Bobbi Pritt, M.D. (00:04):

Hello, I'm Dr. Bobbi Pritt, a clinical microbiologist and laboratory leader at Mayo Clinic, and your host for today's episode. Dr. Bill Morice is back again today to discuss some trending topics. Then I'll be welcoming one of my microbiology colleagues, Dr. Trish Simner, for a deep dive into the use of metagenomic sequencing of cerebral spinal fluid in clinical diagnostics. So Bill, welcome back.

William Morice II, M.D., Ph.D.  (00:28):

Yeah, it's good to be back as always.

Bobbi Pritt, M.D. (00:30):

Yeah, always wonderful to have you here. Well, let's start by visiting what is new and noteworthy in clinical diagnostics. Now, I understand you just got back from Washington, D.C., and have some updates for us.

William Morice II, M.D., Ph.D.  (00:42):

Yeah, that's right. I mean there's a lot that's new and interesting in diagnostics as part of the American Clinical Laboratory Association Board of Directors, for which I'm the chair. But also meeting with people in Congress as well. A big focus is on PAMA, you know, the Protecting Access to Medicare Act (PAMA), and then reform of PAMA or getting relief from PAMA and introducing a new law that would address some of the shortcomings of PAMA.

Bobbi Pritt, M.D. (01:07):

Yeah, we had spoken about that in one of our last podcasts. I'm curious, has there been any developments with the policy?

William Morice II, M.D., Ph.D.  (01:14):

Just to remind people and to ground this, PAMA goes well pre-COVID. It was a system put in place where there was collection of data from a variety of different laboratories ― independent labs, hospital labs, others too ― to basically get market-based price setting for laboratory testing to move away from the traditional clinical lab fee schedule. But there was a flaw in the way the data was collected. It really oversampled independent laboratories like Quest and LabCorp, and underrepresented hospitals, and therefore, skewed to lower reimbursement. The way the law is written, it's still in place. We had multiple delays through COVID on the next round of cuts; we've gone through one. If something's not done with PAMA at the beginning of 2026, the government will set new rates based on 2016 pricing, and you can imagine as well that will certainly not be higher. It will be cuts and laboratories actually will be required to go back and try and collect data from 2019 reimbursement to give to the government. So we need relief from that, and that's really the main focus right now for clinical laboratories, basically all clinical laboratories, both academic and private, and others in Washington, D.C.

Bobbi Pritt, M.D. (02:27):

Wow. And I know we talked a little bit about this in our October 16th episode. Do you think it's going to go through?

William Morice II, M.D., Ph.D.  (02:35):

Well, so there's really three levers, or mechanisms, to kind of get this addressed. One would be to pass a new law, which is the RESULTS Act. We don't have to go into all the specifics, but it really addresses a lot of the shortcomings of both data collection as well as rate setting and PAMA. That would be a new law. And to be honest, with the situation in Congress right now with the shutdown, the likelihood of getting a new law passed before the end of the year is very low. But that's not true just for RESULTS. The other strategy is, can we work with Congress just to get a bridge to say, “Yes, we recognize that this law needs to be changed. We can do some things not just to delay, but a few things that Congress can say to actually start to address so we have more time to consider the RESULTS Act, which does have strong bipartisan support.”

And then the third actually is with the administration. So, it's not in Congress, it's with CMS. CMS actually has the latitude to decide whether or not to take action on the proposed cuts. Those are really the three strategies to try and get this addressed. I would say RESULTS is really more of the long-term fix, very unlikely before the end of the year. Bridge has a real strong chance, but a lot of that depends on how this breaks loose in terms of the shutdown and what will be the focus of Congress as they restart the government. Will there be a place to put something like that in? And then the third would be to work administratively. One of the good news for all labs is that the White House asked for a list of anomalies, meaning things that are anomalies that need to be addressed before the end of the year. And actually, this made it to that list. It's not like it's not getting attention, but it's just a little bit. There's some nail-biting because of just what's going on with the federal government.

Bobbi Pritt, M.D. (04:25):

Right. We're getting close. It's now November. I can't believe it. So we're gonna have to keep a close eye on this Bill, and we can talk about this at a future podcast. Do you think the bridge act has a good chance?

William Morice II, M.D., Ph.D.  (04:36):

I think it does. One thing I'll leave the audience with is to remember, as we've talked about before, that your voice really matters. Right? You know, getting your representation in Congress to recognize that this is an issue to garner their attention when there's a lot of different issues they're trying to think about. So ACLA has a public domain, StopLabCutsNow. I'm sure there's others, your work with CAP. But the most important thing is your engagement; writing letters and participating in some of these things actually makes a big difference. So that's the other thing I would say has a good chance, a reasonable chance, and we can actually increase the odds by being engaged.

Bobbi Pritt, M.D. (05:12):

Yeah, that's such a good point. And all of your professional organizations, like you said ― ACLA, CAP, ASCP ― a lot of them have form letters, and you can just edit it to put in your own thoughts, make it a little bit more powerful. It's pretty easy to do. Everyone should look into that.

William Morice II, M.D., Ph.D.  (05:28):

Yep.

Bobbi Pritt, M.D. (05:29):

Well thanks for joining us for this important update.

William Morice II, M.D., Ph.D.  (05:32):

My pleasure. I'm happy to come back anytime, as you know.

Bobbi Pritt, M.D. (05:35):

Talk to you soon.

Bobbi Pritt, M.D. (05:46):

Since Bill and I recorded our segment, there's been some news about the possibility of a short, temporary delay of PAMA implementation to provide more time to explore options like the ones that Bill mentioned. I know that he will continue to keep us updated on future podcasts, and I encourage you all to stay connected with your professional organizations for more updates. So now, on to today's deep dive with Dr. Trish Simner, who will bring you insights into metagenomic sequencing and clinical diagnostics. Dr. Simner recently joined our Division in Clinical Microbiology at Mayo Clinic and one of her focus areas is metagenomics. Thank you so much for joining me today, Trish.

Trish Simner, Ph.D. (06:27):

Thanks for having me Bobbi.

Bobbi Pritt, M.D. (06:28):

So to begin with, Trish, could you share with our audience what brought you to Mayo Clinic, or I should say, what brought you back to Mayo Clinic since you did your fellowship with us? And also what are some of your clinical interests?

Trish Simner, Ph.D. (06:41):

Yeah, thanks Bobbi. So, as you mentioned, I completed my clinical microbiology fellowship at the Mayo Clinic back in 2013, and I've always aspired to return to the Mayo Clinic later in my career. You know, there's many different reasons why the Mayo Clinic is a special place to me. One is, which might sound a little cliche, is the philosophy of “the needs of the patient come first” has always resonated with me as a Ph.D., clinician, and as a fellow. I've actually brought that throughout my career, even after leaving Mayo Clinic and post-fellowship. The other reasons that Mayo Clinic is special to me is the innovation. I think one of the things that I've been passionate about in my career is being innovative, and Mayo Clinic parallels that passion of mine very closely, being on the forefront of diagnostics to advance medicine and infectious disease.

And it aligns very nicely with the podcast today as we talk about metagenomic NextGen sequencing. The other thing is the people. So, the consultants who I get to work with closely, coming back here as a consultant myself, but have been my mentors in my career, then became my peers post-fellowship. I worked with them very closely on various committees and studies, et cetera. So, coming back here feels very familiar and feels like home. The other thing that is super cool to me is that the medical laboratory scientists that trained me on the bench, you know, they stayed at the Mayo Clinic while I went away for over a decade, and now they're in leadership roles. It's super fun. It feels very familiar from that standpoint as well to see them progress in their careers. And now we're working together too, you know, in terms of management and leadership in the laboratory setting.

So those are the reasons why I came back to the Mayo Clinic. Then in terms of my clinical interests, there are two big interests that I have. One is studying antimicrobial resistance among bacteria, and especially gram-negatives, and translating that information to patient care, whether that be new diagnostics; devising new antimicrobial susceptibility testing methods; guiding therapeutic approaches by understanding risk factors associated with the development of antimicrobial resistance; or understanding the impact of resistance on infection prevention and control. Whether that be knowing you're colonized with a particular organism or how we prevent the transmission or development of disease with that colonizing organism. So that's been one interest of mine. And the second has been developing next-generation sequencing technology for diagnostics to advance infectious disease diagnostics, which again, really aligns with what we're discussing today.

Bobbi Pritt, M.D. (09:19):

Yeah, well, Trish, that's great. And you know, I have to say before we get into more about metagenomics, we are so delighted to have you back, and we're so glad that you consider this home. It's home to us as well. And I love how you mentioned our people. Our people stay with us and you know, some of our technologists celebrated their 40th anniversary here, 40 years of being here at Mayo. So, we are really happy that you came home as well.

Let's transition and talk a little bit about metagenomics. One of the tests that you've been working on since you've been here and now are working on improving is our CSF metagenomics test. Can you tell us a little bit about that one? How does it work? What are the basic steps?

Trish Simner, Ph.D. (10:04):

Yeah, great question, and before I get into that, I'm excited to be back. So super excited to be back here. But talking about metagenomic next-generation sequencing. As I discussed, one of my passions or clinical interests is developing next-generation sequencing. That's a technology. And metagenomics is a method that we utilize next-generation sequencing to be able to provide diagnosis of infection of patients with meningitis or encephalitis because we're focused here specifically on cerebrospinal fluid. We could discuss in the future our expansion to other sources. But when focusing on this methodology, metagenomics is an untargeted approach. You're extracting all nucleic acid within that cerebrospinal fluid specimen, and then you're sequencing all of it. On the back end is when you do your bioinformatics to detect the microbial reads. When we kind of break it down into steps, there's a first step where, as I mentioned, you're extracting all of the genomic information, all of that nucleic acid from that sample. Because it's an untargeted approach, you get both human nucleic acid but also microbial nucleic acid.

From there, we produce two separate libraries, and these are sequencing libraries. These allow us to amplify and sequence all of the fragments of the nucleic acid that we've extracted from that specimen. One library is a DNA sequencing library that allows us to detect our DNA-based microbes that have a genomic consistent of DNA, like bacteria, DNA, viruses, parasites, fungal etiology, mycobacteria, et cetera. But there's a second library that we prepare, which is RNA sequencing library. And the reason we do this is to be able to detect our RNA viruses. What we do on the RNA sequencing side is we actually first do some host depletion. We host deplete some of our ribosomal RNA to kind of enrich our microbial RNA, and then we reverse transcribe the RNA to complementary DNA. Essentially, we make it into DNA, and then we prepare the library from there, which, as I mentioned, allows us to sequence all of those various nucleic acid fragments within that sample.

We get a massively paralleled sequencing of the fragments in these libraries. And then on the backend, once our sequencing run is complete, we then, by bioinformatic means, evaluate the microbial reads. We actually ignore any of the host reads. Remember, I said we're isolating human DNA as well, or human nucleic acid, as well. But really, we're focusing on the microbial reads. We do this by ignoring, pulling out all the host reads, focusing on the microbial reads, and then taxonomically aligning those reads to the lowest taxonomic level possible, ideally to the species level. And we taxonomically classify these using large, well-curated reference databases that previously weren't available that have now become available that allow us to do this method. And then we analyze that data. A doctoral-level microbiologist like myself, Dr. Robin Patel, or Dr. Andy Norgan, then interprets those results in the context of the patient, whether that be our internal patients here at Rochester or external patients. If we have any questions, we'll always reach out to the clinicians at the outside sites to gather further information to kind of guide us.

Bobbi Pritt, M.D. (13:35):

That's a great review of all the different steps. And I love how you focused in on the fact that we're looking at DNA and RNA. Lots of RNA viruses can be important in our CSF specimens, but then also emphasizing that we're trying to look for that needle in the haystack, metaphorically looking for the microbial reads, which may be the minority with the human reads. That's interesting. We can talk about why the human reads might be important in the future as well. But let's stick with CSF metagenomics as we currently have it. When might a clinician consider using a CSF metagenomics test?

Trish Simner, Ph.D. (14:12):

Yeah, I think at this point in time with CSF metagenomics, we want to utilize it when our standard of care methods are unrevealing and when there's still a high concern for infectious etiology, meningitis, encephalitis. You want to make sure you're ordering your standard stains, antigens, cultures, and even some targeted PCRs or panels before you go to metagenomic next-gen sequencing. Because oftentimes the standard of care methods are actually great for detection of the most common etiologies. In my experience, and upon review of the experience here at the Mayo Clinic, metagenomic next-gen sequencing from CSF has the highest value in looking at rare atypical unsuspected etiologies, or alternatively, in cases where the patient might have been on treatment prior to collection of a sample, which reduces our ability to recover the organism in culture. We could still detect the nucleic acid to try to help guide patient care in those instances.

So, it's not a first-line test. It's more of an adjunct to our standard of care test when it's revealing. One of my favorite cases, actually the first case that I was able to diagnose by metagenomic next-gen sequencing, was a case that really highlights the power of metagenomic next-gen sequencing in the diagnosis of cases of meningitis and encephalitis, or when there's no other etiology. This is a case of a 40-year-old from Cameroon, who presented to the neurology clinic with a prolonged history of headaches and fatigue. During her many presentations, she had really extensive work-ups and had found both abnormal autoimmune and infectious disease serologies that were associated with this patient, including Lyme disease at QuantiFERON that was positive for mycobacterium tuberculosis complex. She was ultimately treated for systemic lupus and cryoglobulinemia with some immunosuppressive drugs.

However, the patient continued to experience progressing symptoms, including hearing loss, development of a skin rash. The CSF pleocytosis was still screaming that there was probably some infectious etiology going on here. The MRI findings were progressing despite them trying to broaden therapy as much as possible. At this point, we approved it from a diagnostic stewardship approach to then send it for metagenomic next-generation sequencing, and we identified Trypanosoma brucei. Yeah, and this is perfect for a podcast with you, Bobbi.

Bobbi Pritt, M.D. (16:57):

Yeah, right.

Trish Simner, Ph.D. (17:23):

For the listeners, Trypanosoma brucei is the cause of human African trypanosomiasis, and it's caused by a protozoan parasite that is usually, if left untreated, a fatal disease. Unfortunately, with some of our other pathogens, or fortunately, I should say, with some of our other etiologies that go undiagnosed, what the clinicians do on the frontline is they start to broaden therapy.

So, they start to add broader, broader antibiotics, antifungals, antivirals, et cetera, to try to cover therapy when they don't have a particular etiology. Well, with Trypanosoma brucei, that's not possible. The treatment involves toxic agents. It requires approval from the FDA to get these agents for therapy. I think this really highlights the power of metagenomic next-gen sequencing because in the absence of this finding, we were unlikely to come to the conclusion that the patient had Trypanosoma brucei and would've unlikely been given appropriate therapy for this organism. It was an exciting case to be involved with and to follow up where the patient has near resolution of symptoms now. And so, really highlights the power of metagenomics in these very typical unsuspected cases.

Bobbi Pritt, M.D. (18:15):

That's a great example. And you're right, that's not the kind of thing that you would just prophylax with, you know, your routine antibiotics. That's a very specialized treatment as well. So what do you consider the most important advances in the past few years that have allowed us to make this type of testing available?

Trish Simner, Ph.D. (18:33):

You know, it was back in 2015 when Charles Chiu’s group from UCSF published a successful diagnosis of neuroloptospirosis in the New England Journal of Medicine, applying metagenomic next-gen sequencing. But at that point, it was still in the research setting. We were still trying to understand the methodologies to be able to apply this as a clinical test. It is a complex multistep test. And so, there was a lot of standardization and defining processes and quality control parameters that we needed to structure first before we could bring this into the clinical setting. It's not all that exciting in terms of big things that brought it into the clinical setting. But being able to standardize our methods and optimize development to lock down a method that we could apply and could be reproducible in the clinical setting with defined quality control parameters was one of the big things that allowed us to kind of bring it from the research into the clinical setting.

The other thing is really bioinformatics. Most microbiologists in the laboratory, maybe the older microbiologists, don't have any bioinformatics expertise. I'll say a lot of the newer generation are coming with bioinformatics expertise, which is amazing. However, we've been trained, you know, more from the microbiology standpoint and not the bioinformatics side, handling the large data sets that we get out of sequencing. The development of these automated analytics that allow us to drag and drop the sequencing data, and then it pulls out all the important information and classifies it for us, has been an extremely big step forward for us to bring this into the clinical setting. Otherwise, you needed a bioinformaticist on staff. And the bioinformaticist, especially if they don't have clinical microbiology training, they're also learning a whole new language when it comes from microbiology. I think these automated analytics and these large reference0grade genomic databases have really allowed us to confidently use the data generated from sequencing to try to attempt to establish a diagnosis.

Bobbi Pritt, M.D. (20:41):

That's great, Trish.

Trish Simner, Ph.D. (20:43):

And my biggest wish list for kind of next steps for advancing the method in clinical microbiology lab is really automation. I kind of alluded to this in my previous answer, but the process is complex, multiday. It takes a while to train our staff to be competent in the methodologies. I'm super excited to see more and more automation in the method. It'll be similar to the parallels of PCR in clinical microbiology and in infectious disease diagnostics where, until it became highly automated, we didn't see broad uptake. The more and more automation of these methods that we see, the more and more others outside of large reference labs or academic centers or public health labs will benefit from this technology as well.

Bobbi Pritt, M.D. (21:30):

Yeah. You read my mind because that was actually going to be the next question I asked is, what do you think the next big advances would be? And I know that through the work with our department and as well as some potential collaborators, Mayo Clinic Laboratory is looking at metagenomic tests that use plasma bronchoalveolar lavage specimens to expand metagenomic testing. But I'm curious what other innovations, including, I guess, automation refined bioinformatic systems, should our listeners keep an eye on over the next few years?

Trish Simner, Ph.D. (22:03):

Automation is one that's going to help us implement it more effectively and see broader uptake. I think one thing I'm most excited about with metagenomic next-gen sequencing is, as I mentioned earlier in the program, we get all of this genomic information and a lot of it we just ignore right now. There's so much more we could do with all this data we're generating. And I really believe that, aside from expanding our sources as you discussed, Dr. Pritt, it's also about being able to utilize that data to answer more questions about what's going on in that patient and really becoming a precision medicine diagnostic. What I mean by that is right now we use the microbial reads just to provide an identification of the organism, but we could further study those reads to understand, do they have anti-microbial resistance genes present or absent? Can we get enough coverage of the genome of that organism to try to predict the phenotype to provide therapeutic guidance? Can we learn more about the pathogenesis through virulence factors when we're focusing on those reads associated with the microorganism?

I think the other thing that I'm most excited about is looking at the host reads, and not only the pathogen but the microbiome at sites of infection as we start to expand our sources. The host response can give us information about whether what we're detecting from a microbial etiology, whether the host has a response to it or not, and really is it pathogenic or not? But it could also tell us, “Hey, no. There might be a bystander here, but it's autoimmune related based off of studying the host response.” So we could say a lot more about what's going on in this patient by also looking at the host response.

And then from the microbiome, which is your standard composition of organisms at various sources in your body, here might be a composition of that microbiome that might be more protective or permissive of infection, and we can be utilizing some of that information to understand the evolution or the development of infections at that site. I think there's a lot that we can just do even with the current data being currently ignored. There's so much more potential to that data. And then of course, right now, a lot of our methods from the sequencing standpoint are a little bit slower. There are novel sequencing technologies that are coming that are a lot more rapid, the library preparation is a lot quicker. The sequencing, the analysis can be done in real time. Right now our sequencing, we need to analyze it after the run is complete after 24 hours of sequencing. But there's now new capabilities to kind of analyze that data in real time that can help us reduce the turnaround time for these metagenomic-based assays.

Bobbi Pritt, M.D. (24:57):

Yeah, it's really exciting. It's an exciting field. And who knows, in the future when this becomes faster and cheaper, it could even be a frontline diagnostic. So definitely something to keep an eye on. Well, Trish, thank you so much for joining us. It was such a pleasure to have you on today. And again, welcome back to Mayo.

Trish Simner, Ph.D. (25:15):

Thanks so much, Bobbi. I'm excited to be back.

Bobbi Pritt, M.D. (25:22):

So, let's wrap up with the top takeaways and how to learn even more about the topics we've discussed in our news summary. Dr. Morice shared the latest with PAMA implementation and the RESULTS Act. You can learn more about the RESULTS Act in our October episode that is included in the show notes. And we will also provide updates in future episodes and your professional organizations can be a great resource. So, check those out. And then in our deep dive summary, my colleague Trish Simner, joined us for a deep dive into metagenomic testing and shared where the future of this type of testing is headed. If you want to dive deeper into this topic, there's a link in the show notes to several resources, including a testing algorithm, available tests and articles to learn more. Thank you for joining us today. Our next episode will feature Dr. Chris Garcia. He is the medical director of our biopharma group, and we'll be discussing how the intersection of biopharma and lab medicine is driving advances in healthcare. I hope you'll join us.