Exploring multiplex qPCR as a diagnostic tool for detecting microfilarial DNA in dogs infected with Dirofilaria immitis: A comparative analysis with the modified Knott's test

Current recommendations to diagnose cardiopulmonary dirofilariosis in dogs caused by Dirofilaria immitis involves tandem antigen and circulating microfilariae tests. The modified Knott's test is an important tool in heartworm diagnosis, allowing identification of circulating microfilariae. However, the subjective nature of the modified Knott's test affects its accuracy and diagnostic laboratories usually do not provide a quantitative outcome. Quantitative enumeration of microfilariae enables clinicians to track treatment progress and acts as a proxy for detecting emerging macrocyclic lactone resistance. There is a need for better diagnostic tools suitable for routine use to efficiently and accurately quantify the presence of D. immitis microfilaremia. The aim of this study was to determine whether the quantitative modified Knott's test can be substituted by multiplex quantitative polymerase chain reaction (qPCR) targeting D. immitis and associated Wolbachia endosymbiont DNA in canine blood samples. To do this, genomic DNA samples (n = 161) from Australian dogs, collected as part of a previous 2021 study, were assessed in a TaqMan qPCR targeting DNA of D. immitis, Wolbachia sp. and Canis lupus familiaris. Of the 161 genomic DNA samples, eight were considered positive for D. immitis microfilariae. The qPCR assay demonstrated good efficiency (E = 90 to 110%, R-2 > 0.94). Considering the performance and efficient use of bench time, this TaqMan qPCR assay is a suitable alternative to the modified Knott's test for quantitative enumeration of microfilariae (Cohen's kappa coefficient [kappa]: kappa = 1 using D. immitis qPCR marker, kappa = 0.93 using Wolbachia qPCR marker). The qPCR data demonstrated a comparable result to that of the quantitative modified Knott's test in a 2022 survey of D. immitis in Australian dogs (n = 23) before and after macrocyclic lactone (ML) administration. Improving the detection and diagnosis of canine heartworm infections will assist veterinarians in better managing and controlling disease outcomes and will be valuable for tracking the spread of ML resistance in Australia.