September 21, 2024

Safely Implementing New Diagnostics Platforms Commonly Used in Clinical Laboratories

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Safely Implementing New Diagnostics Platforms Commonly Used in Clinical Laboratories (Presentation Slides)

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Oct. 15, 2024

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Date of session: 03/22/2023

Facilitator

Aufra C. Araujo, PhD

Centers for Disease Control and Prevention

DLSbiosafety@cdc.gov

Didactic Speakers

Christina Egan, PhD

Deputy Director, Division of Infectious Diseases

Wadsworth Center, New York State Department of Health

christina.egan@health.ny.gov

Michael J. Perry, MS, MS Ed.

Associate Director, Biodefense Laboratory

Wadsworth Center, New York State Department of Health

michael.perry@health.ny.gov

Aufra Araujo: Alright, good afternoon, good morning, and good evening, everyone. My name is Aufra Araujo, and I want to extend a warm welcome from the Centers for Disease Control and Prevention in Atlanta, Georgia. I am a PhD Health Scientist in CDC's Division of Laboratory Systems, and I am the facilitator for this Extension for Community Health Care Outcomes, or ECHO, Biosafety session.

Thank you for joining our third session. The topic for this interactive discussion today is Safely Implementing New Diagnostic Platforms Commonly Used in Clinical Laboratories. From the New York State Department of Public Health in Albany, New York, our subject matter experts today are Dr. Christina Egan, the Deputy Director of the Division of Infectious Disease, and Michael J. Perry, the Associate Director of the Biodefense Laboratory.

I briefly stopped sharing the screen because I would like to have a moment where everybody, as much as possible, can turn on their screens and we can get to know each other. Is anyone calling on the phone, or everyone– you see anybody on the phone?

If so, I was going to ask them to introduce themselves, but it doesn't seem like we have anyone calling on the phone. Perfect, so to get things started, I would like for you, as a way of engaging, to enter in chat what's your favorite piece of laboratory equipment so we can have a quick icebreaker.

Biosafety cabinet, yeah. Flow cytometer. Microscope. MALDI-TOF, all the fancy equipment. MALDI-TOF, BSC, centrifuge, yes. Timer, yes I like that. Timer.

If you just joined, the icebreaker question is, what's your favorite piece of laboratory equipment? You can enter it in the chat.

Alright, so Mathews, I see you entered timer. If you could, turn on your camera and unmute yourself and just say hi. Share with us where are you calling from or which lab you work in, and let us know, if you are a biosafety officer, how many– what's your length of time working in biosafety?

Mathews Athiyal: Sure. My name is Mathews Athiyal. I'm working here at the Georgia Public Health Laboratory, and I've been a biosafety officer for about three years now.

Aufra Araujo: Welcome. We are glad you joined. I'm just randomly looking at the response. Erin Bowles– I'm sorry if I messed up your name. Erin Bowles?

Erin Bowles: Hi, there. I do not have a camera that I can turn on. Sorry.

Aufra Araujo: That's OK.

Erin Bowles: But I just think the biosafety cabinet is that thing that always keeps us safe from my first introduction into microbiology way back many years ago. And I really love that we pay more attention now onto proper use of biosafety cabinets. That wasn't even training I was provided when I first started using one. It was kind of just like you got thrown into it, and it was like, set up cultures or do this or that. And so we've come a long way, and I love that.

Aufra Araujo: Excellent. Where are you calling from?

Erin Bowles: I am calling from the Wisconsin State Lab of Hygiene.

Aufra Araujo: Oh, awesome. And how long have you been in the field of biosafety?

Erin Bowles: I have been doing biosafety– I've been here almost 15 years in biosafety outreach to clinical laboratories has been part of the role ever since I started.

Aufra Araujo: Awesome. Thank you so much for joining us. And, Mathews, if I can go back to you, you said your favorite piece of equipment is timer. So can you share with us why the timer?

Mathews Athiyal: Sure, sure. You put on the timer that you have a set time, and when the timer goes off, you know your work is done, and you're excited to see what the results are. So I think that's probably what it is.

Aufra Araujo: I like that. Thank you. One more. I saw MALDI-TOF, so it's such a useful piece of equipment. Leila Filson, sorry if I messed up your name. Please correct me. If you can turn on your camera, come off mute, and share with us why MALDI-TOF and where you're calling from. What's your experience in the field of biosafety? How long?

So, Leila? I wonder, maybe she's not able to unmute? Anybody else that likes MALDI-TOF would like to unmute and just share why MALDI-TOF? Oh, she can't unmute. No problem, Leila. We'll get back with you.

Christina Egan: I think, if Mike could vote, I think he would vote for MALDI-TOF, too.

Michael Perry: Definitely bonus points for everybody that says MALDI-TOF.

Aufra Araujo: Yay. Can we unmute Leila? Or only she can unmute herself, George? No. Yes, doesn't work.

Oh, Leila, it looks like you are off mute. No. No worries. Well, thank you so much. This way, little by little, through all the sections to end of the year, hopefully, we'll know everybody by then. Thank you, everyone, for participating, and let me see. Before we proceed, I want to briefly mention some technical details related to our ECHO Biosafety sessions.

Please use the video capabilities as much as you can. We'd love to see you. All audience microphones are now muted during the discussion. Please unmute yourself to speak.

If you are experiencing technical difficulties during the session, please send a private message to George Xiang, who is labeled as DLS ECHO Tech. He will do his best to respond to your issue, and he's waving to you now. Yay. Hi, George.

If you are connecting to Zoom by phone only at the time of discussion, please announce yourself by name and institution when beginning to speak. Briefly, how do these ECHO sessions differ from other sessions? These sessions are different from expert lecture, teleconference, or webinars in that the discussion of cases or clinical laboratory challenges are the main feature.

Our subject matter experts hope to share some solutions that will be translatable to all of you in your individual laboratories. We also hope you will participate in the discussion by sharing your knowledge and experience.

These ECHO sessions are focused exclusively on clinical and public health laboratories in the United States in the US territories. Once again, we value the discussion amongst all of you and want to encourage you to share your own experience and challenges on the topic. We thank you for your interest and participation. Let me go back to share the slides. Alright. It's moving slower to me than what I see in the camera. Perfect.

Let me get to the next slide. Alright, here is a brief overview of today's session. I introduce our subject matter experts, Dr. Christina Egan and Michael Perry, who will provide a didactic presentation in real case discussion.

Then, Commander Sabrina DeBose, from the CDC Division of Laboratory Systems, will summarize today's discussion. Closing comments and reminders will follow, and we will adjourn this session.

Today's session is being recorded. If you do not wish to be recorded, please disconnect now. Closed captioning will be provided for this session. Please find the link in the chat box. The transcript, audio recording, and slide deck from today's session, as well as other resources, will be posted on the DLS ECHO Biosafety web page.

Now, it is my pleasure to introduce Dr. Christina Egan and Michael J. Perry. Dr. Christina Egan is the Deputy Director of the Division of Infectious Disease, Chief of Biodefense and Mycology Laboratories, and Director of the Biodefense Laboratory at the Wadsworth Center, New York State Department of Health. Christina has been employed by the Wadsworth Center since 1999.

She's currently serving as the Chair of the Association of Public Health Laboratories (APHL) Biosafety and Biosecurity Committee and is a member of the National Science Advisory Board for Biosecurity. Dr. Egan's research interests include improved diagnostics in the field of biodefense using platforms such as real-time PCR, next-generation sequencing, and microspheres immunoassay and nucleic acid arrays.

Michael J. Perry is the Associate Director of the Biodefense Laboratory at the New York State Department of Health Wadsworth Center. Michael has been at the NYS DOH since 2009. Michael serves as a member on the Association of Public Health Laboratories Biosafety and Biosecurity Committee, the APHL Public Health Preparedness and Response Committee, and is the Chair of the APHL Laboratory Operations Workgroup.

Michael is committed to public health service, as is evident by his work through CDC's global health security agenda, in which he has helped develop and deliver trainings on biological safety cabinet maintenance and operation, implementing a risk management program, and implementing a respiratory protection program in many African countries. Christina and Michael, it's a pleasure to have you leading this session today. The floor is yours.

Christina Egan: Thank you very much. Mike is going to start us off today.

Michael Perry: Yeah, so if we can just get the slides up. Perfect. Alright, well, welcome, everybody, and thanks for joining us today. So today, Christina and I will be talking about safely implementing new diagnostic platforms that are commonly used in clinical laboratories. Next slide.

So we have a few things that we would like to cover today, so we're going to start with a little bit of background on MALDI-TOF mass spectrometry and automated microbial identification systems. For each, we'll be discussing facility and safety considerations, examples of high-risk pathogens, and then discussing some clinical cases and best practices.

We hope by the end of today's session, we'll have covered the following two objectives, which are identifying effective biosafety practices that strengthen laboratory systems and advanced laboratory safety, as well as examine biosafety concepts that apply to conducting risk management when performing laboratory activities. Next slide.

So I'd like to start off with a poll question. So has anyone used a MALDI-TOF instrument before?

Aufra Araujo: So we are almost at 80%, so come on, please. Answer the poll question. We'd like to have at least 80%.

Michael Perry: Alright, so it looks like we're pretty split. A little bit more on the yes side of things, so that's good. So I'm going to go into just a little bit of background on the MALDI in case you aren't familiar with it, and even if you are familiar with it, it's always a good review.

So mass spectrometers are really broken out into multiple components– the ion source, the mass analyzer, and the detector. MALDI is considered the ionization source, in which the ions that are created in the sample as a result of pulsed laser irradiation. And then time of flight is the mass analyzer component, in which uniform electromagnetic force is applied to the ions at the same time, and that causes them to accelerate down a flight tube.

So in this case, lighter ions travel faster, arriving at the detector first. So next slide. And so in this slide, this is just a depiction of what I said.

So in this ionization method, the samples are fixed in a crystalline matrix, and then they get bombarded by the laser. The sample molecules vaporize into a vacuum while being analyzed at the same time without fragmenting or decomposing. So potential is applied, and this potential separates the ions by their mass-to-charge ratio. Determines that mass-to-charge ratio by the time it takes for the ions to reach the detector.

So the particles will impinge upon linear detector within a few nanoseconds after ionization, and those higher mass molecules arrive later than the lighter ones. And that flight time measurement makes it possible to actually determine the molecule masses directly, so each peak in the spectrum can say it corresponds to the specific mass of the particle along the time axis starting with the ionization moment. Next slide.

So we have another polling question. One of the responses on here is, if you do not have a MALDI, you can select that one. But where do you spot your MALDI targets?

So it looks like we got a little bit of everything. Majority in the biosafety cabinet, which is great. We have some on the benchtop, some in the– a little bit less than the chemical fume hood, and then about 17% that do not have the MALDI.

And so this is kind of– we're going to go into, a little bit later on, about some of the ones that are on the benchtop. And we didn't ask here to qualify if you use any additional safety protections, but just trying to get an idea of how often they're run on the biosafety cabinet or versus the benchtop.

So when using MALDI for identifying microorganisms recovered from patient specimens, some general safety considerations include the potential for both chemical and biological exposures. So thinking about direct contact with reagents used to prepare samples for analyzing, including matrices and compounds used for actual microorganism inactivation and extraction components.

Looking at the exposure to chemical fumes created during stock solution preparations or target slide cleanings or, again, inactivation procedures. Examining or manipulating the cultured microorganisms, handling the prepared slides or plates before and after MALDI-TOF analysis, and then also just safe handling of primary patient specimens cultured microorganisms just to prevent LAIs. Next slide.

But looking at some of the chemical hazard considerations, MALDI-TOF MS for microbial identification uses a phenolic acid matrix. So typically, the CHCA that is solubilized with organic solvents such as acetyl nitrile and/or ethanol. You want to take into consideration additional chemicals, such as the formic acid or trifluoroacetic acid, which may be used in protein extraction procedures, inactivation protocols, or when you're cleaning reusable target slides.

Generally, small aliquots of matrix and formic acid solution can be handled safely on the benchtop during target slide preparation and on target extraction. However, you still want to be gloved, protective clothing, be in a well-ventilated room.

If you're doing tube extraction or inactivation procedures, including target cleaning, that involves maybe a little bit larger volumes and maybe some more hazardous chemicals, like the trifluoroacetic acid, in this case, you should perform in a class I or type B1 or B2 or a chemical fume hood if you're working with that. Next slide.

So the highest risk of exposure occurs during handling, manipulating, and disposal of primary specimens in cultured microorganisms before analysis. The direct transfer onto a MALDI target can be performed safely on the open benchtop using BSL-2 practices and facilities. However, potentially more hazardous microorganisms should be manipulated using stringent biosafety practices, including, at minimum, the use of a laminar flow BSC until the agent has been inactivated using a validated method or with the addition of a face shield.

So I'm going to stop here, and we're going to do another polling question on the types of targets that are used. So what types of targets do you use– the steel, disposable, or don't have a MALDI?

So that's great. We have a lot of groups that are using disposable targets. So with the disposable ones, some of the hazards you've already got rid of. You don't have to worry about the target cleaning, where you would the steel reusable targets.

And then the matrix alone, it's just something to keep in mind that matrix alone might not be enough to totally inactivate the microorganisms being inactivated. So you need to take that into account, but also taking into account, too, how you're spotting the plates.

So whether the biomass thickness on there, if you have a really thin light layer, or if you have a spot that's a little bit thicker, and whether the organic solutions that you're using are covering the entire spot or just a portion of a spot that are on the plate. Ideally, when you're doing MALDI, you want a thin layer, and you want to cover the entire spot. But if you've done it before, you know that might not always be the case, especially when bringing on new staff and training new folks. But keeping that in mind, you should also adopt and verify some inactivation procedures as well.

So I'm just going to pop up– we're going to have another polling question as well on inactivation procedures. So really, do you have any inactivation procedures in place at your facility– yes, no, I don't know, don't have MALDI?

Oh, again, great. So 54% say yes. 21%, not sure. Actually, 0% said no, which is excellent.

It kind of leads into the next slide, so if we go to the next slide, if you don't have an inactivation procedure, or you're not sure, and you find out later that you don't, really, this next slide is going to help kind of give you some guidance if you're not sure where to start.

So if you're not sure where to start with an inactivation procedure or what has been completed in the past, I have up here an article that was published in 2017, which covers the safety and accuracy of the MALDI-TOF MS for high-risk pathogens. But while this was a limited study, other publications show the same type of results.

So, however, some of the strains were attenuated or surrogate strains, so you want to keep in mind that wild types may act differently. But it is recommended that if a high-risk agent is suspected and needs to be run using the technology, that a tube extraction with filtration is really the best way to go.

It's also important to know the limitations of the library that you are using and be cognizant of which, if any, BT agents are present in the database. So it's advised to incorporate alternative libraries and use MicrobeNet to aid in identification as well. Next slide.

So despite enhanced biosafety education, improved lab practices, better engineering controls, and biocontainment equipment, LAIs continue to pose a risk to personnel working in medical and veterinary labs. If a high-risk infectious agent is suspected, labs should consult with the appropriate reference lab first. Analysis of such isolates should be avoided, as culture manipulation may heighten the risk of environmental contamination and/or personal exposure. Next slide.

So in the event that unsuspecting, high-risk, or select agent is inadvertently analyzed on a MALDI-TOF MS, with or without any type of inactivation step, the incident should be assessed immediately and reported to the lab management in accordance with the lab's biosafety and infection control procedures. The risk management steps should include determining which lab workers were potentially exposed to the agent without appropriate safety measures, potential need for post-exposure prophylaxis, and health monitoring.

So gathering any of those cultures containing the hazard agents, label, sequestering them, prepare contaminated disposables for either onsite or offsite autoclaving, and thoroughly cleaning all the affected bench areas. It's also recommended that, if you have the MALDI instrument, that contains an air intake or exhaust filter that, at minimally, this should also be replaced. Next slide.

So additionally, safety measures are needed because limitations to databases. So depending on the database for the MALDI system, biothreat agents might not be included, so when you analyze an isolate not in the database, you either get an ID of no identification, or you might get another pathogen or closely related species, but with a very low score.

Additionally, the opposite can be true. You can use the information gathered to help direct your identification. So, for instance, if you did not have the secure database for the instrument, which is an additional database that's needed to be purchased for the Bruker system that contains high-risk pathogens and BT agents, then if you were to get an ID of, let's say, Clostridium spirochetes or Bacillus cereus, then you might consider that these could be also potentially Clostridium botulinum or Bacillus anthracis.

You'll want to look at any other early indicators in your assessment, including the Gram stain, any biochemical results, any travel history if that's available. Luckily, for some of the near neighbors, the systems will flag the result and tell you of other potential identification, such as for Bacillus cereus.

There will be a flag saying that the isolate could be potentially Bacillus anthracis, but just being mindful of these flags and make sure that you know where they show up and that you're actually looking at these as well. Next slide.

So now, I'm going to get into some of the good stuff, a little bit of the clinical cases, so next slide. So back in 2015, New York State had four high-risk pathogen cases in a very short period. It was a three-month period, and the cases resulted in 60 laboratory staff being exposed.

The patients traveled abroad, where the disease is endemic. They consumed unpasteurized milk and milk products. The isolates from the clinical specimens were considered slow growing.

So I'll give you the opportunity to get away from the poll question and kind of open it up. You can either unmute and put your guess on what you think this pathogen is, or you can put it in the chat, and we'll just kind of see what you all think it is.

And so, right away, we have one for Brucella in the chat. Another Brucella. Keeping that Brucella alive. Man, I guess I wasn't very sneaky with this, was I? Always Brucella, there we go. So yes, shocker, it was Brucella, so we'll go on to the next slide.

I even blacked out all the stuff in the text, man. Still got the Brucella, though. So surprise, exposure resulted in Brucella isolates being processed in the laboratory. Several of the exposures related to new instrumentation, including the MALDI-TOF.

The public health Laboratory Response Network worked with the labs to provide information on prevention of lab-acquired exposures and infections, and then the LRMs looked to evaluate the MALDI-TOF for biosafety concerns, some of which is in that paper that I referenced a few slides ago.

So we're going to do another polling question. So if you have a Bruker MALDI Biotyper, you have the security database. And then one of the answers in there, too, is yes, you have the security database, but with the Bruker, you can also make your own internal databases. So do you have that as well, the internal database for high-risk pathogens?

That's great. So about half, the groups do have the security database. A couple don't, so just keep in mind if you don't have that security database, some of the biothreat agents will not show up. So if you do happen to put a Brucella on the Bruker system, at least without that security database, there is no indicator on there to let you know that it is a potential Brucella.

And then it's great to see that some labs also have their own, and their own internal database as well. Next slide.

Christina Egan: Mike, there's also a question in the chat. How were the samples processed for these exposures?

Michael Perry: So we're going to actually get into some of these questions. So we're going to go into the case histories now with these, and so as we go through this, if I don't touch on how they're processed, just let me know. But I believe that I will cover it for this section.

So with these cases I mentioned, there were exposures of three laboratories, including our own New York State Department of Health or bacteriology lab. So the crazy part is that all the exposures occurred as a result of one patient. The patient traveled from Mexico to the US, so here at the New York State Department of Health, we receive two isolates in our bacteriology lab.

That first isolate was received from one of the hospitals in which the hospital ID'd the isolate as Haemophilus. So once it was received here, it was subcultured on the bench as our normal procedure for Haemophilus that are received in the laboratory.

So it was prepped on the bench. It was plated for MALDI on the benchtop, and at this point, it wasn't suspected to be Brucella. Then a day later, around the same time, the second isolate was received from a second hospital. This hospital ID the isolate as an unknown Gram-negative coccobacillus.

In this case, it was worked up in the BSC because it was marked as unknown, but initial biochemicals didn't rule in Brucella. So this one was also plated for MALDI, and in this case, the MALDI ended up IDing the isolate as Brucella because we did have the security database.

So at which point, it was moved to our BSL-3 laboratory, where our laboratory confirmed it that it was actually Brucella melitensis. So good job, Darrell, for actually speciating the Brucella exposure as well. Next slide.

So what I want to point out in this one, there's a lot going on. Don't need you to look at everything, but this was the workflow we had here pre-exposure. So the takeaway really is that this is the pre-exposure workflow– a little complicated.

So after this event and reviewing the algorithm, we realized that this current algorithm was just overly complex, and rather than concentrating on the Gram stains, slow growing organisms, the algorithm did not put necessary weight on those criteria. It had listed the expected reactions for all select agents and did not indicate the key tests that should have been highlighted. This has also indicated that we needed to spend some time just retraining individuals on the algorithm.

So the one that– for the first one is, why it was moved to the hood or what prompted that, was the fact that it was an unknown Gram-negative coccobacillus, and so within the algorithm for our bacteriology lab, that alone will trigger it to go into a biosafety cabinet because, in that case, too, it could be potentially other high-risk pathogens. Next slide.

And then the first one, the reason that one was actually done on the benchtop is because they came in as a Haemophilus, and so they were suspecting that the clinical lab that sent it did correctly identify it when it was received. And so that's why that one was delayed.

So really, why did these exposures happen? So the Rouleau algorithm was just too complex. Needed to be simplified. Some of the laboratories were using Bunsen burners to sterilize metal inoculating loops, so that also caused the generation of some aerosols.

Additionally, the MALDI targets were spotted on the open bench without facial barriers at the time. So if you've ever seen a MALDI target, or you've used them, the circles or the spots for your isolates are really small, and I've seen folks that put those spots– have trouble seeing them, and they'll spot them in different ways.

And they'll hold the target directly up to their face as close as possible to see what they're doing. And so you can imagine that this is far from ideal, as the laboratory, and especially keeping it so close, if any aerosols are generated. Next slide.

So some of the proposed changes and the steps– some of them are highlighted in red. And again, it looks complicated, but the main takeaway from this is that, you can see throughout this, the use of the biosafety cabinet for processing until select agents are ruled out. And you can see that all the work starts in the BSC.

Additionally, the change in the algorithm is immediately send isolates to the biodefense laboratory that are slow growing or have suspicious Gram stains. Previously, the isolates were sent to our lab, the biodefense lab, only after performing certain biochemicals. So this will give more rapid detection of if there's a select agent. Next slide.

So fast forward two years to April 2017. Another large network micro lab was involved in a Brucella case. Patient had travel history to Mexico. Blood culture bottles indicated positive after three days. Oxidase catalyst were positive. Those were done on the open bench, not in the BSC.

There were some small faint colonies on a blood plate after 24 hours, and the staff decided to run it on the MALDI, in which the targets were prepared again on the open bench. The MALDI gave no result of identification.

Again, in this case, similar to other ones, the Gram stain from the blood plate indicated a Gram-negative coccobacilli, and the isolate was urease positive. So this laboratory was set up as an open room laboratory. You can imagine, there were many technicians which were considered exposure risks within areas of aerosol generating procedures.

Since this was one of the same hospital labs that had an exposure two years prior, we thought that maybe they weren't kind of listening to the recommendations or they have adjusted anything in their procedures to prevent this, and we met back up with them to see how they were handling these potential high-risk pathogens. Next slide.

But one thing that we ended up doing is that we ended up reviewing multiple things to consider in their workflows and algorithms later in 2017, things such as the ASM sentinel guidelines, going through how your spotting, when your spotting, what the application is, what types of methods do you use for extraction or direct smear.

And then with this same lab who received the positive Brucella specimen exposures the previous year, we received another suspicious isolate. It was all was worked up in a BSC, including the Gram stain and culture subbing. The blood culture bottles were positive. The Gram stain was Gram-negative coccobacillus, so the lab actually didn't do any work when they saw this. Everything– the sub plates and the blood culture bottles were secured in the biosafety cabinet.

And in this case, after getting an early indicator, securing everything, stopping their work between the two hospital labs, both receiving samples from the same patient, I'll put up a poll question on just how many exposures do you think they had this time between the two labs?

So good mix, so we'll go to the next slide. And it looks like that we have some really smart people here, as 44% said that there would be zero exposures, so next slide. The anticipation.

So there were no exposures, so we are very happy not that there was a Brucella, but the fact that there were no exposures because, if you dealt with labs in the past, and you're involved in any Brucella cases, you know that Brucella exposures are constantly occurring. You do your best to try to minimize how many people get exposed and what the exposures are, but to actually have two labs receive specimens, listen to what some of those early identifiers are, stop work, and then end up having no exposures– just, it's a good feeling. Next slide.

And really, what helps with reducing the number of exposures is the risk assessment. It's going to have to be really important when it comes to analyzing specimens using the MALDI, especially if high risk pathogens are suspected.

There are certain things that need to be considered, so what type of spotting or extraction method are you doing? Are you doing the tube extraction or another method of extraction? Is it something that you've validated and shown to be inactivating to microorganisms?

That initial spotting, are you going to do it in the biosafety cabinet? Are you going to do it on a benchtop? If you do it on a benchtop, make sure you're using a face shield or some type of facial barrier.

How are you going to spot it? You don't want to have it directly at your face. You want to have it further away. So if you have folks that have trouble seeing the spot, can you have some type of magnifying light on there that makes it a little bit easier?

Your matrix application, making sure that you're covering the entire spot and not just a portion of the spot. How you're going to transfer or remove the MALDI targets from the instrument or move them to the instrument, and then just the target cleaning itself. Next slide.

So we'll open this one up for a little discussion as well. So what are some of the other things that should be considered in relation to either sample preparation or decontamination? Maybe something that we haven't touched on yet? You can unmute your line and chime in, or if you want to put something in the chat, that's fine as well.

So one of the things that we always think about, too, is just, is there a way, if you're doing a tube extraction, can you implement a filtration step to it? Can you filter out the extract? One thing that we've done in that paper is that we looked at the accuracy of doing filtration versus no filtration.

So if you filter, does that affect the score? So while you might be removing any residual microbial in there, organisms, does it change what the score is? Do you get a lower score with that?

Another great point in the chat, the frequency of the viability study. So is it going to be something that you do one time, or should you continually do it, or multiple times just to make sure that everything is still functioning the same? And that's great to know, too, that the Bruker Biotyper has a kit specific to inactivation.

If you're able to put a link to that in the chat, that would be fantastic, too. That way, everybody who's in here would be able to see that, just in case you're not aware of it. Yeah, take your time. I appreciate you bringing it up. Thank you.

And that's good to know, too. Depending on how you want to look at what system you have, too. Not everybody's using the Biotyper, so if you have the Biomerieux, they have an inactivation kit as well. They also have some of the biothreat agents actually in the databases, so you wouldn't need a separate database.

Perfect. Thank you for putting that link in there. At least you, Tyler, put in there that they learned the hard way and that the VITEK MS is going to accurately identify the Brucella species. It is a hard way to find out, but it's good to know that it does work as well.

I think we can go on to the next slide, and then just some things to consider that we might not have touched on for sample preparation, as I mentioned, filtration steps. How you're going to do the application, what type of matrix is being added, so if you're switching matrices, that's going to have an effect on what it does to your samples that are there.

Do you have a standard biomass that you're playing with as you're spotting, keeping thin layers, and just making sure that you're following correctly the procedural steps? Next slide. And then, just in decontamination, making sure that you're putting in the appropriate PPE, using appropriate disinfectants for what you're working with.

How are you going to decontaminate? Are you decontaminating the area that you're using, the space around the instrument, the sample door of the instrument, making sure that you're changing filters if necessary? Obviously, you do not want to attempt to disinfect or decontaminate the inside of the instrument without consulting the manufacturer, so if you're ever not sure, you can work with the manufacturer to see what types of decontamination is allowed for their specific instrumentation.

And then so, next slide, what I'm going to do is I'm going to now turn it over to Christina for the next portion on the automated microbial identification systems.

Christina Egan: Thank you, Mike. Before we move on, I just want to open it up to see if anyone has any other comments about the use of MALDI. It's interesting because we were very concerned when this system was first being purchased by some of our clinical laboratories about would we start to see an increase in laboratory-acquired illnesses?

And as Mike has mentioned in his talk, we have done our best, but I guess the one thing that I think we have been fortunate is, in New York, we have not– we've seen a number of exposures.

But in the clinical laboratory, we have not seen any illnesses, at least in the last five to seven years. I was just curious if, in any of your states or jurisdictions, have you seen any actual illnesses that may be due to either the MALDI or some other identification system?

And also, we're thinking about new equipment– this is a new technology. For bacterial labs, a lot of our labs in New York do have these systems, but we're starting to see the use more on fungi for yeast and molds. So it is kind of a newer system for those areas of the clinical labs.

And then also, there are protocols for running blood cultures, but there are some labs that are trying to do other specimen types. So it's important for them to do– for clinical labs to do appropriate validation on that.

And Tyler, thank you, says an exposure event related to VITEK MS. Yep, multiple people, prophylaxis, no illnesses seen, at least. And that prophylaxis is pretty tough and no joke. So it does cause people a lot of angst, and the prophylaxis regimen is not easy.

And Carol exposures– yup, open bench Gram stain. Brucella, I think we've all seen that. Aaron, no LAIs that I'm aware of, but exposure to MALDI and rapid blood culture systems. And thank you for Linda. Yes, we're going to be talking about risk assessments, and we do try to stress that as well, that you should be doing risk assessments.

So we'll talk a little bit now about automated microbial identification systems, and Mike talked about one specific type of identification system you can consider, MALDI. But there are many different types of systems. And also, in this part of the presentation, we wanted to look at and talk about another pathogen that we have started to see in New York a little bit more frequently, but still overall infrequent.

And also, identification systems are so frequently used in the clinical laboratory we thought it would be useful to talk about that amongst all of our public health labs because, very often, we're the ones contacted for questions and additional guidance. Usually, it's after they identify a high-risk pathogen.

So can we go to the next slide? And this will be just a brief review. We cannot cover every single type of identification system because there are so many, some being, I think, on the simpler but utilized by probably a larger number of labs from small to large, are these small strips.

One is sold by API, and there's dehydrated substances that can detect enzymatic changes, activity, and fermentation of sugars. And it produces a pH change, which shows a colorimetric change, and then you can see a nice pattern of results.

And you can now– I remember the time that I– I think there's a couple of folks on here as well, that you actually had to go. You had the books, and you look it up. Now, they do it all online, which is a very– these are very simple, but they are identification systems all the way up to the really complex and very automated system.

And an example here is VITEK, but there are many others that are– some of which, I think, are even more complicated and have additional parts that are also identification systems.

And so I would just like to open it up here, too, just to understand what type of automated systems do you have in your laboratories. And you can just unmute or just put it in the chat.

I tell you, so in our laboratory, we actually still use API strips. We do have MALDI, but in New York and in Wadsworth Center, we don't have very many of these really large systems that we utilize, like VITEK or Microscan or all sorts. We do have the bacterial alert systems for our TB, which are growth monitors, which are another type of identification system.

Yeah, and I guess maybe your public health labs do not have many of these larger identification systems either. However, I think that– oh, thank you, Katelyn. Yeah, the MGIT, API strips, MALDI.

We have been contacted by the clinical labs, and I think– and, again, as I mentioned, we do get contacted, usually after they have a potential ID of either a select agent or some other high-risk pathogen. But one of the other labs that we hear from, too, is veterinary labs because they also use these types of identification systems as well.

And their clientele do also get infected with high-risk pathogens as well. And, Tyler, oh, you have the VITEK MS? And you use the Gram-negative for Shigella?

Yep. Yep, so very similar. We started to move to PCR to do the Shigella/E. coli, but used to do that as well. VITEK and MALDI, thanks, John.

So I guess the next question, what types of questions do you– or if you do get questions, do you get– or you hear about from clinical labs about the use of automated systems? Serotyping for Salmonella.

I would say that the top questions we receive are about decontamination. How do you decontaminate the systems, as well as where should we be processing samples and inoculating samples? So a lot of them, I think, can be grouped together in the processing and be the back end of the decontamination. Next slide.

So very similar to what Mike was discussing with regards to MALDI. The highest risk, again, is in the handling and manipulation and cultured microorganisms. Some of the systems are not automated end to end, and so of course, if you do have a cultured microorganism that would put our lab workers at the highest risk.

The other thing for some of these automated systems is preparation of bacterial suspensions to go into some of the systems, and labs really should be using BSL-2 practices and facilities. We have heard, with some of our investigations, there's a lot of preparation of bacterial suspensions on the open benchtop with open vortexing as well as opening tubes up in the general laboratory without kind of any concern, popping tubes off, which causes aerosol generation.

And I'm going to go back a number of years ago, 20 years ago, when I first started at Wadsworth Center, and one of our very seasoned clinical bacteriology members went out to a clinical lab to do a Brucella investigation into a clinical micro lab and actually saw an individual was uncapping tubes and throwing the caps across the lab into the garbage can.

So what we really try to stress with clinical labs to really be careful with these types of activities. Next slide.

Whoops. Oh, you're going back a little? So we kind of saw a few of the high-risk pathogens. I was going to ask a question here. What are some high-risk pathogens that you run into in the clinical micro laboratory or your public health laboratory? You can put them in the chat.

Yes, very important. Neisseria meningitidis is one we really worry about with clinical laboratories. Mike also mentioned Brucella, so I'm sure everyone has that on their mind. Yep, yep. Oh, good, I hadn't even thought about the molds. Excellent. Aaron, yes, rabies, EEE. Very good, yep.

I think we can go to the next slide. I think we've actually covered what I had there. Yeah, I entirely agree, meningitidis is really common, and we actually have had– it was a number of years ago in one of our clinical micro labs in New York, an infection, a laboratory-acquired infection due to Neisseria meningitidis, which was really unfortunate. Oh, and Rickettsia, too.

So listed here are just a few that I put up, and what I wanted to spend a little bit of time now is talking about Burkholderia pseudomallei, which I think is really more of a newer pathogen of concern here in the US, and I'll talk about some recent cases. And I would say from 2000 to at least 2010, I don't think we actually had any Burkholderia pseudomallei coming into our public health laboratory for rule-outs.

But in the last at least five to seven years, we have definitely seen an increase. So can you go to the next slide?

So Burkholderia pseudomallei causes melioidosis. It is a Gram-negative bacillus, and this organism does feed or get nutrition from byproducts of organic breakdown.

And so it loves that soil. Found in the soil and water, and everyone is probably aware that it's found in, and endemic in, northern Australia, in Asia, and then also in the tropics.

And a number of years ago, 2015, 17, we actually had several cases from individuals who were traveling to the Caribbean. Next slide.

And I guess this figure is a little low, too. I think there are on the order of probably five to 10, or five to 20 now, cases annually. But what I really want to point out on this slide is that there are– unlike some of the other organisms that we talk about, there are some risk factors that are really important, and they're especially important when doing investigation exposures in clinical laboratories.

And they're diabetes, chronic liver, and kidney disease immunosuppression because these individuals can become ill and actually can become extremely ill. And there can be different manifestations. You can have a pneumonia, cutaneous infection.

We actually had an individual a number of years ago who was recovering from cancer treatment. Went to the Caribbean and actually got an infection in her breast. She did recover, but you have to be– these risk factors are very significant and also can result infections in sepsis and organ abscesses.

And what's not, I think, still commonly known is that there can be chronic cases, reoccurring, or what they referred to as smoldering. And we, a few years ago, had a case where an individual was diagnosed and treated, and sometimes, it's hard to tell if individuals stay on their course of treatment. But probably about five or six months later, became very ill again, and this individual was seriously ill, but did recover.

The incubation period can be long, and the treatment is IV– can be IV or by oral antibiotics. And the case fatality rate, and it does depend on how quickly the illness is recognized and treatment is initiated. Next slide.

And I think everyone may be aware of the recent cases down in the Mississippi Gulf Coast, where there were several cases of infection by Burkholderia pseudomallei. There were two human cases, and the health department's, and I'm sure the laboratories as well, did some intensive investigations.

And these two individuals, they lived close to each other, but did not have any interaction directly. Weren't the same family. There was no travel history by either of these two individuals, but they became ill.

They were two years apart, and through sequence analysis done by the CDC, they were infected with the same strain. These individuals were hospitalized with sepsis due to pneumonia. They were very ill. They both did have risk factors for this illness.

Luckily, they both recovered, but there was significant sampling done on the households– in the household, the soil and water on the properties. And due to the analysis from the patient in 2020, three of the samples were a match with the actual clinical specimens, leading to determining that, down in the Gulf Coast, that this organism is now endemic there.

And who knows how long before it had been endemic? Don't know. I mean, people really weren't looking for it, but it does show, again, that– and we should all, in public health labs, really be on the lookout, especially in trying to get when people are ill, that travel history because now, in addition to Asia and northern Australia, Caribbean, if they are traveling down in this area.

And then next slide, also, several years ago in 2021– and many of us heard about this through calls through the Laboratory Response Network as well as some of the labs shared with folks represented here were part of the investigation to determine– so Georgia, Kansas, Texas, and Mississippi, there were four individuals that became infected with Burkholderia pseudomallei.

And unfortunately, there were two fatalities– a child and adult. And there was a lot of work went into this investigation to determine how these individuals became ill. Based on sequencing done at CDC, they were able to determine that this was a strain from Asia, and the CDC lab, they have an excellent database of Burkholderia pseudomallei to be able to determine that.

And then through the environmental investigations, identified this room spray that was sold by Walmart, so very unfortunate. Next slide.

So I'm going to talk about several cases that we have had recently. Next slide. So the first was in November of this past year. There was a 67-year-old male who had traveled to Honduras, and right when he got back to the US, presented at the hospital within 24 hours.

Was severely ill. Treated for meningitis. Blood cultures were obtained and were positive the next day.

And unfortunately, work was performed outside of the biosafety cabinet, so not only the MALDI system, but an automated identification system, in this case, I think it was a Microscan. So the actual instrument provided a preliminary result, which was– it was identified at Burkholderia pseudomallei, which I guess, is a good thing.

But I think it's important to note here– so years ago, we used to get calls where, oh, our identification system gave us an ID of Burkholderia pseudomallei, and we would say, really, the chances of this being likely are very low. Send us the isolate, and they never keyed out or confirmed here as Burkholderia pseudomallei.

And now, again, based on the travel, and now, some of these incidents and events that I described, we, I think, have to take these very seriously. So the reference lab did have 13 exposures. The original lab did not have any exposures, and that's because they don't do a lot of this testing on site. They send it to a large reference lab to do that work.

The second case that we had was just a few months ago. It was a 63-year-old female who had traveled to Africa, came back, had three weeks of symptoms listed on the slide.

The lab processed the specimen and utilized automated identification, manipulated the isolate on the bench, and the ID was, again, Burkholderia pseudomallei, resulting in 10 exposures as far as we know.

So along with– as I'm sure folks are aware with these exposures, we have been sending serology down to CDC. And luckily, we have not had any seroconversions, but we're still not very far out with these exposures. Next slide.

So as I mentioned, it was somewhat unfortunate that samples were worked on on the open benchtop, but that the automated systems did provide an ID of Burkholderia pseudomallei. But that's not necessarily always going to be the case.

And I say this because we– with these automated systems, because it's so infrequent in the US to have these isolates, some of these databases contain very few isolates for Burkholderia pseudomallei.

I will say that, back in 2016, we ended up having a isolate that we received from a dog, and the vet lab was excellent and said the automated ID system that they used were saying that it wasn't [B.] pseudomallei. But by morphology, it does look like [B.] pseudomallei.

We got it here, and it was from a dog that was being adopted over here from Thailand. And it ended up that the strain– it was an unusual strain. It actually had a different motility pattern as well as some of the biochemicals.

So keep that in the back of your mind, that with some of these organisms, there's going to be a range of responses to what we consider in our algorithms. And so for pseudomallei, they sometimes will ID as [B.] cepecia and [B.] thailandensis, as well as Pseudomonas [and] Acinetobacter.

And we list two publications on this slide that, if you want to go back and take a look at, it provide some more detail. And just also keep in mind, Burkholderia mallei, that's much less common in the US, but there were studies done to show that, yes, that there are some systems that do incorrectly identify Burkholderia mallei. Next slide.

So if there is an inadvertent analysis of high-risk pathogens, what can you do? Things to keep in mind, to communicate with clinical labs or even our own public health labs. Again, that there are database limitations with some of these automated instruments, as I mentioned before, that there aren't a lot of strains from many of the select agents.

That samples that have a closely related ID or no identification should be moved into the biosafety cabinet. Shouldn't work on the open benchtop. What we have seen in New York, and I'm sure many others, that what happens is that, sometimes, these no identifications or misidentified leads to all this additional testing and additional laboratory exposures.

So we do try to tell labs that they should definitely use travel history. Also, if they do have the ability to do some biochemical testing safely, look at morphology, that this should be done safely, biosafety cabinet, and taking precautions. And use that in addition to the automated ID system.

And let me see, I want to also put up– I missed a polling question. So I just wanted to have the polling question, next polling question. Have you ever had Burkholderia pseudomallei or [B.] mallei exposures in your laboratory?

Oh, that's interesting. 13% occasionally. And I think we're just going to see this increase with agents such as Burkholderia pseudomallei, which we don't see as much as some of the others.

Oh, I see Leah has a question. We've had several Burkholderia pseudomallei— excuse me, pseudomallei positives, but they are all in our BSL-3, yes. Our sentinel labs have had several exposures. Yep, thank you, Leah.

Marcia? Yep, seeing them also in the sentinel labs. I just think we just have to keep this in mind because we are going to see this, I think, increase. And then the final polling question that I would like to ask is, how often do you receive travel histories from patients?

Thank you, Leah, and yeah, largest is sometimes, which is unfortunate. It's too bad that we can't– that we don't get all this information. Hopefully, someday, if we ever get the true electronic medical records, that maybe we would have access and be able to get some of this information.

Yeah, St. John, yep. So this is, I think, an issue that we are all facing here. Can I have the next slide?

And as we were– somebody had written in the chat, risk assessment. So important to do the risk assessment, especially when considering the instrumentation.

It's really unfortunate when you hear about labs that get a piece of equipment in, and they don't realize that, for example, the instrument, if it's a molecular automated instrument, that oh, you can't do pre-lysing So it's really important to do these things before you implement new instrumentation.

The other thing is, if possible, using instruments that are totally automated or allow for front end processing in a biosafety cabinet so that you can have some additional biosafety measures with that processing step. What I find really, really fascinating is some of these– although they're very, very expensive, some of these automated systems that allow for telemicrobiology.

So using the actual instrument that will take pictures of culture plates, and then you can review them. People are reviewing some of these at home, to look at culture plates, so you eliminate that risk of opening and examining plates.

Also consider, although not so critical with Burkholderia, but still growing, we do have Brucella, Francisella, some of the organisms that do have a slower growth rate. And of course, performing, developing your algorithms for working in a biosafety cabinet until you can take those out for the benchtop. Next slide.

So thinking about organisms in general, so categories, Gram-positive, Gram-negative, spore formers, using the growth rate, were there other tests performed other than the MALDI, and using additional characteristics of some of the other morphology and biochemicals.

And finally, too, the ASM lab protocols– are laboratories using these at all? Next slide.

So I'd just like to talk about both types of instrumentation and other biosafety steps that can prevent exposure. So if we go to the next slide, one big thing, venting blood culture vials in a biosafety cabinet. We have had exposures because those were done on an open benchtop.

Mentioned several times, using that biosafety cabinet, reviewing ASM protocols. Reminding labs, contact your LRM lab before you even start to work with a potential high-risk pathogen. Just send it to the LRM lab to do the rule-out and procedures.

And then if you can't take appropriate biosafety steps, limiting the use of automated ID systems or using only particular ones that you have done a risk assessment on. Using additional base protection benchtop shields. Mike spent some time talking about that. Next slide.

And then when labs do call you, and they will, what questions can they ask when implementing new platforms? What are the aerosol generating steps? They should be asking manufacturers, are there aerosol generating steps? The contamination, spills, splashes in the instruments, do they have protocols and procedures?

Do the decontamination methods that you use in your laboratory– are they similar, or is there a different type of disinfection that has to– decontamination that has to occur with these instruments? Concerns with the areas as well as the staff and PPE used.

Have the staff, this is a big one, been appropriately trained? Do they know trigger points, and do you have an inactivation method that is previously verified?

Roland, an excellent comment. Many clinical labs don't have enough biosafety cabinets. Workload is high. Specimen types have to be prioritized to be put in a biosafety cabinet, and we must work to educate about increased risk and other options for engineering controls when a biosafety cabinet is not possible. I couldn't agree more with you about that.

And the other thing, too, is really getting on the infection control folks in the hospital and the infectious disease physicians to provide this information because it's really unfortunate when you're doing this investigation, and you hear the ID doc, well, we had a suspicion, but the clinical lab was never told. So it is very, very frustrating, and especially for the clinical micro labs.

So I wanted to open it up again and see if anyone had any other automated ID instrument type questions or issues or cases that they have had involving some of the automated ID systems that they'd like to share with the group?

The other group, too, that we have also sometimes have to interact with is, sometimes, our academic research labs in our area will be working with some of these organisms, and staff haven't been appropriately trained.

And so that's another group that we have to keep in mind, too, and there are select agent regulations, which has helped, I would say, with kind of ensuring some type of training is going on. But sometimes, if you're outside of a select agent, TB research labs, you do see, sometimes, laboratory-acquired exposures. So if there's no other comments, Sabrina, I'll turn it over to you.

Aufra Araujo: Alright.

Christina Egan: Oh, sorry.

Aufra Araujo: It's OK. Thank you for your presentation, Christina and Michael. This was excellent. I really would like to open the floor for any other ideas or questions that the audience may have. I know, Christina, you were asking that, but before we conclude, I just would like to give another chance. I think this last two slides in particular, it's very rich with several questions on topics to consider when implementing new platforms.

And I don't know if folks are tired, but this is your chance to ask live. And of course, the slides will be available later, but you have Christina and Michael right here, right now, just in case you have any other thoughts or comments or questions to the speakers.

Alright, well, if not, I stop sharing this one. Thank you, again, Christina and Michael. This was an excellent presentation and very rich of information. Let's see.

Thank you all for taking part in our discussion today. We hope you find it valuable in the important work that you engage with in your individual laboratories. We look forward to your participation in future sessions as we dive into specific laboratory biosafety topics.

Soon, you will be receiving an email message containing a post-session survey link. It will take about two minutes to complete that survey. Please do so, as your response will help us inform next steps, other sessions, other ECHO sessions.

Now, let me share my screen again. I'm always so slow to do this to, transition from one to another. Bear with me. My arrow is not moving. Alright, so we are excited to have our next session in April. It will be on Tuesday, April 25 at 12:00 PM.

The topic will be decontamination of laboratory equipment, which will be presented by Dr. Shawn Gibbs from the School of Public Health at Texas A&M University, and Dr. Aurora Le from the School of Public Health at the University of Michigan. Please visit the DLS ECHO Biosafety website to view all upcoming sessions.

If you have any questions, you can reach out to us at dlsbiosafety@cdc.gov. Again, thank you so much to all participants and to our speakers today. Really appreciate you taking the time to attend this session. Now, we will adjourn. Thank you, and have a great day.