False negative antigen tests in dogs infected with heartworm and placed on macrocyclic lactone preventives

Abstract

BackgroundDogs with chronic inflammation, including those with heartworm being managed with macrocyclic lactones and doxycycline (slow kill, SK), may develop immune complexes that block detection of Dirofilaria immitis antigen on commercial tests.MethodsTo determine if SK could result in development of false-negative antigen tests, we collected serum samples from dogs that had been diagnosed with heartworm by antigen detection, with or without confirmation by detection of D. immitis microfilariae, placed on monthly macrocyclic lactones and doxycycline, and that later tested negative on an antigen test, and then tested them for antigen of D. immitis before and after treatment to disrupt immune complexes.ResultsSerum samples from a total of 15 dogs managed with SK were negative for antigen prior to heating on commercial assay (DiroCHEK®, Zoetis) by colorimetric detection and spectrophotometry, but after heat treatment, 8/15 (53.3%) samples converted to positive. Review of the medical records of each dog indicated that, after the heartworm diagnosis, only 7/15 (46.7%) dogs appeared to receive preventive monthly as prescribed, including 3 dogs that had detectable antigen after heating the sample and 4 dogs that did not have detectable antigen after heating. Whole blood was available from 9 dogs; microfilariae of D. immitis were detected in 1 sample.ConclusionsThese data suggest that immune complex formation in dogs infected with heartworm and managed with SK can induce false negative antigen test results, misleading veterinarians and owners about the efficacy of this approach. Moreover, compliance with preventive administration appears poor, even after a heartworm diagnosis. The presence of persistent microfilaremia in at least one dog has implications for resistance selection.

Current trends in canine dirofilariosis in Austria—do we face a pre-endemic status?

“…Yet, even a specific screening system may be susceptible to cross-reactions, as well as false-negative or falsepositive results, as shown in previous studies (Venco et al 2017). Another factor for inconsistency is the pre-treatment with macrocyclic lactones, which can lead to the formation of antigen-antibody-complexes masking the antigen, which could then no longer be detected by commercial test kits (Drake et al 2015). The use of heat-treated serum for inhouse antigen tests can provide a solution to this problem and therefore has been practised in the context of this study since 2015 (Velasquez et al 2014;DiGangi et al 2017).…”

Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis

“…Currently, serological methods are available for detection of specific D. immitis antigens in blood, based on ELISA and rapid immunochromatographic dipstick; however, they are not entirely reliable; thus, more specific and sensitive methods for dirofilariasis screening and serodiagnosis are needed . Overall, due to high sensitivity, specificity and feasibility, serological methods are preferred to parasitological ones . Despite these considerations, many commercial ELISA and dipstick‐based kits are available and routinely used for serodiagnosis of dirofilariasis; however, these assays lack specificity due to cross‐reaction with other common parasite antigens .…”

“…Despite these considerations, many commercial ELISA and dipstick‐based kits are available and routinely used for serodiagnosis of dirofilariasis; however, these assays lack specificity due to cross‐reaction with other common parasite antigens . Previous findings showed that the sensitivity of commercially available heartworm antigen detection kits ranged from 78% to 84%, and the specificity was about 97% but, there are newer studies with much high sensitivity and specificity when used in regions with low infection rates . High sensitivity and specificity serological tests are approved, but low worm burdens and the presence of immature female‐ or male‐only infections can result in low antigen titres, causing false‐negative results.…”

Pig-hunting dogs are an at-risk population for canine heartworm (Dirofilaria immitis) infection in eastern Australia

“…In areas of recent heartworm introduction, newly infected dogs will have no Mff due to the prepatent period of at least six months, single sex infections or burdens with less than two female heartworms. Additionally, the formation of immune complexes can further complicate diagnosis and detection of D. immitis antigen [30,31].…”