What to Do With a Positive 4Dx

What to do with a Positive 4Dx

Leah A. Cohn, DVM, PhD, DACVIM (SAIM) Professor, University of Missouri - College of Veterinary Medicine

Serologic titers detect antibodies or antigen in fluid samples, and these tests have long held an important place in disease diagnosis. In addition to a role in diagnosis, we often employ such tests to screen for infection in apparently healthy animals (e.g., cats for retroviral infections. These screening tests come in a variety of formats and use a variety of technologies; some are “mailed out” and other run as “cage side” tests; some are qualitative, others quantitative. Veterinarians are faced with the choice to screen or not to screen, and if they do screen, they will face questions as to how to handle a positive test in a healthy pet. To respond to those questions, they must understand something of the test itself (e.g., antigen vs. antibody, sensitivity and specificity), the pathogen prevalence in the region (which, in turn, effects predictive value), the pathogen behavior (e.g., is there an effective immunologic response, or is there a chronic carrier state?), and the disease caused by the pathogen (e.g., is the disease simple or difficult to treat, of high or low virulence?)

Antigen vs. antibody matters to interpretation. Tests that detect antigen (e.g., most serologic heartworm tests, FeLV tests in cats), can prove that a pathogen is present in the sample. When these tests are negative, they cannot prove that a pathogen is absent. For example, cats with heartworm infection usually have very low worm burdens (1 or 2 worms is typical), and the antigen test specifically detects antigen in female worms. If a cat with heartworms has only one male worm, the antigen test will always be negative.

As opposed to antigen tests, antibody tests detect the host’s response to antigen encountered at some point in the past. Antibody formation takes some time. This means that titers may be absent during acute infection/illness, which is the reason that convalescent titers (to demonstrate seroconversion or a 4-fold increase in titer) are often required to confirm acute disease such as leptospirosis or RMSF. On the other hand, antibodies may persist long after an infection has resolved, or at least after any threat of illness due to infection has resolved. For example, dogs infected with E. ewingii may remain well or develop acute illness, but chronic illness is not reported. Yet, chronic infection can lead to chronically increased antibody titers even though the (known) risk of illness is past. Antibodies can be cross-reactive, meaning that a positive test result may be due to exposure/infection not with the pathogen of interest, but with a related, cross-reactive organism (pathogenic or not). As an example, the pathogen that causes RMSF is cross-reactive with many other Spotted Fever Group organisms, most of which are not pathogenic; this leads to frequent “false positive” tests. Additionally, certain antibodies may be found after vaccination as well as after natural infection. On occasion, diagnostic tests are designed to “work around” this problem, as is the case with C6 peptide assays for Lyme disease that are positive only after natural exposure to Borrelia burgdorferi and not after vaccination. In fact, all panelists on the ACVIM consensus statement on Lyme borreliosis recommend yearly screening for dogs that have spent time in endemic areas.

An additional set of limitations for serologic tests pertains to the likelihood that a given test accurately reflects pathogen/disease presence or absence. Any test, no matter how good, is subject to both false positive and false negative results. These are reflected in the diagnostic sensitivity and specificity of the test. Diagnostic sensitivity refers to the proportion of tests run on infected animals that are positive, while diagnostic specificity refers to the proportion of tests run on uninfected animals that are negative. It is apparent then that to “rule out” a diagnosis, a test with a high sensitivity is desired, while to “rule in” a diagnosis, a test with a high specificity is desired. Generally for screening exams, a test with high sensitivity is preferred over one with high specificity. Even more important to the clinician than a given test’s sensitivity and specificity are the test’s positive and negative predictive values. The positive predictive value is the probability that an animal that tests positive actually has the infection in question, while the negative predictive value is the probability that an animal that tests negative is free of infection. While positive predictive value certainly is related to the sensitivity and specificity of the diagnostic assay, it is also related to the prevalence of disease in the population of animals tested. This means that a positive test result in a population with a low prevalence of disease, even when the test is very sensitive and specific, has a greater chance of being a false-positive than when the same test is applied to a population with a high disease prevalence. As an example, if a diagnostic test has a sensitivity of 95% and a specificity of 90%, the positive predictive value of that test in a population with a 50% pathogen prevalence would be 90%. This means that 1 out of 10 positive tests would be a false positive. On the other hand, for that very same test applied to a population with a 5% prevalence of pathogen, the positive predictive value would be 33%, meaning that 2 out of 3 positive tests might be false positives.

Finally, understanding the disease that might result from the pathogen exposure identified on a screening test impacts reaction to a positive screening test. If the pathogen frequently results in chronic disease, it makes sense to intervene before disease is manifest. This is very true for pathogens such as heartworm infection – a positive screen should prompt treatment. On the other hand, if the pathogen seldom results in chronic disease, a more “watch and wait” approach may be the responsible approach. There will always be both pro’s and con’s to screening healthy dogs for infection. These are summarized here: Screening Pro’s • May allow treatment before disease occurs • Provides regional prevalence information • May provide sentinel information for both human and animal infections • Informs ectoparasite control practices • May reduce pathogen reservoir for some infections • Alerts to non-tested pathogens carried by the same vectors Screening Con’s • False +’s in low prevalence areas are a real issue • May lead to unneeded treatment • Infection may never become clinical • Past infection may be resolved • Adverse effects; costs; resistance to drugs

• Treatment may not “cure” and doesn’t prevent re-infection • Role of dogs for human infection debatable for some pathogens

So with all this information, what is the optimum response to a positive result on a screening test such as the IDEXX 4Dx in an apparently healthy dog? There is no single answer, but instead all the factors discussed, as well as the owner’s wishes, must be considered. General guidelines are presented here:

Positive Heartworm Screen

1) Evaluate for microfilaria (Knott’s test) 2) Consider confirmatory re-test if microfilaria negative 3) Obtain chest radiographs, CBC, chemistry profile, and UA for disease staging and pretreatment health evaluation. Consider echocardiogram if murmur or cardiomegaly present. 4) Initiate treatment per American Heartworm Society Guidelines using melarsomine (http://www.heartwormsociety.org/veterinary-resources/canine-guidelines.html#8) 5) Initiate heartworm prophylaxis 6) Retest in 6-12 months

Positive Ehrlichia Screen

1) Discuss ectoparasite control with the pet owner. You now have proof their pet was not only bitten by a tick, but that tick transmitted a potential pathogen! Do better on parasite control. 2) Consider geography. In Missouri, it is FAR more likely to be E. ewingii or E. chaffeensis than E. canis causing the positive test. These organisms are far less pathogenic than E. canis. 3) Perform a CBC to look for morulae, thrombocytopenia, anemia, or hyperglobulinemia. If these are identified, antibiotic (doxycycline, minocycline) treatment is indicated. 4) Perform a UA to look for proteinuria. If identified, perform UPC and blood pressure, and consider further evaluation and antibiotic treatment. 5) Perform either a chemistry with creatinine/BUN or an SDMA (much more sensitive) to evaluate renal function. If either are abnormal consider further evaluation and antibiotic treatment. 6) If CBC, renal parameters, and UA are normal, discuss these three options with the owner and let them be part of the decision. The options are: a. Watch and wait (my preferred option for my own dogs in Missouri) i. Educate the owner about the signs of disease ii. Repeat CBC, renal parameters, and UA in one year b. Investigate further i. Perform more specific serologic tests to ID species of Ehrlichia (difficult to find lab to run this commercially) ii. Perform PCR, realizing that a negative test does NOT rule out infection but a positive proves pathogen presence. c. Treat with tetracyclines i. Doxycycline 10 mg/kg daily (q 24 hr or divided BID) PO for 28 days (chronic monocytic ehrlichiosis requires longer treatment than acute, or than granulocytic ehrlichiosis) OR minocycline 10 mg/kg PO q 12h for 28 days.

ii. Realize there are issues of cost, convenience, and adverse effects (dysbiosis, GI upset, hepatopathy, esophageal stricture [cats esp]) associated with tetracycline use iii. Realize that antibiotics may not eliminate the pathogen entirely, potentially allowing delayed illness despite treatment.

Positive Lyme Screen

1) The IDEXX SNAP 4Dx will NOT be positive due to vaccination, but some other tests (Osp-based serologic tests) are. The IDEXX test will only be positive after natural infection. 2) Discuss ectoparasite control with the pet owner. You now have proof their pet was not only bitten by a tick, but that tick transmitted a potential pathogen. 3) Perform urinalysis to look for proteinuria. If identified, perform UPC and blood pressure, and consider further evaluation and antibiotic treatment. 4) A quantitative C6 peptide test is available from IDEXX but the ACVIM consensus statement on Lyme borreliosis does not recommend performing the test as there is no proof that the test is predictive of illness. a. Some panelists on the consensus statement, and the author, do recommend quantitative C6 peptide. Antimicrobial treatment could be instituted if C6 antibody is >30 U/ml. b. If treatment is begun after high titer, can repeat quantitative C6 in 4-6 months time to document new baseline for future comparison. Expect titer to decrease by at least 50%. 5) Four of six panelists on the ACVIM consensus statement on Lyme borreliosis do NOT recommend antibiotic treatment for non-proteinuric, healthy dogs with positive screening tests. i. Watch and wait (Consensus recommendation) 1. Educate the owner about the signs of disease 2. Repeat UA +/- UPC & BP in 3 to 12 months (no consensus on frequency) ii. Treat with antibiotics, especially if C6 >30 U/ml (minority opinion) 1. Doxycycline or minocycline 10 mg/kg q 12 to 24 hr for 30 days 2. Other antibiotics efficacious for Borrelia but not for common coinfection of Anaplasma (amoxicillin, cephalosporins, azithromycine). 3. Realize there are issues of cost, convenience, and adverse effects associated with use of any antibiotic 4. Repeat C6 peptide in 4-6 months to confirm reduced titer resulting from treatment 5. Repeat UA +/- UPC & BP in 3 to 12 months (no consensus on frequency) 6) Discuss pros and cons of Lyme vaccine going forward

Positive Anaplasma Screen

1) Consider geography. In Missouri, a positive test is more likely due to A. platys than A. phagocytophilum. 2) Discuss ectoparasite control with the pet owner. You know have proof their pet was not only bitten by a tick, but that tick transmitted a potential pathogen.

3) Perform a CBC to look for morulae, thrombocytopenia or hyperglobulinemia. If these are identified, antibiotic treatment is indicated. 4) Perform a UA to look for proteinuria. If identified, perform UPC and blood pressure, and consider further evaluation and antibiotic treatment. 5) If CBC and UA are normal, discuss the three options with the owner and let them be part of the decision. The options are: a. Watch and wait (my preferred option) i. Educate the owner about the signs of disease ii. Repeat CBC and UA in one year b. Treat with antibiotics i. Realize there are issues of cost, convenience, and adverse effects associated with tetracycline use ii. Realize that antibiotics may not eliminate the pathogen entirely iii. Doxycycline or minocycline 5 mg/kg PO q 12 hr for 14 to 28 days (ideal therapy little investigated)

Suggested readings

Littman, Meryl P., et al. "ACVIM small animal consensus statement on Lyme disease in dogs: diagnosis, treatment, and prevention." Journal of veterinary internal medicine 20.2 (2006): 422-434.

Diniz P, et al “Ehrlichiosis and Anaplasmosis: An Update”. Veterinary Clinics of North America, 52(6):1225-1266, 2022.

Taber R, et al. “Bartonellosis in Dogs and Cats, an Update”. Veterinary Clinics of North America, 52(6):1163-1192, 2022.

Little, Susan, et al. "Canine infection with Dirofilaria immitis, Borrelia burgdorferi, Anaplasma spp., and Ehrlichia spp. in the United States, 2013–2019." Parasites & Vectors 14.1 (2021): 1-16.

Beall, MJ, et al. “An Improved Point-of-Care ELISA for the Diagnosis of Anaplasmosis and Ehrlichiosis During the Acute Phase of Tick-Borne Infections in Dogs” Topics in Companion Animal Medicine 51, 2022 https://doi.org/10.1016/j.tcam.2022.100735.

24

Companion Animal

Ralph Harvey, DVM, MS, DACVAA

Department of Small Animal Clinical Sciences University of Tennessee College of Veterinary Medicine Knoxville, Tennessee.

26