Podcast: Diagnosing Drug Resistance in Canine Hookworms with Dr. Kaplan

Ray M. Kaplan, DVM, PhD, DEVPC, DACVM (Parasitology), St. George's University, Grenada, West Indies

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In this episode, host Alyssa Watson, DVM, talks to Ray M. Kaplan, DVM, PhD, DEVPC, DACVM (Parasitology), about his recent Clinician’s Brief article, “Diagnosing Drug Resistance in Canine Hookworms via Fecal Egg Count Reduction Test.” Resistant hookworms are no longer uncommon, so Dr. Kaplan joined us to break it down. He details how to diagnose resistance by comparing pretreatment and 14-day posttreatment egg counts using a fecal egg count reduction test. Dr. Kaplan then explains how and when to use “triple therapy” in cases when resistance is confirmed.


Episode Transcript

This podcast recording represents the opinions of Dr. Watson and Dr. Kaplan. Content, including the transcript, is presented for discussion purposes and should not be taken as medical advice. No guarantee is given regarding the accuracy of any statements or opinions made on the podcast. The transcript—which was prepared with the assistance of artificial intelligence—is provided as a service to our audience.

Dr. Watson [00:00:10] Welcome everyone to Clinician's Brief: The Podcast, where we give you a window into the conversations behind all of our content. I'm the host of this program, Dr. Alyssa Watson, and in today's episode, I am absolutely thrilled to be joined by Dr. Ray Kaplan. Dr. Kaplan is an expert in parasitology, as well as the senior associate dean at Saint George's University in Grenada, West Indies. Together, we're going to be taking a deep dive today into the world of drug resistant hookworm infections in dogs. We're going to be talking a little bit about the nuances of diagnosis through something called the fecal egg count reduction test, and we'll also explore some treatment strategies along the way. This topic is totally new to me. I can't say I've ever diagnosed a resistant hookworm infection before, and I've only ever read about this test, so I'm pretty excited to learn some new things today, along with all of you at home. But before we get started, I'd just like to say hello to Dr. Kaplan and give you a chance to introduce yourself to the audience and tell us a little bit about yourself.

Dr. Kaplan [00:01:11] Hi. As you said, I'm currently the senior associate dean at Saint George's University. Before this, I spent 23 years at the University of Georgia, where I did research and taught parasitology. My focus of my research during that time was on drug resistance and parasites, initially working with livestock parasites for drug resistance as the long-standing and severe problem. But given my expertise in drug resistance and parasites, I, a number of years ago, I was made aware of some cases that sounded like drug resistance and hookworm. And so that caught my interest, and I started to pursue it with a PhD student, Pablo Jimenez. And one thing led to another and led to an entire PhD project. And we found out a lot that wasn't known very, very quickly over several years. It's really kind of been changed, how we need to view control and treatment of hookworms.

Dr. Watson [00:02:12] Yeah. This is wonderful. And that's exactly what we're going to be talking about all during today's episode. So just before we dive in, I do want to give a quick shout out to Merck Animal Health for sponsoring today's episode. But let's get in and talk a little bit about this. So, Dr. Kaplan, could you tell us where these multidrug-resistant hookworm infections are most prevalent here in North America? And then for global listeners, is this something that we need to be on the lookout for in other parts of the world, like Europe or Australia?

Dr. Kaplan [00:02:46] I think it's important that in other parts of the world that veterinarians and parasitologist do start doing some surveys to see the extent of drug resistance. I imagine it is there as well. But we have no data, so we have no idea of the extent or of the spread to other countries. In the United States, we did a study in collaboration with IDEXX, who provided us with samples from several regional labs. So we had we had samples from from all over the United States. And what we found was that it was widespread across the United States. Interestingly enough, the prevalence of resistance among among we were only we were only looking at positive samples, so IDEXX sent us positive samples. So in the western United States, they have the lowest prevalence overall of hookworms, so they had fewer samples. But of the samples we tested, they had the highest proportions of resistance out West. And, that's actually not that surprising in that there's less ability to dilute out the resistant worms where you don't have a high level of what we call refugia, or just a background levels of infection. So it is it appears to be quite prevalent across the country. On average about half of all dogs with hookworms were showing evidence of having drug resistant hookworms. And so, so that's really high. A lot higher than we ever imagined it would be.

Dr. Watson [00:04:23] That is is very high and certainly something that is surprising to me. Although, I do practice out west and so, you know, and I I'll say like I did in the intro, I just don't see a lot of hookworm infection. And so maybe that's because it's just less prevalent out here, although the resistant ones, it sounds like our need to be more of a concern for me.

Dr. Kaplan [00:04:44] Yeah. So yeah. West. Yes. There's not a lot of cases, but based upon on data, when you do get a case, there's actually a pretty high probability that there's going to be drug resistance involved.

Dr. Watson [00:04:55] Can you talk to a little bit about kind of the dynamics that contributed or that we think contributed to the rise of these infections? My understanding is they were kind of first noted in racing greyhounds.

Dr. Kaplan [00:05:10] Yes. The, it's been noticed for quite a few years that racing greyhounds, or recently retired greyhounds, have chronic and relatively severe problems with hookworms. So that was not anything new to veterinarians. But as we started to do a deeper dive into the drug resistance, we one of the cases, actually, the first case was that we started investigating was a case from a recently from a greyhound that was, that was recently retired from the track. And, and then as we investigated the racing the greyhound industry a little further, we did we that we visited a few kennels and started collecting samples and, basically essentially every greyhound that we tested was infected with drug resistant hookworms, at, at a relatively high, very high level. So, so we had initial just kind of empirical evidence that that this probably originated with the greyhounds, but but then subsequently, you know, we we did some genetic testing, and we could actually determine what we call the haplotype, which is kind of the, the background genetic profile that the mutation a resistance mutation sits on. And that's a mutation just for benzimidazole resistance, which would be fenbendazole or febantel would be the common benzimidazole that are use for treating hookworm. So we know the mutation that causes that resistance. And so the genetic background that that mutation sits on, we can use that to look at the relationships between, between different, different, samples, different parts of the country and so forth that have this mutation. And what we found was is that the, the, the haplotypes that were most common in the greyhounds were the same ones that were most common just about everywhere, suggesting that it started in the greyhounds and then spread from there. But interestingly enough, there was there was one haplotype that we that was uncommon in the greyhounds less much, much less common in the greyhounds than in dogs. It was a relatively uncommon haplotype in general. But it was still much more common in the pet dogs than it was in racing greyhound, suggesting that there's another origin outside of the greyhounds. So probably probably was from some kennel of some other breed of dog. But highly likely that it was not from a greyhound kennel.

Dr. Watson [00:07:42] Interesting. And then you talk in your article about this concept of larval leakage. And I would just love a refresher on that and kind of give us, you know, the background for why we need to really differentiate this phenomenon of larval leakage from drug resistance whenever we have an animal that where we're seeing signs of persistent infection.

Dr. Kaplan [00:08:08] Yeah, that's a great question. Yeah. Larval leakage is just kind of, it's kind of a weird, cool thing that dogs and hookworms have this relationship that that allows this to happen. Essentially the biology of larval leak is that, you know, dogs can be infected orally, or transdermal, with the larvae, and as dogs start to mature, they become immune to hookworms, relatively immune, not absolutely immune, but relatively immune. So, as all veterinarians know it's hookworms are much more common in puppies than in adult dogs. And so what happens is as dogs develop immunity, when they get infected transcutaneously, those larvae basically don't, don't complete their development, their migration and development into the gut, so the larvae will penetrate through the skin, they'll enter the bloodstream, but instead of going to the lungs and then back down to the gut, which is the normal route that the parasite takes in young puppies, these larvae go into somatic tissues, which can be really most any tissue throughout the body, typically in muscle, and they go into an arrested state, and they can stay there for the life of the dog. And in most cases, that's where they do stay and they never come out. But part of the natural biology of hookworms is when, when intact bitches become pregnant, those larvae become reactivated, and travel to the mammary gland. And that's how we get the transmammary transmission of hookworms, which is really important in the, in the transmission cycle and why puppies have such a high prevalence of infection with hookworm. So that's a natural part of the life cycle. And in most dogs, if they're, if the bitch is spayed or a male dog, those larvae in those somatic tissues will basically stay dormant for the life of that dog because they don't have that, that, hormonal stimulation that happens during, during pregnancy. But there's, it's believed the small percentage of dogs where the larvae kind of just over time, they just continually a small number just continually come back out of arrest out of this encystment and complete their development. And so, so dogs for larva leak are going to constantly have a low level of, of hookworms in, in their gut. And so like every time you do a fecal on them, they have a positive fecal. And so that was, that's been kind of the dogma of larval leak. We don't really know why some dogs have this and others don't. It was believed to be some sort of immune deficit, although that was never demonstrated experimentally. Then so the so layer on top of that though with greyhounds harbor incredible parasite hookworm loads due to the nature of, of of of their husbandry. And so, so greyhounds were being diagnosed with hookworms at a really, really high, high prevalence. And veterinarians and parasitologist were just assuming, well, it's just larval leak, greyhounds just have they're just loaded with hookworms. And so prior to when I got into this, this area, that was the feeling that this is just larval leak, just greyhounds are loaded up with hookworms, and we're just seeing leak. But when I, when I saw some data, it didn't look like larval leak to me, it looked like resistance. And, so it was important then to distinguish those two. And that's where, that's where doing the proper type of testing becomes quite important.

Dr. Watson [00:11:56] And we are absolutely going to talk about that test. But before that, can we just review the different anthelmintics that hookworms that we know currently exhibit resistance against? And then, my understanding is kind of within the hookworm population, there's, there's different levels of resistance across those different classes.

Dr. Kaplan [00:12:21] Yes, absolutely. Well, first thing it's important to appreciate is that drug resistance in parasites is not a black and white thing. It's, there's different, different levels. So a worm isn't just resistant or not resistant. Resistance can get higher and higher and higher level. So, so once you can start, you can have a situation where you have resistance, but the resistance can continue to get worse and worse and worse with higher levels of drug selection. So that's that's an important distinction to make sure that people understand. In terms of the drugs, basically every drug that's approved for use of treatment of hookworms in North America, we've seen resistance at a fairly high level. In our studies in Georgia, in, in the Greyhounds, they had resistance to all classes. So the benzimidazole drugs, which would be febantel and fenbendazole to pyrantel, and to the macrocyclic lactones, so that would include all the ivermectin-type drugs. So there's no there's no ivermectin treatment that's labeled for use in hookworms, but we have, you know, selamectin or milbemycin or moxidectin, and, and we saw in the greyhounds they were resistant to, to everything, including moxidectin, which is moxidectin is going to be the most potent drug overall. And also, it appears, from what we can tell, is that that was the last drug that the hookworms became resistant to. And so it's, so we'll come back to that issue in a minute. But but we're seeing resistance to all of the drugs. The we have a genetic test for benzimidazole resistance, so that's where we did our large, you know, national study with, with, many hundreds of samples from around the country. And we could test them, using DNA sequencing to identify those samples that that carried the mutations for for the resistance to benzimidazole. We don't have any molecular tests for the other drug classes, so the only way we can diagnose those currently is by clinical testing. And that's where the fecal egg count reduction test comes in. So, so the the data that we have on a national scale is just benzimidazole resistance. But from what we saw in Georgia, not only in greyhounds but in other cases of nongreyhounds, we were seeing multiple drug resistance across the board. So we don't all we can say from our data now in terms that the national data is that we have benzimidazole resistance widespread. We don't know how many of those, how many of those worms will have pyrantel resistance or resistance to macrocyclic lactones. My guess would be that high proportion would have resistance to those other drugs as well. But we don't have those data, so we don't really know. That would require, you know, additional studies. And, and it's much more difficult to do those types of studies because since we don't have a molecular test, the only way to determine that would be do clinical studies, which are much more difficult and expensive and time consuming, than just being able to get a bunch of samples in in the lab, test them using a molecular test.

Dr. Watson [00:15:42] So let's talk a little bit about this fecal egg count reduction test. Can you just like in a nutshell explain the test? I think you've already kind of given us a background of why you think it's, you know, so important and really is the most practical approach to diagnosing when we have these worms that we think have multiple drug resistance.

Dr. Kaplan [00:16:04] Yes, absolutely. The fecal egg count reduction test is a test that's been done in farm animals routinely for, for many, many decades to diagnose resistance. One of the great benefits of this test is that it works for pretty much every drug. And, for, for many different types of parasites. And so it and and also it's a clinical test that can be done in house. And so it has a lot of advantages where molecular tests are fantastic. They're convenient. But, we have to have knowledge of the mutations causing the resistance in order to develop a molecular test. And we only have that information currently for the benzimidazole drugs. So. So, like Antech offers a test for drug resistance in their key screen, which is a great test. But but it only test for benzimidazole resistance, which is still appears to be a really good marker. And, and very often I say my guess would be the majority of those cases would have multiple drug resistance to other drugs as well, at least one, if not both other classes of drugs. But but you but you don't know just by doing that, that one test. All you know from that one test is that you have benzimidazole resistant worms. And so the way you can tell if you have resistance to other drugs is by actually testing the, the drugs in the dog, the, the, the the whole principle behind the test is that dead worms don't lay eggs. And all of these drugs, when they were first approved had an efficacy of greater than 99%. And so, so, basically, you shouldn't see any eggs when after you treat a dog. The one exception would be pyrantel the the efficacy in the registration trials was a little bit lower, kind of in the 96-98% range, which means that if you had a high egg count pretreatment, you might see a few eggs posttreatment. But in most cases you would see no eggs even with pyrantel. And so so what happens, so so if you're, if you're testing, if you do an egg count on a dog before treatment and then after treatment, you can actually measure the reduction because we expect there should be no eggs present. Then if we see eggs posttreatment, we can start making clinical inferences on whether we have resistance or not. And that's the whole basis of the fecal egg count reduction test. Of course there's more details about that that we go into in the article. Maybe you want maybe you have questions, to, to get into some of those details. But that's kind of the, the overall, basic idea behind the fecal egg count reduction test where we, you take, do an egg count at the time of treatment, and then you do another egg count 14 days later, and then you just measure the reduction in, in egg numbers between those two egg counts. One thing I should say that's important here is that it's it's relatively uncommon that small, small animal veterinarians do egg counts. They typically do a flotation. And often they're scored qualitatively like one plus two plus three plus four plus. Our research shows that that that does not work. You can't do that. There's, we actually we did we compared qualitative scoring to fecal egg count testing, and the variability was immense. So, so really, I think a flotation is great as a qualitative test. Are there hookworms? Yes or no? And, and maybe you could say there seems to be a lot or there's not many, but that's as far as you can go with it. You can't measure a reduction going that way. So you really need to do a count in order to be able to a quantitative count in order to be able to get any type of good clinical inference on levels of reduction and therefore efficacy.

Dr. Watson [00:20:06] And you said that this can actually be done, you know, in the clinic, but do you need any kind of special equipment to do this to make these counts?

Dr. Kaplan [00:20:15] The only the only special piece of equipment you would need would be the egg counting chamber. Which, the most common one is the McMaster chamber. There's a number of different companies that sell those. They're not that expensive, and they last a long time for years. So they cost somewhere like $35 to be approximate. And they lasts for years and years and years. So the cost per test is is is negligible, really, on the apparatus. There's another type of test called a mini flotac, which is a bit more accurate. But right now, those are difficult to get in, in North America. I used to actually distribute them in my lab. They're they're made by the University of Naples in Italy. And you can still buy them from the University of Naples, but you have to go online. You have to do a bank transfer. It's a bit complicated. Most vets aren't going to want to take all that trouble. So, for for a few years, what I was doing is I was, I had a collaborative agreement with the University of Naples, and my lab would buy, you know, a large number, and then we would then we would resell them at the same price that the University of Naples sold them for, just kind of as a service. But once I left University of Georgia, that, that's that became unavailable again. So we're looking we're looking to find someone else who will serve that, that role. But right now, they're not available in the US directly.

Dr. Watson [00:21:42] In lieu of that, can you send these out to one of our, you know, commercial diagnostic labs and request this test? Request a fecal egg count reduction test?

Dr. Kaplan [00:21:52] Yeah, absolutely. I think all the major laboratories offer that test. Some of the smaller laboratories do as well. And some of the university diagnostic laboratories do as well. You just have to just make sure and be specific, asking if they're doing a fecal egg count reduction test and how they're doing the egg counts. The one thing that you don't want them to be using is centrifugal floatation for the egg count, which so that is one way you can do it. There's, you know, instead of just doing a plus or minus, you could actually count the eggs under the, under the cover slip. But that also has been shown to have a really high level of variability. So that's better than not counting, but using a proper counting method, chamber method has been shown to have much more, a much higher level of of precision and accuracy in getting a good egg count.

Dr. Watson [00:22:47] All right. Well, we're going to take a quick break, and then when we get back, we're going to jump into some of those more specifics about the test.

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Dr. Watson [00:23:29] All right. So I want to go through, you know, you had this really nice, detailed step-by-step in the article on how to perform these tests. And one thing that you talk about, it's very, very important that you perform two separate fecal egg counts both before and after treatment. I was wondering if you could clarify that. When you're talking about doing two counts, are you talking about doing two separate counts, but it's okay for them to be from the same fecal sample? Or do you want two like specific samples from the dog? I was a little confused by that.

Dr. Kaplan [00:24:02] Sure. As a, as a background before I address that directly, when we do the fecal egg count reduction testing in farm animals, we're usually measuring at least six animals. Six, sometimes up to 50 samples in a test.

Dr. Watson [00:24:17] A pool sample, yeah.

Dr. Kaplan [00:24:19] No, no, we do it individually for each animal.

Dr. Watson [00:24:21] Oh, okay.

Dr. Kaplan [00:24:22] But we, but we use multiple animals. And what that does, it greatly decreases the amount of variability because an egg count is a test that has quite a bit of variability in it. Because the worms are not shedding the same amount of eggs at every moment of every day. The same amount of feces is not being is not being produced at every moment of every day. And then when you take your fecal sample, you're just taking a small amount. So there's lots of lots of opportunities for there to be variability in the measurement. And so when we're dealing with a, with a, a patient canine patient, we have one, one animal. And so if we just take one sample pretreatment and one posttreatment, there's going to be variability in both of those measurements. And so by doing two egg counts pre and two egg counts post, we can we can decrease that variability by by 50% essentially. And that will, that will give us quite a bit more accuracy in our measurement of reduction. Now, the regarding the specific question, it, it doesn't have to be a separate sample. It can be the same sample. You just want to do two independent counts from that sample. And, and, studies have shown that due to the just the nature of their variability that doing two samples, doing two counts, separate counts on the same sample versus two separate counts on two separate samples is not going to really improve the situation. So, so you can so you can, you can do the most pragmatic approach and just do two separate counts on the same sample, just independently from each other. And then then you should then you're good.

Dr. Watson [00:26:07] Okay. And then you say, you know, obviously we're doing this because we, we have an animal that we have seen a persistent, you know, positive positive for hookworms on. So, you talk about that it's very important to when you're doing this test to go ahead and then readminister the, the what, whichever anthelmintic they had used previously instead of just like giving another anthelmintic or or jumping to triple therapy, which we'll talk about later. So why is that important that you readminister the one that they had last?

Dr. Kaplan [00:26:46] Well, it's not so much that it's important that you administer the drug that they gave last. But it allows you to start getting a better handle on the clinical case situation. If a dog comes in positive for hookworms and it's been treated somewhat recently with a drug, you wouldn't you wouldn't expect that dog to have hookworms, right? If you gave it a drug and that drug was effective. So if you're trying to determine if that if the dog has drug resistant hookworms or if or, or maybe it was just larval leak because you didn't test soon enough and the worms came back, it seems to me that just to make the most sense that you're going to test that same drug to find out if the drug you've been giving is actually working or not before you just switch to some other drug. So so it's more of a pragmatic decision than, than a requirement. So that way you can determine if the drug that you've been using is going to be effective is effective against, against those worms. Now, and that's where it becomes important to distinguish between larval leak and resistance. I'm not sure if that did you ask that part?

Dr. Watson [00:27:55] We about it a little bit, but yeah, we talked about it a little bit, but yeah. Go ahead.

Dr. Kaplan [00:27:59] So yeah. So so larval leak is just a natural process that's going to occur in many of these dogs, particularly if they harbor a large number of somatic larvae. But it takes, based upon our studies, it takes about a minimum of 3 to 4 weeks from the time of an effective treatment until you'll see eggs again in the feces posttreatment, if it's if it's due to larval leakage. So, so if you if you, treat a dog and it comes back in a month or two months and it's positive for hookworms, you have no idea whether the treatment was effective or not or if it's just larval leak. So to distinguish those, you need to check at two weeks posttreatment because at two weeks posttreatment, there should be no eggs. Because the, any larval leakage that's occurring, those those worms will not be mature and egg laying adults by two weeks of age. So so at two weeks, you're going to be actually measuring the eggs that would be produced by the worms that were present at the time of treatment. So that's where you can get a specific handle on whether there's resistance or not. If you have a reduction, if you, in the efficacy of that treatment at 14 days, then you then you can assuming that, you know, you did your egg counts properly that the drug was a good quality and delivered correctly, right? You have a if you have a substantial reduction efficacy, you can be quite confident that you have drug resistance at 14 days. But once you get past 21 days and especially at past 28 days, then there's no way of telling whether the drug worked or not or if you're just seeing larval leak.

Dr. Watson [00:29:43] So it sounds like that 14 days is really crucial, you know, when we're doing that retest. Are there any, you know, how could it affect the sample if we don't wait a full 14 days? Like, say, we did the retest at at nine days or ten days, would that affect our results as well?

Dr. Kaplan [00:30:01] It definitely can. What we found was with fenbendazole, interestingly enough, in, in worms, they were highly resistant to fenbendazole where we only had about a 20% efficacy against adult worms, we had a 99% reduction in eggs at three days posttreatment. And but then slowly, those eggs started to come back. So, what's happening, and this has been reported in large animals as well, is that worms can be in female worms can be inhibited from producing eggs. They get like a temporary like they get punched in the face type of thing and and they get a headache or whatever. They stop producing eggs temporarily, but they're not killed. And then over, over several days, they, they, they gain they regain the ability to start producing eggs. So what we saw is that you need at least ten days, egg counts for fenbendazole went almost to zero. And then they started coming back up, and, and they continued to come up for quite a while. So if you, if you test before ten days, you'll definitely get a biased result that may that may indicate you have much better efficacy than reality. So 14 days would be our optimal time frame. But at least ten days at least get past go past ten days. And you'll get a reasonably good estimate there. But we saw the egg counts continue to be increasing all the way through the 14 days in those cases.

Dr. Watson [00:31:35] Yep. Sounds like we really want to shoot for that 14-day mark so that we're not testing too soon or too late and skewing our results. Can you talk a little bit about, you know, like I said, we're going to talk about the monotherapy and the and the triple therapy, but could you talk about kind of the guidelines for interpreting the test results, whether or not you're using a single drug versus multiple drugs, you know, in your test?

Dr. Kaplan [00:32:05] Yeah. I think rather than going into the specific numbers because that's in the article, I'll just I'll say more on the on the overall concepts of understanding. Is that when we're going to use different criteria for establishing whether there's resistance, whether we're using a single drug or we or we actually have given multiple drugs simultaneously. So let's start with, with the first drug. So if you're giving one drug, as I mentioned, all of these drugs when they were originally registered should had very high efficacy, so you really shouldn't see any eggs. If you see some eggs, again, you know, that's just where also where it's important to, to do the pre in the post because the pre-egg count could be 100 eggs per gram. It could be 1000 eggs per gram. It could be 10,000 eggs per gram. So if you have 10,000 eggs per gram and you have 99% efficacy, you're still going to have a lot of eggs posttreatment. If you have 100 eggs, 100 per gram pretreatment and you have 99% reduction, you're probably not going to see any eggs posttreatment. So knowing what the pre count is important. So just doing a post count can be very very misleading because there's a huge variability in egg counts in these animals. and so so when you're testing one drug then we expect no eggs. But if you're getting greater than 95% reduction, then that means you have pretty good efficacy, and given the amount of variability that you could expect when testing with, with just, you know, one dog, with, with just even if you do a couple egg counts, we're going to say that there's lack of evidence for resistance against. And if you if you see some eggs, it's a little suspicious. But if you have higher than 95% then things are pretty good with one drug. And then if you fall below 95% in that like, you know, 75 to 95%, that's going to be really suspicious. And less than 75%, that would be really strong evidence of resistance. But the situation is much different when we have three drugs. Okay, so let's say we're using the triple combination. Each drug should have an additive effect. So we should see really zero eggs absolutely zero eggs posttreatment with a triple combination. And let me just let me just provide like a quick example. Even if you had resistance to all three drugs you gave, let's say the efficacy was 80%, which is substantially lower than what you would want for a for a clinical cure. If you gave three drugs at the same time, the first drug would kill 80% there's 20% left. The second drug would kill 80% of that 20%, which is 16 more. That gets you to 96%. There's 4% left. Then you would kill 80% of that, which is which is more than three out of four. So basically three drugs with only 80% efficacy should yield greater than 99% efficacy. And so when you're seeing any eggs posttreatment after triple combination, that's suggesting that you have substantial resistance to to at least one drug and resistance to all drugs. And then obviously, you know, once you start getting, you know, less than 95% efficacy or 98% efficacy with triple combinations, that means that you're going to you have substantial reductions in efficacy across the board in your drugs. So we have to use a much more stringent threshold for, for, for determining whether we're going to have resistance to the triple combination than to any individual drug.

Dr. Watson [00:35:51] Yeah, that makes perfect sense. And also that huge variability that you're talking about is another reason why you want to make sure you do the same number of tests posttreatment and use the same method as you were talking about that you did pretreatment so that you can really have an accurate comparison between the two.

Dr. Kaplan [00:36:14] Right. And one thing I should add while we're talking about the numbers is that the fecal egg count reduction test is a great test for detecting resistance. But it's not a great test for comparing efficacies between different cases when you have high efficacy, there's not there's there's little opportunity for variability, right. And so even so, when there's very, very few eggs posttreatment, there's not going to be opportunity for variability. As the efficacy declines, there's more and more opportunity for variability. And different female worms produce different numbers of eggs. It depends how old they are. Young worms produce less initially produce lower numbers of eggs. Then they start producing more and more. And then as they get old, they start producing less again. And and also, the number of worms that are in the gut can impact the number of eggs that are shed per female worm. So there's lots of things that can cause variability, but those things only only matter once you have resistance. Because once you have reduced efficacy, those factors can can really cause huge variabilities, right, in what you've measured. So whether you measure 20% efficacy or 80% efficacy, that might not actually be any real difference. That could just be due to just random variability due to the nature of the infections and how many worms were there and so forth, and how, you know, what the distribution of the ages of the worms are that are in that dog. But, but the take home is the egg count reduction test has told you whether you have 20% or 80%, you have resistance. You need to do something about that in the management. But but, but but if you have three dogs, one with 20%, one with 50%, one with 80% efficacy, it would be a very, very incorrect interpretation to say, well, the 20% dog has has the worst resistance, and the dog that had 80% efficacy has the lowest resistance. That would not be a correct interpretation.

Dr. Watson [00:38:11] Sure. So let's finish up with some recommendations for clinicians that are managing these cases of persistent hookworm infection. What do we need to do when our standard treatments have shown limited efficacy?

Dr. Kaplan [00:38:25] Yeah. So, I think that's where we need to go to the triple combination, which will include drugs from each drug class. And the reason why we want to use the triple combination is that resistance is not absolute for any single drug. So even if you're only getting 20, 30, 40% efficacy for one drug, when you use in combination you get that additive effect, which can help get you over the finish line essentially to and prevent and decrease the chances that you're going to have eggs surviving the treatment, worms surviving it. Obviously, the eggs are just an indication of it. And. And the reason why we need to use the moxidectin and topical moxidectin is that it's extremely high dose of moxidectin. And what we saw when we did our studies, we our initial studies, we just had three random cases, one from a Greyhound and two from nongreyhounds. The two nongreyhound dogs we tested had resistance to all three drug classes, but they were susceptible to moxidectin, and for the macrocyclic lactones, moxidectin is considered part of the macrocyclic lactone family. But what we we see this that, it's a more potent drug and it will still work when the other macrocyclic lactones do not work. And we see the same thing in some of our large animal parasites. It's temporary though. Once you have resistance to the other macrocyclic lactones and you start using moxidectin, you will get moxidectin resistance fairly rapidly unless you're doing specific things to prevent it, like making sure there's no reinfection occurring by through strict hygiene. Environmental hygiene. But. Well. And but then like the greyhound kennels, they used to use pyrantel, fenbendazole, and ivermectin. Basically the cattle products or horse products because it was cheaper. And so those are those who we once we got resistance to first and the greyhounds didn't start using moxidectin until after they realized that nothing else was working. And so given that, like, 50% of the dogs in the US are carrying drug resistant hookworms, it means that these resistant hookworms have been spreading for probably decades out of the greyhound population and into the pet dogs. Moxidectin resistance is is relatively new. So I would my guess would be the majority of resistant hookworms around the country are still susceptible to topical moxidectin. And the reason why I distinguished topical moxidectin from the other moxidectin products is because it's at a much, much higher dosage. And so and that makes a big difference. And we don't have any evidence that the other, other, other products with lower moxidectin dosages will work against resistant worms. It's possible they will, research would need to be done, but so far, there's no evidence of that being the case. I think it's really that super high dose that we get with the topical that makes the difference. And and so, so that's why we need to use the topical moxidectin and why it often will be, will be effective. However, like I mentioned, we saw in probably the majority of the greyhounds that were coming that were recently coming off the track that moxidectin didn't work either. So that's why we need to do the testing and not just assume the triple combination with the topical moxidectin's going to work. But that's that's where we need to go with with our treatment if we determine conclusively that we have resistance to the drug that we had been typically treating with. Then if the triple combination with the topical moxidectin doesn't work, the only option we have is, is emodepside. Emodepside in the United States is only sold as a topical product for cats. In other parts of the world and Europe in particular, in many countries, it's sold as a canine tablet. And we did a study against our our highly resistant isolate that we got that we obtained from the greyhound, which was, you know, highly resistant to all the drugs, including moxidectin, and we had greater than 99% efficacy with emodepside on the isolate using the canine tablet. But we don't have the canine tablet approved in the United States, so it's unavailable. So we work with some veterinarians, and in a kind of a clinical study, and they use the, the, the, the feline product, and it worked quite well, but there's, there's a bunch of caveats to using it. And we published those in a previous Clinician's Brief article. I think there's probably too, too many to go into now at the time we have. But the most important thing is it does not work topically in dogs. You have to give it orally, and you have to give it at a lower dose than the cat dose. One third the cat dose. Otherwise, it can be toxic in dogs. Because remember the cat is being given that dose topically. We have it doesn't work topically in dogs, we have to give it orally. If we gave the if the cat dose orally to the dogs, we would have a high risk of, of toxicity. So there's a bunch of caveats to using emodepside. And I do highly recommend that anyone who is interested in using emodepside, check out that previous Clinician's Brief article that I published in collaboration with Dr. Pablo Jimenez, which has a lot of those details. The one additional caveat that we didn't know about when we published that paper, which which is an important consideration and will be coming out in a future paper that's going to be published soon by the hookworm task force of the American Association of Veterinary Parasitologist, and that is, is that emodepside does have efficacy against heartworm. So and but it's kind of unpredictable because it's not an approved product. And, and so, there's the potential for, for adverse reactions in dogs that are treated that are, that are heartworm positive, that are treated with the emodepside. And so we recommend testing for heartworm before treating with emodepside. And if that dog is heartworm positive, not using the emodepside because there's potential for adverse reactions that are kind of unpredictable because the studies have not been done. And so there's no way of actually knowing, you know, how how the dog is going to react to the various stages that might be dying due to emodepside, and, and so, so that's, that's an extra, extra caveat that was not in that previous Clinician's Brief article.

Dr. Watson [00:45:03] Well, thank you for sharing that. Certainly there's so many things that we need to be mindful of and consider anytime we're using a medication off label. So, I just want to say thank you so much for walking through all of this. I really learned a ton. I will absolutely be on the lookout for any kind of positive hookworm puppy coming in now, especially because I am on the West Coast, like you said. And so I might have one of these, you know, higher, higher levels of resistance. The final thing we do at the end of our episodes, now that we've answered all these clinical questions and we've worked your brain is to do something a little bit more lighthearted and fun. And I have a couple final questions for you that are just some fun, would you rather questions. A way to kind of lighten things up at the end of the day. Are you ready to play?

Dr. Kaplan [00:45:56] All right, let's see what you got.

Dr. Watson [00:45:59] All right. So if you had to take a long international flight, would you rather be seated next to somebody with a yappy pomeranian under their seat? Or would you rather be seated with someone who wanted to show you all the pictures they had of their dog's poop on their phone?

Dr. Kaplan [00:46:18] I'd probably take the dog poop on the phone.

Dr. Watson [00:46:22] They'd want to hear your expert opinion. If you had to choose, would you rather have to use a microscope that the condenser was stuck and you couldn't adjust the height or the aperture, or your 100x objective was always just slightly out of focus?

Dr. Kaplan [00:46:43] I would definitely take the second because for when we're, when we're looking at parasites under the scope, we don't usually use the 100x objective. That's more so for really, really small stuff. And parasites are big, relatively on the microscopic scale.

Dr. Watson [00:46:59] So we want to have that condenser so we can refract that light.

Dr. Kaplan [00:47:03] Absolutely. That lighting is critical for the big structures.

Dr. Watson [00:47:08] Would you rather find out that the kitten that you were just snuggling has scabies or that the puppy that just licked your mouth has giardia?

Dr. Kaplan [00:47:17] I would much rather have a puppy with giardia licking my mouth. Giardia, really, for the most part, is not super zoonotic. It's possible. There are, there are some bio types that are, but, it's it's quite rare. So that wouldn't concern me all too much.

Dr. Watson [00:47:35] Not worried about it. All right. Last question. Would you rather that all of your students be 10 to 15 minutes late but super engaged and excited about parasitology or always on time, but kind of distracted during your lectures?

Dr. Kaplan [00:47:55] Oh, I would take 10 to 15 minutes late and have them be totally engaged. So yeah, I can, I could, I could, I could teach a lot in, in 30, 35 minutes to students that really are interested rather than, compared to 50 minutes and students that don't really care.

Dr. Watson [00:48:12] Well, that's fantastic. And I'm sure that all your students are really engaged all the time. This was a great conversation. I want to say thank you to you. Thank you so much for pushing through. I know that you had kind of a sore throat, and we really appreciate you being here and sitting down and talking about this topic with us. We also want to just say thank you once again to Merck Animal Health for sponsoring our discussion. And thank you to our audience for listening.

Dr. Kaplan [00:48:37] All right. Well, thank you, Dr. Watson. It's been a pleasure speaking with you.


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