Polymerase chain reaction based detection of fungi in infected corneas

Abstract

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.

Figure 1

Diagram of polymerase chain reaction amplification scheme.

Figure 2

Agarose gel demonstrating sensitivity of PCR assay. Serial dilutions of quantified Aspergillus fumigatus suspensions were assayed with 30 cycles of primary and 30 cycles of species directed PCR amplification. Far left lane shows 100 bp DNA ladder, (200 bp fragment obscured by loading dye). Lane 1: Aspergillus fumigatus 500 fungal elements. Target 214 bp product DNA band visible, as predicted for A fumigatus directed primers. Lane 2: 100 elements, target DNA band present. Lane 3: 25 elements, target DNA band present. Lane 4: 5 elements, target DNA band present. Lane 5: 0 elements, no target DNA. Lane 6: template A fumigatus DNA, positive control.