Six different respirators were evaluated; because of their scarcity, most of them were obtained from two local hospitals after they had been grommeted for fit testing. They included three molded and three pleated types. Molded types included the 1860, 8210 (3M Company, St. Paul, MN) and 1510 (Moldex, Culver City, CA) models; the pleated included the Aura 1870, Vflex 1804 (3M Company, St. Paul, MN) and Pleats Plus 1054 (Aearo Company, Indianapolis) models.
Heat Treatment of Respirators
To create a heating chamber akin to a hospital blanket warmer in a high containment (BSL-3) laboratory, a two shelved, 57 L Model BD 56 standard incubator (BINDER Inc., Bohemia, NY) with its temperature set at 70°C was used. A small pan (6 inch X 6 inch, 2 inch depth) filled with water was placed below the bottom shelf the night before the experiment to sustain relative humidity (RH) at the highest level achievable passively. Temperature and RH were recorded using EL-USB-2 Temperature & Humidity Data Logger, (Lascar electronics, Erie, PA). For fit testing and integrity testing, whole respirators were exposed to the moist heat by placing them on the shelves and leaving them there for a continuous 6 hours.
Quantitative Fit Testing
Quantitative fit testing was performed in a small room (500 ft3) using a PortaCount® Pro+ Respirator Fit Tester Model 8038 and FitPro+™ Fit Test software (TSI Incorporated, Shoreview, MN). An ultrasonic room humidifier (Honeywell, Charlotte, North Carolina) was used to generate aerosol particles for the testing. The quantitative fit test was performed as per CSA Z94.4-18 protocol, where the ratio of particles inside the respirator to the number of particles outside the respirator was determined to calculate the fit factor by the software. Seven well defined exercises were performed as part of this standardized test: normal breathing, deep breathing, turning head side to side, moving the head up and down, reading a standardized passage aloud, bending up and down, and normal breathing. A respirator that scores a fit factor of minimum 100 for each of the exercises and an average of 100 more was considered a pass[5,6].
Filter integrity testing
Metal grommets used for fit testing were sealed with glue on the outer and inner surfaces before testing. Filtration efficiency testing was performed using the NIOSH sodium chloride (NaCl) aerosol method on a TSI 8130A Automated Filter Tester[7,8]. Respirators were fastened to a 3mm thick aluminum disk using 3M 3792LM hot melt glue, and allowed to fully set for 20 minutes before being loaded into the TSI 8130A. Respirators were challenged for 5 minutes at a flow rate of 85±4 L/min with an aerosol of NaCl particles at a concentration not exceeding 200 mg/m3.
Assessment of SARS-CoV-2 Inactivation with Dry and Moist Heat Treatments
To determine whether several hours of exposure to dry or moist heat at 70°C would inactivate SARS-CoV-2, small swatches cut from each of the 6 respirators were surface contaminated with SARS-CoV-2 virus inoculum. The inoculum was prepared by mixing the virus in a standard tripartite organic soil load (bovine serum albumin, tryptone, and mucin) as per ASTM standard to mimic body fluids. Ten µl of the inoculum estimated to contain approximately 5.0 log TCID50 of SARS-CoV-2 was spotted onto the outer surface of each respirator swatch at 3 different positions. Following 60 minutes of drying, they were placed on the two shelves of the incubator and left there for 6 uninterrupted hours. Corresponding positive control respirator swatches were concurrently spotted with the same viral inoculum, dried under the biosafety cabinet for an hour, and processed for virus titer determination to account for the effect of drying on virus recovery.
Following heat treatment, virus was eluted from the respirator material by excising the spotted areas on each respirator swatch and transferring each into 1 ml of virus culture medium (DMEM with 2% fetal bovine serum and 1% penicillin-streptomycin). After 10 minutes of soaking and elution of the material by repeated pipetting, the entire eluate from each excised coupon was transferred to each well of a Vero seeded 6-well plate. Plates were incubated for up to 1 week for signs (CPE) of viral growth. Eluates recovered from positive control coupons were used for viral titer determination in TCID50 per Reed and Muench. Results for each treatment indicate mean + standard deviations of three biological replicates.
Temperature and RH recorded inside sterilization pouch and paper bag
In real-world application methods, individual N95 respirators would be placed in steam sterilization pouches or paper bags before placing in the warming cabinets. To mimic this, a temperature/RH logger was placed inside several types of bags in order to ascertain that a N95 respirator in a bag or bags in a warming cabinet would be exposed to the appropriate temperature-humidity profile. The first logger was placed inside a steam sterilization pouch (Chex-All® II Instant Sealing Pouch, Propper Manufacturing Company, Long Island City, NY) that was sealed before placing in the warming cabinet. Similarly, another logger was placed inside a single paper grocery-size bag (brown single-layer 140 GSM Kraft paper), its opening folded once and a piece of tape was used to keep the fold in place. In the third configuration, a logger was placed inside two bags; first by bagging in a lunch-size bag (brown single-layer 120 GSM Kraft paper), the opening of which was once folded closed, then placing the smaller bag inside another paper grocery-size bag as before, the opening of which closed in the same manner as before (figure 1).