Financial support for this project came from Servei Veterinari de Bacteriologia i Micologia from the Universitat Autònoma de Barcelona.

Malassezia pachydermatis is part of the normal microbiota of canine skin and external ear canal, and is also associated with otitis externa in dogs. Laboratory detection of Malassezia otitis relies on the presence of elevated numbers of the yeast on cytologic examination of otic exudate. Although cytology has high specificity, it has low sensitivity, resulting in false-negatives and posing a challenge for clinicians to accurately diagnose Malassezia otitis. We developed a quantitative PCR (qPCR) to detect and quantify M. pachydermatis yeasts and validate the method with swabs from external ear canals of dogs. Our qPCR uses the β-tubulin gene, a single-copy gene, as a target. The limit of quantification was established as 0.18 ng/reaction, equivalent to 2.0 × 10 genome equivalents (gEq). Swabs from healthy dogs yielded quantification values of ≤2.7 × 10 gEq in the qPCR, whereas swabs from dogs with otitis yielded quantification values of ≥2.5 × 10 gEq. Our qPCR assay provides accurate quantification of M. pachydermatis yeasts from swab samples from dogs, is more sensitive than cytology, and could be used to monitor response to treatment. Our assay could also be valuable in a research setting to better understand the pathogenesis of M. pachydermatis.