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T. Chan, X. Sun, S. Chee, C. Chee, K. Song; PCR-Based Detection of Bacteria and Fungi Implicated in Severe Ocular Infections . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1843.
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Purpose: To evaluate polymerase chain reaction (PCR)-based assays to detect bacteria and fungi in ocular samples from patients with severe ocular infections. Methods: Nested-PCR and real-time PCR assays were developed to amplify portions of the bacterial 16S or fungal 18S ribosome gene. Forty ocular samples from patients with presumed infectious endophthalmitis, keratitis or scleritis, including 27 vitreous humor, 5 aqueous humor, 6 corneal, 1 scleral, and 1 conjunctival samples, were tested by nested-PCR assays, as well as by standard microbiological techniques. The results of the two diagnostic techniques were compared. Real-time PCR was also performed on 12 vitreous samples from infected eyes. Vitreous samples from 30 non-infected eyes were also examined, serving as negative-controls. Results: PCR and culture results matched (were both positive or both negative) in 14 (35%) of the 40 samples from infected eyes. Twenty-six (65%) samples were PCR-positive but culture-negative. No culture-positive samples were PCR-negative. Among the 36 PCR-positive samples using nested-PCR, 29 were positive for bacteria and 7 for fungi. Of the bacterial organisms, 18 were identified as Gram-positive bacteria, 11 as Staphylococci, 5 as Streptococci and 9 as Gram-negative bacteria. More specific identification to the species level was obtained in 15 samples, namely: S. pneumoniae (n=1), S. agalactiae (n=1), P. acnes (n=2), K. pneumoniae (n=4), P. aeruginosa (n=3), M. tuberculosis (n=2) and C. albicans (n=2). Of the 30 negative-control samples, none were positive for bacteria or fungi by either culture or PCR. Positive detection of bacteria was obtained in 9 of 12 infected vitreous samples using real-time PCR, with the quantitative pathogenic load being approximately 1000-30000 CFU per ml of vitreous. Conclusions: PCR-based assays offer overwhelming advantages over conventional culture techniques in the detection of bacteria and fungi implicated in severe ocular infections. Real-time PCR is also a potentially useful tool, in view of its quantitative and rapid diagnostic capability.
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