Evaluation method of mask filtration efficiency
Prevention of infection with airborne pathogens and exposure to airborne particulates and aerosols (environmental pollutants and allergens) can be facilitated through use of disposable face masks. The effectiveness of such masks for excluding pathogens and pollutants is dependent on the intrinsic ability of the masks to resist penetration by airborne contaminants. This study evaluated the relative contributions of a mask, valve, and Micro Ventilator on aerosol filtration efficiency of a new N95 respiratory face mask.
Viruses and cell lines
Influenza A (H1N1) virus, strain A/PR/8/34 was obtained from Charles River Laboratories (Horsham, USA). Rhinovirus type 14, strain 1059 (ATCC VR-284) was obtained from the American Type Culture Collection (Manassas, USA). The Madin-Darby canine kidney cell line (MDCK, ATCC CCL-34) and the H1-HeLa cell line (ATCC CRL-1658) were also obtained from the American Type Culture Collection.
The test mask is an N95-rated (16) respiratory face mask comprised of the following layers, from outer to inner (Figure 2): an outer layer constructed of hydrophobic non-woven polypropylene that prevents external moisture from entering the mask material, followed by two layers of melt-blown non-woven polypropylene that capture oil and non-oil based particles through four key mechanisms. These include: (I) inertial impaction; (II) interception; (III) diffusion; and (IV) electrostatic attraction (16). The next layer is a modacrylic support layer that provides rigidity and adds thickness to the mask, giving it more structure and adding to the feel of comfort. The innermost layer is another hydrophobic non-woven polypropylene layer which minimizes moisture within the mask from entering the mask material and adversely impacting filtration efficiency. The mask samples were supplied to INSPEC Certification Services (Greater Manchester, UK), and Nelson Laboratories (Salt Lake City, USA) by Innosparks Pte Ltd. (Singapore) for a portion of the testing, while RB (China) provided mask samples to Microbac Laboratories (Sterling, USA) for additional testing.
Assessment of influenza A virus and rhinovirus type 14 penetration
An aerosol filtration test apparatus (Figure 3) assembled at Microbac per modified ASTM F2101-14 (12) was used for the influenza and rhinovirus penetration study. Test masks (the mask with operating Micro Ventilator, the mask with operating Smart Valve but without Micro Ventilator, or the mask with the Smart Valve covered/sealed) were placed between upstream and downstream chambers. The virus filtration efficiency test was performed in triplicate (N=3). For each run, a six-jet Collison nebulizer (Mesa Labs, Butler, USA) was filled with a measured amount of virus suspended in 0.1× Minimum Essential Medium (MEM) and the virus was aerosolized and delivered into the upstream chamber using high-pressure air. A downstream vacuum was turned on to create an air flow (28.3 L/min) through the mask that was intended to mimic human breathing (17). After the delivery of the aerosol, the upstream air pressure was turned off and the downstream vacuum pump was left on for another minute to pull residual aerosol from the chambers into the one-stage Andersen sampler. Virus aerosol that passed through the mask was captured on a Petri dish containing semi-solid medium (5% gelatin/minimal essential medium). The collected sample was liquefied at 36±2 °C for approximately 10 min. The resulting samples were divided into separate portions for ribonucleic acid (RNA) extraction and for infectivity assays (18).
Viral infectivity was measured on the basis of cytopathic effect (CPE) generated in MDCK cells (for influenza virus) and H1-HeLa cells (for rhinovirus). For the infectivity assay, a ten-fold dilution series of the samples collected as described above was prepared in a dilution medium. The sample dilutions were then inoculated onto the host cells. After 4–9 days of incubation, the CPE was scored under a phase-contrast light microscope. Viral titers were calculated in units of log10 50% tissue culture infectious doses (TCID50) per mL according to Spearman-K?rber (19).
For the qRT-PCR assay, RNA was extracted using a Qiagen QIAamp? Viral RNA Mini kit following the manufacturer’s instructions. The RNA was analyzed using primers and probes specific to each virus. For influenza H1N1 virus, the forward primer was 5’-GAC CRA TCC TGT CAC CTC TGA C, the reverse primer was 5’-AGG GCA TTY TGG ACA AAC GTC TAA, and the probe was 5’-(FAM) TGC AGT CCT CGC TCA CTG GGC ACG (BHQ). For rhinovirus 14, the forward primer was 5’-GAG GTG TGC TGT GTG CTA CT and the reverse primer was 5’-GAC TTG GTT GGC GTG TTG AC.
Assessment of S. aureus and bacteriophage ΦΧ174 penetration
Bacterial and phage filtration efficiencies for the masks without Micro Ventilator were determined at Nelson Laboratories per EN 14683:2014 (14) and ASTM F2101-07 (13), respectively. The purpose of this testing was to assess the filtration efficiency of the filter material used in the test mask (Figure 2).
The bacterial filtration efficiency test compared the upstream bacterial control counts to downstream counts (i.e., counts attributed to passage of bacteria through the face mask). A suspension of S. aureus was aerosolized using a six-jet Collison nebulizer and delivered to the face mask (N=5 replicate measurements) at a constant flow rate (28.3 L/min) and challenge delivery [1.7–2.7×103 colony forming units (CFU)] with a mean particle size (MPS) of 3.0±0.3 ?m. The aerosolized droplets were drawn through a six-stage Andersen sampler for collection.
The viral filtration efficiency test compared the upstream bacteriophage control counts to downstream counts (i.e., counts attributed to passage of bacteriophage through the face mask). A suspension of bacteriophage ΦΧ174 was aerosolized using a six-jet Collison nebulizer and delivered to the face mask (N=5 replicate measurements) at a constant flow rate (28.3 L/min) and challenge delivery [1.1–3.3×103 plaque forming units (PFU)] with a MPS of 3.0±0.3 ?m. The aerosolized droplets were drawn through a six-stage Andersen sampler for collection.
Assessment of paraffin oil and sodium chloride penetration
Filter penetration by the paraffin oil method for the test masks with operating Micro Ventilator was evaluated at INSPEC Certification Services per EN 149:2001 + A1:2009 (15) using a modified Phoenix SG-20 aerosol generator with detection using a photometer. The purpose of this evaluation was to provide evidence that the test mask configured with each of the features designed to enhance comfort would satisfy the requirements of this Standard. Per the Standard, the median diameter of the generated particles must be 0.6 ?m (15). Three replicates of the mask were evaluated under “as received”, “simulated wearing”, and “mechanical strength and temperature conditioning” conditions per the Standard (15). The methodology is equivalent to that specified by the National Institute for Occupational Safety and Health (NIOSH) for rating masks. For instance, an N95-rated mask removes 95% of a 0.3-?m (mass median aerodynamic diameter) particle sodium chloride aerosol and is not resistant to oils (16).
Single-factor analysis of variance (ANOVA) was used to determine the statistical significance of differences in the mean filtration efficiency values obtained for different test microorganisms, or in particulates testing, the differences between “as received” and other conditions (see above). A P value <0.05 was considered to be statistically significant. The significance of differences among mean values for different test mask configurations for a given virus was evaluated using a two-tailed t-test.