3. Results

3.1. Nebuliser APS Readings
All nebulisers produced particles in the respirable size range, as measured by the APS, Table 2. MMAD values tended to be smaller for each nebuliser at the lower RH range, but this was not statistically significant (p > 0.05). Across all RH values, aerosol particles with the lowest MMAD were produced from the PARI Sprint Star, compared to the Omron (p = 0.0012) and the 1 inch SLAG (p = 0.0132); no other MMAD results were significant. The PARI SprintStar also had the lowest level of variance in the size distribution, calculated by geometric standard deviations (GSD) (ca. 1.6); indicating production of mono-disperse aerosols of ca. 1.2 µm diameter from this nebuliser. The nebuliser which produced the widest distribution of particle sizes was the 1 inch SLAG (GSD of ca. 2); indicating high polydispersity. This was only significant compared to the GSDs of the Omron (p = 0.0018) and Sprint Star (p = 0.0134).

3.2. Nebuliser PFU Counts
Viable virus was consistently recoverable from the AGI and from all six nebuliser reservoirs following aerosolization for 5 min, across three different relative humidity ranges. The total PFU collected as an average (with standard deviation) across all RH values, were, in descending order: PARI SprintStar 2.76 × 105 (s.d. 5.06 × 104), SLAG 90 mm 2.26 × 105 (s.d. 3.92 × 105), Collison 6-jet 1.49 × 105 (s.d. 1.41 × 105), Collison 3-jet 1.21 × 105 (s.d. 3.47 × 104), Omron MicroAIR U22 1.00 × 105 (s.d. 8.99 × 104) and SLAG 1 inch 1.39 × 104 (s.d. 2.16 × 104). From these data, VF values of SARS-CoV-2 aerosols have been calculated, to estimate the impact on viability of physical forces experienced during nebulisation, with lower values indicating a larger impact on viability. Figure 2 shows that median VF values across all RH values were between 6.7 × 10−6 (Collison 6 jet) to 2.6 × 10−2 (90 mm SLAG). Overall, the SLAG nebulisers resulted in higher VF values (suggestive of their gentler mode of action on aerosol generation), compared to the four other nebuliser types, though only the 90 mm SLAG results were significantly higher than the 3-jet and 6-jet Collisons (p = 0.010). Humidity did not significantly impact VF values (p = 0.177), but values were slightly less variable at lower RH, with coefficients of variation for high, intermediate, and low RH of 206.3%, 235.2%, and 125.5%, respectively.

3.3. Spray Factors
Spray factor values were also calculated across all RH values; the PARI Sprint Star produced the highest SF, with the 3- and 6-jet Collisons and the Omron within 1 log10 of this; SF for the SLAGs were the lowest of all. For each nebuliser the median and standard error are given below (in descending order): PARI SprintStar 2.77 × 10−6 (1.83 × 10−5); Collison 6-jet 2.23 × 10−6 (1.97 × 10−6); Collison 3-jet 1.85 × 10−6 (8.70 × 10−7); Omron MicroAIR U22 1.66 × 10−6 (1.20 × 10−6); 90 mm SLAG 6.45 × 10−7 (4.31 × 10−7); 1 inch SLAG 3.19 × 10−8 (7.5 × 10−8). The SF generated from the PARI SprintStar was significantly higher than that from the 1 inch SLAG, (p = 0.012); but no other SF’s differed between nebulisers. Humidity did not significantly impact SF values (p = 0.609).

3.4. Andersen Sampling Efficiency
The novel virus collection methods employed on the Andersen sampler were comparable to each other in overall efficiency, and particle size fractionation capability, when SARS-CoV-2 was aerosolised from a Collison 6-jet nebuliser. Figure 3 shows the comparison in biological sampling efficiency for the three sampling methods employed. For all three, the majority of SARS-CoV-2 particles were collected on the lower stages of the sampler, <3.3 µm. The liquids in the AGI samplers, collected from the Collison 6-jet (first study), contained 4.48 × 103 pfu/L, (standard deviation 2.39 × 103), which is comparable to the PFU/L collected by the Andersen methods: total PFU recovered across all stages, per litre of air sampled (with standard deviations) were: 27mL cMEM, 3.8 × 103 pfu/L (s.d. 2.7 × 103); 7 mL cMEM, 7.1 × 102 pfu/L (s.d. 6.9 × 102); and Gelatine, 2.0 × 102 pfu/L (s.d. 1.4 × 102). The total PFU/L across the three Andersen methods and AGI sampler were not significantly different (p = 0.241).

3.5. Andersen MMAD Results
The PFU collected across each stage allowed the calculation of MMAD for each media type. These numbers correspond well to the MMADs as measured by the APS (Table 3). The GSD, as measured by the APS, denotes polydispersity of particles around 1.7 µm, from the 6-jet Collison, which corresponds to the earlier measurements at different RH ranges (Table 3). The collection methods are not statistically different from each other, in terms of MMAD.