key: cord-350236-7fit8q1s authors: Wan, Bin; Zhang, Xinlian; Luo, Dongxia; Zhang, Tong; Chen, Xi; Yao, Yuhan; Zhao, Xia; Lei, Limei; Liu, Chunmei; Zhao, Wang; Zhou, Lin; Ge, Yuqing; Mao, Hongju; Liu, Sixiu; Chen, Jianmin; Cheng, Xunjia; Zhao, Jianlong; Sui, Guodong title: On-site analysis of COVID-19 on the surfaces in wards date: 2020-08-18 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2020.141758 sha: doc_id: 350236 cord_uid: 7fit8q1s Abstract SARS-Cov-2 has erupted across the globe, and confirmed cases of COVID-19 pose a high infection risk. Infected patients typically receive their treatment in specific isolation wards, where they are confined for at least 14 days. The virus may contaminate any surface of the room, especially frequently touched surfaces. Therefore, surface contamination in wards should be monitored for disease control and hygiene purposes. Herein, surface contamination in the ward was detected on-site using an RNA extraction-free rapid method. The whole detection process, from surface sample collection to readout of the detection results, was finished within 45 min. The nucleic acid extraction-free method requires minimal labor. More importantly, the tests were performed on-site and the results were obtained almost in real-time. The test confirmed that 31 patients contaminated seven individual sites. Among the sampled surfaces, the electrocardiogram fingertip presented a 72.7% positive rate, indicating that this surface is an important hygiene site. Meanwhile, the bedrails showed the highest correlation with other surfaces, so should be detected daily. Another surface with high contamination risk was the door handle in the bathroom. To our knowledge, we present the first on-site analysis of COVID-19 surface contamination in wards. The results and applied technique provide a potential further reference for disease control and hygiene suggestions. In early 2020, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) spread across the globe, causing more than 20 million infections and 700,000 deaths as of Aug 13, 2020 (Dong et al., 2020) Coronavirus can spread through the air and survives on various surfaces for considerable periods. On June 18, Beijing reported new incidents related to the Xinfadi Market cluster (Owen, 2020) COVID-19 was discovered on the surface of chopping boards used for imported salmon at the Xinfadi food market, providing that COVID-19 could survive on material surfaces. Previous researches reported that the COVID-19 virus can inhabit the surface of materials in wards. (Guo et al., 2020; These researchers confirmed the virus by real time reverse transcription polymerase chain reaction (RT-PCR), which typically performs deactivation, nucleic acid extraction, and RT-PCR amplification of the collected samples. However, nucleic acid extraction risks nucleic acid losses and places high demands on the detection limit. Furthermore, the whole nucleic-acid extraction and amplification process requires approximately 2.5-4 hours for one batch of detection. Therefore, a rapid detection method should be applied J o u r n a l P r e -p r o o f Journal Pre-proof for on-site COVID-19 identification in the environment. Loop-mediated isothermal amplification (LAMP) has achieved brilliant performance in pathogenic virus detection, accomplishing amplification within 45 minutes (Liu et al., 2018) . LAMP assay also performs nucleic acid amplification without requiring nucleic acid extraction (Lalli et al., 2020) , thus preventing RNA damage through a tedious process. For these reasons, we applied LAMP in our present report on surface-contamination detection. Patients with confirmed COVID-19 are retained for over two weeks in rooms with many living and medical apparatuses. Nosocomial transmission plays a major role in viral spread and infection, especially in wards. Confirmed patients living in the ward can spread viruses through coughing or even shortness of breath (Ghinai et al., 2020) . This experiment aimed to determine the concentration of surface contaminants in wards of the Chengdu Center of Disease Control (Chengdu CDC), which has been designated for the treatment of COVID-19 patients during the disease outbreak. Samples were collected from seven sites: 1) bedrail; 2) bedside cupboard; 3) chairs; 4) door handles of the bathroom; 5) light switches; 6) remote controller or beeper; 7) fingertip of electrocardiograph (ECG) monitoring. The samples were collected on 18th, 19th and 24th Mar, 27th and 28th Apr of 2020. The sampling site was illustrated in Figure 1 (a). The correspondences between patient clinical information and collected samples were listed in Table S The surface contamination samples were collected as described Index A of the Hygienic Standard for Disinfection in Hospital (Chinese National Standard, GB 15892-2012). Briefly, a 5 cm × 5 cm standard scale board was placed on the surface of sampling material, and the surface was evenly rubbed within the 25-cm 2 area with a cotton swab wetted with 0.9% sodium chloride. After swiping, the cotton swab was immersed in 1 mL 0.9% sodium chloride solution prepared for nucleic-acid amplification detection. The collected surface-contamination samples were immediately transferred to the BSL-2 laboratory next to the Chengdu CDC wards for analysis. Viral contamination was detected by a nucleic acid extraction-free isothermal detection kit, specifically, a novel coronavirus real-time isothermal amplification kit (Cat.No. PCSYHE), acquired from Shanghai Fosun Long March Medical Science Co., Ltd and certificated by 7 The detection process was performed following by manufacture's instructions. For each batch, we prepared sufficient reaction reagent for n tested samples plus two control samples. That is, n × 1 μL of Bst enzyme, n × 1 μL of RT II enzyme, and n × 18 μL of COVID-19 gene reaction reagent were added to a centrifuge tube, mixed by shaking, and centrifuged at low speed for a few seconds. After separation, 20 μL aliquots were pipetted into PCR reaction tubes. The reaction tubes could be placed at 2~8 °C for 3 hours at most after separation. Next, 5 μL of the samples were added to different PCR reaction tubes. A 5-μL Negative Control and 5 μL-Positive Control were added to the control wells. The five positive samples from the handles were 100% associated with positive results from the ECG fingertips positive results. Four positive cases from the handles were also associated with positive results from bedrails, and three cases were associated with positive results from cupboards and light switches. Therefore, a surface contamination sample from the door handle can be interpreted as an ultra-high risk label. In daily monitoring, the bathroom door handles should be swabbed and analyzed. When the samples from a door handle report positive results, the corresponding ward poses an enhanced hygienic challenge. Three of the collected samples (samples #0324-27, #0324-11, and #0427-L5) presented on six contaminated surfaces. All of these confirmed cases came from J o u r n a l P r e -p r o o f outside mainland China and presented positive symptoms. On a 1-4 scale of clinical severity, where type 1 denotes mild cases and type 4 represents the severest cases, sample #0324-27 was classified as type 2, and samples #0324-11 and #0427-L5 were classified as type 3. The clinical symptoms might influence the contamination degree and infection risk. Further research on these relationships is ongoing. We successfully applied an extraction-free SARS-Cov-2 isothermal amplification detection method to on-site analysis of surface contamination by COVID-19 patients in wards. For each confirmed case, seven sites in the ward were collected and analyzed. The detection process is efficient and labor-saving, as desired for on-site COVID-19 contamination detection. Among 31 cases collected from 18 March to 27 April of 2020, 72.7% reported positive amplifications on the ECG fingertip, indicating that this surface is an important hygiene site. The correlation results also confirmed that bedrails should be regularly monitored, as contamination on bedrail surfaces is relevant to many other contaminated surfaces. Although the analysis can be performed by standard real-time PCR instruments, simpler isothermal amplification fluorescent instruments are suggested for on-site analysis, as they are less expensive, smaller in size, and more easily transported than standard PCR instruments. In future works, we will analyze the relationship between different clinical symptoms and surface contamination, which may reveal the transmission mode of COVID-19 in wards. validated the method. Yuqing Ge, Hongju Mao, Sixiu Liu, Jianmin Chen, Xunjia Cheng, Jianlong Zhao, and Guodong Sui: conceived the idea, coordinated the project, and wrote the manuscript. -First report about on-site detection of SARS-CoV-2 in wards. -The whole detection process could accomplish within 45 minutes without nucleic acid extraction. -The presence of SARS-CoV-2 in wards was confirmed by nucleic acid isothermal amplification. J o u r n a l P r e -p r o o f Prevention and Control of Major Infectious Diseases such as AIDS and Viral Hepatitis" (2018ZX10732401-003-016); the Science and Technology Commission of Shanghai Municipality (Nos. 17JC1401000, 19441903700 and 18DZ1113000) ，the National Natural Science Foundation of China An interactive web-based dashboard to track COVID-19 in real time First known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the USA Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric A sample-to-answer labdisc platform integrated novel membrane-resistance valves for detection of highly pathogenic avian influenza viruses Covid-19: WHO raises concerns about new cases in Beijing Environmental virus surveillance in the isolation ward of COVID-19 SARS-CoV-2 RNA detection of hospital isolation wards hygiene monitoring during the Coronavirus Disease 2019 outbreak in a Chinese hospital The authors declare no competing interests.