Sessions

Dirofilaria immitis (heartworm, D. immitis), a vector-borne disease, poses an increasing risk to both domestic and wild canines worldwide, with climate change contributing to its spread. Rapid and reliable diagnostics are vital for prevention and control, especially in veterinary contexts where large-scale testing is needed. Various methods exist, including visual tests such as smears or the modified Knott’s test, serological assays (such as ELISA), and molecular techniques like PCR and real-time PCR. While each has advantages, they also face limitations in terms of sensitivity, specificity, cost, or technical demands, which can hinder their use.

To address these challenges, we designed and tested five new primer sets targeting the cytochrome oxidase subunit I (COI) gene to create a loop-mediated isothermal amplification (LAMP) assay for detecting D. immitis in DNA isolates from dog blood samples (n = 40). The performance of each primer set was evaluated against real-time PCR results, which served as the reference standard to assess sensitivity, specificity, and diagnostic utility. Among them, Primer Sets 1 and 44 showed strong amplification with high specificity and sensitivity, with only a single false negative value. In contrast, Sets 15 and 32 performed poorly, producing false negatives, while Primer Set 2 had the best sensitivity but reduced specificity, likely due to cross-reaction with Dirofilaria repens (skin worm).

Overall, our results demonstrate that extensive preliminary screening of the designed primer sets is necessary to achieve adequate sensitivity and specificity, resulting in a promising and cost-effective LAMP test, a scalable alternative for heartworm detection, capable of distinguishing closely related filarial parasite species. With further improvements, particularly in simplifying DNA extraction and with the development of a similar detection method for D. repens, an optimized and validated LAMP assay has great potential as an on-site diagnostic tool to support large-scale surveillance and control of dirofilariasis amid shifting epidemiological patterns.