key: cord-0960360-caaozv93
authors: Meister, Toni Luise; Dreismeier, Marielen; Blanco, Elena Vidal; Brüggemann, Yannick; Heinen, Natalie; Kampf, Günter; Todt, Daniel; Nguyen, Huu Phuc; Steinmann, Jörg; Schmidt, Wolfgang Ekkehard; Steinmann, Eike; Quast, Daniel Robert; Pfaender, Stephanie
title: Low risk of SARS-CoV-2 transmission by fomites – a clinical observational study in highly infectious COVID-19 patients
date: 2022-05-05
journal: J Infect Dis
DOI: 10.1093/infdis/jiac170
sha: ce024b99ead64a06535f1aa800d7115c507d47aa
doc_id: 960360
cord_uid: caaozv93

BACKGROUND: The contribution of droplet-contaminated surfaces for virus transmission has been discussed controversially in the context of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic. Importantly, the risk of fomite-based transmission has not been systematically addressed. Therefore, the aim of this study was to evaluate if confirmed hospitalized COVID-19 patients can contaminate stainless steel carriers by coughing or intensive moistening with saliva and to assess the risk of SARS-CoV-2 transmission upon detection of viral loads and infectious virus in cell culture. METHODS: We initiated a single-center observational study including fifteen COVID-19 patients with a high baseline viral load (CT value ≤ 25). We documented clinical and laboratory parameters and used patient samples to perform virus culture, quantitative PCR and virus sequencing. RESULTS: Nasopharyngeal and oropharyngeal swabs of all patients were positive for viral RNA on the day of the study. Infectious SARS-CoV-2 could be isolated from 6 patient swabs (46.2 %). While after coughing, no infectious virus could be recovered, intensive moistening with saliva resulted in successful viral recovery from steel carriers of 5 patients (38.5 %). CONCLUSIONS: Transmission of infectious SARS-CoV-2 via fomites is possible upon extensive moistening, but unlikely to occur in real-life scenarios and from droplet-contaminated fomites.

The emergence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent 2 of the Coronavirus Disease-19 (COVID-19), has raised the general awareness towards different hygiene 3 and prevention measures to limit viral spread. Although SARS-CoV-2 is mainly transmitted via 4 respiratory droplets and aerosols exhaled from infected individuals (e.g., upon breathing, speaking, 5 coughing or sneezing [1]), droplet-contaminated surfaces (fomites) have also been widely perceived as 6 another potential route of transmission. In particular, different studies reported that SARS-CoV-2 can 7 persist on inanimate surfaces for days under controlled laboratory conditions [2-4] and genomic material 8 of SARS-CoV-2 has been detected on diverse surfaces and materials in hospital, private and public 9 settings [5] . Consequently, a clinically significant risk of SARS-CoV-2 transmission by fomites has been 10 assumed and extensive hand hygiene and disinfection procedures have been initiated early during the 11 pandemic worldwide. Although recent studies suggest a low risk of viral transmission by fomites for most 12 instances [1,6], it is still considered possible given a timely order of events (e.g., direct contamination of a 13 surface by an infected individual followed by timely skin contact by another individual and direct contact 14 towards susceptible mucosae) [7] . However, most efforts to study surface transmission of SARS-CoV-2 15 have either focused on the detection of viral RNA via RT-qPCR rather than direct detection of infectious 16 viral particles and/or employed lab-grown viruses which do not recapitulate the specific infectivity of 17 patient-derived SARS-CoV-2 particles. Consequently, these findings do not necessarily allow to 18 adequately estimate the potential of SARS-CoV-2 transmission from directly contaminated surfaces. 19

To examine the risk of transmission by surfaces directly after contamination by individuals infected with 20 SARS-CoV-2, we performed a clinical observational study, including hospitalized patients with high viral 21 loads (CT ≤ 25, up to 2.03×10 9 RNA copies). The aim of this study was to evaluate if confirmed 22

hospitalized COVID-19 patients can contaminate stainless steel carriers by coughing or intensive 23 moistening with saliva and to assess the risk of SARS-CoV-2 transmission upon detection of viral loads 24 and infectious virus in cell culture. 25

Hospitalized patients (age > 18 years) treated at St. Josef-Hospital Bochum, Germany, with confirmed 3 SARS-CoV-2 infection with a high virus load (RT-PCR from combined nasopharyngeal and 4 oropharyngeal swab (swab) with a cycle threshold (CT) ≤ 25 on admission) were included in this study. 5

The initial viral load was determined using Allplex™ 2019-nCoV Assay (Seegene Inc., Seoul, Republic of 6 Korea) targeting three SARS-CoV-2 specific genes (E gene, RdRP gene and N gene) with a sensitivity of 7 100 copies per run. Exclusion criteria were acute myocardial infarction, current need of ventilation 8 support systems (e.g., high flow-or non-invasive ventilation), current treatment in an intensive care unit, 9 evidence of drug or alcohol abuse, acute psychiatric disorders and any clinical or mental disorder that 10 might deteriorate the patient's condition during the standardized procedure of sampling (such as 11 dysphagia), as per investigator's judgement. Collected clinical data included medical history, current daily 12 medication, laboratory results, blood gas analysis, and results of x-rays or computed tomography (to 13 define the "clinical classification of COVID-19-infection" following the recommendation of the World 14

Health Organization (WHO) [9] and Robert Koch Institute (RKI) [10]). 15

After written informed consent, two combined nasopharyngeal and oropharyngeal swabs were collected 17 from each patient. Then, patients were asked to forcefully cough two-times on a pre-defined surface area 18 containing nine standardized steel-carriers, each with a one-centimeter diameter ("cough"), using a 19 specially designed tripod with a defined distance of 15 centimeters (Appendix Figure 1 ). In addition, 20

patients were asked to moisten nine steel carriers with saliva for ten seconds within their mouth 21 ("moisten"). After defined time points at room temperature (1 min, 5 min, 15 min, 30 min, 45 min, 90 22 min, 120 min and 240 min), the steel-carriers were placed in containers containing 2 mL cold Dulbecco's 23 modified Eagle's medium (DMEM complete, supplemented with 10 % (v/v) fetal calf serum, 1 % non-24 essential amino acids, 100 IU/mL penicillin, 100 µg/mL streptomycin and 2 mM L-Glutamine) and 25

transported on ice to the biosafety level three laboratory of the Ruhr-University Bochum. The study was 1 conducted between November 2020 and April 2021. VeroE6 cells were seeded at 3×10 5 cells/well in a six well cell culture plate and incubated for at least four 5 hours at 37 °C and 5 % CO 2 . Hereafter, the medium was replaced with 1.8 mL of patient swabs, "cough" 6 samples or "moisten" samples and 2.5 µg/mL amphotericin B was added. Over a maximum period of ten 7 days, cells were monitored daily for the appearance of a cytopathic effect (CPE), indicating productive 8 virus infection. Upon visible CPE, cells were harvested for RT-qPCR and the supernatant (SN) was 9

collected for viral titration and RT-qPCR. Viral titers in the SN were quantified by endpoint-dilution and 10 the 50 % tissue culture infective dose (TCID 50 /mL), calculated according to Spearman and Kärber [11] . 11

SARS-CoV-2 RNA was isolated from the supernatant using AVL buffer and the QIAamp Viral RNA Kit 13 (QIAGEN®, www.qiagen.com) according to the manufacturer's instructions. RNA was directly subjected 14 to one-step quantitative PCR (RT-qPCR) running a GoTaq Probe 1-Step RT-qPCR System (Promega®, 15 www.promega.com). Total RNA was purified from VeroE6 cells using the RNeasy Mini Kit (QIAGEN®, 16 www.qiagen.com). Subsequently, 500 ng of total RNA were reverse transcribed using the PrimeScript™ 17 RT Master Mix (Takara®, www.takarabio.com) and subjected to two-step RT-qPCR running a GoTaq 18

Probe 2-Step RT-qPCR System (Promega®, www.promega.com). RT-qPCR was performed as described 19

previously [12] using a light cycler LC480 to quantify the M-Gene abundance. 20

Clinical patient parameters are expressed as mean ± SD or n (% of total). Results are expressed as means 22

(± SEM). Clinical characteristics were screened for correlations using Spearman's correlation coefficient. 23

Statistical significance was defined as α=0.05. Statistical analysis was performed using GraphPad Prism 24 version 8.0.0 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com). 25

Sample size calculation was performed using G*Power Version 3.1.9.6 for windows [13] . The study was conducted according to the revised principles of the Declaration of Helsinki and was 2 approved by the ethics committee of the Ruhr-University Bochum (registration number 20-7065) in 3

November 2020. All patients gave written informed consent. 4

Sequencing and strain assignment 5 RNA of the initial swaps was isolated using the NucleoSpin RNA kit (Macherey & Nagel) followed by a 6 reverse transcription utilizing the SuperScript IV together with Oligo dT and random hexamer primer 7 (Thermo Fisher) according to the manufacturers' instructions. Subsequently, the cDNA was subjected to 8 deep sequencing. Sequencing libraries were prepared from 4.5µl cDNA using NEBNext® ARTIC SARS-9

CoV-2 Library Prep Kit for Illumina sequencing platforms (New England BioLabs® Inc., neb.com, 10 catalog #E7650). Concentration and size of the cDNA amplicons and libraries were assessed using Qubit 11 fluorometer and Tapestation (High Sensitivity D1000 ScreenTape), respectively. High throughput paired 12 end sequencing was performed using Illumina MiSeq sequencer and MiSeq Reagent Kit v2 (500-cycles) 13

following the manufacturer's recommendations. Raw reads were quality checked, trimmed and mapped to 14 the SARS-CoV-2 reference sequence (NCBI Reference Sequence: NC_045512) using QIAGEN CLC 15

Genomics Workbench 21.0.5. After removing duplicates, partially full length consensus sequences were 16 extracted and samples were assigned to respective lineages using the pangolin tool [14] ( 

factors: 1 (6.7 %)). Body temperature on admission was 37.0 (±0.9) °C. On the study day, 9 (60 %) 1 patients were categorized with a mild COVID-19 disease according to STACOB criteria (adaptation 2 following the WHO Therapeutics and COVID-19:living guideline [9]). Nasal oxygen support was 3 required by seven (46.7 %) patients. Mean peripheral oxygen saturation on admission was 93.4 % (SEM ± 4 7.4 %). None of the included patients were vaccinated against SARS-CoV-2. 5

On the day of the study, most patients had only mild symptoms (n= 9/15, 60 %) and were categorized to 6 mild COVID-19 disease. Follow-up revealed a clinical worsening in 10 patients and 3 died (2 patients 7 died of COVID-19, 1 patient died with COVID-19). Consistent with literature [16] , a high level of lactate 8 dehydrogenase (r=0.53, p=0.044), leucocytes (r=0.69, p=0.0056), but also C-reactive protein (r=0.54, 9

p=0.035) on admission was significantly correlated with a more severe COVID-19 infection (Appendix 10 Figure 2 ). No significant correlation was found for COVID-19 severeness and other described severeness 11

predictors including age (r=0.44, p=0.11), alanine aminotransferase (r=0.31, p=0.26), aspartate 12 aminotransferase (r=0.07, p=0.81), procalcitonin (r=0.53, p=0.47), or D-dimers (r=0.45, p=0.11). 13

The patients' viral load before sample acquisition as determined with the combined nasopharyngeal and 15 oropharyngeal swabs are displayed in Table 2 . Mean CT-values in RT-qPCR analyses were E-Gene 15.3 16 (SEM ± 2.7), S-Gene 18.1 (SEM ± 4.9) RdRP-Gene 17.2 (SEM ± 3.7) and N-Gene 19.4 (SEM ± 5.5). 17 Viral variants included supposed wildtype (n=10, 66.7 %), variant of concern (VoC) Alpha (n=4, 26.7 %) Table 2 ). 22

Two patient samples were excluded from the study due to bacterial/fungal contamination within the 23 cultures (Appendix Table 1 ). Viral RNA could be detected from all (n=13/13) combined nasopharyngeal 24 and oropharyngeal swabs (inoculum) and viral RNA could be successfully detected within the inoculated 25 cell cultures (Figure 2, Appendix Figure 3 ). After inoculation with the "swabs", viral loads in the cells 26

ranged from 2.23×10 1 to 2.03×10 9 RNA copies/50 ng and in the supernatant from not detectable to 1 6.58×10 7 RNA copies/mL (Appendix Table 1 ). Infectious virus ("Infectivity"), determined as TCID 50 /mL, 2 could be recovered from the nasal-oropharyngeal swabs from n=6/13 (46.2 %) patients (Figure 2 ; P1, P2, 3 P3, P5, P8, P10). Of note, despite inclusion criteria defining a high viral load (CT ≤ 25, Table 2 contribute to the spread of other common respiratory pathogens [18, 19] , including experimental studies 1 examining the transfer of infectious influenza viruses and/or respiratory syncytial virus between hands and 2 surfaces [20] . However, current evidence points towards a low risk of SARS-CoV-2 transmission in this 3 scenario [1,21,22], requiring a timely order of specific events [7] . To examine this potential risk of SARS-4

CoV-2 surface transmission, we assessed the amount of SARS-CoV-2 genomic material and infectious 5 viral particles after contamination by individuals infected with SARS-CoV-2 over time. The results of the 6 present study highlight that viral contamination via coughing on surfaces does not represent a major risk 7 of transmission. 8

Our study cohort was characterized by non-vaccinated, hospitalized, mostly elderly patients with multiple 9

comorbidities. Since the vaccination program in Germany started in December 2020 (during the study 10 period), none of the patients were vaccinated against SARS-CoV-2. This cohort may therefore be quite 11

representative for hospitalized patients during the first and second COVID-19 wave in most countries 12 consequently, follow-up mortality in the present study. However, the present cohort is characterized by 20 high viral load. Hence, a high transmission rate can be assumed [26], supporting the main conclusion of 21 the study. Of note, several laboratory parameters on admission significantly correlated with a severe 22 outcome. However, the present study was not designed for this analysis and therefore, these correlations 23 need to be considered exploratory. 24

Infectious virus could be recovered from the combined nasopharyngeal and oropharyngeal swabs and 25 steel-carriers contaminated via intensive "moistening" from a significant number of patients. For some 26

patients, infectious virus could be recovered for up to 240 min (Figure 2 ). This demonstrates that 1 infectious virus can be transferred from saliva by moistening onto surfaces from patients and can be 2 recovered for several hours. As described previously, we did not observe differences of the viral stability 3 between the wildtype and VoCs (Alpha and Beta), implying a comparable environmental stability [27] . 4

The stability of SARS-CoV-2 on surfaces is likely determined by a combination of factors, including the 5 initial amount of infectious virus deposited, possible presence of antibodies within the sputum and 6 environmental parameters. Given the controlled laboratory conditions for virus recovery as herein 7

presented (e.g., large inoculums, small surface area, no UV exposure), the viral survival observed might 8 therefore differ from real-life scenarios, necessitating careful interpretation. For example, a recent study 9 observed a low transfer efficiency between different surfaces and fingertips following an initial drying of 10 an inoculum with a low viral titer (1×10 4 TCID 50 /mL) [22] . Hence, even if sufficient viable virus is 11 deposited on a surface, a timely contact and high transfer efficiency are required to transfer an infectious 12 dose, which subsequently needs to be exposed towards susceptible tissues (e.g., mucosa, eyes). 13 Importantly, we did not observe the recovery of infectious virus after patients coughed onto a surface, 14

implying that droplet-contamination of surfaces does not present a major transmission route for SARS-15

CoV-2. Given that non-hospitalized and pre-symptomatic, asymptomatic, and mildly symptomatic Dowell and coworkers [8] took smears from hospitalized SARS patients, placed those smears on surfaces, 20 then assayed for viral RNA and infectious virus. While they did find viral RNA, no infectious virus was 21 recovered from the surfaces, which is in line with the results obtained in this study. 22

Our study encompasses several limitations. Patients were encouraged to forcefully cough twice to 23 contaminate surfaces. However, we cannot exclude that potentially repeated coughing over a prolonged 24 time results in a more effective virus transfer compared to our controlled conditions. Moreover, sneezing 25 can produce significantly more infectious droplets potentially containing infectious particles, therefore, we 26 The present study provides evidence that fomites may not be as critical in the transmission of SARS-CoV-8 2 as initially suspected. However, the present study also provides evidence that infectious SARS-CoV-2 9

can be found on some fomites for a relatively brief period of time after contamination with extensive 10 amounts of saliva. Therefore, common hygiene practices (e.g., coughing/sneezing into elbows, hand 11 hygiene) should still be considered to avoid surface contamination and virus transfer. Face masks may 12 further mitigate the risk of fomite transmission. Collectively, our findings suggest that fomites 13 contaminated with coughing are unlikely to be an important source of SARS-CoV-2 transmission. 14

Ethics approval and consent to participate 16

The study was conducted according to the revised principles of the Declaration of Helsinki and was 17 approved by the ethics committee of the Ruhr-University Bochum (registration number 20-7065) in 18

November 2020. All patients gave written informed consent. 19

The study is published with patients consent. 21 22

The datasets used and/or analyzed during the current study are available from the corresponding author on 24 reasonable request. . CT = cycle threshold resulting from RT-PCR performed at St. Josef-Hospital Bochum. VoC = variants of concern. *VoC-Analyses started in the midst of the study period in February 2020 and was performed via melting curve analysis. # no sufficient material available. bold: characteristic mutations associated with VOC according to RKI [17] . with the patient material and monitored on a daily basis. Upon the emergence of cytopathic effects, the 10 supernatant was collected to determine viral loads by RT-qPCR (RNA copies/mL; indicated by 1 ) and 11 viral titers by an endpoint-dilution assay (TCID 50 /mL, indicated by 3 ). Additionally, RNA was isolated 12 from the cells and subjected to RT-qPCR to determine viral loads (RNA copies/50 ng total RNA, 13 indicated by 2 ). For each patient (P1-P10), three panels were designed. The top small panel includes 14 exclusively data regarding the patient swabs, while the larger middle panel shows the data for the 15 "moisten" samples and the lower panel the data collected from the "cough" samples. For "moisten" 16 and "cough" samples viral loads and infectivity at nine different time points were determined. The 17 color indicates the amount of virus being detectable in each sample, with light grey being the lower 18 limit of detection to dark blue resulting in 10 10 RNA copies/mL, RNA copies/50 ng or TCID 50 /mL. 19

The visible CPE was rated two dimensionally, with light grey being "no visible CPE" and dark green 20 being "visible CPE". 

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A realistic transfer method reveals low risk of SARS-CoV-2 15 transmission via contaminated euro coins and banknotes

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Deliberation, Dissent, and Distrust: Understanding distinct 19 drivers of COVID-19 vaccine hesitancy in the United States

Outcomes Among Patients with Breakthrough 21 SARS-CoV-2 Infection After Vaccination