key: cord-0708025-nepypdoq authors: Yang, Minghui; Li, Liang; Huang, Ting; Li, Shaxi; Zhang, Mingxia; Yang, Yang; Jiang, Yujin; Li, Xiaohe; Yuan, Jing; Liu, Yingxia title: SARS-CoV-2 Detected on Environmental Fomites for Both Asymptomatic and Symptomatic Patients with COVID-19 date: 2021-02-01 journal: Am J Respir Crit Care Med DOI: 10.1164/rccm.202006-2136le sha: 5a29d427bc51d2aeb6bf2f7a74032d7e0812b180 doc_id: 708025 cord_uid: nepypdoq nan through environmental fomites (1) . Nosocomial transmission of SARS-CoV-2 was reported in hospitals (2) . Surface and air contamination of SARS-CoV-2 was also reported in clinical areas (3) . The majority of the SARS-CoV-2-infected population is asymptomatic, and human-to-human transmission from asymptomatic patients has been documented (4, 5) . However, there is a lack of information on the viral transmission potential and environmental fomites for asymptomatic patients, especially when compared with symptomatic patients. Recent studies on the potential environmental fomites of coronavirus disease (COVID-19) confirmed that symptomatic patients can spread the virus to their surroundings (6) (7) (8) . However, for patients who stayed asymptomatic throughout the disease course, little is known on their viral spread potential through environmental fomites, although these patients can be easily neglected and stay within the general population (8) . We thus performed viral detection on various environmental fomites from asymptomatic and symptomatic patients with COVID-19. We aimed to determine potential environmental risk factors to provide information for public sanitation strategies. Study design. Sixty-six patients with confirmed COVID-19 hospitalized in Shenzhen Third People's Hospital were enrolled, including 20 asymptomatic and 46 symptomatic patients. Nasopharyngeal swabs were collected for SARS-CoV-2 detection. "Asymptomatic patients" were defined using similar criteria in a previous report (4) . Briefly, these people were afebrile without chills or cough. Physical examination and other laboratory results, except for the positive viral RNA diagnosis in their nasopharyngeal swabs, were unremarkable. They did not experience any symptoms during the entire period from admission until the viral RNA detection turned negative. This study was approved by the ethics committee of Shenzhen Third Positive patients represent the patients whose environmental fomites were detectable with SARS-CoV-2. x Positive patients represent the patients whose environmental fomites were detectable with SARS-CoV-2 k One positive sample from a symptomatic patient's pillow was detected. ¶ One positive sample from a symptomatic patient's glasses was detected. **One positive sample from a symptomatic patient's infusion pump was detected. † † Except for two positive samples of mouth/nose contacts collected from the same patient, for all the rest of the positive environmental samples, each of them was collected from a different participant. People's Hospital (No. 2020-165). Written informed consent was obtained from all participants. Environmental surface sampling. Dacron swabs premoistened with viral transport medium (Yocon) were used for sufficient contact with environmental surfaces. In patients' rooms, samples from environmental fomites such as N95 masks, water cups, and cell phones were collected before and after disinfection by 75% (vol/vol) alcohol with repeated rinsing and wiping. Ward floor, toilet floor, ward door handles, water taps, and other room surfaces were sampled before and after disinfection by spray using 2,000 mg/L chlorine-containing disinfectant followed with repeated water rinsing and wiping. Environmental sampling was conducted the same day when the participant tested positive for SARS-CoV-2 RNA in a nasopharyngeal swab. qRT-PCR. Viral RNA extraction and qRT-PCR for SARS-CoV-2 were conducted as described previously (9) . The sample was considered SARS-CoV-2 positive when: 1. The cycle threshold (Ct) value was <38. 2. The Ct value fell between 38 and 40, and a repeated qRT-PCR gave a Ct value <40. Statistical analysis. The quantitative data was described as the mean 6 SD, or as the median (min to max). Each type of fomite from the same patient was grouped together. With at least one positive viral RNA detection within the grouped environmental samples, the type of fomites examined is considered contaminated for the particular patient. For each fomite type, the positive rate is calculated as (positive patient number)/(total patient number), unless otherwise stated. Patient characteristics were compared using the x 2 test or Fisher's exact test for categorical data, and the Student's t test for continuous data. P value ,0.05 was considered statistically significant. All the analysis was performed using SPSS Version 22.0 (SPSS) and GraphPad Prism 6.0 (GraphPad Software, Inc.). SARS-CoV-2 RNA was detected within the environmental samples collected from both asymptomatic and symptomatic patients with COVID-19. One hundred seventy-nine environmental specimens were collected from 20 asymptomatic patients, and 376 specimens were collected from 46 symptomatic patients with COVID-19, with the sampling details and SARS-CoV-2 RNA detection results presented in Table 1 . Samples from squat toilets showed the highest positive rate in both asymptomatic (28.6%) and symptomatic (33.3%) groups, followed by samples related (COVID-19) . Positive rate of collected environmental fomites (EFs) represents the ratio of the number of positive types of EFs collected from a patient to the total number of types of EFs collected from the specific patient. The positive EFs include the area around a squat toilet, water cup, breathing exerciser, straw, oxygen catheter and atomizer, inside the mask, hand/foot, cell phone, pillow, and glasses as shown in Table 1 . Within both the asymptomatic patient group and the symptomatic patient group, patients were further divided according to the cycle threshold (Ct) values of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA qRT-PCR from nasopharyngeal swab samples, as the high-viral-load group for Ct ,30, and as the low-viral-load group for Ct >30, determined from the distribution of patient nasopharyngeal sample Ct values. Positive rates of collected EFs were compared between the high-versus low-viral-load patient groups for the number of positive EFs/the number of total tested EFs. P , 0.050 is considered statistically significant. Spearman rank coefficient correlation analysis showed that the positive SARS-CoV-2 RNA detection rate of EFs from both symptomatic and asymptomatic patients were highly correlated with the specific patient's nasopharyngeal viral load assessed by Ct values (asymptomatic: r = 20.630, P = 0.0070; symptomatic: r = 20.600, P = 0.0003). with mouth/nose contacts (water cup, breathing exerciser, straw, oxygen catheter, and atomizing nozzle) (asymptomatic: 15.0%; symptomatic: 16.7%), and third samples from inside of the mask (asymptomatic: 8.3%; symptomatic: 20.0%). Age factor showed no significant influence on the viral spread potential to the surroundings. Both symptomatic and asymptomatic patients can potentially spread SARS-CoV-2 environmentally. SARS-CoV-2 detection rate in environmental samples was significantly correlated with the patient nasopharyngeal viral load, despite the symptoms. By further dividing patients according to their nasopharyngeal viral loads, we found a significantly higher SARS-CoV-2 detection rate in the environmental samples from patients with higher viral loads in the symptomatic group. Similarly, in asymptomatic patients, the average positive rate of environmental samples was 18.2% in the group with higher viral loads, whereas it was 0.96% in the group with lower viral loads, although the difference is not statistically significant (Figure 1 ). Spearman rank coefficient correlation analysis showed that the positive SARS-CoV-2 RNA detection rate of environmental samples from both symptomatic and asymptomatic patients were highly correlated with the specific patient's nasopharyngeal viral load assessed by Ct values (asymptomatic: r = 20.630, P = 0.0070; symptomatic: r = 20.600, P = 0.0003). Environmental disinfection limits SARS-CoV-2 spread. Four hundred fourteen environmental samples were collected before disinfection and 141 after disinfection. The positive detection rate of samples was 5.95% (10/168 samples) in the asymptomatic group and 8.94% (22/246 samples) in the symptomatic group before disinfection. SARS-CoV-2 RNA was negative in both the asymptomatic and symptomatic groups after disinfection. Here we provide an evaluation of potential environmental fomites for SARS-CoV-2 comparing a large number of samples from symptomatic and strictly asymptomatic (no symptoms throughout the disease course) patients with COVID-19. We showed that patients with higher nasopharyngeal SARS-CoV-2 loads seem more likely to spread the virus to the surroundings, regardless of symptoms, although standard disinfection procedures effectively clear viral residues. A previous study revealed potential fecal-oral transmission of COVID-19 (10), and our study further suggests the importance of hand hygiene and disinfection of public toilets. A limitation of our study is that we only used qRT-PCR for viral detection, not viral culture, because of technical difficulties. Positive detection of SARS-CoV-2 RNA indicates viral contamination on the environmental samples, indicating a potential risk of viral residue. In conclusion, SARS-CoV-2 environmental contamination presents in both asymptomatic and symptomatic patients. Environmental disinfection is vital for reducing SARS-COV-2 spread. Monitoring symptoms is insufficient to identify SARS-CoV-2 spreaders. n Central Airway Tree Dysanapsis Extends to the Peripheral Airways To the Editor: Dysanapsis refers to a mismatch between airway tree and lung size (1) . Dysanapsis of the central airway tree, readily quantified by multidetector computed tomography (MDCT), is associated with chronic obstructive pulmonary disease (COPD) risk independent of cigarette smoking (2), but the mechanisms are uncertain. Dysanapsis is believed to arise early in life (1), and airway tree morphogenesis occurs via an interactive branching program from trachea to terminal bronchioles (3). We hypothesized that central airway tree dysanapsis would extend to the terminal bronchioles in the absence of clinical respiratory disease. Lung donors without clinical respiratory disease (n = 10) were recruited from the University of Pennsylvania Hospital and the Katholieke Universiteit Leuven, and image analyses were performed at the University of British Columbia. All procedures were approved by the ethics and biosafety committees at the institutions involved. Lung imaging and quantitative assessment. The preparation of lung specimens is detailed elsewhere (4, 5) . Briefly, each lung explant was inflated with air to a transpulmonary pressure of 30 cm H 2 O and then deflated to 10 cm H 2 O, was frozen with liquid N 2 vapor, and then underwent MDCT imaging (Siemens Sensation 16 or GE Discovery CT750 HD system). Frozen lung specimens were then sliced in the transverse plane and 5-6 cores of tissue per explant were extracted by random sampling for cryo micro-computed tomography imaging (Nikon HMX-225ST) (6) . Apollo Software (VIDA Diagnostics) was used to segment the airway tree and lung specimen volumes by a trained reader unaware of other participant information (7) . Central airway tree dysanapsis was assessed using airway lumen diameters measured at standard central anatomic locations (lobar, segmental, and subsegmental airways) and lumen diameter reference equations to calculate the mean percent-predicted central airway lumen diameter (hereafter, percent-predicted central airway tree caliber) (2) . This single continuous value quantifies the deviation of observed central airway tree caliber from what is expected based on donor age, sex, height, and explant lung volume. A percent-predicted central airway tree caliber of 100% represents the "expected" mean central airway tree size. A value below 100% represents a smaller central airway tree than predicted ("hypoanapsis"), and a value above 100% represents a larger central airway tree than predicted ("hyperanapsis"). Terminal bronchioles, defined as the last purely conducting airway, were identified and lumen diameters were measured perpendicular to the local long axis. The mean lumen diameter for all terminal bronchioles per core sample was computed (4). Other variables. Total central airway count per milliliter of lung was defined as the sum of all segmented airways detected by MDCT divided by the explant lung volume. Terminal bronchiole count per milliliter of lung was defined as the sum of all terminal bronchioles identified by micro-computed tomography within a core divided by the core volume (8) . A simplified calculation of central and peripheral airway tree dysanapsis was also performed, defined respectively as the ratio of mean central airway lumen diameters to the cube-root of lung volume (hereafter, central airway-to-lung ratio), and the ratio of mean terminal bronchiole diameter to the cube-root of lung volume (hereafter, peripheral airway-to-lung ratio). Statistical analysis. The primary analysis was a linear regression model of terminal bronchiole lumen diameter fitted using generalized estimating equations to account for multiple randomly sampled core samples per explant and adjusted for explant lung volume and pack-years. A secondary analysis was restricted to donors with ,1 pack-year (n = 4). Sensitivity analyses included the unadjusted model, and models additionally adjusted for total central airway count per milliliter, terminal bronchiole count per milliliter, and comparing the central to peripheral airway-to-lung ratios. Analyses were performed using SAS 9.4 (SAS Institute). The significance threshold was 0.05 and two-side P values are reported. Donor characteristics of the 10 explants included in the analysis are summarized in Table 1 . The mean percent-predicted central airway tree caliber was 94.5 6 12.5% (range, 76.0-113.0% predicted), and among 541 terminal bronchioles identified, the mean lumen diameter was 570 6 130 mm. 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