Top 5 Tips for Interpreting Heartworm Test Results

Andrew R. Moorhead, DVM, MS, PhD, DACVM (Parasitology), University of Georgia

Cassan N. Pulaski, DVM, MPH, PhD, University of Georgia

ArticleLast Updated March 20226 min readPeer Reviewed
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It is commonly believed that heartworm disease can be diagnosed based on a simple positive or negative in-clinic test result; however, this is not always true when testing dogs and is less accurate in cats. Interpretation of a heartworm antigen test is only one component of an accurate heartworm diagnosis; a variety of testing modalities may be required. Testing for the presence of antigens and microfilariae is recommended in dogs; diagnosis in cats usually requires additional testing (eg, antibody testing, radiography; Table).

Following are the top 5 scenarios, according to the authors, encountered when diagnosing heartworm disease in dogs and cats.

1. Microfilariae-Positive; No Antigen Detected

Occult infections (ie, presence of adult worms without circulating microfilariae) can occur in up to 20% of dogs with heartworm disease1 and are likely a result of immune-mediated clearance of microfilariae or drug treatments. Dogs can also be microfilariae-positive but have no detectable antigens. This can occur for various reasons, including infection with a different filarial parasite, blood transfusion from an infected dog, or antigen blocking, in which the heartworm antigen is presumably bound by host antibodies, masking the antigen from antibodies in the testing kit.2 In these patients, a fresh sample should be collected and submitted for further investigation. 

Additional testing may include a modified Knott’s test or molecular modalities to differentiate Dirofilaria immitis from other species (eg, Acanthocheilonema reconditum) or antigen testing with heat-treated serum at a reference laboratory (high heat can break antigen–antibody complexes, leaving free antigens for detection).3 If a microfilariae-positive, antigen-negative dog seroconverts to antigen-positive after heat treatment, then heartworm adulticide therapy is warranted. Because of these phenomena, testing dogs for both antigen and microfilariae is recommended.






Microfilariae testing


Detects microfilariae via direct examination of a drop of fresh blood or concentration (ie, modified Knott’s test)

Direct examination may be insensitive if low microfilariae count; modified Knott’s testing requires formalin and time

Antigen testing

Dog, cat

Detects antigen produced by adult female worms or dying male and female worms

Does not detect larvae or immature worm infections; rarely detects male-only infections; results may be affected by antigen–antibody complex formation

Antibody testing


Detects antibodies produced by a cat in response to presence of heartworm larvae, immature adults, or adult worms

Studies have shown significant false-negative results

Thoracic radiography

Dog, cat

Detects vascular enlargement, pulmonary parenchymal inflammation; reliable method of assessing severity of cardiopulmonary disease in dogs

Radiographic signs can be subjective and affected by clinical interpretation


Dog, cat

Detects echogenic walls of immature or mature worms, providing definitive evidence of heartworm infection; also allows for assessment of cardiac anatomic and functional consequences of disease

Not an efficient method of diagnosis, particularly in lightly infected patients; accuracy rate influenced by ultrasonographer experience with heartworm detection

2. Antigen-Positive After Melarsomine Treatment

Although treatment with melarsomine is typically highly effective, a small number of worms may survive and cause the host to remain antigen-positive, despite adherence to the recommended 3-dose adulticidal protocol. In addition, some dogs may have been given a dose that is too small (patient weight and drug dose should be checked before each injection) or an inappropriate melarsomine regimen may have been used (eg, treatment timing was incorrect, 2 doses were given instead of 3). 

Dead and dying worms can release detectable antigens, which can persist for some (unknown) time following treatment.3 The American Heartworm Society (AHS) currently recommends retesting dogs 9 months posttreatment; the 2-dose melarsomine regimen (ie, 2 doses 24 hours apart) is recommended if the patient is still antigen-positive.3

3. Slow-Kill/Salvage Treatment in Antigen-Negative Dogs

Although the AHS treatment protocol using melarsomine is recommended in dogs with heartworm disease, long-term administration of a macrocyclic lactone and doxycycline is an alternative, extra-label adulticidal treatment. The salvage protocol requires a significantly extended treatment timeline compared with the AHS protocol, and, although it can be effective, time needed to kill or eliminate adult worms varies between protocols and can be prolonged.4 It is uncertain when dogs receiving the salvage protocol are considered heartworm-free, as antigens can be bound by the host’s antibodies, resulting in no antigen detected. This has been documented in dogs undergoing a salvage protocol5; however, in a study, no adult worms were found during necropsy in patients in which no antigen was detected after heat treatment of serum.6 No antigens should be detected after heat treatment before patients are considered truly negative.

4. Positive or Negative Heartworm Infection in Cats

Determining heartworm status of a cat can be difficult, and multiple clinical aspects and diagnostic approaches should be considered, including patient history, physical examination, serum chemistry profile, hematology, immunodiagnosis (both antigen and antibody testing), thoracic radiography, and echocardiography.7 Testing for microfilariae is not always helpful or recommended because of the transient nature of microfilaremia in cats; >95% of heartworm infections in cats are occult.8 Heartworm antigen testing in cats has similar (and additional) challenges as with dogs, including reduced test sensitivity and the possibility for antigen–antibody immune complex formation.3 

A combination of antigen (possibly using heat-treated serum if the patient shows clinical signs but results are discordant) and antibody (when available) testing is currently recommended, but a negative antigen test result does not exclude infection, and a positive antibody test result only indicates exposure to heartworms (ie, larvae).7 A positive antigen test result (before or after heat treatment) is a strong indicator of active heartworm infection and may warrant supportive treatment (eg, corticosteroids, antihistamines).9

5. Newly Antigen-Positive

In both dogs and cats, a positive antigen test result should be confirmed prior to diagnosing heartworm disease and beginning treatment. The easiest way to confirm a positive antigen test result is with detection of D immitis microfilariae; however, microfilariae may not be present in a heartworm-positive dog (and are highly unlikely to be present in a heartworm-positive cat). In these patients, additional antigen testing should be pursued for confirmation using a fresh blood sample and an alternative testing platform (eg, different manufacturer, reference laboratory). Heat treatment is not needed in antigen-positive cats. Heat treatment is, however, recommended in cats with clinical signs but no antigen detected or microfilariae-positive and antigen-negative cats.


Diagnosing heartworm disease can be challenging. It is critical to use all resources available and seek continued education on updates to the AHS guidelines.