Study design

Participants were recruited year-round from March 2013 through May 2016 in a general outpatient clinic of a private hospital in Hong Kong. As routine practice, clinic staff screened all individuals attending the clinics for respiratory and any other symptoms regardless of the purpose of the visit at triage. Study staff then approached immediately those who reported at least one of the following symptoms of ARI for further screening: fever ≥37.8 °C, cough, sore throat, runny nose, headache, myalgia and phlegm. Individuals who reported ≥2 ARI symptoms, within 3 d of illness onset and ≥11 years of age were eligible to participate. After explaining the study to and obtaining informed consent from the participants, a rapid influenza diagnostic test, the Sofia Influenza A + B Fluorescent Immunoassay Analyzer (cat. no. 20218, Quidel), was used to identify influenza A or B virus infection as an incentive to participate. All participants provided a nasal swab for the rapid test and an additional nasal swab and a separate throat swab for subsequent virologic confirmation at the laboratory. All participants also completed a questionnaire to record basic information including age, sex, symptom severity, medication, medical conditions and smoking history. In the first phase of the study from March 2013 to February 2014 (‘Influenza Study’), the result of the rapid test was used to determine eligibility for further participation in the study and exhaled breath collection, whereas in the second phase of the study from March 2014 to May 2016 (‘Respiratory Virus Study’), the rapid test did not affect eligibility. Eligible participants were then invited to provide an exhaled breath sample for 30 min in the same clinic visit.

Before exhaled breath collection, each participant was randomly allocated in a 1:1 ratio to either wearing a surgical face mask (cat. no. 62356, Kimberly-Clark) or not during the collection. To mimic the real-life situation, under observation by the study staff, participants were asked to attach the surgical mask themselves, but instruction on how to wear the mask properly was given when the participant wore the mask incorrectly. Participants were instructed to breathe as normal during the collection, but (natural) coughing was allowed and the number of coughs was recorded by study staff. Participants were then invited to provide a second exhaled breath sample of the alternate type (for example if the participant was first assigned to wearing a mask they would then provide a second sample without a mask), but most participants did not agree to stay for a second measurement because of time constraints. Participants were compensated for each 30-min exhaled breath collection with a supermarket coupon worth approximately US$30 and all participants were gifted a tympanic thermometer worth approximately US$20.

Ethical approval

Written informed consent was obtained from all participants ≥18 years of age and written informed consent was obtained from parents or legal guardians of participants 11–17 years of age in addition to their own written informed consent. The study protocol was approved by the Institutional Review Board of The University of Hong Kong and the Clinical and Research Ethics Committee of Hong Kong Baptist Hospital.

Collection of swabs and exhaled breath particles

Nasal swabs and throat swabs were collected separately, placed in virus transport medium, stored and transported to the laboratory at 2–8 °C and the virus transport medium was aliquoted and stored at −70 °C until further analysis. Exhaled breath particles were captured and differentiated into two size fractions, the coarse fraction containing particles with aerodynamic diameter >5 μm (referred to here as ‘respiratory droplets’), which included droplets up to approximately 100 µm in diameter and the fine fraction with particles ≤5 μm (referred to here as ‘aerosols’) by the G-II bioaerosol collecting device12,15,19. In the G-II device, exhaled breath coarse particles >5 μm were collected by a 5-μm slit inertial Teflon impactor and the remaining fine particles ≤5 μm were condensed and collected into approximately 170 ml of 0.1% BSA/PBS. Both the impactor and the condensate were stored and transported to the laboratory at 2–8 °C. The virus on the impactor was recovered into 1 ml and the condensate was concentrated into 2 ml of 0.1% BSA/PBS, aliquoted and stored at −70 °C until further analysis. In a validation study, the G-II was able to recover over 85% of fine particles >0.05 µm in size and had comparable collection efficiency of influenza virus as the SKC BioSampler19.

Laboratory testing

Samples collected from the two studies were tested at the same time. Nasal swab samples were first tested by a diagnostic-use viral panel, xTAG Respiratory Viral Panel (Abbott Molecular) to qualitatively detect 12 common respiratory viruses and subtypes including coronaviruses (NL63, OC43, 229E and HKU1), influenza A (nonspecific, H1 and H3) and B viruses, respiratory syncytial virus, parainfluenza virus (types 1–4), adenovirus, human metapneumovirus and enterovirus/rhinovirus. After one or more of the candidate respiratory viruses was detected by the viral panel from the nasal swab, all the samples from the same participant (nasal swab, throat swab, respiratory droplets and aerosols) were then tested with RT–PCR specific for the candidate virus(es) for determination of virus concentration in the samples. Infectious influenza virus was identified by viral culture using MDCK cells as described previously21, whereas viral culture was not performed for coronavirus and rhinovirus.

Statistical analyses

The primary outcome of the study was virus generation rate in tidal breathing of participants infected by different respiratory viruses and the efficacy of face masks in preventing virus dissemination in exhaled breath, separately considering the respiratory droplets and aerosols. The secondary outcomes were correlation between viral shedding in nose swabs, throat swabs, respiratory droplets and aerosols and factors affecting viral shedding in respiratory droplets and aerosols.

We identified three groups of respiratory viruses with the highest frequency of infection as identified by RT–PCR, namely coronavirus (including NL63, OC43, HKU1 and 229E), influenza virus and rhinovirus, for further statistical analyses. We defined viral shedding as log 10 virus copies per sample and plotted viral shedding in each sample (nasal swab, throat swab, respiratory droplets and aerosols); the latter two were stratified by mask intervention. As a proxy for the efficacy of face masks in preventing transmission of respiratory viruses via respiratory droplet and aerosol routes, we compared the respiratory virus viral shedding in respiratory droplet and aerosol samples between participants wearing face masks or not, by comparing the frequency of detection with a two-sided Fisher’s exact test and by comparing viral load (defined as log 10 virus copies per sample) by an unadjusted univariate Tobit regression model, which allowed for censoring at the lower limit of detection of the RT–PCR assay. We also used the unadjusted univariate Tobit regression to investigate factors affecting viral shedding in respiratory droplets and aerosols without mask use, for example age, days since symptom onset, previous influenza vaccination, current medication and number of coughs during exhaled breath collection. We investigated correlations between viral shedding in nasal swab, throat swab, respiratory droplets and aerosols with scatter-plots and calculated the Spearman’s rank correlation coefficient between any two types of samples. We imputed 0.3 log 10 virus copies ml−1 for undetectable values before transformation to log 10 virus copies per sample. All analyses were conducted with R v.3.6.0 (ref. 22) and the VGAM package v.1.1.1 (ref. 23).

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.