Molecular testing to diagnose feline infectious diseases (Proceedings)

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Molecular testing to diagnose feline infectious diseases (Proceedings)

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Apr 01, 2010




Simplified, there are two arms of the immune response: the innate immune response and the adaptive immune response. Realistically, these two arms are intimately entwined and difficult to separate. For many diseases, both are key factors in resistance to and survival of infection. These immune responses can be used to diagnose clinical diseases. The innate immune system consists of those responses that are not specific to the invading organism and do not require previous exposure. The most obvious component of this is the physical barrier to infection: the skin and mucous membranes. However, cellular responses include natural killer cells as well.

The adaptive response is organism specific and consists of the humoral and cell mediated immune response. As before, these two arms of the immune system are not clearly delineated but intertwined and rely on each other for adequate performance. Measurement of these two immune responses has been the mainstay of the diagnosis of infectious diseases. The humoral response is most easily measured and is usually associated with the so-called "titer" of disease. Actual physical methods for measurement of antibodies to a particular organism and a basic knowledge of these is necessary to understand the strengths and pitfalls associated with relying on the results for diagnosis. Enzyme linked immunosorbent assays, or ELISA's, can be used to detect antigen or antibody in a patient sample. A "cartoon" of one type of the methods is below.

The advantages of this, as well as a similar technique, the IFA, in which a slide is substituted for the well and the secondary antibody is labeled with a fluorescent reporter, is that it is relatively inexpensive, requires a small volume of sample, and can be adapted for high throughput. However, the antigen and antibody have to be standardized and the results depend on a single antigen antibody interaction. Non-specific binding may result from the reliance upon a single biologic reaction for a result, making specificity occasionally a problem in these assays. However, the ability to automate these reactions, minimal sample volume, and relative low cost make these excellent candidates to be used as screening assays.

Similarly, western blots rely on an antigen-antibody interaction; however, individual reactions are visualized on a paper, allowing for evaluation of multiple antibody-antigen interactions, improving specificity. Similar advantages are that minimal sample volume is needed, however, it is much more labor intensive and is more difficult to automate, resulting in increased cost. These are much more appropriate as a confirmatory test given the higher specificity but increased cost.

Any antibody titer simply represents exposure to the antigen, not active infection. However, certain disease courses have been studied experimentally and allow for diagnosis of active infection based on the presence of a particular antibody or level of response. This is not true for all diseases. Therefore, ideally, demonstration of the organism with evidence of an immune response to infection or pathologic markings would be used. Demonstration of the organism is obvious when associated with cytology or identification using histopathologic examination. However, these are insensitive methods and rely on large sections of tissue or other sample. The advent of the polymerase chain reaction, or PCR, greatly improved the sensitivity of detection of infectious organisms. PCR relies on nucleic acid specific primers and a proprietary enzyme to amplify minute amounts of nucleic acids from an infectious organism amidst a sample taken from the host. Detection of nucleic acids does not necessarily imply active infection either as non-viable organisms can be detected as well. Additionally, great care must be taken in selecting the laboratory that performs the assay: because of the exponential amplification false positives can be a problem and strict quality control must be maintained. Finally, the assays can be expensive as licensing fees must be paid to holders of the patent on the reaction, adding to expense of the assay.

The use of PCR, or rt-PCR (reverse transcription PCR, used to detect RNA, as the RNA must first be reverse transcribed prior to use in the PCR reaction), in feline medicine offers the clinician another tool for diagnosis. However, as with methods to detect and immune response to organisms, results must be interpreted in light of appropriate clinical signs and history. A positive result only implies presence of the organism, not necessarily a disease associated with active infection. Feline herpes virus is a perfect example of this. In the individual animal, a positive PCR result for FHV-1 can suggest either a carrier of the organism with a concurrent disease and no active infection, an animal with an unrelated disease and a secondary recrudescence of FHV-1, or a disease syndrome associated only with FHV-1. The clinician must then take this result, examine clinical signs and history, and determine if further diagnostics for additional diseases are needed.