Evaluation of the effectiveness of antigen testing in the diagnosis of canine dirofilariasis

Abstract

Dirofilariasis is a dangerous parasitic disease, the spread of which among dogs worldwide and in Ukraine is increasing, necessitating effective diagnostic methods for timely detection and treatment. Accurate diagnosis is critical to preventing severe complications, particularly cardiovascular pathologies caused by Dirofilaria immitis. In our study, we used a method involving preheating of blood samples prior to antigen testing with immunochromatographic tests, which significantly enhanced detection sensitivity. The aim of the study was to assess the effectiveness of the proposed approach in combination with well-known methods for immunochromatographic detection of antigens from nematodes D. immitis extracted from dogs. The research was conducted between 2022 and 2023, analyzing 192 serum samples from dogs aged 1 to 14 years. To detect D. immitis antigens, the commercial Heartworm Ag (Vet Expert) test system was used. Before testing, samples underwent thermal treatment: they were heated to 100°C for 5 minutes in a thermal block, causing protein coagulation. After centrifugation, the supernatant was tested according to the manufacturer’s instructions. This technique increased the likelihood of detecting antigens even at low concentrations of parasitic proteins. Additionally, a modified Knott’s method was employed to identify microfilariae in the blood, including staining with methylene blue, and the species composition was confirmed using PCR. Morphometric characteristics of filariae were assessed using microscopy. When testing blood serum, 14 (7.3 %) samples tested positive for D. Immitis antigen using the standard method, while this rate increased to 49 (25.5 %) after preheating. The Knott’s method identified microfilariae in 23 (21.7 %) animals: 7 cases were D. immitis, 11 were D. repens, and 5 dogs exhibited coinfection with both species. The number of microfilariae in the blood varied depending on the species: 120–2400 microfilariae/mL for D. Immitis and 10–870 microfilariae/mL for D. repens. The average length and width of D. Immitis microfilariae were 310.2±6.9 μm and 5.10±1.68 μm, respectively, while for D. repens, these measures were 360.12±2.82 μm and 7.85±1.23 μm. The obtained results highlight the necessity of preheating of serum samples to enhance the sensitivity of antigen testing for D. immitis. This approach also facilitates the identification of coinfections with D. repens. The proposed method is an important complement to standard diagnostic procedures and can minimize the occurrence of false-negative results.