Study Design and Participants.
This was an open-label, randomized (1:1) clinical trial to assess the effectiveness of a comprehensive intervention to prevent SARS-CoV-2 spread during an indoor live concert. Study participants were recruited from a list of subscribers to news related to live music events; a call for enrolling in the study was performed through non-official media like WhatsApp, Telegram, or email. Eligible participants were adults aged 18 to 59 years with a negative result in an Ag-RDT performed on a nasopharyngeal swab collected immediately before entering the event. Participants with known COVID-19 diagnosis within the 14 days before the event, relevant comorbidities, or living with older people were excluded. Supplementary Appendix 2 provides a complete list of selection criteria.
SARS-CoV-2 screening and randomization
From 9 hours before starting the event, the healthcare staff collected nasopharyngeal swabs from all eligible participants. The same nasopharyngeal specimen was used to perform in situ Ag-RDT (Panbio™ COVID-19 Ag Rapid Test, Abbott) and a transcription-mediated amplification test (TMA, Procleix Panther®, Grifols). The TMA result was reported 24 to 48 hours after ending the event. All TMA-positive samples were re-tested by RT-PCR. The day after releasing the result, all TMA-positive individuals were contacted by phone for a structured interview by a physician, and their electronic medical records were screened to identify the exact date of a prior positive SARS-CoV-2 diagnostic test. All swabs with positive TMA results were assessed for viral isolation on cell culture. All study participants were visited 8 days after the event for nasopharyngeal swab collection and TMA test (follow-up day 8 test).
Study participants with a negative result on Ag-RDT were randomly assigned 1:1 to either enter the indoor live event (experimental arm) or not entering the event and returning to normal life (control arm). The computer-generated block randomization (REDCap™ module) was stratified by age, gender, and previous COVID-19 in the questionnaire.
Indoor event procedures
All participants installed two smartphone applications (apps). The “Radar Covid” (a contact tracing app) was intended to capture close contacts of subjects potentially infected during the concert. The “Test-Wallet” app was used to confidentially report the study test results (i.e., Ag-RDT, TMA, and PCR) and fill a health questionnaire before and ten days after the event, and a satisfaction questionnaire for those attending the concert (Supplementary Appendix 3). Data generated by the Test-Wallet app were encrypted using SHA-1 encryption and with 256-bit SSL security certificate. All SARS-CoV-2 positive results were reported into the public health electronic system and triggered quarantining measures and contact tracing studies.
All participants received an N95 mask at the venue entrance. Mask wearing was mandatory during the entire event. No physical distancing was required in the concert room (with a capacity of 900 persons); singing and dancing were permitted. A smoking area was set outdoors; the area had 20 people capacity and strict control of crowding and physical distancing.
Drinks―including alcoholic beverages―were served only in the bar zone, located in a supplementary room with a capacity for 1,600 people. Participants were asked to remove their face mask only when drinking. People movements inside the venue were previously defined and signaled. Securities personnel oversaw all movements and took actions―if necessary―to prevent queues in and around the venue foyer and toilets. Hydroalcoholic hand sanitizer gel was provided at multiple points in the venue.
The temperature of the dancing room and bar were maintained between 19.3 and 20.4 ºC during the event to facilitate wearing the mask and coats (the cloakroom was closed to avoid queues in front of it). Average CO2 measurements before starting the event were 440 ppm in the dancing hall and 417 ppm in the bar room, both similar to those typically obtained on open air in the city. Public health safety guidance in force at the time of the event recommended not exceeding 1,000 ppm during the event.
The total surface of the venue was 1.024 m²: 228 m² for the dancing hall, 381 m² the bar hall, and 157 m² in the lobby. There were no outer windows in the two respective halls; however, all access and exit doors remained open during the event, allowing additional fresh air from the inner courtyard.
The event, held in the Sala Apolo (Barcelona, Spain) on December 12, 2020, lasted for five hours and included four performances: two DJ sessions and two live music acts. Besides the study participants and artists, 58 staff members (organizers, security, sound, light technicians, and bartenders) were inside the venue during the event. All of them were tested for SARS-CoV-2 using Ag-RDT at the same time points as study participants
Nasopharyngeal specimens were collected with flocked swabs in a viral universal transport medium (Deltalab S.L, Barcelona, Spain). Samples were received at the laboratory and were processed immediately, inactivated, and analyzed by TMA. All TMA-positive results were confirmed by RT-PCR assay to determine the cycle threshold (Ct) values (AllplexTM® SARS-CoV-2, Seegene) using the software designed by the company. Leftover positives samples were conserved at -80°C.
Nasopharyngeal specimens with positive SARS-CoV-2 TMA results despite testing negative on Ag-RDT were analyzed for viral isolation on cell culture. Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen®). Two individuals with negative SARS-CoV-2 RT-PCR, and two viral stocks previously isolated were cultured in triplicates as negative controls as previously described12. DMEM supplemented with FBS and Pen/Strep were supplied to cells and inspected every 2 days for cytopathic effects. On day seven, cell supernatants were assayed with a high-sensitivity quantitative ELISA for SARS-CoV-2 nucleocapsid protein (ImmunoDiagnostics®).
The study was approved by the Ethics and Clinical Research Committee of the Hospital Universitari Germans Trias in Badalona, Spain. All subjects signed electronically an informed consent (Signaturit®). The study was conducted according to the Declaration of Helsinki and local legislation and is registered at ClinicalTrials.gov: NCT04668625.
Anonymized data are fully available upon reasonable request from the corresponding author after approval by the hospital Ethics Committee.
The primary efficacy endpoint was the difference in incidence of RT-PCR-confirmed SARS-CoV-2 infection at 8 days between the control and the intervention groups. It was assessed in the full analysis set, which included all participants who were randomly assigned, attended the event (in the experimental arm), and had a valid result in the day 8 follow-up SARS-CoV-2 test.
We used a Bayesian beta-binomial model to analyze the number of infected cases in each group. This approach allows prior information to be included, which is useful when estimating the probability of rare events13,14.
The seven days cumulative incidence observed in the city of Barcelona when the second RT-PCR was carried out was around 1.3 per thousand people, according to official data.15 However, this value underestimates the true rates due to the difficulty in recording asymptomatic cases. On the other hand, the study population had some exclusion/inclusion criteria that could influence this value. For the control group, the prior distribution chosen was a Beta(1.1, 400), with median 0.002, and the probability of values greater than 0.01 is approximately 2%. Uncertainty about the probability of infection among the experimental group was higher, and a Beta(1, 28.4) was chosen, with a probability of obtaining values greater than 0.1 around 5% as a prior distribution. For each group the posterior median and the highest posterior density interval were calculated. In addition, to compare the probabilities of infection between groups, the difference in their probabilities and its credible interval (CI) were calculated.
A Bayesian analysis was performed to estimate the negative predictive value of the antigen test vs the RT-PCR test and versus a viral cell culture16. For the prevalence, a Beta(1.1, 400) was chosen again as a prior distribution, and the sensitivity and specificity are given with a non-informative Beta(1 1) priors. The median and the highest posterior density CI were calculated for the negative predictive value.
All analyses were performed using R version 4.0.3.