Hello, it's Anthony Chadwick from the webinar that welcoming you to another one of our evening webinars, this time kindly sponsored by Carros Animal Health. We're going to be talking about the role of biomarkers in canine chronic inflammatory enteropathy. Where are we now?
And we're really, really fortunate to have Professor Sila Salavati with us today, . Professor Salavati graduated from the University of Giessen, where she also completed her doctoral thesis which kindled her interest in small animal gastroenterology. I was with her last week, although I didn't.
Spotter at the ECBIM conference in Maastricht and she's actually a ECBIM companion animal diploma in small animal internal medicine. She completed her PhD at the Royal Veterinary College investigating the effects of symbiotics in canine chronic enteropathy, and then she moved to the University of Edinburgh in 2016. Where she later became head of the Small Animal Internal Medicine Service, she has a particular interest in gut motility, immunology, probiotics, the microbiome, which is very much in fashion at the moment and er a really exciting area, and faecal microbiota transplants.
She published extensively on these topics, both in the form of 40 research articles and several book chapters. Due to this she is also holding the RCBS recognised specialist title in small animal gastroenterology. And I'm also really pleased to have Lucy Williams, on the line as well, who is from Carras Animal Health.
Lucy is a vet, and she's the marketing manager at Carros, and she will be talking a little bit about the. Brand new, innovative and very exciting product that they have available to help us in diagnosing and er helping us to, to Have a better understanding of the disease, which I'm, I'm not gonna spoil the fun about Lucy, you can reveal all in about 40 minutes' time, so Silka, thank you so much for agreeing to do this talk, I'm really looking forward to understanding it as a dermatologist, the gastrointestinal tract is just an imagination of the skin, so as a dermatologist I'm sure I'm gonna learn and enjoy, so over to you Sila. Yes, thank you very much, Anthony, for, for the kind introduction and also for the general invitation to, to speak today.
So I'm just gonna share my screen now and hope that, you will be able to see this, and also, obviously from myself, also, thanks to Karo's Animal Health for, you know, making the connection, with the webinar vet and, sponsoring this. This webinar, so, obviously as you've just heard, I'm very, excited about anything that has to do with the gastrointestinal tract, and, I think particularly biomarkers, you know, is something that maybe has been a bit quiet, in the, in the last few years but is now sort of picking up, and that's, you know, the reason why we will be talking about, this. Particular topic and as just said and also shown here in my slide, we're gonna talk about this in the context of canine chronic inflammatory enteropathy, previously, probably better known as inflammatory bowel disease or or IBD .
And this is a little overview of what we're going to cover as part of this webinar. So as the title suggests, we're gonna heavily focus on the biomarkers first a sort of little general introduction, and then we're gonna do some comparison, briefly where, where we can, and then of course focus on, canine chronic enteropathy and, it, it, it would not be possible in. In the time that we have to talk about all the potential investigated and available biomarkers in the wider context, so we're going to focus on a few of those, as you can see, we're going to talk a little bit about .
Dysbiosis, inflammation, autoimmune markers and functional markers, some of them might be more familiar, to, to all of you than, than others, and then I'm also gonna show you some, unpublished data on some novel markers that, some of them I was, you know, involved in, in establishing or, or looking into, . What you might not see on this slide in terms of topics that we're going to talk about is what the definition of chronic enteropathy is, how we diagnose and and treat it. So for the purpose of this particular talk, I'm just assuming that, hopefully, most if not all of you know, what I'm.
Talking about, but if you do have questions about this, you know, afterwards, then I'm, I'm happy to enter into the, the discussion of, of, of that as well with, within reason, because it is a big topic and we can, we can talk about this for many, many hours. Well, at least I can, you might not want to, and that's, that's fine as well, . Nola I've now just lost my little.
Way of pointing, hang on a second. There we go. OK, so let's start with a sort of slightly dry things, but always good to, to start with this in terms of so that we all know what we're talking about and on the same page in terms of definitions and, believe it or or not, there are actually papers and, sort of consensus and statements about what a biomarker actually is and there is this, this group or this publication as you can see, American.
Driven and, you know, particularly by, the, the National Institutes of Health and, and the FDA, about, exactly that, you know, what are biomarkers, and, and other tools, generally speaking when it comes to And the terminology and, and diagnostics, and it is something that I don't think I've really thought about that much before actually preparing this, this talk, but, there can be a lot of different things and hence it is quite important that we make these sort of distinctions, and there's also papers about their definitions and, and applications just in the general context of, mostly human health or, or, or human medicine. But sort of as a little, I guess bottom line or or take home from the definition side of things, it is basically anything that can be measured as either an indicator of a normal process, biological that is a pathogenic process or a response to an exposure or intervention, and I think that's why. You know, in their discovery and utilisation, they can be very variable and it can have something to do if you wanted to translate that into a medical terms with diagnosing something, prognosticating something or predicting something, or, you know, sort of.
Utilising for follow-ups for the relapses for it, it honestly can be pretty much anything and obviously also the substrate itself is not defined, so it can be any biological substance fluid tissue that you can, you can potentially think of and based on these . Papers or these sort of consensus statements if you like, especially the one I just told you here, the best. I've tried to make a little visual summary of the most common areas of where in human medicine, and hence probably also in veterinary medicine going forward biomarkers are.
Used and as I just said, some of them have to do with, you know, assessing risk or susceptibility to specific conditions, some are used as diagnostic or monitoring tools, or maybe responses to particular treatments, prognosis I've already mentioned some. Maybe also markers of an intervention, safety or drug toxicity. They can be surrogates for other biological processes, and, and some of them can even be digital, although I, I'm not planning on going into this much because I am.
Not the right person to talk about all the advantages and applications of AI for, for example, so, so that, you know, I'm, I'm going to stick to the more traditional sense of, of the ease. And what we luckily have is already a few papers that are specific to the use of biomarkers in canine chronic enteropathy. This is one, from Romi Halman from, a few years ago, and I think again, maybe mirroring, what we've just seen, you know, these sort of different categories of biomarkers, and then some of the things that maybe mean a little bit more to us in the sort of practical sense sort of allocated to these, to these groups.
And again, as you can see, it's sometimes more about, you know, where it is measured, is it a micro, is it a biochemical variable or has it something to do with the a microbiome or is it a genetic marker, but sometimes also, you know, has it something to do with a specific function or metabolic pathway that we, we are, you know, looking into. Or, you know, are, are they sort of, surrogates for specific cells that we might, be interested in. So that's a sort of broad, you know, overview, and this is obviously changing and evolving all the time, and I've just put these little stars next to the few that I've picked, and that I think are currently of, of interest and maybe more importantly also available to us because a lot of these.
Tests or biomarkers will, will have been established or assessed in a research setting but are actually really hard to, to replicate or might not be available from, commercial laboratories and, and that's why I made, you know, a little, selection of, of, of those things here. And so, you know, in no particular order, simply maybe because it is the one that is the first one on, on this little biomarker wheel here, I would want to start with looking at the dysbiosis index. Now, Some of you might be very familiar with this, and some of you might not have heard of, the dysbiosis index before, just about how it, a little bit about how it came to be.
So that is, a publication from, the Texas A&M, GI lab, mainly, driven by Jan Sokodolski, who, I think it's probably safe to say is the sort of veterinary. Expert in anything that has to do with the microbiome, specifically in dogs, but also cats, And they have this, his group has published this paper and basically what the intention was here is to saying that microbiome analysis in the more classical sense as in sequencing, or, or, you know, metagenomics is complex, very expensive, and you need a lot of, you know, bioinformatic expertise to even interpret what is going on. And so what they basically wanted was a sort of simpler tool that would be easier, and I guess also in a way cheaper to run, but also to interpret that could well mark differentiate between dogs that have Chronic enteropathy and healthy dogs, and this is what you can see here on the right, you can always see the healthy dogs and the chronic enteropathy dogs sort of side by side, and basically what they've done is they run an awful lot of, individual quantitative PCRs on faecal samples, For specific bacteria, and had a very complicated algorithm to then basically identify a number of them, and you can see the names here and these are obviously the same as, you can see on in these panels that are in their combination, most likely to differentiate, healthy from chronic.
Enteropathy, and you could see that, you know, for example, the total bacterial count, is not particularly different, but then when you look at some of these, you can see that for example things like, fecali bacterium and, posterium hiranonis, actually now called Pep Acetobacter hiranonnis are for example. On average, lowered, so decrease in chronic enteropathy and some, some others, for example, streptococcus and E. Coli are increased.
And then once again they put that into a complicated formula and that spits out an overall dysbiosis index and as you can see, That was basically the first step of establishing a reference range. So in healthy dogs, this dysbiosis index tends to be in the negative, you know, values versus anything that is sort of, you know, zero or in the positive, seems to be indicating dysbiosis based on this index and, is, consistent with, chronic, enteropathy. And, and this is, available as a, as a diagnostic test, so you can either send it, directly to Texas A&M or you can send it to, you know, IDE as, the, the commercial lab that, that offers this test, and if you do, then this is often, the sort of output that you get, so you get the overall, number, and as long as you remember that it should be, in the negative, then you have a, a good idea as to, what is normal, so this would be considered normal here, and then you get these, different bacterial species here and you can, you also get a sort of rough reference interval if that's what you would like to call it, so you can assess even maybe if the value here is OK, whether one of them is maybe not quite as abundant as you would like, or the other way around, if this is abnormal, let's say it would be +5, then you can see which of these bacteria, drives, this particular dysbiosis, and you might think, well, I don't know what any of this means, but what we're gonna talk about in just a moment is, mostly, you know, regarding clostridium or pep Acetobacter, hiranonis, what sort of functional consequences that had and maybe potentially what that could also mean for, for treatment, of dogs with chronic enteropathy, and you will get a, you know, a spiel of interpretation.
With that, as well, that'll help you, remember these, these things, and they have, you know, shown that this is a relatively good, biomarker, if you like, because that's what we're talking about, for distinguishing between healthy and chronic enteropathy, but I also would like to, you know, point out to you that not every dog with chronic enteropathy has, an abnormal dysbiosis index, and this is something that you will see again and again. None of these biomarkers will separate, you know, healthy from disease perfectly, and so we cannot use them as an adjunctive. Diagnostic too, just because your desbiosis index is normal doesn't mean that this dog doesn't have chronic enteropathy, and to me, this probably means that, this is an umbrella term for maybe a few different, phenotypes of the disease that we haven't really characterised molecularly very well, and maybe the dogs down here are slightly different to the dogs that are in the grey zone and are yet again different from the dogs that are very dysbiotic, but we just don't know yet what that is or whether you have to take a few of these different biomarkers and combine them all to sort of subclassify these dogs, better.
The other thing that I want to briefly mention that the dysbiosis index can be useful for if you are considering or maybe already performing, sort of maybe more novel treatments for chronic enteropathy like faecal microbiota transplants, because what is also interesting, and you see a little bit of this here, but even more in this follow-up study with 116. Sort of clinically and, his, you know, from the history, healthy dogs, that there is a small percentage of supposedly healthy dogs that are not healthy from the microbiome perspective, and not only do they have abnormal dysbiosis index, but they also have significantly reduced numbers of these bacteria that are, you know, associated with, with health, so it might be also good for screening for donors for faecal microbitter transplantation, etc. So now let's talk a little bit more about what this actually means functionally when we sort of remain within the, the microbiota, and this is an important mechanism or yeah, I guess functional consequence that has recently.
On the back of, these microbiota analysis come to light, that not only does it mean that these bacteria are shifted, but also that there is an abnormal function of the microbiome as well, because you could say, you know, does it really matter what these bacteria are are called, as long as they do their job, and maybe there's a lot of likely, there's a lot of redundant. In the microbiome as well. Well, you know, if you look at one of the functional markers, which is, faecal bile acids, so that's different from the bile acids obviously that we measure in the, in the blood, right?
So that is the bile acids that the bacteria in the large intestine make from the 5% of bile acids that do not get recycled in the . Enterrepatic, circulation, and, bear with me here, it'll become clearer hopefully in a second, but I think what you can see here is that healthy dogs, have a very high percentage of so-called secondary bile acids versus if you look at dogs with chronic. There seems to be nearly like a bit of a split.
There is a population here that looks very similar to healthy dogs. There's a few that are sort of little stragglers here in the middle, but there is a proportion of dogs with chronic enteropathy that seem to have hardly any, secondary bile acids. What does that actually mean or why would we care about this, and that is, the, the reason we care about this is from what we know about consequences of bile acid disturbances in human inflammatory bowel disease.
So let's start looking at these sort of healthy subjects. So normally what happens, as you obviously know, liver or . You know, biliary epithelium, secretes bile, and that ends up where, well, in via the duodenal papilla in the small intestine, and then what happens is, most of it, as I just said, gets recycled and taken up in the ileum again.
But a small percentage makes it into the large intestine where the bacteria here will change, most of these primary bile acids, and sulfated bile acids into secondary bile acids, and the, sort of biggest sort of, I guess, chemical, difference between secondary and the other acids is that these are less inflammatory, they're less irritating to the gut epithelium and to the, to the mucosa, OK, so they have even been shown to reduce inflammatory signalling, and also have a sort of inhibitory effect on some, pathogenic bacteria as well. And what we know in IBD in people, because there's dysbiosis, there's less of the normal microbiota that can actually make these secondary bile acids. So what we have is, you know, a slightly, lower percentage of secondary bile acids, but most importantly, you know, an increase of the unmetabolized, if you like, primary and sulfated.
Bile acids, and they cause irritation and inflammation of the gut epithelium, and that creates this inflammatory loop. So in a way, not only is dysbiosis, especially if it results in bile acid disturbances, a consequence of IBD, it also perpetuates the inflammation that goes on, in the gut. And here is where it becomes interesting in terms of who is actually responsible for this.
Well, the major bile acid converter that has been identified in people, but also subsequently in dogs and in cats is our friend, used to be called Clostridium hiranonnis, has been renamed as Pept Acetobacter hiranonnis. So what I just showed you a couple of slides before, the lack of, this particular bacterium directly translates into dysfunctional, impairment, so this is really, really important. And in fact what I've just learned at ECVIM, congress that Anthony was talking about is that there are now already some of my colleagues, you know, gastroentero veterinary gastroenterologists that are actually looking into, using Pep Acetobacter here are known as, as a probiotic and basically manufacturing it to give it back to, to these animals, and obviously that would be very exciting, but we're not quite there yet.
So what else, sort of do we know? When we look at the dysbiosis index and it's sort of correlation with, Hiranonnis here, well, you know, that our good old friend metronidazole, and I don't know if, if you know this, but me and a lot of colleagues, that are, you know, working in the gastroenterology field in small animals, we are quite passionate about the fact that we should no longer use metronidazole, or in fact, a lot of the other antibiotics that we. To so readily, you know, give to animals with suspected chronic enteropathy because it does maybe make them better clinically, but we also know that as soon as we stop the antibiotics, the clinical signs will come back.
But what we have now shown here based on the dysbiosis index and the amount of Clostridium here are known, is that when you give healthy animals metronidazole, you make them incredibly dysbiotic and you completely deplete them. Of, of her unknownness, so there's again this direct correlation, but what is even more important is what is here in this little circle is that look at after, you know, a total of, 12 weeks, most of them. You know, the animals, you know, are still.
sorry, here in the dysbiosis index, are still or again normal, but there are some that are now positive in the abnormal, number of the dysbiosis index, and that correlates with the continuous reduction or depletion of clostridium hiranonis. So the hiranonis itself could also serve, I guess, as a, as a marker, and it is obviously part of the dysbiosis index, so there is a strong, correlation between, between that. And just to show you a little bit more about this, if you do consider or perform faecal microbiota transplants, these were dogs that were given.
Thylazin, and you can see how that dysbiosis index shoots up with thylazin, so it's not only metronidazole and the clostridium herononis goes down, and then they, were given, an, an FMT here and you can see how these things, you know, really markedly normalised, and I mean, I'm not gonna go into the details, but there's lots and lots of publications by now that have shown these significant correlations between the secondary bielastic production and. The closterium here are known as numbers, so we really think that this is, one of the bugs, one of the major bio acid converters, and we really want to, you know, keep it in the gut because it is important for, for health. So that was a little bit here about the, the microbiome as a sort of marker of health or, or disease, but we're gonna have to for time reasons move on to look at some of the other, you know, values or, or biomarkers that are available.
I'm gonna very briefly talk about C-reactive protein or CRP, which is a very old marker, especially in the context also of, of gut disease. I do remember that there was an article, a long, long time ago in, In a veterinary newspaper that said, wow, this has now revolutionised . Diagnosing inflammatory bowel disease as it was as it was then called in dogs, because if you've got a dog with chronic diarrhoea, you simply measure its CRP and if it's high, you have made the diagnosis of inflammatory bowel disease.
And that is obviously, you know, not only a simplification, but simply untrue, and I think we all know, that CRP is a fantastic inflammatory marker, but also that nearly anything anywhere in the body. I always say to my students, you probably can have an ingrown toenail and you probably have a slightly elevated. CRP and the same is true for, for our dogs, so it is by no means particularly specific.
So again, like it is with all of those, or a lot of those biomarkers, they are not a substitute for making a diagnosis the traditional way. They may be able, like from this study to help you differentiate. Whether your animal falls into a specific subcategory and how they respond to treatment.
So for example, on average, the value of CRP was higher in dogs that needed, steroids or immunosuppression to respond, and get better from their chronic enteropathy as opposed to dogs with food responsive or antibiotic responsive enteropathy. And again, if they were complete responders, you know, versus partial or non-responders, you can see that on average, which is that line here in, in, in the middle, you know, there there might be a difference, but you can also see that the spread and that there are lots of animals that are overlapping with the other categories, so it is a statistics game which is hard if you want to apply this to an individual animal. An interesting, molecule, and you're gonna hear a little bit more about this, also from Lucy is, calprotectin that has been, around for a while, and if you know a little bit about human inflammatory bowel disease, then you know that this is the inflammatory marker, both in faeces and in serum that is used, quite wide, widely, as part of sort of, disease activity staging or recognising if there are flare-ups of IBD.
So it's basically a molecule that gets mostly made from neutrophils and from neutrophils in the gut, and if there's more inflammation, then you've got more of that in your faeces, and it does also work in serum, although maybe not so well in, in dogs and cats compared to, to humans. And here you can see some of the earlier studies where, faecal calprotectin was looked at and again very similar to the CRP you can see that there's a difference between severities I guess or respond types of response as well as complete versus partial or no response in the sort of level of faecal calprotectin. And in this particular study they identified a cutoff of about 15.2.
That separated these two, these two groups, and the sensitivity and specificity is somewhere around 75 to 80%. That sounds high, but we can talk about that later if you like, whether that is, you know, acceptable in, in, in this sort of context. I'll also want to point this out because there is maybe a problem, with these sort of low, values here, and I'll come, come on to that, in, in just a second.
Now my own sort of journey about with fickle carrotectin basically started because I had a student project that you know, we were trying to use a sort of commercially available bedside test for, faecal cyprotectin, and, we very quickly, figured out that this didn't work because the cutoff was so high, it would only become positive with these very high values, and you've just seen that a lot of dogs will be much, much lower, for, for that, so we then moved on to a more, I guess traditional sort of large, wet chemistry. Lab way of, of measuring, faecal carrotectin, in, in dogs, and you're gonna, gonna hear a little bit more about maybe one of the more, you know, veterinary appropriate, bedside test for, for that, and just to, you know, to let you know, I have, you know, have tested, or used, the GI quest for, in, in a few animals, and, I have done a correlation with side by side measuring with the sort of wet. Lab tests, as you can see, very few data points, but it's always a start, and there seems to be, a relatively good, correlation, which is, which is nice to see, but obviously you, you would need much more data points to be sure that that, that statement holds up.
Looking at faecal carprotectin, with this wet lab test, which is, the, the Bullmann, test, and we have now established that in our lab here, so I can use that. And, for clinical cases, we all can use that if we want to, and there's an interesting sort of observation here, so, I'm just gonna show you healthy controls, dogs with chronic enteropathy, dogs with protein losing enteropathy, and dogs with gastrointestinal neoplasia, because I thought, you know, it's always nice to compare animals with, you know, that have a specific disease with healthy animals, but you really want to know how these tests perform with, with what I would consider. Chronic enteropathy mimics, right?
They look the same, but they might be different and you might want to treat them differently. And as you can see here, there is, Quite a bit of significant increase, particularly in the PLE and the GI neoplasia, group, but there's no statistical difference, in our little pilot study here, between, enteropathy and, and healthy controls, and you can take that a little bit further by including, potentially even more, GI things like acute hemorrhagic diarrhoea syndrome or colitis, and interestingly, that was, you know, the, the one that was the highest of all of these groups, and I also even included some dogs that had, some level of diarrhoea, but not a diagnosis associated with the. In the end, for example, dogs with sepsis or dogs with pyrexia of unknown origin, as you can see, some of them have very, very high values, and I think for me, while this still needs a lot of, you know, verification and, and higher numbers, because you can see in some of these categories there's only like maybe 2 or 3 or 4 dogs.
For me, this is important because it tells me that this is a marker of gut inflammation, not a marker of chronic enteropathy, and that there's lots of other diseases in the gut that can cause elevated values. Moving on to some other inflammatory biomarkers that you might have heard of, and I'm just going to briefly glance over them because they are not commercially available, faecal IgA as such, you know, there's some evidence that maybe German shepherd dogs have a decrease in their IGA production, and that's why they are more prone to both infectious but also other inflammatory diseases, but you know, it's, it's very, there, there's not an awful lot of studies, going on about that. And then other, working groups have tried to look at, more specific markers, as a proxy for specific inflammatory cells in the gut like eosinophilic infiltration or even mast cell, correlation, which unfortunately, you know, it's very preliminary and we don't have enough data to, to, to recommend them, so really more, more studies would, be needed for those.
What about autoimmunity, markers? You might have heard about, Pianca, which is, perinuclear anti-neutrophilic cytoplasmic antibodies, quite the mouthful, and that is a very interesting marker, and several studies available, particularly associated with protein losing disease, both enteropathy and nephropathy in soft coated wheat terriers like these little chaps here, because it is a, Interesting biomarker in the sense that it is elevated about 2 years before you can see that these dogs have actually decreases in their serum albumin, so it could be a massive help for screening and maybe breeding out these types of diseases because it is such an early marker. It's very, very hard to run this test because you need, to have live neutrophils from the, patient to do this, and that's probably why there isn't a commercial lab that I'm aware of that is currently offering that, which limits its use in, in our diagnostic sort of, you know, everyday routine.
There's a relatively recent study that has looked at other autoimmunity markers, and I do not pretend that I fully understand why they have chosen these particular markers and what that really actually means from a sort of mechanistical standpoint, but they looked at things like, IGA again in total, anti-flagellin, which is a bacterial protein, anti, polymorph nuclear leukocyte, anti-gliadin, which is, you know, also relevant with the, gluten hypersensitivity or, or, in people, etc. And you can see here you've got IBD group, non-IBD group, and normal group. Again, a lot of these markers seem to be elevated, but it's the same picture that I mentioned to you before, there's always a group of IBD dogs, that are very similar to the, to the control groups, and again, so we need to, you know, be able to use this in the context of phenotyping these animals a bit better.
And they, they conclude, they conclude that, you know, a vari variable percentage of dogs with IBD show, you know, these sort of autoimmunity markers, and they suggest that some of them maybe even in combination could be helpful in in diagnosing these diseases. OK, moving on to some maybe of the more familiar markers, if you, if you like, particularly looking at the functional biomarkers, and I'm not gonna talk about all of them in, in, in, you know, vast amount of, of detail, particularly permeability. And absorptive function tests are really difficult and again, even in a research setting, you know, very cumbersome to do, but we have some other things available like faecal alpha-1 proteinase inhibitor, cobalamin, methylmalonic acid, and folate, and we can talk about those briefly.
So, faecal alpha-1, proteinase inhibitor was for a long time actually available to be measured in the faeces, at the Texas A&M lab as well, but it's, for a while not available, because of one of the reagents being no longer manufactured, and I don't think that that has, changed recently, which is a, which is a real shame because. Kind of similar to the Pianca, it is, in a way an, a marker of early gastrointestinal protein loss is very sensitive, and, you know, it, it, it has a sort of, important role in maybe trying to detect protein losing enteropathy, early, but because it isn't available, you know, we, we can't really say much about it at the moment. Then the sort of, you know, long standing conundrum or, .
I guess problem with folate and cobalamin. This is what you find in the textbooks. This is literally directly taken from, from Ettinger, that if you combine the measurements of serum folate and serum cobalamin, you can actually distinguish between malabsorption and sort of dysbiosis or what was in the old texts called small intestinal bacterial overgrowth, and the idea behind that was that if you have true intestinal damage, whether that's inflammation or maybe even you know, neoplastic infiltration, that you can't absorb folate, you can't absorb cobalamin, so both will be low in your serum, and opposed to this, if you, if, if your intestine is generally fine, but you have some sort of shift in your microbiota or overgrowth, you might have excessive, excessive production of folate by bacteria, while they like to use and bind the cobalamin for themselves and their own metabolism, so you would have this, You know, slightly different pattern where the serum folate would be high and the serum comalment would be low, and that has been advocated as a marker of dysbiosis.
Now, I didn't quite believe that because I think from a clinical perspective, I particularly with folate, it seems to do whatever it wants in, in these sort of conditions. So, one of our former interns now also boarded an internal medicine specialist, did a study while he was with us where. He looked at all the folates that were ever measured in our hospital over a certain period of time and were grouped into chronic enteropathy, other GI diseases, and non-GI diseases, and as you can see, there's absolutely no difference in the average values across those groups, as also evidenced by this P value.
And then we thought, well, OK, but what about if we look at only the chronic enteropathy dogs in their sort of subgroups, food responsive, antibiotic responses, steroid responsive, PLE, and yet again, as you can see, there's no difference and there's no statistical, significance here either. So really, folate does indeed what it, what it wants, and this was subsequently also. Shown by other working groups, and this mainly by Stan Marks in the in the US and they also wanted to look at folate concentrations as a marker of chronic, as a biomarker of chronic enteropathy, and their conclusion is that having low folate is inferior as a biomarker compared to having low cobalamin, and so we really don't really care so much, I think, about what folate does.
Cobalamin, however, in itself is a very important marker. The prevalence is very variable if you look at the different pieces of literature. It has been associated with a negative prognosis, so it is even a prognostic biomarker if you like, and approximately 50% of animals with low serum cobalamin will also have metabolic consequences, and what that mainly means is, either a low methylmalonic acid, or, .
Oh, it's not, it's on the next slide, it's just fallen out of my head, but in any case, again, you can see, you know, that, there is a percentage of dogs, again, not all of them, that will have this as a helpful sort of biomarker of, of malabsorption or loss, really. You can measure urine methylmalonic acid in different labs, for example, you know, abroad, there's also some, I believe, in the UK that offer it. It's frozen urine, and it's also always a bit cumbersome to be sent.
And then, you know, you have to ask for a sort of organic acid screening. Sorry, this is in German because it is a German lab, but here it actually says, significantly increased urinary excretion of methylmalonic acid in this particular, and that that is a sign of cobalamin malabsorption. I think you can measure serum, methylmalonic acid, also at the Texas, A&M, lab, and, here they tell you what, they, they need, need.
It also needs to be frozen, which can also be a similar, challenge, and here it is the other, you know, marker, of, cobalamin deficiency could be, plasma homocysteine. And of course if you have, congenital, cobalamin deficiency, like Muslimsbeck syndrome, there's a genetic test for that as available as well. So this is from Romi Halman's paper.
This is just, I think, and I'm not gonna go into detail here, but a very nice summary of a lot of those and some more biomarkers, and I think what is important for us is to look at the sensitivity and specificity. And you will see that, you know, in a lot of cases it isn't very good, so the sensitivity particularly, but in, in some of them, even the also the specificity is, is really, really poor. So it's much more likely than having one biomarker that we're going to probably move forward to, you know, having to look at a panel and subclassify dogs according to that, which at this point in time, we don't quite know yet how to do and how that translates into, maybe differences in their treatment.
Moving on to some of the sort of maybe newer molecules, I don't know if you've already heard about microRNAs, these are small noncoding RNAs, and you know, they, they have been found, you know, to be increased in a lot of diseases, both in people and and animals, but this one is the one that is most consistent in human IBD, and this has also been on a research level. Investigated in dogs and so maybe this is something for the future that we're going to get panels of these microRNAs that we can either measure in serum or in in faeces, and that might also help us to understand disease pathogenesis a bit better. I'm not going to go into detail here, but this is from this paper and you can see again, you know, there is overlap, but some of them are better than others in differentiating healthy versus disease.
And what is also interesting subsequently, that other studies have shown that might also be able to actually distinguish gastrointestinal cancer from chronic enteropathy, and particularly if you look here at the top panel, you can see that some of them are very high in cancer and sort of moderate to similar in healthy controls versus chronic enteropathy. So that might be a very interesting application for those going forward. And what I wanted to show you at the end, is simply, the things that we have been working on, and that comes from, some work that has been going on here in Edinburgh, in the, in the human, hospital dealing with Crohn's disease, patients, the Institute for Regeneration and Repair, and they have developed, an essay called IBD Sense, which is basically, an essay that looks at a different molecule, .
Involved in in inflammation in the faeces and it looks at actually its activity not only abundance, and there is a little bit of this of information on this on, on their website, so it's a fluorescence based technology and they think it is better than faecal carprotectin in people to actually look for disease and inflammation markers and relapse and disease activity in in IBD. And so that molecule is called, what they're looking at, is activity of gran enzyme B. It is, a, a protease that is expressed in lots of immune cells, and what is different in that compared to the calprotectin, for example, is that it isn't from neutrophils, it is actually from lymphocytes, which I think makes it very attractive, for dogs, particularly because we know that the most common.
Inflammatory cells that we see in, in, Crohn's in chronic enteropathy is actually lymphocytes, and plasma cells, and we have a larger project ongoing with that, is a residency project and where we have presented some preliminary data as a, as a, as a poster just at, the aforementioned ECVIM congress, a couple of weeks, ago. It is a said, fluorescent, essay that measures actually activity, so gran granzyme B. Activity in the from the faecal sample, and this is the first data that we had from from dogs where we used, you know, a handful of dogs with chronic enteropathy and one or two healthy controls, and we saw a massive signal in these dogs with disease and hardly anything in healthy dogs, so we got very excited about this, and since then we have been able to measure this in .
A few more dogs and again we've got healthy controls, chronic enteropathy and gastrointestinal neoplasia, and you can see this lovely sort of nearly sort of disease categorization here, the highest in neoplasia, second highest in enteropathy, and sort of lowest, and you can see this similar level of overlap here which is also interesting to see. So we would have to define. A cut off which we haven't yet done.
And what is also interesting if you look at the subgroups of chronic enteropathy here, that it does seem to be the highest in the food responsive group, which is not necessarily something that we would have expected, and then it goes a little bit down in immune responsive and it is even a little bit lower in protein losing enteropathy. So we're currently investigating this. We need more numbers in this, and we also want to compare it to faecal carrotected because in, in that we seem to see an opposite trend.
So we're even considering whether, you know, there's maybe a ratio, granzyme B to carprotectin ratio or the other way around where, that could really help to separate out these different disease, groups or, or subcategories. So that was our little whistle stop tour through biomarkers in the context of chronic enteropathy in, in dogs. As you can see, a lot of things have happened, you know, in, in this field in recent years.
Some of them are more familiar to us, than others and are available as commercial essays and have sort of real life applications, but there's lots more that have promised, and might be available. Some point for us to use in in everyday diagnostics and their application is as variable as we would expect from the sort of broad category of of biomarkers and obviously, you know, there's lots of people also working on untargeted sort of omics technologies that probably will throw up even more discoveries. So this is a really exciting time to, to look into this topic.
So this brings me to the end of my talk. Thank you very much for your, attention and my details are here. So if you also want to contact me later, my email address is here at the bottom for you, and, with this, I am very happy to take your questions and later handing back to Anthony.
Thank you. Ah thank you so much Silka, that was excellent, we've got a couple of questions, but we'll leave them till after Lucy's presentation, so if you're listening in from exotic places. Like Edinburgh, it's always good to know where people are listening in from, so do pop that into the chat and then any questions put into the question and answer, and we will come to those at the end of Lucy's presentation, as I say, Lucy works with Cariss Animal Health who kindly sponsored and and partnered with us on this webinar, and Carys also were very, very kind to get involved in the veterinary green discussion forum with partnership which allowed, some university people come to our veterinary green discussion forum in June, so thank you all so much for that, Lucy, and, I'll leave it over to you to explain about the new test.
Perfect, thank you so much and thank you to Sia for such an interesting presentation. I'm just gonna take a few minutes to tell you about our new faecal cowprotectin test that Sia kindly mentioned called GI Quest. As Silca has already mentioned, just a brief overview, faecal calprotectin is a sensitive and non-invasive biomarker of gastrointestinal inflammation.
As Sila said, it's found primarily in the cytosol of neutrophils, and so when they disintegrate, the calprotectin is released into the GI tract, hence why it corresponds so well with GI inflammation. It's stable in faeces and resistant to bacterial breakdown, so it's reliably found in the stool, and it's in the NICE guidelines for humans for IBD and is being increasingly used in veterinary medicine as well. As faecal calprotectin is so closely correlated with inflammation of the GI tract, we see an increase in it with many diseases, such as chronic inflammatory enteropathies in both dogs and cats, food responsive enteropathies, long-term NSAIDD use, dysbiosis, and many other diseases as well.
So why would we use GI Quest? GI Quest is an in-house lateral flow test, so it allows you to have the results in 15 minutes. It's stress-free for patients, non-invasive, and it can help guide clinical decision making, monitor treatment response, and it also helps with client communication because of its semi-quantitative nature.
It has a 25 pounds list price to a vet practise, so pricing to owners even at 100% or 120% markup would only be 50 pounds or 60 pounds. Obviously this does very much depend on your practise and practise markups, but it's usually really cost effective. GI Quest underwent clinical validation study with the University of Bristol, where it was found to be highly effective in differentiating dogs and cats with inflammatory enteropathy from healthy controls, as well as correlate with the clinical improvement in dogs with food responsive enteropathies in a diet trial.
And also detect NSAID induced gastrointestinal side effects even in asymptomatic animals. It has a high sensitivity and specificity, making it a reliable test for GI inflammation. If you want to see all of the data and graphs, you can find them on our website or email us and we can send you all the information.
GI quest can be used in practise in lots of different ways. You only need a really limited sample size, and it's non-invasive and cost effective, so it can be utilised in multiple points of the diagnostic journey. Some practises, have said they already like to run the test prior to a kind of expected chronic GI appointment, so they already have the data going into the.
Consult with the owners, and due to the small amount that is needed for the sample, some are even using the stool from gloves they've performed a rectal examination with, so the owner doesn't have to come back in. So lots of different ways to use it. You don't need temperature control and it's stable for at least 3 days, so if it's been left on the side for a day or 2 before being brought into the practise, it's not a problem.
As I said, it's also really effective at supporting owner compliance and understanding for both the diagnos diagnostic stage, but also the monitoring of the treatment. I've just put together a couple of clinical scenarios of where GI Quest is currently being used or could be used in practise. Of course it makes sense, we would use it for cases with chronic GI signs to help inform the next steps.
As well as if owners decline diagnostics, whether it's due to their worry about invasiveness for things like biopsies or financial barriers, it can then provide the objective data that can help guide our empirical therapies. As I've said, it can be used to monitor treatment or diet trials and monitor the GI side effects in NSAID use. It's also, as I said, a really useful tool in helping client communication, it takes something that, Is generally quite hard to quantify and puts it into numbers for them.
So if we're saying to an owner, we started at a 6, we've started treatment, and we're now at a 2, it's really clear to them that the treatment has been working effectively. GI Quest can also have a really strong place in contextualised care. It can be used in a really pragmatic way to help overcome owner hesitation about even starting a diagnostic process, and it can help if they have limited finances and or are worried about invasive testing.
As I said, it can help the communication challenges with many different stages of the diagnostic process, and it can also help owners who are concerned, for example, those animals that have had GI flare-ups before, and they're particularly vigilant and they say that they're, Animal just doesn't seem themselves, it can be a really good way to have a quick check if there's any sign of inflammation, quickly and non-invasively. Just to show you how simple the test is to run, it's very similar to the COVID tests we all became so familiar with. The whole procedure is contained, so there's, you know, cleanliness in the lab or prep room.
You just take the stick out of the tube, insert the end into the stool a few times, replace it and screw the lid back on. You then shake it for a couple of minutes so it mixes with the reagent. Take off the red part at the bottom, discard the first drop, wipe the bottom, and then pop two drops into the cassette, again, exactly like we did with the COVID test, and you just leave it at room temperature for 15 minutes, and then you can read the scorecard.
Reading the test is also super simple, you have the control line and the test line in the cassette, and you use the scorecard in the box which has lines in a colour gradient, and you just read across the, the number on the line that colour matches the one on the cassette. In dogs, a scorecard reading of 3 or above is positive for GI inflammation, with increasing number being increasing. Amount of inflammation, and 2 is borderline, so very much depending on the clinical scenario, we'd often recommend retesting in 2 weeks.
And then with cats, exactly the same process, but the numbers are just slightly different, with 1 being borderline and 2 and above being positive for inflammation. So in summary, GI Quest is a rapid in-house test for GI inflammation. It's non-invasive, cost effective, and it can really help aid clinical workflow.
It can be used for both the diagnosis and monitoring of various diseases and treatments, and it can be used at different points of the diagnostic process. It's validated by the University of Bristol and has a high sensitivity and specificity. It's stocked at the main wholesalers, so NVS, MWI and Coviatris, and if you want any more information or as I said, any of the graphs or data, just pop onto our website or contact us via email and we can send it all over to you.
Thank you so much. Thank you so much, Lucy, just while we're getting those couple of questions in. We've got somebody listening in from Cambridge.
From Canada, Iraq. New Delhi in India, Portugal, Switzerland, Netherlands, Cyprus. So people listening in, er even from exotic born er Portsmouth, Brazil, Romania, Italy.
Portugal and India again, so great, and Argentina, so great hearing people listening from all over, and. Remain is asking will the presentation be available as a PDF. It's actually recorded Remains, so this will be available within about 24 hours on the site, you'll be able to look at your leisure.
Also, Bianca listening in from Mexico, so great seeing everybody listening all over, but my understanding, Lucy, sadly is that the test is just available in the UK at the moment, but are there plans to take it, into a more international audience? Yes, certainly plans, as you said, at the moment it's unfortunately only in the UK but we're certainly looking to expand, so watch this space. Brilliant, I'm.
Silka, there are a few questions if I can er go through those with you. How can new biomarkers help CE? Can early diagnosis help support a specific treatment or drug?
That's from an anonymous attendee. Yeah, so, I, I think it is obviously a very broad question, you know, if you think about the different variety and different types of biomarkers that we've, we've just, Just talks about, but I think the, the one of, if I sort of paraphrase this into which one of those are most clinically useful, . I will say a lot of them at the moment from my perspective have a reasonably limited use, and I reserve them for cases that either there's emerging evidence that it might have an, have an influence on treatment.
One example would be a recent study on the dysbiosis index that seems to show that. Weirdly and slightly counterintuitive that dogs with chronic enteropathy that have a reasonably normal dysbiosis index respond to faecal microbiota transplantation better, clinically speaking, than the ones that have very abnormal indices. I think we have to be careful in taking, Take that with a pinch of salt because, you know, it might be that they just need a different regime, they might need it more frequently, or, you know, it's always a matter of whether the donor's good for one dog and maybe not so good for for the others.
This is all very new and we don't know enough about this, but. Sometimes I also use some of those, including the dysbiosis index, for similar reasons it was what Lucy mentioned, if an owner is simply not convinced that there's anything wrong, and you're just like, here is a number, and that number shows, and you know, no, we don't want to give the 5th course of metronidazole because look what metronidazole has already done to your dog's gut, and it's all very horrible, and, you know, I know that. But sometimes people need to see a visual or numerical representation of, of, of that.
So sometimes a little bit of psychology comes in that as well, and you, as you have seen with the examples for some of the others like the studies, about faecal car protectant, even though the cutoffs were obviously quite different to maybe what other faecal calprotecting protecting test might do is that you might not be able to establish. A diagnosis, but you might have an inkling that maybe this dog needs more aggressive treatment because it's a very high value, or maybe this dog might be more likely to be only partial responder, or if you're very unlucky, even a non-responder, so it might, again, I'm, I'm very hesitant applying population. Medicine to individuals.
I think that's a problem in both humans and, and dogs, but if the owner wants to have, is there anything that we can have to predict? Is my dog likely or unlikely to respond to A, B, or, or C, then maybe some of those markers. Are useful and I know for example the Pianca test has been used by at least in the UK by the by the breeding clubs for soft coated wheat and terriers to try and identify dogs that might go on to develop protein losing disease and to exclude them from.
The breeding programmes as well early on, so that it has a sort of more breed health, implication, there as, as well, so I don't want to drag this out too much, but there are certain applications, but some of them are quite specific, I would say. Yeah. Obviously there were some photographs there of cats within presentations, but my understanding is the test at the moment is mainly limited.
To dogs, but is there a level, again another anonymous question, er, it's always the great thing about webinars, you don't have to stand up and be brave and ask a question when everybody's looking at you, there's never such a thing as a stupid question, but the, the person saying, is there a level for cats that may be suggestive of something more sinister than inflammatory enteropathy, and I'm presuming talking about er potential neoplasias and so on. But I think still could you want to answer that. A lot of, I, I see, you know, a, a lot of these questions, in the, in the question and answer box here, and a lot of them are about further differentiating subcategories of disease or linearity testing.
What does it mean if it goes down, or I mean in my head, what does it mean if it doesn't go down? I think it's, it's, it's a more possibly more difficult to answer question. And I think the simple answer for a lot of these things is we don't know at this stage.
We don't have large studies that have serial measurements, and we don't know if even we need these values to go down, right? If we think of the histological findings, we have quite a number of studies that have before and after treatment biopsies in dogs with chronic enteropathy. And yes, mostly these times are relatively short, maybe 6 weeks or 12 weeks after start of treatment, but the inflammation is still there, even though the animals are clinically better, and that seems.
To be a repetitive theme over and over again, and we don't have any studies that I'm aware of that are really longitudinal as in years after the initial diagnosis, so we don't know what to expect should the inflammation be away after 6 months, after, after a year, is it ever gonna go away, or is it more important that we actually look at the animal and look at, you know, the clinical signs and as long as they are controlled, do we, do we care what these values are? Are, are doing, I, I don't think we have studies that will, can answer that at this point. And I suppose what they are is they help with our diagnosis, but often you would go on to do more invasive tests.
Like . You know, sort of er endo endoscopy and so on to, to try and get to that definitive diagnosis, wouldn't you? Yeah, I, I think, I think there's a yes and a no in, in, in that particular question because, I think we have to be very careful to use a single value that is a marker of information to make a very specific diagnosis and I've shown you some of the data that I have in, you know, that we have done internally where pretty much.
Diarrhoea causes certain values to go up, and I mean that's a very expensive way of confirming what you already know and your dog has diarrhoea, so you could be very cynical about this and go like, well, you know, is this really, is this really helpful at all, but I think we are managing more and more of these chronic cases without biopsies and without, doing invasive tests for, for several reasons, maybe because, you know, biopsies are not. That helpful unless they show cancer, and so we don't really care, is this lymphoplasma cytic or is it eosinophilic, is it mild, moderate, or severe? Is it only in the duodenum and also in the, in the ileum none of this for the clinical treatment plan that you come up with really matters, and I think we are more.
Inclined to take biopsies when there is really significant evidence of malabsorption like a hypercoalabinemia or low albumin, then we go like, oh, I really rather rule out cancer sooner rather than than later, but it, it is no longer part, you know, of the routine, we take biopsies in every, in every animal, so a lot of these chronic enteropathy dogs can be managed reasonably conservatively with empirical. Treatments a lot of the time, but, but not metronidazole or Tylazine. I, I always say I take home.
Yeah, when there was still Twitter, I'm no longer on whatever the platform is called, but, you know, the hashtag metronidazole is not an anti-diarrheal. I was really trying to push that, but I think it was a bit, too complicated and, and, and too long, but, it, it really is a lot of antibiotics are really harmful to the gut microbiota, so we really should not use. In any case, and I mean there are new guidelines even for acute diarrhoea, where it very clearly says antimicrobials have no role in in treating that unless you have identified a really specific pathogen.
I mean, you know, the example for the chronic ones, granulomatous colitis, you know, initially. Diagnosed in, in boxes and in Frenchies most, most commonly, that is still a disease that needs antimicrobials, but you want to do this based on a tissue biopsy that you cultured and have an, and, and a sensitivity testing there. Don't just chuck random antibiotics at, and, you know, these dogs either because they're becoming more and more resistant around the world already.
So we want to be really practising good antimicrobial stewardship here. We are in fact helping rumour, which is the responsible . Use of Medicines Alliance and the small animal and equine, they're having a conference on the 11th of November, a virtual conference in the morning and the afternoon on antimicrobial products and also antiparasit parasiticides, so do make a little note in your diary because, you know, we as vets should not be using antibiotics unless there's a really good reason.
There's my, my, studies have shown me that a million people die every year because of antimicrobial resistance, and we really need to keep these drugs for serious issues rather than for ones that really aren't very, significant like a diarrhoea, I mean most of the time. You don't need to use antibiotics for diarrheas, do you, silica? No, and I think, you know, that's where dermatology and gastroenterology, you know, are, are very, very similar.
It, it used to be the land of antibiotics and we're becoming more and more reluctant to, to use those. And even if we just change the order of things, if we say we try diet and we now know we have to use at least 3 dietary trials, and then we may be moving to probiotics and then we may be moving to other microbiome modification treatments like faecal transplants and you know. And then only as a sort of, if everything else, you know, even the immunosuppressants haven't worked, then we might consider as a last resort, antibiotics.
Even that already has reduced the number of animals that will ever receive antibiotics to such a small amount that it would make a massive difference to the overall antibiotic use. And obviously doing culture before you just throw antibiotics at them anyway. Well that's the problem, isn't it, in the gut that really doesn't work because what are you going to culture like E.
Coli. And, and it doesn't really mean much in the context of that, and I think a lot of European countries have, for a number of years now, obviously quite, more stringent rules that you can only use antibiotics if you do have a sensitivity profile, and for the gut that makes no sense, sensitivity of what, you know, there's trillions of different bacteria in there, you can pick and choose whatever you can culture, so yeah, they really, they really shouldn't be used. Silka, Lucy, thank you so much for those brilliant presentations, thanks obviously to Carris for the work that you're doing in this area, but also for partnering with us on this webinar.
It's always interesting at Webinar Vet to see lovely new innovative products coming out that just give us more opportunities and more options. With patients that present to us, so, thank you so much, thanks everyone for listening, on a Tuesday night when I'm sure there are other things that you could be doing, so thank you, do tell your friends about it if, if you think they may be interested in watching it, it will be available. Usually tomorrow but at at worst by Thursday, so .
Thank you so much everybody, er again, Silka and Lucy, you never hear the tumultuous applause on a a webinar, but people are Stephanie's saying thank you so much, this was really fab. Veronica, thank you so much. Susannah, thank you, great webinar.
Christinnell, thanks so much for the presentation, Jorge, thank you so a round of applause to you both and thanks for your time and looking forward to seeing everyone on another webinar very soon. Take care, thanks again, bye bye. You.
