Detection and isolation of infectious SARS-CoV-2 omicron subvariants collected from residential settings

Figures & data

Figure 1. A comparison of results from field samples in three categories: Total - (dark gray), PCR-positive - (light gray), and infectious samples (white). Swab samples are from high-touch surfaces, and all other samples are from air samplers. (a) Shows sample counts by Instrument. The BC-251 contains two cyclone and one filter sample collection stages. 66 individual BC-251 samples are shown here as 22 since all three stages from a single sampler were combined. Only the second BC-251 cyclone stage (1-4 μm particles) collected detectable virus. BioSpot-1 and BioSpot-2 refer to two separate instruments of the same model which, along with VIVAS, employ water-based condensation particle growth technology (b) Depicts sample positivity rates by Sampler Type. BioSpot-1, BioSpot-2, and VIVAS comprise the “Condensation” group based on their common particle collection mechanism. BC-251 samples fully comprise the “Non-Condensation” group. By Fisher’s exact tests, significant difference exists between these rates based on type of air sampler (p ≪ 0.05).

Figure 2. SARS-CoV-2 detected by RT-qPCR according to the Sampler Type used to collect air samples, depicted with (a) all data included and samples with non-detectable virus shown as zero-values, and (b) only samples where virus was detected. Significant p-values according to Wilcoxon rank sum tests are shown with asterisks, and “ns” indicates no statistically significant difference.

Table 1. Results from mixed effects hurdle models.

		Step One		Step Two
Concentration	Explanatory Variable	Odds Ratio	P-Value	Rate Ratio	P-Value
$\frac{\mathbf{GE}}{\mathbf{L} \mathbf{Air}}$	Sampler Type (Condensation)	0.0245	0.00164*	0.616	0.739
	Location (Secondary)	1.63	0.592	3.72	0.114
	Temperature	1.19	0.738	1.09	0.884
	Relative Humidity	1.46	0.540	0.652	0.600
	Variance due to Volunteer	0.810		1.20
$\frac{\mathbf{PFU}}{100 \mathbf{L} \mathbf{Air}}$	Location (Secondary)	0.813	0.761	2.01	0.332
	Temperature	0.896	0.754	0.463	0.0462*
	Relative Humidity	1.08	0.833	0.868	0.785
	Variance due to Volunteer	0.145		0.864

* p ≤ 0.05

The reference category is Non-Condensation for Sampler Type and Primary for Location. Temperature and Relative Humidity have been scaled and centered to correct for skewing caused by the magnitude of the raw data. PFU/L was multiplied by 100 to correct for flow-adjusted concentrations < 1 and facilitate use with the model. Step One of each model assessed the likelihood of failing to detect virus in a sample. Therefore, a smaller odds ratio means that including the parenthetical categorical variable or increasing a quantitative variable (Temperature, Relative Humidity) increases the chances of detecting virus. Step Two of each model estimated the impact of covariates on the average concentration of virus detected. Therefore, the rate ratio means that including a categorical variable or increasing a quantitative variable increases the average concentration of virus by the indicated amount.

Table 2. Wilcoxon rank sum tests results following pairwise comparisons within categorical explanatory variables.

Concentration	Data Considered	Explanatory Variable	Comparison	P-Value
$\frac{\mathbf{GE}}{\mathbf{L} \mathbf{Air}}$	All Data	Sampler Type†	Condensation – Non-Condensation	2.27E-05*
		Location	Primary – Secondary	0.518
		Instrument	BC-251 – BioSpot-1	1.81E-04*
			BC-251 – BioSpot-2	4.00E-03*
			BC-251 – VIVAS	3.26E-04*
			BioSpot-1 – BioSpot-2	0.287
			BioSpot-1 – VIVAS	0.683
			BioSpot-2 – VIVAS	0.187
	Only Detectable Samples	Sampler Type	Condensation – Non-Condensation	0.616
		Location	Primary – Secondary	0.941
		Instrument	BC-251 – BioSpot-1	0.667
			BC-251 – BioSpot-2	0.905
			BC-251 – VIVAS	0.629
			BioSpot-1 – BioSpot-2	0.628
			BioSpot-1 – VIVAS	0.927
			BioSpot-2 – VIVAS	0.610
$\frac{\mathbf{PFU}}{\mathbf{L} \mathbf{Air}}$	All Data	Location	Primary – Secondary	0.601
		Instrument	BC-251 - BioSpot-1	8.27E-05*
			BC-251 - BioSpot-2	1.00E-03*
			BC-251 - VIVAS	8.58E-07*
			BioSpot-1 – BioSpot-2	0.361
			BioSpot-1 - VIVAS	0.798
			BioSpot-2 - VIVAS	0.153
	Only Culturable Samples	Location	Primary-Secondary	0.153
		Instrument	BioSpot-1 – BioSpot-2	0.556
			BioSpot-1 - VIVAS	0.413
			BioSpot-2 - VIVAS	1.000

^† Indicates significant results according to the hurdle models, *p ≤ 0.05.

Annotated Sampler Type and Location results expound on significant results from hurdle models. Sampler Type is not shown for viable virus because only condensation samplers collected virus that was isolated. Instrument results are included because that category could not be included in the hurdle models due to collinearity with Sampler Type.

Figure 3. SARS-CoV-2 detected by RT-qPCR according to the occupancy frequency of the sampling location, depicted with (a) all data included and samples with non-detectable virus shown as zero-values, and (b) only samples where virus was detected.

Figure 4. Viable SARS-CoV-2 isolated in Vero E6 cells according to the occupancy frequency of the sampling location, depicted with (a) all data included and samples with non-detectable virus shown as zero-values, and (b) only samples where the culturing of viable SARS-CoV-2 was successful.

Figure 5. Resultant concentration of SARS-CoV-2 detected by RT-qPCR in surface swab samples, shown with all sample data included and undetectable virus concentrations plotted as zero-values (All Samples) and with only samples containing detectable virus concentrations (Positives).

Table 3. Detection of SARS-CoV-2 and culture of viable virus from surface swabs.

Volunteer	Type	Item(s)	Area (cm^2)	Location	GE/cm^2	PFU/cm^2
1	Single	Mobile Phone	93.5	Primary	2.32E + 03	ND
1	Single	Computer	232.3	Primary	4.09E + 02	ND
4	Single	Mobile Phone	90.3	Primary	4.29E + 04	1.83
5	Composite	Mobile Phone & Computer Desk	158.0	Primary	4.12E + 03	ND

Of the 38 total swab samples, only the four samples containing virus detectable by RT-qPCR are shown. “ND” indicates that infectious virus was not found in the sample.