Rapid Point-of-Care Detection of Dirofilaria immitis and Dirofilaria repens in Canine Blood Using Two Direct Closed-Tube LAMP Assays

Canine dirofilariasis, caused by Dirofilaria immitis and Dirofilaria repens, is an emerging vector-borne disease of increasing veterinary and zoonotic importance. Rapid and species-specific detection is essential for effective clinical management and epidemiological surveillance. This study aimed to develop and diagnostically evaluate two novel species-specific loop-mediated isothermal amplification (LAMP) assays for the direct detection of D. immitis and D. repens in canine whole blood, performed in parallel in separate reactions, with emphasis on simplified and potentially near-point-of-care applicability. Primers targeting mitochondrial COI and NADH gene regions were designed and validated. In silico specificity analysis against 13 filarioid species confirmed the absence of non-specific primer binding. A direct closed-tube LAMP protocol using sodium hydroxide-Chelex-100 lysis was optimized, enabling amplification without conventional DNA extraction while reducing contamination risk and processing time to under 60 min. Relative diagnostic performance was evaluated relative to quantitative real-time PCR (qPCR) results. Using purified DNA, the D. repens assay achieved 100% relative sensitivity and relative specificity, whereas the D. immitis assay showed 94.5% relative sensitivity and 100% specificity. In direct whole-blood assays, relative specificity remained 100% for both targets, while sensitivity decreased to 90.9% for D. immitis and 77.42% for D. repens, with most false-negative reactions associated with high qPCR Ct values (>30). These findings demonstrate that the proposed assays provide a rapid and practical molecular diagnostic approach with potential applicability for point-of-care veterinary testing.