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S.L. Vanderveldt, S.H. Yoo, D. Miller, E.C. Alfonso; Real Time PCR Identification of S. aureus and P. aeruginosa in Bacterial Keratitis Specimens . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1919.
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Pseudomonas aeruginosa and Staphylococcus aureus are the two most common isolates recovered in bacterial keratitis samples. Rapid identification of the responsible pathogen may aid in the appropriate selection and tailoring of antibiotic therapy. This study will assess the feasibility of identification of Pseudomonas aeruginosa and Staphylococcus aureus by real time PCR analysis of corneal ulcer specimens, and determine the sensitivity and specificity of this technique for the identification of these specific bacterial pathogens in corneal tissue.
Patients with a clinical diagnosis of infectious keratitis were enrolled. A sterile blade was used to inoculate a culture swab which was stored at 0 °C. Additional culture plates were inoculated in the standard fashion and the isolates were identified by the Bascom Palmer Eye Institute Microbiology Laboratory in the usual manner. The collected and stored culture swab samples were re–suspended in lysis buffer and the bacterial DNA was isolated. This template DNA was used for SYBR–green real time PCR analysis with probes for the sa442 gene (specific for S. aureus) and the oprI gene (specific for P. aeruginosa).
Forty (40) infectious keratitis specimens were collected. The culture results of these specimens are as follows: 18 culture negative, 11 P. aeruginosa, 8 S. aureus, 2 S. pneumoniae, and 1 S. marcescens. Using the sa442 gene as the amplification product, all 8 S. aureus samples were identified by real time PCR. There were no false positives. The average cycle threshold was 22.3. Using the oprI gene as the amplification product, all 11 P. aeruginosa samples were identified with no false positives and an average cycle threshold of 22.9.
The identification of both S. aureus and P. aeruginosa by real time PCR is feasible in human cornea tissue by this protocol. The sensitivity and specificity of these protocols in this sample size are 100 percent. Real time PCR may allow more timely diagnosis of the responsible pathogen in bacterial keratitis for accurate tailoring of antibiotic therapy.
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