Development of Real Time Quantitative PCR in Diagnosis of Acanthamoeba Keratitis

Abstract

Abstract: : Purpose: The bad prognosis of Acanthamoeba keratitis justified a rapid and sensitive method of diagnosis. Clinical signs often lack of specificity particularly at the very beginning of the infection or when the patients had been treated with various antibiotics some of them with an amoebicidal activity. Classic PCR assay has been showed as a sensitive tool for routine diagnostic of Acanthamoeba keratitis. To improve sensitivity, specificity and rapidity of PCR, we evaluate a real time PCR method on 293 corneal scrapings. Methods: A real time PCR with TaqMan® probe was carried out inwith Smart Cycler (Instrumentation Laboratory) and ABI PRISM 7700 SDS (Applied Biosystems). PCR targets regions of the subunit 18S ribosomal RNA gene of Acanthamoeba. Corneal specimens from patients with clinical signs suggesting Acanthamoeba keratitis or with a keratitis and an history of recurrent infection without improvement though antibiotic or antiviral treatments were routinely investigated for Acanthamoeba species. So, 293 corneal scrapings were tested by direct real time PCR, direct examination and culture. The parasital load has been estimated by « absolute » quantification with serial dilutions of Acanthamoeba cysts. Results: We were able to detect and quantify Acanthamoeba in a few time with real time PCR. Twenty–four corneal scrapings were PCR positive. Only two samples were positive by direct examination. They were PCR positive too. No sample was positive by culture. PCR is higher sensitive than conventional methods for Acanthamoeba detection. Conclusions: The use of probes in real time PCR increases the sensitivity and specificity of the detection compared to classic PCR. Amplification and detection are performed in one step, so the time of the PCR is reduced to less than 4 hours. Our results confirmed the sensitivity of PCR for Acanthamoeba detection and the ability to detect and quantify few amount of Acanthamoeba in a few time. Sequence analysis of 18S rRNA gene identified 13 Acanthamoeba genotypes, with a majority of T4 genotype in Acanthamoeba keratitis. More experiments have been necessary to determine the usefulness of both Acanthamoeba quantification and genotyping for the prognosis and the choice of therapy in Acanthamoeba keratitis.

Keywords: Acanthamoeba • keratitis • clinical laboratory testing