Turning Up the Heat on Heartworm Diagnosis

Antigen testing for Dirofilaria immitis has been a foundational component of model preventive veterinary care for many years, particularly for dogs. For privately owned pets, the results of such testing guide prevention strategies and, in the event of a positive result, treatment for heartworm disease. In shelter populations, the results are often a key determining factor in the management of a dog throughout its stay in the shelter system, including its likelihood of a live release.

The Heartworm Hotline column is presented in partnership between Today’s Veterinary Practice and the American Heartworm Society (heartwormsociety.org). The goal of the column is to communicate practical and timely information on prevention, diagnosis, and treatment of heartworm disease, as well as highlight current topics related to heartworm research and findings in veterinary medicine.

Although the accuracy of commercially available D immitis antigen test kits has been widely studied, recent reports have sparked renewed interest in the effect of antigen–antibody immune complexes on test results.^1–6 Such complexes can interfere with antigen detection, resulting in “no antigen detected” (NAD) test results in infected animals, and should be considered when NAD test results conflict with clinical expectations.

What Are Immune Complexes?

Immune complexes represent soluble antigen bound to endogenous antibody, forming an insoluble unit that remains in circulation. Such complexes are a normal component of a functioning immune system and are cleared by phagocytosis when the balance of antigen to antibody in circulation is maintained. Excess immune complexes in circulation can result in tissue deposition, leading to local inflammatory responses and a variety of autoimmune diseases. Vasculitis, glomerulonephritis, pneumonitis, and arthritis are common sequelae of immune complex deposition in the respective affected organ system. In the case of diagnostic testing methods that rely on soluble antigen for detection (eg, enzyme-linked immunosorbent assay, lateral flow assays), antigen bound in an immune complex may not be available for detection, leading to an NAD result despite the presence of antigen in the test sample.

A variety of techniques can be used to dissociate circulating immune complexes in a diagnostic sample. Such techniques rely on denaturing proteins within the complex, allowing for precipitation of antibodies and subsequent freeing of the antigen (Figure 1). In laboratory settings, proteolytic enzymes (eg, pepsin), acid treatment (eg, ethylenediaminetetraacetic acid, citric acid), heat (104°C for 10 minutes), or a combination of these methods is frequently used for immune complex dissociation (ICD).

FIGURE 1. Immune complex dissociation with sample pretreatment. Figure used with permission from IDEXX Laboratories, Inc. Ag, antigen; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay.

Heat pretreatment (HPT) of serum samples was standard practice in veterinary diagnostic laboratories through the mid-1990s and is still available upon request. However, the demand for simple, cost-effective, commercially available, point-of-care test kits led to its decreased use.

How Does Heat Pretreatment Affect Diagnosis of Heartworm Infection?

In recent years, the effect of ICD in the form of HPT has been studied in diagnostic samples from cats and dogs.^1–6 Such reports have demonstrated substantial increases in antigen detection in both species, resulting in greater diagnostic sensitivity (Table 1); however, the antigen detected cannot be identified as coming from living or dead heartworms. It follows that in dogs with heartworm disease that have received adulticidal therapy, a positive result on an antigen test with ICD does not indicate that the therapy was unsuccessful. Regardless of whether HPT is used in post-treatment testing, dogs should be tested for both antigen and microfilariae 6 to 12 months after completion of adulticidal therapy to assess treatment efficacy.

TABLE 1 Effect of Heat Pretreatment on Antigen Detection

[ultimatetables 2 /]

Many factors can affect detection of heartworm antigen in samples, leading to NAD results in truly infected dogs. They include the stage of infection, concurrent medications (including doxycycline and heartworm preventives), and microfilarial status of the animal (Table 2).

TABLE 2 Effect of Heat Pretreatment on Clinical Factors

[ultimatetables 3 /]

Stage of Infection

Although detection of D immitis is typically expected 7 months after infection (with earliest detection 5 months after infection),⁷ some evidence suggests that HPT may allow earlier antigen detection.

—  In one study of experimentally infected dogs, 100% of infections were detected after HPT of test samples obtained 4 months after infection.⁸ When samples were obtained 5 months after infection, only 42.6% of infections were detected in samples without HPT, while 100% of those infections were identified after HPT.⁸

—  In another study of experimental infection in dogs, positive antigen test results were obtained 31 to 36 days sooner in heated (days 127 to 132 after infection) versus unheated (day 163 after infection) serum samples.⁹

— In a study of experimental infection in cats, detection of heartworm antigen was possible as early as 5.5 months after infection when samples underwent HPT.⁵

The potential for earlier detection of heartworm antigen after HPT of samples from recently infected animals is theorized to be the result of a more robust immune response early after infection, along with the lower level of antigen produced by nongravid female worms, which both lead to greater immune complex formation and subsequent antigen blocking.

Heartworm Treatments

Several studies have suggested that the administration of macrocyclic lactones and/or antibiotic therapy (eg, doxycycline) can affect immune complex formation and subsequently interfere with antigen testing.

—A study of 19 naturally infected dogs being managed with monthly topical 10% imidacloprid plus 2.5% moxidectin along with oral doxycycline (10 mg/kg q12h for 30 days, every 6 months) demonstrated a substantial variation in timing of antigen detection between HPT and non-HPT samples.¹⁰ Among dogs that initially tested negative with non-HPT samples, antigen was detected in 50%, 95%, and 100% of dogs tested with HPT after 6, 12, and 18 months of therapy, respectively.

— Another report of 29 shelter dogs with antigen detection after HPT demonstrated 3.8 times greater odds of immune complex interference with test results when a history of macrocyclic lactone administration was reported.²

— Fifty-three percent of a cohort of privately owned dogs that had a negative antigen test result and received monthly macrocyclic lactones and doxycycline had detectable antigen after HPT of serum samples.³

There are a few possible explanations for the influence of macrocyclic lactone and doxycycline administration on immune complex interference with diagnostic test results. Although doxycycline has some anti-inflammatory activity, administration of macrocyclic lactones and doxycycline in dogs with active heartworm infection is not adulticidal in the short term. The persistence of live heartworms allows for continued antigenic stimulation, subsequent inflammatory response, and antibody production in the face of a decreasing antigen load. Secondarily, use of these medications often results in sterilization of the female worms and subsequent decrease in antigen release. These factors could contribute to a relative antibody excess, disrupting the normal clearance mechanisms and promoting continued immune complex creation.

Presence of Microfilariae

The presence of detectable circulating microfilariae in canine blood samples also appears to influence the interference of immune complexes with antigen detection. Circulating microfilariae contribute to the chronic inflammatory response of heartworm disease and are therefore theorized to contribute to continued antibody production, allowing continued immune complex formation. One report of 26 shelter dogs with circulating microfilariae demonstrated 32 times greater odds of converting to a positive antigen test result after HPT compared with dogs that tested antigen negative both before and after HPT.² Other reports have also identified circulating microfilariae in dogs whose test results converted from negative to positive after HPT.^1,3,4 Interestingly, one of these reports included microfilariae of D immitis, Dirofilaria repens, and Acanthocheilonema reconditum,¹ which suggests the potential for decreased specificity of antigen tests after HPT.

Can I Perform Heat Pretreatment in My Clinic?

A variety of veterinary diagnostic laboratories offer HPT panels upon request. In general, costs are minimal (<$30) and results are available within 1 to 3 days. Perhaps the biggest benefit of using diagnostic laboratories for HPT is the consistency and reliability of sample handling and testing techniques. Testing methods are typically validated and concomitantly run with positive controls to ensure accuracy of results.

Diagnostic laboratory services are preferred, but when these are not available or feasible, a simplified HPT protocol has been successfully used (Box 1, Figure 2). In a study of shelter dogs, 616 samples underwent the simplified HPT protocol.² Of these, 13 samples could not undergo repeat testing after heating. Five “untestable” samples were presumed to be directly related to inconsistencies in the application of heat, resulting in solidification of the serum sample. The remaining 8 samples were untestable for a variety of reasons presumably unrelated to the heating protocol (eg, insufficient serum, invalid controls).

Box 1. Simplified Heat Pretreatment Protocol

1. Dilute serum sample with approximately equal volume of 0.9% NaCl.
2. Place ~250 mL tap water into 500-mL glass beaker.
3. Microwave beaker to the point of boiling (approximately 2 min in 1000 W).
4. Remove heated water from microwave and place tube with diluted sample in the heated water for 10 minutes. (Note: Place serum in a glass collection tube and remove the rubber stopper before heating. Some warping of tube is expected.)
5. Repeat antigen test.

FIGURE 2. Diluted serum samples undergo a simplified heat pretreatment protocol. Note the use of glass tubes and the absence of rubber stoppers.

When Should I Consider Heat Pretreatment?

Heat pretreatment is likely not indicated in most heartworm screening test scenarios. It should be considered when screening test results conflict (eg, antigen negative and microfilariae positive), when patients are receiving an alternative treatment protocol (eg, macrocyclic lactone and/or doxycycline), when the patient has known chronic inflammatory diseases (eg, pyoderma, otitis, endoparasitism), or when dogs test negative but originate from a known heartworm-endemic region and have a history of lapsed or no preventive administration. An NAD result on a pretreated sample by a diagnostic laboratory can also rule out suspicion of heartworm infection.

In short, clinicians should consider HPT—as well as clinical staging with physical examination, complete blood count, blood chemistry analysis, urinalysis, and radiography—whenever there is a strong clinical suspicion of heartworm disease in the presence of negative screening test results. One report identified heartworm antigen in 64.7% of “negative” samples from patients for which the veterinarian’s clinical suspicion strongly supported heartworm infection.

The bottom line: There is no substitute for the clinical acumen of a veterinarian.

Summary

Immune complex interference is one factor clinicians should consider when interpreting the results of diagnostic tests that rely on antigen detection, especially when screening test results do not match clinical suspicions. Recent research has provided some insight into factors that can affect screening test results while identifying specific scenarios that may justify the added step of HPT of serum samples. These findings highlight the importance of adhering to the American Heartworm Society’s diagnostic testing recommendations. Annual screening for both antigen and microfilariae is the best way to identify heartworm infection as early and as consistently as possible.