Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR

A protocol for the rapid detection of fungal DNA in ocular samples, derived from three species, Candida albicans, Aspergillus fumigatus, and Fusarium solani, has been developed. Two novel panfungal primers complementary to 18 S rRNA sequences present in all three species were designed, Panfungal PCR was followed by three nested PCRs utilizing species-specific primers. PCR s ensitivity ranged from 50 to 100 fg of free DNA and between one and two C. albicans organisms. In addition, we also developed a rapid and reliable DNA extraction protocol. This protocol minimized DNA loss during extraction, w hilst removing compounds from vitreous and aqueous fluids that have previou sly been shown to have inhibitory effects on PCR. Preliminary results obtai ned after testing the protocol on three patient samples support culture res ults and medical history. However, one patient was PCR positive but culture negative, suggesting that the sensitivity of this protocol may exceed that of traditional culture techniques. This system, therefore, constitutes an additional protocol that may significantly aid patient management in cases where fungal endophthalmitis is suspected.