Canine Heartworm Antigen Tests

Does a negative test mean a dog does not have heartworm and does a positive test mean the dog is infected with heartworms?

By Dr. Michael W. Dryden

Canine Heartworm Disease (CHD) caused by Dirofilaria immitis continues to be a commonly diagnosed parasitic disease in the United States. It is now considered at least regionally endemic in all states, except Alaska, with over 250,000 dogs being diagnosed annually.

Given the large number of dogs being infected annually and the known medical risks of infection (6 – 13” long worms in the pulmonary arteries and right heart), routine annual screening is commonly conducted in practices. Additionally, dogs presenting to the clinic with symptoms indicative of CHD (cough, exercise intolerance, rapid breathing, ascites, cyanotic mucous membranes, ataxia, heart murmur, tachycardia, etc..) must have the presumptive diagnosis confirmed by appropriate diagnostics.

For at least two decades the most commonly used diagnostic tests have been antigen tests. All currently available antigen tests (ELISA, Immunochromatographic and Hemagglutination) are designed to detect heartworm antigen circulating in the blood. While the specific antigens that are being targeted are proprietary, it is reported that all antigen tests detect a protein produced by mature female worms (likely a uterine antigen) and currently there are no USDA licensed serologic tests that can detect male D. immitis.

While currently available antigen tests are highly sensitive and highly specific, there are limitations that must be taken into consideration before telling a dog owner that their dog is or is not infected with D. immitis.

1. Circulating heartworm antigen appears in the blood as early as five months post-infection in a small percentage of dogs, but most dogs are not antigen positive until seven months post-infection. Yes, contrary to popular belief, a dog infected six months previously can be negative on an antigen test.
2. A low worm burden can markedly affect a test’s sensitivity. These tests will detect dogs infected with one – two female worms only 60 – 70 percent of the time. Such dogs, while often asymptomatic, can either have negative tests, positive tests or inconsistently positive tests.
3. Recent studies have documented that antigen tests may not test positive in up to 7% of dogs due to the occurrence of “antigen-antibody complexes” that are formed in the dog’s blood. These complexes bind the circulating antigen so that it is “unavailable” to react on the antigen tests. These dogs will only test positive after specific heat or acid treatment is applied to the dog’s serum sample to dissociate the immune complexes, neither of which can be accomplished in a veterinary practice.
4. False positives, while uncommon can also occur a) in well-type tests due to inadequate washing, b) due to residual circulating antigen post-adulticide treatment, and c) cross reaction with Spirocerca lupi or other unknown antigens. While the esophageal nematode S. lupi is most commonly encountered in the Southern states, such as Louisiana, this author has recently consulted on cases in Kansas, Nebraska and Missouri, one of which was positive on an antigen test.

Now even with the abovementioned limitations, parasitologists are not recommending that practices stop using these tests for clinical diagnosis or routine screening. They are the best diagnostic tests available. What is being recommended is that veterinarians learn to appreciate the limitations of the tests and augment their diagnostic procedures.

For routine annual screening we need to be conducting an antigen test and also go “old school” and look for circulating microfilariae (Knott’s test, filter test, or even a direct smear) in the blood.

For dogs with clinical signs indicative of CHD, conduct not only an antigen test, but also a test to recover microfilariae, chest radiographs and a CBC. If both the antigen and microfilariae tests are negative (remember 20+% of dogs with CHD do not have circulating microfilariae - occult infections) in a dog with clinical signs of CHD, consider submitting 1.5 to 2ml of serum to a diagnostic service that will conduct heat (KSVDL and others) or acid (IDEXX) immune complex dissolution.

Remember that the American Heartworm Society (https://www.heartwormsociety.org/) does not recommend testing a dog with an antigen test until at least six months post-treatment due to residual circulating antigen.

It is recommended that in antigen-positive, microfilariae-negative asymptomatic dogs, adulticide therapy should not be instituted until the antigen result is verified on a different manufacturer’s antigen test.

Finally, if the heartworm antigen test is negative this does not always mean the dog is not infected with D. immitis. What the test is telling us is that there is not enough circulating antigen to be detected, which could be because the dog is truly negative, only male worms present, low female worm burden, immature (prepatent) infection, test error or immune complexes binding the antigen.

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