key: cord-294069-7zr77r71
authors: Hu, Xiaowen; Ni, Wei; Wang, Zhaoguo; Ma, Guangren; Pan, Bei; Dong, Liyan; Gao, Ruqin; Jiang, Fachun
title: The distribution of SARS-CoV-2 contamination on the environmental surfaces during incubation period of COVID-19 patients
date: 2020-09-30
journal: Ecotoxicol Environ Saf
DOI: 10.1016/j.ecoenv.2020.111438
sha: 
doc_id: 294069
cord_uid: 7zr77r71

Roles of environmental factors in transmission of COVID-19 have been highlighted. In this study, we sampled the high-touch environmental surfaces in the quarantine room, aiming to detect the distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the environmental surfaces during the incubation period of coronavirus disease 2019 (COVID-19) patients. Fifteen sites were sampled from the quarantine room, distributing in the functional areas such as bedroom, bathroom and living room. All environmental surface samples were collected with sterile polyester-tipped applicator pre-moistened in viral transport medium and tested for SARS-CoV-2. Overall, 34.1% of samples were detected positively for SARS-CoV-2. The positive rates of Patient A, B and C, were 46.2%, 0 and 61.5%, respectively. SARS-CoV-2 was detected positively in bedroom and bathroom, with the positive rate of 50.0% and 46.7%, respectively. In contrast, living room had no positive sample detected. Environmental contamination of SARS-CoV-2 distributes widely during the incubation period of CCOVID-19, and the positive rates of SARS-CoV-2 on environmental surfaces are relatively high in bathroom and bedroom.

Since a novel human coronavirus, named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected in Wuhan, China, in late 2019, the coronavirus disease 2019 caused by this virus has been reported drastically all over the world. As of May 25, 2020, about 5,408,301 confirmed COVID-19 cases including 345,064 deaths have been reported in 188 countries (JHU, 2020) . So far, compared with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV), a relatively low fatality (about 7%) has been summarized from SARS-CoV-2 infected cases, however, it is obvious that SARS-CoV-2 is much more contagious as evidenced by its spread to 188 countries across the globe within a very short time.

Roles of environmental factors in transmission of COVID-19 have been highlighted before. Several impressive published works on environmental roles revealed the potential risk of environmental factors on the transmission and prevalence of COVID-19 pandemics, such as climate change, fomites, water transfer, air and food (Eslami & Jalili, 2020; Qu et al., 2020; Wigginton & Boehm, 2020) . Kampf et al. emphasized that inanimate surface contact is an important way of transmitting the SARS-CoV-2 (Kampf et al., 2020) . SARS-CoV-2 can survive on surfaces for hours to days, which can live on inanimate surfaces such as metals, glass and plastic at room temperature increasing the opportunity for transmission via touch (Kampf et al., 2020; van Doremalen et al., 2020) . Eslami and Jalili concluded that reducing the frequency of touching surfaces by hands and disinfecting surfaces can J o u r n a l P r e -p r o o f reduce the amount of coronavirus load on surfaces and the rate of transmission (Eslami & Jalili, 2020) . Although transmission from contaminated surfaces and fomites are emphasized as an indirect transmission route of SARS-CoV-2, detection of the virus on the environmental surfaces is largely unknown.

From March 18 to 24, 2020, three Chinese oversea students returned to Qingdao, China. After tested negative nucleic acid without any symptoms at the entry quarantine, they were then transferred to the hotel for 14 days quarantine. During quarantine period in hotel, they presented initial symptoms or were tested positive SARS-CoV-2 as a presymptomatic person. Meanwhile, they were transferred to the local hospital for further diagnosis and treatment. In addition, we synchronously sampled the high-touch environmental surfaces in the quarantine room, aiming to detect the SARS-CoV-2 distribution on the environmental surfaces during the incubation period of COVID-19 patients. Our findings would extremely address the importance of touching the contaminated environment transmission, and help extend an effective protocol to interrupt the indirect environmental transmission of SARS-CoV-2, limit its spread, and mitigate its risks.

Three COVID-19 patients, as Chinese oversea students in America and England, returned to Qingdao from March 18 to 24, 2020. They had no fever and other symptoms at the entry quarantine, and transferred to a hotel for 14 days quarantine.

During the quarantine, their nasopharyngeal swabs were collected every one to three J o u r n a l P r e -p r o o f days. If the swab was tested negatively for SARS-CoV-2, the student would be still in quarantine in hotel, whereas if the swab was tested positively for SARS-CoV-2, or presenting initial symptoms of COVID-19, such as fever, cough, myalgia and fatigue, etc., they would be immediately transferred to the hospital for further diagnosis and treatment. Personal, clinical, and radiological characteristics onset of illness were obtained with standardized data collection forms from the interview, field reports as well as electronic medical records. Additionally, their frequency of face washing, hands washing, tooth brushing, bathing and excrement during the quarantine were interviewed by telephone after discharged from hospital.

This study was approved by the Ethics Commission of Municipal Centre of Disease Control and Prevention of Qingdao and written informed consent was waived considering the emergency of infectious disease.

Environmental surface samples were collected with sterile polyester-tipped applicator pre-moistened in viral transport medium. Fifteen sites were sampled from the quarantine room, including light switch, bathroom door knob, inner wall of toilet, towel, inner surface of washbowl, sewer inlet, floor (0.5-1.5meters from the bed), bedside table surface, pillow, sheet, duvet cover, television (TV), TV remote controller, telephone, and bay window. All sampling sites were marked in Fig. 1 . The surface of the entire item was swabbed except for pillow, sheet, and duvet cover by swabbing 10 times vigorously from 2 directions (horizontally and vertically) in an about 100cm 2 area that they contacted. All sites were sampled within 4 hours after the J o u r n a l P r e -p r o o f 6 positive nucleic acid test of the patients.

All samples after sampling were transferred to the Qingdao Municipal Center for Disease Control and Prevention for SARS-CoV-2 detection. Tests were carried out in biosafety level 2 facilities, using a commercial Novel Coronavirus Nucleic Acid Detection Kit (Shanghai BioGerm Medical Technology Company) in a total reaction volume of 25μL, targeting SARS-CoV-2 virus frame1ab (ORFab1). Viral RNA was extracted from sample material and collected in elution buffer, and then underwent real-time reverse-transcription-polymerase-chain-reaction (RT-PCR) with SARS-CoV-2-specific primers and probes. Reverse transcription was performed at 50°C for 10 min, 95°C for 5 min, followed by 40 cycles of RT-PCR analysis at 95 °C for 10 s, and annealing/elongation/fluorescence detection at 55°C for 40 s. A cycle threshold (Ct) value were used to approximately reflect the viral loads (inversely related to Ct-value) in the respiratory tract, and lower Ct values indicated higher viral loads and vice versa (Xu et al., 2020; Zhu et al., 2020) . According to the Technical

Commission the People's Republic of China (Chinese CDC, 2020), a Ct value less than 37 was defined as a positive test, and a Ct value of 40 or more was considered as a negative test. An equivocal result, defined as a Ct value between 37 to 40, required confirmation by retesting. If the repeated Ct value was less than 40 and an obvious peak was observed, or if the repeated Ct value was less than 37, the result was deemed positive.

Quality controls in SARS-CoV-2 detection was conducted as follows:

environmental surface samples were at the room temperature for no more than 2 hours before laboratory detection; viral RNA was isolated from sample material by automatic nucleic acid isolation instruments (MagNA Pure 96, Roche Diagnostics GmbH, Germany) to minimize the possibility of laboratory contamination; Novel

Coronavirus Nucleic Acid Detection Kit recommended by National Health

Commission of China was adopted, and laboratory steps were performed according to the instruction of the manufacturer. In addition, SARS-CoV-2 Medium Quality

Control and Negative Quality Control (TMNQC, RANDOX, UK) were used for RNA isolation to conduct quality controls. After comparing with stability and sensitivity of products from six manufacturers, we purchased the most precise primers from Shanghai BioGerm Medical Technology Company. According to the stability of weak positive quality control results (Synthetic SARS-CoV-2-RNA, Twist Bioscience, #102024), the effectiveness of primers was monitored in real time.

Three patients returned to Qingdao, China, during March 18 and 24, 2020, and their characteristics were shown in Table 1 . Patient A and C were onset of illness (fever, cough or fatigue) during the quarantine time. When presenting initial symptoms, they immediately reported to professional medical staffs stayed in hotel.

Then, medical staffs stayed in hotel immediately sampled their nasopharyngeal swabs and environmental surfaces, and transferred them to the hospital for further diagnosis Additionally, the frequency of washing behaviors of patients at the quarantine room, including face washing, hands washing, tooth brushing, bathing and excrement, were shown in Table 2 .

All sampling sites were marked in Fig. 1 . In total, forty-one samples were collected from the quarantine rooms of the three COVID-19 patients. As shown in Fig.   3 As shown in Fig. 1 and Table 3 , the quarantine room was divided into three functional areas. Bedroom and bathroom were tested positively for SARS-CoV-2, with the positivity rate of 50.0% (7/14) and 46.7% (7/15), respectively. In the bathroom, all the six sites were positive for RT-PCR tests, including light switch, bathroom door knob, inner wall of toilet, towel, inner surface of washbowl and sewer inlet, and the lowest Ct value was detected from inner wall of toilet. In the bedroom, all the five sites were positive for RT-PCR tests, including floor, beside table surface, pillow, sheet and duvet cover, and the lowest Ct value was detected from pillow. In contrast, living room was negative for virus, and all sites, such as TV, TV remote controller, telephone and bay window, were negative for RT-PCR test.

In addition, all environmental sites came from five types of material. As shown in Table 3 , the positive rate was highest in cotton sites (60.0%, 6/10), followed by ceramic sites (40.0%, 2/5), metal sites (40.0%, 2/5), wood sites (33.3%, 2/6) and plastic sites (16.7%, 2/12)

Although researchers enhanced the importance of environmental contamination on the route of SARS-CoV-2 transmission (Faridi et al., 2020; Guo et al., 2020; Ong et al., 2020; Yung et al., 2020; ) , there has been limit data revealing the contribution of Temperature, humidity and surface material types may be the important environmental factors for the survivability of SARS-CoV-2 on surfaces (Aboubakr et al., 2020; Biryukov et al., 2020; Ren et al., 2020) . In our study, we found that environmental samples from bedroom and bathroom were detected positively for SARS-CoV-2 with close positive rates (50.0% and 46.7%, respectively), and the average Ct value in bathroom (32) was lower than that in bedroom (35) Several limitations should be known in our study. Firstly, we did not sample any indoor air, which should be strengthened in future study. Compared with other sampling sites in the room, the frequency of touching main door knob may be relatively low. However, main door knob should be still as necessary sampling site.

Unfortunately, we did not sample it in all rooms, which should be paid for attention in our future study. Although we used central heating to describe the situation of room environment, other environmental factors, such as room temperature and humidity,

were not detected accurately in the quarantine rooms. In addition, our study only analyzed the environmental contamination in the quarantine room based on a small size of COVID-19 patients, and further research on a larger size should be performed to evaluate the contribution of environmental contamination to the transmissibility of COVID-19.

Environmental contamination of SARS-CoV-2 distributes widely during the incubation period of CVOID-19, and the positive rates of SARS-CoV-2 on environmental surfaces are relatively high in bathroom and bedroom. Our findings would extremely address the importance of touching the contaminated environment transmission, and help extend an effective protocol to interrupt the indirect environmental transmission of SARS-CoV-2, limit its spread, and mitigate its risks.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conceptualization, writing-original draft.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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We are deeply thankful to all health-care workers involved in the diagnosis and treatment of patients in Qingdao.

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: