliable method for the quantification of SARS-CoV-2 RNA in aerosols. Such a COVID-19 airborne surveillance could have a wide range of applications including early detection and surveillance of emerging SARS-CoV-2 variants. . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.19.21250064 doi: medRxiv preprint The effects of room ventilation on SARS-CoV-2 RNA concentrations in aerosols were investigated under different conditions. Initially, measurements were performed in several rooms in two COVID-19 isolation units. The risks of viral contamination as well as the intensity of ventilation were estimated for each room. Subsequently, three locations of general interest were investigated in a high endemic area (State of Baden-Württemberg, Germany): a concert hall, an outpatient endoscopy facility, and a shopping mall. Aerosol sample collections were performed by using simple, non-powered cold traps (Aeroprotektor Twin Tower, Scientifixx, Germany). The cold trap consists of two standardized 350 mL cold packs covered by a removable stainless-steel surface. The cold packs are frozen overnight in a standard freezer (-20°C). In order to prevent any contamination, the covered cold packs are frozen and transported in sealed plastic bags. At the point of interest, the frozen and covered cold packs are vertically fixed in a metal box collecting all condensed water (1-10 mL within 4-6 hours). 200 µl of condensed water is then pipetted into a nuclease free microcentrifuge tube for further analysis. Alternatively, the collecting box can be sealed containing the thawed cold packs, fixing elements, and the condensed water for immediate transportation to a specialized laboratory. . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.19.21250064 doi: medRxiv preprint Prior to nucleic acid amplification, SARS-CoV-2 RNA was isolated from 50 µl condensed water (Viral Xpress Kit, Merck Millipore, Darmstadt, Germany) and diluted in 50 µl AE Elution Buffer (5 mM TRIS/HCl pH 8.5, Macherey-Nagel, Düren, Germany). Alternatively, condensed water was briefly centrifuged for 1 minute at 10 .000 g and pipetted directly into the qRT-PCR reaction mix. For SARS-CoV-2 quantification, 2 µl template (isolated RNA or centrifuged condensed water) was added to a 8 µl of ready-to-go CTT one-step qRT-PCR mix containing SARS-CoV . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.19.21250064 doi: medRxiv preprint Aerosol sample collection was performed for 6 hours within an isolation room and in the corridor next to the door of the isolation room. The isolation room was Within a 4.000 sqft concert hall 10 cold traps were placed around an orchestra and on audience rows 3 and 5 during a 3 hours rehearsal. In one of these . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 26, 2021. 10 cold traps we found measurable amounts of SARS-CoV-2 RNA (6.000 copies/mL). The contaminated cold trap was located on a loudspeaker next to the radiator at the outer wall of the concert hall. Between October 2020 and January 2021, aerosols were randomly collected by cold traps over a period of 6 hours in 2 endoscopy rooms, at the patient reception, and in a doctor´s room. In the first phase of the experiment, 12 measurements were performed. Significant amounts of SARS-CoV-2 RNA were detected in 6 collected aerosol samples. The highest SARS-CoV-2 concentration (12.000 copies/mL) was found in an endoscopy operation room, although the central ventilation system exclusively supplies outdoor fresh air avoiding any recirculation. In the second phase of the experiment, additional fresh air ventilation was initiated. As often as possible a window was on tilt in each room. Despite the additional fresh air ventilation, 4 out of 7 follow-up aerosol collections remained positive for SARS-CoV-2, but RNA concentrations were on a low level (< 2.000 copies/mL). Finally, additional fresh outdoor air ventilation was further intensified by completely opening the window between each patient in both endoscopy operation rooms. Follow-up measurements did not reveal any detectable SARS-CoV-2 RNA in the collected aerosols. . CC-BY-NC-ND 4.0 International license It is made available under a 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.19.21250064 doi: medRxiv preprint On a Friday 14 cold traps were placed in a shopping mall from 10 am to 3 pm. Points of interest were highly frequented areas (escalators, kiosk, checkout, and fitting room at the ground level; 3 checkouts on the 2 nd floor; refrigerated section, checkout as well as meat and fish counter on the 3 rd floor), a non-ventilated store at the ground level, and 2 staff areas (break room and administrative department). Out of these 14 cold traps, 5 contained significant levels of SARS-CoV-2 RNA. Surprisingly, the highest SARS-CoV-2 concentration (5.400 copies/mL) was found on the ground level between the escalators. The cold trap at the kiosk located next to the escalators was also positive for SARS-CoV-2 (2.000 copies/mL). Moreover, SARS-CoV-2 RNA was found in 1 out of 3 checkout areas on the 2 nd floor as well as on the 3 rd floor next to the fish counter. Both cold traps contained 2.000 copies/mL SARS-CoV-2 RNA and were located near to an inflow of the central 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.19.21250064 doi: medRxiv preprint on a Monday, we found only half of SARS-CoV-2 RNA in collected aerosols compared to Friday before (2.700 copies/mL). Finally, a fashion store located on 4 floors was tested for SARS-CoV-2 RNA aerosol contamination on a Friday. Compared to the shopping mall, customer frequency within the fashion store is generally less than 25%. Cold traps were placed at areas with a high customer frequency (checkout areas, escalator, fitting rooms) or less ventilated zones. However, none of the nine cold traps contained any significant amounts of SARS-CoV-2 RNA. The role of SARS-CoV-2 airborne spreading in the current pandemic is still under debate, although there is growing evidence that susceptible individuals might be infected by this route of transmission [7] [8] [9] 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 January 26, 2021. ; https://doi.org/10.1101/2021.01.19.21250064 doi: medRxiv preprint 12 identification of infected groups of people as well as an early detection and surveillance of emerging SARS-CoV-2 variants even at public places. However, collecting viable SARS-CoV-2 aerosols is a rather sophisticated process requiring saturation of small virus particles prior to condensation. The BioSpot-VIVAS TM Bioaerosol Sampler has been recently demonstrated as an effective tool collecting viable SARS-CoV-2 by encapsulating airborne particles into liquid droplets followed by a deposition onto a liquid surface 11 . The method presented herein is comparably simple but the underlying mechanisms of SARS-CoV-2 collection is yet unknown. However, saturation of small virus particles into droplets capable for condensation most probably also occur. Within the first hour of the collection period, the cold traps do not contain any SARS-CoV-2 particles (data not shown). Subsequent SARS-CoV-2 aerosol saturation and condensation obviously need 2-3 hours mediated by ambient temperatures and wet surfaces which are maintained by thawing of the cold packs. Although this method needs further optimization, it can be readily used for SARS-CoV-2 airborne surveillance. The procedure is easy to handle, cost efficient and suitable to be used worldwide even in emerging and developing countries. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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CC-BY-NC-ND 4.0 International license It is made available under a 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 January 26, 2021. 1-3 Negative probes - 1 Aeroprotektor is a registered trademark of Scientifixx Corp., Gaggenau, Germany 2 ++ = contamination > 2 hours, + = contamination 1-2 hours, -= contamination < 1 hour 3 ++ = permanent ventilation with 1-2 windows on tilt, + = intermittent ventilation , -= no ventilation