Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease”) primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.