1. Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease of 2019 (COVID-19) has been a causative factor in the deaths of more than of 5.6 million people worldwide [1]. There is currently no firm consensus on its routes of transmission, as evidence exists that pathogenic SARS-CoV-2 aerosol particles across a distribution of sizes, play a major role, and that both droplets and aerosols are implicated [2,3,4,5,6,7,8]. “True” aerosol transmission is considered to occur from droplet nuclei, particles < 5 µm, which can remain suspended in the air indefinitely and can penetrate the lower lung [9]. Particles between 5 and 10 µm can travel over shorter distances (metres, depending on surrounding air currents), and be inhaled into the upper respiratory tract; whilst even larger particles from 10 to 100 µm, most likely play a role in either direct transmission [10], where droplets land directly onto mucosal membranes from the respiratory secretions of another person; or in indirect (fomite) transmission, where they settle onto surfaces subsequently touched, and contamination is transmitted to the mucosal membranes of the face, causing infection [11]. Previous work investigated the survival of SARS-CoV-2 on different surfaces [12,13]. The work reported here focuses on the generation and size characterisation of SARS-CoV-2 in aerosol particles.
Further understanding of the viability and particle deposition profiles of SARS-CoV-2 will add knowledge to the transmission potential of the virus. In addition, these data will aid in the development of more reliable animal models to recapitulate COVID-19 disease in humans; to aid understanding of disease diagnosis, pathology, and to evaluate new therapeutics. Thus far, the majority of SARS-CoV-2 animal studies have employed inoculation of mucous membranes to cause disease: intranasally, intratracheally, orally, intraocularly, or a combination of routes [14,15,16,17,18]. Whilst these studies have resulted in successful infection, they reflect only direct and indirect contact transmission, and not aerosol-acquired infection. Infection route has been shown to influence disease severity for SARS-CoV-2 [19,20], as well as other respiratory diseases [21,22]. Thus, methods of producing stable aerosols of virus with known particle size distribution is key to the development of more informative animal model of SARS-CoV-2 infection by the aerosol route of transmission.
Our studies investigated the viability and size distribution of particles created by a number of different nebuliser types; 3- and 6-jet Collison nebulisers; two medical nebulisers (normally used to deliver therapeutics) [23,24,25,26,27]; and two sparging liquid aerosol generators (1 inch and 90 mm SLAGs). In terms of nebuliser function, the “gold standard” Collison, created in 1973 [28], generates aerosols by applying an airflow (at around 26psi) to a liquid suspension of microorganisms; creating a vacuum which draws the suspension up a tube into which are engineered jets (between 1 and 24, depending on model type). The liquid suspension exits the jets at high force, creating particles which impact onto the surrounding glass jar and break up into smaller particles, for delivery into the test system. This method is designed to deliver near mono-disperse aerosols, in the size range of 1 to 3 µm [29]; but is considered “harsh” on microorganisms, applying both shearing and impaction forces which can render a proportion of the organisms non-viable. It also re-circulates the liquid suspension meaning that over longer spray times, the viability of the suspension can decrease. As SARS-CoV-2 is an enveloped virus, it is thought to be more prone to damage from the environment than unenveloped viruses. We therefore investigated nebulisers employing different aerosolization methods for comparison.
Of the medical nebulisers employed, the Omron MicroAIR U22 contains a finely perforated mesh, across which an electric current is applied. The current causes vibration of the mesh at 180 kHz, which aerosolises the liquid suspension sitting atop it, to produce fine particles for delivery to the lower respiratory tract [30]. The Pari LC Sprint Star utilises the Venturi principle, where compressed air draws liquids through a narrow orifice to impact on the inside of a tube. This impaction creates particles of varying sizes; the larger particles are removed by baffles, to generate a fine particle aerosol [31].
Sparging Liquid Aerosol Generators (SLAGs), are considered to be one of the “gentlest” forms of nebulisers, as they are thought to mimic natural aerosol generation in the respiratory tract more closely through the bursting of bubbles [32]. SLAGs function at low pressure by applying air to a perforated disc, wetted by the microbial suspension, creating bubbles; bursting of the bubbles generates the desired aerosols, with no shearing or impaction forces, nor any recirculation of the liquid. The larger disc diameter version allows for larger volumes of aerosol delivery [33].
An additional part of the study investigated whether an Andersen size-fractionating impaction sampler using novel virus collection methods, cell culture medium and gelatine filters, could efficiently measure the particle size distribution of aerosols of SARS-CoV-2 generated by a 6-jet Collison nebuliser.
The aims of these studies were to determine the survival of SARS-CoV-2 in aerosols generated by these nebulisers and to characterise their sizes; to allow the assessment of the risk of transmission, and generate data for future in vivo studies that use aerosol infection as the route of delivery.