Fungal polymerase chain reaction testing in equine ulcerative keratitis

Abstract

To assess the diagnostic utility of fungal polymerase chain reaction (PCR) in forty-three horses with naturally acquired corneal ulcers presenting to a private practice. Routine evaluation of cytologic, histologic, and microbiologic samples was performed. Two PCR approaches were compared - generic and specific fungal nested PCR followed by sequencing and quantitative PCR (qPCR). PCRs were applied to pure control fungal cultures, corneal tissue from ulcerated eyes and in a subset of 9 horses, to swabs from contralateral normal eyes. The expected fungus was identified by nested PCR and qPCR in all control fungal cultures. In all fungal culture-positive affected eyes (10/43), one or more fungi were identified by nested PCR and 4/10 were positive by qPCR. In 6/10 animals, the same fungus was identified by nested PCR and culture. Of these 6, only three were positive by qPCR. Fungal agents were identified by morphology in 8/10 horses. Diagnosis of fungal keratitis was reserved for only those cases in which the same fungus could be identified by PCR, culture, and morphology (5 horses). In 33/43 culture-negative affected eyes and in 6/9 unaffected eyes, one or more fungi were identified by nested PCR in 26 samples and by qPCR in 2 samples. Apart from Aspergillus spp, similar fungi were identified in affected and control eyes. Most eyes harbored mixed bacterial and fungal agents. Nested PCR results confirmed all cytologically positive cases of fungal keratitis. Nested PCR identified a greater spectrum of agents than either culture or qPCR.