Specific detection of fungal pathogens by 18S rRNA gene PCR in microbial keratitis

Abstract

Background: The sensitivity and specificity of 18S rRNA polymerase chain reaction (PCR) in the detection of fungal aetiology of microbial keratitis was determined in thirty patients with clinical diagnosis of microbial keratitis.

Methods: Corneal scrapings from patients were used for Gram stain, culture and PCR analysis. PCR was performed with primer pairs targeted to the 18S rRNA gene. The result of the PCR was compared with conventional culture and Gram staining method. The PCR positive samples were identified by DNA sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. Main outcome measures were sensitivity and specificity of PCR in the detection of fungus in corneal keratitis.

Results: Combination of microscopy and culture gave a positive result in 11 of 30 samples of microbial keratitis. PCR detected 10 of 11 samples that were positive by conventional method. One of the 19 samples that was negative by conventional method was positive by PCR. Statistical analysis revealed that the PCR to have a sensitivity of 90.9% and specificity of 94.7% in the detection of a fungal aetiology in microbial keratitis.

Conclusion: PCR is a rapid, sensitive and useful method to detect fungal aetiology in microbial keratitis.

Figure 1

Schematic representation of the fungal ribosomal 18S rRNA gene and ITS regions with primer binding locations. ITS- Internal transcribed spacer region.

Figure 2

Specificity of 18S rRNA PCR in yeasts, filamentous fungi and bacterial strains. Lane 1, 100 bp plus marker; lane 2,Candida albicans; lane 3, Penicillium sp.; lane 4, Fusarium sp.; lane 5, Alternaria sp.; lane 6, Aspergillus versicolor; lane 7, Aspergillus fumigatus; lane 8 Staphylococcus aureus; lane 9 Enterococcus sp.; lane 10 Pseudomonas aeruginosa; lane 11, Haemophilus influenzae; lane 12, Human DNA; lane 13, negative control and lane 14, 100 bp plus marker.

Figure 3

Sensitivity of PCR at the DNA level using 10-fold dilutions of Aspergillus versicolor DNA. Lane 1, 100 bp plus marker; lane 2, 10 ng; lane 3, 1 ng; lane 4, 100 pg; lane 5, 10 pg; lane 6,1 pg; lane 7, 100 fg; lane 8, 10 fg; lane 9, 1 fg; lane 10, negative control and lane 11, 100 bp plus marker.

Figure 4

Detection in clinical samples using 18S rRNA PCR. Lane M1, 100 bp plus marker; lane N, negative control; lane P, positive control; lane 1–14, clinical samples and lane M2, 1 kb marker.