September 21 Results of Disinfection Methods for PPE Intended for Reuse

 September 21, 2020 Report

Experimental Approach

The process for data generation will involve laboratory-scale controlled disinfection efficacy testing and post-disinfection, material-functionality testing of selected PPE items exposed to various disinfection/cleaning methods. During the initial phases of the study, small coupons of the PPE material with inoculated viruses will be tested for efficacy of disinfection. The effective techniques will then be subjected to full scale PPE items and include wear testing and evaluation of functional integrity. The project will be conducted in the following successive phases:

Phase 1: Method Development. Development of virus surrogate inoculation technique(s) for contamination of selected PPE will be optimized for PPE items including investigating the use of soil loads/stabilizers to maintain adequate viral loads on the PPE items to evaluate disinfection efficacy. Method development will also evaluate sampling methods (e.g., extraction vs. cloth wipes) that will be used to quantify the virus on the PPE material and neutralizer effectiveness for quenching disinfection at the appropriate time points in the study. Quenching/neutralization of the disinfectant at the time of sample collection is necessary so that disinfection does not continue to occur for variable lengths of time while samples are being processed and analyzed.

Phase 2: Evaluation of selected PPE disinfection methods for reuse. The virucidal efficacy of selected disinfection technologies and different disinfection chemistries of commercial off-the-shelf (COTS) products will be evaluated on PPE materials representing different classes of protective equipment. In line with EPA’s Product Performance Test Guideline 810.2200: Disinfectants for Use on Environmental Surfaces Guidance for Efficacy Testing, a 3 Log reduction in virus will be used as the disinfectant performance metric in this study. Post-disinfection functionality testing will be performed using multi-step visual assessments and standard industry methods. Wear cycles simulating post-disinfection use will be included for selected PPE items.

Phase 3: Development of visual aids. After selection of disinfection technologies offering the best balance between efficacy and PPE material compatibility, a series of visual aids will be prepared to explain our experimental procedures and results to the general public.

Testing will be performed using two viruses - bacteriophages MS2 and Phi6 (Table 1). The SARS-CoV-2 virus is an enveloped virus, like Phi6, but infects eukaryotic cells not bacterial cells. Six different disinfection/cleaning technologies will be evaluated in this study including: Low Concentration Hydrogen Peroxide Vapor (LCHPV), laundering (simulated at bench scale initially and then followed by fully scale experiments), a combined ozone/Ultra Violet (UV) technology, and three COTS product categories: a diluted bleach spray (Bleach-COTS), 70% isopropyl alcohol wipes (IPA-COTS), and a quaternary ammonium compound based spray (QAC-COTS). These techniques will be coupled with up to nine different PPE items including N95 filtering facepiece respirators, homemade face mask/coverings, procedure masks, face shields, safety glasses, Tyvek^® coverall suits, medical scrubs, denim street clothing, and shoes depending on the known material compatibility of each item or capacity of each test method (Table 2). For example, surfactants are known to damage the filtration performance of the materials used to make N95 respirators and therefore they will not be tested with the laundering or COTS products. The test matrix was assembled with input from an interdisciplinary team of emergency responders, health care professionals, and researchers. The general testing procedure  will include small coupons of each PPE item being inoculated with the viruses and allowed to dry at ambient conditions followed by application of disinfection/cleaning methods according to manufacturer instructions, followed by neutralization of disinfectant and quantification of virus remaining.

Table 1: Virus Types used in this Study

Virus	Virus Description	Viral Host	Analysis Method
MS2 (ATCC 15597-B1)	Non-enveloped virus; bacteriophage	Escherichia coli	Plaque assay, E. coli C-3000 (ATCC 15597)
Phi6	Enveloped virus; bacteriophage	Pseudomonas syringae	Plaque assay, P. syringae LM2489

Table 2: PPE-Disinfection Method Test Matrix

PPE Category/Item	Disinfection method: LCHPV	Disinfection method: Laundering	Disinfection method: Bleach-COTS	Disinfection method:  IPA-COTS	Disinfection method: QAC-COTS	Disinfection method: Ozone/ UV
Specialized Respiratory Protection/N95 filtering facepiece respirator	x					x
Non-specialized Respirator Protection/Homemade face mask/covering	x	x	x	x	x
Non-specialized Respirator Protection/Procedure mask	x		x	x	x	x
Face and Eye Protection/Face shield	x		x	x	x	x
Face and Eye Protection/Safety glasses	x		x	x	x	x
Specialized Body Protection/Tyvek® suits (coveralls)		x	x	x	x	x
Non-specialized Body Protection/Medical scrubs		x				x
Street clothing (denim material)		x				x
Non-specialized Body Protection/Shoes	x	x	x	x	x	x

PPE coupons that undergo successful disinfection efficacy testing will be followed by functionality testing of full-sized PPE items. Standard methods for functionality testing do not exist for all items, but where applicable United States or international standards will be used. In brief, the functionality tests will consist of visual assessments (e.g., notation of discoloration, cracking, tearing, stretching, odors, and hazing) and technical assessments for the specialized PPE items. For example, this will include particulate filter efficiency testing (modified NIOSH Standard Test Procedure TEB-APR-STP-0059), tensile strength testing, and static advanced head form fit evaluation for N95 respirators as well as water resistance testing using hydrostatic pressure testing AATCC TM127-2017(2018)e for Tyvek^® coveralls.

Current Results

Method development is complete for the viral stability of Phi6 on denim, N95s, face shields, procedural masks, Tyvek^®, and shoe PPE material surfaces using liquid inoculation of the Phi6 virus. Small 2 x 4 cm coupons (triplicate for each PPE material) were inoculated with 10 μL of 2 x 10⁸ PFU/mL of Phi6 (liquid inoculation method) in 0.22 μm filter sterilized 10% beef extract in a phosphate-buffered saline (PBS) solution (for a target concentration of 2 x 10⁶ PFU/sample). The droplet was spread across the material surface. After 2 hours of drying time at ambient conditions, in individual covered petri dishes, samples were extracted in 10 mL of 10% Dey-Engley (DE) neutralizing broth and vortexed for 2 minutes continuously prior to plating using a soft top agar overlay method[1] using P. syringae as the bacterial host. Results in Table 3 demonstrate that considering a 1 Log detection limit, excluding shoes, it was possible to maintain a stable viral load of Phi6 on these materials that will allow for disinfectant testing that can demonstrate at least a 3 Log reduction in viral load. Tests for the remaining PPE items are still in progress in addition to aerosol inoculation methods.

Table 3: Phi6 2-hour Stability on PPE Materials

Material	Average Phi6 Sample Recoveries (Log PFU)	Standard Deviation Phi6 Sample Recoveries (Log PFU)
Inoculum Control	6.7	0.04
Stainless Steel Control	5.5	0.38
Denim	5.0	0.05
N95	5.1	0.54
Procedural Mask	6.5	0.05
Tyvek®	6.3	0.14
Face Shield	6.4	0.08
Shoe	3.4	0.44

Neutralizing method development is also complete for the Bleach-COTS, IPA-COTS, and QAC-COTS products as well as for the laundering method. The diluted Bleach-COTS solution was prepared using the U.S. Center for Disease Control’s (CDC’s) recommended recipe (80 mL household bleach [7.5% sodium hypochlorite] in 3.8 L) and was sprayed directly into 10 mL of 10% DE Broth using a general household-use cleaning bottle. Surface-based neutralization testing was adopted to mimic the decontamination procedure of small PPE coupons for QAC-COTS, IPA-COTS and laundering. In brief, this involved applying the products per manufacturer instructions to PPE items followed by extraction in 10% DE Broth and spiking the extracts with liquid inoculum at a target concentration of 2 x 10⁶ PFU/sample of Phi6, vortexing for 2 minutes and immediately plating using the soft agar technique. For all disinfectants, Phi6 concentrations remained stable indicating adequate neutralization of any residual disinfectants using 10% DE Broth. Phase 1 disinfection testing has been completed for PPE items in Table 3, paired with COTS disinfectants identified in Table 2. The diluted Bleach-COTS was spray-applied using a general household-use cleaning bottle, the QAC-COTS was a spray-based product applied using the manufacturers container (0.1% alkyl [50% C14, 40% C12, 10% C16] dimethyl benzyl ammonium saccharinate in 58% ethyl alcohol), the IPA-COTS was a wipe-based product with 70% IPA, and the laundering involved a delicate detergent (no enzymes, bleaching agents, or phosphates) for the Tyvek and a general household detergent for the other PPE items. Detergents were applied at manufacturer specified concentrations. The disinfectant contact time was 30 minutes (for the case of the IPA wipe, the residual from wiping was allowed to interact for 30 minutes). The results of these tests are provided in Table 4.

Table 4: Phi6 Liquid Inoculation PPE-Disinfection Results

PPE Item	Volume of disinfectant delivered per sample (mL/sample)	Positive Control Phi6 Recoveries (Log PFU)			Disinfected Coupons Phi6 Recoveries (Log PFU)		Phi6 Reduction* (Log PFU)
		Average		Standard Deviation	Average	Standard Deviation
Bleach-COTS
Stainless Steel Control	2.8a	6.0	0.08		Non-detect	Non-detect	≥ 5.0
Face Shield		5.5	0.20		Non-detect	Non-detect	≥ 4.5
Procedural Mask		5.6	0.27		Non-detect	Non-detect	≥ 4.6
Tyvek®		6.1	0.48		Non-detect	Non-detect	≥ 5.1
IPA-COTS
Stainless Steel Control	Not available  (1/4 of IPA wipe)b	5.2	0.2		2.5	0.8	2.7
Face Shield		5.7	0.2		4.4	0.2	1.3
Procedural Mask		5.7	0.1		4.8	0.3	0.9
Tyvek®		5.5	0.1		4.1	0.9	1.4
Shoe		3.9	0.2		2.8	0.7	1.1
QAC-COTS
Stainless Steel Control	0.51c	5.2	0.2		Non-detect	Non-detect	≥ 4.2
Face Shield		5.7	0.2		Non-detect	Non-detect	≥ 4.7
Procedural Mask		5.7	0.1		2.9	1.8	2.8
Tyvek®		5.5	0.1		1.2	0.3	4.3
Shoe		3.9	0.2		Non-detect	Non-detect	≥ 2.9
LAUNDERING
Stainless Steel Control	40d	4.7	0.34		Non-detect	Non-detect	≥ 3.7
Tyvek®		5.6	0.14		Non-detect	Non-detect	≥ 4.6
Shoe		3.8	0.51		Non-detect	Non-detect	≥ 2.8
Denim		4.5	0.1		Non-detect	Non-detect	≥ 3.5
a determined volumetrically prior to testing (n=3) b surface of each coupon was wiped two times (top to bottom); then wipe was folded and surface was wiped from top to bottom again c determined gravimetrically prior to testing (n=3); specific gravity of QAC-COTS spray is 0.8967 g/cm 3 at 25°C, per safety data sheet. d total volume of hot and cold wash detergent and water rinse cycles for simulated laundering using an orbital shaker *detection limit is 1 Log PFU

Table 4 indicates that the bleach-COTS product provided >3 Log reduction compared to controls for all PPE items tested thus far. The IPA-COTS product resulted in < 3 Log reduction for all PPE materials tested. The QAC-COTS product also provided viral reduction for all items tested, but log reductions were lower than 3 Log for the procedural masks and shoe items. For QAC-COTS with shoes, the maximum observable log reduction was limited to 2.9 Logs due to detection limits and initial viral load. All simulated laundering samples achieved >3 Log reduction except the shoe material. For simulated laundering with shoe material, the maximum observable log reduction was limited to 2.8 Logs due to detection limits and initial viral loads.

Several important observations occurred during Phase 1 method development tests. The first was that the spray method of disinfectant application provided a large volume of bleach in relation to the size of the PPE coupon swatches used (i.e., visual pooling. Volumes/sample is listed in Table 4). Future experiments will verify the efficacy of a smaller spray volume of bleach. The second observation was that when used under realistic use conditions, the wipe-based IPA-COTS product did not saturate the PPE coupons. This may have contributed to lower viral disinfection and conclusions should not be drawn regarding the efficacy of liquid IPA from these wipe results. Safety concerns for spraying IPA (flammability) limit its application to this research project. These results will be used in planning Phase 2 experiments. Immediate future work is planned to complete stability, neutralization, and disinfection for the remaining PPE and disinfectants listed in Table 2.