Amie: Me too. There is so much I wanna ask, and I know, as a clinician who works in this space, and all of those that are listening, I feel like microbiome testing is still a landscape and territory that people are exploring with excitement, but also it feels a bit like uncharted territory at times, and so really gaining knowledge and confidence in this area is an ever-emerging space, because there is so much to know, and of course there’s far too much to know that we could ever cover in a single podcast, but what I wanna talk with you today about is some of those areas that I think practitioners get a bit tripped up on, and perhaps because this isn’t something that was in the naturopathic curriculum at the time when we trained, it’s really one of those postgraduate areas that we’re all, I guess, catching up in. And I suppose where I’d like to start is looking at the various methodologies that can be applied when it comes to assessing what’s going on in the microbiome, and what the key differences are. Can you start there for us?

Tom: Absolutely, yeah. We do get a lot of questions about that as well. So, it turns out, when practitioners are considering what sort of methods, what sort of tests may be best for assessing the microbiome and gut health, we know that there are several options out there, of course. But I think one of the starting points for practitioners to be aware of is it’s really about comparing apples and oranges. We’re not always comparing apples to apples, so it’s not sort of…there’s a lot of questions about, you’ll see these in social media, which gut tests, which microbiome tests do you like the best, which do you think is the best for clinical practice? And so, it is important to understand that they’re really apples-to-oranges comparisons. And so we wanna look at what are those differences among the tests, and there’s quite a few. So, a lot of it boils down to methodology. So, when it comes to the methodology, really, the two main, current methods that are out there, these are both DNA-based methods. One of them is referred to as metagenomic sequencing. One of them is referred to as PCR, in particular something called quantitative PCR.

So, I’ll start with metagenomic sequencing. So, that’s a method that’s commonly used currently in research. So, a lot of the research scientists who are studying the microbiome like to use metagenomic sequencing because they can get this big-picture view of the microbiome. They’re studying the community. There’s a lot of research questions that you can answer with that. Also, there’s a lot of data, so it’s certainly a big deal to try to figure out how to analyze all that data. Oftentimes, it’s sort of in the context, for example, of understanding what patterns of microbes may be related to a given disease or given environmental change, or even dietary factors. But when you really look at that pipeline, so research, I think we all know, when it comes to the point of research, often it is for clinical applications. So, there’s the idea of basic research, and sort of that preclinical research. Then there’s clinical research, for the development of diagnostic type tests. So, that’s really one of the big differences, is metagenomic sequencing was really developed for research purposes, because there’s a lot of data to deal with. A lot of the microbes that come up on metagenomic sequencing, you’ll see these lists of hundreds of microbes in the microbiome. We don’t really know what most of them do. They haven’t really been well-studied. And that’s really important when it comes to clinical interpretation. When you’re looking at microbes that can inform clinical decisions, you wanna know something about those microbes. You wanna know what they’re linked to. You wanna know something about what you can do about them, what sorts of things they’re doing in the gut.

So, oftentimes, with that metagenomic sequencing and research, the ultimate goal is to identify a small subset that can be used in a clinical type test. And those clinical tests will often use PCR. So, it’s really more of a diagnostic type test when you’re talking about PCR. Of course, that was something that I think became better now during the COVID epidemic, pandemic, where suddenly a lot of clinicians became much more informed about testing and the types of testing for COVID. The gold standard for detecting these types of pathogens is quantitative PCR. So, that’s really one of the main differences there. And so, again, you’re casting a wide net with metagenomic sequencing. You can look at things like diversity, and sort of all the different microbes that are there, up to a point. One of the other advantages, though, of quantitative PCR, other than it allows you to focus in on the microbes that are most clinically relevant, based on the research, often using metagenomic sequencing, is there’s really kind of a long list of advantages. One of them is rapid turnaround time. So, metagenomic sequencing, often there’s several weeks before you can get the results. But oftentimes, you want those results quicker, to be able to make decisions for patients.

The other things come down to really, a lot of it has to do with specificity and sensitivity. So, when it comes to specificity, metagenomic sequencing is really good at identifying kind of the big picture. But when it comes down to specifics, specific pathogens, for example, specific opportunists, it’s not always as good at doing that, for several reasons. One is, a lot of these important microbes that we think of as pathogens and opportunists are low-abundance. So, they can be present at amounts in the microbiome that are far lower than the typical commensal microbes. And yet they can still cause a lot of problems, even though they may be present at a million times lower in concentration, because they can do kind of a lot of damage in the gut. That’s where qPCR shines. It was specifically developed to be able to zero in on these particular pathogens and opportunists, and other important microbes, so you can kind of identify those needles in the haystack. So, that’s how you can really think of this quantitative PCR. You’re not really looking at the whole haystack. You’re trying to identify, really precisely and quantitatively, these needles in the haystack.

So, I’ll kind of turn it back to you, because there’s a lot of subtopics we can talk to, or talk about here, in terms of the differences, but a lot of it has to do with clinical and diagnostic versus more sort of research-focused. And then also the specifics of really carefully quantitating and identifying the microbes that you’re looking for. We can certainly get into some of the details about methodology, and why there are these differences, but that’s kind of a little bit of the high level, in terms of the differences.

Amie: Yeah, thank you for clarifying that, because I think there are so many different ways to approach any problem or assess data, and there’s always limitations, and pros and cons. And I suppose if I was to summarize what you just shared, then, is that metagenomic sequencing provides just a huge volume of data, but at this stage, based on what we know as far as clinical application of that data goes, it’s quite a small fraction of that. And thereby, quantitative PCR testing is allowing us to cut straight to the chase for the things that we need, in a much quicker timeframe, but also focusing on those things that are clinically relevant, based on the scientific literature that we have today. And I suppose that, really, and you can correct me if there’s more to this, but answers the question around which method is most accurate. And I’m sure it’s a little bit more nuanced than what I just said, but it’s not that either method is inaccurate, but there is a difference in application, and the way that we pull that information together, and why. And if we’re considering what’s more relevant to clinicians, at this stage, I can see how PCR testing is going to give us the quickest access to the data that’s most clinically relevant. But in terms of microbiome functions, whether it’s LPS production or something else, is there anything further we need to understand, either around the accuracy of the testing, or which methods are more important to understand the functional aspects of what’s going on?

Tom: Absolutely. Yeah. So, there’s different ways you can look at that, and that’s actually one of the sort of points that often the metagenomic sequencing type companies tend to promote, is that they can look at a range of different functions. Technically, you can do the same thing with PCR, as long as you target those functions. You can do it at the species level, and look at specific butyrate-producing bacteria, or LPS-producing bacteria. I think the key there is there are different ways of looking at functions. You can group microbes by their functionality. And you can look at butyrate-producing bacteria, LPS-producing bacteria. Ultimately, though, you wanna know a lot more about that context. So, context is important. So, if you’re just, say, doing a metagenomic sequencing test, and they’re giving you sort of a score for LPS production, that doesn’t really tell you some of the information you’d wanna know. That can still be useful, but you wanna know who’s producing that. Because, for example, LPS can be produced by microbes that are commensals. E. coli is one of the sort of best-known commensals that produces LPS. Commensal E. coli is actually generally harmless. It can be overgrown in certain scenarios, but it’s not the same thing as detecting, say, salmonella. Salmonella is closely related. It also produces this inflammatory-type LPS. It’s very similar to E. coli, but you wanna know, is it salmonella or is it E. coli? And again, with qPCR, you can be much more confident, because salmonella is present, usually at low levels. qPCR is able to detect down to 10 to 100 cells per gram of stool. And that’s out of, on average, about 100 billion microbes per gram of stool. So, again, it’s really the needle in the haystack.

Sequencing is not able, generally, the type of sequencing that’s done commercially, sort of these direct-to-consumer-type tests, do what’s called shallow sequencing. So, you’re basically only able to look at the more abundant microbes. So, important pathogens, like salmonella, would not generally be detected as reliably, or not detected at all. So, again, when it comes to functions, you can look at functions either way. You can look at it based on groupings, based on genes. Ultimately, though, you wanna know who is involved in that function, and what is the context.

Amie: I think that’s such an important point. And I suppose that really establishes why quantitative PCR is so important in terms of specificity, as well as the actual quantity, because lipopolysaccharides, yes, they can generate inflammatory responses in the gut wall, and of course, systemically from there. But knowing that they’re present doesn’t give you much direction around what needs to be done about it, because the distinction between commensals that might be producing them, versus pathogens, is a big one. And also, the volume of that, you know. Are they present? Sure. But in what quantity, and is this a quantity we need to do something about? So, I think, from a clinical perspective, that really can guide a practitioner really directly, in terms of what needs to be done, if anything needs to be done. But I guess one of the other challenges, then, that can come up with quantitative PCR is, because it is so sensitive, we also can get a report that highlights low-level pathogens that might otherwise be missed. And I would love to hear from you about the clinical significance of these kinds of results that come through.

Tom: We get that question quite a bit as well. As you can imagine, because of the sensitivity, we do pick up low levels of opportunists and pathogens that often raises questions. And that, of course, a lot of that has to do with just knowing how to interpret the test properly, so education is a big piece, part of that solution. When you’re looking at the presence of, say, a pathogen… So, there are differences in terms of levels, of course, quantitative levels. And that’s really, again, where PCR shines. We do wanna know, is this just sort of a very small amount? You might have just had a minor exposure. And then the patient isn’t necessarily symptomatic. So, symptoms are obviously very important to assess in terms of the relevance. Once again, it does come down to context. So, when it comes to pathogens, we know that their presence sometimes can be indicative of some of those protective factors being less robust than they should be, even though they may not be developing the clinical signs and symptoms of a full-blown infection, and so therefore may not necessarily need to be treated for that pathogen. We still know that if it’s present, for example, stomach acid might be insufficient. Or the protective commensal microbiome might be insufficient. So it’s still kind of a clue for us to look for these other imbalances, and ask the question, well, why is this pathogen there in the first place?

So that’s one of the reasons why they’re still reported, even though they may not always be at a level that’s causing symptoms. And I think that’s a concept that really kind of gets misinterpreted. There’s a lot of misconceptions around there. You’re gonna hear practitioners refer to this term “false positives.” I think some of that sort of misunderstanding comes around this idea that, oh, I see salmonella, or I see C. difficile on the test. My patient doesn’t have those symptoms at all. So, what does that mean? Is that a false positive? In most cases, of course, the vast majority of cases, that’s not the case. PCR, again, is considered very specific, very sensitive. It can pick these up. But it does mean, in most cases, that it’s there, but it’s likely to be an asymptomatic infection. And that’s an area that’s becoming better-studied, now that we have these more sensitive techniques like PCR, quantitative PCR. So we know that many of these pathogens we can get exposed to, and some individuals may go on to develop an infection, if their immune system is compromised, if they don’t have a good amount of commensals, etc. So, once again, there’s these individual differences, and it’s good to be aware of those differences, and that that influences how you interpret the results there. We even see a few cases where patients might have higher levels of pathogens, and yet they’re still asymptomatic. So that would suggest, and oftentimes in those cases, we’ll see that their commensal microbes are actually at a really good level. And so, presumably, and based on the research, their microbiome is able to basically kind of keep those pathogens in check, which is one of their important roles in the gut.

Amie: Thank you for sharing that, because I think, as we develop more sensitive testing across the board, in medicine, and we also learn more and more about the interface between the innate and the adaptive immune system, we’re really seeing the meeting of our innate immune system’s ability to protect against infection. And when it comes to pathogens, for example, it’s not a binary situation, where you either have an infection or you don’t, meaning there’s an absence of those microbes. It’s that we have a whole ecosystem that works together. Using an example with skin, you can pick up staph on, let’s say, a sample of 100, but only maybe 20% have an actual staph infection, because their hydrolipid film, their antimicrobial peptides that are produced at the skin, and their commensal skin flora are staying on top of the other things that could otherwise become pathogenic in the event the immune system goes down. And I think when we’re looking at the gut, that the sheer volume of what comes through there, in terms of antigenic material, we’ve got secretory IgA, which is part of the mucosal glycocalyx, plus the commensals, and those other things, like stomach acid production, bile acid production. So, what we’re really seeing with these really sensitive tests is we’re all of a sudden lifted the lid on what else is in there, on a more, you know, a smaller scale. But that doesn’t necessarily mean there is an infection, or even that there’s necessarily been a past infection, or maybe it could be. But what we’re really identifying is, or really witnessing, is the innate immune system at work, and managing the presence of those things. And is that clinically relevant or is it not? That’s gonna depend on the case history, so many other factors. But in terms of that coming up, probably the question that I’ve seen come up the most is around Helicobacter pylori. And I think that’s because it’s one of those pathogens that can cause pretty serious symptoms when there is an infection present. If an infection is present, usually treatment is pretty rough, and has some collateral damage around the gastrointestinal system. And I think a question on some practitioners’ minds are, like, when it pops up on a PCR, in this case with the GI-MAP, where do you make the distinction around what needs to be done? Is it a collation of symptoms, history? Are there any other variables we have to take into consideration in addition to those?

Tom: So, you definitely hit the nail on the head, that context is always really important. So, it does come down to clinical judgment, but also, we provide the additional information for a reason. So, of course, the quantitative amount of H. pylori does make a difference. But keep in mind that these are not sort of, as you kind of alluded to, the cutoffs, that it’s either positive or negative in some of the older tests. It’s not like you meet some magical threshold level, and suddenly it’s a problem. Right? So, for some individuals just below that threshold, it could still be a problem. For other individuals who have a healthier gut, they can probably tolerate higher levels, and it’s really not as much of a problem for them. But it’s still a good guide overall. I mean, you wanna know, overall, especially if you’re treating, or you’re doing something with the gut, and you’re comparing it before and after, if you see that it’s down, say, tenfold, then that means it might still be present, because a lot of people still have low-level H. pylori, that may not be a problem, but that tells you that your protocol is certainly going in the right direction.

There is, of course, a correlation with symptoms. So, you alluded to these more serious symptoms. That gets into sort of more of the conventional medical sort of view of H. pylori as a cause of ulcers, and can even cause stomach cancer. So, certainly very serious outcomes for some individuals. The virulence factors certainly play a key role there. They’re considered risk factors. So, like any risk factor, if something’s present, that could be potentially serious, it does not necessarily mean you’re gonna get that condition. But it’s a piece of information for the clinician that says you might wanna weigh this a little bit more. If you’re kind of on the fence about “should I use antibiotics, should I just treat with an herbal protocol, or even not treat?” there’s not a lot of symptoms, presence of virulence factors can basically weigh in on that decision.

And there’s also, in our field, functional integrative medicine. Of course, we look at things in a different way beyond just kind of the conventional medicine scenario, where, okay, so the patient doesn’t have ulcers, doesn’t necessarily have a family history risk for cancer. Is there a problem here with this H. pylori if it’s still high? We do know that H. pylori, for example, can affect stomach acid. And that may not be something that in the conventional medicine field they’re too concerned about. But certainly in our field, we know that optimizing digestion certainly can be one of the goals when you’re working with patients. So, some practitioners may decide to treat, usually typically more with herbal treatments, in those scenarios where you might be more concerned about some of these less-serious scenarios, but scenarios that might still be affecting patient health, like hypochlorhydria.

Amie: Yeah, thank you for pointing that out. Because I think this is where applied functional medicine is, where it really steps into the into the fore. And when we sort of zoom out and look at germ theory versus terrain theory, and how those two meet, when it comes to assessing patients, for someone who has low-level stress, who’s asymptomatic, who appears to be producing…you know, doesn’t have any digestive issues whatsoever, that doesn’t mean it’s necessarily something we need to treat. But if another patient had the exact same test result, but we can see their ALP is low, which means their zinc’s down, which means their stomach acid isn’t optimal, you know, their protein and creatinine scores are down, we’re seeing signs of malabsorption or elevated muscle turnover. And any of those secondary signs that are indicative either, you know, catabolism issues, stress issues, adrenal, you know, poor sleep, maybe co-infections of other sorts that are obvious, then, to me, that would say the weight of, like, the pathogenic information is, this person’s overall system, innate immune system, isn’t particularly strong. They’ve already become weakened in other areas, and therefore, this potential pathogen is more of a threat than it is for someone whose terrain is very strong.

Tom: Exactly. Yeah, and I actually wanted to expand upon that just a little bit, in terms of the terrain. I think that’s a great term overall. In the research, there’s just sort of this growing focus, not just on the microbiome. In the early years, that was the focus, using metagenomic sequencing and other ways, to kind of characterize the microbes that are there. And then they would see there are these differences in different diseases and conditions. And then of course, eventually, the question is, why is that the case? So, looking at the gut environment is really important. It’s not just a one-way effect, where the microbiome affects gut physiology. Certainly it does. But the physiology in turn affects the microbiome. And so that’s another key piece of the puzzle that you really wanna have in a clinical test, when you’re looking at gut health and the microbiome. To the point where, I think it was just three or four years ago, in the research community, they actually redefine the term “microbiome” to not just include the microbes in cells, which we refer to as the microbiota, but to also include gut physiology, the environment. That that’s equally important because the two are sort of integral. So, you mentioned the immune system. We certainly know that inflammatory microbes, pathogens, LPS-producing microbes, can influence inflammation. But in turn, one of the reasons they do that is inflammation actually creates a favorable environment for those pathogens and opportunists, and it’s also detrimental to the commensals. So it’s sort of like they’re kind of tipping the balance in their favor by promoting inflammation. Sometimes you can have inflammation, of course, starting for other reasons. We even know that actually, antibiotics, under certain circumstances, can start that process of low-grade inflammation. Gut infections, poor diet, stress, to some extent. So if you’re looking for these root causes, what might be influencing microbes, and why you have these imbalances, it’s really essential to have access to that information on digestion, immune function, intestinal barrier health, etc. That’s incredibly important for interpretation.

Amie: Yes, it’s a, as you say, context is everything. And you can’t just look at the microbiota out of context with all of the other elements of the physiology that make up a good gut. And, again, zooming out the elements of physiology in a human that sets up gut health for better or for worse, and I think that’s one of the challenges of applied functional medicine, is you have to take into consideration all of those moving parts and how they fit together. And ultimately, I think this brings us back to that naturopathic principle of really treating the human in front of you, and not just the data that you’re looking at. You have to find a match in terms of appropriate treatment response. But, because we are now in a day and age of more data, and I think it’s very sexy and very exciting and very interesting, I think another thing that is challenging, that I’d love to explore a little more with you, is the impact of live organisms versus dead organisms, and also what’s being picked up as far as genetic material in quantitative PCR or metagenomic sequencing. And when I think about this from a clinical point of view, I understand that even dead microbes, their DNA, and their pathogen-associated molecular patterns, or PAMPs or DAMPs, can still trigger toll-like receptor activity. But again, in the context of the physiology, where is the weight sitting in terms of disease versus something that we don’t have to worry about so much? But I think, now that we have access to these more sensitive tests in a clinical setting, they’ve left the research, academic labs, and arrived in our clinics, when we look at these DNA methods, where is the line in terms of what it’s picking up, of organisms alive versus dead, and what does that mean clinically in terms of infection indications?

Tom: It’s really an interesting topic. We got this question pretty frequently when I first started with Diagnostic Solutions a while back, because there’s this idea that, just as you mentioned, DNA can be released by dead microbial cells, and do these methods detect that, and is that still clinically relevant, to detect dead microbes? So, one of the overall considerations is, once again, you have to look at the context. So, the short answer is yes. DNA methods, whether it’s sequencing or PCR, can pick up DNA as long as it’s still intact, right? So, you need a sufficiently intact amount of DNA. If it’s all kind of degraded, and the pieces are too small, then neither of those methods are gonna work to reflect that. So you’d have to have fairly intact DNA present. That’s really one of the keys is, is there intact DNA present? Some estimates suggest that when you’re sampling stool with DNA methods, again, either sequencing or PCR, somewhere in the range of 20% to 30% is from microbes that are basically no longer intact. The question is, though, is that from past infections, or is that just because microbes died an hour before? Right? Because microbes have this very rapid lifestyle, and that’s likely still relevant if they died just a few minutes or a few hours before.

But mostly what you wanna think of, because we hear this all the time, I think people have this concept that we’re thinking more kinda, or confusing the concept with antibodies. Antibodies that are detected in your blood or your serum, to pathogens, those can reflect past infections. And that’s kinda the definition of, you know, kind of a principle for how vaccines work, etc. But, ultimately, when you’re thinking of the gut, the material in the gut, of course, is passing through. The vast majority of, even microbes, we lose a pretty large percentage of microbes every day, just through your normal bowel habits. And of course, that’s kind of continually moving through the GI tract. And an average transit time, I know it varies quite a bit between people who have fast or slow transit, but the rough average is somewhere around 28 hours. So, at the very least, for the most part, you’re not gonna be picking up any microbes that are weeks or months old, because they’ve already been flushed out of the system. There’s this idea that maybe they’re hanging out in biofilms. If they’re actually in biofilms, almost by definition, they’re still active, because they have to be able to form the biofilm. They have to be able to still be in the biofilm and stay alive, because they’re kinda just protecting themselves temporarily.

So, I don’t wanna get too in the weeds here, but it’s important to think of microbes in the gut as live, dormant, and dead. So, dormant’s kind of a category we don’t hear too much about, but a lot of the microbes actually are thought to be present in dormant or semi-dormant states, because they’re only gonna grow when their particular food is available. And of course, generally, we’re not eating 24/7, right? So we’re gonna have a period of fasting, where the microbes don’t have access to their favorite food sources, for example. The ones that thrive on mucus, they’re probably gonna be a little bit more steady. But the question is, is, you know, once again, it comes down to clinical relevance. So, for example, when you compare DNA methods to, you may have heard the term metatranscriptome. These are expression-based type tests. Those are only gonna detect microbes that are essentially active at the time that the microbes were sampled, because you have to be active in a live cell to be able to express your genes. Whereas DNA, again, could be around for a while, at least a few hours or so.

But the idea there, though, is that you can detect with DNA methods microbes that may be dormant in stool, but were clinically relevant when they were in the small intestine or in the stomach. So, that’s essentially the principle, for example, behind detection of H. pylori. So, without getting in, again, in too much detail, we know that generally, H. pylori present in stool is dormant. It’s present in a different form of cell, that’s still alive, but not active like it would be in the stomach, because once it’s out of the stomach, that’s just not its favorite environment anymore. But it can stay alive. So, long story short is a lot of these clinically relevant microbes, like H. pylori, Giardia…we know Giardia causes problems in the small intestine, and yet we pick it up in stool. So, If you were just to go with one of these metatranscriptomic tests, chances are you wouldn’t see those at all because they’re no longer really very active. With DNA, you can pick them up, coming from these higher places, higher up in the GI tract. So, I think it’s important to distinguish that temporally, where something coming from the stomach, like H. pylori, that, again, takes several hours to get down into the stool, but it’s not from a past infection that’s once in the past. So, that was a lot of information there, but it’s not as simple of a topic as you might initially think. So, it’s important, I think, to have some of that context around it.

Amie: Yes. I think so. And I think that is something we really need to be aware of as clinicians, is the limitations and the context for pathology testing, and that is, in the case of metatranscriptomic testing, if you’re actually taking a sample directly from the stomach, or a sample directly from the small intestine, and you’re looking for microbes that are actually active in those areas, then it becomes more clinically relevant. But if what you’re doing is sampling a stool that has been through an entire life cycle of roughly 28 hours, or whatever it is, and has gone through pH changes, and undergone various different mechanisms of digestion, you have to view the data that you’re getting in the context of that. And again, it’s not this binary conversation that you either have an infection or you don’t, or the microbes are active or they’re dead. You have this dormant state, when they are not in the environment in which they actually come alive. There’s also, in terms of the volume, there’s a threshold after which quorum sensing switches on, and pathogens can actually become pathogenic, as opposed to just the little guys that are hanging out, not really bothering anybody.

Tom: Right.

Amie: And I think, yeah, whenever we’re looking at this data, we’ve got to look at it in the context of all of those aspects. So, thank you for sharing that, because I do think there is, you know, some people might view dead DNA, first of all, not to have any effect on the immune system, which of course it can, but it also doesn’t mean that it’s an old infection, or is irrelevant to the situation, because the bulk of the stool is microbes, and it’s being sheared off the gut wall within the last 28 hours or so, give or take, which means it’s still contextually relevant to today, or the last few weeks, not something that happened years ago, that’s still kicking around. I think the only exception to that might be where someone has done a stool test post a massive bowel and gastrointestinal protocol, that’s included shearing off plaques and old material that maybe has collected there, but typically, when clinicians are running a GI-MAP, it’s after an initial presentation, and typically before a protocol. It’s being used to guide a protocol. So, in the way in which it’s administered, typically, that information can be considered current, rather than a reflection of something old that’s been sitting around in the gut. But I think what that leads me to, then, is the next question around how it can be used to diagnose things. And you’ve given us a really good explanation around how to filter the information that we’re given, regarding Helicobacter pylori, but I know that SIBO is another big thing that comes up. And, from a clinical point of view, where do you see the GI-MAP informing what we know about SIBO?

Tom: Actually, just before we dive into that, though, because to me, that’s a very interesting question. We get that one, probably it’s in our top three, four questions we get. One of the things I wanted to add a little bit, though, about the DNA thing, because it is fairly nuanced, is this idea of biofilms or plaques in the gut. Certainly, plaques, biofilms, they might be the same concept, but it’s actually known that biofilm is dynamic, right? So, biofilm itself is constantly remodeled by the organism, so even, you might think biofilm is this thing that’s just sitting there. And there might be certain organisms that are maintaining that biofilm. So if you were to basically detect that in stool, or sample it directly over time, you still see those same microbes, but they’re dividing and changing over time, the biofilm’s changing. And they’re attached, typically, to the mucus or the underlying epithelium. That’s constantly turning over. So, what we think of, and it seems to be kind of this constant thing, “Oh, you have biofilm, it’s been there for quite a while,” it actually is very dynamic, so I just wanted to kind of make that point that, once again, what we’re looking at in stool is still not really reflecting the past, very far back, because even biofilms are constantly changing, so that it’s…

Amie: That’s a good point.

Tom: Yeah, that’s something I think that’s just come out of research in the last few years, and it takes things a while to kind of filter down, but we could probably have a whole podcast just on that whole topic, biofilms.

Amie: Yes.

Tom: But as far as this question we get a lot, can you use stool testing, and a particular GI-MAP, to “diagnose SIBO,” or even to get some clues, does the patient maybe have SIBO? So, in terms of certainly diagnostic, there are definitions out there for how to diagnose SIBO, based on either taking a sample directly from the small intestine, and then using culture, and then essentially counting the colonies on the culture, to get a colony-forming unit, or CFU count. So, there are definitions there. But the most common, of course, is breath testing. So, breath testing is certainly different from stool testing, and that’s considered the accepted way, or at least the most common way, to assess SIBO. But keep in mind, though, that that definition there is based on gases, when it comes to the breath testing. So, it’s based on microbes that produce gas.

When you look at GI-MAP, what we just talked about, that we can get, detect DNA from microbes that are coming from higher up in the GI tract. So it’s giving us clinical insights into some of these key microbes that we know from research live or cause problems in different parts of the GI tract. And that’s certainly true for the small intestine. So, we talked about Giardia. Giardia is well-known to primarily infect the small intestine, Cryptosporidium as well. And a number of the opportunistic microbes tend to overgrow in the small intestine. Now, they’re not necessarily synonymous with “SIBO,” again, because SIBO’s based on gas-producing microbes. We know that most of those opportunists on GI-MAP either don’t produce those types of gases, they’re not hydrogen producers, they’re not hydrogen sulfide producers, methane producers, or they don’t produce sufficient amounts, because they’re present at really low levels. And yet we know they’re clinically significant. So, the short answer to this question is no, it’s not technically SIBO, but you’re looking at it, the small intestine, potentially in a different way. We would call that potential small intestinal dysbiosis. And it depends on the microbes and sort of what’s known about them from research, and where they live. Pseudomonas is another classic example. Research shows, in multiple studies, that that’s largely a resident of the stomach and small intestine, not really a resident of the lower GI tract. We know from research that that’s linked to everything from IBS to food sensitivities.

So, we’re getting a lot of insights. And then on top of those microbes, we’re also getting insights into digestion, which of course takes place in the small intestine. We’re getting some insights into food reactions, based on the anti-gliadin marker, and also the eosinophil protein X, which is the eosinophil activation protein. Those tend to be, those immune responses to foods, tend to happen primarily in the small intestine. So, again, we’re getting some additional insights that is very different from what you would get from a breath test. When you’re looking at testing, there are cases where patient might have some issues in the small intestine, but it’s not SIBO. And yet we can see things on GI-MAP, frequently, that could still indicate problems in the small intestine, but it’s just not technically SIBO.

Amie: So, in terms of when we’re looking at that information that comes through, we’re getting a much broader range of information that tells us what’s happening in the small intestine, or potentially. And certainly we can see patterns that could indicate SIBO, by its classic definition, and also other indications of dysbiosis in the small intestine that aren’t “SIBO,” in air quotes. And I suppose, clinically speaking, it would allow us to perhaps see, okay, there’s a lot of other activity going on in the small intestine that could actually be contributing to these symptoms, SIBO-type symptoms, or, actually, there’s some red flags here that this might actually be SIBO in its purest definition, and therefore the next step would be to look at breath testing, to confirm that that’s the case. Is that how you would approach it?

Tom: Yeah. And there’s also just different scenarios. So, we often will get cases where patients had kind of gone down the SIBO path. They had symptoms that the practitioner kind of assumed were SIBO. They went and did breath testing, and confirmed from that cutoff that they had SIBO, treated. And either the SIBO symptoms came back, or they never really responded that well in the first place, so then, often, they’ll follow up with a test like GI-MAP, to see, well, what else might be going on? There’s a lot of things that can go on. I would say one of the most common things we see is high H. pylori. So, of course, SIBO doesn’t really take into account potential H. pylori. We see candida is pretty common in those scenarios. So, you’re getting a lot of these other pieces of information that can lead to symptoms that seem like they might be SIBO. And if you think about some of those symptoms, like bloating. Bloating is such a common symptom. Most infections, parasite infections, candida, etc., can also cause bloating. So you really want wanna, a lot of people will start with something like GI-MAP, to get kind of that broader picture, and get a differential diagnosis sort of take on things.

One of the other things that should be mentioned is, you may be aware there’s some critiques about breath testing. Certainly, there has been some reviews by prominent IBS researchers about breath testing and some of the drawbacks. One of them is the potential for false positives, due to rapid transit of the sugars that are used in testing to the colon. So you get this early peak, but it’s actually not taking place in the small intestine for some people. If they have rapid transit, then that can be, basically, the early peak is happening in the colon. There’s a lot of research on the gas producers in the colon, and those are on GI-MAP. So, if you wanna know about the hydrogen producers that are in the colon? So, again, that could be one of the reasons why some patients that were thought to have SIBO [inaudible 00:47:11] go down that path of treating SIBO, maybe didn’t get the response that you were looking for. In some cases, it might just be that that actually was happening in the colon. And we can get great insights into methane producers in the colon, hydrogen producers in the colon, and then hydrogen sulfide producers as well.

Amie: I think that’s so helpful to know. I can see how it would be so useful to do a GI-MAP first, and then perhaps add in those additional tests. But I think, conversely, for clinicians who’ve gone, “Oh, this is absolutely SIBO,” and then they run a breath test, and then that doesn’t come up, then, where to from there? And I think that’s where the GI-MAP can fill in all the rest of those gaps, because it’s such a broad range of information that you get. And not just on the microbial side, but also the immune function, and some of the terrain in terms of secretory IgA as well. So, I guess the short answer is it can’t be used to diagnose SIBO per se, but it can absolutely give us some wonderful insights into the small intestine, including microbial patterns that could point to SIBO and would justify ordering a SIBO test, and also can save us from ordering a SIBO test unnecessarily, when we can see other microbes that are actually contributing to that bloating and those SIBO-type presenting symptoms, which I think is just so helpful, especially when we, again, zoom out and consider how are we treating the whole patient. We always have to think of whether or not they have SIBO or dysbiosis of some other type. How did they get there? Is it an innate immune system issue? Does that stem back to chronic stress, which reduces blood flow to the gut, which then set this up in the first place? Was it a course or multiple courses of antibiotics? Whatever the case may be, is the secretory IgA still down? Well, you can treat antimicrobially all you want, but if you don’t bring that back up, you’ll just see relapsing infections. So, it does, I think, give us a much broader ability to approach things when we can see all of the pieces on the table.

Tom: Exactly. And I would add to that list, probably at the top, would be digestion. So, whether it’s… Of course, GI-MAP doesn’t have, there really aren’t any gut tests that have direct indicators of stomach acid. You have to triage that, and get a picture together to see, of course, based on symptoms as well. With GI-MAP, we know we talked about H. pylori is a potential cause of low stomach acid. There are low stomach acid-related patterns, very well-known and defined in the research, including high levels of Streptococcus. Also high levels of staphylococcus, Enterococcus, those types of overgrowth microbes, that are in that section on opportunists, those are fairly closely related to reduced digestion. So that can be a sign that maybe you wanna look at digestion. But then, of course, we have elastase, which is a direct marker for pancreatic enzyme production. And I would say that’s one of the most commonly deficient markers in patients that have gone down this SIBO path. So, and we get this repopulation of patients in our consults, that have been down that SIBO path, and you’re looking at GI-MAP. Again, the top ones tend to be H. pylori, Pseudomonas, staphylococcus, evidence of food reactions, and then poor digestion. So, just to your point, why not start there, and look at those? Because these are all different factors for many patients, that kind of covers the bases, and that can explain why they have those symptoms. And they may not need to go down the SIBO route.

Amie: Yes. You can start with improving digestion, and allow the innate immune system just to get back on top of what’s going on microbially. And I think, sometimes, going back to those basics, we overlook, in the name of fancy testing and fancy protocols. And certainly, one of the things that I find myself saying a lot in practice is “there are no teeth in the stomach.” If we’re gonna take it to the bare basics, people often don’t chew their food properly. They’re often eating standing up, or running out the door, or while they’re driving 100 miles an hour on the freeway. And there are just so many things that we’re doing that put extra pressure on our digestive system, that need to be addressed before we dive into a fancy protocol. But certainly, picking up those things, like elastase, as to how well our organs are currently doing their job, is so powerful, because then it also, I find, and it’s understandable that people are very data-driven. And I think telling a patient, “You need to do X, Y, and Z,” they can be a bit non-committal sometimes, or perhaps not consider some of those basic pieces of advice to be of particular importance. But when you can actually point to a marker, sort of say, “This is what that means,” it gives them a reason to be doing what you’re sharing with them also. And you’ve mentioned some really interesting markers already, and I wanted just to circle back to the eosinophil activation protein, because I think this is one of particular interest to me, because I’m in the environmental medicine space, and looking at some of those external drivers of eosinophil activation. But I’d love to hear from you, when a clinician’s looking at it on a GI-MAP, what is that informing them about, and what should they do about that?

Tom: That’s a great question. So, we do get a lot of questions about that. It’s not one of the markers that comes up frequently. It’s fortunately not that common. I would say maybe 10% of patients tend to have elevated, but it’s something that’s much more commonly elevated when patients have food reactions. So, there’s a lot of research on the role of eosinophils in food reactions, particularly food allergies, and also the interactions between eosinophils and mast cells. So, these are kind of both your key cells that are picking up on what’s going on in the mucosa. Are we being exposed to toxins? Are we being exposed to certain microbes, allergens, etc.? So, they’re kind of these sentinel cells in the gut. So, when it’s activated, of course, that’s telling you, by definition, that eosinophils are activated. Studies actually show that most eosinophils in the gut tend to be in the upper GI tract, particularly in the small intestine, to some extent in the stomach. We certainly know they’re not commonly in the esophagus, but they certainly can be in eosinophilic esophagitis. But they’re actually even just by default present in the small intestine. So, the key there is, are they activated or not, because they have normal functions in the gut, where they’re actually helping to maintain the intestinal barrier and immune balance. But once basically something triggers them, they detect something that they need to fight off, then they become activated. And so that’s really kind of your key marker to let us know that eosinophils are activated. It’s a different type of inflammation than the type of inflammation that’s reflected in calprotectin. So, that’s really one of the advantages of having more than one immune activation marker. Calprotectin is kind of more your classic inflammation, that’s involved in inflammatory bowel disease, infections in the gut, etc.

So, that’s really a little bit about eosinophil activation protein. And one of the things I think that’s not as well-recognized is how the gut sort of interacts as a whole. I know we kind of touched on this a little bit, sort of systems-level approach. So, just to kind of give you one really important example, of course, with GI-MAP, you can assess the commensals, some of those key commensals, like Akkermansia, Faecalibacterium. So, Faecalibacterium is one of those important butyrate producers. In fact, it’s significantly important enough that overall butyrate levels track the Faecalibacterium. Well, it turns out that butyrate is one of the factors that can help keep eosinophils, mast cells, and other immune cells from being overly activated. So, if you see that eosinophil activation protein high, patients having a lot of food reactions or other types of reactions, sometimes it’s things like mold, environmental exposures, one of the things you potentially can do to reduce that is work on the commensals, if you see that those are deficient.

Amie: Yeah, it’s a great point.

Tom: So, again, it’s hard to kind of put all those pieces together, but once you start to learn how to connect these dots, it gives you a lot more that you can act on. And you start to see how these things are connected, and gives you new avenues for basically helping to support patients.

Amie: I think that’s such a good distinction. And I think also, when we’re looking at that eosinophil activation, where you’re having food reactions, you can then also filter through, is the secretory IgA appropriate, and therefore mucosal biofilm okay? Or is it low, and therefore the gut wall is vulnerable and more reactive, and would perhaps settle down if we could raise that? Or is it highly elevated, meaning there’s probably some sort of infection that the body’s trying to respond to, that then’s having a domino effect into the rest of the body? And certainly, for me, I would also pop in there exposure to mycotoxins as well, when we see an increase in eosinophils as a potential issue. And the other thing I wanted to ask you about was occult blood, in terms of that either it’s present or it’s not, or looking at it through the lens of a more of a spectrum of a reference range, and what clinicians should be interpreting that data as?

Tom: That’s a great question. So, of course, with any reference range, the cutoff level is meant to indicate a level below which it’s less likely to be an issue, and a level above which, when it’s flagged as high, that it’s more likely to be an issue. Of course, on GI-MAP, that cutoff is right around 10. So, certainly it doesn’t necessarily mean that if you have a 9, there’s no problem at all, and if you have 11, it’s really bad. You have to take into account the context of the patient. A lot of practitioners do get concerned if they see any level of occult blood detected, because they feel that in a healthy gut, that shouldn’t really be the case. It’s more just to give you an indicator of, overall, based on the reference range and a lot of the scientific information that kind of goes into determining that, are the levels detected more likely to be benign sources, such as hemorrhoids, etc., or just minor inflammation? Or is it more likely to be something that you really wanna pay closer attention to? So, that’s the whole point of a reference range. If we just reported it as positive and negative, and you didn’t know that there was any number there at all below 10, which is considered the cutoff for high, you wouldn’t know to kind of pay attention to that, right? So a lot of practitioners see, say, a 5, something is, okay, I don’t need to necessarily act on this right away, unless the patient has symptoms that would cause her concern. But I wanna keep an eye on that, right? So I might wanna recheck that in a few weeks, a month or so, and just make sure that’s not increasing. [inaudible 00:59:19] similar to this idea of the low-level pathogens. You know, do you wanna know that they’re there or not? It sort of makes it simpler if you don’t report the low levels, because it’s like, okay, I don’t…a little bit of the ignorance-is-bliss thing. We don’t know it’s there, we’re not gonna worry about it. But having the information does tell you something. It’s telling you there might be something going on here, less likely to be a big concern, but do monitor it, just to make sure that it doesn’t become a big concern.

Amie: Yes. I think, you know, again, that this is the challenging aspect to more and more sensitive pathology testing, which we’re so grateful to have as practitioners, that it is, you know, by detecting, you know, more sensitive levels of things, it does then become a little murky. It’s no longer, you know, see this, do that, kind of situation. There is a bit of a gray area that people are operating in. And I think, just sort of going back to the Helicobacter pylori conversation again, I know we’ve touched on it a couple of times already, and gone into it fairly in depth, but just thinking out loud about what we were talking about to do with SIBO, and whether or not the information that comes through suggests, okay, it would be worthwhile investing in a SIBO test, based on what we’re seeing here. Where do you stand as far as the reference ranges for Helicobacter go, in terms of, I know we’ve discussed, there’s virulence factors to consider, and there’s also what is the overall volume. And of course, that’s always gonna be looked at through the lens of, is this patient exhibiting any symptoms that we know to be either directly or indirectly related to Helicobacter infection? But, in terms of, you know, when we see that come through on the GI-MAP, just like with SIBO, the GI-MAP is not a replacement for breath testing. GI-MAP is not a replacement for antigen testing or breath testing, or even biopsy with Helicobacter. How do you see those pieces fitting together clinically, for someone in practice?

Tom: I think it really depends on sort of, to some extent, what the purpose is for using the different tests. So, when it comes to a tool like GI-MAP, that covers a lot of bases, right, it depends on really what the symptoms are, and sort of what you’re looking for. But it’s a great screening tool. So, if you see that H. pylori is present, but it’s not high, you know, it’s maybe in that E2 range. So, it’s there, but it’s, patient may not have any symptoms consistent with H. pylori, some practitioners will just leave it at that, and they’ll focus on other imbalances. Others may decide, well, based on this patient’s presentation, you know, we’re seeing evidence of low stomach acid or something, that might cause them to decide to treat. The test itself, of course, doesn’t dictate whether [inaudible 01:02:15] practitioner treats or not. That’s clinical judgment. If it’s above that cutoff level, again, the cutoff levels are meant as a little bit of a red flag, to say, “Hey, this may be more significant. Pay a little more attention to it, because it could be a cause of the patient’s symptoms.” It’s not the test itself telling you, “Yes, the patient’s symptoms are definitely caused by H. pylori, because it’s above that number.” So, they’re really meant to be guidelines. They’re not meant to really be strict, “As soon as it’s above this level, you must do, X, Y, or Z.” That clinical judgment is really very important, and clinical judgment, of course, depends on a lot of factors, including how well-educated you are in interpreting the test. So that’s really a big focus area for Diagnostic Solutions Lab, is really to help educate practitioners, so you have the science-based background, the evidence-based background, to help you decide what you feel is best for your patients.

Amie: Yeah. And I think that perhaps addresses some of the concern around over-amplification and over-treatment. And I think with the amplification, that has more to do with, you know, the PCR methodology, but also whether having these things appear on the GI-MAP is going to result in over-treatment. And I think, you know, for better or for worse, it really comes back to the clinician being responsible for their own understanding and experience, and being able to use their clinical judgment when looking at data in the context of the patient in front of them, their symptomatology, what their other pathology is also telling them, in terms of what direction they’re going to go in next. Is that something that you agree with, or is there anything further to add there?

Tom: No, I think you definitely hit the nail on the head. And one of the other analogies I like, it’s not a perfect analogy, because… When it comes to blood sugar, for example. Imagine that you got a patient’s blood sugar result that just said, in range or out of range, right? You didn’t get a number. So, you might say, “Okay, it’s in range. We’re good to go.” Or “It’s out of range. We need to do something about this.” Having the actual number for blood sugar, it could be sort of high normal, and you know that, all right, we need to try to optimize this further. It could be normal, low normal, etc., or just a little bit out of range, versus if it’s, you know, a fasting blood sugar of 180, then you know that’s a pretty significant issue. So, I think that’s, in some ways, a good analogy, because having those numbers and having a reference range gives you an idea of sort of how you wanna approach it, how aggressively you wanna approach it, what’s the context, again, versus just sort of this positive, negative, it’s there, it’s not there.

Amie: Yes. Yeah, I think that’s so important. Again, it comes… It’s not that binary, you have it or you don’t. Or it’s a problem or it’s not. Where do you sit on the spectrum? And when you look at it in the context of symptomatology, and what else is going on, that we can see clinically and, you know, pathology-wise, putting those pieces together. Everything tells a story, doesn’t it? And can you interpret that story properly and translate that into what does this patient actually need to feel better and get better?

Yeah. Well, look, I know I’ve picked your brains an awful lot in this conversation, and I’m not gonna keep you for too much longer, but I am really curious. I know this is such an area, like, a passionate area of expertise for you, and given you are in this, working in this area of the microbiome and its connection to chronic disease and aging, are there any insights that you can give us, not necessarily in the context of what we can see on the GI-MAP already, but what’s sort of to come in the research and academic space around what the gut can inform us around aging and how well we’re going, or what we might need to do to age better?

Tom: That would be a whole additional webinar.

Amie: A whole other episode? Yeah.

Tom: [crosstalk 01:06:32] Yeah, I mean, that’s a really fascinating area of research. It’s kind of just at the early stages, so… I’m not sure if you’ve heard of this concept of hallmarks of aging. People who’re in the longevity field, that’s something that’s sort of a big focus area, kind of almost a framework. So, the latest iteration is that there are 12 hallmarks of aging that have been identified. That was updated from a version, say, 10 years ago. Dysbiosis is one of the new hallmarks that was added, so they’re now kind of recognizing this set of research over the last 10, 15 years, that certainly there are characteristic imbalances that happen with aging. And they are likely related to the other hallmarks of aging. So, one of the other newly-added hallmarks of aging is chronic inflammation, right? So, we know that’s a feature of most chronic inflammatory diseases, age-related diseases. It’s also a feature of aging, to the point where there’s this term, “inflammaging.” So, it turns out that there’s evidence that this dysbiosis that happens with aging, which is often characterized not so much differently from what we typically think of, so, a lack of the good guys, and then an increase in, “the bad guys,” the opportunists, is pretty common. Those sorts of things can start to drive chronic inflammation. So, there’s evidence that that may be one of the factors that’s sort of contributing to this age-related increase in inflammation.

But it kind of cuts across the board. I mean, we can get into all kinds of details, which I’m sure we don’t have time for now. But one of the ones that to me was fascinating, there’s kind of a subset of the longevity medicine field, and the sort of healthy longevity field, that has to do with this regenerative medicine concept. Right? So, that’s involved stem cells. We think of them in terms of stem cell injections and those sorts of approaches. It turns out that the microbiome, now, is known to play a role, certainly in the gut, in regeneration. So, the microbiome balance has a big influence on that epithelial turnover. Right? So, when we’re talking about gut healing, everybody thinks about leaky gut, intestinal permeability. They’re not so much thinking about this turnover of all the cells lining the gut, completely turning over every five days. That, to me, is really the essence of gut healing. You want that process to go well. And so, the microbiome influences that process. If you have dysbiosis, that process is not going well. And there’s a lot of research around that.

But even in terms of, you know, there’s more focus now on muscle health with aging, for example. In its relation to this muscle loss, called sarcopenia, it turns out that we talked about butyrate. Butyrate can help kind of tamp down on these inflammatory cells, and kind of influence that balance. That process is known, what happens in the gut influences this muscle regeneration process. So, in distant tissues now, we’re finding that the gut microbiome has these influences. So I think it’s, just fascinating to see these different concepts coming out of research for how just this simple concept of balance of your microbiome and gut health balance can influence our systemic health in so many different ways, including in aging.

Amie: I just think that’s so fascinating, and just brings us back to Hippocrates’ age-old quote, “All diseases begin in the gut.” And I think, yeah, the, you know, the information we get from the GI-MAP really can quite literally be the key to not just gut health, but for the rest of our body, the rest of our wellbeing, and for the rest of our life. It’s just fascinating.

Tom: Absolutely.

Amie: Well, I’d certainly love to have you back for another conversation on another time, Tom, but thank you so much for being so generous with your knowledge with us today, and just so appreciate you taking us through all things microbiome.

Tom: It’s been my great pleasure, so thank you so much for inviting me.

Amie: Thank you so much, Tom. And thank you, everyone, for joining us today. And remember, you can find all the show notes and other podcasts on the Designs for Health Australian website. I’m Amie Skilton, and this is “Wellness by Designs.”