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Keratitis is an inflammation of the cornea most commonly caused by an infection with bacteria, viruses, fungi or Acanthamoeba. Patients with keratitis present with an infected eye and have moderate to severe pain, which may or may not be associated with oedema, cellular infiltration, vascular invasion and scarring, and the treatments depend on the accurate diagnosis of the aetiologic agent. Early diagnosis and treatment are essential for improving the visual outcome of patients. 1
Most of the trials comparing the performances of diagnosis of keratitis were conducted using the qualitative polymerase chain reaction (PCR) technology, where the amplified products had to be electrophoresed through 2% agarose and the results assessed as positive or negative by ultraviolet transillumination. The assessment of the end points (visual exam of bands on a gel or immunoassays) does not permit the accurate determination of the partial inhibition provoked by specimens. The development of the real-time PCR technology made quantification of a targeted DNA possible under routine conditions because it created relationships based on the number of cycles elapsed before achieving detectable fluorescence (Ct), and because the threshold is dependent on the starting target-DNA copy number, the total or partial inhibition of a reaction can be automatically detected if the Ct is delayed.
Because samplings for microbiological diagnosis are carried out after corneal staining with fluorescein or topical administration of anaesthetics, we studied the potential interference of oxybuprocain and fluorescein on the performances of the PCR carried out routinely to detect three different herpesviruses and Acanthamoeba associated with eye diseases.
METHODS
Viral or acanthamoebal suspensions serially diluted in transport media with or without oxybuprocain or fluorescein were extracted manually by a column-based extraction kit (QIAamp, Qiagen, Courtaboeuf, France) or by the automatic Magnapure total nucleic acid isolation system (Roche, Meylan-Cedex, France).
The primers and labelled probes-the same as those used in the lab for routine diagnosis-were validated with international standards. 2 Calibrated samples containing titrated viruses purchased from the European Union Concerted Action on Quality Control of Nucleic Acid Amplification Program (EQCP-Glasgow, Glasgow, UK) were tested pure and diluted in distilled water before DNA extraction to assess the sensitivity and detection limits of each assay. Real-time PCRs for each agent were carried out in a final volume of 25 μl...