Canine Heartworm Testing

Heartworm (Dirofilaria immitis) disease in adult dogs in the United States increased 21.7% from 2013 to 2016, according to a recent American Heartworm Society (AHS) survey,¹ yet only about one third of the more than 70 million owned dogs are tested for the presence of adult D immitis.² Heartworm testing and prevention is an important component of preventive care in dogs, and proper completion of screening and diagnostic testing is essential. Dogs should be started on preventives no later than 8 weeks of age.

Animals acquire infection through the bite of an infected mosquito that deposits stage 3 larvae (L3) at the wound site. Within approximately 3 days after infection, the L3 molts to stage 4 larvae (L4) in the subcutaneous tissue. After weeks of migrating through tissue, larvae become sexually immature adults. Worms then enter the vascular system and reach the heart and pulmonary arteries. (See Figure 1.) Final maturation and mating occurs within the pulmonary vessels.³

Canine heartworm tests can detect infection with adult heartworms and immature larvae (microfilariae). Antigen tests detect proteins produced by adult female heartworms, whereas the modified Knott test is used to identify microfilariae in a blood sample. No test is currently available to detect adult male heartworms.

FIGURE 1

The heartworm life cycle. Printed with permission of the American Heartworm Society (heartwormsociety.org)

Testing Guidelines

Antigen tests detect a protein produced by adult female heartworms using an antibody specific to D immitis. Antigen tests are highly sensitive and specific and should be performed annually in dogs 7 months of age or older.³

The 2014 AHS Guidelines for the Prevention, Diagnosis, and Management of Heartworm (Dirofilaria immitis) Infection in Dogs recommends that heartworm antigen test results be recorded as positive or as no antigen detected rather than negative.³

Methods for microfilariae detection and identification include examination of fresh blood under a coverslip, presence of movement within a microhematocrit tube just below the buffy coat, and concentration testing (ie, modified Knott test, filtration).⁴ The modified Knott test, which is sensitive enough to yield consistent results even when concentrations of microfilariae are low, is the preferred concentration method.^4,5 The test may be performed in-house or sent to a reference laboratory.

To positively identify and confirm the microfilariae species, a modified Knott or filter test is required. Direct blood smear and the buffy coat method are unacceptable techniques for proper identification. 

Patients receiving year-round macrocyclic lactones (ML) should also be tested for microfilariae annually. False negative results on antigen tests can be seen in some dogs when immature worms or only a few worms are present and too little antigen is being produced to be detected. Antigen may also be hidden, even if enough is being produced, because antigen-antibody complexes within the sample can mask detection. Performing both tests annually helps identify infections that may not have otherwise been discovered.

Testing is warranted if a patient’s preventive treatment has lapsed or the administration history is unknown. Seven months is considered a reasonable interval when determining the predetection period or when infection may have occurred, so testing patients prior to 7 months of age is not necessary.⁴ Antigen tests can be positive as early as 5 months postinfection, whereas the same result for microfilariae may take 6 months.⁴ A patient receiving preventive could take up to 9 months to become antigen-positive.<sup4sup>

Not all heartworm-positive dogs have microfilariae. Every patient with a positive antigen test should be tested for microfilariae at the same time, especially those already receiving ML preventives.⁴ Microfilariae detection and identification validates a positive antigen test, allows for the identification of dogs at risk for a reaction to treatment with microfilaricides, and establishes which dogs are reservoirs for infection.⁴    

If antigen test results are positive but no microfilariae are detected, the antigen test should be repeated using another brand of kit.⁴  

If clinical signs of heartworm disease (eg, cough, exercise intolerance, weight loss) are present, additional diagnostics, including imaging studies (ie, radiography, echocardiography) may be warranted. A patient’s history should also be considered because other conditions such as congestive heart failure can cause similar signs.

Step-by-Step Instructions

Antigen Tests

Two types of point-of-care test kits are available: enzyme-linked immunosorbent assay (ELISA) and immunochromatographic.^6,7 Instructions for use vary, so the package insert should always be reviewed before performing the test. Keep the following tips in mind when using these antigen test kits:

• Store test kits appropriately. Double-check package instructions for storage of both test and conjugate. Some items may be stored at room temperature while others must be refrigerated. Check test kit expiration dates regularly and dispose of expired kits.⁷

• Use the appropriate sample for testing. Tests may be run using anticoagulated whole blood, serum, or plasma. Serum or plasma can be used fresh or stored at 35°F-46°F (2°C-8°C) for up to 1 week.^6,7

• Follow proper testing procedures. Some tests require that all components be at room temperature (64°F-77°F [18°C-25°C]) before use.^6,7 Make sure that conjugate lot numbers match lot numbers of tests being used—conjugates are not interchangeable.⁴ Set the timer for the correct amount of time and read the test promptly when the timer sounds. Failure to follow the instructions can lead to false results.^6,7

Modified Knott Test

The AHS recommends using the modified Knott test to detect and identify microfilariae. (See American Heartworm Society, above.) The test can be performed in-house or sent to an outside laboratory. 

For proper identification, the D immitis microfilariae (295-325 µm) should be measured using the calibrated ocular micrometer. Because of their likeness to the nonpathogenic Acanthocheilonema reconditum (250-288 µm), the filarid formerly known as Dipetalonema reconditum, the 2 must be differentiated. 

The following supplies are required^4,5:

• Personal protective equipment (eg, gloves) 

• 1 mL blood (in EDTA or heparin) 

• 9 mL 2% formalin 

• Centrifuge tubes 

• 1 drop methylene blue 

• Glass slide

• Coverslip

Follow these steps^4,5:

• Put on gloves.

• Add 1 mL anticoagulated blood (in EDTA or heparin) and 9 mL of 2% formalin to a new centrifuge tube. Cap the tube using a rubber stopper or parafilm, then mix the blood and formalin by inverting the tube several times.

• Remove the cap, place the tube in a centrifuge, and spin at 1500 rpm for 5 minutes, using a balance tube if necessary. 

• Pour off the liquid, leaving only the sediment. 

• Add 1 drop of 0.1% methylene blue to the sediment and use a pipette to mix the stain with the sediment.

• Place the stained sediment on a glass slide and cover with a coverslip.

• Use the 10× objective lens of a microscope to examine the slide for microfilariae. If microfilariae are noted, use the 40× objective to observe the specific characteristics of the larvae and the calibrated ocular micrometer to measure the length of the larvae. 

  • D immitis: tapered head, with straight body and tail, 295 to 325 µm

  • A reconditum: blunt head and curved tail, 250 to 288 µm

Conclusion

Dogs infected with D immitis often display no clinical signs. Annual testing can identify heartworm infection in its early stages when treatment provides a more positive prognosis. Veterinary nurses should be familiar with the indications, timing, and proper steps for performing each type of heartworm test to ensure that test results are accurate and patients receive the appropriate treatment.