Novel diagnosis of fungal endophthalmitis by broad-range real-time PCR detection of fungal 28S ribosomal DNA

Abstract

To detect the fungal genome in the ocular fluids of patients with fungal endophthalmitis by using a novel broad-range polymerase chain reaction (PCR) system. After informed consent was obtained, ocular fluid samples (aqueous humor or vitreous fluids) were collected from 497 patients (76 patients with infectious endophthalmitis including clinically suspected bacterial and fungal endophthalmitis and 421 patients with infectious or non-infectious uveitis). Forty ocular samples from non-infectious patients without ocular inflammation were collected as controls. Fungal ribosomal DNA (28S rDNA) was measured by a quantitative real-time PCR assay. Fungal 28S rDNA of the major fungal species, such as Candida, Aspergillus, and Cryptococcus, were detected by novel broad-range real-time PCR examination (>10(1) copies/ml). Fungal 28S rDNA was detected in the ocular fluids of 11 patients with endophthalmitis or uveitis (11/497, 2.2%). All 11 positive samples were detected in the infectious endophthalmitis patients (11/76, 14.5%). These PCR-positive ocular fluids had high copy numbers of fungal 28S rDNA (range, 1.7 × 10(3) to 7.9 × 10(6) copies/ml), which indicated the presence of fungal infection. Of the 11 patients who were PCR positive, further examinations led to a diagnosis of fungal endophthalmitis in ten patients. The fungal 28S rDNA was detected in one non-infectious case (a false-positive case). In addition, there were two PCR false-negative cases that were clinically suspected of having fungal endophthalmitis. This novel quantitative broad-range PCR of fungal 28S rDNA is a useful tool for diagnosing endophthalmitis related to fungal infections.