2.7. Data Analysis
Virus titre in samples collected from nebulisers, AGI and Andersen samplers was calculated by taking averages of technical replicates (plaque assay performed in duplicate), to give PFU/mL; this was then multiplied by the total liquid volume of the sample to give total PFU.
Viable fraction (VF) values were calculated for each nebuliser assessed. The VF is the relationship of viable virus particles arising from the total number of particles nebulised. It is found from the total PFU collected in the AGI, divided by the total number of particles counted by the APS during the 5-min spray. The units are PFU/particle.
Spray Factors (SF) were calculated for each nebuliser assessed. The SF is a unitless ratio that defines a relationship between the viability of a challenge suspension in the nebulizer (Cneb, PFU/L) and the concentration of viable virus in the circulating aerosol (Caero, PFU/L) and is commonly used in animal aerosol challenge models [23,29,36]. The value obtained is system dependent, but it allows comparisons of stability between different agents used:SF=CaeroCneb
The concentration of virus in the circulating aerosol (Caero) is calculated by the formula:Caero=Cimp× VimpQimp× texp where Cimp is the concentration of virus (PFU/L), Vimp is the volume of collection fluid in the AGI (mL), Qimp is the sample flow rate of the AGI (L/min), and texp is the exposure time (min). Virus concentrations in Caero are converted to PFU/L to allow calculation against Cneb in PFU per litre of air sampled.
Calculation of Mass Median Aerodynamic Diameter (MMAD) size from Andersen data: Total PFU was used to calculate (in Microsoft Excel) cumulative percentage collected at each stage 1 to 6 as:Cumulative % of stage (n)=Σ[total pfu of stages (n→6)]Σ[total pfu stages (1→6)]×100
These percentages were plotted against the size cut-off for each stage, here the cut-offs for stages 1 to 6 are 7 µm, 4.7 µm, 3.3 µm, 2.1 µm, 1.1 µm, and 0.65 µm, respectively; and log10 transformed on both axes. A linear regression line was added and the particle size at 50% is then taken as the MMAD for that run. This took place in GraphPad Prism v9.