Corneal Pathogens Identified by Rapid Molecular Methods

These pathogens cause corneal ulcers and this methodology is much faster than standard methods.

The laboratory test, using real-time polymerase chain reactions (RT-PCRs) on corneal scrapings, was compared to the standard culture method. The main feature of the RT-PCR procedure is that the amplified DNA is detected as the reaction progresses in real time rather than at the end of the process, and it is completed within two hours. Culture and smear examination of the corneal scrapings are time consuming and dependent on expertise in microscopy.

Corneal scrapings were taken from 40 eyes of 40 patients suffering from bacterial keratitis, fungal keratitis, and Acanthamoeba keratitis. All these conditions can lead to corneal opacity, deteriorating visual acuity, and loss of sight. The samples were tested by the two methods and the results were evaluated and compared. The RT-PCR method adopted used the cycling probe Cycleave PCR produced by Takara Bio Inc. (Shiga, Japan).

The real-time PCR assay for the six commonest corneal ulcer pathogens delivered results within two hours, whereas the cultures could only be examined after 48 hours.

Of the 40 eyes, 20 had the same pathogens--Staphylococcus aureus, S. pneumoniae, Pseudomonas aeruginosa, methicillin-resistant S. aureus (MRSA), and Candida species were detected by both methods. Six showed negative results by both methods. Results of both methods disagreed in 14 eyes: 11 eyes had positive results for one of the six pathogens only on RT-PCR, two had positive results on culture only, and one eye had positive results for two different pathogens. The magnitude of the bacteria and fungi infections could be calculated by the RT-PCR method. The results were published in May 2010 in the Archives of Ophthalmology.

Motoki Itahashi, M.D., Ph.D., and colleagues of Kinki University School of Medicine, (Osaka-Sayama, Japan) acknowledged that RT-PCR has a high risk of false positivity and the number of patients were limited in this study. However, the scientists who developed this technology plan to use it as a basis for a diagnostic kit for pathogen-specific detection in the busy ophthalmic clinical practice.

Related Links:
Takara Bio Inc.
Kinki University School of Medicine