Detecting parasites key to countering rising hookworm anthelmintic resistance
Molecular tests can read parasites’ inner making by accessing the DNA code in a highly specific way
Anthelmintic-resistant parasites have long been recognized as a significant health problem in large animals (e.g. sheep, horses) and appear to be an increasing issue for companion animals, too. Indeed, a recent study using Companion Animal Parasite Council (CAPC) data collected from 2012 to 2018, including more than 39 million fecal tests, showed hookworms (Ancylostoma caninum) in dogs had jumped by almost 50 per cent during the study timeframe.1
Developing hookworm multi-anthelmintic drug resistance (MADR) is a likely component of this increase in dog parasite fecal numbers, as well as a significant sign of antimicrobial resistance (AMR) concern. In addition to being an antimicrobial stewardship issue, parasite MADR is also a One Health concern, due to a number of these parasites (like hookworm) being zoonotic, as well as the impact of fecal contamination of the environment with drug-resistant parasites.
Through targeted research on anecdotally persistent canine hookworm infections in adopted greyhounds, a molecular marker for one major anthelmintic drug class (i.e. benzimidazoles [BZ], like fenbendazole) was identified and functionally linked to hookworm persistence. Subsequently, more detailed studies discovered these hookworm strains were, in fact, multi-anthelmintic drug resistant (MADR) against three major anthelmintic drug classes. Further—and alarmingly—MADR hookworm persistence is no longer restricted to greyhounds or isolated to Florida, as was previously believed.2,3,4
While detecting MADR in the lab has, traditionally, been a laborious, lengthy, and expensive endeavour, this past year has seen advancements in molecular testing for the genetic markers related to anthelmintic resistance. This has led to the creation of sophisticated and specific real-time polymerase chain reaction (PCR) tests (referred to as qPCRs), which are fast (typically 24-hour turnaround), as well as affordable for the pet owner.3
The test, Antech Diagnostics’ proprietary KeyScreen GI Parasite PCR, contains a genetic A. caninum marker (specifically for identifying BZ resistance in the canine hookworm) as part of its 20-parasite PCR detection panel, along with a marker that detects Giardia strains with zoonotic potential. In March 2022, this test became available to veterinary clinics in the United States and Canada for pet parasite wellness fecal screening (for puppies, kittens, and adult dogs and cats), which is in line with expert- and evidence-based guidelines from CAPC and the Canadian Parasitology Expert Panel (CPEP).
When a selected sample of the test’s first year of submissions were analyzed, KeyScreen detected parasites in more than 25 per cent of submitted fecal tests—with co-infections (more than one parasite found) detected in over a quarter of them.5 Shockingly, this same data sample revealed a never-seen-before picture of the distribution and frequency of resistant hookworm in North America. In this cohort of more than a quarter-million canine fecal submissions, BZ resistance was detected in A. caninum was detected at a rate of 11.2 per cent across the U.S. and Canada, with the highest frequency in America’s Western region (13.4 per cent) and the lowest frequency in Canada (4.0 per cent).4 However, in certain geographies, BZ resistance frequencies ranged as high as 25.1 per cent, which is in line with rising anecdotal reports from veterinary clinicians and specialists on cases across the U.S. and Canada. Since that time, more dogs with BZ resistant A. caninum have been described in Canada (some that have been imported from the U.S., and some dogs lacking an importation history), indicating BZ resistance is endemic in Canada at this time.4
Photo © iStock / Getty Images Plus/Oksana Restenko/1454618373
A unique approach
Antimicrobial resistance is a rising concern that necessitates a need for thoughtful antimicrobial use—a.k.a., antimicrobial stewardship (AMS). Unfortunately, like antibiotic resistance, anthelmintic resistance is growing quickly and has moved beyond a single form of BZ resistance (that is to say, it is now MADR).
To help clinicians more effectively and efficiently treat individual pets and meet AMS needs, the KeyScreen parasite panel has been updated with a second benzimidazole genetic marker to detect up to 25 per cent more resistant hookworms.6 The new marker was first reported in June of 2022 at the American Association of Veterinary Parasitology (AAVP) conference in Snowbird, Utah.7,8 For the veterinary industry, this is a remarkably agile translation of new research findings into an actionable and timely clinical test, which significantly improves treatment decisions and health outcomes for pet dogs and their owners, and is also in line with AMS and One Health.
Likewise, qPCRs aid in accurate parasite surveillance and reporting. Indeed, examination of nearly one year of processing samples collected in 2022 revealed a concerning number of positive tests for BZ-resistant hookworm. Specifically, while BZ resistance and MADR were initially believed to be confined to greyhounds, analysis of compiled data revealed greyhounds to be only one of 84 dog breeds to carry hookworms with the resistance marker.3 When analyzed by dog breed, greyhounds had the highest hookworm infection rate (21.6 per cent), of which almost a quarter (22.4 per cent) were carrying the benzimidazole resistance marker; however, mixed-breed dogs were also observed to be frequently infected with hookworms (19.1 per cent), with 15.1 per cent carrying resistant hookworm.
Image provided by BIOVET, a division of Antech Diagnostics, part of the MARS Petcare family
Aspects of One Health
The pandemic heightened awareness of the human-animal bond, as well as the implications of disease transmission. Further, factors such as climate change, antimicrobial overtreatment in livestock populations, and loss of wildlife habitat spaces increase human exposure to disease and zoonotic infectious agents.
According to the World Health Organization (WHO), 60 per cent of emerging infectious diseases in humans are zoonotic, accounting for one billion cases of illness and millions of deaths each year. While the risk of humans contracting disease from pets continues to be low, there is a growing need for improved tests able to assess the zoonotic potential of a parasite.
Giardia offers a unique example of this zoonotic concern due to its complex set of genetically distinct and host-adapted species. Divided into assemblages from A through G with individual host preferences, A and B Giardia assemblages, which can carry zoonotic potential, can be found in companion animals and other species; however, conventional tests used in veterinary medicine (e.g. centrifugation-flotation, antigen detection) do not have enough resolution to differentiate between these assemblages. This makes it nearly impossible for veterinarians to know if an individual Giardia-positive pet could be a threat to human health or not. While immunocompetent people may be able to resist infection or clinical signs, risk of infection and subsequent medical concern may be higher in children and in older or immunocompromised adults.
Additionally, if a Giardia-detected dog infected with a non-zoonotic assemblage has no clinical signs, antimicrobial treatment (as in line with AMS principles) may not be indicated. Considering the inability of current fecal tests (e.g. flotation, antigen) to identify potentially zoonotic Giardia assemblages, antimicrobial treatment (albeit well-intentioned) could follow a positive test in some situations, which runs counter to the veterinary profession’s commitment to antimicrobial stewardship.
As such, the ability of parasite tests to detect potential Giardia by zoonotic assemblages allows veterinarians the opportunity to inform on human health risk, aids AMS efforts, and allows education of pet owners, particularly in risk assessment in vulnerable human groups. If a dog in a reduced-risk environment is not showing clinical signs of disease due to Giardia, veterinarians can feel more confident in their decision not to treat, supporting antimicrobial stewardship.
What molecular tests can do for pets
Molecular diagnostics using PCR testing bring new opportunities to veterinary medicine. These tools are uniquely positioned to expand parasite testing to previously un- or under-detected parasites, as well as address new threats, such as emerging (and evolving) drug resistance, and pinpoint zoonotic risk.
A qPCR can read the genetic codes of parasites, which goes beyond simple dentification on a fecal test. Indeed, PCR can not only read genetic presence of drug resistance and zoonotic risk, but also expand understanding of life cycles and the research of parasites’ biology—this in turn, can result in new diagnostic opportunities and innovations. In the past two decades, molecular research on parasites led to the re-naming of parasites into new families, has subsequently proven to be of increasing sensitivity than other diagnostic tools, such as fecal flotation (O&P), and allowed for the formulation of novel control strategies and prevention of disease.
Parasitic infections are more common in cats and dogs than estimated in the recent past. The higher sensitivity of qPCR molecular tests, combined with the ability to detect parasites invisible and undetectable by microscopy, paints a picture of more frequent parasites in our pets—ones which were already present, but simply not visible to the human eye. Today’s parasites, characterized by rapid mutations and developing and expanding drug resistance, have successfully outpaced traditional screening and diagnostic capabilities, requiring more sophisticated tests that can go beyond simple identification. This further highlights the need for advanced testing, which allows assessment of a parasite’s drug susceptibility and zoonotic potential.
Molecular tests are best suited, as they can read the parasites’ inner making by accessing the DNA code in a highly specific way. Further, when these tests are designed with veterinary clinicians in mind (i.e. rapid turnaround time, small sample size, and increasingly affordable), they can lead the way to better identify and treat pets effectively, relieve staff of the work and time of performing fecal tests, and provide the highest quality of care on multiple levels for pets and their devoted owners.
Christian M. Leutenegger, BSc., DrMedVet, PhD, FVH, is vice-president of research and diagnostics at Antech Diagnostics and Imaging (Mars Pet Care Science & Diagnostics). A graduate of the University of Zurich Veterinary School of Medicine, Dr. Leutenegger completed a doctoral thesis on developing novel DNA vaccines for feline immunodeficiency virus (FIV). During his PhD, he developed a strong interest in molecular immunology and virology and started to develop early protocols for DNA quantification. In 1999, he founded the Molecular Core Facility at the University of California, Davis (UC Davis) School of Veterinary Medicine. In 2019, Leutenegger joined Antech Diagnostics to expand into molecular genetics and cancer diagnostics, and to advance its infectious disease detection platforms.
References
1 Drake J, Carey T. Seasonality and changing prevalence of common canine gastrointestinal nematodes in the USA. Parasites Vectors. 2019;12:430. doi:10.1186/s13071-019-3701-7 https://pubmed.ncbi.nlm.nih.gov/31488192
2 Jimenez Castro PD, Durrence K, Durrence S, Gianechini LS, Collins J, Dunn K, Kaplan RM. Multiple anthelmintic drug resistance in hookworms (Ancylostoma caninum) in a Labrador breeding and training kennel in Georgia, USA. J Am Vet Med Assoc. 2022;261:342-347. doi:10.2460/javma.22.08.0377 https://pubmed.ncbi.nlm.nih.gov/36520649
3 Leutenegger CM, et al. Emergence of Ancylostoma caninum parasites with the benzimidazole resistance F167Y polymorphism in the US dog population. Int. J. Parasitol. Drugs Drug Resist. 2023;14:131-140. https://www.sciencedirect.com/science/article/pii/S2211320723000015?via%3Dihub
4 Evason, MD, et al. Emergence of canine hookworm treatment resistance: Novel detection of Ancylostoma caninum anthelmintic resistance markers by fecal PCR in 11 dogs from Canada, Am J Vet Res (published online ahead of print 2023): https://doi.org/10.2460/ajvr.23.05.0116
5 Leutenegger CM, et al. Frequency of intestinal parasites in dogs and cats identified by molecular diagnostics. ACVIM abstract, Philadelphia, June 2023
6 Leutenegger CM, et al. Association of the novel benzimidazole resistance marker Q134H with F167Y in dogs with Ancylostoma caninum. ACVIM abstract, Philadelphia June 2023
7 Gilleard J. A novel Q134H isotype-1 Beta-tubulin putative benzimidazole resistance mutation is widespread in Ancylostoma caninum populations from pet dogs and greyhounds across the USA. University of Calgary. 67th annual meeting, AAVP. 25-28 June 2022.
8 Venkatesan A, Jimenez Castro PD, Morosetti A, Horvath H, Chen R, Redman E, Dunn K, Collins JB, Fraser JS, Andersen EC, Kaplan RM, Gilleard JS, 2023. Molecular evidence of widespread benzimidazole drug resistance in Ancylostoma caninum from domestic dogs throughout the USA and discovery of a novel b-tubulin benzimidazole resistance mutation. PLoS Pathogens, 2 March 2023. https://doi.org/10.1371/journal.ppat.1011146
