SARS‐CoV‐2 environmental contamination associated with persistently infected COVID‐19 patients

Abstract Background Severe COVID‐19 patients typically test positive for SARS‐CoV‐2 RNA for extended periods of time, even after recovery from severe disease. Due to the timeframe involved, these patients may have developed humoral immunity to SARS‐CoV‐2 while still testing positive for viral RNA in swabs. Data are lacking on exposure risks in these situations. Here, we studied SARS‐CoV‐2 environmental contamination in an ICU and an isolation ward caring for such COVID‐19 patients. Methods We collected air and surface samples in a hospital caring for critical and severe COVID‐19 cases from common areas and areas proximal to patients. Results Of the 218 ICU samples, an air sample contained SARS‐CoV‐2 RNA. Of the 182 isolation ward samples, nine contained SARS‐CoV‐2 RNA. These were collected from a facemask, the floor, mobile phones, and the air in the patient room and bathroom. Serum antibodies against SARS‐CoV‐2 were detected in these patients at the beginning of the study. Conclusions While there is a perception of increased risk in the ICU, our study demonstrates that isolation wards may pose greater risks to healthcare workers and exposure risks remain with clinically improved patients, weeks after their initial diagnoses. As these patients had serum antibodies, further studies may be warranted to study the utility of serum antibodies as a surrogate of viral clearance in allowing people to return to work. We recommend continued vigilance even with patients who appear to have recovered from COVID‐19.


| BACKG ROU N D
The outbreak of coronavirus disease 2019  has strained the capacity of hospitals worldwide, placing healthcare workers at significant risk of exposure. Air and surface contamination with SARS-CoV-2 has been detected in hospital settings where newly diagnosed COVID-19 patients are cared for. [1][2][3] SARS-CoV-2 has also been shown to have a prolonged presence in saliva and stool samples and an environmental stability greater than SARS-CoV-2 on surfaces. [4][5][6][7] Therefore, the risks of nosocomial infections are likely significant.

COVID-19 patients typically test positive for SARS-CoV-2 RNA
for extended periods of time, weeks in some cases, necessitating prolonged hospitalization or isolation. 8,9 Patients who have recovered from severe COVID-19 can also continue to test positive. Since these patients have been hospitalized for extended periods, it is possible that they have developed humoral immunity to SARS-CoV-2 while still testing positive for viral RNA in swabs. The extent of environmental contamination by these patients in healthcare settings is unknown but these data are particularly relevant to inform measures to prevent exposure of healthcare workers. They are also relevant due to the considerations of using the presence of serum antibodies as a surrogate marker of viral clearance in allowing people to return to work. Therefore, it is important to determine whether environmental contamination with SARS-CoV-2 can still be associated with patients with serum antibodies.
To address these concerns, we collected air and surface samples from the intensive care unit (ICU) and an isolation ward of The First Affiliated Hospital of Guangzhou Medical University (FAHGMU), which is a designated hospital for the treatment of critical and severe COVID-19 pneumonia cases in Guangdong Province, a large province in southern China. Two air samplers were used: a sampler developed by the US National Institute of Occupational Safety and Health (NIOSH) that fractionates airborne particles into three size fractions and a cyclonic aerosol particle liquid concentrator. Overall, environmental contamination in the ICU was minimal. Environmental contamination was greater in the isolation ward, in which SARS-CoV-2 RNA was detected in multiple samples, including air samples taken in the patient room and bathroom. All patients in this study have serum IgG titers against SARS-CoV-2. Therefore, COVID-19 patients and individuals that have recovered from severe COVID-19 could still be shedding virus into the air and environment weeks after illness onset.

| Collection of surface samples
Surface samples were collected according to the "World Health Organization Surface sampling of MERS-CoV in health care settings, June 2019". 10 Samples were collected using 15-cm sterile flocked plastic swabs (Shenzhen Mairuikelin Company). Swabs were wetted with viral transport medium (VTM) prior to sample collection and then placed in 15-mL tubes containing 3 mL VTM. 11 Samples were collected between 8 am and 11 am.
In the ICU, swabs were taken from areas proximal to four patients showing the highest viral loads by quantitative RT-PCR prior to sampling and in areas used by healthcare workers. The locations of swabs taken from patient-specific areas were the floor less than one meter away from patient head, the bed rail, the patient's clothing, the bedsheet, the control panel of the ventilator, and the ventilator outlet valve (samples E01 to E06, respectively). The locations of swabs taken from areas not associated with individual patients were the changing room door handle, the floor of changing room, the faucets at the handwashing station, and the keyboard of shared computer (samples E07 to E10, respectively) ( Table 1).
In the isolation ward, patients were placed in separate rooms with their own bathrooms. Swabs were taken from the rooms of five patients and the bathrooms of two patients. Patients with the highest viral loads in respiratory or stool samples prior to sampling were selected. The locations of swabs taken from patient rooms were the floor less than one meter away from patient head, the floor greater than one meter away from patient head, the bed rail, the bedside table, the patient's mobile phone, the bedsheet, the patient's facemask, and the television remote control (samples E01 to E08, respectively). The locations of swabs taken from patient bathrooms were the toilet, the bathroom door handle, and the faucet handles Conclusions: While there is a perception of increased risk in the ICU, our study demonstrates that isolation wards may pose greater risks to healthcare workers and exposure risks remain with clinically improved patients, weeks after their initial diagnoses.
As these patients had serum antibodies, further studies may be warranted to study the utility of serum antibodies as a surrogate of viral clearance in allowing people to return to work. We recommend continued vigilance even with patients who appear to have recovered from COVID-19.

K E Y W O R D S
coronavirus, COVID-19, intensive care unit, SARS-CoV-2, transmission on the sink (samples E09 to E11, respectively). It should be noted that squat latrines were in the isolation ward bathrooms ( Table 1).
The locations of swabs taken from areas not associated with individual patients were the changing room door handle, the floor of changing room, and the cleaner's mop handle (samples E12 to E14, respectively) ( Table 1).

| Collection of air samples
We collected air samples using two cyclonic sampling devices: a two-stage cyclonic bioaerosol sampler developed by the NIOSH 10,11 (NIOSH,Centers for Disease Control and Prevention) and an aerosol particle liquid concentrator (model W-15, Beijing In both the ICU and isolation ward, the NIOSH sampler was placed on a tripod at the head of the bed within one meter of the patient's head at a height of 1.3 m (sample N01, Figure 1 and Table 1). In the isolation ward, the NIOSH sampler was also used in the bathroom by mounting it on an infusion support near the sink, less than one meter from the toilet (sample N02, Table 1). A portable analyzer that recorded temperature and humidity was also mounted on the tripod. Air was collected for four hours continuously at a flow rate of 3.5 L/min into three size fractions: >4 μm (collected in a 15-mL tube), 1-4 μm (collected in a 1.5-mL tube), and <1 μm (collected in a polytetrafluoroethylene (PTFE) membrane filter with 3.0 μm pore size). After each collection, the 15-mL and 1.5-mL tubes were detached and 1 mL of VTM was added. The filter was removed and immersed in 1 mL VTM.
The DingBlue sampler was placed at the head of the bed within one meter of the patient's head on the opposite side of the bed to the NIOSH sampler (sample D01). In the isolation ward, the DingBlue samplers were also used to collect air samples from the patient bathroom (samples D02 and D03, Table 1). The sampler was placed on the bathroom sink at a distance of less than one meter from the toilet. Air samples were collected at a flow rate of 14 L/min for 30 minutes into a 5-mL tube containing 3 mL VTM. Air samples were collected in the bathroom by instructing the patient to turn on the DingBlue air sampler before using the toilet. Medical staff would then collect the sample from the machine after sampling. Other air samples were collected by medical staff.

| RNA extraction and quantitative RT-PCR
All liquid samples were subjected to 30-min heat inactivation at 56°C prior to RNA extraction as part of national biosafety requirement. RNA was extracted from 0.28 mL of the VTM containing the air and surface samples using the QIAGEN vRNA mini kit (QIAGEN) according to the manufacturer's instructions. RNA samples were screened for the presence of SARS-CoV-2 RNA encoding the ORF-1 or N genes using the "New Coronavirus 2019-nCoV nucleic acid detection kit (Fluorescence PCR method)" (Sansure Biotech Inc) and an ABI 7500 real-time PCR machine (Thermo Scientific). The viral copy numbers in patient specimens were calculated using a standard curve established by the diagnostic laboratory of The First Affiliated Hospital of Guangzhou Medical University.

| SARS-CoV-2 Spike and Nucleocapsid IgG ELISA
Recombinant SARS-CoV-2 spike (S) (encompassing the extracellular domain, S1 and S2 subunits) and nucleocapsid (N) proteins (Sino Biological) were used to coat 96-well plates at 0.5 μg/mL overnight at 4°C. After washing and blocking, serially diluted sera (at a starting dilution of 1:100) were added to the plate and incubated for two hours at 37°C. Plates were washed, and specific antibodies were detected using an anti-human IgG horseradish peroxidase-conjugated secondary antibody (Sigma). Colorimetric reaction was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Gibco Technologies). Reactions were stopped using 0.5 mol/L sulfuric acid and absorbance read at 450 nm.
End-point titers were determined to be the last reciprocal dilution with a positive/negative optical density (OD) ratio ≥ 2. Assay specificity for S and N proteins, tested using non-COVID-19 sera (n = 203), was comparable at 97.5% and 97.0%, respectively (data not shown).

| Pseudovirus antibody neutralization assay
Neutralizing antibody titers against SARS-CoV-2 in patient serum samples were determined using a pseudovirus assay as previously described. 3 Briefly, SARS-CoV-2 pseudoviruses were generated by co-transfection of a lentiviral packaging plasmid,

| Minimal environmental contamination with SARS-CoV-2 RNA was detected in the ICU
We collected 218 air and surface samples from the ICU of The This patient already had serum antibody titers to the SARS-CoV-2 before this positive sample was collected and had subsequently developed virus neutralization antibody titers detected using a pseudoparticle assay after our study was completed (Table 5). Aside from the collection of respiratory samples and an anal swab, no other aerosol-generating procedures were performed on the day that this sample was collected ( Figure 3A). Overall, little environmental contamination with SARS-CoV-2 RNA was detected in the ICU.

| Detection of environmental contamination with SARS-CoV-2 in the isolation ward
The population of the isolation ward consisted of five patients that had recovered from severe COVID-19 but were still returning samples that tested positive for SARS-CoV-2 RNA. These patients had been hospitalized for 37, 39, 40, 45, and 47 days when we commenced sampling (Table 3) (Table 4 and Figure S1). Using a less stringent cutoff (Ct < 45), two other samples collected from this patient; a facemask (Ct = 44.9) and an air sample collected in the patient's room (Ct = 44.7) were positive.
Consistently, high viral loads were detected in lower respiratory tract (LRT) samples collected from this patient during the sampling period  (Table 4). This patient showed high serum IgG titers against SARS-CoV-2 S and N proteins and high virus neutralization antibody titers detected using a pseudoparticle assay before and after our sampling was conducted (Table 5). RNA. Two of the three patients had serum IgG titers to the SARS-CoV-2 S and/or N proteins before these samples were collected and one had neutralizing serum antibody titers detected using a pseudoparticle assay (Table 5). After sampling, all patients had serum IgG titers to the SARS-CoV-2 S and N proteins and neutralizing serum antibody titers detected using a pseudoparticle assay (Table 5).
These findings suggest that persistently infected, clinically improved patients may still shed virus into the environment and URT samples may be poor indicators of shedding potential for these formerly severely ill patients. Frequencies and dates when these procedures were performed are described in Figure 2.

| D ISCUSS I ON
b Refer to Table 1 for a description of the samples. the environmental contamination associated with patients that had been hospitalized for weeks post-COVID-19 diagnoses. Due to the timeframe involved, these patients may also have some humoral immunity to SARS-CoV-2. Therefore, we also measured serum antibodies to determine whether their presence mitigated SARS-CoV-2 environmental contamination. This is particularly relevant as the presence of serum antibodies is being considered as a surrogate of viral clearance to allow people to return to work.
SARS-CoV-2 has been found in hospital settings, in patient wards and in the ICU. [1][2][3] However, these studies were conducted in wards where newly diagnosed patients were being cared for. The focus of our study was to assess SARS-CoV-2 environmental contamination F I G U R E 2 Summary of viral loads in patient swabs, clinical care procedures performed during sampling, and outcomes of environmental sampling. Timeline shows samples collected from patients and testing results for SARS-CoV-2 RNA by quantitative real-time PCR in the (A) intensive care unit and (B) in the isolation ward. Environmental samples that tested positive for SARS-CoV-2 RNA by quantitative real-time PCR are also shown in hospital settings caring for patients with prolonged COVID-19.
Two broad types of patient were studied here: (a) Severely ill COVID-

patients in an ICU and (b) individuals whom have recovered from
severe COVID-19 have been discharged from the ICU, but must remain in the hospital under isolation as they are still testing positive for SARS-CoV-2 in swabs. The patients in this study had been hospitalized for as long as 57 days.
We did not find much evidence of SARS-CoV-2 contamination in the ICU, even though these patients, particularly patients 10 and 14, still had high viral loads and underwent procedures that are likely to generate aerosols. We did detect a high Ct value in one air sample, indicating that SARS-CoV-2 RNA may potentially have been present in the air. Taken together, this suggests that the infection control measures and practices of the ICU did not generate F I G U R E 3 Layouts of hospital rooms. Samples were collected in the intensive care unit (ICU) (A) and in an isolation ward (B). In the ICU, airflow originated in the ceiling above the foot of each patient bed and was extracted through vents in the wall at bed height. Placement of the NIOSH and DingBlue air samplers and bed numbers are shown. The red triangle indicates the handwashing station that was sampled.
In the isolation ward, each patient was isolated in different rooms with their own bathrooms. The locations of the NIOSH and DingBlue air samplers are shown in their positions relative to the patient's bed, toilet, and bathroom sink. Diagrams are not to scale. Refer to Table 1  Therefore, it is not known whether these samples could be capable of infecting a healthy individual. Due to the national biosafety regulation and limited resources during the outbreak, we were unable to sample more frequently or consistently. Lastly, the Ct values we detected in this study were in the lower limits of the detection threshold. This could be due to two potential factors; these patients had been infected for a long time and did not have high viral loads, particularly in the URT, or the heat inactivation step performed as part of the national biosafety requirement could have increased the Ct values of the samples. 18 Therefore, we applied a less stringent Ct cutoff compared to the standard clinical diagnostic criteria to our qPCR results.
The discovery of SARS-CoV-2 RNA in air samples in the bathroom on multiple days suggests that virus-laden particles are generated by toilet usage. Therefore, the risk of nosocomial infection with SARS-CoV-2 may be greater in bathrooms and in wards caring for mild patients compared to the ICUs, where we found minimal evidence of viral presence. Although the greatest transmission risks are still likely posed by patients early in infection when they have high viral loads, our study shows that infected patients may still pose a risk weeks after their initial diagnosis. 18,19 Further, as these patients had serum antibodies, further studies may be warranted to study the utility of serum antibodies as a surrogate of viral clearance. We recommend that healthcare workers continue to be vigilant even with patients who appear to have recovered.