Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified from a cluster of patients with pneumonia of unknown cause in Wuhan, China, in December 2019. SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus and the seventh member of the coronavirus family that infects humans. It is distinct from both severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) . Infection with SARS-CoV-2 may result in a wide range of clinical manifestations from asymptomatic infection to critical illness. As of August 11, 2020, COVID-19 has caused a worldwide pandemic, with nearly 20 million confirmed cases and 0.74 million deaths (World Health Organization, 2020a).
SARS-CoV-2 is believed to spread efficiently, but the exact transmission routes, especially the fomite, airborne (or aerosol), and fecal-oral routes, remain under debate (World Health Organization, 2020b) . Viral RNA has been detected from various surfaces of isolation rooms and intensive care units in which COIVD-19 patients are treated, but detection from air has been relatively difficult Ong et al., 2020) . The probable explanation is either that patients released limited virus during the sampling period or that patient's exhaled breath (EB) is readily diluted by the room's ventilation before it can be sampled, which is consistent with the proximity effect in the transmission of respiratory infections (Liu et al., 2017) . Although SARS-CoV-2 may be transmitted by transmission routes similar to those of other coronaviruses, such as large droplets, aerosols, or contact, the potential of transmission via each route requires systematic investigation.
Nosocomial transmission of the virus has been reported in China: more than 3000 medical staff members have been infected during medical practice (Zhang, 2020) . According to a single-center J o u r n a l P r e -p r o o f case series involving 138 hospitalized patients in Wuhan, China, nosocomial transmission was suspected to occur via 29% of infected medical staff and 12.3% of hospitalized patients . Similarly, the transmission of virus from a single health care worker to several other patients and health care workers was assumed to be responsible for the hospital outbreak of SARS-CoV-2 in the University Hospital of Münster, German (Schwierzeck et al., 2020) . An accumulating body of evidence suggests that although COVID-19 patients are mostly infectious during the early stage of infection, the high viral loads in their sputum and feces specimens during later stages of infection may still pose a risk to healthcare workers (Zheng et al., 2020) . This hypothesis has yet to be systematically evaluated.
In this study, we aimed to investigate the EB and the hospital environmental contamination by COVID-19 patients in the later stage of infection. To that end, we sampled the EB, exhaled breath condensate (EBC), surfaces of isolation wards, and personal necessities of 21 patients with laboratory-confirmed COVID-19 and hospital drainage systems from 12 to 47 days after symptom onset and subjected the samples to SARS-CoV-2 detection by quantitative reverse transcription polymerase chain reaction (RT-qPCR).
Twenty-one patients with laboratory-confirmed COVID-19 who were hospitalized in the First Affiliated Hospitals, College of Medicine, Zhejiang University, from February 13, 2020, to March 5, 2020, were enrolled in this study. We collected samples of EB, EBC, size-segregated aerosols from the air, frequently touched surfaces, and the drainage system. Patients' sputum and feces specimens were collected daily by the hospital staff for purposes of routine diagnosis. The patients' J o u r n a l P r e -p r o o f clinical data were obtained from hospital electronic medical records. This study protocol was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University.
An exhaled aerosol collection system was developed ( Figure S1 ), and the patients were asked to breath normally through a mask for 30 min, during which they were asked to perform 10 forced coughs. The mask did not interrupt the patient's oxygen inhalation. The exhaled air was sampled by the NIOSH bioaerosol sampler, which collected air at 3.5 L/min (105-L air was sampled during the 30-min sampling) and separated virus-laden aerosols into three size fractions: <1 µm, 1-4 µm, and >4 µm (Lindsley et al., 2010) . After collection of EB samples, the NIOSH bioaerosol sampler was disassembled in a biosafety cabinet, and 1 mL of viral transport medium was added to each collection tube and PTFE filter to suspend virus particles.
The EBC samples were collected using a sterile laboratory-made EBC collection system comprising a 15-mL centrifuge tube with the bottom cut off and a 50-mL centrifuge tube ( Figure   S2 ). The patients were asked to blow into the 15-mL centrifuge tube for 10 min. Approximately 200-500 μL of EBC was collected from each patient for further analysis.
For sampling of isolation room air, a NIOSH sampler was placed on a tripod 1.2 m in height and 0.2 m away from the bed at the side of the patient's head ( Figure S3 ). The sampling duration was 30 min, and a total of 105-L room air was sampled.
Frequently touched public and private surfaces in isolation rooms were sampled, as illustrated in Figure S3 and Table S1. The standard sampling area was 10 × 10 cm 2 (swab applied in horizontal J o u r n a l P r e -p r o o f followed by vertical and diagonal sweeps). For surfaces smaller than 10 × 10 cm 2 , the entire touchable surface was swabbed. After sampling, the swabs were immediately placed into 1.5 mL of viral transport medium.
Virus-laden aerosols may be generated during flushing in the toilet and in sewage pipes, accounting for potential fecal-oral transmission of SARS-CoV-2. For lavatory bioaerosol sampling, a NIOSH sampler was placed on a tripod 1.2 m in height and 0.5 m away from the toilet bowl in the lavatory of the isolation rooms; the 30-min sampling period began when the patient used the toilet for defecation. After the patient exited the toilet, the toilet bowl and floor drain were also swabbed for sampling. In addition, water samples were taken in 15-mL centrifuge tubes from the main sewage pipe and wastewater pipe of the building in which the COVID-19 patients were isolated ( Figure S4 ).
Viral RNA was extracted from all collected clinical or environmental specimens using MagNA Pure LC 2.0 (Roche, Basel, Switzerland). RT-qPCR was performed using a China Food and Drug Administration-approved commercial kit specific for SARS-CoV-2 detection (Da'an Gene Co., Ltd., Guangzhou, Guangdong, China) according to the manufacturer's protocol. Specimens with quantification cycle (Cq) values of 40 or lower were considered positive for SARS-CoV-2 RNA. Specimens with Cq values higher than 40 were tested again, and those with repeat Cq values that were higher than 40 or undetectable were considered negative for SARS-CoV-2 RNA. The 
A total of 21 moderately to critically ill patients with COVID-19 (aged 13-72 years; median, 61 years) were recruited. Sampling was performed 12 to 47 DAO (median, 29 days). The patients' details are listed in Table 1 . All recruited patients were in the recovery period with normal body temperature and improved lung infection conditions according to computed tomography scan.
Viral loads in the sputum specimens were detected varying from negative to 1.2×10 10 copies/mL (median, 1.3×10 5 ), and those in feces specimens from negative to 1.9×10 9 (median, 7.1×10 3 ).
Of the 254 samples (9 EB samples, 8 EBC samples, 12 bedside air samples, 202 public/private surface samples from isolation rooms, and 23 feces-related air/surface/water samples), 14 tested positive, as summarized in Table 2 , suggesting the potential for nosocomial transmission of SARS-CoV-2 via multiple routes. The viral RNA concentrations were 1112 copies/m 3 and 745 copies/m 3 respectively in the <1 µm and >4 µm fractions of the only positive air sample. The maximum viral RNA concentrations were 38 copies/cm 2 in sampled private surfaces of COVID patients (excluding the toothbrush) and 3092 copies/mL in sewage/wastewater samples. These findings prioritize areas for effective environmental disinfection practices to reduce virus transmission and support the need for strict adherence to personal hygiene. However, the positive detection rates and virus contents in these positive samples were relatively low, indicating that patients release limited virus particles into the environment more than 10 DAO.
J o u r n a l P r e -p r o o f Respiratory droplets, or bioaerosols, are carriers of pathogens and are responsible for the transmission of respiratory infectious diseases. They are produced during expiratory activities such as coughing, sneezing, talking, and normal breathing. To test the potential of respiratory bioaerosol in transmitting SARS-CoV-2, 9 EB and 8 EBC specimens were collected from 15 patients between 13 and 43 DAO in the present study. The aerosol collection system in this study directly sampled patients' EB before it was highly diluted by the room air, but all samples in three size fractions tested negative for viral RNA [positive detection in sputum samples; see Table 2 ], though 10 forced coughs were performed by each patient. Two of the eight EBC samples tested positive for the virus (Patients 9 and 17), with the virus concentration as 216.0 copies/mL and 222.0 copies/mL, respectively. This detection rate was likely attributable to the high collection efficiency of both fine and coarse aerosols and the low possibility of dilution by air or viral transport medium.
Furthermore, 1 (Patient 9) of the 12 air samples from the patients' bedsides (24-43 DAO) tested positive (sputum viral loads between negative and 4.5×10 5 copies/mL); RNA was detected in two size fractions from that air sample, with virus concentrations of 1111.9 copies/m 3 and 744.6 copies/m 3 in the <1 µm and >4 µm fractions, respectively. One of the two positive EBC samples and the positive bedside air sample belonged to Patient 9; his sputum viral load values on the sampling date were 6.3×10 5 copies/mL and 3.2×10 5 copies/mL, respectively. These findings are consistent with a sampling event in two Wuhan hospitals where viral RNA in aerosols detected in isolation wards and ventilated rooms was very low . In another sampling event with NIOSH samplers, viral RNA was readily detected from the air of isolation rooms where patients were at 5 D.A.O.; the highest concentration was 1384 copies/m 3 and 2000 copies/m 3 in the 1-4 µm and >4 µm fractions, respectively, and no viral RNA was detected in <1 µm size fraction .
Although some COVID-19 patients evaluated during the later stage of infection in our study had a relatively high viral load in their sputum specimen, shedding of virus particles from the respiratory tract into the environment was limited, which is in agreement with previous findings that viral load in nasal or throat samples is high in the early stage of infection and decreases to a low level at around 9 DAO (Woelfel et al., 2020; Zou et al., 2020) . Virus-laden respiratory droplets from the deep lung tend to deposit and accumulate into the respiratory mucous, but difficult to follow the respiratory airflow to enter the indoor environment (Kleinstreuer and Zhang, 2010) . The transmission characteristics of SARS-CoV-2 differ from those of SARS because the viral loads in the respiratory specimens of SARS-infected patients peak at 10 DAO (Peiris et al., 2003) , and considerable positive rates of surface swabs have been detected in SARS patients' rooms at 5-15
DAO (Dowell et al., 2004) . However, the virus-shedding course of SARS-CoV-2 is similar to that of influenza virus (Tsang et al., 2015; Zou et al., 2020) , and the NIOSH bioaerosol sampler has also successfully detected influenza virus in samples of 32 out of 38 influenza-positive patients, 60% of whom had symptom onset within 3 days (Lindsley et al., 2010) . This finding highlights the importance of early control measures for COVID-19 patients and suggests a relief for medical staff working in isolation rooms and intensive care units with recovering (later stage) patients. Studies have reported much high positive rates from public surface samplings near COVID-19 patients within 5 DAO Ong et al., 2020) . However, no viral RNA was detected from samples taken from frequently touched public surfaces in the isolation rooms over 7h after routine sanitization, further indicating limited transmission potential via the fomite route during our sampling period. In previous studies, viral RNA were readily detected from public surfaces in isolation rooms of patients in the early stage of infection Ong et al., 2020) . The viral loads of the sputum and feces specimen of 12 patients from the isolation rooms (Table S1 ) in our study were negative-4.5×10 5 copies/mL and negative-3.7×10 4 copies/mL, respectively, on the sampling date.
J o u r n a l P r e -p r o o f COVID-19 patients have a long virus shedding course in feces specimen, and viral RNA, even viable virus particles, has been detected in the feces-related specimens of patients with confirmed COVID-19 (Guan et al., 2020; Tian et al., 2020) . Moreover, previous studies have suggested that the viral loads of feces specimens peak late after the symptom onset and may last for a longer duration compared with those of respiratory specimens (Xing et al., 2020) . Thus, fecal-oral and fecal-aerosol transmission have been suspected as potential routes of SARS- CoV-2 (McDermott et al., 2020; Tian et al., 2020) .
We sampled lavatory air, toilet bowl, floor drain and sewage, apart from the public lavatory surfaces as described above and in Figure S3 . Two of the six samples from toilet bowl after defecation and toilet flushing tested positive for the viral RNA, namely 4 copies/cm 2 and 2 copies/cm 2 , respectively (viral loads for positive detection in the feces specimens, 2.1×10 5 copies/mL-5.0×10 6 copies/mL). Further, one of six floor drain samples tested positive, with a viral concentration of 2 copies/cm 2 , indicating that virus-laden aerosol transmission by the drainage system (as illustrated in Graphical Abstract) is probable. None of the six lavatory aerosol samples tested positive; although patients' feces specimen were test high in viral loads, the toilet didn't produce detectable amount of virus-laden aerosols in our sampling event. Moreover, four of the five water samples from the drainage system tested positive. The viral RNA concentration of the sewage is as high as 3092 copies/mL, but the sewage treatment station worked well to reduce it below the detection limit ( Figure S4 ).
The detection of SARS-CoV-2 in the toilet bowl and sewage samples in this study is consistent with that finding, indicating high viral loads in the patients' feces-related specimens. Notably, one
sample from the floor drainage tested positive, which was probably contaminated by the virusladen bioaerosols from the drainage system. The toilet flushing process produces large numbers of J o u r n a l P r e -p r o o f bioaerosols in the sewage pipe, which may re-enter vertically-aligned lavatories in the same building through the floor drain. This likely explains the SARS cluster outbreak that occurred in Amoy Garden in 2003 (Yu et al., 2004) .
Our findings provide important health implications for the control of nosocomial infection and personal protection: (1) ensure good ventilation for each isolation ward to reduce the potential risk of transmission via aerosols;
(2) in addition to routine disinfection practices, perform frequent and thorough environmental cleaning and disinfection of areas not easy to clean; (3) perform ventilation and disinfection after using the toilet to reduce aerosolization of virus and contamination of surrounding surfaces, and preserve tap water seals to prevent the re-entry of bioaerosols from pipes; (4) do not share personal necessities, and disinfect and replace them regularly. However, the major limitation is that we only detected the presence of the nucleic acid J o u r n a l P r e -p r o o f
The authors declare no competing financial interest. 