Using a polymerase chain reaction (PCR) test to diagnose benzimidazole-resistant hookworms

Veterinarians can use a polymerase chain reaction (PCR) test to diagnose benzimidazole-resistant hookworms and reduce the danger of a One Health crisis that could impact animals and humans.

By Emily Singler

As I wrote about in an earlier article on the diagnosis of multi-anthelmintic drug resistant (MADR) hookworms, surveillance testing and research have revealed that what was once thought of as a problem only in Florida, and only with greyhounds, is much more far-reaching in terms of both geographical locations and breeds of dog.   

According to Michelle Evason, BSc, DVM, DACVIM, MRCVS, global director of veterinary clinical education for Antech (part of Mars Science and Diagnostics), MADR hookworms have been found in multiple breeds of dog and locations throughout the United States and are now endemic in parts of Canada as well.1,2  

This is concerning because MADR hookworms can wreak havoc on both companion animal and farm animal species as well as humans, making it a true One Health crisis. For this reason, research has focused on ways to identify and treat cases of MADR hookworms in animals. 

What is benzimidazole resistance?  

Benzimidazoles are a class of anthelmintics that include febantel, fenbendazole, oxfendazole, and flubendazole, among others.  

The marker for benzimidazole resistance, named F167Y, was found in farm animal species first and later identified in dogs. The selection pressure on bovine and ovine species (among others) of being housed in large groups and repeatedly dosed with anthelmintics as a herd instead of based on individual weights is similar to what is widely believed to have happened in racing greyhounds, where MADR hookworms were first documented.   

The benefits of PCR testing  

A polymerase chain reaction (PCR) test has now been developed that can identify markers for benzimidazole resistance in hookworms. Keyscreen, a PCR test created and offered by Antech diagnostics, can identify the benzimidazole resistance markers F167Y and Q134H in fecal samples, as well as genetic material from 20 common canine and feline intestinal parasites, and a marker for the potentially zoonotic giardia assemblage A &B. 

When compared to other available fecal parasite tests, the PCR test has several advantages, Evason explains.  

The KeyScreen PCR test is a rapid, commercially available, and affordable test that can identify benzimidazole resistance, and is also a superior test to centrifugal fecal flotation (O&P) for detecting hookworms and other intestinal parasites in general.3  

“I think we now all appreciate that passive fecal flotation is a poor test,” Evason said, “because of the high potential for false negatives and misidentification.”  

While a fecal test that involves zinc sulfate centrifugation increases the accuracy of the results, it still doesn’t compare to PCR. Tests that involve visual identification of eggs or the parasites themselves carry risks of uncertainty, including false negatives and false positives.  

PCR, on the other hand, is “super helpful,” Evason said, because “either the genetic material of the parasite is there, or it’s not.”  

Evason recommends following CAPC recommendations and AAHA guidelines for routine fecal testing/screening schedules in adult dogs and cats, along with puppies and kittens.  

She notes that many practices have moved to using the Keyscreen test for routine fecal screening and as a diagnostic test for patients with clinical signs like diarrhea. 

Where further work is needed 

At this time, many markers for resistance to other classes of anthelmintics have not yet been identified—and, aside from KeyScreen, are not commercially available—although this is an area of active research.  

Evason believes that finding additional markers and making these easily available to veterinarians will not be a straightforward or rapid process.  

“It is nothing short of miraculous to have a commercial test option for benzimidazole resistance,” she said, calling the discovery of additional markers for resistance a “pretty steep hill for researchers and labs.” 

Emodepside resistance 

Some cases of resistance to emodepside, the anthelmintic whose extra-label use has been described to successfully treat some cases of MADR hookworms, have been reported. This is hugely concerning, as there are only so many drugs available to treat these resistant worms in animals and humans.  

Accordingly, research is ongoing to look for other drug options that may work. However, this will take time.  

At the practitioner level, “the biggest thing is awareness of hookworm resistance,” Evason said. Veterinary professionals need to have MADR on their radar, no matter where they practice or which breed of dog they are treating.  

They also need to continue to practice responsible antimicrobial stewardship in their use of anthelmintics. This means dosing anthelmintics appropriately for their patients, It also means judicious use of drugs like emodepside, and use of a clinical decision-making algorithm 4,5 or connecting with experts, to avoid selecting for resistance to in parasites that affect both animals and humans.  

With the discovery of hookworm resistance, Evason said it makes sense for us to be nervous about the ramifications and to stay vigilant.  

The research, advocacy, education, and preventive medicine techniques we practice in veterinary medicine will be key to safeguarding human and animal health.  

“We as a profession have to do our part.”  


  1. Leutenegger CM, et al. “Screening for the Ancylostoma caninum Benzimidazole resistance marker F167Y reveals widespread, geographic, seasonal, age and breed distribution in North America,” International Journal for Parasitology, vol. 24 (April 2024):
  2. 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 (vol. 84: Issue 9):
  3. Leutenegger CM, et al. “Comparative Study of a Broad qPCR Panel and Centrifugal Flotation for Detection of Gastrointestinal Parasites in Fecal Samples from Dogs and Cats in the United States,” Parasites & Vectors (16: Article 88, 2023):
  4. Jimenez Castro PD, Kaplan RM. “Persistent or suspected-resistant hookworm infections,” Clinician’s Brief (Updated July 2020. Accessed November 16, 2023):
  5. Jimenez Castro PD, Kaplan RM. “Persistent hookworm infections in dogs,” Clinician’s Brief (Updated July 2020. Accessed November 16, 2023): 


Further reading 

Hookworm Anthelmintic Resistance: Novel Fecal Polymerase Chain Reaction Ancylostoma caninum Benzimidazole Resistance Marker Detection in a Dog (JAAHA 

Hook Before you Treat! Drug-Resistant Hookworms in North America (Today’s Veterinary Practice) 


Cover photo credit: © baranozdemir E+ via Getty Images Plus  

Disclaimer: The views expressed, and topics discussed, in any NEWStat column or article are intended to inform, educate, or entertain, and do not represent an official position by the American Animal Hospital Association (AAHA) or its Board of Directors. 




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