Study design and participants
This study was designed as an open-label, randomized controlled trial. Participants were registered from September 16, 2020 to November 19, 2020 at a hospital admitting symptomatic COVID-19 patients and an accommodation facility housing asymptomatic SARS-CoV-2-infected people. Symptomatic COVID-19 patients or patients with comorbidities were admitted to the hospital after confirming a positive polymerase chain reaction (PCR) test result from their nasopharyngeal specimen. Asymptomatic SARS-CoV-2-infected people were sent to the accommodation facility after a positive antigen test result. The antigen test was performed at the quarantine station at a nearby airport. At the hospital, participants stayed in negative-pressure rooms (5.5 air changes per hour; area, 14.5 m2; and volume, 36 m3). At the accommodation facility, the participants stayed in private rooms (air changes per hour, unknown; area, 12 m2; and volume, 30 m3).
The presence of SARS-CoV-2 RNA on linens before and after washing or disinfection was evaluated on the day that participants were admitted to the hospital or facility (day 1) and on day 3 after admission. Also, to assess the potential infection risk among people handling linens in the rooms where COVID-19 patients were staying, air and PPE were sampled and analyzed for the presence of SARS-CoV-2 RNA.
The participants were assigned one of the five types of washing and disinfection methods shown below for each facility and laundry opportunity (days 1 and 3) using static simple randomization. Blinding was not performed.
Assessment of linen contamination and evaluation of washing or disinfection
The linens used by SARS-CoV-2-infected participants (sheet; pillowcase; duvet; cloths (upper and lower part); cloths bottom; bath towel; and face towel) were changed and sampled after 1, 3, 5, and 7 days of use to test for SARS-CoV-2 RNA. Linens were made of either cotton or a cotton-polyester blend. The linens collected on days 1 and 3 were washed or disinfected using one of the five randomly assigned methods: (1) washing in tap water (15–25 °C), (2) washing in tap water (15–25 °C) with a commercially available polyoxyethylene laundry detergent (Attack Zero®, Kao Corporation, Tokyo, Japan), (3) washing in tap water (15–25 °C) with a commercially available softener (Humming®, Kao Corporation), (4) disinfection by immersion for 30 min in a 250-ppm sodium hypochlorite solution, and (5) disinfection by immersion for 10 min in hot water (80 °C). The presence of SARS-CoV-2 RNA was tested from rinse water samples.
Samples from linens used by the SARS-CoV-2-infected participants were collected using 10 × 87-mm sterile flocked plastic swabs (Eiken Chemical, Japan). Before sample collection, the swabs were moistened with viral transport medium containing 2% heat-inactivated fetal bovine serum (FBS), 100 μg/mL gentamicin, 50 μg/mL amphotericin B, 8 μg/mL tylosin, and 10 μg/mL levofloxacin (VTM; Hanks’ balanced salt solution). The moistened swabs were inserted into 21.9 × 93.6-mm tubes containing 8 mL VTM. Samples were collected by wiping the swab in three directions over a 100 × 100 mm2 area near where the participant’s face had been on the sheet, pillowcase, and duvet: on the collar for the nightgown top, near the feet for the nightgown bottom, and near the middle for the bath and face towels. After washing or disinfecting the linens, 500 mL of rinse water was collected in sterilized bottles and stored at 4 °C. Prior to analysis, the samples were filtered with a drip filter to remove debris, and then the filtered samples were weighed and 5% polyacrylic acid (MW 25 kDa) dissolved in 0.4 M NaOH to a final concentration of 0.13% was added. After repeating mixing and standing at room temperature for 30 min, the samples were centrifuged at 11,000×g and the pellets were collected. Finally, the supernatants were obtained by resuspending the pellets in 2 mL of PBS (-) and centrifuging at 6000 rpm for 3 min.
Presence of airborne SARS-CoV-2
To assess the risk of droplet and airborne transmission while people handled SARS-CoV-2-contaminated linens, air samples were collected before and during or after linen changes. A Sartorius MD8 Airscan sample device (Sartorius AG, Göttingen, Germany) was placed at a height of 100 cm and 50 cm away from the bed. Sterile gelatin filters (80-mm diameter; 3-μm pore size, T3 phage-capture rate 99.94%; Sartorius AG) were used to filter 2000 L of air at a flow rate of 50 L/minute. The patients remained in the room during linen changes and while air was sampled. After air sampling, the gelatin membrane filter was fragmented and inserted into a 100-mL centrifugation tube with 5 mL VTM. The filter was dissolved by shaking at 37 °C for 15 min and then stored at − 80 °C until RNA extraction.
To assess the potential risk of contact transmission while people handled SARS-CoV-2-contaminated linens, samples were collected from the surfaces of their PPE immediately after changing the linens. Fifty-two samples were collected from each PPE item (N95 mask, goggles, upper gown, and lower gown) by wiping the swab in three directions over a 50 × 50-mm2 area: on front surface of the mask, front surface of the goggles, near the neck for the gown top, and near the feet for the gown bottom.
SARS-CoV-2 RNA detection and virus isolation
All samples were transported to the National Institute of Infectious Diseases (NIID) and examined using the real-time reverse transcriptase (rtRT) PCR method developed by Shirato et al.16 Viral RNA was extracted from samples, including nasopharyngeal, linen, and PPE swabs, using a MagMAX CORE nucleic acid purification kit (Thermo Fisher Scientific, Tokyo, Japan) on the KingFisher Flex purification system (Thermo Fisher Scientific) according to the manufacturer’s instructions: each 60-µL RNA elution was prepared from 200-µL samples. The rtRT-PCR mixture, including the N2 primer set, was prepared using a QuantiTect Probe PCR kit (QIAGEN, Venlo, Nederland) in a final volume of 20 µL. This comprised 5 µL extracted RNA, 0.5 µM forward primer, 0.7 µM reverse primer, and 0.2 µM TaqMan probe. The reaction was performed, using a LightCycler 480 system (Roche, Basel, Switzerland) with reverse transcription at 50 °C for 30 min followed by denaturation at 95 °C for 15 min. Next, 45 cycles of amplification were performed using a thermal cycling profile of 95 °C for 15 s and 60 °C for 1 min. The viral copy numbers in patient specimens were calculated using a standard curve established by the NIID. The limit of detection of cycle threshold (Ct) values was set at 40 based on a report by Buchan et al. and the NIID protocol17.
The specimens used for virus isolation were prepared as inoculation material that had undergone sterile filtration using a centrifuge filter unit (Ultrafree-MC, 0.22 μm; Merck Millipore, Burlington, MA, USA). Vero E6/TMPRSS2 cells (5% FBS DMEM) cultured in 12-well plates were inoculated with 100 μL of the specimen and cultured at 37 °C and 5% CO2 for 7 days to check for a cytopathic effect (CPE). A total of 100 µL of the first culture supernatants was transferred to a new monolayer cell plate and cultured under the same conditions as described above. After three serial passages, we judged positivity/negativity by CPE.
For demographic data, summary statistics were shown as medians (interquartile range or range), frequency distributions, or proportions. Meteorological parameters were shown as mean (± standard deviation). The difference in RNA detection proportions between the accommodation facility and hospital was evaluated by Fisher’s exact test. Longitudinal changes in RNA detection proportions were assessed with the Cochran-Armitage test, and a p value less than 0.05 was considered statistically significant. A 95% confidence interval was used to determine whether there were statistically significant differences in the percentages of positive samples among the different groups of cleaning and disinfection methods. All statistical analyses were performed with SAS V.9.4 (SAS Institute, Cary, NC, USA).
We explained the risks and benefits of the study to all participants and obtained their informed consent. All methods were performed in accordance with the relevant guidelines and regulations, and this study was approved by the Institutional Review Boards of the NIID (approval No. 1167) and International University of Health and Welfare (approval No. 20-Nr-066).