Persistence of SARS-CoV-2 on N95 filtering facepiece respirators: implications for reuse

Methods Collection

Virus propagation

• Virus used: Severe Acute Respiratory Syndrome (SARS)-Coronavirus CoV-2 strain USA-WA1/2020 (WA1), obtained from BEI Resources (Manassas, VA).
• Working stocks had titers of approximately 106 tissue-culture infectious doses 50% [TCID50] per milliliter.
• Material stored as single-use vials at ≤ -80⁰

Test coupons

• Six NIOSH Approved® FFR models
  • Four NIOSH Approved and FDA-cleared Surgical N95 FFRs
    • 3M 1860 and VFlex 1804
    • Moldex 1512 and 2200
  • Two NIOSH Approved N95 FFRs
    • 3M 8210 and 8511
  • Non-porous controls – glass coupons (microscope cover slides)
  • Rectangular coupons (2×5 cm) taken from unused FFRs.

Coupon contamination and storage conditions

• Virus suspended in
  • Complete cell culture medium
  • Human saliva
• Ten droplets (10 µL each droplet) applied to coupons under ambient conditions (20⁰C–22⁰C and 30%–50% RH).
• Total deposition of SARS-CoV-2 was approximately 1×10⁵ TCID50 across the outer surface of the FFR coupon.
• Coupons were allowed to dry under ambient conditions.
• Transferred to brown paper bags and stored at 20⁰C–22⁰C and at 20% (15%–25%), 45% (30%–50%), and 75% (70%–75%) RH.
• Virus on the FFR coupons and glass slides were extracted after 0- , 1- , 24- , 48- , 96- , and 120-hr timepoints.
  • Additional assessments were performed after 4- , 6- , and 12-hr post-drying when more data points were needed.
  • Virus persistence was evaluated in triplicate for each tested surface, timepoint, and condition.
• Some conditions were not tested for all FFR models due to limited supply.

Virus extraction and analysis

• Coupons were removed from the paper bags and placed into individual 50-mL conical tubes containing a 10-mL extraction buffer.
• Starting volume was concentrated to approximately 0.5 mL.
• Media was added to equilibrate all washed retentates to approximately 2 mL.
• Virus viability was assessed by TCID50 assay in Vero E6 cells.
  • Samples inoculated in quintuplicate onto a single 96-well plate at 70% cell monolayer confluency.
  • Plates were incubated at 37 ± 2⁰C and 5 ± 2% carbon dioxide for 72 ± 4 hr.
  • Observed microscopically for cytopathic effects (CPE, visible morphological changes in cell cultures caused by viral infections).
  • Used to quantitatively calculate the viral titer for each sample.
• Extraction efficiency was assessed relative to a direct spiking and quantification of the extraction medium for
  • NIOSH Approved and FDA-cleared surgical N95 FFR (3M 1860)
  • NIOSH Approved N95 FFR (3M 8511)
  • Glass control surface