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Polymerase chain reaction based detection of fungi in infected corneas
P A Gaudio1, U Gopinathan, V Sangwan
1Yale Eye Center, New Haven, CT 06520, USA.
Aims:
To evaluate a polymerase chain reaction (PCR) based assay to detect fungi in scrapings from infected corneas.
Methods:
A PCR assay was developed to amplify a portion of the fungal 18S ribosome gene. Corneal scrapings from 30 patients with presumed infectious keratitis were evaluated using this assay, as well as by standard microbiological techniques, and the results were compared. Conjunctival swabs from each patient's healthy, fellow eye were also evaluated by PCR.
Results:
PCR and fungal culture results matched (were both positive or both negative for fungi) in 22 (74%) of 30 scrapings from infected corneas. Three (10%) of 30 samples were PCR positive but fungal culture negative; two of these appeared clinically to represent fungal infections, and the third was clinically indeterminate. Four (13%) scrapings were positive by PCR but also by bacterial and not fungal culture. One specimen (3%) was PCR negative but fungal culture positive. Of the conjunctival swabs from each patient's healthy fellow eye, five (17%) of 30 were positive by PCR, and the opposite, infected eye of all five of these harboured a fungal infection.
Conclusions:
PCR is promising as a means to diagnose fungal keratitis and offers some advantages over culture methods, including rapid analysis and the ability to analyse specimens far from where they are collected.
Insights
Polymerase chain reaction (PCR) effectively detects fungi in corneal scrapings, showing promise for diagnosing fungal keratitis. This molecular method offers faster results and greater flexibility than traditional fungal cultures.
Area of Science:
- Ophthalmology
- Mycology
- Molecular Diagnostics
Background:
- Infectious keratitis is a significant cause of visual impairment globally.
- Fungal keratitis diagnosis often relies on labor-intensive and time-consuming culture methods.
- Rapid and accurate detection of fungal pathogens is crucial for timely treatment and improved patient outcomes.
Purpose of the Study:
- To assess the efficacy of a polymerase chain reaction (PCR) assay for identifying fungal DNA in corneal scraping samples.
- To compare the diagnostic performance of the PCR assay against standard microbiological culture techniques.
- To evaluate the utility of PCR in detecting fungal infections in patients with presumed infectious keratitis.
Main Methods:
- Development of a PCR assay targeting the fungal 18S ribosomal gene.
- Analysis of corneal scrapings from 30 patients with suspected infectious keratitis using both PCR and fungal culture.
- Comparison of PCR results with culture outcomes and clinical diagnoses.
- Evaluation of PCR on conjunctival swabs from healthy fellow eyes.
Main Results:
- The PCR assay demonstrated a 74% concordance with fungal culture for infected corneal scrapings.
- PCR identified additional fungal infections missed by culture (10% of cases).
- PCR detected fungal DNA in 17% of healthy fellow eyes, correlating with fungal infection in the contralateral eye.
Conclusions:
- Polymerase chain reaction (PCR) shows significant potential for diagnosing fungal keratitis.
- PCR offers advantages over culture, including speed and the ability to process samples remotely.
- The assay's sensitivity warrants further investigation for clinical application in ophthalmology.