Performance of serologic tests used to detect heartworm infection in cats

OBJECTIVE: To compare heartworm serum antibody (Ab) and antigen (Ag) test results, using commercial laboratories and in-house heartworm test kits, with necropsy findings in a population of shelter cats. DESIGN: Prospective study. ANIMALS: 330 cats at an animal shelter. PROCEDURE: Between March and June 1998, 30 ml of blood was collected from the cranial and caudal venae cavae of 330 cats that were euthanatized at a local animal shelter. Results of heartworm Ab and Ag serologic tests for heartworm infection were compared with necropsy findings in this population of cats, using commercial laboratories and in-house test kits to measure serum Ab and Ag concentrations. RESULTS: On necropsy, adult Dirofilaria immitis were found in 19 of 330 (5.8%) cats. Combining results from serum Ab and Ag tests achieved higher sensitivities than using serum Ab and Ag test results alone (i.e., maximum sensitivities of 100% vs 89.5%, respectively, whereas use of serum Ag and Ab test results alone achieved higher specificities compared with the use of a combination of serum Ab and Ag results (i.e., maximum specificities of 99.4% vs 92.9%, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of our findings, if a cat has clinical signs that suggest heartworm disease despite a negative heartworm serum Ab test result, an alternative heartworm Ab test, a heartworm Ag test, thoracic radiography, or two-dimensional echocardiography should be performed.     

Comparison of three immunodiagnostic tests for experimental Heterophyes heterophyes infection in dogs

The aim of this study was to compare the performance of three in-house diagnostic tests, i.e. counter current immunoelectrophoresis (CCIE), intradermal (ID) and indirect fluorescent immunoassay (IFI), for the diagnosis of Heterophyes infection. One hundred and twenty puppies were randomly divided into eight groups (n=15/group). Heterophyes heterophyes infections were established in these puppies by administering a dose of 50 H. heterophyes encysted metacercariae/puppy by gavage. Forty puppies of similar age and sex were divided into eight groups, of five puppies each and were used as negative controls. Sera pooled from separate infected and uninfected groups were tested against H. heterophyes antigens, weekly for 8 weeks post-infection (PI). The ID assay detected infected puppies sooner than any of the serological tests. Sero-conversion was first detected 2 weeks PI by ID assay and 1 week later by CCIE and IFI assays. ID test performed well till the end of the experiment (sensitivity and specificity: 100% and 90%, respectively). Both IFI and CCIE assays had a sensitivity of 40% and 20%, respectively for early detection of antibody, which was less sensitive than ID but both assays were more specific (100%) than the ID assay. The lowest agreement was between ID and IFI tests (40.3%), whilst the highest was observed between CCIE and IFI tests (67.2%). Of the three evaluated methods, the ID test could be recommended for scientific and laboratory diagnosis of heterophyosis in naturally infected animals. However, since none of the investigated method are optimal (i.e, offers 100% specificity and sensitivity), the choice of test employed must depend on the aim of the study.     

Comparison of serologic tests for detection of antigen in canine heartworm infections

In 30 random-source dogs, we determined sensitivity and specificity of 5 serologic tests for detection of canine heartworm antigens. Seventeen of the dogs were infected naturally with adult Dirofilaria immitis, and 4 of the infected dogs were amicrofilaremic. The ability of the serologic tests to predict whether a dog was infected or uninfected (overall test accuracy) ranged from 73 to 97%. Sensitivity was not affected by circulating D immitis microfilariae, but was markedly influenced by the number of adult D immitis present. False-positive reactions were rare and were not associated with intestinal parasites or Dipetalonema reconditum microfilariae. Modifications of some of the test procedures were necessary to maximize test accuracy and reproducibility. These modifications and other technical details might limit the usefulness of some of the tests in a veterinary practice.     

Incidence of positive heartworm antibody and antigen tests at IDEXX Laboratories: trends and potential impact on feline heartworm awareness and prevention

Data from the IDEXX Laboratories Reference Laboratory Network were retrospectively examined for feline heartworm testing trends in testing frequency, geographic bias, and prevalence for the years 2000--2006. Examination of the data supports the commonly held view that veterinarians do not embrace heartworm disease testing or prevention in cats to the same degree they do in dogs. Despite significant awareness and adoption of heartworm testing and prevention in dogs, we hypothesized that heartworm testing rates are lower for cats than for dogs despite a significant prevalence of feline infection. This is important because a perceived low rate of infection in cats is likely to manifest in a low adoption of testing and prevention. In reality, the overall feline heartworm antigen-positive rate is significant--on average 0.9% over the period studied--and in some regions was estimated to be as high as 4.6%. This compares with an average canine heartworm prevalence rate of 1.2%, a feline leukemia virus prevalence of 1.9%, and a feline immunodeficiency prevalence of 1.0%. Based on the low rate of testing and these prevalence rates, practitioners are routinely missing cases of adult feline heartworm infections and the recently defined heartworm-associated respiratory disease (H.A.R.D). Increased antigen testing would result in detection of a significant number of positive cases. In addition, this population of infected cats would represent the "tip of the iceberg" relative to the greater number of cats that have early infection or are at risk for infection.     

Comparison of serological tests for the detection of natural heartworm infection in cats

Serological tests were performed on 380 cats with necropsy-confirmed heartworm disease to compare the performance of currently available commercial laboratory and point-of-care heart-worm serological tests in a heartworm-endemic area. Overall, antigen tests detected 79.3% to 86.2% of heartworm infections and were highly specific. Most cats with false-negative antigen tests had a single male worm. Antibody tests detected 62.1% to 72.4% of heartworm infections and had a wider range of false-positive results (1.4% to 19.1%) than antigen tests (0.3% to 2.0%). Serological tests for feline heartworm infection varied in diagnostic performance. Combining results from antigen and antibody tests achieved greater sensitivity than using either test alone.     

Evaluation of efficacy of heartworm preventive products at the FDA

The Center for Veterinary Medicine, U.S. Food and Drug Administration (FDA/CVM) has authority under the United States Code 21 under Section 514.80 to monitor for adverse experiences of approved animal products. Although veterinarians voluntarily report suspect drug-related events to manufacturers, firms that market FDA-approved animal products must report serious events to the FDA within 15 working days of the veterinarian or pet-owner's call to them. Under the present regulations, canine heartworm preventatives are approved for 100% efficacy after testing in laboratory and field conditions. The report of lack of efficacy against heartworm larvae is a serious adverse drug event because the resulting condition or the treatment of the condition is life threatening. Information on lack of effect that are deemed possibly, probably, or definitely drug-related available for review under generic product on the FDA/CVM website Surveillance of these reports indicates there are some failures for virtually all heartworm prevention product categories. Most failures have been reported in heartworm-endemic states. At this time, it is unclear whether these are representative of the rare occurrences of failure that have been in existence for a long time, but not reported regularly or promptly, or whether there is a true increase in complaints of ineffectiveness and real variability between products. This paper discusses methods, personnel, and procedures in place in the Division of Surveillance that will aid the FDA to better assess heartworm preventive treatment failures. It discusses scoring paradigms presently utilized by FDA/CVM to assess severity of complaints of lack of efficacy against heartworms, and welcomes audience input as to how to improve existing processes. Results suggest that more comprehensive reporting will provide FDA/CVM more accurate surveillance information regarding efficacy problems. Such practices will permit FDA/CVM to better interpret both incidence and severity of in-effect and possible patterns of emerging resistance and to convey this in any necessary updated labeling. It also indicates that as part of that process, practitioners should return to a more conservative testing schedule.     

Comparison of heartworm antigen test kit performance in dogs having low heartworm burdens

Sensitivity and specificity of four in-clinic heartworm antigen test kits, AbboScreen (Abbott Laboratories), Snap PF (IDEXX Laboratories), Solo Step (HESKA Corporation), Witness (Synbiotics Corporation) and two heartworm antigen microwell plate assays, DiroCHEK (Synbiotics) and PetChek PF (IDEXX) were compared in a blinded study using serum or plasma drawn from 237 random source dogs, including 140 with necropsy-confirmed, low worm burden infections (minimum 1 worm, maximum 10, mean 2.3, median 3) and 97 confirmed heartworm-free at necropsy. In general, microwell format tests were more sensitive than membrane format tests and tests using ELISA technology were more sensitive than tests using lateral flow immunochromatographic technology. Percent sensitivity and specificity, respectively, were PetChek PF 76 and 97, DiroCHEK 71 and 94, SNAP PF 67 and 98, Solo Step 60 and 98, and AbboScreen 52 and 96. The Witness test protocol was changed by the manufacturer midway through the study, and the newer version of this test kit arrived containing a package insert alerting the user to a change in procedure, which purportedly resulted in improved sensitivity. PetChek was significantly more sensitive than all other tests except DiroCHEK and the new version of Witness. DiroCHEK was significantly more sensitive than all tests except PetCheck, SNAP and the new version of Witness. Snap was more sensitive than AbboScreen and the old version of Witness. Differences in specificity were not significant (P>0.05).     

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.     

Pulmonary manifestations of heartworm disease

The clinical signs associated with heartworm disease are the result of changes in the pulmonary arterial system. These clinical signs are the result of either pulmonary hypertension or lung parenchymal disease associated with vascular changes. An increase in pulmonary arterial pressure produces an increase in right ventricular afterload, which may lead to exercise intolerance, syncope, and right-sided congestive heart failure. Coughing, dyspnea, and hemoptysis are the results of pulmonary parenchymal disease.     

Testing for occult heartworm infection

Heartworm infection in dogs is endemic in southern Ontario but occurs only sporadically throughout the remainder of Canada. The disease may either be associated with microfilariae in the patient's blood, a patent infection, or it may be occult. This paper describes a case of occult dirofilariasis in a dog, with emphasis on the diagnosis. A patent infection could be missed if the clinician tests an insufficient amount of blood. He should perform multiple concentration tests using either the modified Knott's technique or a filtration method. Occult infections occur in prepatent or unisexual infections, when the worms become sterile following therapy, or when the host produces antibodies that result in the destruction of the microfilariae. The recent release of a kit which detects the presence of antibodies to the adult heartworms now enables veterinarians to make an accurate diagnosis in the vast majority of dogs.