Wipe Disinfection of Reusable Elastomeric Half-Mask Respirators for Health Care Use

Microbes

Sets of three EHMRs (Honeywell model RU8500) were inoculated with organisms chosen as surrogates representative of different classes of health care-acquired infections (HAIs), including gram-negative bacteria, spore-forming bacteria, and viruses. Although respirator use protects against respiratory pathogen exposure, common HAI organisms are still present during respiratory disease outbreaks. Therefore, surrogate organisms in this study were chosen for two reasons: (a) to represent respiratory pathogens that might be expelled onto a HCP’s respirator by a patient during care and (b) to represent HAIs that are commonly present in the health care environment and can pose a risk of transfer to respirators via contaminated hands and gloves during handling. In laboratory studies, P. aeruginosa, itself a cause of HAIs, was chosen to also represent gram-negative HAIs such as Klebsiella and E. coli. Bacillus atrophaeus was chosen to represent the gram-positive spore-forming pathogen Clostridioides difficile. Two bacteriophages served as surrogates for human viruses. Φ6 was a surrogate for enveloped respiratory viruses such as SARS-CoV-2 and MS2, a non-enveloped virus, was chosen as a conservative surrogate to represent human viruses that are more resistant to disinfection and environmental stressors. In the volunteer study, Raoultella terrigena was chosen as a surrogate that represented gram-negative HAIs while posing minimal risk to study participants handling respirators.

There is variation in methods for respirator disinfection testing across studies; methods in this study were adapted from Lawrence et al. (Lawrence et al., 2017). EHMRs were dried at room temperature for 30 min before disinfection. Microbes were suspended in trypticase soy broth, (target concentration 10⁷–10¹⁰ plaque forming units/milliliter [PFU/mL] for viruses and 10⁸ colony forming units/milliliter [CFU/mL} for bacteria). Four sites were inoculated with 50 µL of suspension on each site: nose 2 cm directly above the vent, center back of the outward-facing side of the strap, and top edge of the right and left filter covers. These sites were chosen as locations where respiratory droplets from a coughing or sneezing patient might land (nose, filter covers), and where a person would be likely to touch the respirator during wearing or handling (filter covers, strap). The 50 µL was placed on each of the sites in ten 5 µL drops to simulate the deposition of droplets on the respirator surface. The suspension was inoculated in droplets on discrete locations on the respirator to simulate the expected deposition of respiratory secretions from a cough or sneeze, which would most likely not uniformly coat the respirator surface. EHMRs were dried at room temperature for 30 min before disinfection to simulate microbes sitting on the respirator surface for the length of a patient care encounter.

Disinfecting Wipes

Wipes were EPA-approved hospital disinfectants with combinations of hydrogen peroxide (HP), alcohols, and quaternary ammonium compounds (QAC): 0.5% HP (Oxivir^® TB Wipes; Virox Technologies Inc.), 0.76% didecyldimethylammonium chloride+7.5% ethanol+15% isopropanol (CaviWipes1; Metrex Research), 0.4% benzalkonium chloride (The Big Ones; Allegro Industries), and 0.9% sodium chloride+benzalkonium chloride (Hygéa Sterile saline Wipes; Professional Disposables International, Inc.). Respirators were disinfected by the same person, using a standard procedure with five wipes. They sat at room temperature for manufacturer-recommended contact time (1 min for 0.5% HP and 0.76% didecyldimethylammonium chloride+ethanol+isopropanol). There was no manufacturer-provided contact time for sodium chloride+benzalkonium chloride, so 2 min was used based on contact time for 0.4% benzalkonium chloride.

Recovery and Assay

Sites were sampled post-disinfection by polyester swab premoistened with neutralizing buffer. Swabs were suspended in a neutralizing buffer, vortexed for 30 s, and plated on trypticase soy agar for P. aeruginosa and B. atrophaeus. Φ6 and MS2 were enumerated by the single agar layer method with hosts P. syringae and Escherichia coli (ATCC#15597). All were incubated overnight at 37°C.

Statistical Analysis

Results were expressed as log₁₀ reduction, a standard measure for testing the effectiveness of disinfectants against microbes. Log reductions measure the remaining viable microbes on a respirator site relative to the starting concentration of viable microbes at that site. The reduction was expressed as log₁₀(N1/N0), where N1 is the contaminant concentration on the respirator after disinfection and N0 is the concentration in the inoculum applied to the respirator before disinfection. The detection limits of experiments were the maximum observable reduction of microbes in an experiment, calculated as log₁₀(1/N0) for each organism from each EHMR. The use of the detection limit makes it possible to quantify the theoretical log reduction from an experiment where no viable microbes are remaining on a surface. This reflects the fact that the measurable reduction of a microbe by a disinfectant is always relative to the starting quantity. For example, if 500,000 viable organisms (5.7 log₁₀) are inoculated at a site, the smallest measurable number remaining at that site is one organism. Therefore, the maximum log₁₀ reduction that can be experimentally measured is 5.7 log₁₀. If there are no microbes remaining, then the reduction achieved is ≥5.7 log₁₀. This allows the calculation of a predicted number of surviving microbes on a respirator from any starting concentration. The chi-square tests were used to compare the number of times organisms were recovered across sites, disinfectants, and organism types. Log-rank tests were used to compare the reductions. All analyses were performed using R.

Subjects

Subjects were nurses and nursing students from the Emory Healthcare system and Emory University School of Nursing. This study was reviewed and approved by the Emory University Institutional Review Board (STUDY00002519; See 45 C.F.R. part 46.114; 21 C.F.R. part 56.114). Subjects completed a demographic survey including their nursing role, time in role, education level, and previous experience wearing EHMRs and other respirators.

Microbes

For each participant, one “test” respirator was inoculated on the nose, strap, left filter cover, and right filter cover with 50 µL of tryptic soy broth suspension containing Raoultella terrigena (surrogate for gram-negative HAI microbes, target 10⁷–10⁸ CFU/mL) and MS2 (target 10⁷–10⁸ PFU/mL). The respirator was dried at room temperature for 30 min and given to the participant. One “control” respirator per day was matched to all test respirators run that day. The control was inoculated identically to the test respirators, dried for 30 min, and sampled in the same manner. All sampling was done by the same investigator. EHMRs were used only once per day; at the end of the day they were disassembled, cleaned, and disinfected (Bessesen et al., 2015).

Disinfection

Based on the laboratory study, 0.5% HP wipes (Oxivir^®) were used because they demonstrated the largest log₁₀ reductions. Subjects were randomized into three cohorts: naive, two-wipe, and five-wipe. The naïve cohort was not given instructions on a number of wipes or how to wipe. They were told to disinfect the respirator, in the same manner they would if they had just removed it after patient care, and to remove the splash guards to disinfect the outside of the filter covers. For the other cohorts, there were two sets of step-by-step instructions: one using two wipes and one using five. Instructions were piloted iteratively for clarity with five people unfamiliar with the project. Subjects were filmed. A facilitator observed whether two- and five-wipe subjects followed instructions, noted departures on a checklist, and informed them in real time if they missed a step. To represent a scenario where an HCP might be disinfecting a respirator after one patient care encounter ends and they are moving on straight to the next patient, no precleaning of the respirator was done, only wipe disinfection.

Recovery and Assay

Inoculated sites were swabbed with a polyester sponge (3M Corp) premoistened with neutralizing buffer (BD Life Sciences). The sponge and 25 mL of phosphate-buffered saline+0.1% Tween were stomached in a bag for 2 min. The liquid was centrifuged at 2500×g for 15 min and supernatant assayed for MS2 by the double agar layer method. The pellet was resuspended in phosphate-buffered saline, assayed for R. terrigena by spread plating on a MacConkey agar, and incubated overnight at 37°C. The reduction was expressed as log₁₀(N _test /N _control ), where N _test is the concentration (R. terrigena CFU/mL, MS2 PFU/mL) recovered from the test respirator and N _control is the concentration recovered from the control respirator.