Comparison of Three Rapid Commercial Canine Parvovirus Antigen Detection Tests with Electron Microscopy and Polymerase Chain Reaction

Abstract

Different antibody-based tests for rapid detection of Canine parvovirus antigens in feces are commercially available, allowing quick diagnosis in a clinical setting. However, the diagnostic accuracy of these tests compared with standard methods has not been evaluated so far. In the current study, 3 commercial tests were compared with immune-electron microscopy (IEM) and polymerase chain reaction (PCR). Dogs were divided into 3 groups: group A, samples from dogs with acute hemorrhagic diarrhea (n = 50); group B, dogs with chronic diarrhea (n = 10); and group C, dogs with no evidence of gastrointestinal disease (n = 40). Specificity of all 3 commercial tests versus PCR and IEM was good to excellent (92.2–100%). Sensitivity, in contrast, was poor: 15.8–26.3% versus PCR and 50–60% versus IEM. In group A, 10 dogs were positive by IEM and 24 dogs were positive by PCR. Positive PCR results were also obtained from animals in control groups (group B, 1 dog; group C, 5 dogs). No dog in group B or C was positive by IEM. In conclusion, the rapid tests are useful to diagnose canine parvoviral enteritis, but they do not rule out parvovirus infection in an animal with typical clinical signs. In addition, a small percentage of healthy dogs and dogs with chronic diarrhea showed positive PCR results; this may be due to asymptomatic/persistent infection or intestinal passage of virus. The significance of this finding remains unclear.

Canine parvoviral (CPV) enteritis is an acute life-threatening infection. It is highly contagious and represents one of the most common causes of acute hemorrhagic diarrhea in pet dogs. 5 Therefore, quick and reliable diagnosis is important. Virus is generally shed extensively for 7–12 days, but long-term excretion may occur as well. 1,3

Antemortem diagnosis can be made by immune-electron microscopy (IEM) 8 or polymerase chain reaction (PCR) of fecal samples. 4,7 The sensitivity of EM is believed to be relatively low due to large quantities of virus required for a positive test result. 2 However, IEM may enhance sensitivity. Polymerase chain reaction has been described as both sensitive and specific for the detection of CPV enteritis. 7 Because of the short incubation time, serologic tests are not relevant for the diagnosis of CPV enteritis. Moreover, serum antibodies can persist for months and the serologic response to infection cannot be discriminated from antibody titers induced by vaccination. Thus, serologic tests are not considered practical for the diagnosis of CPV enteritis.

Several fecal antibody-based antigen tests are available as rapid screening tools in a suspicious patient. The objective of the current study was to assess sensitivity and specificity of 3 commercial CPV antigen tests in comparison with IEM and PCR.

Fecal samples from 3 groups of dogs were analyzed. Group A consisted of 50 fecal samples from 48 dogs that presented with acute hemorrhagic diarrhea. Group B dogs (n = 10) presented with chronic diarrhea of more than 3 weeks' duration. Group C included 40 animals with no evidence of gastrointestinal disorders based on history and physical examination. Rapid tests were performed in randomized order for each fecal sample, and a part of each sample was submitted for IEM and PCR analysis.

The following commercially available tests for the detection of CPV were performed according to the manufacturers' instructions: SNAP parvo antigen test, a FASTest parvo strip, b and the Witness parvo card. c

The SNAP parvo antigen test is based on enzyme-linked immunosorbent assay (ELISA) technology. The FASTest parvo strip is a lateral flow immunochromatographic test (sandwich system with 2 different antibodies, 1 of which is membrane-fixed and 1 bound to gold particles). The Witness parvo card is also based on immunomigration (radioimmunomigration technology), where antigen-antibody complexes are visualized by a color change of a presensitized band on a nitrocellulose strip.

Immune-electron microscopy was carried out according to standard operating procedures at the Institute of Virology (Giessen, Germany). The test comprised dilution of samples in phosphate-buffered saline, ultrasonication in a closed system on ice, low-speed centrifugation, and incubation with antiserum. d Negative staining was performed using 2% (w/v) methylamine tungsten for 30 sec. Finally, the sample was examined by IEM e for at least 10 min. A sample was considered positive if aggregated particles with a size of about 20 nm morphologically resembling members of the Parvoviridae were detected.

For detection of CPV DNA by PCR, a conventional nested PCR protocol was used. DNA was prepared, f and primers targeting a conserved region within the VP1/VP2 gene of the CPV were used. 6 In a positive case, PCR products of 536 bp (first round) and 492 bp (second round) were observed. Sensitivity and specificity for each rapid CPV test were calculated separately against the results of PCR and IEM.

In group A, only 3 dogs reacted positive in all 5 tests. Of 7 additional samples with positive IEM and PCR results, 4 reacted negative in all rapid tests, whereas 3 samples were positive in only 2 of the rapid tests. Twenty-two dogs from group A were tested positive in PCR but negative in IEM. Of these, 18 showed negative results in all 3 rapid tests, 2 were positive in all 3 rapid test systems, and 2 were positive with the Witness card only. For the remaining 18 dogs of group A, negative results were obtained both by PCR and IEM; 3 of the latter dogs tested positive with the Witness card.

In groups B and C, 6 dogs showed positive results in canine parvovirus PCR. None of the dogs in these groups tested positive by IEM or any of the rapid tests.

Because there is no gold standard method to detect parvovirus in fecal samples, comparison of the rapid tests with IEM and PCR was done separately in the present study. Compared with IEM, sensitivities for the Snap Test, the FASTest, and the Witness card were 50%, 40%, and 60%, respectively. Specificity for the tests was 97.8%, 97.8%, and 92.2%, respectively. When comparing the rapid tests to PCR, sensitivity dropped to 18.4%, 15.8% and 26.3%, whereas specificity increased to 100%, 100% and 95.2%.

With regard to general aspects of the test systems evaluated, each of them was easy to perform and the results were easy to read. There were no invalid test results in all 3 tests. With PCR used as a reference test, 3 of 13 positive samples (23.1%) obtained by the Witness parvo card test were false positive, but none of the other rapid tests were. Six of 50 dogs in the 2 control groups were positive by PCR. It remains unclear whether this is due to carrier state or merely viral DNA.

This study showed that the evaluated rapid tests for the detection of CPV in feces have a high specificity and poor sensitivity compared with PCR and IEM. Some dogs with chronic diarrhea or without gastrointestinal signs may also have positive CPV PCR results. The significance of this finding remains unclear. Not much is known about the prevalence of CPV shedding in dogs with chronic diarrhea. Because of altered intestinal mucosal barrier function, an infection with viruses may be facilitated. An influence of a nongastrointestinal disease on immunity leading to a higher susceptibility for viral infections can also not be excluded.

A positive fecal PCR result alone is not sufficient for diagnosis of CPV enteritis. Instead, diagnosis should be based on a combination of history, clinical signs, laboratory parameters, (especially leucopenia) and positive fecal test results.

In general, when a positive rapid test result is obtained, diagnosis of CPV enteritis is likely to be correct. However, a negative test result does not rule out parvovirosis as a differential diagnosis in a dog with hemorrhagic diarrhea. In such cases, additional tests should be considered, ideally examination of fecal samples for CPV by IEM or PCR.

Acknowledgements. The authors thank the manufacturers of the 3 rapid parvovirus tests for donating the test kits for this study.