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J Vet Diagn Invest 5:647-648 (1993)
Comparison of three diagnostic tests for Dirofilaria immitis
in a low-incidence area
Lois Roth, Lynne Brown, Susan Brum, Linda Foster
Martha Nelson, David Reczeck, Deana von Schantz
Currently, prevention of canine Dirofilaria immitis infection
relies on use of therapeutic agents that effectively eliminate
infective larvae inoculated by mosquitoes. 5 These agents
also kill circulating D. immitis microfilariae. 5 Because adverse
reactions may occur in microfilaremic dogs given larvacides,
it is important to determine that dogs are parasite
free prior to beginning seasonal prophylactic medication. 5
For many years, the modified Knotts test, a procedure to
detect microfilariae, has been used as a screening test to
determine if dogs could be given prophylactic treatment for
D. immitis. 5,6 Because of concern for sterile or unisexual infections,
in which no microfilariae are produced, serologic
tests to detect antigens produced by adult D. immitis were
developed. 2,3,6,8,9 The serologic test kits, using enzyme-linked
immunosorbent assay (ELISA) techniques, are commercially
available. They are considered specific for D. immitis somatic
antigen and are reported to be a sensitive indicator of infection
in dogs having worm burdens > 10. 3,5,6,9
Most testing of the commercially available kits has been
done under laboratory conditions or in regions considered
hyperendemic for D. immitis infection. 1-3,6,8,9 A more recent
survey has been done in a hypoendemic region. 7 In lowincidence
areas, D. immitis may still cause serious clinical
disease in infected animals. Additionally, apparently healthy
dogs harboring D. immitis may serve as a reservoir of infection
for other animals. During the height of the 1991 heartworm
testing season in the northeastern United States (March-
July) 0.41% (13/3,148) of dogs tested for the presence of D.
immitis microfilariae in the Clinical Laboratory at Angell
Memorial Animal Hospital were positive. Occult heartworm
tests were not done routinely, but 4 of 45 serum samples
tested were positive for adult D. immitis antigen. Microfilariae
were detected in none of these. Only 1 of these dogs
was treated with D. immitis adulticide. The other 3 were
considered clinically healthy and given monthly doses of
ivermectin or milbemycin oxime throughout the summer
and early fall. These data prompted a more systematic assessment
of occult heartworm testing in this non hyperendemic
area, which was done during part of the 1992 heartworm
season.
All blood samples submitted to the Clinical Pathology Laboratory
at Angell Memorial Animal Hospital for “heartworm
checks” between May and July 1992 were evaluated for microfilariae
by filtration techniques and for adult D. immitis
antigen using 2 commercially available kits. a,b Samples from
1,359 dogs were tested, and the results were compared. If
discrepancies among test results occurred or a positive result
From the Clinical Laboratory, Department of Pathology, Angell
Memorial Animal Hospital, Boston, MA 02130.
Received for publication December 4, 1992.
647
Based on physical examination and radiographs,
the clinicians strongly suspected that 2 of these 3
dogs had few adult D. immitis in their right ventricles, and
the dogs were treated with adulticides before beginning preventative
therapy. The presence of adult D. immitis infection
in the right ventricle in the third dog was questionable, yet
the owner preferred to have the dog treated with adulticide
before preventative therapy was begun.
Although the number of infected dogs detected in this study
was small, infection in 5 of 12 animals would have gone
undetected if only a filter test, accompanied by acid phos-
was obtained, tests were repeated by a different technologist.
Microfilaria identification using the alkaline phosphatase
technique was done to differentiate between D. immitis and
Dipetalonema reconditum on all samples in which microfilariae
were observed. Clinical follow-up was obtained for 11
of the 12 dogs that had any positive test result by reviewing
the medical record or discussing the case with the clinician.
The results of the laboratory tests are summarized in Table
1. Samples from 1,347/1,359 dogs (99.12% of samples submitted)
were negative for microfilariae and adult D. immitis
antigen using both commercial kits. Microfilariae, subsequently
identified as D. immitis, were observed in blood
samples from 7 dogs. Four of these dogs also had adult D.
immitis antigen detected by both kits. Two microfilaremic
dogs were negative for D. immitis adult antigen with both
kits. Blood from 1 dog that was positive for microfilariae
was serologically positive for adult antigen with only 1 of the
commercial kits. Five dogs were serologically positive for
adult D. immitis antigen but negative for microfilariae.
The 5 dogs that had positive tests for adult D. immitis
antigen but negative filter tests were considered otherwise
healthy. They had no evidence of circulatory or respiratory
compromise. Adulticide treatment was recommended for each
of these dogs, but owners chose to administer ivermectin or
milbemycin oxime monthly to their pets. Owners were advised
to observe their dogs carefully and to restrict their pets’
exercise for at least 2 days following the administration of
the monthly heartworm preventative. Adverse reactions were
not reported in any of these dogs. Six of the 7 dogs that tested
positive for D. immitis microfilariae underwent treatment
for the elimination of adult D. immitis and microfilariae,
regardless of the results of the adult antigen test. Additional
information could not be obtained for 1 dog that was positive
on all 3 tests. Treatment in 1 dog that had positive results
with all 3 tests was initiated as an emergency procedure when
the dog developed acute respiratory distress. Two dogs that
were microfilaremic were adult antigen negative using both
kits, and 1 microfilaremic dog was antigen negative with 1
kit but antigen positive with the other. Such results are likely
to occur if the adult worm burden is low. In these cases, the
sensitivity of the tests for detection of adult antigen is decreased.
2,3,6,8,9

648 Brief communications
Table 1. Results of a filter test for Dirofilaria immitis microfi- tion. 4 Therefore, accurate detection of D. immitis infection
lariae and 2 ELISA kits for adult Dirofilaria immitis antigen in 1,359 is clinically important, even in areas where heartworm disdogs.
ease is relatively uncommon. In our hospital, only a filter
concentration test is suggested for dogs given daily heartworm
preventative medication during the “heartworm seaventative
son”; an annual occult heartworm test alone is considered
appropriate for dogs receiving year-round monthly treatments.
Both tests are recommended for a dog given monthly
preventative medication for only part of the year prior to
initiating a preventative therapeutic schedule for the new
“heartworm season.” A thorough physical examination should
be an integral part of the diagnostic procedure. The dog’s
history, use, and previous as well as future heartworm prethought
medication regime are considerations when seson”;
lecting the appropriate diagnostic tests for D. immitis infecphatase
to be best suited for this study. There are several
staining, was used. The modified Knotts test and the
concentration filter technique used in this study are considtemporarily.
Other therapeutic regimes may also cause
ered sensitive for detection of D. immitis microfilariae. 5 The
concentration filter test is a common method for microfilaria
detection used by private practitioners in the area and is
causes of antigen-positive, micrifilaria-negative infections that
might explain these results. Continued use of monthly heart-
worm preventatives may mask the presence of adults in dogs
harboring D. immitis by rendering the adults sterile, at least
tion in dogs.
Acknowledgements. We appreciate the donation of diagnostic
kits for Dirofilaria immitis adult antigen by Idexx Lab-
oratories, Westbrook, ME, and by Symbiotics Corp., San
Diego, CA, for use in this study.
Sources and manufacturers
Dirochek ® , Symbiotics Corp., San Diego, CA.
Petchek ® , Idexx Laboratories, Westbrook, ME.
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