- 1 Direct Microscopic Examination
- 2 Test Considerations
- 3 Collection Technique and Sample Transport
Direct Microscopic Examination
Microscopic examination of fluid or tissue samples from patients suspected of having a viral infection is unlikely to contribute to the diagnosis. Because viruses are small and generally intracellular particles, neither light microscopy nor viral culture techniques are used in the practice setting. Some commercial and academic laboratories do offer electron microscopy of tissue (cells), fluids, or feces from infected patients, which may allow for direct visualization of virus particles. Results depend on the quality of the sample evaluated, the type of equipment available, and the experience of the individual performing the microscopy.
Various laboratory techniques are currently available for the identification of viral infections in dogs and cats. Excellent qualitative testing platforms are commercially available for in-practice use. Molecular diagnostic tests, viral culture, histopathology, and serology, all of which are routinely available to veterinarians, require that samples be submitted to commercial laboratories for assessment.
Among the in-hospital test systems used to identify virus, the enzyme-linked immunosorbent assay (ELISA) is the most common testing platform used. ELISA testing can be performed quickly (minutes) with little or no patient preparation and with relatively high sensitivity and specificity. Virus (antigen) detection tests are available as point-of-care tests for FeLV antigen in blood or serum and canine parvovirus (CPV) antigen in feces. In addition, these point-of-care tests for viral infections are capable of identifying patients that have not been exposed, enabling the clinician to rule out infection and viral shedding.
Test sensitivity refers to the likelihood that a patient with known infection will have a positive test result (a test with high sensitivity is expected to have few false-positive results).
Test specificity refers to the likelihood that a patient that is free of the infection will have a negative test result (a test with high specificity is expected to have few false-negative results).
In addition, many commercial and point-of-care nonquantitative serologic assays are available that detect antibody to many of the viruses that affect dogs and cats. However, the positive predictive value of antibody tests is typically lower than that of antigen tests. For example, a positive antibody test result to a particular viral pathogen typically does not constitute a diagnosis of infection, especially in the absence of clinical signs. It may merely reflect recent vaccination (e.g., feline immunodeficiency virus). On the other hand, a negative antibody test result generally does indicate that the patient has had no prior exposure to the virus (or vaccine).
Serology refers to the use of serum to detect the concentration of antibody and is widely used in veterinary medicine. The value of antibody titers in diagnosing a viral infection is dependent on a number of factors, including the infecting virus, vaccination history, and time since exposure. Use of acute and convalescent antibody titers in a patient suspected of having an acute viral infection can be a reliable diagnostic tool if a fourfold or greater increase in titer can be demonstrated over 2 to 4 weeks. Acute and convalescent viral titers in individual patients are rarely performed in veterinary medicine.
Virus isolation, however, is a valuable diagnostic tool that is underused in veterinary medicine, perhaps because of the limited number of commercial and university laboratories that provide viral isolation services and the increased availability of molecular diagnostic testing services. Diagnosis of viral upper respiratory infection in cats (herpesvirus 1 and/or calicivirus) is perhaps among the situations for which virus isolation can be most useful, especially in cluster households where many shedding carrier cats exist and kittens may be at risk.
To obtain a sample for viral isolation from the oral cavity of a cat, quickly insert a sterile cotton swab into the oral cavity to the level of the tonsil or oropharynx. By rolling the swab across the epithelium, it is possible to harvest cells and virus from infected cats. Immediately place the swab into a virus transport medium (usually provided by the laboratory). Antibiotics added to the solution prevent bacterial overgrowth of the sample. For short-term transit (5 days or less), hold specimens for viral isolation at 4° C rather than frozen. On reaching the laboratory, the specimen will be inoculated into a suitable tissue culture. Within a few days it is usually possible to establish, based on the cytopathic effect on the tissue culture, whether a virus infection is present. Fluorescent antibody testing can be done subsequently to confirm the isolate.
Although availability is limited, direct assessment of specimens (e.g., feces for CPV or canine or feline coronavirus) can be accomplished by electron microscopy. These methods can be useful for infections in which the virus concentration in the specimen reaches 106 to 107 organisms per milliliter. Specimens such as feces, vesicle fluid, brain tissue, urine, or serum can be submitted for electron microscopy.
Tissue specimens and exfoliative cytologic preparations can be submitted for viral identification by histopathology, immunohistochemistry, and direct fluorescent antibody testing. Such testing has limited application in patients with active disease because of the limited availability of these types of services and the time required for samples to be processed and reviewed by a pathologist. These tests can be particularly useful in postmortem diagnostics when multiple animals are potentially at risk.
Molecular diagnostics refers to the use of nucleic acid-based tests for the detection of viral DNA or RNA. Polymerase chain reaction (PCR) is a laboratory technology that offers exceptional test sensitivity. Through its ability to amplify trace amounts of DNA or RNA from pathogenic organisms millions of times, PCR facilitates identification of the “target” sequence of nucleic acid and therefore the infecting organism. This technology is also available commercially for the detection of DNA from selected bacteria and rickettsiae. PCR technology is particularly useful in the very early stages of a viral infection, when the level of antibody has not yet reached levels that are detectable with conventional antibody tests. In addition, PCR testing may detect healthy virus carrier animals that pose a risk to a larger population of susceptible animals yet cannot be identified by conventional virus isolation or identification technologies. It should be noted, however, that PCR technology is still subject to false-positive and false-negative test results. Therefore such testing is not necessarily indicated as a primary or exclusive test method for an individual patient.
Collection Technique and Sample Transport
Serum, plasma, or other fluids (e.g., cerebrospinal fluid [CSF]) can be tested for the presence of antibodies to selected pathogenic viruses. Whole blood samples should be allowed to thoroughly clot and retract (or the sample should be centrifuged) before serum is collected. Samples are submitted in a leak-proof vial. Refrigeration is appropriate for samples that must be held for several hours before testing.
Histology and Immunohistochemistry
Samples are limited to tissue obtained during surgical biopsy. As with conventional histopathology, samples (no more than 5.0 mm thick) should be placed in 10% buffered formalin and submitted in a leak-proof vial. It is recommended that the volume of formalin used be at least 10 times greater than the tissue sample submitted.
Fluorescent Antibody Testing
Testing can be performed on tissues collected during surgical biopsy or from tissue impressions (exfoliative) made from tissue imprints on a clean microscope slide. It is recommended that tissue impressions on slides be fixed in alcohol or acetone before submission. Fresh tissue is submitted on wet (not dry) ice and is not subjected to formalin fixation.
Small amounts of tissue suitable for electron microscopy should be no larger than 1×2 mm thick. Fixation in 2% to 4% glutaraldehyde for 24 hours at 20° C is required. Feces and body fluids collected for electron microscopy should be submitted fresh, not frozen or fixed in preservative. If shipping is required, feces and body fluids may be refrigerated or shipped on wet ice. Samples should be viable for 48 to 72 hours.
Sterile swabs may be used to collect samples for viral culture and isolation. Samples should be inoculated into a sealed vial containing viral transport medium (usually provided by the laboratory). Samples should not be frozen or fixed in preservative.
Polymerase Chain Reaction Testing
Laboratories offering PCR testing typically accept serum or anticoagulated (ethylenediaminetetraacetic acid [EDTA]) whole blood in leak-proof vials. Samples should be refrigerated and shipped on wet ice. Samples should not be frozen.