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Making sense of it all: Understanding and interpreting patient-side diagnostic tests (Proceedings)

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Nov 01, 2009

Numerous patient-side tests are available to aid in the detection and diagnosis of various parasites and tick-borne pathogens. These immunoassays are based on the detection of either antigen or antibody in the blood or feces of the patient. There are 3 types of immunoassays in use: (1) ELISA; (2) Immunochromatographic; and (3) Agglutination. For parasites, the first two comprise the bulk of the tests currently available.

ELISA stands for Enzyme Linked Immunosorbent Assay. ELISAs can take many forms, but, the simplest consists of a solid phase or test surface, sample, conjugate, wash solution and substrate (chromagen). The conjugate is usually an antibody that has been bound or conjugated to an enzyme. The substrate is a reagent that changes color when exposed to the enzyme. For commercial kits, except for the test sample, all or most of the components, are generally supplied in the kit. In the kit will be a test device. Highly specific antibodies or antigens have already been coated onto (solid phase) or embedded into (test surface) the device. The patient's sample is placed on the solid phase of the assay or mixed with the conjugate and then placed in the assay. For heartworm tests and Giardia coproantigen test, the ELISA is detecting antigens in the sample. If the sample contains the antigens, they bind to the antibodies that are immobilized in the device. The conjugate also binds the antigens present resulting in a sandwich with the antigen sandwiched between the immobilized antibody and the conjugate. Unbound material is washed away and conjugate then interacts with the substrate. The resulting enzymatic reaction provides a visible color change. If the antigen is not present in the patient's sample, then the sandwich does not form, the conjugate is washed away and the substrate has nothing to interact with. Therefore, there is no color change. For antibody tests, such as Borrelia burgdorferi, Anaplasma phagocytophilum and Ehrlichia canis, the process is the same except that antibodies are detected rather than antigens.

Immunochromatographic assays can also be used to detect antigen or antibody in the test sample. The method is based on the same principles as the ELISA; however, latex or gold particles replace the enzyme and substrate and there is no wash step. The method uses a solid phase, the test sample, and colloidal gold or latex conjugate with or without flow solution. As with the heartworm ELISA, highly specific antibodies are coated onto the patient line and heartworm antigen is coated onto the positive control line. An absorptive pad contains antigen-specific antibodies conjugated to the gold or latex particles. The sample is placed on an absorptive pad and, if necessary, flow solution is added. If antigen is present, the gold or latex conjugate attaches to it and flows down the test strip. The antibody that is immobilized at the patient line then attaches to the antigen/gold or antigen/latex complex and color is produced. If no antigen is present, these complexes do not form and the gold/latex conjugated antibody flows past the patient line and no color is formed. There is an excess of gold/latex conjugate present so that, regardless of whether antigen is present in the test sample or not, there is sufficient conjugate present to react with the positive control line. As the gold/latex conjugate flows past the control line, it combines with the antigen that is immobilized there and color is produced.

Although the sensitivity and specificity of these tests has greatly improved over the years, they are not 100%. Therefore, false positives and false negatives can occur. Some reasons for false positive results include improper sample quality, inadequate washing (ELISAs), over-incubation, and defective test. All test kits come with quite specific instructions as to the timing of the test and when to read the results. Prolonged incubation can result in non-specific binding of test components resulting in the appearance of a positive result. Proper washing is also quite important. Removal of all unbound conjugate is necessary to, again, prevent non-specific binding and the appearance of a positive result. Some reasons for false negatives include inadequate sample volume, improper storage of sample, wrong sample type, insufficient incubation time, and expired or improperly stored test kit. Acute infections may also return negative results as there has been insufficient time for the production of a detectable antibody response. For heartworm antigen tests, all male infections or only 1 or 2 females present may also produce false negative results.

Interpretation of test results requires the "big picture" approach. These tests are not the definitive diagnostic tests(s) for determining whether or not a pet has the disease and must be interpreted in light of all available patient information. For the antibody tests, a positive result means the pet has been exposed but does not necessarily mean the pet has an active infection. Positive results in the absence of clinical disease generally require further confirmatory testing. If confirmed, treatment may or may not be required, depending on which organism is present. Positive results for any of the tick-borne diseases does clearly indicate poor tick control. Furthermore, positive results indicate humans are at risk of infection as well. While people do not get the organisms from the dog, they are exposed to the same ticks as they hike, walk, and, otherwise, play with their canine companion in the tick-friendly environment. Consequently, humans run the same risk of infection.

References available upon request