Evaluating apparent discrepancies in heartworm test results

A 10-year-old female dog presented to its veterinarian for routine exam, vaccination and heartworm testing. It was estimated that the dog had been off heartworm preventative for more than six months.  In-clinic testing revealed the presence of microfilariae on blood drop analysis, but was negative on an in-house antigen test. TVMDL received whole blood (EDTA) and serum for further testing which included performing a Modified Knott’s test on the whole blood and a heartworm antigen test on the serum. The Modified Knott’s test revealed the presence of numerous microfilariae (approximately 4 per LPF) that were confirmed to be Dirofilaria immitis. TVMDL’s standard procedure upon finding microfilariae is to perform morphological analysis to determine whether the larvae are those of Dirofilaria immitis or Acanthocheilonema reconditum (formerly Dipetalonema reconditum), as larvae from either parasite can be found in the blood. A. reconditum, another filarioid, is considered non-pathogenic. The adults live in the skin while the microfilaria circulate in the blood stream. Information in the chart below can be used to help distinguish the two species of larvae.

TVMDL uses a well-based heartworm antigen ELISA test kit to detect D. immitis antigen in both dogs and cats. Antigen tests are designed to detect antigens of mature female heartworms. Dogs usually have a higher adult worm burden than cats and, unlike cats that frequently have a single-sex infection, dogs typically have a mixed sex infection. A positive antigen test is expected in dogs that harbor adult Dirofilaria immitis, yet TVMDL’s ELISA heartworm antigen test was negative, appearing to support the results of the in-house test performed at the referring clinic.

An occult heartworm infection (i.e. positive antigen test with no observable microfilariae) is a relatively common finding, either due to the elimination of microfilariae through the continued use of macrocyclic lactone preventatives, or because of the cyclic nature of microfilaremia in which larvae are not always observed in peripheral blood analysis.¹However, it is rare to have confirmed D. immitis microfilariae without adult heartworms, unless adults have recently died off, such as when a dog has undergone adulticide treatment. Because of the test result discrepancies in this case, TVMDL performed a heat-treated analysis of the serum which proved to be positive for heartworm antigen. This result was confirmed by another laboratory which routinely performs the heat-treatment procedure.

Previously, heat-treating serum samples was a manufacturer required step when using ELISA heartworm antigen test kits. However, for various reasons, manufacturers dropped this requirement and routine testing is now performed on non-heat-treated serum samples.² Heat treatment of samples has made a comeback within the last several years, primarily in response to the widespread use of the “slow-kill” heartworm treatment method. This “treatment method”, which is not endorsed by the American Heartworm Society, advocates the use of macrocyclic lactone preventatives in heartworm positive dogs, not only to prevent future D. immitis infections, but to eventually eliminate both microfilaria and adult worms with continued use over several years. It has been theorized that the slowed death of the adult worms and ongoing inflammatory response associated with this practice promote the creation of antigen-antibody complexes. These complexes result in antigen-binding in situ, in essence blocking the antigen, and rendering it incapable of reacting with the assay. Heat treating the sample prior to testing helps to break these complexes, allowing the antigen to be freed and to bind with the reagents of the test.³

In accordance with the American Heartworm Society guidelines⁴, TVMDL does not routinely heat-treat serum samples used to screen for heartworm antigen.  However, in those cases where adult heartworms are strongly suspected (i.e. presence of microfilariae, clinical signs, etc.) but a patient tests negative by conventional antigen testing, heat-treating the serum sample may be beneficial in confirming a diagnosis of heartworms.

For more information about TVMDL’s test catalog, visit tvmdl.tamu.edu.

REFERENCES

1. AM Ionica, et al., Dirofilaria immitis and repens show circadian co-periodicity in naturally co-infected dogs, Parasites & Vectors, 2017; 10:116 (Published online 2017 Feb 28. doi: 10.1186/s13071-017-2055-2.
2. B Blagburn, 15^th Triennial Heartworm Symposium, Sept 2016; www.American Heartworm Society.org
3. J Drake, et al., False negative antigen tests in dogs infected with heartworm and placed on macrocyclic lactone preventatives, Parasites & Vectors, 2015; 8:68 (doi: 10.1186/s13071-015-0698-4).
4. American Heartworm Society, Current Canine Guidelines for the Prevention, Diagnosis, and Management of Heartworm (Dirofilaria immitis) infection in Dogs, 2018.