Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections

Citation
C. Ferrer et al., Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections, J CLIN MICR, 39(8), 2001, pp. 2873-2879
Citations number
46
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Clinical Immunolgy & Infectious Disease",Microbiology
Journal title
JOURNAL OF CLINICAL MICROBIOLOGY
ISSN journal
0095-1137 → ACNP
Volume
39
Issue
8
Year of publication
2001
Pages
2873 - 2879
Database
ISI
SICI code
0095-1137(200108)39:8<2873:DAIOFP>2.0.ZU;2-6
Abstract
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.