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The ABCs of Diagnosing Infectious Diseases
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Accurate diagnosis of infectious disease is increasingly important given the expanding geographical distribution of many, particularly vector-borne, diseases. Though some pets with infectious disease present with clinical signs many do not. This is especially true with vector-borne disease in dogs in which the majority are subclinical. Thus, screening for VBD in healthy patients is important especially given that many of these diseases can also affect humans and dogs may act as sentinels of emerging disease in a specific region.
Serology assesses humoral immunity and is the mainstay of screening for infectious disease as it is relatively easy to perform and can be done pet side. High sensitivity is important in screening tests to minimize false negatives. Serology can detect antibody or antigen. A positive antibody result indicates exposure to the pathogen (or cross-reaction from vaccine). To determine if active disease is present a combination of clinical signs, laboratory findings, magnitude of antibody titer, or additional testing, e.g., polymerase chain reaction may be required. A negative antibody result indicates lack of exposure or acute exposure (there is lag time to seroconversion). A positive antigen result confirms infection. A negative antigen results usually excludes the diagnosis. The cellular immune system is mediated primarily by T cells (CD4 and CD8) and plays a major role in defense against intracellular organism (e.g, Rickettsia spp., viruses). Serology may be negative in infections in which cellular immunity predominates.
All positive screening tests should be confirmed by follow-up testing. An ideal confirmatory test is highly specific thereby minimizing false positive results. As an example, a positive result or ‘blue dot’ on a SNAP test for VBD in a clinically normal dog should be followed by assessing for laboratory changes consistent with VBD (thrombocytopenia, hyperglobulinemia, proteinuria) and possibly PCR testing or obtaining a titer (amount of antibody, below). As another example, a positive FeLV antigen test should be followed by a second antigen test and quantitative PCR to confirm and stage the infection.
A titer is a quantitative measure of antibody. It is obtained by performing serial 1:1 dilutions with the ‘endpoint’ titer reported as the highest dilution at which antibody is detected. Comparing Leptospira canicola titers 1:100 and 1:1600, 1:1600 (‘one to sixteen hundred’) is the higher titer indicating more antibody. For most infectious disease 10-14 days are required for seroconversion after exposure. Hence the recommendation to obtain repeat or ‘convalescent’ titers 2 weeks after an initial low or negative result in an acutely ill patient. A 4-fold or greater increase in titer confirms disease. Titers to many VBD persist for months to years (lifelong in some) and therefore are not useful in monitoring response to treatment.
PCR is a molecular diagnostic tool the identifies DNA (or RNA) of a pathogen and is highly sensitive and specific for confirming infectious disease. Quantitative or real-time PCR allows determining the number of DNA copies which can be useful in differentiating vaccine from infection and in staging disease, e.g., leishmania, FeLV. Whole blood is the most common sample for diagnosing infectious disease, but PCR can also be run on urine (with whole blood for leptospirosis), aspirates of bone marrow, lymph nodes, spleen, and other tissues. A positive result indicates the organism is present and may be causing disease. A negative result usually excludes the diagnosis. However, prior treatment with antibiotics and/or organism in tissue not sampled can lead to ‘false’ negative results.
Serology and PCR testing are complementary, and it has been shown that performing both, sometimes more than once, maximizes diagnosis compared to performing one or the other. Co-infections are common with VBD and PCR panels that include multiple infectious agents are useful in this setting, especially in pets that present with severe or atypical clinical signs or that fail to improve within 3-5 days of therapy.
References
- Creevy KE, Grady J, Little SE, et al. 2019 AAHA canine life stage guidelines. J Am Anim Hosp Assoc 2019;55:267-290.
- Little SE, Levy JK, Hartmann K, et al. 2020 AAFP feline retrovirus testing and management guidelines. J Fel Med Surg 2020;22:5-30.
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