Introduction: Pseudomonas aeruginosa is a corneal pathogen and may cause corneal ulceration. The goal of this study was to determine the potential of PCR for detecting P. aeruginosa in corneal specimens from patients with keratitis.
Study Aims: 1) To establish a specific real-time PCR assay to detect P. aeruginosa. 2) To determine a secondary target for P. aeruginosa that may provide a universal target for other bacterial pathogens. 3) To validate both assays for diagnostic testing with true positive and true negative clinical samples.
Methods: 1) Analytical studies were conducted by testing P. aeruginosa and other bacteria isolated from patients with keratitis with a PCR assay designed to amplify the ecfX gene of P. aeruginosa. The outcome parameters were limit of detection, and amplification efficiency. 2) Similarly, P. aeruginosa isolates were tested for the 16S rRNA gene using the same parameters. 3) Validation of both assays was done by testing 20 cornea samples known to be positive for P. aeruginosa and 20 clinical samples known to be negative for P. aeruginosa DNA. Descriptive statistics were determined. PAGE analysis was performed to confirm the presence of amplified product.
Results: 1) Amplification efficiency of the ecfX assay was 96.6%, with a limit of detection of 33.6 copies of target DNA/µl. All 21 P. aeruginosa isolates were detected, with no detection of the 35 non-P. aeruginosa isolates. 2) Amplification efficiency of the 16S rRNA assay was 103.4%, with a limit of detection of 8.12 copies /µl. All 21 P. aeruginosa isolates were detected. 3) The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency for the ecfX and 16S rRNA assays were, [75%, 95%, 94%, 79%, and 85%], and [70%, 100%, 100%, 77%, and 85%], respectively. PAGE analysis supported specificity of the DNA amplified products.
Conclusions: Both real-time PCR assays used in this study detected P. aeruginosa DNA from keratitis patient samples. These results indicate that aside from culture, PCR may be a useful adjunct method in the diagnosis of keratitis patients.
Public Health Relevance: Real-time PCR can be used to detect P. aeruginosa from patients with keratitis to help preserve vision.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04122010-203551 |
| Date | 28 June 2010 |
| Creators | Hillenbrand, Maria Elizabeth |
| Contributors | Regis P. Kowalski, MS (M)ASCP, Robert M.Q. Shanks, PhD, Robert M. Wadowsky, ScD, Velpandi, Ayyavoo, PhD, Jeremy J. Martinson, DPhil |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-04122010-203551/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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