Evaluation of the larval migration inhibition assay for detecting macrocyclic lactone resistance in Dirofilaria immitis

Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L-3). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L-3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC50 values ranged from 1.57 to 5.56 M (p >= 0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L-3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.