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Viable SARS-CoV-2 detected in the air of hospital rooms of patients with COVID-19 with an early infection

Open AccessPublished:November 07, 2022DOI:https://doi.org/10.1016/j.ijid.2022.11.003

      Highlights

      • Viable SARS-CoV-2 can be detected in the air around patients with an early infection.
      • Airborne viable SARS-CoV-2 is associated with high levels of airborne viral RNA.
      • Airborne SARS-CoV-2 is not related to nasopharyngeal viral RNA and viable virus.

      Abstract

      Objectives

      This study assessed the concentration of SARS-CoV-2 in the air of hospital rooms occupied by patients with COVID-19 who had viable SARS-CoV-2 in nasopharyngeal (NP) samples in early infection.

      Methods

      Between July and October 2021, NP swabs were collected from 20 patients with early SARS-CoV-2 infection admitted to a tertiary hospital in Japan. Air samples were collected from their rooms, tested for SARS-CoV-2 RNA, and cultured to determine potential infectivity.

      Results

      The NP swab samples of 18 patients were positive for viable SARS-CoV-2 (median concentration: 4.0 × 105 tissue culture infectious dose 50/ml). In the air samples, viral RNA (median concentration: 1.1 × 105 copies/m3) was detected in 12/18 (67%) patients, and viable virus (median concentration: 8.9 × 102 tissue culture infectious dose 50/m3) was detected in 5/18 (28%) patients. The median time between illness onset and sampling was 3 days. The RNA concentration was significantly higher in samples wherein viable SARS-CoV-2 was detected than in samples in which viable virus was not detected (P-value = 0.027).

      Conclusion

      Viable SARS-CoV-2 can be detected in the air surrounding patients with early SARS-CoV-2 infection. Health care workers should pay attention to infection control when caring for patients with early SARS-CoV-2 infection.

      Keywords

      Introduction

      COVID-19 is caused by the highly transmissible SARS-CoV-2. Understanding the role of surface and air contamination near patients with COVID-19 in the transmission of SARS-CoV-2 is essential to ensure the prevention of SARS-CoV-2 transmission. SARS-CoV-2 RNA has been detected on surfaces and in the air in hospitals (
      • Dinoi A
      • Feltracco M
      • Chirizzi D
      • Trabucco S
      • Conte M
      • Gregoris E
      • et al.
      A review on measurements of SARS-CoV-2 genetic material in air in outdoor and indoor environments: implication for airborne transmission.
      ;
      • Gonçalves J
      • da Silva PG
      • Reis L
      • Nascimento MSJ
      • Koritnik T
      • Paragi M
      • et al.
      Surface contamination with SARS-CoV-2: a systematic review.
      ). However, only a few studies have recovered viable SARS-CoV-2 from the air collected near the patients with COVID-19 (
      • Lednicky JA
      • Lauzard M
      • Fan ZH
      • Jutla A
      • Tilly TB
      • Gangwar M
      • et al.
      Viable SARS-CoV-2 in the air of a hospital room with COVID-19 patients.
      ,
      • Lednicky JA
      • Lauzardo M
      • Alam MM
      • Elbadry MA
      • Stephenson CJ
      • Gibson JC
      • et al.
      Isolation of SARS-CoV-2 from the air in a car driven by a COVID patient with mild illness.
      ;
      • Vass WB
      • Lednicky JA
      • Shankar SN
      • Fan ZH
      • Eiguren-Fernandez A
      • Wu CY.
      Viable SARS-CoV-2 Delta variant detected in aerosols in a residential setting with a self-isolating college student with COVID-19.
      ).
      Patients with COVID-19 have higher SARS-CoV-2 viral loads in clinical samples in the early phase of the illness (
      • Kim MC
      • Cui C
      • Shin KR
      • Bae JY
      • Kweon OJ
      • Lee MK
      • et al.
      Duration of culturable SARS-CoV-2 in hospitalized patients with Covid-19.
      ). A previous study showed that surface samples that yielded viable SARS-CoV-2 were collected from patients’ environments within 5 days of illness onset (
      • Kotwa JD
      • Jamal AJ
      • Mbareche H
      • Yip L
      • Aftanas P
      • Barati S
      • et al.
      Surface and air contamination with severe acute respiratory syndrome coronavirus 2 from hospitalized coronavirus disease 2019 patients in Toronto, Canada, March–May 2020.
      ). However, few studies have investigated the load of viable SARS-CoV-2 in air samples from patients who are hospitalized. Therefore, we hypothesized that viable SARS-CoV-2 may be detectable in the air surrounding patients with COVID-19 during the early phase of infection. This study aimed to determine the concentration of SARS-CoV-2 in the air of hospital rooms occupied by patients with COVID-19 who had viable SARS-CoV-2 in nasopharyngeal (NP) samples.

      Material and methods

      Study protocol

      This prospective study was conducted from July 20 to October 31, 2021, in Hiroshima University Hospital, Hiroshima, Japan. We included adult patients who were hospitalized in general wards during working hours and who had laboratory-confirmed COVID-19 based on a positive reverse transcription-quantitative polymerase chain reaction (RT-qPCR) SARS-CoV-2 test result performed on an NP swab (NPS). Patients who have had COVID-19 symptoms for ≤7 days, admitted to a private or multipatient room with no other patients who were not under negative pressure, and have not received treatment for COVID-19 before the time of sampling were included. The rooms of the patients were air-conditioned and maintained at a temperature of approximately 26°C. Relative humidity was not controlled or measured.
      NPS samples were collected before air sampling on the day of air sampling, using 1 ml of sterile saline. Demographic and clinical data were collected through participant interviews and chart reviews. The data collected included the Charlson comorbidity index (
      • Charlson ME
      • Pompei P
      • Ales KL
      • MacKenzie CR.
      A new method of classifying prognostic comorbidity in longitudinal studies: development and validation.
      ), the interval between illness onset and sample collection, the presence or absence of symptoms (fever [temperature >37.5°C], cough, sore throat, runny nose, and diarrhea), and the requirement for supplemental oxygen at the time of air sampling. We also recorded whether patients were wearing surgical masks at the time of air sampling.
      This study was approved by the Ethics Committee for Epidemiology of Hiroshima University (approval number: E-2157). Written informed consent was obtained from each participant.

      Air sampling

      Air samples were collected using an MD8 Airport (Sartorius AG, Göttingen, Germany) with a water-soluble gelatin membrane filter (80 mm in diameter and 3 μm pore size) at a fixed rate of 50 l/min for 40 minutes, corresponding to a final sample volume of 2 m3 of ambient air. In the patient's rooms, samplers were placed approximately 1 meter from the patient's head and anterior to the mouth and nose. The windows of the room were closed during the air sampling.

      Laboratory procedures

      All samples were transported and processed immediately at Hiroshima University. All work with SARS-CoV-2 was conducted in a biosafety level 3 laboratory. NPS samples were vortexed for 30 seconds before aliquoting. The gelatin membrane filters used for air sampling were placed in 5 ml of sterile saline and vortexed for 30 seconds before aliquoting.

      Reverse transcription-quantitative polymerase chain reaction

      RNA extraction and RT-qPCR were performed as previously described (
      • Nomura T
      • Kitagawa H
      • Omori K
      • Shigemoto N
      • Kakimoto M
      • Nazmul T
      • et al.
      Duration of infectious virus shedding in patients with severe coronavirus disease 2019 who required mechanical ventilation.
      ). Before RNA extraction, the air samples were prewarmed to 37°C until the gelatin had dissolved completely. RNA extraction was performed using the Maxwell Viral Total Nucleic Acid Purification Kit (Promega Corporation, Madison, WI, USA), and RT-qPCR was performed using the One-Step PrimeScript III RT-qPCR mix (Takara Bio Inc., Shiga, Japan), which targets the SARS-CoV-2 nucleocapsid (N) gene. Samples with cycle threshold (Ct) values ≤40.0 were considered positive. A known quantity of SARS-CoV-2 RNA was used as a positive control, and the viral load was calculated from the Ct values.

      Cell and viral culture

      Vero cells expressing transmembrane serine protease 2 (TMPRSS2) (VeroE6/TMRPSS2 cells, procured from the Japanese Collection of Research Bioresources Cell Bank, Japan) were used to culture SARS-CoV-2 (
      • Matsuyama S
      • Nao N
      • Shirato K
      • Kawase M
      • Saito S
      • Takayama I
      • et al.
      Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.
      ). The viral culture methods were performed as described previously (
      • Kitagawa H
      • Nomura T
      • Nazmul T
      • Omori K
      • Shigemoto N
      • Sakaguchi T
      • et al.
      Effectiveness of 222-nm ultraviolet light on disinfecting SARS-CoV-2 surface contamination.
      ). The cells were observed daily for 5 days to check for a cytopathic effect, and the infection titer was determined using the median tissue culture infectious dose (TCID50) method. If no cytopathic effect was observed within 5 days, the culture was considered negative. The RT-qPCR assay described previously was used to confirm SARS-CoV-2 isolation from the supernatant.

      Mutant strain test and whole genome sequencing

      Primer/Probe N501Y (SARS-CoV-2) and Primer/Probe L452R (SARS-CoV-2) Ver.2 (Takara Bio Inc., Japan) were used to detect the N501Y mutation in the Alpha variant of concern and the L452R mutation in the Delta variant of concern in the NPS and air samples. These tests were performed using the same procedure as that used for RT-qPCR, according to the manufacturer's instructions. Whole genome sequencing for SARS-CoV-2 isolated from NPS samples was performed as described previously (
      • Ko K
      • Nagashima S
      • E B
      • Ouoba S
      • Akita T
      • Sugiyama A
      • et al.
      Molecular characterization and the mutation pattern of SARS-CoV-2 during first and second wave outbreaks in Hiroshima, Japan.
      ). The identification of SARS-CoV-2 variants in the isolates was performed using the Global Initiative on Sharing All Influenza Data database (https://www.gisaid.org).

      Statistical analysis

      The Wilcoxon rank-sum test was used to test for statistically significant differences in the continuous variables between the groups, and the chi-square tests or Fisher's exact test were used to test for statistically significant differences in the categorical variables between the groups. All statistical analyses were performed using JMP 16.0 (SAS Institute Inc., Cary, NC, USA). P-values of <0.05 were considered statistically significant.

      Results

      A total of 20 patients were enrolled in the study. NPS samples were collected from each patient and air samples were collected from each patient's room. The virus cultures of the NPS samples from two patients were negative. Therefore, their data were excluded from the analysis. No viral RNA or viable virus was detected in the air samples of these two patients.
      Demographic, clinical, and virological data from the 18 participants are shown in Table 1. The participant characteristics and the detailed results of the NPS and air samples collected are shown in Supplemental Table 1. Of the 18 participants, five (28%) were male and the median age was 61 years (interquartile range [IQR]: 46-78 years). The median interval between the onset of illness and sampling was 3 days (IQR: 2-4 days). The median RNA concentration in the NPS samples was 2.3 × 107 copies/ml (IQR: 1.0 × 107-2.4 × 108 copies/ml), and the median viable virus concentration was 4.0 × 105 TCID50/ml (IQR: 3.5 × 104-3.0 × 106 TCID50/ml). Viral RNA was detected in 12 of the 18 (67%) air samples, and viable virus was detected in five (28%) air samples. The median RNA concentration in the air samples was 1.1 × 105 copies/m3 (IQR: 1.5 × 102-4.7 × 105 copies/m3), and the median viable virus concentration in the air samples was 8.9 × 102 TCID50/m3 (range: 5.8 × 102-1.0 × 104 TCID50/m3).
      Table 1Demographic, clinical, and virological data of patients with COVID-19.
      Patient characteristicsPatients with viable SARS-CoV-2in air samples (n = 5)Patients without viable SARS-CoV-2in air samples (n = 13)P-value
      Age (years), median (range)65 (56-94)56 (26-92)0.14
      Sex, number, (%)0.58
       Male2 (40)3 (23)
       Female3 (60)10 (77)
      Charlson comorbidity index, n (%)
       Moderate-severe (≥3)2 (40)3 (23)0.58
      Symptoms at time of air sampling, n (%)
       Fever (>37.5°C)5 (100)9 (69)0.28
       Cough5 (100)8 (62)0.25
       Sore throat1 (20)2 (15)>0.99
       Runny nose3 (60)4 (31)0.32
       Diarrhea0 (0)3 (23)0.52
      Wearing mask at the time of air sampling, n (%)2 (40)11 (85)0.10
      Nasopharyngeal swab samples
       Ct value, median (range)19.9 (15.9-23.5)21.2 (14.0-24.7)0.55
       Ct value ≤203 (60)3 (23)0.27
       SARS-CoV-2 RNA (copies/ml), median (range)1.1 × 108 (4.3 × 106-1.6 × 109)2.0 × 107 (2.7 × 106-5.5 × 109)0.62
       Viable SARS-CoV-2 (TCID50/ml), median (range)4.0 × 105 (2.0 × 104-1.1 × 107)4.0 × 105 (6.0 × 103-2.0 × 108)>0.99
      Air samples
       Ct value, median (range)27.9 (25.0-31.0)39.1 (29.2-40.0)
      Only samples with Ct values ≤40.0 (n = 7) were analyzed.
      0.027
       SARS-CoV-2 RNA (copies/m3), median (range)5.5 × 105 (6.7 × 104-4.8 × 106)2.4 × 102 (1.3 × 102-2.3 × 105)
      Only samples with Ct values ≤40.0 (n = 7) were analyzed.
      0.027
       Viable SARS-CoV-2 (TCID50/m3), median (range)8.9 × 102 (2.8 × 102-1.5 × 104)N/A
      Ct, cycle threshold; N/A, not applicable; TCID50, median tissue culture infectious dose.
      a Only samples with Ct values ≤40.0 (n = 7) were analyzed.
      Age, sex, Charlson comorbidity index, the interval between the onset of illness and sampling (median days [range] 3 [2-4] vs 3 [1-7], P-value = 0.68), symptoms, mask wearing, the Ct value of the NPS samples (Ct >20 vs Ct ≤20), and the SARS-CoV-2 RNA and viable SARS-CoV-2 concentrations of the NPS samples did not differ significantly between patients, with and without detectable viable SARS-CoV-2 in the air samples. At the sampling, only one patient with detected viable SARS-CoV-2 in the air samples required oxygen through a face mask. Other patients did not require oxygen. In the air samples in which SARS-CoV-2 RNA was detected, the RNA concentration was significantly higher in samples in which viable SARS-CoV-2 was detected than in samples in which viable virus was not detected (median RNA concentration 5.5 × 105 copy/m3 and 2.4 × 102 copy/m3, respectively; P-value = 0.027).
      The relationship between SARS-CoV-2 RNA concentration in NPSs and air samples is shown in Figure 1, whereas that of viable SARS-CoV-2 concentration in NPSs and SARS-CoV-2 RNA concentration in air samples is shown in Figure 2. The relationship between SARS-CoV-2 RNA concentration and viable SARS-CoV-2 concentration in air samples is shown in Figure 3. Viable SARS-CoV-2 in air samples was associated with a high concentration of viral RNA in air samples; however, it was not associated with the concentration of NP viral RNA or viable virus.
      Figure 1
      Figure 1Relationship between SARS-CoV-2 RNA concentration in nasopharyngeal swabs and air samples of 18 participants. TCID50, median tissue culture infectious dose.
      Figure 2
      Figure 2Relationship between viable SARS-CoV-2 concentration in nasopharyngeal swabs and SARS-CoV-2 RNA concentration in air samples of 18 participants. TCID50, median tissue culture infectious dose. *This dot includes the data of two samples.
      Figure 3
      Figure 3Relationship between SARS-CoV-2 RNA concentration and viable SARS-CoV-2 concentration in air samples of 18 participants. TCID50, median tissue culture infectious dose. *This dot includes the data of three samples.
      All SARS-CoV-2 strains isolated from the NPS samples and air samples were negative for the N501Y mutation and positive for the L452R mutation, suggesting that they were the Delta variant. Whole genome sequencing was performed on 12 of 18 SARS-CoV-2 strains isolated from NPS samples (Supplemental Table 2), which confirmed that all strains belonged to the B.1.617.2 lineage (Delta variant).

      Discussion

      In our study, SARS-CoV-2 RNA and viable SARS-CoV-2 were detected in 12/18 (67%) and 5/18 (28%) air samples, respectively. The viable virus concentration in the air was 2.8 × 102 to 1.5 × 104 TCID50/m3, which is similar to the results of a previous study showing viral concentrations in the air ranging from 6.0 × 103 TCID50/m3 to 7.4 × 104 TCID50/m3 (
      • Lednicky JA
      • Lauzard M
      • Fan ZH
      • Jutla A
      • Tilly TB
      • Gangwar M
      • et al.
      Viable SARS-CoV-2 in the air of a hospital room with COVID-19 patients.
      ). To the best of our knowledge, the study by
      • Lednicky JA
      • Lauzard M
      • Fan ZH
      • Jutla A
      • Tilly TB
      • Gangwar M
      • et al.
      Viable SARS-CoV-2 in the air of a hospital room with COVID-19 patients.
      is the only other study that has isolated viable SARS-CoV-2 from air samples in a hospital setting. In the study, the air samplers collected airborne particles using a water vapor condensation method, and the samplers were stationed 2.0-4.8 meters away from the patients. In other settings, viable Delta variant SARS-CoV-2 has been detected in the air collected by air samplers positioned 3 meters away from an infected individual experiencing mild symptoms in a self-isolating room (
      • Vass WB
      • Lednicky JA
      • Shankar SN
      • Fan ZH
      • Eiguren-Fernandez A
      • Wu CY.
      Viable SARS-CoV-2 Delta variant detected in aerosols in a residential setting with a self-isolating college student with COVID-19.
      ). In the study, the same air samplers as that in the study of
      • Lednicky JA
      • Lauzard M
      • Fan ZH
      • Jutla A
      • Tilly TB
      • Gangwar M
      • et al.
      Viable SARS-CoV-2 in the air of a hospital room with COVID-19 patients.
      were used. Viable SARS-CoV-2 was also detected in the air of a car driven by an individual with COVID-19 (
      • Lednicky JA
      • Lauzardo M
      • Alam MM
      • Elbadry MA
      • Stephenson CJ
      • Gibson JC
      • et al.
      Isolation of SARS-CoV-2 from the air in a car driven by a COVID patient with mild illness.
      ). In our study, an air sampler with a gelatin membrane filter (3 μm pore size) was used. Because previous reports showed that viable SARS-CoV-2 was detected from samples collected by air samplers, which can collect airborne particles even smaller than 3 μm (
      • Lednicky JA
      • Lauzard M
      • Fan ZH
      • Jutla A
      • Tilly TB
      • Gangwar M
      • et al.
      Viable SARS-CoV-2 in the air of a hospital room with COVID-19 patients.
      ,
      • Lednicky JA
      • Lauzardo M
      • Alam MM
      • Elbadry MA
      • Stephenson CJ
      • Gibson JC
      • et al.
      Isolation of SARS-CoV-2 from the air in a car driven by a COVID patient with mild illness.
      ;
      • Vass WB
      • Lednicky JA
      • Shankar SN
      • Fan ZH
      • Eiguren-Fernandez A
      • Wu CY.
      Viable SARS-CoV-2 Delta variant detected in aerosols in a residential setting with a self-isolating college student with COVID-19.
      ), the actual concentration of SARS-CoV-2 in the air around patients with COVID-19 may be even higher than that found in our study.
      Several previous studies, which used different methodologies to measure airborne SARS-CoV-2, detected SARS-CoV-2 RNA from the air around patients with COVID-19 but failed to detect viable SARS-CoV-2 (
      • Dinoi A
      • Feltracco M
      • Chirizzi D
      • Trabucco S
      • Conte M
      • Gregoris E
      • et al.
      A review on measurements of SARS-CoV-2 genetic material in air in outdoor and indoor environments: implication for airborne transmission.
      ). To the best of our knowledge, our study is the first to detect viable SARS-CoV-2 from air samples collected by air samplers with gelatin filters. However, our methodology, including the use of an air sampler with gelatin filters, the sampled air volumes, and the distance from the air sampler to the patient was not specific.
      The relatively high proportion of positive air samples in our study may be attributable to the short interval between symptom onset and sampling and the high NP viral load of the participants (
      • Kim MC
      • Cui C
      • Shin KR
      • Bae JY
      • Kweon OJ
      • Lee MK
      • et al.
      Duration of culturable SARS-CoV-2 in hospitalized patients with Covid-19.
      ). However, the detection of viable SARS-CoV-2 and SARS-CoV-2 RNA in the air samples was not necessarily associated with the concentration of NP viral RNA or viable virus (Figures 1, 2). These results suggest that the detection of viable SARS-CoV-2 and SARS-CoV-2 RNA in air samples is also associated with a multitude of factors other than the patient's viral load. These include the patient's behaviors, such as coughing, sneezing, talking, mask wearing, and environmental circumstances. Previous studies reported that the highest number of SARS-CoV-2 RNA was emitted by singing, followed by talking, and the least was from breathing (
      • Alsved M
      • Nygren D
      • Thuresson S
      • Medstrand P
      • Fraenkel CJ
      • Löndahl J.
      SARS-CoV-2 in exhaled aerosol particles from COVID-19 cases and its association to household transmission.
      ;
      • Coleman KK
      • Tay DJW
      • Tan KS
      • Ong SWX
      • Than TS
      • Koh MH
      • et al.
      Viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in respiratory aerosols emitted by patients with coronavirus disease 2019 (COVID-19) while breathing, talking, and singing.
      ). In addition, mask wearing reduces emitted airborne SARS-CoV-2 RNA (
      • Adenaiye OO
      • Lai J
      • Bueno de Mesquita PJB
      • Hong F
      • Youssefi S
      • German J
      • et al.
      Infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exhaled aerosols and efficacy of masks during early mild infection.
      ). In this study, the median interval between the onset of illness and sampling was 3 days, which is shorter than that in previous reports (
      • Kotwa JD
      • Jamal AJ
      • Mbareche H
      • Yip L
      • Aftanas P
      • Barati S
      • et al.
      Surface and air contamination with severe acute respiratory syndrome coronavirus 2 from hospitalized coronavirus disease 2019 patients in Toronto, Canada, March–May 2020.
      ;
      • Lebreil AL
      • Greux V
      • Glenet M
      • Huguenin A
      • N'Guyen Y
      • Berri F
      • et al.
      Surfaces and air contamination by severe acute respiratory syndrome coronavirus 2 using high-flow nasal oxygenation or assisted mechanical ventilation in intensive care unit rooms of patients with coronavirus disease 2019.
      ;
      • Zhou J
      • Otter JA
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      • Cimpeanu C
      • Meno Garcia D
      • Kinross J
      • et al.
      Investigating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface and air contamination in an acute healthcare setting during the peak of the coronavirus disease 2019 (COVID-19) pandemic in London.
      ). A previous study found that viable SARS-CoV-2 could be detected in samples from surfaces near patients within 5 days of illness onset, but viable virus in air samples could not be detected (
      • Kotwa JD
      • Jamal AJ
      • Mbareche H
      • Yip L
      • Aftanas P
      • Barati S
      • et al.
      Surface and air contamination with severe acute respiratory syndrome coronavirus 2 from hospitalized coronavirus disease 2019 patients in Toronto, Canada, March–May 2020.
      ). Our results suggest that the risk of SARS-CoV-2 transmission in patients who are hospitalized early during their disease course may be high, and attention should be paid in controlling the transmission.
      In addition, we used VeroE6/TMRPSS2 cells, which can bind and cleave the SARS-CoV-2 spike protein more efficiently and facilitate early surface-mediated cell entry and viral fusion. VeroE6/TMRPSS2 cells have been shown to enhance SARS-CoV-2 isolation (
      • Hoffmann M
      • Kleine-Weber H
      • Schroeder S
      • Krüger N
      • Herrler T
      • Erichsen S
      • et al.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ;
      • Matsuyama S
      • Nao N
      • Shirato K
      • Kawase M
      • Saito S
      • Takayama I
      • et al.
      Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.
      ). Therefore, a more sensitive culture assay using VeroE6/TMRPSS2 cells may facilitate a higher rate of positive viral culture in air samples.
      Previous studies have suggested that individuals infected with the Delta variant have a higher viral load than those infected with the wild-type strain (
      • Li B
      • Deng A
      • Li K
      • Hu Y
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      • et al.
      Viral infection and transmission in a large, well-traced outbreak caused by the SARS-CoV-2 Delta variant.
      ;
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      Clinical and virological features of SARS-CoV-2 variants of concern: a retrospective cohort study comparing B.1.1.7 (Alpha), B.1.315 (Beta), and B.1.617.2 (Delta).
      ;
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      • Delagrèverie H
      • Visseaux B
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      • Ferre V
      • Marot S
      • Jary A
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      • Boutolleau D
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      • Calvez V
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      ). Based on the whole genome sequencing, the timing of the study period, and the mutations identified, the SARS-CoV-2 isolates in the NPSs and air samples are all likely the Delta (B.1.617.2) variant.
      This study showed that the viable SARS-CoV-2 in the air samples was associated with a high viral RNA concentration in air samples; however, it was not associated with the concentration of NP viral RNA or viable virus. This study included only patients with early SARS-CoV-2 infection who had viable SARS-CoV-2 in the NPS samples. Therefore, these results suggest that the presence of viable SARS-CoV-2 in a patient's NPS sample is not the only condition for detecting viable SARS-CoV-2 from the air surrounding the patient. Two air samples in which SARS-CoV-2 RNA was detected and viable SARS-CoV-2 was not detected contained 1.8 × 105 and 2.3 × 105 copies/m3 of SARS-CoV-2 RNA, respectively. The concentration of SARS-CoV-2 in these samples was higher than that in some air samples containing viable SARS-CoV-2. We were unable to determine whether the failure to culture the virus from air samples was due to the low level of viable SARS-CoV-2 in the air samples (below the sensitivity of the virus culture), viable virus being inactivated during the air sampling and viral culture process, or whether only nonviable viral RNA was collected. The virus particles collected by various air sampling devices can become inactivated during the air sampling process (
      • Pan M
      • Lednicky JA
      • Wu CY.
      Collection, particle sizing and detection of airborne viruses.
      ).
      Our study had several limitations. First, owing to the small sample size, we were unable to identify risk factors associated with the detection of viable SARS-CoV-2 in the air samples based on participant characteristics or behavior. Among the five patients who did not wear a mask during air sampling, five (100%) and three (60%) were positive for SARS-CoV-2 RNA and viable SARS-CoV-2, respectively. However, in our study, we did not formally investigate whether masks can prevent the shedding of viable SARS-CoV-2 virus. In addition, during the air sampling, patients were not monitored to see if they continued to wear a mask. The severity of coughing and sneezing as well as the behavior of the patients with COVID-19, such as talking, were not assessed. Viral emission may depend on a multitude of factors, such as viral load; patient's behaviors, such as coughing, sneezing, talking, shouting, and mask wearing; and environmental circumstances. Therefore, further studies are needed to determine the conditions under which viable SARS-CoV-2 can be isolated from the air. Second, air samplers were placed adjacent to participants, 1 meter in front of their heads. Therefore, the air sampler likely harvested both droplets and aerosols. However, our air sampling method did not differentiate particle size, and we were unable to distinguish droplets from aerosols (<5 µm). Third, we did not investigate the association between the presence or quantity of viral RNA or viable viruses in the air samples and the risk of transmission. Fourth, we did not perform whole genome sequencing on all SARS-CoV-2 isolates to determine the types of variants. Moreover, the genetic relationship between SARS-CoV-2 strains isolated from the patient and air sampling was not investigated as in a previous study (
      • Kotwa JD
      • Jamal AJ
      • Mbareche H
      • Yip L
      • Aftanas P
      • Barati S
      • et al.
      Surface and air contamination with severe acute respiratory syndrome coronavirus 2 from hospitalized coronavirus disease 2019 patients in Toronto, Canada, March–May 2020.
      ). However, based on the timing of the study period and the mutations identified, the SARS-CoV-2 isolates are all likely the Delta variant. The possibility that emission may differ for other more contagious SARS-CoV-2 variants, such as Omicron, is likely, but further studies are required.
      In conclusion, viable SARS-CoV-2 can be detected in the air surrounding patients with COVID-19 with a high viral load. Health care workers should pay attention to droplet and airborne infection control when caring for patients admitted shortly after the onset of symptoms. Further studies are needed to investigate the conditions under which viable SARS-CoV-2 is frequently and best isolated from air samples and what factors determine its emission efficacy.

      Funding

      This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

      Ethical approval

      This study was approved by the Ethics Committee for Epidemiology of Hiroshima University (approval number: E-2157).

      Author contributions

      HK designed the study. HK, TN, YK, MK and TN participated in data acquisition. HK, TN, KO and NS contributed analysis and interpretation of data. HK drafted the manuscript. TS and HO critically revised the manuscript for important intellectual content. All authors read and approved the final submitted manuscript.

      Data availability

      The characteristics of the participants and the detailed results of the collected samples are shown in Supplemental Table 1. The 12 full genomes sequence data of SARS-CoV-2 included in this study are deposited in GISAID (https://www.gisaid.org) and GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The accession numbers are provided in Supplemental Table 2.

      Declaration of competing interest

      The authors have no competing interests to declare.

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