Pilot studies
Medical masks were tested as per protocols developed through two pilot studies in Sydney Australia.
Pilot study 1 (laboratory testing)
The aim of this pilot study was to identify areas of maximum virus concentration on the surface of masks. Medical masks were donned on a simple mannequin in a laboratory setting and fluorescent particles (UV Glow powder) were sprayed front on and side on from a distance of approximately 1 m using a spray bottle. We performed three experiments from the front and three experiments from the sides of mannequin. UV light was used to quantify the density of particles on mask surface and to identify area of maximum concentration. In all three experiments, most particles were concentrated on upper right, middle and left sections of the masks (Figs. 1 and 2).
Pilot study 2 (clinical testing)
The second pilot study was conducted in two tertiary referral hospitals in Sydney Australia to develop testing methodology. Twelve HCWs (doctors and nurses) from the infectious diseases, respiratory/ chest wards and intensive care unit (ICU) participated in the study. HCWs were asked to wear medical masks for a shift (minimum 30 min) used masks were tested in the Virology Research Laboratory, University of New South Wales and Prince of Wales Hospital Sydney Australia. If a respirator was indicated due to airborne inflictions, HCWs were excluded from the study and were allowed to use a respirator.
Medical masks were divided into six sections as shown in Fig. 3. Samples were taken from upper three sections of masks i.e. 36 samples were tested in total (12 masks X 3 samples). The outer layer of the mask was removed using sterile tweezers. The mask layer was placed into a 15 ml falcon tube containing 700 μl of Phosphate buffered saline and vortexed for 20 s. After 10 min incubation the mask was placed in a custom made filter tube inside an eppendorf tube and centrifuged briefly. The filtrate was then transferred to 1.5 ml Eppendorf tube. Total nucleic acid was extracted on the Kingfisher Flex (Thermo Scientific) using the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) according to the manufacturer’s instructions. Presence of respiratory viruses was detected using the Seegene Allplex™ Respiratory Panel Assays 1,2,3 (Seegene).
Main study
The main study was conducted in respiratory wards and fever clinics of three selected hospitals in Beijing China from December 2017 to January 2018. Doctors and nurses from selected wards were invited to participate in the study. Participants include nursing and medical staff aged > 18 years working full time in the ward who were able to provide written and informed consent. Participants with pre-existing respiratory, medical illness or pregnancy were excluded. As we did not test the participants, detail history on respiratory symptoms was taken to rule out contamination of masks by participants themselves.
HCWs from the participating wards were asked to wear medical masks for a shift (6–8 h), or as long as they could tolerate the masks with no adverse event. Three layered standard medical masks were used. If HCWs used more than one mask during their shift, first sample was collected and tested. Used medical masks were collected at the end of the day and were stored immediately in zip-lock bags. HCWs were advised to store masks in in zip-lock bags while they take off the masks during break time. All masks samples were labelled with participants’ ID and hospital ID. At the end of the study, HCWs were asked to complete a short survey to collect information on mask use in routine (type of mask used, number of masks used and situations when masks were normally used) and during the study period (wearing time, number of patients seen, situations when masks were used, aerosol generating procedures performed and hand hygiene during donning and doffing). Participants reported “number of masks used” and “number of patients seen” in absolute numbers. “Duration of mask use” was recorded in hours as, < 1 h, 1 to 2 h, 2 to 4 h, 5 to 6 h, 7 to 8 h, > 8 h. “Situations when masks were used” were categorized into: “used continuously”, “used continuously except during breaks”, “used only during patients’ encounters” and “used only high-risk patient encounters”.
Mask testing for the main study
Medical masks were tested in the Beijing CDC laboratory. All masks were collected immediately after use in zip-lock bags and kept at − 80 °C until testing. Pilot studies showed that upper sections of masks were more contaminated (Figs. 1 and 2). The outer layers of upper right, middle and left mask were separated with a same size, placed into separated tubes containing 700 μl PBS buffer (Gibco, USA), vortexed for 1 min, and finally aliquoted 50 μl for viral testing. We performed three tests on upper right, middle and upper left sections of the masks on around a quarter mask sample (26%) and performed one test on the remaining mask samples (74%). For one testing, outer layers of upper right, middle and left section of mask were separated and placed into the same tube. Viral DNA/RNA was extracted using KingFisher Flex 96 viral DNA/RNA purification kit (Thermo Fisher, USA) according to the manufacturer’s instructions. The reverse-transcription polymerase chain reaction was performed to amplify 15 viral target genes, including influenza A/B virus, influenza A(H1N1) and A(H3N2), parainfluenza viruses 1–4, rhinoviruses, bocavirus, human metapneumovirus, adenovirus, respiratory syncytial virus, coronaviruses OC43, 229E, NL63 and HKU1 using a commercial multiplex combined real-time PCR detection kit for Respiratory virus, which is developed by “Jiangsu Uninovo Biological Technology Co. Ltd.” in China.
Sample size
Currently there is very limited data on testing of masks surface for presence of pathogens. In previous studies influenza virus was detected on over 50% of the fomites tested in community settings during influenza season [17]. The rate is expected to be higher in the healthcare setting and moreover other viruses will also be tested. Assuming 25% higher positivity rate in the healthcare setting, the required sample size would be 134 masks, with 80% power and two-sided 5% significance level for detecting a significant difference. Some HCWs might not be able to provide mask samples, we aimed to recruited 145 HCWs in total for this study.
Analysis
Descriptive analysis was conducted, and rates and frequencies were calculated. Univariate analysis was performed to identify the factors associated with mask positivity. Logistic regression was used to calculate odds ratio (OR) and 95% confidence intervals (CI) Data were analyzed in SAS (SAS Institute Inc., USA) version 9.4.
Ethics and consent to participate
Ethics approval for pilot study was sought from South Eastern Sydney Local Health District (SESLHD). Ethics approval for the main study was sought from Human Research Ethics Committee UNSW (HC16703) and IBR China. Written consent as obtained from all participants.