PURPOSE: To compare polymerase chain reaction (PCR) to microbial culture for the detection and identification of bacterial and fungal pathogens in microbial keratitis. DESIGN: Prospective cohort study. METHODS: A total of 108 consecutive corneal ulcers were cultured and analyzed by PCR using pan-bacterial and pan-fungal primers. PCR products were cloned, sequenced, and compared to culture results using standard bioinformatics tools. RESULTS: Of the 108 samples, 56 were culture-positive, 25 for bacteria and 31 for fungi; 52 were culture-negative. After eliminating false-positive PCR products, 94 of 108 were positive by PCR, 37 for bacteria and 57 for fungi. Nineteen of 25 bacterial culture-positive samples were positive by PCR, and 29 of 31 samples culture-positive for fungi were positive by PCR. The majority of sequenced PCR products matched the positive culture results. Of the 52 culture-negative samples, 46 (88%) yielded pathogen deoxyribonucleic acid (DNA) PCR products, 18 bacterial and 28 fungal. These represented a variety of species, including at least three novel previously uncultured microbes. CONCLUSIONS: PCR detects microbial DNA in the majority of bacterial and fungal corneal ulcers, and identifies potentially pathogenic organisms in a high proportion of culture-negative cases. Yield and concordance with culture are higher for fungal than bacterial ulcers. Practical use of the technique is limited by artefactual amplification of nonpathogenic organisms. PCR may be used as an adjunct to culture to identify potential pathogens in microbial keratitis.
PURPOSE: To compare polymerase chain reaction (PCR) to microbial culture for the detection and identification of bacterial and fungal pathogens in microbial keratitis. DESIGN: Prospective cohort study. METHODS: A total of 108 consecutive corneal ulcers were cultured and analyzed by PCR using pan-bacterial and pan-fungal primers. PCR products were cloned, sequenced, and compared to culture results using standard bioinformatics tools. RESULTS: Of the 108 samples, 56 were culture-positive, 25 for bacteria and 31 for fungi; 52 were culture-negative. After eliminating false-positive PCR products, 94 of 108 were positive by PCR, 37 for bacteria and 57 for fungi. Nineteen of 25 bacterial culture-positive samples were positive by PCR, and 29 of 31 samples culture-positive for fungi were positive by PCR. The majority of sequenced PCR products matched the positive culture results. Of the 52 culture-negative samples, 46 (88%) yielded pathogen deoxyribonucleic acid (DNA) PCR products, 18 bacterial and 28 fungal. These represented a variety of species, including at least three novel previously uncultured microbes. CONCLUSIONS: PCR detects microbial DNA in the majority of bacterial and fungal corneal ulcers, and identifies potentially pathogenic organisms in a high proportion of culture-negative cases. Yield and concordance with culture are higher for fungal than bacterial ulcers. Practical use of the technique is limited by artefactual amplification of nonpathogenic organisms. PCR may be used as an adjunct to culture to identify potential pathogens in microbial keratitis.
Authors: Lawson Ung; Paulo J M Bispo; Swapna S Shanbhag; Michael S Gilmore; James Chodosh Journal: Surv Ophthalmol Date: 2018-12-24 Impact factor: 6.048