Comparison of results of three commercial heartworm antigen test kits in dogs with low heartworm burdens

OBJECTIVE: To compare results of 3 commercial heartworm antigen test kits performed on serum samples from dogs infected with low numbers of adult female heartworms. DESIGN: Blinded laboratory evaluation. Sample Population-Serum samples from dogs (n = 208) proven at necropsy to be infected with 1 to 4 adult female heartworms and from dogs (32) without heartworms. PROCEDURE: Samples were sequentially tested with each test kit, following the manufacturers' instructions, by licensed veterinary technicians in private practice who were not aware of infection status of the dogs. The order of test kit evaluations was randomly chosen. For each test kit, sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were evaluated. RESULTS: All tests yielded some false-negative results, and there were significant differences among tests in regard to ability to detect low heartworm burdens. Sensitivity of the test kits ranged from 78 to 84%. For all test kits, sensitivity increased as number of female heartworms increased. All 3 test kits had high specificity (97%). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that sensitivity of the 3 commercially available heartworm antigen test kits ranged from 78 to 84% when used to test serum samples from dogs with low heartworm burdens, and that sensitivity varied among test kits. For all 3 test kits, specificity was 97%. All 3 test kits yielded false-positive and false-negative results for some dogs with low heartworm burdens.     

Sensitivity and specificity of the indirect-fluorescent antibody test and two enzyme-linked immunosorbent assays in canine dirofilariasis

Ninety dogs naturally infected with Dirofilaria immitis and 103 noninfected dogs, as determined by necropsy, were used to compare the sensitivity and specificity of the cuticular and somatic reactions of the indirect fluorescent antibody test (IFA-C and IFA-S, respectively) and 2 enzyme-linked immunosorbent assays (ELISA). In microfilaremic and amicrofilaremic heartworm-infected dogs, negative results were common for all serotests. In dogs without adult heartworms at necropsy, 32% to 49% were positive, using 1 ELISA, 27% to 29% were positive with the other ELISA, 15% to 36% were positive with the IFA-S, and 0% to 1% were positive using the IFA-C, depending on the classification of borderline reactions. The prevalence of false positive serotests was probably not due to the detection of precardial stages of D immitis in dogs obtained from areas of low endemicity. Until the causes of the false-positive tests are resolved, the use of currently available serotests for routine diagnostic screening or as criteria for instituting treatment is not recommended.     

Evaluation of a new in-clinic method for the detection of canine heartworm antigen

Canine heartworm is endemic in many parts of the world, and veterinarians rely on rapid in-clinic antigen tests to screen for this infection. Recently, an in-clinic, instrument-based rotor employing a colloidal gold agglutination immunoassay was launched in the marketplace (VetScan VS2(?) Canine Heartworm (HW) Antigen Test Kit; Abaxis, Inc.). Because of the widespread use of heartworm prevention and possible false negative test results in dogs with low heartworm burdens, the performance of the VetScan VS2(?) HW test and a commercially available in-clinic, membrane-based ELISA test (SNAP(?) Heartworm RT Test; IDEXX Laboratories) was compared using samples from dogs with low heartworm burdens and/or low levels of circulating antigen. Ninety serum samples were evaluated using the two methods. Testing was performed according to the manufacturer's product insert by personnel blinded to sample status. The samples were derived from two populations: dogs with necropsy-confirmed heartworm status (40 with 1-4 female worms, 30 with no worms), and field dogs (20) confirmed positive for antigen by microtiter plate ELISA (PetChek(?) Heartworm PF Antigen Test; IDEXX Laboratories). All 40 dogs with heartworms on necropsy were also confirmed to have circulating antigen by the PetChek HW ELISA. In necropsy-negative dogs (n=30), neither the VetScan VS2 HW nor SNAP HW tests detected heartworm antigen. Of the samples testing positive for antigen by PetChek HW (n=60), the VetScan VS2 HW and SNAP HW tests detected antigen in 15 and 56 samples, respectively. Percent agreement (plus 95% confidence interval) for each test relative to the PetChek HW qualitative result was 50% (40-60%) for VetScan VS2 HW and 96% (89-98%) for SNAP HW. Relative to the presence or absence of female worms at necropsy, agreement was 61% (50-72%) for VetScan VS2 HW and 99% (92-99.6%) for SNAP HW tests. It is clinically important that dogs with low heartworm burdens and/or low levels of circulating heartworm antigen be correctly identified by veterinarians in order to ensure prompt treatment, and the VetScan(?) VS2 HW test does not appear to be as accurate as the SNAP HW or PetChek HW tests when performed on this subset of patients.     

Effects of treatment with ticlopidine in heartworm-negative, heartworm-infected, and embolized heartworm-infected dogs.

Ticlopidine hydrochloride was evaluated for its effectiveness in inhibiting platelet aggregation and serotonin release in 5 laboratory Beagles before and after heartworm implantation with 7 adult Dirofilaria immitis, and after embolization with 7 dead heartworms to mimic what happens after heartworm adulticide treatment. Five other laboratory Beagles, similarly implanted and embolized with heartworms, were used as nonmedicated controls. During the heartworm-negative stage, the dosage of ticlopidine that inhibited adenosine diphosphate (ADP)-induced platelet aggregation in 5 dogs by at least 50% after 5 days of treatment was 62 mg/kg of body weight once a day. In the same dogs implanted with 7 adult heartworms 21 days previously, mean (+/- SD) ticlopidine dosage required to obtain similar results was 71 (+/- 13) mg/kg given once daily. During the 21 days after dead heartworms were implanted in heartworm-infected dogs, mean ticlopidine dosage was 108 (+/- 35) mg/kg (range, 62 to 150 mg/kg). Ticlopidine treatment was associated with increased platelet numbers in all 5 dogs during the heartworm-negative stage and in 4 of 5 dogs during the heartworm implantation and heartworm embolization stages. Mean platelet volume tended to decrease as platelet numbers increased. At necropsy, gross and histologic pulmonary lesions were less severe in ticlopidine-treated dogs than in nonmedicated control dogs.     

Canine Dirofilaria immitis infection in a hyperenzootic area: examination by parasitologic findings at necropsy and by two serodiagnostic methods

A total of 174 dogs from an area hyperenzootic for Dirofilaria immitis were grouped into 4 age categories and necropsied; information was obtained on adult D immitis infections and on the presence of microfilariae. Serum samples from these dogs were examined by an enzyme-linked immunosorbent assay (ELISA) for antibody to adult D immitis and by an indirect fluorescent antibody test (IFAT) for antibody to microfilarial surface antigens. In dogs less than or equal to 5 months of age, necropsy demonstrated no evidence of infection; however, positive serologic results indicated that some of these dogs had prepatent infections. The percentage of dogs with ELISA titers (positive) increased with age, as did the percentage of dogs with adult D immitis infections. The IFAT results were positive in some dogs in each age category. Sera from all 29 dogs with occult infections were positive by ELISA. Sera from 6 of 20 dogs with occult dual-sex heartworm infections and 1 of 9 dogs with occult single-sex heartworm infections were positive by IFAT. For diagnosing occult dirofilariasis, the ELISA had a positive predictive value which increased with age of the dog to a maximum of 65.0% in dogs greater than or equal to 12 months of age; ELISA had a negative predictive value of 100% in all age groups. In contrast, positive and negative predictive values for the IFAT decreased with age of the dog to 60% and 37.5%, respectively, in dogs greater than or equal to 12 months of age.     

Interaction between canine platelets and adult heartworms: platelet recognition of heartworm surfaces

An interaction between blood platelets and adult heartworms was examined in vitro. Surfaces of glutaraldehyde-fixed heartworms, which were taken from infected dogs washed, and incubated in platelet-rich plasma (PRP), were evaluated by scanning electron microscopy. Adherence of platelets to heartworms occurred only with PRP from infected dogs. Aggregation to epinephrine and adenosine diphosphate of PRP incubated with heartworms was monitored. Seemingly, platelet activation to heartworm membranes occurs in dogs with heartworm disease. The increased platelet reactivity was also observed in dogs with occult heartworm disease, indicating that the presence of circulating microfilaria was not important for this process. The ability to transfer the reactivity to heartworm-negative platelets by suspending them in heartworm-positive plasma indicated that this reactivity resided in the plasma. The processes leading to platelet activation may be responsible for the platelet-associated vascular disorders of canine heartworm disease.     

Prevalence of Dirofilaria immitis in a stray dog population in western New York.

In a survey in 1973 undertaken to determine the prevalence of Dirofilaria immitis in 100 dogs from a public pound in western New York, 2 dogs were found to be positive. Microfilariae of Dipetalonema sp were not present. Hearts and pulmonary blood vessels were dissected for adult worms, and blood samples were examined for microfilariae, using a modified Knott technique. Although adult heartworms and microfilariae have been recovered from wild foxes, coyotes, and wolves in New York, this is the first report of heartworm in domesticated dogs in the state.     

Two-dimensional echocardiography for diagnosis of dirofilariasis in nine dogs

Two-dimensional echocardiography (real-time ultrasonography) of the heart was used to detect adult Dirofilaria immitis in the right ventricle and atrium of a 2-year-old Labrador Retriever with occult heartworm disease and in 8 microfilaremic dogs. Adult worms were easily identified as parallel echogenic lines separated by a hypoechoic region. Images of worms fixed in formalin after recovery from dogs with experimentally induced heartworm infection had an identical appearance. The sensitivity of this imaging procedure was tested on the 8 dogs with naturally acquired infection, and worms were observed in the right ventricle of 7 of the dogs. Two-dimensional echocardiography appears to be a sensitive noninvasive procedure, which can be used in combination with thoracic radiography and serotests to improve accuracy of diagnosis of canine occult heartworm disease.     

Evaluation of a single injection of a sustained-release formulation of moxidectin for prevention of experimental heartworm infection after 12 months in dogs

OBJECTIVE: To evaluate the efficacy of a single injection of a sustained-release formulation of moxidectin in preventing heartworm (Dirofilaria immitis) infection for 12 months in dogs. ANIMALS: 14 healthy dogs. PROCEDURE: Group A (nontreated control dogs; n = 6) received sterile vehicle administered SC, and group B (treated dogs; n = 6) received a sustained-release formulation of moxidectin administered SC. All dogs were housed in a heartworm-endemic area for 11.5 months, and heartworm antigen and modified Knott tests were performed monthly. All dogs (including 2 additional control dogs [group C]) were then inoculated with infective-stage larvae (L3) of D. immitis, and 4.5 months later, all dogs were euthanatized and post-mortem examinations were performed. Adult D. immitis were counted and measured, and their age was estimated. RESULTS: All dogs in groups A and C were infected with young (4- to 4.5-month old) adult male and female D. immitis. No dogs in group B were infected with heartworms. CONCLUSIONS AND. CLINICAL RELEVANCE: The age of heartworms recovered suggests that infection was the result of experimental inoculation and not natural exposure to mosquitoes during the 11.5-month period the dogs resided in a heartworm-endemic area. A single SC injection of a sustained-release formulation of moxidectin was effective in providing protection against heartworm infection after 12 months in dogs. This formulation is a convenient method of heartworm prophylaxis that could eliminate the problem of poor owner compliance.     

Evaluation of a covered-rod silicone implant containing ivermectin for long-term prevention of heartworm infection in dogs

OBJECTIVE: To evaluate use of covered-rod (CR) silicone implants containing ivermectin for long-term prevention of infection with Dirofilaria immitisin dogs. ANIMALS: 145 adult male and female dogs. PROCEDURES: Dogs received implants of different sizes, and ivermectin concentrations and serum ivermectin concentrations were monitored for 16, 57, and 56 weeks, respectively, in 3 preclinical dose selection studies. Ability of implants to prevent infection with D immitis was evaluated in 2 further studies; dogs were challenged with 50 infective third-stage larvae 52 weeks after implant administration and necropsied 145 days after challenge, and the total number of adult heartworms was counted. A field study was then undertaken in which client-owned dogs received an implant and plasma samples were collected at intervals until week 52 for ivermectin analysis and heartworm antigen determination. RESULTS: Use of the implants resulted in maintenance of an ivermectin concentration > or = 0.2 ng/mL for 12 months. In challenge studies, no treated dogs had adult heartworms, in contrast to untreated dogs, which all had adult heartworms at necropsy. In the field study, dogs treated with an implant had negative results of heartworm antigen testing for 12 months. CONCLUSIONS AND CLINICAL RELEVANCE: The CR silicone implant containing 7.3 mg of ivermectin was 100% effective in preventing experimental infection with D immitislarvae and resulted in negative results for heartworm antigen in a field trial. This product has the potential to alleviate poor owner compliance with monthly prevention regimens.     

Indirect fluorescent antibody test in occult dirofilariasis.

Indirect fluorescent antibody titers to Dirofilaria immitis microfilaria (IFA-mf) and peripheral eosinophilia were recorded from 15 to 52 months in ten experimentally infected dogs with occult dirofilariasis (heartworm infection without microfilaremia). Five dogs which were experimentally sensitized with D immitis microfilaria did not exhibit microfilaremia after inoculation with infective-stage larvae. In three other dogs, microfilaremia suddenly ceased after 4 to 7 months. In these three dogs, antimicrofilarial antibodies were detectable by IFA-mf test as soon as microfilaremia ended. In the remaining two dogs, which exhibited spontaneous occult dirofilariasis, antibodies were detected at the end of the prepatent period of 6 months. The presence of adult worms was confirmed by angiocardiography. Significant IFA=mf titers (greater than or equal to 1:8) persisted after successful treatment with an adulticide. Reinfection of treated dogs reestablished occult dirofilarasis. Eosinophilia was present in all dogs and peaked at about 3, 6, and 9 months after they were inoculated with infective-stage larvae. At necropsy, the ten dogs harbored gravid, reproducing adult worms in the heart and pulmonary arteries.     

Diagnostic criteria for an enzyme-linked immunosorbent assay for occult heartworm disease: standardization of the test system in naturally exposed dogs

The development of criteria for interpreting and reporting the results of an occult heartworm enzyme-linked immunosorbent assay to practitioners is described. The antigen is a saline extract of adult female Dirofilaria immitis. The cutoff absorbance A400 nm values were estimated, using 106 dogs free of infection. Any A400 nm value less than 0.526 is considered negative and values greater than or equal to 0.784 are positive. Intermediate A400 nm values are interpreted as suspect. Absorbance values for serum samples from 13 client-owned amicrofilaremic dogs revealed a bimodal distribution consistent with presumptive diagnosis based on clinical signs, which indicates that the test may be used to support a diagnosis of occult heartworm disease. The present serotest, however, is unable to distinguish microfilaremic dogs from noninfected dogs. Serum from dogs infected with other common helminths failed to crossreact with the D immitis antigen, with the exception of Dipetalonema reconditum.     

Nine-month evaluation of dogs after open-heart surgical removal of heartworms

Adult heartworms were surgically removed from 4 infected dogs by use of intracardiac techniques during cardiopulmonary bypass. The number of worms removed ranged from 12 to 14 per dog. Observation for 9 months after surgery gave no clinical evidence of active adult heartworm infection, in spite of the consistent finding of circulating microfilariae during the follow-up period. There were no significant early or late postoperative complications. Results of postoperative hematologic and biochemical studies were unremarkable. At necropsy (following euthanasia, 9 months after surgery) the heart and pulmonary arteries of all dogs were free of adult heartworms; pathologic changes attributable to residual infection were not found.     

Myocardial fluid balance in dogs with naturally acquired heartworm infection

OBJECTIVE: To determine the effect of naturally acquired heartworm (Dirofilaria immitis) infection on myocardial fluid balance as indicated by myocardial water content and the dynamics of transepicardial fluid flow. ANIMALS: 7 dogs infected with adult heartworms and 8 dogs free of heartworm infection. PROCEDURES: Infected dogs had heartworms in the right ventricle, pulmonary artery, or both but no evidence of cardiovascular disease on physical examination. A hemispheric capsule was attached to the epicardial surface of all dogs for determination of transepicardial fluid dynamics and permeability of the epicardium to water and protein. Myocardial water content and hydroxyproline content were assessed at necropsy. RESULTS: Myocardial water content was significantly lower in heartworm-infected dogs. No differences in myocardial hydroxyproline content, transepicardial fluid flow, or epicardial water or protein permeability were detected. Conclusions and CLINICAL RELEVANCE: Heartworm infection significantly altered myocardial fluid balance in dogs, possibly because of a change in the myocardial interstitial pressure-volume relationship. These changes may be associated with increased vulnerability to cardiovascular stressors in heartworm-infected dogs.     

Surgical transplantation of adult Dirofilaria immitis to study heartworm infection and disease in dogs

Heartworm infections were established in dogs by surgical transplantation of adult heartworms into the external jugular vein. The heartworms were obtained from donor dogs that had been infected with large numbers of infective larvae. This heartworm model standardized the infection with a known number of heartworms of a known age and sex. This paper presents the methods for obtaining heartworms from donor dogs, for selection of recipient dogs, and for transplantation of heartworms.     

Efficacy of combined chemotherapy against gastrointestinal nematodes and Fasciola hepatica in cattle

Direct blood smear examination (using 0.05 ml of whole blood) detected 168 (80.9%) of 204 microfilaremic canine blood samples as determined by the modified Knott test for microfilariae (mff) of Dirofilaria immitis (using 1 ml of whole blood). Direct smear examination detected all of 134 microfilaremias greater than 50 mff ml(-1), but only 31 of 70 (44.3%) microfilaremias having less than 50 mff ml(-1). In a separate retrospective query of a database of 963 dogs with necropsy-confirmed heartworm infections, 834 (86.6%) were positive by the DiroCHEK heartworm antigen test, and 504 (52.3%) were microfilaremic by the modified Knott test. Only 2 (0.4%) of the microfilaremic dogs were DiroCHEK negative and another 18 (3.6%) were very weak positives. Although these microfilaremic dogs were not tested by direct smear, only one of the two DiroCHEK-negative and six of 18 weakly DiroCHEK-positive dogs had microfilaremias so low that a direct smear may have given a false negative result. Significant adverse reactions to either diethylcarbamazine or the macrolide endectocides have not been reported for microfilaremias less than 500 mff ml(-1), thus substitution of the direct smear for a concentration test for mff, such as the modified Knott test or membrane filtration, does not appear to increase the risk of an unexpected adverse reaction to heartworm prophylactic drugs. Such a substitution results in only a very slight decrease (on the order of 0.1%) in the overall sensitivity of heartworm screening, provided a test for mff is run concurrently with an antigen test. If a test for mff is the only screening test used, then substitution of a direct smear for a concentration test may decrease the sensitivity of heartworm screening by nearly 20%, depending on the prevalence of low level microfilaremias in the population of dogs tested.     

Dirofilaria immitis: worm burden and pulmonary artery proliferation in dogs from Michigan (United States)

Despite the ability to prevent heartworm disease, infection with Dirofilaria immitis continues to be a major problem for domestic dogs. To determine worm burden in heartworm-positive dogs from three county animal shelters in the state of Michigan in the United States and to assess the relationship between gross intimal proliferation and worm burden, necropsy was done on 176 heartworm-positive dogs. Adult heartworms were found in the heart and pulmonary artery of 170 of the 176 (96.6%) dogs examined. Mean worm burden was 14 +/- 13 (range 0-85). Fifty-nine percent of dogs had < or =10 heartworms. In contrast, 52% of dogs in a published report from the southern US (Florida) had worm burdens >10 [C.H. Courtney, Q.Y. Zeng, The structure of heartworm populations in dogs and cats in Florida, in: Proceedings of the American Heartworm Symposium, 1989]. These data suggest that mean worm burden in northern areas may be < or = that in warmer areas. Also, since diagnostic tests are less sensitive with lower worm burdens, diagnosis of heartworm infection in Michigan and other surrounding more northern states may be a greater challenge than in areas with higher worm burdens.     

Prevalence of naturally occurring Dirofilaria immitis infection among nondomestic cats housed in an area in which heartworms are endemic

OBJECTIVE: To determine prevalences of heartworm exposure (ie, positive heartworm antibody test results) and heartworm infection (ie, positive heartworm antigen test results or identification of mature heartworms at necropsy) among nondomestic cats housed in an area in rural North Carolina where Dirofilaria immitis is known to be endemic and among nondomestic cats housed in areas with a low prevalence of dirofilariasis or in an area considered to be free from heartworms. DESIGN: Cross-sectional prevalence survey. ANIMALS: 97 nondomestic cats in North Carolina (study population) and 29 nondomestic cats in Colorado; Queensland, Australia; or Auckland, New Zealand (control population). PROCEDURE: Results of serologic tests and postmortem examinations were reviewed. RESULTS: Results of heartworm antibody tests were positive for 57 of 75 (76%) study cats and 1 of 29 (3%) control cats. Male study cats had a significantly higher risk of heartworm exposure than did female study cats (relative risk, 1.3). Results of heartworm antigen tests were negative for all 47 study cats and 16 control cats that were tested. Postmortem examinations were performed on 21 study cats, and 1 (5%) was found to be infected with heartworms. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that nondomestic cats housed outdoors in the southeastern United States are at risk for heartworm exposure and infection, with male cats having a greater risk of exposure than female cats.     

RADIOGRAPHIC AND 2-D ECHOCARDIOGRAPHIC FINDINGS IN EIGHTEEN CATS EXPERIMENTALLY EXPOSED TO D. ZMMZTZS VIA MOSQUITO BITES

Eighteen cats were exposed to Dirofikria immitis infected mosquitoes. Thoracic radiography was performed prior to exposure and at 5, 7, and 9 month intervals following exposure. Immunologic testing for adult heartworm antigen was performed on days 168,195,210,224,237,254 and 271 post infection. Necropsies were performed on all cats. Adult heartworms were found in 61% of the exposed cats. Radiographic findings in heartworm positive cats included bronchointerstitial lung disease, lobar pulmonary arterial enlargement and pulmonary hyperinflation. In most heartworm positive cats, lobar arterial enlargement resolved as the disease progressed while pulmonary hyperinflation progressively became more common. Pulmonary patterns in heartworm positive cats remained abnormal throughoutthe study while abnormal pulmonary patterns resolved in over 50% of the heartworm negative cats. Cardiomegaly was seen in less than 50% of the cats with adult heartworms at necropsy. This study suggests that the radiographic appearance of heartworm disease is variable and radiographic changes are dependent on the time post infection at which cats are evaluated. Echocardiographic examinations were randomly performed on 16 of 18 cats. Heartworms were identified in 7 cats. No false positive identifications were made. Persistent pulmonary disease accompanied by resolving vascular disease in heartworm cats with pulmonary hyperinflation may be difficult to distinguish from cats with feline allergic lung disease. Echocardiograms may be helpful in identifying adult heartworms in cats in which the radiographic signs or immunodiagnostic data are insufficient to provide a diagnosis.     

Use of echocardiography for the diagnosis of heartworm disease in cats: 43 cases (1985-1997)

OBJECTIVE: To determine the usefulness of echocardiography in the diagnosis of heartworm disease in cats and to compare this modality with other tests. DESIGN: Retrospective study. ANIMALS: 43 cats with heartworm infection that had echocardiographic examinations at 2 veterinary teaching hospitals between 1985 and 1997. Twenty-two of these 43 cats also underwent radiography of the thorax and heartworm antibody and heartworm antigen testing. PROCEDURE: Cats were determined to be infected with Dirofilaria immitis infection on the basis of 1 or more of the following findings: positive modified Knott or antigen test result, echocardiographic evidence of heartworm disease, or confirmation of the disease on postmortem examination. The percentage of echocardiographs in which heartworms were evident was compared with the percentage of radiographs in which pulmonary artery enlargement was evident and results of antigen or antibody tests in cats in which all tests were performed. RESULTS: Overall, heartworms were detectable by use of echocardiography in 17 of 43 cats, most often in the pulmonary arteries. In the 22 cats in which all tests were performed, antibody test results were positive in 18, antigen test results were positive in 12, and pulmonary artery enlargement was evident radiographically and heartworms were identifiable echocardiographically in 14. Heartworm infection was diagnosed exclusively by use of echocardiography in 5 cats in which the antigen test result was negative. CONCLUSIONS AND CLINICAL RELEVANCE: Although echocardiography was less sensitive than antigen testing, it was a useful adjunctive test in cats that had negative antigen test results in which there was a suspicion of heartworm disease. The pulmonary arteries should be evaluated carefully to increase the likelihood of detection of heartworms echocardiographically.