Development, optimization, and validation of a multiplex real-time PCR assay on the BDMAX platform for routine diagnosis of Acanthamoeba keratitis.

Maubon, Danièle; Richarme, Claire; Post, Lucie; Robert, Marie Gladys; Bernheim, Diane; Garnaud, Cécile

The reported number of cases of Acanthamoeba keratitis (AK) is continually increasing. Molecular diagnosis has become the first choice of ophthalmologists for identifying and confirming this clinically problematic diagnosis. However, in-house molecular procedures are time-consuming and may not be compatible with the urgency of the situation. In this study, we adapted a previous in-house AK-PCR, on the BDMAX, a full-integrated automated platform for molecular biology (Becton Dickinson, Heidelberg, Germany), for the rapid routine diagnosis of Acanthamoeba keratitis. We compared different protocols to optimize DNA extraction from Acanthamoeba cysts. We evaluated the analytical parameters of the AK-BDMAX-PCR. Thirty-two samples were simultaneously tested with the AK-BDMAX PCR and the original AK-PCR from which it was developed. A thermal-shock pretreatment protocol was validated. The analytical parameters were similar to those obtained with the previous in-house AK-PCR method. We then assessed the performance of the AK-BDMAX PCR for routine testing on 40 clinical samples, mostly corneal scrapings. Frozen ready-to-use in-house PCR premixes were stable over eight months. Overall, 34 (85%) of the 40 clinical samples were considered to be true negatives, four (10%) were considered to correspond to probable AK and two (5%) were considered to correspond to possible AK.