The goal of this study was to determine whether sequence analysis of intern
al transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect funga
l pathogens in patients with ocular infections (endophthalmitis and keratit
is). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were ampli
fied by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fu
ngal species (yeasts and molds) were used to test the selected primers and
conditions of the PCR. PCR and seminested PCR of this region were carried o
ut to evaluate the sensitivity and specificity of the method. It proved pos
sible to amplify the ITS2/5.8S region of all the fungal strains by this PCR
method. All negative controls (human and bacterial DNA) were PCR negative.
The sensitivity of the seminested PCR amplification reaction by DNA diluti
ons was 1 organism per PCR, and the sensitivity by cell dilutions was fewer
than 10 organisms per PCR. Intraocular sampling or corneal scraping was un
dertaken for all patients with suspected infectious endophthalmitis or kera
titis (nonherpetic), respectively, between November 1999 and February 2001.
PCRs were subsequently performed with 11 ocular samples. The amplified DNA
was sequenced, and aligned against sequences in GenBank at the National In
stitutes of Health. The results were PCR positive for fungal primers for th
ree corneal scrapings, one aqueous sample, and one vitreous sample; one of
them was negative by culture. Molecular fungal identification was successfu
l in all cases. Bacterial detection by PCR was positive for three aqueous s
amples and one vitreous sample; one of these was negative by culture. Ampli
fication of ITS2/5.8S rDNA and molecular typing shows potential as a rapid
technique for identifying fungi in ocular samples.