Optimizing heartworm diagnosis in dogs using multiple test combinations

BACKGROUND: Various heartworm (HW) diagnostic testing modalities detect products of, or reactions to, different life cycle stages of Dirofilaria immitis. Microfilariae (Mf) can be directly visualized in blood, antigen (Ag) from immature and adult heartworms may be detected on commercial assays, and antibody (Ab) tests detect the host immune response to larval stages. Ag and Mf tests are commonly used in dogs, which frequently carry adult HW infections, but Ab tests have only been validated for use in cats. In some HW-infected dogs, Ag is blocked by immune complexing leading to false-negative results. Heat-treatment (HT) to disrupt these complexes can increase the sensitivity of HW Ag tests. The aim of this study was to compare different methods for diagnosing HW infection in dogs at high risk using individual and paired diagnostic tests, including an exploration of using Ab tests designed for cats to test canine samples. METHODS: One hundred stray adult (≥ 2-year-old) dogs in Florida shelters were tested using Mf, HW Ag, and HW Ab tests (feline HW Ab tests currently not commercially validated/approved for use in dogs); two versions of each test platform were used. RESULTS: Fourteen dogs tested positive using point-of-care (POC) Ag tests; an additional 2 dogs tested positive with microtiter well assay, and an additional 12 dogs tested positive using HT Ag testing. For individual tests, Ag test sensitivity/specificity compared to HT Ag was 50–57%/100%, and Ab tests were 46–64%/82–94%. Sensitivity estimates for individual tests were higher when comparing to non-HT Ag. Pairing POC Ag tests with Mf tests improved sensitivity without loss of specificity, while pairing POC Ag and Ab tests modestly increased sensitivity at the expense of specificity. CONCLUSIONS: Screening dogs for HW infection using both POC Ag and Mf detection, which is recommended by the American Heartworm Society, improved diagnostic performance in this study compared to single Ag test use, but may have missed more than one in four infected dogs. The need to improve access to highly accurate, rapid, and inexpensive large-scale HW testing for dogs in animal shelters remains largely unmet by current testing availability. The development of practical and validated protocols that incorporate heat or chemical treatment to disrupt Ag-Ab complexes in POC testing or decreasing the cost and time required for such testing in reference laboratories might provide solutions to this unmet need. Similar studies performed in countries where the prevalence of parasites such as D. repens or A. vasorum is different to the USA could potentially yield very different positive predictive values for both HT and non-HT Ag tests. GRAPHIC ABSTRACT: [Image: see text]