Viral diagnostics: What do the results mean? (Proceedings)

Diagnosis of viral infections is an important component of small animal patient care. Viral pathogens may affect any animal at any age, and clinical signs alone are often not enough to confirm the diagnosis. In these cases, virus-specific testing is needed. Testing for viral pathogens is as varied as the viruses themselves, and results can be confusing. But confirmation of the diagnosis can affect treatment, prognosis, and control measures.

There are two basic strategies for diagnosing a viral infection: "Look for the bug, or look for antibody to the bug." Within these two strategies are many methodologies, with varying sensitivities, specificities, turn-around times, and expense. This discussion will address some of the methodologies most commonly used for viral diagnostics.

Virus detection

Identification of the virus from the infected animal is often considered the gold standard for diagnosis of viral infection. Both nonspecific and specific assays are available. Nonspecific assays include virus culture and visualization by electronmicroscopy. Specific assays generally involve either detection of specific viral proteins (antigen detection) or specific genetic material (e.g. polymerase chain reaction, or PCR).

Virus isolation. Virus culture, or isolation is the propagation of virus in cell culture and can be done on a variety of samples. It's nonspecific, in that, theoretically, any virus that is present in the sample will be propagated and ultimately identified. It is usually more sensitive than antigen detection, but the turn-around time can be significant (1-2 weeks). For isolation to be successful, the virus must remain viable from the time it is collected from the animal until it reaches the laboratory. For some hardy viruses, like feline calicivirus, this is generally not a problem; but for other, more labile viruses, like feline herpesvirus, rapid transport and chilling are required. Not all viruses propagate well in the laboratory, like the virus of feline infectious peritonitis, and even if the virus does "grow", the relationship of the virus isolated to the disease in the animal is not always causal. Generally, though, virus isolation is sensitive and diagnostic.

Electronmicroscopy. This procedure is most commonly done on fecal samples from animals with enteritis. It is nonspecific in that any virus present in the sample can be visualized and identified; thus enteritis due to rotavirus, coronavirus, or parvovirus can be distinguished. The organism does not need to remain viable for identification and the turn-around time is rapid. Drawbacks are the relative low sensitivity of the assay (though in the acute phase of viral enteritis, this is not a concern, as the animal is shedding large amounts of virus), expense, and its limited availability. The equipment is expensive, and expertise is required; thus EM is usually only available from laboratories associated with academic institutions.

Antigen detection. Antigen detection involves the identification of virus protein through the use of virus-specific antibody. As with virus isolation, a variety of biologic samples can be used. Swabs of affected areas (e.g. conjunctivitis), tissue impressions, cells pelleted from a fluid (e.g. tracheal wash), can all be used; the assay detects the virus within infected cells. One of the most common techniques is immunofluorescence assay (IFA), where the antigen-specific antibody is labeled with a fluorescent dye. This test is generally inexpensive and fast. However, sensitivity is not high, thus a positive result rules in, but a negative result does not rule out a virus. And since it is a specific assay, it will not detect other agents. For example, an IFA for feline herpesvirus from a conjunctival swab will not detect feline calicivirus; this would have to be done separately, at an additional charge.

A common antigen detection assay used in many veterinary practices is the FeLV ELISA. This assay detects FeLV protein in the serum of an infected cat. The virus-specific antibody is anchored to the membrane, which binds the antigen in the serum sample. A soluble virus-specific antibody with an enzyme attached is then added, which binds the viral antigen, and is not washed away. Addition of the substrate leads to the color change, seen as a blue dot, for example. This assay is rapid and easy. The sensitivity is relatively high, and specificity is good. However, positive results, especially in an asymptomatic cat should be confirmed with a different test, as false positive results can occur. The canine parvovirus ELISA is another example of an antigen detection ELISA. Here, the anchored and soluble antibodies are specific for parvovirus, and the assay is done on fecal samples. Again, sensitivity and specificity are good.

Other antigen detection assays are available, such as immunohistochemistry and immunoperoxidase staining (often done on formalin-fixed tissue), but all have as their basis virus-specific antibody binding to the viral antigen in the sample; they simply vary in the antibody tag, and the sample type.

Genetic detection. The most common method for detecting viral genetic material is polymerase chain reaction (PCR). With this technology, small pieces of a virus' gene are repeatedly synthesized, effectively amplifying the original genetic material, allowing its detection. This methodology requires virus-specific targeting of the genetic material, thus it is a specific assay, with separate assays for each virus. Some PCR assays are termed multiplex, which means that multiple viruses are specifically targeted in a particular assay. For example, a multiplex assay for feline herpesvirus and calicivirus can detect both viruses specifically in a single sample. The product of PCR is identified through use of a genetic probe or other method that confirms the identity of the amplification product. Real-time PCR is a variation of conventional PCR in that the amplification can be followed in "real-time". Generally, it has increased sensitivity and specificity as compared to conventional PCR, and is also more rapid. PCR assays may be done on a variety of biologic samples, depending on the assay. For example, for canine parvovirus, feces can be used; for upper respiratory tract infection, swabs may be used; and for FIV, blood is used.

One key aspect of PCR to remember is that it can be exquisitely sensitive, a double-edged sword. Very small amounts of genetic material are amplified repeatedly, leading to millions of copies. Because of this, subclinical, even latent infections can be detected; thus, positive results must be interpreted in light of the other clinical parameters. Contamination can also be a problem, leading to false positive results. Stringent controls and experienced personnel are required for accurate results.

Loss during transport can negatively affect results. While PCR does not reflect the viability of the organism (inactivated virus can be detected), degradation of the genetic material can occur. To prevent degradation of the genetic material during transport, chilling and rapid shipment is generally recommended. This is particularly true for those viruses whose genome is RNA instead of DNA, as the former is very labile.

False negative results can occur as a consequence of viral genetic variation. The assay uses gene-specific priming for the DNA synthesis; this priming is based on the nucleotide sequence of the target virus. With viruses whose genetic sequence varies significantly among strains, this may lead to lack of DNA synthesis and amplification, and false negative results. This is a problem, for example, with FIV in cats. PCR has been used in an attempt to identify infection in vaccinated cats (for whom the antibody ELISA is thus ineffective); however, because of the significant strain variation of different FIV isolates, PCR is often negative, even in infected cats. Thus, as with any diagnostic assay, the results must be interpreted carefully.

Antibody detection

An alternative to "looking for the bug" is "looking for antibody to the bug" in a patient. Antibody detection gives the "immunologic history" of the animal – it reveals whether or not an animal has been infected in the past (recently or distantly) with a particular pathogen. It does not however determine when in the past that infection occurred. Some assays allow quantitation of the antibody, giving a numeric titer to the result. Others give only positive vs negative results, with no antibody quantitation. Methodology of antibody assays also varies, and standardization among laboratories does not exist; therefore, comparisons cannot be made between labs, or assays. Cutoff values for positive results also may vary. As a consequence, numeric results do not, in and of themselves, reveal infection status. That is, a high titer does not necessarily mean current infection, nor does a low, or even negative titer necessarily mean no infection.

Direct detection of antibody. With these assays, the virus is used to "capture" the patient's antibody in the serum sample. This virus is anchored to a solid substrate such as a glass slide, or membrane. The serum, or serum dilutions are added, and if antibody is present, it will bind to the anchored virus. This binding is then visualized by the addition of antibody to the patient's antibody (e.g. anti-cat IgG) which is purchased commercially. This second antibody is tagged (fluorescent dye, enzyme, etc) and after binding to the now-anchored patient antibody, it allows detection the patient's antibody. Immunofluorescence assays are used in many laboratories, and are often done with increasing serum dilutions, in order to determine the antibody level – the highest serum dilution that fluoresces is reported as the antibody titer.

These methodologies using antibody to the patient's antibody for detection are among the most common types of serologic assays. For example, the FIV ELISA uses virus anchored to a membrane to "capture" the cat's antibody in the serum sample. Soluble antibody to the cat's antibody is then added. The enzyme tagged to this secondary antibody leads to a color change (blue dot) after the addition of substrate. Because they generally require a commercially-produced anti-antibody, they are usually only available for the common domestic animals such as dogs and cats. To avoid the need for these anti-antibodies, other options are available for some serologies, described below.

Detection of antibody-facilitated activity. These assays use antibody-antigen binding to facilitate an activity that can be visualized. For example, virus antigen linked to latex beads will be bound by the patient's antibody leading to the clumping of the latex beads (latex agglutination assay). Other assays take advantage of the fact that antibody bound to antigen will precipitate out of solution at appropriate concentrations. The best example of this is the Coggins test for Equine Infectious Anemia (EIA), an agar gel immunodiffusion test. Virus and patient's serum proteins (including antibody) placed in separate wells in agarose diffuse into the agarose; where they meet in optimal concentration, a line of precipitation forms that can be visualized.

Detection of antibody-inhibited activity. In some assays, the binding of antibody to the virus will inhibit an activity that can be visualized. For example, hemagglutination inhibition assay for antibody to canine parvovirus: canine parvovirus has the ability to clump certain red blood cells (RBC); this activity is exploited in this laboratory assay. Virus is mixed with the patient's serum. If antibody to parvovirus is present in the serum, it will bind the virus. When RBC's are added to the well, binding of the virus by the patient's antibody will inhibit virus clumping of the RBC's. The RBC's thus settle out of solution in a pellet. Another example is serum virus neutralization. In this assay, virus is again incubated with the patient's serum; if antibody is present it will bind to the virus. The solution is then added to cell culture – if antibody is present, the virus is "neutralized" and does not infect the cells, thus no cell death occurs. If there is no antibody, the virus is free to infect and kill the cells, which can be seen as cell death. This assay takes several days to complete.

Interpretation of serology. As stated above, the presence of antibodies does not necessarily indicate current infection. The exception to this is viruses which cause lifelong infection, such as FIV – the presence of antibody thus indicates current infection, as the virus is never cleared. For the majority of viruses, current infections can only be confirmed with paired serology documenting a rising titer. This is not commonly done; because of this, caution must be used to avoid over-interpreting the results.

With acute infections, or virus infections where disease is seen soon after exposure, antibody levels may be low or even negative. Conversely, after infection and clearance of the virus, antibody levels may remain significantly elevated for extended periods, months, even years. Vaccination can also complicate matters, as many of the common pathogens are included in routine vaccines, thus most animals are seropositive.

Sensitivity and specificity will vary with different assays. Generally speaking, western blot is very sensitive and specific; ELISA's are also sensitive and specific; IFA's are moderate in sensitivity and specificity; SVN's and HI's are quite sensitive and specific. Most are rapid (SVN is the exception) and inexpensive; many are also quantitative, providing antibody levels with the results (ELISA's and western blot are the exceptions with most available assays). With all serologic assays, the results must be interpreted in light of the other clinical data. Unless paired titers are done, current infection status cannot be determined with a single assay regardless of magnitude, unless the infections are invariably lifelong, as with FIV.

For in-house testing for any virus, an awareness of the capabilities and limitations of the assay is critical. For tests sent to outside laboratories, the best advice for submitting samples for and interpreting the results of any assay is to communicate with the testing laboratory. For accurate assessment of the results, communication is critical.

Kennedy, MA. 2005. Methodology in Diagnostic Virology. In: Vet Clin Exot Anim. 8(7-26).