Diagnosis of Canine Heartworm Infection
Margaret C. Barr, DVM, PhD; Elizabeth P. Boynton, DVM; Peggy L. Schmidt, DVM, MS, Diplomate ACVPM; Frank Bossong, DVM; and Gary R. Johnston, DVM, MS, Diplomate ACVR
Dirofilaria immitis, the causative agent of canine heartworm disease, induces significant cardiopulmonary pathology in dogs in the United States and worldwide.
Although highly effective methods are available to prevent infection in domestic dogs, D immitis has continued to spread throughout the U.S. Recent estimates of prevalence in dogs presented for veterinary care, based on a national clinic-based survey, range from 0% in a few Western and Midwestern U.S. areas to almost 7% in some regions of the Southeastern states along the Mississippi River and the Gulf Coast.1
Factors influencing the spread of canine heartworm (CHW) include:
- Increased movement of domestic dogs across the U.S.
- Fewer mosquito and vector control programs in response to concerns about pesticide safety
- Lack of compliance with prevention programs
- Increased infection of nondomestic canid reservoir hosts, such as the American coyote.2
While dogs are considered to be definitive hosts for heartworms, D immitis may infect many different species, including indigenous canids, such as wolves, foxes, and coyotes; many species of felids; and occasional incidental hosts, such as deer, ferrets, raccoons, horses, and primates.
The noncanid species are typically dead-end hosts; however, even a single heartworm in some animals may cause significant pulmonary disease, especially in cats.2,3 In humans, dying adult worms are associated with the development of pulmonary granulomas, which may be misdiagnosed as lung cancer on thoracic radiographs.3
DIAGNOSIS OF INFECTION
In-clinic diagnosis of CHW infection is typically accomplished through detection of circulating microfilaria or soluble CHW antigen, and is dependent upon allowing adequate time from potential infection date to test date to account for the prepatent period (see Life Cycle of Heartworms). Because of this lag time, a general rule of thumb is to wait until a dog is 7 months of age before testing for the first time.4
Life Cycle of Heartworms
1. The life cycle of a patent CHW infection starts with introduction of L3 larvae from an infected mosquito into the canid host.
2. In the dog, the larvae mature into L4 larvae and then into adult worms that localize in the pulmonary arteries and occasionally in the right cardiac ventricle.
3. Following a prepatent period of 6 to 7 months, microfilariae are produced via sexual reproduction and are released into the dog’s bloodstream, where they may remain viable for months to years.
4. Mosquitoes feeding on the infected host ingest the microfilaria, which then migrate to the Malpighian tubules where they mature into first-stage larvae.
5. Ultimately, the larvae mature to the L3 stage in the salivary glands of the mosquito and become infective, thus completing the heartworm life cycle.
When to Test
Periodic testing, usually yearly, is recommended for all dogs, and it is advisable to retest whenever changing chemoprophylaxis agents or starting prophylaxis after a period of noncompliance. When changing heartworm preventive medication, additional testing 4 months after switching products is required to evaluate the efficacy of the original product. This process is especially important in light of recent suspicions of growing CHW resistance to macrocyclic lactones in some regions of the U.S. (see Prophylaxis Update).
Chemoprophylaxis is the primary weapon available to prevent heartworm infections in individual animals and controls spread of this parasite by reducing infections in the primary reservoir population. Macrocyclic lactones (ivermectin, moxydectin, selamectin, and milbemycin oxime) are the most commonly used heartworm preventives. The American Heartworm Society’s current guidelines4 for control of CHW infections in dogs include:
- Begin CHW prophylaxis in puppies as early as possible, usually before 8 weeks of age.
- Year-round chemoprophylaxis (even in areas where cold weather interrupts the CHW life cycle) is highly recommended to increase compliance.
- Although collies and some other dogs are sensitive to macrocyclic lactones, commercially available products for dogs used at the recommended doses are considered to be safe in all breeds.
- Reports of lack of efficacy of macrocyclic lactones in some areas of the U.S. are being investigated; currently, there is no strong evidence of widespread emergence of resistant strains of CHW. Lack of treatment and lack of compliance are of much greater concern.
The issue of apparent lack of efficacy is yet to be resolved. A recent re- port detailed genetic changes in some populations of heartworms that may confer greater resistance to chemoprophylactic agents.5 Whether or not these changes translate to the emergence of macrocyclic lactone-resistant strains of heartworms in endemic populations remains to be seen.
Microfilaria may be visualized in whole blood using a direct smear technique or from blood concentrated using a modified Knott’s test or membrane filtration. Compared to other methods of heartworm detection, blood smears and concentration techniques are relatively insensitive; therefore, a test should not be considered negative until at least 1 mL of blood has been examined.4 However, the sensitivity of the modified Knott’s test is greater than that of the direct smear method when samples contain < 50 microfilaria per 1 mL of whole blood6 and the test provides enhanced ability to distinguish D immitis from other canine microfilaria.
Microfilaria tests are now viewed as supplemental assays, secondary to the more sensitive heartworm antigen tests. Microfilaria detection can be used to:
- Validate a positive antigen test
- Determine if the dog is a potential reservoir for infection
- Identify dogs at risk for severe reaction to treatment with microfilaricides.
Limitations of microfilaria tests include:
- The fact that about 20% of heartworm-infected dogs are not microfilaremic (occult infection), usually because only 1 sex of worm is present or the infection is prepatent.4
- The use of macrolide preventives may result in clearance of microfilaria from the blood of treated dogs after several months of use.2,4
Enzyme-linked immunosorbent assay (ELISA) technology (Figure 1) was used in the first in-clinic CHW antigen tests; however, several current in-clinic CHW antigen tests are immunochromatographic (ICT) assays (Figure 2).
1. Anti-CHW antibodies (Ab) linked to an enzyme are mixed with whole blood, plasma, or serum, allowing them to bind to heartworm antigens (Ag) in the test sample.
2. The conjugate–sample mixture is then applied to the sample port (S) and allowed to flow across a membrane on which spots of additional anti-CHW Abs have been bound to detect low (L) and high (H) antigen levels.
3. A wash solution is used to remove unbound enzyme conjugates. The wash is followed by a substrate solution that acts with the bound enzyme to produce a color change, indicating a positive test.
4. Positive (C) and negative (N) control dots are also included on the membrane. Presence of a color change in the positive control is required for a valid test, and sample dots must also have stronger color development than negative control.
5. When no positive control dot develops or the negative control has high reactivity, the test is considered invalid and must be repeated.
- A drop of the sample (whole blood, serum, or plasma) is added to the sample port (S) of the test device.
- The sample drop reconstitutes the conjugate of gold particles linked to anti-CHW antibodies (Ab) dried on a pad in the test device. A chase buffer is often used to improve flow.
- The CHW antigen (Ag) in the sample and the reconstituted conjugate flow down the test strip over 2 specific areas.
- The first area is a line containing anti-CHW Ab (test line, T), which is immobilized on the strip. If CHW Ag is present, the anti-CHW Ab will capture it along with bound particle conjugate, causing a visible line to form.
- Excess conjugate migrates past this line and over the control (C) area, where conjugate-specific antibodies capture the particle conjugate causing a visible line to form.
Positive samples must produce obvious lines in both the test and control sections of the strip for the test to be valid. Negative samples produce an obvious line only in the control section. Invalid tests, indicated by a line in the test area but not the control area, or by no lines in either area, must be repeated.
Even though they are more sensitive than microfilaria detection, CHW antigen tests:
- May give false negative results in animals that are infected only with male worms or with very few female worms; antigen testing fails in this case because the monoclonal antibodies used in the tests primarily detect female-specific antigens7
- Are not always precise with regard to correlation between the amount of heartworm antigen in the blood and the number of adult female worms.
Commercial CHW antigen tests evaluated during 2001 to 2002 were found to reliably detect at least 84% of CHW infections in which 3 or more female adult worms were present, but sensitivity decreased when fewer than 2 adult females were present.8,9
In addition to detection of microfilaria and/or heartworm antigen, other tests may be useful in diagnosing CHW infection, especially in microfilaria-negative dogs or dogs with low worm burdens. Recently, polymerase chain reaction (PCR) technology has been used for the detection and identification of specific microfilaria in peripheral blood samples.
- Amplification of DNA from circulating microfilaria in a multiplex PCR assay gave reliable positive results for samples containing as few as 4 microfilaria per mL and distinguished D immitis infections from D repens infections.10
- Another multiplex PCR assay was used to distinguish between 6 different species of microfilariae in dogs and resolve discrepancies in morphologic diagnosis by 2 diagnostic laboratories.11 Molecular speciation identified microfilaria from several samples as D immitis, differing from an original morphologic diagnosis as Acanthocheilonema (Dipetalonema) reconditum.
Currently, the clinical relevance of PCR testing for CHW is low because it is not available through large commercial laboratories.
Ultrasound and radiology can be useful tools for diagnosing and staging CHW disease. Radiographic findings in a heartworm positive dog can be variable due to several factors such as:
- Length of infection and corresponding disease progression
- Size of the population of worms present
- Worm location in the cardiopulmonary system.12
In an animal with recent CHW infection, there may be no radiographic changes present; however, as the duration of infection increases, common changes include (Figure 3):
- Right ventricular enlargement
- Pulmonary vasculature dilation
- Parenchymal lung infiltrates.
In dogs with severe disease, the pulmonary arteries can appear tortuous or have nodular opacities. Hepatomegaly and pleural effusion may also be seen radiographically if the progression of the CHW infection leads to right-sided heart failure.12
On ultrasound, adult worms can appear as parallel hyperechoic lines present in the pulmonary artery and occasionally in the right ventricle (Figure 4). Echocardiography can also be a useful tool in detecting the extent of right heart enlargement and functional impairment due to CHW infection.4,13 The presence of worms in the orifice of the tricuspid valve in dogs with hemoglobinuria is indicative of caval syndrome.4
EVALUATION OF CHW ANTIGEN TESTS
Given that in-clinic antigen assays are now the primary method for detecting CHW infection in dogs, it is important to know how well current tests are performing. In the past 10 years, some tests have been reformulated and new tests have been introduced.
We evaluated the performance of 4 immunoassays currently available for point-of-care diagnosis of CHW infection. Samples from a total of 114 dogs were tested, including 51 negative and 10 positive ethylenediaminetetraacetic acid (EDTA) whole blood samples, and 15 negative and 37 positive serum or plasma samples (Table 1).
Table 1. Overview of Samples Used in This Study
|No. of Samples||Sample Source||Sample Type||Classification|
|51||CHW screening clinic (low risk)*||Whole blood||Negative|
|9||Annual CHW screening (low risk)*||Plasma||Negative|
|1||Laboratory (uninfected)**||Whole blood||Negative|
|10||Laboratory (experimental infection)**||Whole blood||Positive|
|37||Laboratory (experimental infection)**||Serum/plasma||Positive|
Tests were designated A, B, C, or D. Concordance among the tests was excellent.
- The only discordant test was a weak positive on Test A that was negative on all other tests. The sample, which was whole blood from a healthy, low-risk dog, was rerun on all tests with the same results using both whole blood and plasma.
- None of the 114 samples run on Tests A, C, and D were invalid; 2 samples run on Test B did not develop a positive control line and were declared invalid. The same samples were repeated successfully on additional Test B units.
The ability of the CHW tests to correctly identify CHW diseased dogs (sensitivity) and CHW disease-free dogs (specificity) was determined using the described criteria for identification of diseased and nondiseased samples (Table 2). Tests B, C, and D each demonstrated 100% sensitivity and 100% specificity, while Test A demonstrated 100% sensitivity and 98.5% specificity.
Table 2. Sensitivity & Specificity of CHW Tests
|A||VetScan Canine Heartworm Rapid Test
|B||Solo Step CH
|C||Canine Heartworm Test
|D||SNAP Canine Heartworm Antigen Test Kit
Potential Study Bias
Some potential for bias existed in the performance of diagnostic tests in this study.
- Heartworm infected and noninfected animals were selected from distinctly different populations rather than from a spectrum of diseased and nondiseased subjects.14
- CHW infected animals were all artificially infected. Of these 47 dogs, we received information on the infection dose for 25 dogs, with necropsy data available on 6 dogs.
- The worm burdens in the artificially infected dogs were similar to the mean number of worms reported for naturally infected dogs in Michigan, higher than those reported for Maine, and lower than those reported for Florida.8,15 However, marginally infected animals (worm burden of < 2 adult females), which can represent the largest numbers of false negative animals, were underrepresented and, therefore, our reported sensitivity values may be artificially elevated.
On the other hand, reported specificity values may be artificially elevated due to incorporation bias within the CHW negative group (see Standards for Heartworm Testing).14 The accepted gold standard is absence of adult worms at necropsy, but our disease-free population was defined by alternative criteria that included previous negative CHW antigen test results. This can lead to significant bias and artificial elevation of test specificity.
Standards for Heartworm Testing
The accepted gold standard for CHW testing is presence or absence of adult worms in the pulmonary arteries and/or heart on necropsy examination.7-9 However, necropsy as a gold standard for diagnostic assays presents difficult procedural and ethical challenges. Therefore, our evaluation of positive samples for gold standard status was based on partial verification.
We included a limited number of necropsy-confirmed samples obtained from a laboratory involved in CHW research. In addition, we included blood from dogs that were experimentally infected with CHW. Samples from healthy dogs that were highly likely to be CHW-negative due to geographic location and previous negative CHW antigen test results and/or history of preventive medication were considered to be negative for the purposes of this study (Table 2).
The likelihood of bias was minimized through selection of traits that reduced the likelihood of these subjects to be infected including:
- Disease-free clinical presentation (all dogs)
- Residence in a low-risk area (all dogs)
- History of administration of CHW preventative (most dogs)
- Previous negative test results (multiple times in some dogs).
However, because the previous negative test results were determined by 1 of the 4 test kits (Test D) utilized in this study, the potential for bias, although small, is still present.
In most cases, diagnosis of CHW infection is relatively straightforward and easily accomplished through in-clinic testing. However, an understanding of the heartworm life cycle and recognition of the limitations of current diagnostic procedures are critical to successful diagnosis.
Two major limitations of most CHW tests are:
- Inability to detect infection during the long prepatent period
- Decreased sensitivity of detection when very few or no female worms are present.
Commercial CHW antigen tests have high sensitivity and specificity but performance may be compromised when these limitations are encountered.
We gratefully acknowledge veterinary technicians Kathryn Gresco, Eva Jaeger, and Kimberly Holt for their help with the CHW screening clinic. We also thank Dr. Brenda Knowlton and student representative Bridget Morton of Pfizer Animal Health for helping plan and organize the clinic.
CHW = canine heartworm;
EDTA = ethylenediaminetetraacetic acid;
ELISA = enzyme-linked immunosorbent assay;
ICT = immunochromatographic test;
PCR = polymerase chain reaction
Partial funding for this study was provided by SafePath Laboratories (safepath.com).
- Bowman D, Little SE, Lorentzen L, et al. Prevalence and geographic distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in the United States: Results of a national clinic-based serologic survey. Vet Parasitol 2009; 160(1-2):138-148.
- Bowman DD, Atkins CE. Heartworm biology, treatment, and control. Vet Clin North Am Small Anim Pract 2009; 39:1127-1158.
- Lee ACY, Montgomery SP, Theis JH, et al. Public health issues concerning the widespread distribution of canine heartworm disease. Trends Parasitol 2010; 26(4):168-173.
- American Heartworm Society. 2005 guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria immitis) infection in dogs. Vet Parasitol 2005; 133:255-266 (revised January 2010). Revised guidelines available at: heartwormsociety.org/veterinary-resources/canine-guidelines.html.
- Bourguinat C, Keller K, Blagburn B, et al. Correlation between loss of efficacy of macrocyclic lactone heartworm anthelmintics and P-glycoprotein genotype. Vet Parasitol 2011; 176:374-381.
- Courtney CH, Zeng Q-Y. Relationship between microfilaria count and sensitivity for the direct smear for diagnosis of canine dirofilariosis. Vet Parasitol 2001; 94:199-204.
- Weill GJ. Dirofilaria immitis: Identification and partial characterization of parasite antigens in the serum of infected dogs. Exp Parasitol 1987; 64:244-251.
- Courtney CH, Zeng Q-Y. Comparison of heartworm antigen test kit performance in dogs having low heartworm burdens. Vet Parasitol 2001; 96:317-322.
- Atkins CE. Comparison of results of 3 commercial heartworm antigen test kits in dogs with low heartworm burdens. JAVMA 2003; 222(9):1221-1223.
- Gioia G, Lecova L, Genchi M, et al. Highly sensitive multiplex PCR for simultaneous detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood. Vet Parasitol 2010; 172:160-163.
- Rishniw M, Barr SC, Simpson KW, et al. Discrimination between six species of canine microfilariae by a single polymerase chain reaction. Vet Parasitol 2006; 135:303-314.
- Bahr RJ. Heart and pulmonary vessels. In Thrall DE (ed): Textbook of Veterinary Diagnostic Radiology, 5th ed. St. Louis: Elsevier, 2007, pp 583-584.
- Atkins C. Canine heartworm disease. In Ettinger SJ, Feldman EC (eds): Textbook of Veterinary Internal Medicine, 6th ed. St. Louis: Elsevier, 2005, pp 1124-1125.
- Mower WR. Evaluating bias and variability in diagnostic test reports. Ann Emerg Med 1999; 33(1):85-91.
- Kaiser L, Williams JF. Dirofilaria immitis: Worm burden and pulmonary artery proliferation in dogs from Michigan (United States). Vet Parasitol 2004; 124:125-129.