GI-MAP Stool Test Interpretation

Dr. Hedberg: Well, welcome, everyone to Functional Medicine Research. I’m Dr. Hedberg, and I’m looking forward today to my conversation with Dr. Thomas Fabian. He is a PhD., and he’s a clinical laboratory consultant, translational science expert, functional nutrition practitioner, educator, and speaker. He is a former biomedical research scientist and deep expertise in the role of the human microbiome and health, chronic disease and aging. As a leading expert in translational applications of microbiome research and functional medicine and integrative health settings, Tom’s primary focus is on providing educational resources and consulting services for practitioners and scientific advisory and consulting services for clinical testing laboratories. Dr. Fabian, welcome to the show.

Dr. Fabian: Thanks so much, Nik. It’s great to be here today, and I’m looking forward to the conversation.

Dr. Hedberg: Excellent. So, we’re gonna be talking about the diagnostic solutions, lab, GI-MAP test, and we’re gonna cover interpretation, you know, what these markers mean. And so, for all the practitioners listening, they’ll have a strong idea of how to approach this test and how to use these things clinically. So, why don’t we start with…take it from the top in the pathogen section? And I wanted to ask you specifically about C-diff. There’s toxin A and toxin B Clostridium difficile markers on this test. And what is your interpretation of this if it’s positive and the patient is symptomatic, and then you treat them, and then they’re no longer symptomatic, but the toxin still shows up on the stool test? Can you elaborate a little bit on that type of presentation?

Dr. Fabian: Sure. No, I haven’t personally seen that particular scenario but, in general, it’s important to keep in mind a lot of people can be carriers of C-diff. So, the majority of the time that we see it detected positive, whether it’s low levels or high levels, typically, patients don’t have the classic symptoms. So, that suggests that they’re probably just a carrier. And there’s, sort of, kind of, a gray area in between where there still may be some effects of C-diff. Of course, that’s one of the purposes of looking at the markers on GI-MAP like calprotectin, zonulin, etc. to see if there seems to be any evidence that may be have an impact, even if there aren’t symptoms. So, we’re also learning a lot more from research about factors that can control or influence the ability of various pathogens to thrive and also whether or not they can cause infection or if they have their, you know, typical pathogenic effects. So, that’s essentially factors that influence virulence.

So, one of the first things I want to mention is all the microbial markers on GI-MAP are assessed based on detection of DNA. So, when you’re looking at DNA, you’re looking at detection of the organisms or the genes but not necessarily whether the genes are being expressed. And that’s definitely true for toxins. So, lots of research has been coming out in research years in terms of, again, as I mentioned, things that regulate toxins, and it’s very specific. So, pathogens tend to only express those toxins under very specific conditions when conditions are favorable for them. So, for example, if you’ve detected C-diff and it really syncs up with what’s going on with the patient, symptomatically, and you decide to treat, and then on a retest, symptoms are better, but the C-diff is still detected, that’s telling you that it’s still there. But you may have improved the gut environment to the point where they’re not expressing their toxins at that point. So, they may just at that point become carriers.

Dr. Hedberg: Yeah, that just makes me think about, you know, some of these patients because we can’t or I can’t necessarily say that it’s the C-diff, you know, that was causing the symptoms because then these other patients, they had other pathogens and dysbiosis and other issues. And so, that could have also been the reason why they were symptomatic.

Dr. Fabian: Absolutely.

Dr. Hedberg: Yeah. So, that can make it difficult to really get a clear picture of what exactly is causing the symptoms if there’s multiple issues there. So, that makes sense. And, you know, for the practitioners listening, so when it says less than DL, that’s less than the detectable limit. Correct?

Dr. Fabian: Correct. Yeah.

Dr. Hedberg: And then, if we see a number there but it’s in the black, can we assume that it has been identified, it’s just not at a high enough level to be flagged in the red?

Dr. Fabian: Correct. Yeah. We do see that pretty frequently. And, of course, through our extensive validation that’s required as a CLIA-certified laboratory, those are, you know, definitely true positives. So, it is there when they’re present at low levels. And the type of method that we use called quantitative PCR is highly sensitive and also highly accurate in terms of its quantitation. So, you can have a high level of confidence in those numbers when you see them at low levels. But there is a lot of confusion or misunderstanding, I should say, among clinicians about these low levels because that’s, essentially, kind of, a new way of looking at pathogens. Because in the past, most of the methodology, whether it’s culture or standard PCR, for example, those methods generally have higher cutoff levels, in many cases, due to lower sensitivity.

And so, essentially, what you’re seeing in those tests is when it’s positive, it’s at a much higher level. So, clinicians, if they’re coming from that sort of background and using those sorts of tests, they assume if it’s “positive,” then it’s likely to be high and produce symptoms.

And so, this looking at pathogens at a low level is, kind of, a new scenario. And, of course, it’s a gray area. In some cases, it does appear that they still can cause symptoms for some patients. For example, with H. pylori, we do often see that below that cutoff for being considered high. Clinicians have reported anecdotally that, in some cases, it fits with the symptoms. So, they’ve decided to go ahead and treat, even though it wasn’t officially high. And in many cases, they reported that symptoms improved. So, that suggests, at least for some patients, that lower levels may still be relevant for that particular patient.

There are other things, though, that you can potentially get insights from just by the presence of pathogens, whether they’re high or levels. And, again, this is all coming out from relatively recent research that indicates that the gut environment, as you can imagine but there’s really a lot of research now supporting this, that the gut environment influences whether or not pathogens can basically thrive in the GI tract, you know, again, at low or high levels. Just to, kinda, cut to the chase, common ones that have emerged from research that we also see clinically would be low stomach acid. So, for example, it’s commonly known that proton pump inhibitor use can lead to an increased risk for various infections, including C-diff. Also, poor digestion, potentially, can contribute, as well. So, there’s a lot of growing research around the idea that, particularly for pathogens that are in the colon, that typically thrive in the colon, that they often depend heavily on the presence of amino acids, the availability of amino acids, which in a healthy colon are present at very low levels.

And that’s due to, when digestion is working as it should, digestion absorption, very little protein gets into the colon. And then the protein that does get in the colon is broken down and then quickly used up by the normal, healthy bacteria to fuel their growth. So, when you have certain types of disruptions like antibiotics, etc., or even a really poor diet that lacks fiber, that potentially can increase the risk for pathogens because it leads to less competition for those amino acids. And then the pathogens can use those amino acids to fuel their growth. C-diff is a classic example. That one, actually, has been shown to thrive on a number of amino acids, in particular an amino acid called proline, which happens to be present, and ironically in high levels, in collagen and, of course, products that have collagen in them. Also, bone broth, for example, collagen supplements. And this is something that we actually do see occasionally clinically, that patients who have relatively high levels of C-diff may or may not be symptomatic. But when I asked a clinician, “Are these patients on any sort of collagen supplement?” oftentimes, the answer is, “Yes.” And, again, that really fits with what we know from research.

Dr. Hedberg: Yeah, interesting. You brought up some really great points. And, I mean, that’s one of the things I like about the GI-MAP. As you said, it allows for a gray area rather than just a binary, you know, positive or negative. And that leaves a lot of interpretation to the clinician, because if all you did was just report the so-called positive and then nothing else, then maybe, you know, they could have say 5, 6, 10 different pathogens but they’re all just at a low level. But they’re just not technically positive. And so, I like that. And that would allow me to do things like make some indirect interpretations. And you brought up a really great one, which is hypochlorhydria, low stomach acid, which is just so common these days in so many people.

Dr. Fabian: Exactly.

Dr. Hedberg: So, I do like that aspect. And one of the other ones, as far as markers go, I just wanted to ask you about, because I get a lot of questions about this from practitioners, are the viral pathogens, the adenovirus, and the norovirus because we’ll see these in people who are asymptomatic. And so, it’s my understanding that, you know, I mean, if the gut is relatively healthy, these viruses will just, kinda, take care of themselves with the gut immune system. Is that, basically, how that works when we see those positive?

Dr. Fabian: Generally, yes. Those are markers that we don’t see very often. So, they’re fairly rare. When we do see them, of course, the first question is, does the patient currently have gastroenteritis symptoms? And if not, have they had any recent bouts of gastroenteritis? And in cases where the patient, you know, basically says, “No, I haven’t had any obvious symptoms recently,” then, clearly, in that case, it would be a subclinical type infection. So, not necessarily at a high enough level that’s triggering symptoms. There may be some differences in terms of specific strains, for example. So, it’s possible they may have a strain that’s less pathogenic.

But, of course, once again, the environment in the gut is such a big factor. And with almost any pathogen, based on what we know so far, the gut environment, both the physiology and the rest of the normal microbiome and the state of the microbiome, can really influence the impact of those pathogens. So, that’s really where you want to assess the whole picture. And, typically, with various type of pathogens, and this is actually known for norovirus, which is one of the best-studied GI viruses, is that when you have certain types of dysbiosis, kind of, the typical opportunistic overgrowth that tends to be more on the inflammatory side, that can predispose to norovirus infection.

And then, on the other side, kind of, of the coin is, additional research is starting to show that even though a lot of these pathogens, especially the ones on the first page, we tend to think of them as causing acute infections. You know, they cause symptoms for a few days or maybe a week or two, but then, they eventually resolve in most cases. And then, the patient’s no longer symptomatic, and the infection goes away. We’re finding that in some patients that various pathogens can persist long-term. So, you know, they may not necessarily cause obvious symptoms, but they still might contribute to some subclinical pathology. So, for instance, leaky gut, low-grade inflammation, and persistent dysbiosis are one of the sets of, kind of, consequences from these long-term infections.

And then, the other, sort of, wrinkle to the whole picture is patients can also continue to have symptoms even after infections have been resolved. And that’s, kind of, a growing recognition, based on the fact that there are persistent changes in the immune system and also with the microbiome after a number of different types of GI infections. And then, those changes can go on, potentially, and contribute to things like inflammatory bowel disease, IBS, even autoimmune conditions.

Dr. Hedberg: Right. Yeah, I’ll see Yersinia enterocolitica sometimes lasting for a while, especially in thyroiditis patients, Hashimoto’s. And then, I’ll also see giardia and cryptosporidium sometimes hanging out for a while. Sometimes, if they have a dog and they let the dog lick their lips, you know, the face a lot, I’ll see giardia issues with that sometimes. So, yeah, that’s a good point. Some of these can last for a while. And you kind of have to hunt down the source of reinfection. Are you seeing anything similar like that in any of these markers?

Dr. Fabian: We do, and I would say the most likely one that we see that, kind of, applies to that scenario would be H. pylori. So, that is one of the markers that oftentimes when clinicians treat, they decide that it’s, you know, likely to be causing symptoms, they treat… Even with cases where they see symptom improvement, they may do a retest and see if it’s still there. Now, of course, it’s important to pay attention to the levels. So, oftentimes, they’ll start off saying, “Well, it looks like this protocol was not effective.” But when you compare the before and after, the numbers show that it was reduced in many of those cases, you know, by a factor of 10 or more. So, it is significantly reduced. And that could be why the symptoms are better, but it’s still there.

And that begs the question of, “Is that okay? At a low level is H. pylori, you know, really not causing problems and it’s not necessary to eliminate it?” That’s a bit of a gray area, but, again, a number of lines of research that have come out in recent years suggest that H. pylori actually can be fairly easily transmitted from person to person. And it’s even thought to be an example of a foodborne microbe. So, patients may, potentially, be re-exposed from certain foods. Also, some research indicates that for some patients, it can also exist in the oral microbiome as, kind of, a reservoir. So, of course, typical treatments are targeting the infection in the gut. But if they’re not really doing anything about oral health, then that could just basically end up reseeding the gut after treatment. So, there are a number of different, sort of, lines of evidence.

One other, sort of, set of studies I’ve come across, I haven’t seen a lot of research on this recently so I don’t know really… It’s mostly coming out of a couple different labs. So, of course, with most research, you want to see a lot of replication to make sure that it’s, you know, truly valid. But there’s some hints that H. pylori can actually exist inside of candida cells, that candida can harbor H. pylori, particularly under harsh conditions. So, it may be a reservoir, again, which suggests that if you see candida along with H. pylori, then, of course, you may want to consider addressing that, as well.

Dr. Hedberg: Yeah. H. pylori’s a really interesting microbe. And I’m glad you brought up the point about it living in the oral microbiome because that’s a really important point. And that makes it easy to transmit from person to person. And so…

Dr. Fabian: Exactly.

Dr. Hedberg: I mean, the way I approach this H. pylori is if it’s detected but it’s not in the red and there doesn’t appear to be any major, you know, autoimmune connections or symptoms, then it’s not something that I’m going to necessarily aggressively go after. However, if it is just detected and in the black and there is a strong clinical connection to it, then I will go ahead and do something about it. And then, of course, if it’s in the red, then I usually will do something to address it, even if there’s just the slightest hint of a clinical connection there. So, what do you think about that approach, and would you make any adjustments to that approach?

Dr. Fabian: I would definitely say, based on everything we know from research and also what clinicians report, that really seems to be spot-on. So, certainly, there are cases where we do see H. pylori when it’s in the red so it’s officially high, where it doesn’t appear to be causing any problems for a given patient. And that may be due, for example, again, to the specific strain or strains that are present, or the overall state of, you know, their GI health that’s, sort of, mitigating potential negative effects. But, kind of the flip side is that even when it’s on the low end, say in the E2 range, which is, kind of, in that moderate level range, a lot of people have that level. I think our stats indicate that, at least in our patient population, that I think somewhere around 75% of patients have E2 levels of H. pylori. So, it’s pretty common, and it doesn’t appear to be causing problems for most patients. But because of, again, all this emerging research, we know a lot more about H. pylori than even 5 or 10 years ago.

And we know that low levels for some patients may still have enough of an impact on stomach acid that that can then have some downstream consequences. So, of course, symptoms are important to assess. But also, I think you mentioned before, that in the scenario of C-diff, that oftentimes you’ll see, kind of, broader dysbiosis picture. We refer to that… So, we recognize three different, common dysbiosis patterns. That’s, kind of, one of the differentiators of GI-MAP. We don’t have, sort of, a generic dysbiosis index. But there are really subtypes of dysbiosis that can be very informative. And so, when you have poor digestion, especially due to things like low stomach acid, you can end up with a bit of an overgrowth type pattern. And you can see that pattern potentially in the normal bacteria section where some of the markers may be elevated. Also, in the opportunistic bacteria section, you’ll often see that. And even candida and various parasites may be part of that pattern as well. We tend to see them more commonly when there’s a general overgrowth pattern and also evidence that patients may not be digesting well.

So, it’s really important, you know, once again, to look at the big picture. And that really gives you a lot of great insights, you know, into what’s going on more on the, sort of, broader ecosystem, you know, gut physiology integrative level. And that can suggest, you know, additional things that you may want to consider as part of your treatment approach versus just, sort of, the, you know, breaking up the antimicrobials, which is often needed and often effective. But, you know, we see this a lot with SIBO, for example, that patients that have been treated repeatedly for SIBO either they just don’t improve, or they improve for a while and then they relapse. If the clinicians decide to run a GI-MAP at that point, we’ll often see this type of overgrowth pattern and especially specific opportunists that just seem to be particularly problematic like H. pylori, pseudomonas candida.

Dr. Hedberg: Yeah. So, I’ll see, you know, low-grade detectable H. pylori and I see correlations with the low pancreatic elastase and also, you know, clinical signs of low stomach acid. And then, restoring that and, of course, dealing with other issues with the patient tends to resolve the H. pylori without necessarily, you know, doing anything for it. And so, because it is going to have a more difficult time in the stomach if stomach acid is adequate. And so, that’s just one of the approaches that I take. What do you think about that?

Dr. Fabian: Yeah. Once again, I think that’s definitely very in line with what we know from research and also what we see clinically. And, kind of, with any aspect of the GI tracts, of course, the lower GI and the oral cavity are the best study. But the idea is pathogens, opportunists generally, don’t take hold, don’t overgrow, as long as everything is in balance. And that has to do, of course, with physiology and then, also, the microbiome. So, in the stomach, as you mentioned, if stomach acid is normal, that can be one key factor that helps keep H. pylori in check.

But also, even in the stomach which, you know, we know from relatively recent research, actually is not really sterile like you might think, based on the high acid levels, that there is a microbiome typically in a healthy stomach. And there are species like Lactobacillus species, streptococcus, that are fairly acid-tolerant that can thrive to some extent in the stomach. And certainly, with Lactobacillus, there’s a lot of research showing that that, normally in a healthy stomach, helps keep H. pylori in check. And, of course, we know lots of people are deficient in these various types of beneficial bacteria. And that’s where fermented foods, probiotics, and other factors may also help to keep H. pylori in check.

Dr. Hedberg: And moving onto the normal bacterial flora section, I mean, is there anything in this section that would alter the actual treatment for the patient? Any of these normal bacteria being high or low that would really push you in one direction or the other?

Dr. Fabian: Absolutely, yeah. So, as I mentioned, we do recognize three common dysbiosis patterns. And that, again, is very well-supported by what we know from research. So, just real quickly, we have the inflammatory dysbiosis pattern, the insufficiency pattern, which is lack of key beneficial bacteria, and then the overgrowth/digestive dysfunction type pattern. So, those are, kind of, the three ones. And then, there’s lots of inter-individual variations in those patterns. But within the normal bacteria section, you potentially can see evidence of all three of those patterns in that section because of the types of microbes that we included. So, that section, of course, is not the entire microbiome. They’re just a representative sampling of the normal microbiome. But they’re carefully selected to give you insights into these different types of patterns.

So, examples would be some of the key beneficial groups would be…probably top of the list would be faecalibacterium prausnitzii, which is a major butyrate producer. Low levels of it link to a pretty broad range of diseases and conditions. And so, that’s really thought to be a keystone species, and it’s important to see that, you know, in healthy ranges. So, even if you see that low normal, E3, E4 levels, and if that’s consistent with what’s going on with the patient, that can be significant.

Really a key example would be autoimmune conditions. So, there’s, kind of, an emerging picture that one of the key immune response imbalances that’s really characteristic of a long list of autoimmune conditions is increased pro-inflammatory bacteria and pro-inflammatory responses, and then a decrease in beneficial bacteria and anti-inflammatory responses. And there’s a direct connection there because we know those beneficial bacteria, especially the ones that produce butyrate, can promote, for example, T-regulatory cells that can help keep autoimmune-type reactions in check. So, we do see that type of deficiency pretty frequently in patients that have chronic inflammatory and autoimmune conditions, as well as in conditions like food allergies, food sensitivities, etc.

Within that same section, there are two that are also potentially inflammatory and/or a part of an inflammatory dysbiosis pattern. And that would include the Escherichia group. And the main species there is, of course, the well-known E. coli. That’s actually what the E in E. coli stands for is the Escherichia. Then, there’s the Enterobacter group. Both of those are part of this general group of proteobacteria. That’s one of the key phyla in the microbiome that tend to be generally more pro-inflammatory. They produce factors like LPS, for example, that can be especially inflammatory. In normal levels, they actually have very key roles in keeping a healthy microbiome. But when they’re out of balance, they can both contribute to inflammation, again, because of the higher production, for example, of LPS, but also an inflamed environment say during an infection, IBD, etc. that can create a better environment for them, less competition with the other bacteria, so they overgrow. So, you often will see, for example, high Escherichia in inflammatory dysbiosis.

And then the third thing within that section would be, kind of, a general overgrowth pattern related to reduced digestion. Markers that you’ll often see elevated in that scenario would be high firmicutes, even high Akkermansia. That seems to be one of the more common ones. For example, when H. pylori is elevated, you’ll often see Akkermansia elevated and a few others like the clostridia group, etc. If you see an overgrowth pattern there and an overgrowth pattern on the next page in the opportunistic section, you know, that suggests considering whether the patient may have some digestive issues.

Dr. Hedberg: Right. Yeah, when I get worried is when I see the firmicutes quite high. And I’ll usually see that in people who were C-section babies, if they weren’t breastfed. And then, especially, if they had antibiotics the first three years of life, that seems to really drive the firmicutes up and set up…

Dr. Fabian: Oh, that’s interesting.

Dr. Hedberg: Yeah, and set up some issues as an adult. An antibiotic, the first three years of life, that’s a pretty devastating drug to have to take. And I see a lot of correlations there. And then, of course, they’re usually insulin-resistant and inflamed, as you said, seeing that elevated.

Dr. Fabian: Right.

Dr. Hedberg: So, you mentioned the next section of the test, which is the opportunistic bacteria, dysbiosis, overgrowth. Mainly, I mean, I’m looking this as…I’m just, kind of, globally looking at it. Are there any particular bacteria on here that if you see it high that shifts the way that you would approach the patient?

Dr. Fabian: Yeah. A key example would be on page three. One of the opportunists is called Pseudomonas. We have both markers at the genus level and then, also, a marker specifically for the species Pseudomonas aeruginosa. That’s a really interesting species and it appears to be something that was kind of overlooked for many years because there just wasn’t as much research on its role in the gut. Especially with Pseudomonas aeruginosa, that’s mostly tied to the lung infections and even pretty serious internal infections, in some cases. But the role in the gut, it’s been known to be in the gut for a while. But what its function was, what it was doing was really not well-established.

A series of studies that have come out in the last few years show that it actually, mostly, thrives in the upper GI tract. So, that’s, kind of, another important aspect of using DNA-based testing like GI-MAP uses with QPCRs that you can detect organisms that may not actually be thriving in the colon but may be thriving higher up such as H. Pylori. And so, Pseudomonas has been found to typically inhabit the stomach, and especially the upper small intestine, so the duodenum.

Actually, when it’s at high levels and it expresses certain virulence factors, then it’s been linked to, generally, increased incidence of food sensitivities and especially to gluten reactivity and celiac disease. It’s not necessarily thought to be a “cause” of celiac, based on what we know so far. Of course there’s that strong genetic component there, for example. But it may be one of the triggers that triggers onset, or may worsen symptoms for patients that have celiac. So, there’s a growing amount of evidence there. And oftentimes, we’ll see…so it wasn’t really until I’d come across that research that we didn’t notice that there was this connection on our test between presence and especially high levels of Pseudomonas and then the anti-gluten marker that we have in the intestinal health section, which, of course, is, kind of, a general marker for gluten reactivity and sometimes can even be elevated due to reactivity to cross-reactive foods, gluten cross-reactive foods, that have similar proteins to gluten.

We often will see that even just in the high-normal range, but especially elevated along with high Pseudomonas. So, if patients are, you know, continuing to consume gluten and they continue to have GI symptoms, seeing Pseudomonas along with the anti-gliadin being elevated suggests not just, for example, gluten avoidance, which is often, kinda, you know, the typical approach, but also looking more carefully at the dysbiosis pattern and whether or not it may be actually helpful to address Pseudomonas in those cases.

Dr. Hedberg: And the methanobacteria, some people will ask, “Any connections there with methane-dominant SIBO?” Do you see that at all?

Dr. Fabian: We do. Now, we have to point out that our test is, of course, not in any way diagnostic for SIBO particularly because, you know, we’re looking at a stool sample that mostly reflects colon but gives us some insights into organisms higher up. The methanogens though and the vast majority of methane-producing bacteria or I should say the kind of bacteria, like, they’re actually called archaea, they tend to exist in the colon. And that’s mostly because they’re strictly anaerobic-type microbes. And, of course, the colon is the part of the GI tract that is anaerobic. Plus, for their ability to thrive and metabolize, they depend heavily on fermentation by bacteria, and especially the production of hydrogen gas by bacteria that ferment. And they take that hydrogen and they convert it into methane. And so, that’s thought to be one of the key reasons why they’re so predominant in the colon because most of the fermentation in the gut by bacteria happens in the proximal colon. Particularly if there’s adequate fiber in the diet and other fermentable carbs, that really drives fermentation activity, that then can fuel the growth of methanogens.

So, in that context, you know, we often do see that patients who have evidence of slow motility, constipation, that they have high levels of methanogens or even just high normal. So, on our test, high normal ranges for the methanogens, that’s the methanobacteriaceae family on page three, we would say that, kinda, roughly based on what we see clinically, high levels may be, kind of, in the upper E8 range and higher. So, when we see levels in that range, patients are more likely to have constipation but certainly not all. We’ve seen a few cases that have even relatively high methanogens, and yet, they don’t have constipation.

So, that may, of course, mean that there are other factors that are, sort of, coming into play that, you know, influence overall motility. But mostly, aside from the methane production and linked to constipation, especially, again, at the high normal to officially high levels, they’re confirming that there’s higher-than-normal fermentation activity. So, that gives you a lot of insights, then, into does this patient have issues with digestion? Are they consuming, you know, too many carbohydrates? You know, there’s some reason why they’re having excessive fermentation. So, it’s important to look into that because that may be relevant to their symptoms.

Dr. Hedberg: And then, the autoimmune triggers section, this is a list of bacteria that have been shown in the literature to be connected to various autoimmune diseases. Anything that you wanted to say about this section, other than understanding that connection?

Dr. Fabian: Absolutely, yeah. So, the main reason, as you mentioned, that they’re included in that section is there have been some links in the literature, mostly associations. In a few cases, some kind of mechanistic insights that they may actually contribute to those autoimmune conditions. But that’s really why most of those were selected, is based on those associations with various autoimmune conditions. Now, it turns out that most of the ones in that group are also, generally, potentially inflammatory. So, you’ll also see them in many inflammatory-type scenarios. And you’re looking to see whether there’s evidence of inflammatory dysbiosis. That’s a key section to consider. So, for example, Citrobacter, Klebsiella. Probably, in that section Klebsiella would be the most common one that we see linked to inflammation. And there’s a lot of growing research on that one linking it to inflammatory bowel disease, potentially a range of autoimmune conditions such as ankylosing spondylitis, rheumatoid arthritis.

It’s also a really interesting microbe, especially Klebsiella pneumonia, from the standpoint that it typically, from what we know, is coming from higher up. So, it’s known to be present in the upper GI tract. It can even be in the bile duct. It’s common in the respiratory tract. And it can even be in the oral cavity. Recent research shows that if patients have gum disease, for example, and then they have higher levels of Klebsiella in their oral microbiome, then, of course, they have higher levels of Klebsiella in their mouth. Then, when they swallow, and especially if they have low stomach acid, then you can end up with more getting into the lower GI tract.

And if they have, sort of, a series of things that predispose them to, for example, low beneficial bacteria, then they’re more likely to end up with lower GI inflammation. So, this is really, kind of, coming back to making sure that you’re taking, you know, overall, kind of, a systems holistic integrative view of the whole GI tract instead of just, sort of, looking at one aspect and one microbe. With all this new information we have, you know, you see Klebsiella on the test, you might see calprotectin elevated, for example. But another question to ask is, is their stomach acid okay, and do they have issues with their oral health? And all of those can, potentially, be quite helpful.

Dr. Hedberg: Excellent. And then, the fungi yeast section, we’ve got, you know, candida and a few other species there. The way that I’ve used this is, you know, some of these could just be transient. If it doesn’t seem to fit the picture clinically, you know, some patients, if the candida comes back high in the red, you know, they’ll ask, “Do I have candida?” And I don’t think we can necessarily say that just from a positive indi. So, how do you approach the yeast section?

Dr. Fabian: Essentially, pretty much along the lines of what you described. So, candida is far and away the most commonly clinically-relevant among the ones that we have in that section. The other ones are actually known mostly to come from environmental sources and particular food. So, when you see them detected, and especially if they’re, kinda, moderate to high levels, that wouldn’t be coming just directly from food. To reach those levels, they would have to be growing in the gut. But even then, it’s relatively uncommon for us to see scenarios where they’re contributing to symptoms. And that’s the non-candida fungi. So, it does not appear common that they’re obviously causing symptoms in most cases. If you look at the information that’s out there on those types of fungi, they tend to be issues more for patients that are immunocompromised.

So, when you have that combination of an immunocompromised patient with really high levels and they have symptoms that don’t seem to be attributed to anything else, then certainly it could be causing problems. It just doesn’t appear to be very common. So, we see them pop up here and there, mostly low levels, probably coming from food. But typically, one of the things that it’s likely telling you is just like any other opportunist, they’re reflecting…the fact that they’re there and, kind of, eking out an existence is reflecting that the gut environment has been disrupted and out of balance, and that normally what keeps it in check… You know, once, again, low stomach acid is a common one, lack of beneficial bacteria, etc., all the typical things. You often see evidence of that, and that would suggest, you know, you may not need, for example, to use an antifungal agent but maybe just need to rebalance what’s going on with the gut, whereas Candida’s really a different scenario.

It is a fairly common resident. Most patients do have candida. Research, once again, indicates that. So, candida, of course, can exist in two, sort of, general forms. I think there’s a few others that are thought to potentially exist, but primarily yeast form, or the form that actually creates what are called hyphae. These are, sort of, long projections that can extend through the epithelium into the lamina propria below. And that can stimulate an immune response, an inflammatory immune response. Some studies have indicated that candida’s one of the most inflammatory microbes in the gut when it’s in that form. So, our test doesn’t differentiate between those two different forms. So, just as you noted, just because candida is there doesn’t necessarily mean it’s causing problems. Once again, you know, careful assessment of the patient is really important and symptoms.

But if you see, generally, a disrupted microbiome and, in particular, of course, you see high calprotectin, those may be indicators that there’s some issues. Clinically, we see candida mostly associated with that poor digestion, overgrowth pattern. So, once, again, suggesting that reduced digestion may be part of the picture and contributing to its overgrowth. Also, common clinical observations with candida is, kind of, a range of sensitivity issues, food sensitivities. Especially when it’s really high, it seems to be more common that clinicians will be dealing with patients that just have a broad level of sensitivity or report that they’re just, sort of, sensitive to everything. And we tend to see that more commonly with candida. And that could be because of the combination of…in that case, it may have transformed into the hyphae form. It may be causing some leaky gut. It may be causing some inflammation, that they’re all contributing to that.

Dr. Hedberg: Yeah. And that could also be driving TH2 polarization, which would increase allergies, and histamine, and things like that. So, yeah, that makes sense. Now, the virus section…so, we have two viruses, cytomegalovirus and Epstein-Barr. These are both herpes viruses, some connections with them and autoimmune diseases, inflammatory bowel. And something like Epstein-Barr, you know, that can be active in a specific organ or a specific area in the body and not necessarily have a systemic effect. So, can you comment on these two viruses and how a clinician would use these clinically?

Dr. Fabian: Sure. So, with these viruses, number one, we don’t see them commonly at all. They’re pretty rare. And based on what we know from research and also what we’ve seen clinically, you know, they can be present and not necessarily related to symptoms. But if they’re especially high, they certainly can be. So, they’re somewhat different scenarios. So, with EBV, it is known from research that in some patients, EBV can be latent. So, it often exists in a latent form, nonactive. It can be latent in the intestinal epithelium. And in certain scenarios, whether it’s immunosuppression or an inflammatory scenario, may result in its reactivation. So, that’s, kinda, the first consideration is if you see it, it’s been reactivated, it’s replicating, and it’s in the gut. So, you know, that’s indicating that maybe the patient is immunocompromised or has some sort of aspect of the immune system that may be suppressed or the opposite. Inflammation has also been shown to trigger growth of some of these or replication of some of these viruses.

So, as far as consequences in the gut, it’s not really well-defined yet. But some studies suggest that EBV, when it’s reactivated, because it’s, you know, reactivated in those epithelial cells, can contribute to dysfunction in the intestinal barrier. So, if you see, you know, evidence from symptoms or other markers on the test suggesting that the intestinal barrier is compromised, that may be a contributor. Also, then, with CBV or CMV, it’s a little bit different. That one is primarily latent in the immune cells such as monocytes. So, if patients have, say, an existing inflammatory bowel disease such as ulcerative colitis, that general inflammatory scenario and/or the immune suppressants that a lot of those patients are eventually put on can cause that virus to be reactivated.

And one of its key organs, that it tends to target is the colon. So, then, those monocytes basically hone in on the colon. And then, they can develop into pro-inflammatory macrophages. And meanwhile, this CMV is replicating in those macrophages. And just based on research indicating what they’re doing in the macrophages, they’re contributing to a more inflammatory phenotype. So, at the very least, it’s fairly well-characterized that CMV in the gut could be worsening patients that already have a diagnosed condition like ulcerative colitis.

As far as contributing to other inflammation scenarios and sort of, separately from ulcerative colitis, we really don’t have much research on that. I have occasionally seen it, but, again, it’s not a virus that we see very commonly. So, there really aren’t a lot of cases that I can draw upon, you know, to talk further about that one. But inflammation of the gut, you know, is really a common consequence when it’s reactivated.

Dr. Hedberg: Right. Yeah, I don’t see these very often either. But they are helpful, I think, especially if you’re dealing with a lot of inflammatory bowel in your practice. So, the last microbiology section is the parasites. And the way I look at this is if I see a lot of these, then I’m thinking low stomach acid and dysbiosis and inflammation. And then, you know, because a lot of these are just hand-to-mouth, contaminated food or water, they get them. They should just clear, you know, from the body, unless it’s something really difficult like a tapeworm or a hookworm or something like that. What should practitioners be thinking when they see these? I mean, obviously, if it’s in the red, then there’s a strong indication there to treat. But what if you just see a bunch that are detected but not in the red? What should clinicians be thinking about these?

Dr. Fabian: Yeah, that’s a great question. So, parasites definitely, I think, create a lot of, kind of, diverse opinions and confusion. And I think what you mentioned is really our understanding as well, that certainly what we see commonly and clinically with GI-MAP is that, by and large, they’re mostly present, especially when you see them at low levels, when there’s general evidence of dysbiosis and, again, kind of, that overgrowth, poor digestion-type pattern indicating that there’s probably some connection, just like we see with, say, candida and other opportunists that those gut conditions are out of balance and then creating a favorable opportunity for them to, kind of, eke out an existence.

It may or may not really be causing any significant issues. Kind of, interestingly, that list on page four is organized alphabetically, but it turns out that it’s, sort of, roughly in order of their likelihood to contribute to symptoms. So, the ones, kind of, at the lower end like endotrichomoniasis, you can look up the information on those, and they’re widely regarded as pretty harmless parasites that generally don’t need to be treated. May cause symptoms in some patients. You know, once again, typically more in the immunocompromised patient scenario.

So, that, kinda, begs the question of, you know, a lot of clinicians really do tend to just treat parasites when they see them. And that’s, oftentimes, just from their training that they’re, you know, often encouraged to treat parasites. So, some clinicians see those as being more significant. We really don’t see that pan out so much, you know, with the scenarios that we see with GM-MAP. They just don’t seem to be directly causing symptoms. As you mentioned before, that can be a little bit difficult sometimes because it’s almost always within the context of broader dysbiosis and other things going on. You may see low elastase… So, it could be that that whole picture is really what’s causing symptoms, not so much the individual organism in that case, whereas high levels…

And, again, that’s, you know, one of the reasons why we really focus on the benefits of QPCR, that provides clinicians with precise quantitation, along with these cutoff indicators to, kind of, guide you when levels are high enough to be more likely to cause symptoms and cause problems. And clinicians do use those quite a bit as a guide to determine whether or not to treat. But, as always, that’s within the context of symptoms and the patient assessment. So, it is a bit of a gray area. And I know that’s just, kind of, a challenge for busy clinicians. It’s like we want everything to be, you know, very cut and dried and as simple as possible. And if you see something, you want, kind of, a simple answer as, should this be treated or not be treated? But really, clinical judgment comes into play quite a bit because there are some gray areas. And that’s a section where there’s definitely some significant gray area.

Dr. Hedberg: Right. And, like, if you have a parasite like Blastocystis hominis and the patient has Hashimoto’s disease, there was a recent paper that found if you treat the Blastocystis hominis in Hashimoto’s patients that does bring down their TH-17 polarization. So, IL-17 is elevated in those patients driving the autoimmunity. And there are improvements in the patients and their antibodies if you treat that particular parasite. So, sometimes, you’ll see a specific connection there that would warrant definitely addressing it. But I definitely agree, it just seems like getting the patient’s diet right and strengthening digestion and maybe some probiotics, I mean, that alone tends to deal with a lot of these. And you don’t have to necessarily go in, you know, with the herbal bulldozers and knock them out.

Dr. Fabian: Right. Yeah, that’s a very interesting set of research that you mentioned. I haven’t actually come across that related to Blasto. So, I’ll have to check that out.

Dr. Hedberg: Yeah. I’ll send you that paper after the interview. So, the last section is the intestinal health. I find these to be fairly straightforward [inaudible 00:53:10], you know, you’re looking at fat in the stool, and then, the elastase. I want to get your opinion on the elastase because, you know, the interpretive guide and then, there’s, you know, clinician opinion and things like that. And some will say, “Well, it really needs to be above, you know, 400. If it gets below that, then you might want to consider treating it,” even though on the lab it says normal is greater than 200. So, can you comment on when you might actually want to use HCL and/or pancreatic enzymes based on this particular number?

Dr. Fabian: Yeah. Absolutely. So, of course, with any marker, I mean, you know, we do give the cutoffs as a guideline for really understanding when it’s especially out of range and more likely to contribute to symptoms and require treatment. But there’s also the idea of the functional ranges. So, we consider optimal with GI-MAP, in general, to be, kind of, roughly around 500 and above. Clinically, we tend to see that when patients have levels that are down closer to say 300, 350 range, that’s starting to get into a range where that’s more consistent with GI symptoms and other evidence that they may not be digesting very well. Plus, clinicians often report that if they decide to supplement with betaine or with digestive enzymes when they’re kind of low normal that, often, they will get symptom improvement. So, that all suggests that, kind of, when you see it in that range and below, it’s still potentially suboptimal and worth considering whether or not patients may benefit from support, even though it may not be officially low.

Dr. Hedberg: Yeah. Did you say 400 or 500?

Dr. Fabian: Roughly around 500 is considered…

Dr. Hedberg: Five hundred.

Dr. Fabian: …to be optimal. Four hundred, generally, most clinicians don’t really do much when it’s say 400, 450. But once it drops into the 300s, especially the lower 300s, that seems to start to get into an area that’s more likely to be consistent with clinical signs and symptoms.

Dr. Hedberg: And the Beta-glucuronidase, I mean, I have to admit I don’t really use this that much to make clinical decisions because it’s usually just elevated in patients with a lot of dysbiosis and compromised digestion. And I say that in relation to just the classic approach for this when it’s elevated is just to give the patient calcium D-glucarate. And so, I mean, do you see any real advantages to how you would change, how you treat the patient if this is high? I know there’s connections with estrogen reabsorption and things like that. But do you have anything to add there?

Dr. Fabian: Yeah, definitely. So, with Beta-glucuronidase, it’s mostly generated or produced by a subset of bacteria in the microbiome. And they generally tend to be, for the most part, the types that we would consider more dysbiotic. There are some significant exceptions like the main members of the Bacteroidetes phylum, which are normal bacteria, can also produce it. But you’ll especially see it elevated in conditions where there may be also evidence of inflammatory dysbiosis. But because there’s a range of bacteria that produce it, not all of them are inflammatory. So, it’s, kind of, a catch-all marker for dysbiosis, so, you know, particularly when it’s really elevated. If it’s just a little bit elevated, you know, it really may not be causing too many issues or a sign of too many issues. But once you see it well into the 3,000 range and higher, that’s often a pretty significant indicator of dysbiosis.

And I think that’s important in the sense that with our normal bacteria section, as I mentioned earlier, we only have a representative sampling of the microbiome. We certainly don’t have the entire microbiome represented there. So, sometimes you can, of course, have dysbiosis that’s not reflected as much in that section. We see that occasionally with some patients where they have high Beta-glucuronidase, which is likely indicating imbalances. But we really don’t see a lot of evidence on the test itself.

The same with secretory IGA. That’s, kind of, the opposite scenario where it’s a good catch-all marker for lack of beneficial bacteria. Typically, you know, it does involve ones that are on our test. They’re frequently…you’ll see it elevated when we really don’t see any evidence of the ones that are low in that section. But we do know that secretory IGA, in order for it to be at normal levels, requires stimulation from beneficial bacteria. So, they’re, kind of, opposite markers.

And you’ll see that in some cases where Beta-glucuronidase is especially high and secretory IGA is especially low. And that could be a good, sort of, confirming factor to the overall extent of dysbiosis. And that can be informative for how clinicians approach things. Ultimately, to get the Beta-glucuronidase back down into the normal range, that does require rebalancing the microbiome. So, that’s, ultimately, sort of, the treatment approach that’s needed. So, the calcium D-glucarate is a great, kinda, interim measure to, basically, mitigate the effects of high levels.

And, as you mentioned the other, sort of, significant consequence when Beta-glucuronidase is elevated is it’s really involved in interfering with the typical process of eliminating toxins and hormone metabolites through the GI tract. So, if those levels are high, that can cause them then to persist, both in the gut and then especially they can get reabsorbed and persist systemically. So, high levels of Beta-glucuronidase, for example, then links to inflammation and also colon cancer in the gut. And that may be due to the fact that it just increases the toxin burden. So, you know, again, depending on the patient scenario, some patients just have compromised liver function, compromised detoxification. And this would just add an extra, sort of, burden onto the liver in those cases. And then, you mentioned the hormone connection, which is actually quite common, especially for women in terms of estrogen. So, we do see those, kinds of, clinical scenarios pretty frequently.

Dr. Hedberg: And then, the anti-gliadin antibody, you had mentioned this earlier. And I find this very useful, especially in helping patients understand that they need to avoid certain foods, especially gluten. And also, as an example, I had a patient and he wasn’t really responding. And according to him and his diet review, he was gluten-free. But on retesting, it kept coming back positive. And then, finally, we figured out that the restaurant he was going to, pretty much every day for lunch, the salad dressing had gluten in it. And he was told that it was gluten-free. And we tracked it down to that one thing. And so, it can be really useful in that regard. And then, you mentioned cross-reactive gluten foods. And so, I have a long list of those. What is your understanding of some of the cross-reactive gluten-associated foods?

Dr. Fabian: That does seem to be quite common. We often will refer patients to, for example, the list for one of the Cyrex labs assays just as one, you know, good example of established cross-reactive foods. But there are others as well. I don’t know how well some of those have been established. I’ve seen things like coffee, sesame, etc., on some lists but not others. So, I think that there is, kind of, a core that tend to be more likely. Surprisingly, dairy is one of them, dairy protein because there is some structure to, I believe it’s casein, that’s similar to gluten. And, then certainly, the other grains, proteins from the other grains can be pretty common. Seeds, nuts, those are, kind of, among the more common cross-reactive sources. I don’t think there’s a whole lot of research, in general, on that topic though, not to my knowledge. So, there probably are foods that potentially, you know, can cross-react they just are not yet identified.

So, I think the central issue there though… So, of course, the food elimination aspect, being more careful about gluten sources and all that can be quite helpful. But also understanding, kinda, the underlying contributors and root causes to that scenario and what that’s telling you. So, typically, when you see reactivity to any particular food, of course that’s likely to also indicate that patients may have broader reactivity to foods. And so, even though one marker is really only reflecting reactivity to gluten, possibly the cross-reactive foods, that’s likely a general indicator that they’re just more reactive to foods. So, the question is why is that the case. Once again, a lot of it comes down to microbiome balance. Lack of beneficial bacteria has been one of the key factors that has been shown to influence oral tolerance, make oral tolerance less effective. So, they’re more likely to react.

Some microbes, actually, can contribute to the breakdown of gluten in good or bad ways. So, I mentioned Pseudomonas is one of them. It can break down gluten in a way that makes it result in higher levels of symptoms whereas Lactobacillus species actually can do the opposite. So, that may be one other approach when you see that elevated. And then, kind of, a third area is just the general picture of not digesting well. So, there are a number of studies showing that, particularly with low stomach acid, that can also predispose to food reactivity for, kind of, the obvious reason that we need adequate stomach acid, especially for proper protein breakdown. When those proteins are not really broken down efficiently, that means all the way down into amino acids, they can retain their ability to stimulate the immune system. And that’s called immunogenicity. So, there’s a lot of factors there to consider. But when you see that elevated, we often see that consistent with the general poor digestion-type pattern.

Dr. Hedberg: And you mentioned the high Beta-glucuronidase and the low secretory IGA pattern as potentially indicating inflammatory dysbiosis. Was that correct? And did you have anything else to add about secretory IGA?

Dr. Fabian: Absolutely, yeah. So, that one, we most commonly see it low versus high. Once again, as I mentioned, that’s mostly thought to be related to lack of beneficial bacteria, and, in particular, butyrate-producing bacteria. So, that can often be something that’s low in a variety of conditions, particularly autoimmune conditions, chronic inflammatory conditions that feature lack of beneficial bacteria as part of the overall imbalances. So, again, you know, even though certain supplements can help compensate or help stimulate secretory IGA like Saccharomyces boulardii, glutamine, Vitamin A, colostrum, etc., those can be quite helpful in the interim.

But, ultimately, you want to, once again, focus on balancing the microbiome. So, you’ll often see that low in autoimmune conditions. Also, especially in scenarios where patients may be more reactive to food antigens, in particular. Because one of the functions, normal functions of secretory IGA is to actually help coat or bind to food antigens to help reduce their reactivity or their ability to react to the immune system or with the immune system. So, they play some important functions there.

Now, on the other end of the spectrum, you know, of course, sometimes you’ll see that elevated as well. It’s somewhat less common, but that’s indicating that at the time of…when the patient collected the sample that they were reacting to something, typically either some sort of opportunistic or pathogenic microbe. On our tests, probably the most common ones would be parasites and candida, and then, also, once again, broader reactivity to foods. So, if they had consumed a meal or certain foods in the days prior to the test where they were especially reactive, that may cause the secretory IGA to be elevated.

Dr. Hedberg: So, that would be a good thing. If you see it elevated, then you know that we’re having a normal immune response to a particular pathogen or food.

Dr. Fabian: To a point, yes. I mean, of course, there can be excessive reactions or…

Dr. Hedberg: No, not a food but a pathogen, yeah.

Dr. Fabian: Right. Yeah. I mean, certainly, any immune response certainly has its range of, you know…kind of where it’s meant to help restore balance. But, of course, things can be excessive, or immune responses can be chronic as well.

Dr. Hedberg: And then the calprotectin, which is the last marker to cover, I mean, is there anything more that we can really derive from this, other than it’s showing us inflammation? I mean, I know there’s connections with high calprotectin and colon cancer. Anything else to add there, if it’s elevated?

Dr. Fabian: There are a number of different things that can cause it to be elevated. Now, that’s typically when it’s elevated modestly. So, say, the cutoff is around 173. Say you see a patient that has 200 or 250, you know, it’s possible that they might have IBD or something like that. But also, it’s, kind of, in those high ranges that are not super high, then it can also be things like infections, potentially even certain drugs like non-steroidal anti-inflammatories, NSAIDs. It can cause inflammation and it can cause calprotectin to be elevated. But when it’s really elevated, most commonly we see that in patients that are diagnosed with inflammatory bowel diseases. And I’ve seen cases where patients will have levels 3,000, 5,000, and I think in a couple cases, I’ve seen it close to 10,000. So, that, kinda, gives you, kind of, a perspective there that those are very high levels that are clearly indicative of, you know, very high levels of inflammation. Often, you’ll see occult blood in those situations, as well. Whereas these lower levels, it’s a bit of a… You know, like a lot of things, there’s a gray area when they’re, kind of, in the in-between levels.

And it’s really important to consider a couple things, what may be causing that. Sometimes you’ll see evidence directly on the test, say a pathogen, on the first page. C-diff, for example, enterohemorrhagic E. coli., [inaudible 01:09:26]. Especially with patients that are symptomatic, you’ll often see levels in that, sort of, 200 to 500 range. Other than that, there may be inflammation for unknown reasons. It may require further investigation. One thing to consider though is that calprotectin is a fairly volatile marker. So, there can be things that just transiently elevate it. So, high levels don’t always mean that there’s a chronic problem. It could just be something they were exposed to, for example.

So, retesting when it’s especially elevated is certainly something that we would emphasize. And if you do see things on the test that, you know, say there’s no concerning symptoms from the patient, you don’t see any occult blood, etc. overall, you know, the clinician concludes there’s a less worrying scenario. And certain things that are, kind of, obvious for them to work on with their patient such as dysbiosis, they may do those things first and then do a recheck to see if it’s still elevated. And, of course, if it is, despite, kind of, doing the normal gut health type approaches, then that suggests further evaluation that’s probably necessary.

Dr. Hedberg: Right. Right. Excellent. Well, this has been a really comprehensive review. I think we’ve covered everything in detail. So, I appreciate you coming on. And is there anywhere you’d like people to find you online?

Dr. Fabian: Generally, I would direct you to the Diagnostics Solutions Laboratory website. I am listed in their staff section if you want to find out a little bit more about my background. I do have a course that’s currently not available because I’m in the process of revising it, and that’s microbiomastery.com. I’m hoping that that course will be available sometime in the spring. It’s been a little challenging just to carve out enough time to get that wrapped up. Currently, the schedule’s pretty busy. So, again, currently, that’s not available but eventually, that course will be available. But clinicians that want to know more about GI-MAP, we can certainly direct you to our website. And then under the clinician’s tab, we have a learning resource section, which is where we have things like our interpretive guide, whitepaper, and a long list of webinars, previously-recorded webinars, that can help clinicians get more out of GI-MAP and learn how to really interpret the various markers and patterns properly.

Dr. Hedberg: Excellent. Well, I use the GI-MAP test. I find it very useful clinically. It’s very easy to do for patients because it’s just one sample, rather than multiple samples. And I’ve just gotten excellent results. So, again, thanks for coming on. And everyone listening, go to drhedberg.com and just do a search for GI-MAP. And there’ll be a full transcript of this interview, in case you missed anything. We went into a lot of detail so I think that will be helpful. So, take care, everyone. This is Dr. Hedberg, and I will talk to you next time.