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M. Rosenblatt, L. A. Oliveira, I. R. Schwab, R. T. Kashiwabushi, M. C. Z. Yu, R. Sampath, L. B. Blyn, M. J. Mannis, L. B. de Sousa, D. J. Ecker; High-Throughput Molecular Diagnostics for the Rapid Detection of Pathogens in Corneal Ulcers. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3645.
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To evaluate the ability of a novel molecular diagnostic technique for the identification of bacterial pathogens in patients with bacterial keratitis.
After informed consent, standard cultures and smears were obtained as well as an extra corneal scraping for molecular analysis. Test sample swabs were placed in liquid media which was then immediately frozen and stored for later analysis. For those patients with positive bacterial cultures, nucleic acids from thawed samples were extracted and subjected to PCR using 16 primer pairs designed against genetic regions broadly conserved across bacterial species. PCR amplicons were then rapidly analyzed for base composition, and quantity using electrospray ionization mass spectrometry (PCR/ESI-MS). Bioinformatics analysis of primer pair and base composition allowed for the unique identification and quantification of the bacterial species. Bacterial load greater than 104 was graded as a positive test.
Eleven eyes of eleven patients had positive bacterial cultures and specimens for these eyes were subjected to further molecular analysis. All patients with cultures positive for Moraxella spp. (2), Serratia marcescens (1), Haemophilus influenzae (1), and Streptococcus spp. (1) also had positive identification using PCR/ESI-MS. One patient with a positive culture for coagulase negative staphylococcus (CNS) had molecular results positive for Serratia. Three patients with CNS, two with Corynebacterium, and one with a mixture of these organisms by culture had negative molecular results.
A molecular diagnostic approach which utilizes serial PCR and mass spectrometry effectively identified the most virulent pathogens in a series of patients with culture positive bacterial keratitis. The molecular approach did not detect the pathogens in patients with less virulent (and perhaps non-pathogenic) bacteria detected by standard culture techniques. Molecular diagnostic approaches to bacterial keratitis may rapidly identify virulent pathogens in the cornea, and allow for the early implementation of tailored anti-microbial therapy.
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