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P.L. Goldschmidt, L. Batellier, S. Degorge, M. Sullam–Rea, D. Gleize, T. Bourcier, E. Zito, C. Alouche, L. Laroche, C. Chaumeil; Diagnosis of Keratitis Induced by 3 Herpesviruses and Acanthamoeba: Effects of Topical Anaesthetics and Fluorescein on the Real–Time Dna–Amplification Techniques . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3067.
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Patients with keratitis present an injected eye and moderate to severe pain associated or not with oedema, cellular infiltration, vascular invasion and scarring. The treatments of keratitis depend on the accurate diagnosis of the etiologic agent. Because samplings for microbiological diagnosis are performed after topical administration of anaesthetics and/or corneal staining with fluorescein, we studied the potential interference of oxybuprocain and fluorescein on the performances of the real–time PCR to detect 3 different Herpesviruses and Acanthamoeba.
DNA from viral or acanthamebal suspensions serially diluted in transport media with or without oxybuprocain or fluorescein were extracted by the Qiagen method. The primers and labelled probes –the same as those used for routine diagnosis– were validated with international standards.
+: No difference between the sample and the positive control without product; I: inhibition (difference with positive controls ≥1 log DNA copies/reaction); +/–: partial inhibition (difference with positive controls ≥0.5 but <1 log DNA copies/reaction)
Oxybuprocain and Fluorescein solutions used in clinical settings for diagnosis of keratitis inhibit the detection of 3 Herpesviruses and Acanthamoeba by real–time PCR. The molecular diagnosis of keratitis by techniques based on genomic amplification produce inaccurate results (false negative) or underestimates the microbial load if the corneal specimens contain fluorescein or oxybuprocain. Before sampling, ophthalmologists should be aware to rinse intensively the eye surface with appropriate solutions in order to avoid the simultaneous introduction of the residual fluorescein or the topical anaesthetics into the tubes containing the corneal specimens to be analysed by PCR.
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