key: cord-301079-n1nytr6k
authors: Tan, Li; Ma, Boyi; Lai, Xiaoquan; Han, Lefei; Cao, Peihua; Zhang, Junji; Fu, Jianguo; Zhou, Qian; Wei, Shiqing; Wang, Zhenling; Peng, Weijun; Yang, Lin; Zhang, Xinping
title: Air and surface contamination by SARS-CoV-2 virus in a tertiary hospital in Wuhan, China
date: 2020-07-27
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.07.027
sha: 
doc_id: 301079
cord_uid: n1nytr6k

Abstract Background Few studies have explored the air and surface contamination by SARS-CoV-2 virus in healthcare settings. Methods We collected air and surface samples from the isolation wards and intensive care units designated for COVID-19 patients. The clinical data and tests result of nasopharyngeal specimens and serum antibodies were also collected from the sampling patients. Results A total of 367 air and surface swabbing samples were collected from the patient care areas of 15 mild and 9 severe/critical COVID-19 patients. Only one air sample taken during the intubation procedure tested positive. High-touch surfaces were slightly more likely contaminated by the RNA of the SARS-CoV-2 virus than low-touch surfaces. Contamination rates was slightly higher near severe/critical patients compared to those near mild patients, although not statistically significant (p <0.05). Surface contamination was still found near the patients with both positive IgG and IgM. Conclusions Air and surface contamination of the viral RNA was relatively low in healthcare settings after enhancement of infection prevention and control. Environmental contamination could still be found near seroconverted patients, suggesting the needs of maintaining constant vigilance in healthcare settings to reduce healthcare associated infection during the COVID-19 pandemic.

Since its first emergence in December 2019 in Wuhan city, China, the novel coronavirus SARS-CoV-2, has spread to over 200 countries and regions within five months (Phelan et al., 2020) .

As of 30 June 2020, the total cases of COVID-19 infection have reached over 10 million globally, and the death toll were nearly 500,000 (2020). Similar to two previous coronaviruses, SARS-CoV (Ip et al., 2004) and MERS-CoV (Hunter et al., 2016) , this newly emerged virus has caused outbreaks in healthcare settings (Chan et al., 2020) . The fast spread of COVID-19 infection J o u r n a l P r e -p r o o f could have been facilitated by transmission of mild, pre-symptomatic and even asymptomatic cases (Kam et al., 2020 , Rothe et al., 2020 , suggesting that early detection of cases might be a challenge in healthcare settings. Studies have shown that viral shedding could peak soon after symptom onset (Wölfel et al., 2020) , and viral loads of asymptomatic COVID-19 patients could be as high as those of symptomatic cases (Zou et al., 2020) .

Similar to SARS-CoV, the aerosols of novel coronavirus SARS-CoV-2 could survive in air up to three hours, on plastic and stainless steel surfaces up to 72 hours in a controlled experimental environment (van Doremalen et al., 2020) . The RNA of SARS-CoV-2 has been detected in respiratory specimens, faeces, blood, and urine samples , Young et al., 2020 . Previous studies have reported that viral shedding of SARS-CoV-2 peaked soon after symptom onset, and peaked within one week (Wolfel et al., 2020) . Most patients were seroconverted within two weeks, and seropositivity of IgG appeared slightly earlier than those of IgM . The current evidence suggests that serum antibody levels might not be associated with disease severity, but it remains unclear whether viral shedding could be lower among those with both elevated IgM and IgG .

Here we collected air and surface samples from isolation wards and ICU units of a tertiary hospital in Wuhan, with the aim to evaluate environmental contamination after enhancement of infection prevention and control measures (IPC) during the COVID-19 pandemic. We also assess the association of patients' disease severity, seroconversion status and environmental contamination.

J o u r n a l P r e -p r o o f 

The whole hospital areas were classified into low-and high-risk areas with different IPC measures implemented. The latter included triage stations, fever clinics, outpatient clinics and wards of respiratory and infectious diseases, and emergency department. The rest were classified as low-risk areas. HCP including doctors, nurses, and ward assistants were required to put on a full set of personal protective equipment (PPE) when working in highrisk areas, whereas only surgical masks were required for those working in low-risk areas.

The detailed requirement for PPE can be found in Supplementary Table 1 There was no airborne infection isolation rooms (AIIR) in this hospital. To reduce the risk of airborne transmission, the central air conditioning system was turned off and natural ventilation was used in isolated wards. Windows were kept open for 30min at least twice per day, and one electronic fan was installed on the top of windows in each inpatient ward to increase ventilation. If there was no patients inside the room, ultraviolet lights (wavelength 253.7 nm, Shuangsheng Medical Ltd, SX-01A) were used to disinfect empty isolation rooms for at least one hour. In clean areas (green zone in Supplementary Figure 1 ), it was followed by 3% hydrogen peroxide spray and closed for two hours' disinfection.

Surfaces of premises and floors were disinfected twice per day using sodium hypochlorite at 1000mg/L. In the incident of spillage, sodium hypochlorite at 5000mg/L was used to disinfect soiled premise or floor. In the event of large spillage by blood, vomits and other body fluids, soiled premise or floor were immediately covered by sodium hypochlorite at 5000mg/L for 30min followed by disinfection of sodium hypochlorite at 1000mg/L.

During 14 -29 March 2020, the IPC team of the OVB hospital conducted a comprehensive investigation on environmental contamination of SARS-CoV-2 virus. Selection of patients in our study was subject to the consent given by patients, and availability of manpower. To investigate the contamination risks of aerosol generating procedure, we particularly selected at least one patient who was receiving one of the following ways of oxygen therapy at the time of sampling: oxygen supply via nasal cannula, invasive ventilation via tracheostomy, invasive ventilation via endotracheal intubation, and ECMO. Additional J o u r n a l P r e -p r o o f numbers of mild and severe patients were then recruited from different patient rooms given the availability of manpower and testing kits. A total of 24 patients, 15 in the general isolation wards and 9 in the ICU, were selected from eleven wards given the availability of manpower and testing kits. The anonymized demographic and clinical data of these patients were collected from the electronic medical records of the OVB hospital.

In the general isolation wards, three patients stayed in one room and were advised not to walk around except going to bathroom. Most severe/critical patients stayed in single rooms in the intensive care unit (ICU). The individual patient data and a floor plan of sampling sites in the ICU can be found in Supplementary file. Surface samples were taken from before daily decontamination procedures. Experienced infection control nurses who wore full PPE swabbed selected high-touch surfaces, including patients' mobile phones, bedrails, door handles, light switches, side tables, and medical instruments in patient wards, as well as low-touch surfaces including floors, chairs in the corridor. Surface sampling was conducted before the routine clean procedure. Each surface was sampled by two pre-moistened sterile cotton swabs simultaneously, both were immediately put into one tube of viral transport media (VTM, Yocon Ltd, Beijing, China). Air samples were taken by placing an air sampler within one meter of patient head, which continuously filtered air and trapped small virus particles by a membrane at the speed of 5L/min. After one hour the membrane was removed and cut into small pieces to be stored in VTM for further tests. The air sampler was placed at the same height of (or slightly lower than) an electronic fan installed on top of windows to expel the air from wards to outside. Air samples were taken from patient rooms, the corridor outside patient room, and nearby nurse stations.

Hand swabs were collected from both hands of mild patients. We did not swab the hands of severe and critical patients due to their conditions. The outer and inner layer of surgical masks worn by these patients was cut into small pieces that were immediately kept in VTM for laboratory tests. The body fluid samples of sputum and alveolar lavage fluids were also taken from some severe and critical patients, and saliva taken from additional 31 mild/moderate patients who were not sampled for environmental contamination. The nurses and doctors who were taking care of these patients were also invited to participate into this study. Infection control nurses swabbed the surfaces of their PPE, including coveralls (front and arm side), facepiece (front surface), gloves and bottom of shoe covers.

Hand swabs were also collected from some HCP before they did hand hygiene. An average of five samples were taken from each HCP. Nurses also recorded the time since donning, exposure to aerosols, and incidence of spillover, if any.

Samples stored in VTM were immediately transported on ice to the laboratory of the BGI Medical Diagnostics Company (Wuhan) for RT-PCR tests of the open reading frame (ORF) 1a/b genes of SARS-CoV-2. RNA was extracted using the QIAamp Viral RNA Mini Kit and then proceeded with the RT-PCR kit (BGI Biotechnology, Wuhan) using the SLAN® realtime PCR system by Hongshi technology (Shanghai, China). The test results were also divided by highand low-touch surfaces near mild and severe/critical patients. Blood samples were taken from the patients on the same day for tests of SARS-CoV-2 specific antibody IgM and IgG, using the kits of the Wondfo Biotech Co., Ltd (Guangzhou, China), which had sensitivity of 86.4% and specificity of 99.6% for IgG and IgM (Wondfo, 2020) . The titer higher than 10AU/ml was regarded as positive. The classification of mild, severe and critical infections J o u r n a l P r e -p r o o f followed the national diagnosis criteria (China, 2020b). Surface contamination rates were compared between the mild and severe/critical patient groups, and between seroconversion groups (IgM or IgG positive) using the Fisher exact test. The significance level was set to 0.05.

The ethical approval has been obtained from the ethics committee of the Tongji Hospital in Huazhong University of Science and Technology.

A total of 355 surface swabbing samples were collected from low-/high-touch surfaces near patients, hands and masks of patients, PPE of HCP while taking care of these patients. The detailed sampling sites are listed in Supplementary Table 2. One sample was found positive for SARS-CoV-2 in low-touch surfaces and 9 in high-touch surfaces. High-touch surfaces near severe/critical patients had a slightly higher contamination rate than those near mild patients (5.7% versus 2.4%, Table 1 (Table 1) .

Of 40 environmental samples from fever clinics and ICU common areas (corridor and nursing stations), none tested positive. All of 20 swabs of door handle and keyboards and 17 air samples in clean areas also tested negative. We collected twelve air samples from patient rooms, with one near the air exhaust fan on the window and the rest within one meter of patients' head. Only one sample was positive for SARS-CoV-2, which was collected within 10cm of a female patient while undergoing endotracheal intubation for invasive mechanical ventilation. One sample of cooling water from ventilator circuits was positive;

suggesting regular thorough clean is needed for ventilator. Two of nine severe or critical patients with sputum and saliva tested positive, and one saliva sample of 31 mild/moderate patients tested positive. All three also had SARS-CoV-2 detected in their throat samples on the same day.

None of 36 surgical masks from 18 patients (14 mild and 4 severe/critical) had the RNA of SARS-CoV-2 detected, though some patients have worn the same mask for 24 hours. For swabs from gloves, gowns, facepieces and bottom of boot covers worn by HCP inside dirty areas, all of 54 samples tested negative. None of the hand swabs from HCP were tested positive.

In this study we collected a large number of surface swabs from various sites of isolation wards and ICU after enhanced standard and transmission-based precautions. We also compared environmental contamination of low-and high-touch surfaces, patient hands and PPE of HCP, and the results were also linked to clinical data of sampling patients. A small J o u r n a l P r e -p r o o f proportion of samples (2.8%) were positive for SARS-CoV-2 in RT-PCR, which was much lower than those reported in an emergency field hospital in Wuhan, China . The reasons could be the stringent IPC measures adopted in the OVB hospital.

Nevertheless, a slightly higher contamination rate was observed in high-touch surfaces than in low-touch surfaces, suggesting that environment decontamination shall focus more on these high-touch surfaces.

We observed that severe/critical patients were slightly more likely to contaminate their surroundings, as compared to mild ones. Most of these patients were 20 days after symptom onset, and 10 out of 24 patients (41.7%) still tested positive for SARS-CoV-2 using throat swabs on the day of sampling. The serology tests prior to or on the sampling date showed that nearly all were seroconverted (23/24 IgG > 10Au/mL, 18/24 IgM > 10Au/mL).

This echoes the findings of a recent study in Germany (Wölfel et al., 2020) , which found that viral shedding continued after seroconversion. A recent study reported the RNA of SARS-CoV-2 virus could be detected in faces as long as 47 days (Wu Y. et al., 2020) . Unfortunately, we could not find whether viruses detected on surfaces were still viable, due to the lack of laboratory capacity for viral culture and quantitative PCR. Therefore, it is unclear whether environmental contamination was correlated with viral loads of patients.

The RNA of SARS-CoV-2 virus could be detected in saliva and sputum of three patients (one severe/critical patient had both saliva and sputum positive), which is consistent with the previous reports (Pan et al., 2020) . To our surprise, none of surgical masks worn by patients had positive results. Another study found only 1 out of 14 surgical masks worn by mild and severe COVID-19 patients tested positive for SARS-CoV-2 . This low positive rate is not statistically different from ours. We speculate the reason of negative results J o u r n a l P r e -p r o o f could also be due to low virus titers from these patients, as most of them were 30-40 days after symptom onset when sampling. Laboratory studies showed that SARS-CoV-2 virus titers peaked 5-6 days post symptom onset and decreased to an undetectable level 8-10 days post symptom onset in most patients (He et al., 2020 , Pan et al., 2020 , Wölfel et al., 2020 . Several studies have reported a longer period of detecting RNA by RT-PCR in biological samples (particularly feces), compared to detecting viable viruses by viral culture (Wölfel et al., 2020 , Wu Yongjian et al., 2020 . In this study, only one patient had diarrhoea, but none of the samples from the patient were positive, including five samples from bathroom surfaces.

A study by Liu et al collected air samples from different areas in one tertiary hospital and one Fangcang shelter hospital in Wuhan, the latter of which served as quarantine centers for mild COVID-19 cases with limited medication treatment . They detected the RNA of SARS-CoV-2 at low concentrations in the Fangcang hospital, but not in the patient rooms of the tertiary hospital . Another study in the AIIR of a tertiary hospital in Singapore also did not detect any virus in air samples (Ong et al., 2020a) .

Similarly, in our study, only one air sample that was collected near patient during the endotracheal intubation procedure had SARS-CoV-2 detected. No virus was detected in additional 17 air samples from clean areas (staff offices), although isolation wards were not under negative pressure. Our findings could support that natural ventilation together with extra air exhaust fans could efficiently reduce virus aerosols in patient rooms. It is of note that five surface swabs from the front side of facepiece and gloves of HCP who conducted aerosol-generating procedures (AGP) for this were all negative. This highlights the importance of wearing proper PPE in AGP. Although we collected a large number of surface swabs from different parts of PPE, including the bottom of boot covers, none were positive in PT-PCR. Another study also found a high contamination rate in three swabs from shoe sole of HCP, but none of the samples from other parts of PPE were positive . Interestingly, studies in Singapore detected the SARS-CoV-2 virus in surface swabs of front of shoes worn by HCP, but not in other parts of PPE as well (Ong et al., 2020a , Ong et al., 2020b . The transmission risk from HCP to patients appears low, since none of the PPE samples (except shoes) in these studies was found positive. But more frequent floor disinfection might still be necessary to further reduce the transmission risk in healthcare settings.

Two patients were found with hand contamination of the SARS-CoV-2 virus, which highlights the importance of hand hygiene education for patients. We placed one bottle of alcoholbased hand rub (ABHR) near each ward entrance, and taught patients how to properly wash or rub their hands when they were admitted. If resources allow, ideally each patient should have one bottle near their bedside. None of the HCP were found to have their hands contaminated, which could be due to regular audits on hand hygiene compliance by the IPC team.

There are several limitations in our study. First, this is a single center observational study; therefore, the result and protocol might not be generalized to other healthcare facilities, especially those with limited resources. Second, although we collected a large number of samples, the number of patients recruited was relatively small. As the result, statistical power might not be enough for comparison across patient groups. Third, the sensitivity and specificity of RT-PCR tests on surface contamination samples might not be same as those from human specimens. Hence, false negative and positive results might have occurred in our samples. Last but not least, it is unclear whether the virus was still viable on surfaces, since we did not culture the positive specimens.

Environmental contamination of the SARS-CoV-2 viral RNA could be found even in seroconverted patients in healthcare settings, and the contamination risk was higher in high-touch areas near severe/critical patients. Enhanced standard and transmission-based precautions should be maintained during the entire COVID-19 pandemic period, to minimize the infection risk of HCP. 

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Total surface included: low-touch surfaces, high-touch surfaces, hands of patients, masks, ventilator circuit and PPE of HCP. Fisher exact test p-value = 0.577 between mild patients and severe/critical patients; p-value = 0.358 between patients with both antibodies positive (IgM+ / IgG+) and only IgG positive (IgM-/ IgG+)

LT, BM, XL, LY and XZ originated and designed the study. JZ, JF, QZ, SW, ZW and WP contributed to sample collection. LT, BM, XL contributed to data entry and clean. LH and PC