Sessions

Canine dirofilariosis is an important mosquito-borne parasitic disease with zoonotic potential. The main causative agents are Dirofilaria immitis, which causes heartworm disease, and Dirofilaria repens, which primarily causes subcutaneous dirofilariosis. Accurate differentiation between the two species is important for appropriate treatment and prevention. PCR and qPCR provide reliable species identification; however, their time and equipment requirements may limit their use in veterinary clinics and field settings. Loop-mediated isothermal amplification (LAMP) can be performed at a constant temperature and may therefore provide a suitable basis for simplified molecular diagnostic approaches.

In this study, we developed a direct, closed-tube LAMP assay and evaluated its diagnostic performance for the detection of D. immitis and D. repens directly from canine whole blood, without conventional DNA extraction. A total of 60 EDTA-anticoagulated whole blood samples were examined. Following NaOH–Chelex® 100-based sample preparation, amplification was performed using species-specific primer sets. Results were evaluated by closed-tube fluorescence detection under blue-light illumination and compared with duplex qPCR results.

Duplex qPCR identified 23 D. immitis monoinfections, 12 D. repens monoinfections, 14 coinfections, and 11 samples negative for both species. The relative sensitivity of direct LAMP was 91.89% for D. immitis and 84.62% for D. repens. No false-positive results were observed for either species, resulting in a relative specificity of 100% for both assays.

Our results indicate that direct, closed-tube LAMP may provide a rapid and simplified approach for the molecular detection of D. immitis and D. repens. Eliminating the conventional DNA extraction, combined with isothermal amplification and closed-tube detection, may reduce equipment requirements as well as the risk of cross-contamination. Following further optimization and validation in a larger sample cohort, the method may also be suitable for near-point-of-care diagnostic applications.