>> So welcome to today's webinar on Assessment of Elastomeric Respirators in Healthcare Delivery Settings, Randomized-controlled Elastomeric Studies, Disinfection Safety and Effectiveness or RESponSE. And I'm Adam Hornbeck. I'm, a nurse practitioner here at the National Personal Protective Technology Laboratory. So today's objectives will be to provide an overview of NPPTL and EHMRs. What I'm going to summarize is EHMR hospital demonstration three-part study, and share study findings from the two sites that completed this portion of the study. So the National Personal Protective Technology Laboratory or NPPTL, that's a division of NIOSH, which is the National Institute for Occupational Safety and Health, and that's within the Centers for Disease Control and Prevention or CDC. It was established in 2001 at the request of Congress, and the mission here is to prevent disease, injury, and death for millions of workers who rely on personal protective technology. One of the primary responsibilities is to test and approve respirators. This ensures standard level of quality and filtration efficiency for all who use them. NPPTL also conducts research on numerous other personal protective equipment. Some of those include hearing protection, gloves, and safety glasses just to name a few. EHMRs are tight fitting respirators that can be synthetic or rubber material and can be repeatedly reused, equipped with replaceable filters and may have disposable components. They are NIOSH-approved and have an OSHA-assigned protection factor just as N95 FFRs do. Fit testing is also required. NIOSH has been exploring the use of EHMRs in healthcare since around 2014. EHMRs are designed for long-term reuse with the ability to be cleaned without damage to the respirator. This study chose to use the Honeywell RU85004 which has a speech diaphragm, filter covers, and exhalation valve redirection cover. The need to fit test is required and must be done for that specific make model and size. And then anytime a change in style is made, fit testing must be completed again. And listed here is 29 CFR 1910.134, the respiratory protection standard, which I'm sure many of you are familiar with. So there are three parts of the study as a whole. The first part was JET FIT, looked at the time, resources, and training to rapidly fit test and train a large group of staff. Today's webinar on response looked at EHMR disinfection with wipes and organisms both common to a healthcare setting. And then the final part, reuse, which findings will be coming in the near future, was a three-to-six-month study following hospital workers as they used the EHMRs while caring for patients. This looked at concerns, any barriers to use, and best practices for implementing and using EHMRs in healthcare. So the purpose of this study, it's just because there are a few studies on the EHMR disinfection. They are a reusable alternative for N95 FFRs, can also be used during public health emergencies or a viable alternative to keep if you're stockpiling pandemic supplies. And here's some of the references. And with that, I'm going to turn it over to Dr. Casanova. She's going to share her slides and findings from her portion of the study, so take it away, Dr. Casanova.

>> I am Leslie Casanova. Thank you, everybody, for coming. I am going to present on our portion of this EHMR study, which consists of a two-phase study. One is a laboratory-scale study of disinfection of EHMRs, and the other is a human volunteer study. So I'd like to, at the beginning, acknowledge everyone who participated in this. It was a large team effort and collaboration between GSU Emory and NIOSH, and I'd particularly like to acknowledge Dr. Kimberly Erukunuakpor, who performed the laboratory study as part of her doctoral dissertation. So a big part of what I'm presenting here is work that she did as part of her doctorate. So, Adam gave a great recap about EHMRs, and I assume because you're all here to hear us talk about respirators that you know the fundamentals of N95s and reusable respirators. And of course, many people have been thinking for many years, well, what would happen if theoretically, we had a huge pandemic and we ran out of N95 respirators? What would we do? What are the options in that situation? And of course, we ran headlong into that, and it forced many healthcare systems and facilities to make some very difficult choices about what they were going to do when confronted with shortages of single-use N95 FFRs for their healthcare workers. And so what are your options in precisely that situation? And there were two main options that most healthcare facilities pivoted towards. The first, which was probably the most widespread option, was exploring ways to reuse single-use respirators or reuse N95 FFRs, but another option is reusable respirators. The elastomeric half-mask respirator is what we're going to talk about here, and you already saw an Adam's short presentation, a picture of what one of these looks like, but here's one here. This is in fact, the one that we used in our study. Whichever one you choose, whether it's reusing N95 FFRs or switching to something like an EHMR, you have one big issue, and that is that the surface of the respirator can become contaminated by pathogens during patient care. So there is the potential for the respirator when it is reused multiple times or over multiple patient care encounters, that the respirator itself may become a fomite, and if it is touched in the process of donning or doffing, particularly doffing, that it can result in hypothetically transfer of contaminating pathogens to the healthcare worker's hands. So if you look in the literature, there have been a number of studies both before and after the COVID-19 pandemic that examined options for disinfecting and decontaminating N95 FFRs so that they could be reused multiple times over multiple patient care encounters, and people explored a lot of different options for decontaminating single-use N95s to allow for multiple use. This included hydrogen peroxide, so vapor hydrogen peroxide, ethylene oxide, using hospital sterilizers, the same kinds of systems that you would use, for example, for surgical instruments and things like that, so using preexisting hospital sterilization systems that used hydrogen peroxide or ethylene oxide, the use of ozone, the use of ultraviolet light. And when looking at options for disinfecting EHMRs, you have the possibility of using wipes on the impervious surfaces, and also all of these other disinfection options, but one of the challenges is that if you look at the literature, there are many good studies of these options, but there's no standardized method. There's no method that is called for by regulation, and currently, no standardization body has put out a method that would be one that everyone would adopt. So there's still this question of well, if we are going to choose among these options that require decontamination, what do we do? And how do we make those choices? So, kind of diving more into our options, and the two main ones, reuse of FFRs or pivoting to EHMRs, the advantage of N95s is that healthcare workers are experienced in wearing them. They are the most widespread type of respiratory protection. The disadvantage of reusing them is that it's possible that they might lose fit over time, and you need to figure out how to decontaminate them but there is a dual challenge in decontaminating N95s, namely that the decontamination method itself has to be effective in reducing pathogens, but also whatever decontamination method is chosen cannot affect the filtration efficiency of the N95. And it is completely possible to evaluate the filtration efficiency of an N95 and see how it might change over time, but you need someone with the expertise and the resources or equipment to be able to do that. And if you have questions about that, I'm going to bounce you to the people from NPPTL to explain how that works. With EHMR option, theoretically, they are reusable as long as they maintain their physical integrity. They might be able to maintain fit over multiple uses more than an N95. However, again, they have challenges as well. There's the logistics of rollout, which were part of another -- part of this study, and so I can bounce questions about that to Dr. Colleen Kraft. The surface of the EHMR is a more complex surface. There are more parts of it to clean and disinfect, and since it's a much less used option, many healthcare workers aren't used to wearing one, and so there are a lot of questions to answer about healthcare workers' experience of wearing this type of respirator and whether it's sustainable over time, and whether it can be adopted in widespread way. So in this study, this is a two-phase study to measure the reduction of microbes on the surfaces of EHMRs. And this again, in these two phases, we started with a controlled laboratory study using a standardized procedure to disinfect the surfaces of EHMRs, and the purpose was to compare a set of commercially available disinfectant wipes that are used in healthcare settings for their ability to reduce pathogens on the surface of the EHMR. So the idea behind our disinfectant selection here is that it is looking at disinfectants that any hospital theoretically would be able to acquire easily and immediately, or that might already be present in the hospital. For the second phase, we took the results from the first phase to narrow down our options from a range of disinfectant wipes down to one that had acceptable performance, and we used that one type of wipe in a study where we had volunteer healthcare providers test how different wiping methods reduced microbes on the surfaces of an EHMR. So after choosing a disinfectant in a controlled laboratory study, we then examined how this performed under simulated-use conditions where real healthcare workers tried to disinfect EHMRs. So I'm going to go through each of the parts of this study describing the two phases, first the laboratory study and then the volunteer study. So I'm going to go through the methods for both, and then I'm going to talk about the results of both, so we'll start with the laboratory study. Again, this is Dr. Erukunuakpor's work. We tested EHMRs in sets of three and this is the exact model, the pictures that are that you saw earlier. We chose for the test microbes, bacteria and viruses representative of healthcare-acquired infections. So these are thought to be general surrogates of the different classes of microbes that we might be concerned about in the healthcare environment. We chose Pseudomonas aeruginosa as a representative of the gram-negative bacteria. We used Bacillus atrophaeus as a surrogate for spore-forming bacteria because when we talk about disinfection of EHMRs, and particularly in the context of the COVID-19 pandemic, we might think about the problem of viruses contaminating the surfaces of EHMRs, but it's important to remember that even in a pandemic situation, if we're reusing respirators, we still have the same old hospital-acquired pathogens that we've always had, and we need to worry about them possibly contaminating respirators. So it's possible that you could be wearing an EHMR for respiratory protection with COVID patients, but people still have C. diff just like they always have, so that's why we're trying to cover a range of pathogens here. We have a gram-negative. We have a spore-former. And for viruses, surrogates for viruses, we used two bacteriophages, MS2 and phi-6, and the reason we chose two, MS2 is a surrogate for the non-envelope viruses. Norovirus would be an issue sample of a non-envelope [inaudible] virus of concern. Phi-6, like SARS-CoV-2, is an enveloped virus. So we're looking at the response of all of these to a suite of disinfectants. We used these certain organisms to artificially contaminate respirators. Our target concentration for each was quite high. Here, we're not necessarily trying to simulate the numbers of organisms that a patient would deposit onto the surface of an EHMR. Rather, we're trying to simulate -- or we're trying to have the numbers be high enough that we can follow a large log reduction or we can measure a large log reduction under these experimental conditions. So to try to simulate what an actual patient care encounter might be like, we contaminate the respirators, and then we let them dry at room temperature for 30 minutes before disinfection because this simulates maybe the length that organisms might sit on the respirator during an actual patient care encounter prior to disinfection. So here, you can see the parts of the respirator that we contaminated. So we made a cocktail of organisms. They were deposited on the surface of the respirator in very small droplets, so we chose these spots for a couple of reasons. First, on the front of the respirator, they're the ones that might be most likely to become contaminated, say, if you're exposed to respiratory secretions, body fluids, something like that, during a patient care encounter. They're also probably ones that might be likely to be touched the most often, and that's why we chose to put organisms on the back of the strap of the respirator because we theorized that that's a spot that's likely to be touched as people attempt to take the respirator off, and therefore, it could be a risk for contamination. So we have these four sites on the respirator artificially contaminated with organisms, allowed to dry for 30 minutes, and then disinfected. And the way this was done was that a test respirator and a control respirator were both inoculated in identical manner, allowed to dry in an identical manner, and then one of them was disinfected. Both of them were sampled, and the reductions of organisms were measured as log 10 reduction by comparing the amount remaining on the test respirator to the amount remaining on the control respirator. So what we used, EPA-approved hospital disinfecting wipes. So again, these are things that we thought would be readily available to hospitals. And they covered some of the widely used hospital disinfectant, so hydrogen peroxide, quaternary ammonium compounds, and alcohol. So these are the brand names, if anyone is interested, and these all came as wipes. So what we did was when these were disinfected, they were held at room temperature, and they were held for the manufacturer-recommended contact time. So these are what -- these are the contact time we use. We're talking about short contact times, one minute to two minutes. So this is the standard practice that was used, and these were all done by the same person for consistency, so they were all done by the same person using a standard procedure, which I'll show you in a moment. The standard procedure used five wipes (and we're going to return to that in the human volunteer portion of this study), moving from the top to the bottom of the respirator and disinfecting the strap last. And I'm not going to go through this in a lot of detail, but what I want you to take away about the basic sequence is that it uses multiple wipes. One wipe is used to clean the top half. A second wipe is used for one filter cover, and then it's folded over and used for the other filter cover. A third wipe is used for the lower half, and a fourth and fifth white are used for the strap. And then the disinfectant is allowed to sit on the surface for the manufacturer's recommended contact time. So we go through multiple wipes, each wipe going with a different part of the respirator and we're going to come back to this idea of different surfaces. So the way this is done -- again, we're going to come back to this in the human volunteer study, and I have some pictures here, and I'm glad to come back to these in the Q&A, because I'm not going to go over them in a huge amount of detail, but the idea is that you would be wearing gloves. You would have this respirator sitting in front of you on a surface. You'd be wearing gloves, and you would go through the system where you would move from one wipe to another, folding over wipes so that each part of the respirator gets two passes, as you can see here. And moving from the top to the bottom of the respirator as you change wipes. So top of the respirator, filter covers, bottom of the respirator, and concluding with a strap. So this is the basic way that you would go through cleaning the respirator, moving through wipes as you do this. And it would take five wipes, so that's what we did for the laboratory study, and we're going to carry this forward the human volunteer study, and we're going to come back to some of these same methods, so I'm going to go through the methods and then talk a little bit about our data. So based on the laboratory study results, which I'm going to show you in a moment, we chose single disinfectant, and this was a hydrogen peroxide disinfectant, .5% hydrogen peroxide, and we chose it based on comparing the log reductions with different disinfectants in the laboratory portion of the study. One of the other -- and this is from a practical standpoint -- one of the advantages perhaps of using hydrogen peroxide is that it doesn't leave a lingering odor on the respirator after disinfection, which you can imagine might be one of the issues with, say, bleach. So what we did was we recruited about 50 nurses and nursing students from the Emory healthcare system. What we did was for each person, we took a what we call a test respirator. It was inoculated at four sites, the same four sites that were inoculated in the laboratory study and it was inoculated in the same way where 50 microliters of suspension were distributed in small drops at each of the sites, so the inoculation was identical to what took place in the laboratory study. For this one, we used Raoultella terrigena because it's a representative of the gram-negative bacteria and also because it's acceptable to IRBs when we are proposing a project that could potentially expose individuals to this organism, Raoultella is an acceptable choice for that particular scenario. And we also chose MS2 which is harmless to people. It is a conservative surrogate for human viruses. And again, we used large -- a large number of organisms in order to be able to measure large log reductions. So we inoculated it, let it dry at room temperature for 30 minutes, and then it was given to the volunteers, so nurses and nursing students from Emory healthcare. We randomized them into what we call three cohorts, and I'm going to keep coming back to this in the results. We have a two-wipe cohort, a five-wipe cohort, and the naive cohort. So the two-wipe cohort was given a set of instructions for how to disinfect the respirator step-by-step using two wipes during the process. The five-wipe cohort was given instructions for how to disinfect step-by-step using five wipes in the process. And what we call the naive cohort was handed a package of wipes and a respirator, and they were not given instructions for how to disinfect. They were told to disinfect the respirator as they would do after patient care, and it was left up to them how to do that. Everyone was observed during disinfection. For those who are given instructions, part of it was observing whether they deviated from them, but everyone was observed. So the naive cohort, they weren't told, "You should use X number of wipes. This is how you do it." They were just told, "Disinfect the respirator however, you would if you had just removed it after patient care." The only specific thing they were told was to remove the splash guards, which is an important step. So this is -- and I'm happy to come back to this if there are questions, but this is the protocol for disinfecting respirators, and it's similar to the one we used in the laboratory study in that you move from the sort of top to the bottom of the respirator, one site on the respirator at a time, using wipes and folding wipes as you move through the process with the idea that you cover every surface of the respirator including the strap. So, as I said, I'm happy if we have time during the Q&A to come back to specific questions about the methods because there's a lot going on there, but I want to get to the results. And again, I'm going to examine these in two phases, the laboratory and the volunteer study. So we're going to start with the laboratory study, and I kind of want to go through this in two ways. The first I'm going to present here is our test organisms and the percent of surfaces, the percent of total surfaces tested that still had organisms on them after disinfection. And this is of interest, because we do want to know are some parts of the respirator more challenging to disinfect than others? Do some parts of the respirator experience less log reduction, perhaps because they're harder to reach, perhaps because it's harder to get people to follow the correct steps? There are a lot of reasons why that might be, but how many parts of the respirators still have organisms on them after disinfection is a thing of interest and how that works with different disinfectants. So I think the takeaway here is that there's no perfect disinfectant, but some are better than others. QAC-only and quaternary ammonium, only a quaternary ammonium compound with saline, generally performed not quite as well as a QAC/alcohol or hydrogen peroxide. One of the things that you can see here is that we tend to have more spores leftover on the surface, which is not surprising. Phi-6 and Pseudomonas aeruginosa were quite susceptible. So we're going to come back to this idea about disinfecting sites after when I get to the human volunteer study. So this is probably what we are most interested in is what are what did the log reductions of different organisms look like against different disinfectants in the lab study? And again, what we can see here is that there perhaps is no disinfectant that is absolutely the best one in every scenario, and there's overlap between disinfectants, but overall, looking at the different log reductions and trying to choose the optimal one that has the highest log reduction for all these organisms, we settled on the Oxivir Tb wipe, which is the .5% hydrogen peroxide wipe. It had very good activity against Phi-6 and Pseudomonas aeruginosa, very good activity against MS2, less activity against spores, but that was typical of all the disinfectants tested. So we move forward with hydrogen peroxide wipes to the human volunteer study. Overall, in the big picture of the laboratory study, there's just a couple of takeaways. Organisms were more frequently remaining after disinfection on the filter covers, less so on the nose and strap. And there are again, some possible reasons for this. If you look at the filter covers, you can see that they're more of an irregular surface, so they seem more likely to have organisms remaining on them after disinfection, but the recovery of the organisms didn't statistically significantly differ across sites, so there wasn't one site that had significantly worse disinfection or better disinfection than the others. So that's an important observation, because we do want to know that there -- whether some sites are more difficult to disinfect than others, because that might affect what we tell people to do. The proportions of surfaces that still had organisms left, it was greatest when people used QAC and saline, and least for hydrogen peroxide, which is one, and QAC/alcohols, so this is one of the reasons we chose hydrogen peroxide moving forward. Not surprisingly, recovery differed by organism. Bacillus atrophaeus and MS2 were recovered more frequently than the bacteria, which is kind of not surprising because spores and non-envelope viruses overall tend to be more resistant to disinfection than gram- negative bacteria and envelope virus. We know this very well. And we achieved a medium log reduction of about four log 10 that we can consider that fairly good. There's not necessarily a regulatory benchmark or a bright line that we're comparing this to, but we do want to maximize disinfection. So when we look at recovery by organism, we may see that with different disinfectants, there are going to be trade-offs. There's not one disinfectant that stands out as it's much better, it's significantly better across the board than any other disinfectant. So we chose again, hydrogen peroxide as the disinfectant to move forward with to the volunteer study, and you can see that there's sort of an order of effectiveness of the quaternary ammonium compounds with alcohol seeming to increase the effectiveness of this disinfectant somewhat. So when we look at reductions by organisms, and I'm happy to come back to this and answer questions, but these are just the general ideas moving forward into the human volunteers study, Pseudomonas aeruginosa overall was highly susceptible, as was phi-6. This is not surprising. MS2 and Bacillus were less susceptible. Not surprisingly, spores were the least susceptible. But again, we got pretty good performance with hydrogen peroxide against both of these organisms, and there was a trend that quaternary ammonium compound by itself and quaternary ammonium compound and saline had the poorest performance. So we carried forward the hydrogen peroxide wipes into the volunteer study. One thing here I want you to see is across all the participants, and remember, we have three cohorts, one had instructions to use two wipes, one that had instructions using five wipes, one given no instructions, and we looked at the number of wipes that they used. People who were instructed to use two wipes and five wipes sometimes ended up using more. Interesting thing here in the naive cohort with no instructions, there was a tendency across participants to use only one wipe, except for some really determined people who used actually seven wipes, but it is kind of interesting here that there were a lot of people in the no-instruction cohort who chose to use only one wipe. So this is, I think, the one figure with the biggest takeaways that I want you to have. This is median log reductions of MS2 and Raoultella terrigena across all participants and all respirator sites, so this is how well the disinfectant performed when participants in each cohort used it. And what I want you to do is I want you to focus on the little dots. Now the bars are the median reduction, so you can see that the median log reduction that we achieved tended to land somewhere between three and five log 10. But if you focus on the little dots, kind of what I want you to see here is that there is a lot of between subject variation and between cohort variation. So each of these dots is an individual subject, and this, I think, is one of the things that as a microbiologist, is of big interest to me is a variation as a characteristic of the data, and we can see here that when we have people doing this under actual simulated-use conditions, there is a lot of variation among individuals, even in the same cohort, and within cohorts where people were given instructions. So here, it's not necessarily that some number of wipes or some kind of instructions, reduce the amount of variation. This is still a quite variable phenomenon in terms of the log reduction that we achieve if we look at all individuals, and if we just look at means or medians, you can easily kind of miss the amount of variation in the data, and I think this has important implications for the overall results. What I want you to see here is that if we do look at means and standard deviations, we can see that there's a lot of overlap in between cohorts, within cohorts, across sites, and also between organisms, so here we're looking at Raoultella terrigena. So if you just focus on the bacteria, which is on your left, you can see that, again, the mean log reductions tend to float between three and five log, but there are -- the standard deviations are quite wide, and overall, there's a lot of overlap. So, here again we can see that what happens when people disinfect under single -- under simulated-use conditions is that you get a lot of variation, but we are getting, if we look at the means and medians, between three and five log reduction. So just an observation about time is that there wasn't really, again, like we said, the naive cohort, there was a tendency to use one wipe, but overall, I think the take-home in time, with time is that there wasn't a statistically significant difference in the times -- total time spent disinfecting among cohorts. It was roughly two minutes, but the one takeaway that I think is important here is that time was not a significant predictor of log 10 reduction. So time, the time spent was not related to how effective the disinfection was. So I'm just going to point out a couple of the big takeaways, I think, from this data that I'd like you to remember. There was a lot of variation in log reduction within and between subjects. There was variability within cohorts. There was variability across sites for both organisms, yet time was not a significant predictor of log reduction. So log reductions were greater overall for the gram-negative bacteria than the non-envelope virus. That's not surprising, but again, this idea of variation as a characteristic of this data, I think is very important because it has implications for how we think about this going forward, so overall, we found that hospital disinfectant wipes can reduce bacteria on average three or four log reductions, bacteria and viruses on respiratory surfaces. These can definitely be achieved under controlled laboratory conditions. It can also be achieved by volunteers under simulated-use conditions, but again, there's this important idea that there is a lot of between-participant variation. Even across cohorts that have different numbers of wipes, cohorts that received instructions, cohorts that didn't receive instructions, so hospital disinfectant wipes appear to be a viable option, but what we're seeing is that this idea of variation, I think, has some pretty important implications for how we think about this. One of the things that if we look at between organism variation, between disinfectant variation, we can try to -- we can choose the optimal disinfectant, but there are going to be tradeoffs of each one. One disinfectant didn't absolutely stand out as the best one across all organisms, and their -- whatever disinfectant we choose, it has to be compatible over time with the components of the EHMR, which is not something we measured in this study. So another one of the big takeaways, I think, is that going forward, this is just an initial study. There are still a lot more questions we can ask about the disinfection of EHMRs moving forward, but one of the things that we definitely want to think of is how do we teach people to do this? How do we train people to do this, so that we achieve optimal disinfection and reduce variation? And that, I will bring this to a close and turn it over to Dr. Pompeii, and thank you very much, everyone, for listening.

>> Okay, so my name is Lisa Pompeii, and I am presenting work pertaining to disinfection of elastomeric respirators in healthcare settings that was led by an infectious disease epidemiologist at the University of Texas, including doctors Eric Brown and Charles Darkoh and I am presenting in their stead. The objective of the study was to determine the effectiveness of commercial disinfection wipes available to healthcare personnel to disinfect elastomeric respirators (also called EHMRs) in a healthcare setting. This study consisted of three phases. The first phase was a laboratory disinfection study. In this order, we inoculated elastomeric respirators (or EHMRs) with inactive organisms. We then disinfected these inoculated respirators with commercially available wipes, and we then tested the respirators to determine the effectiveness of the wipes. The second phase of the study was primarily conducted in the field, where a study coordinator inoculated the EHMRs with inactive organisms, which were then disinfected by healthcare personnel using commercial disinfectant wipes. We then moved back into the laboratory where we tested the effectiveness of these wipes on disinfecting the respirators. In prior studies, we have instructed participants on respirator disinfection using either video instruction or face-to-face instruction. So the third phase of this study was we tested the effectiveness of just using a step-by-step written instruction guide only for the elastomeric disinfection training. Workers were asked to review step-by-step written instructions that included pictures on how to disinfect the elastomeric respirator using a two-wipe and a five-wipe method. So for the first phase of the study, again, which was in the laboratory, we selected various types of microbial organisms typically found in healthcare settings, which Lisa Casanova provided a nice overview of why the different classes or groups of organisms were selected. And we have some overlap with her study, but then also some that she didn't use, or they didn't use. These organisms also different in cell wall structure and we thought it was important with regard to testing the effectiveness of the disinfection wipes and how they performed. So the four organisms that we used included Bacillus subtilis which is a bacterial spore, Mycobacterium fortuitum which is a gram-positive bacteria, Pseudomonas aeruginosa which is a gram-negative bacteria, and Bacteriophage MS2, which is a virus. And again, Lisa went over some of this in her presentation. We used similar wipes. I believe we used all the same wipes in the lab disinfection phase. So these four commercially available wipes Oxivir with an active ingredient of hydrogen peroxide, CaviWipes with the active ingredient (excuse me) of isopropanol and ammonium chloride, Allegro wipes with a active ingredient of benzalkonium chloride, and then we also used sterile saline. So with regard to the study methods under sterile conditions in the lab, we inoculated the elastomeric strap, nose piece and filter cap (which we've illustrated here) with 20 microliters of each organism. This was conducted on separate EHMRs for each organism. So more specifically, five respirators were inoculated with each organism for a total of 20 inoculated respirators. Once they were inoculated, each mask was left to dry for two hours at room temperature. After the inoculated sites dried, they were then disinfected for one minute with one of the four wipes. Each mask was disinfected with a different wipe just for clarification. So for example, one EHMR inoculated with MS2 was disinfected with a CaviWipe only for one minute. One inoculated respirator for each of the study organisms was left untreated, and those respirators served as study controls. So after the disinfection process, each inoculated site on the respirator was swabbed and plated on a sterile petri dish and incubated aerobically at 37 degrees Celsius for 24 hours. Colony-forming units (or CFUs), or plaque-forming units (PFUs) were then counted. The percent reduction was calculated for each sample relative to the untreated control for each organism and respirator site. The same process was carried out for the four respirators that served as controls, where they were inoculated with the study organism again but not disinfected. All four types of wipes were very effective at reducing microbial contamination across most pathogens and respirators sites. So on this slide, we present findings from the Oxivir wipes and the CaviWipes. So for the Oxivir wipes, there was a range of a 99.5% to 100% reduction across all pathogens and respirator sites except for the B. subtilis on the nose piece, which was reduced to 97.7%, and the filter cap which was 98.7% CFU reduction. For the CaviWipes, there was a range of 96% to 100% reduction across all pathogens and sites, except for the nosepiece trials for B. subtilis, which was 87.6% reduction, and the MS2 which was 72.7. On this slide, we present the findings for the Allegro wipes and the saline wipes. So slightly -- Allegro wipes were slightly less effective than the Oxivir and the CaviWipes. There was greater than 94% reduction across all pathogens and sites except for filter cap trials for the B. subtilis, which was 89.4%, and MS2 which was 75.2% reduction. For the saline wipes, they were least effective but still worked surprisingly well. There was greater than a 91% reduction of pathogens across most sites with the exception of MS2 at all sites. So moving into the second phase of our study, in this phase this involves the disinfection of elastomeric respirators in the healthcare setting. In a prior study, we observed that healthcare personnel are capable of learning and performing a standard operating procedure for cleaning an elastomeric respirator with little or no mistakes. However, prior studies have not assessed the reduction of microorganisms on the surface of respirators that were disinfected by healthcare personnel in the healthcare setting, with the exception of course of Lisa's study that she is presenting today. So the second phase of the study involved two of the four organisms from the laboratory study including the B. subtilis, and the Pseudomonas. So for the commercial wipes, we selected the two that we found most effective in the laboratory study which included Oxivir wives and the CaviWipes. We also include the sterile saline wipes to test their effectiveness in this real-world work setting, assuming that if a worker was -- didn't have wipes with them, that they would possibly just wipe down their respirator with water. So we recruited 10 healthcare personnel from Memorial Hermann Hospital in Houston, Texas, via email invitations and flyers, which included seven respiratory therapists, one nurse, one speech pathologist and one occupational therapy assistant. Participants were asked to complete an online recruitment survey and a consent form and they were then selected -- they were then scheduled (excuse me) for the in-person study session. So in preparation for this phase, 16 EHMRs were inoculated with 20 microliters of the sample on the strap, nosepiece, and filter cap. The inoculated EHMRs were left to dry at room temperature before participant -- before the participant disinfection trials were conducted. So the B. subtilis dried for at least 60 minutes, and the Pseudomonas for at least 90. Three disinfection stations were set up for each participant. So each station included two pads were placed with the left pad designated as dirty. The dirty side and the right side was designated as clean. One inoculated respirator was initially placed on the dirty side. Disinfectant wipes were provided with a different wipe at each station. And then the other disinfectant supplies -- disinfection supplies were also provided such as gloves and hand sanitizer. Participants were initially asked to watch a three-minute video in which they were instructed on how to disinfect the elastomeric respirator. As outlined here, they received step-by-step instructions on disinfection, from donning gloves to disinfection to allowing the EHMR to dry. After the video instruction, the participants were led to the disinfection area. They were asked to disinfect three respirators that were inoculated with the same organism, but each station involving disinfecting -- but each station involved disinfecting with only one of the three disinfectant wipes. After they completed the disinfection, the respirator was set to dry for one minute and then swabbed. Those swabs were then stored for transfer to the lab. In the lab, each sample was plated on a sterile petri dish and incubated aerobically at 37 degrees Celsius for 24 hours. Similar to our lab study, we followed the same procedure. CFU and PFUs were determined by manually counting the colonies and plaques. Percent CFU and PFU reduction was calculated for each sample relative to the untreated control for each organism and respirator site. So for our findings, we found that the Oxivir wipes were effective against both pathogens at most respirator sites. We conducted unpaired t-tests and found statistically significant differences in the reduction of pathogens for Oxivir wipes relative to the controls. For the Oxivir wipes, there was a 98.8% to 100% reduction except for Pseudomonas on the nose piece, which was at 96.7, and B. subtilis on the filter cap which was 97.4. For the CaviWipes, they were also very effective with a range of 96.2% to 100% reduction across all sites for all pathogens. And then again, for the saline wipes, both pathogens were reduced at all sites by an range of 94.4% to 99.9%, except for the Pseudomonas on the strap, which was reduced to 86%. So moving on to the third and final phase of the study, we tested if workers could properly disinfect the respirator using written instruction guide rather than being instructed in a face-to-face or video format. So we recruited 40 healthcare workers. 20 workers were asked to follow the two-wipe disinfection instruction. Again, this is the process that we've used in all of our other elastomeric studies. We -- and then 20, workers were asked to follow the five-wipe disinfection. Participants were provided with a step-by-step written instruction guide that included pictures of the EHMR disinfection process, and we included just one page of that here so you could see how it was set up. This included either the two-wipe method, which we also refer to as the sandwich method. The two-wipe method is similar again to the wipe method that we used in our other studies and including phase two of this study. With this method, the participant handles the elastomeric between two disinfectant wipes and wipes down the respirator parts of the respirator. For the five-wipe method, this included the use of five separate wipes, one used for each section or area of the respirator. So workers were asked to initially review the written disinfection guide, and were allowed to ask questions if needed. They then used the document as a guide to disinfect the elastomeric respirator twice. During the first attempt, they were allowed to ask the study coordinator questions. On the second attempt, they had to rely on only the disinfection guide. So each step that was conducted correctly was worth one point, and zero points if it was incorrect. The two-wipe method consisted of 17 steps, and the five-step method consisted of 20 steps. We also timed how long the disinfection process took each worker. So in this initial finding slide, the initial steps for the EHMR disinfection was similar across the two-wipe and the five-wipe methods, so we combined the data here. So for most of the steps, participants improved from the first to second attempt with respect to conducting hand hygiene, donning gloves, setting up the cleaning pad, all the way to removing their surgical mask from the exhalation valve. So again, there was improvement across each of these steps. For the two-wipe method -- two-wipe disinfection method, participants performed well and typically better on the second attempt, with the exception of the first two steps where they needed to perform hand hygiene and to place the second wipe on top of the respirator before picking up. But overall, the scores were higher for each step on the second attempt. And overall, they were high. I mean, I feel confident about using this type of method to educate workers with regard to this two-step method. For the five-wipe disinfection method, again, which involved using five separate disinfection wipes at each -- so a different wipe at each step of the cleaning process, they also performed well in the second attempt. This process is significantly more detailed than the two-wipe method and workers still performed well. These findings suggest that using the handout education method would work for instructing workers on how to disinfect. With regard to the two-wipe method -- disinfection method, the mean time to disinfect ranged from five minutes and 25 seconds in the first attempt, to four minutes and 42 seconds for the second attempt. And for the five-wipe method, the duration of time was similar to that of the two-wipe. And you probably ask why are we timing this, but the timing is important. We've looked at this across a lot of different elastomeric studies of how long does it take to disinfect because this is part of the worker process and the time that it takes for them to be using that respirator -- the step that they have to take, either before they store that respirator, or they pick it up to use it again, I mean, so the timing of this is important of how long does this take? So the study has several strengths and some limitations. The study investigated various types of microbial exposures commonly found in the healthcare setting, while previous studies have only investigated EHMRs inoculated with the influenza virus, again, with the exception of Lisa Casanova's study, The study included a field phase to assess the efficacy of EHMR disinfection by healthcare personnel in a hospital environment while previous studies on EHMR disinfection have only been conducted in laboratory settings. The disinfection method tested in this study using commercially available disinfecting wipes can be easily adapted by healthcare organizations implementing the use of EHMR respirators by healthcare personnel. So, there are some limitations. The circumstances in the field may result in longer inoculation times of microorganisms on the respirator surfaces before disinfection, though this is mitigated by significantly larger inocula tested in this study than what would possibly be encountered in the field. When 100% CFU or PFU percent removal of inocula was found, it is possible to trace amounts of microorganisms remaining quantities below detection threshold -- below the detection threshold of our methods used in the study. So again, these could be trace amounts of organisms that we did not detect. And the field portion of the study only included two microorganisms rather than four. And I think Lisa Casanova, also, you know, she provided a nice summary of where we need to go next, and also some of the other limitations of testing this really in the laboratory or in a controlled field environment. So in conclusion, we concluded that EHMRs can be effectively disinfected by healthcare personnel in the hospital setting. It seems that they're quite capable of doing that. Oxivir and CaviWipes seem to be the most effective disinfectant wipes in both the laboratory and hospital settings across most pathogens and respirators sites. And the disinfection training using handout with photo education tool may be a viable method for training workers on EHMR disinfection. So I know that we are right at 2:00. I have my references here, and my contact information, and I'm happy to answer any questions or share any of this information outside of the time that we have for this, so I'll end there, and I'll stop sharing my screen.

>> Yeah, since we are at the end of our time for today, if you did ask a question and would still like it answered if we didn't get to it as we went through the slides, please feel free to email myself or one of the speakers if you have their email. We will get that answered for you. I'd like to thank everyone for participating today. Thank you, Dr. Casanova and Dr. Pompeii for your wonderful presentations today. And with that, we will come to an end. Thank you, everyone. Have a great day