key: cord-0909033-3bwqbe5n authors: Dumont-Leblond, Nathan; Veillette, Marc; Bhérer, Luc; Boissoneault, Karine; Mubareka, Samira; Yip, Lily; Dubuis, Marie-Eve; Longtin, Yves; Jouvet, Philippe; McGeer, Alison; Duchaine, Caroline title: Positive no-touch surfaces and undetectable SARS-CoV-2 aerosols in long-term care facilities: an attempt to understand the contributing factors and the importance of timing in air sampling campaigns date: 2021-02-12 journal: Am J Infect Control DOI: 10.1016/j.ajic.2021.02.004 sha: 957b4d2cc98bf6ada387dcaa953b0f4e097bbeb3 doc_id: 909033 cord_uid: 3bwqbe5n BACKGROUND: Long-term care facilities (LTCF) are environments particularly favourable to coronavirus disease (SARS-CoV-2) pandemic outbreaks, due to the at-risk population they welcome and the close proximity of residents. Yet, the transmission dynamics of the disease in these establishments remain unclear. METHODS: Air and no-touch surfaces of 31 rooms from 7 LTCFs were sampled and SARS-CoV-2 was quantified by real-time reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: Air samples were negative but viral genomes were recovered from 20 of 62 surface samples at concentrations ranging from 13 to 36,612 genomes/surface. Virus isolation (culture) from surface samples (n=7) was negative. CONCLUSIONS: The presence of viral RNA on non-touch surfaces is evidence of viral dissemination through air, but the lack of airborne viral particles in air samples suggests that they were not aerosolized in a significant manner during air sampling sessions. The air samples were collected 8 to 30 days after the residents’ symptom onset, which could indicate that viruses are aerosolized early in the infection process. Additional research is needed to evaluate viral viability conservation and the potential role of direct contact and aerosols in SARS-CoV-2 transmission in these institutions. . People above 65 years old were 5 to 13 times more likely to be hospitalized and 90 to 630 times more likely to die from the disease than individuals between the age of 18 and 29 years old(3). The oldest and most frail seniors require hours of daily assistance and many reside in long-term care facilities (LTCFs) where outbreaks of viral respiratory (influenza) and gastrointestinal tract infections (norovirus) are common (4) . SARS-CoV-2 transmission in LTCFs has also been reported worldwide, including in the United States (5), the Netherlands (6) and Canada (7) . SARS-CoV-2 outbreaks are more likely to happen in confined/crowded congregate living spaces like LTCFs, nursing homes and prisons than traditional living spaces (8, 9) . People living in LCTFs generally have limited mobility, live in close proximity to each other, and require close contact with care personnel, leading to increased number of potential transmission events. Knowledge of SARS-CoV-2 spread is incomplete and it is still not clear how the virus is transmitted in LTCFs, particularly when recommended infection prevention strategies appear to be properly applied. Public health organizations recognized respiratory droplets and aerosols as major transmission routes for the virus (10, 11) . Although SARS-CoV-2 virus preserves infectivity for days on various surfaces (12, 13) and in the air (14) , no specific report clearly supports that COVID-19 can be transmitted via fomites, and it is not considered to be the main route of transmission (15, 16) . On the other hand, it was suggested that aerosols (short or long distance transmission) could be involved in the transmission of COVID-19 (17) (18) (19) (20) (21) . Indeed, it was reported that coughing, sneezing, talking or even breathing can lead to emission of SARS virus aerosols in both respirable and inhalable sizes (22) (23) (24) . Aerosols from various sizes (inhalable, thoracic and respirable) can be produced and enter the respiratory tract (25) (26) (27) (28) (29) . Since both SARS-CoV-1 and SARS-CoV-2 are phylogenetically highly similar, it seems possible that COVID-19 may also be spread by small particle aerosols (30) . Nonetheless, it is not clear how the emission of SARS-CoV-2 aerosols is modulated in both symptomatic and asymptomatic infected people. It seems that the earlier stages of COVID-19 is associated with emission rates as high as 10 5 viral RNA copies per min (31) . Accurate information about airborne concentrations of SARS-CoV-2 is still sparse and no standardized or reference sampling and detection methods have been validated for this purpose (32) . Published reports have used various approaches of air and no-touch surface sampling but experimental parameters are sometimes ill-defined, such as particle sizes and concentrations, air sampling and downstream processing (type of sampler, sampled volume, nucleic acid purification, real-time reverse transcription polymerase chain reaction (RT-qPCR)), environmental parameters (high/low risk areas, air exchange rates, sampler position/location), and presence and type of aerosols generating procedures (AGP). In addition, the contribution to viral aerosolization of common interventions in LCTF such as the use of continuous positive airway pressure (CPAP) machines or toilet flushing were not described in the COVID literature, nor was the impact of poor ventilation. All these limitations and differences in experimental approaches complexify interpretation and makes it difficult to generalize published knowledge to long-term-care facilities (32) . Nonetheless, these studies report positive air 5 samples and no-touch surfaces in healthcare settings (≈8% to 100% positive) (31, (33) (34) (35) (36) (37) (38) , suggesting SARS-CoV-2 may be aerosolized in COVID-19 LCTFs. Other than control and prevention measures such as personal protective equipment (PPE) and personal hygiene, appropriate ventilation should limit the spread of COVID-19 (39) . de Man et al. reported that inadequate ventilation in a nursing home building led to an outbreak that stemmed from aerosol transmission of SARS-CoV-2 (40) . It was also stated in the press that broken ventilation (100% recirculation) could have allowed the aerosols to concentrate and spread in the building causing at least 200 cases and 64 deaths among the 236 residents (41) . This prospective study was conducted to determine air and no-touch surface contamination by SARS-CoV-2 in LCTFs with COVID-19 outbreaks during spring 2020. Air and no-touch surface samples were simultaneously taken in COVID-19 positive patients' rooms. Contributing factors to the presence of SARS-CoV-2 aerosols in these healthcare settings were investigated and the outbreak calendar was obtained after the sampling visits. This paper adds knowledge that could help to limit propagation of COVID-19 among resident and healthcare workers in LTCFs. Seven LTCFs in major cities of the province of Quebec were visited during spring 2020 on a convenience basis. The willingness of the establishments to allow sampling guided these choices. Rooms of patients diagnosed with COVID-19, placed under droplet/contact isolation precaution, and cohorted in a dedicated -red‖ zone, were sampled. These red zones were floors or wards hosting only positive residents. The LTCFs observed no visitors allowance policy as well as standard infection control practices. No Table 1 ). Since these are living environments, as opposed to hospitals and critical care settings, symptoms monitoring was not rigorous, and these data could not be reliably included in this article. Only the LTCFs II, III, and V had a central ventilation system with intake and extraction vents in the corridor, but not inside the rooms (Supplementary Table 1 A total of 93 samples from 31 rooms hosting a patient were included in this study. Three samples were collected simultaneously for each room, one air sample and two surface samples. Table 1 illustrates the number of rooms sampled for each LTCFs, as well as additional information regarding the number of days since diagnosis of the patient and the number of days since the first case was confirmed in the LTCF in comparison to the sampling date. Air sampling was performed using an IOM Multidust sampler (SKC, Eighty Four, PA, USA) loaded with a 3 m gelatin filter (Sartorius Stedim Biotech, Gottingen, Germany). The samplers were attached to a portable pump Gillian Air 5 (Gillian, Sensidyne, USA) and calibrated at 3 L/min. Sampling was performed for 4 hours (total volume of air=720 L). They were put on furniture, at least 1.5m above the ground and placed approximately 2m from the resident to limit the capture of droplets. Filters were eluted on the day of sampling and stored at -80 ˚C until RNA extraction. Gelatin filters were dropped in 900 µL of viral transport media (VTM) (Redoxica, Little Rock, USA) at 37 ˚C until complete dissolution (less than 5 minutes). The solution was also divided in 400 µL aliquots and frozen immediately at -80˚C. Two surfaces of approximately 10 cm 2 were sampled for each room using flocked swabs (Puritan, USA). Swabs were humected in 1mL of VTM (Redoxica, Little Rock, USA) prior to sampling. They were eluted in the remaining liquid after surface sampling. The swabbed surfaces were unfrequently cleaned. Most swabs took a very dark color from the dust they collected. The elution liquid was divided in 400 µL aliquots and froze at -80 ˚C until RNA extraction. The top of the door frame inside the room of the resident and the top of a shelving unit were sampled. These areas are located between 2 and 4 meters from resident if bedridden and are considered not touched or cleaned on a frequent basis, which would limit the inference of surface contamination by residents or workers and act as a proxy for viral propagation in the environment through air. The sample treatment and quantification were described in Dumont-Leblond et al (42) . Briefly, the 400 µL aliquots of each type of sample was directly extracted using the MagMAX Viral RNA Isolation Kit (Applied Biosystems, Vilnius, Lithuania), following the manufacturer's instructions. The final elution volume was of 50 µL. Purified RNA was immediately quantified by RT-qPCR. Extraction controls (no template controls) were performed for each extraction batch. Nominal variables were expressed with frequencies and percentage (%) and were analyzed using Fisher's exact test. Continuous variables were reported as mean ±SD and analyzed using Student's t-test. The normality assumption was verified with the Shapiro-Wilk tests on residuals from the statistical model. The Brown and Forsythe's variation of Levene's test statistic was used to verify the homogeneity of variances. A mixed model, looking into the number of genomic copies, was performed to compare the viral yield of sample swabs between shelving units and door frames. A first factor was linked to the comparison between doors and shelves and was analyzed as a repeated-measure factor with the use of a generalized covariance structure. The rooms were analyzed as a random factor. We used residual maximum likelihood as the method of estimation and the Kenward-Roger method to estimate denominator degrees of freedom for the tests of the fixed effect. The normality assumption was verified with the Shapiro-Wilk tests after a Cholesky factorization on residuals from the statistical model. The Brown and Forsythe's variation of Levene's test statistic was used to verify the homogeneity of variances. Statistical analyses were adjusted for the number of days since the diagnosis or the beginning of the outbreak. Data were log-transformed to respect these assumptions. The results were considered significant with p-values < 0.05. All analyses were conducted using the statistical package SAS v9.4 (SAS Institute Inc., Cary, NC, U.S.A.). The source of aerosolized particles that led to no-touch surfaces contamination could be the patients themselves through respiratory droplets generation. However, other mechanism may be involved such as aerosolization through fecal matter manipulation during diaper changes/flushing toilets (43) . In addition, virus found in settled dust on no-touch surfaces can be re-aerosolized in the environment and deposited elsewhere from a surface to another. Patient-emitted aerosols and re-aerosolized particles cannot be differentiated in this study. When they are present, airborne particles can be inhaled and the virus can reach the respiratory tract (44) . SARS-CoV-2 could not be detected in any of the air samples. The residents' room were sampled from 8 to 30 days after they were first diagnosed with COVID- 19 . Evidence suggests that replication-competent virus in mild disease decreases after the symptom onset and that transmission happens more frequently within 5 days since the first symptoms (45) (46) (47) (48) (49) (50) . Concentration of the virus RNA in the upper respiratory tract is also known to decline after symptoms onset (49) (50) (51) (52) (53) . The air sampling campaign might have missed the window of time in which aerosols were more highly present in the rooms due to stronger resident shedding. Since aerosolization from patients may mostly rely 13 on sporadic events such as cough, the relatively short sampling time (4 hours) might have missed these events. Also, the possibility of underestimation caused by viral degradation during the sampling process cannot be completely discarded. However, a similar methodology was deployed in actively ventilated hospital rooms where airborne particles were detected (42) . A combination of poor timing and viral degradation may explain the lack of detection of aerosols in the presence of the patients, even in poorly ventilated rooms. Currently no standardized protocols for the study of airborne SARS-CoV-2 have been proposed or validated, leading to limited ability to compare studies. The sample collection protocol in this work was guided by previous literature and expertise on the study of viral bioaerosols and on the very few published articles at the beginning of the pandemic (38, 42) . To date, a consensus regarding a reproducible air sampling approach has not been reached even considering the various studies published to date (32) . However, these slight variations may obscure our ability to compare results. The difficulty to rapidly deploy sampling teams in this context of outbreaks constitutes a major limitation to this study, since they might have missed the pic level of viral aerosol production and exposition risks. Epidemiological data reporting long-distance transmissions of COVID-19 have yet to be published. The level of contribution of the airborne route of transmission of SARS-CoV-2 is still to be defined and new models of a broader airborne model involving inhalable aerosols for SARS-CoV-2 transmission in low-risk health care settings is to be considered (32) . Cumulative data and positive air and no-touch surface samples found in healthcare facilities suggest that airborne transmission does not occur only for smaller aerosols, but that some larger particles normally classified as droplets can remain airborne and be transported inside building such as LTCFs (32) . In addition, contamination of no-touch surfaces likely involve larger particles or droplets given their ability to settle. In that context, improper ventilation could contribute to viral accumulation in these environments (32) . SARS-CoV-2 could not be detected in air samples from residents' room in 7 different LTCFs from 8 to 30 days after symptoms onset. However, viral genomes were recovered from settled dust on no-touch surfaces, suggesting viral dissemination in the environment through air had happened previous to sampling. This could be an indication of the importance of timing between the patients' stage of infection and air sampling deployment. The collaboration of LTCFs is deemed crucial in future work in order to access these establishments in a timely manner and to allow the collection of environmental data in the potential peak of exposition risks. The authors have no conflict of interest to disclose. World Health Organization. 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