Parasitology-Mycology Laboratory, Infectious Agents Department, Grenoble Alpes University Hospital, Grenoble, France; TIMC-IMAG, Centre National de la Recherche Scientifique, Université Grenoble Alpes, Grenoble, France.
Parasitology-Mycology Laboratory, Infectious Agents Department, Grenoble Alpes University Hospital, Grenoble, France.
J Mol Diagn. 2020 Dec;22(12):1400-1407. doi: 10.1016/j.jmoldx.2020.09.001. Epub 2020 Sep 22.
The reported number of cases of Acanthamoeba amebic 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 diagnostic procedures are time-consuming and may not be compatible with the urgency of the situation. In this study, a previous in-house AK-PCR technique was adapted for use on BD MAX (Becton Dickinson, Heidelberg, Germany), a fully integrated, automated platform for molecular biology, for the rapid routine diagnosis of AK. Different protocols were compared to optimize DNA extraction from Acanthamoeba cysts. The analytical parameters of the AK-BD MAX PCR were evaluated. Thirty-two samples were simultaneously tested with AK-BD MAX PCR and the original AK-PCR from which it was developed. A thermal-shock pretreatment protocol was validated. The analytical parameters obtained with BD MAX were similar to those obtained with the previous in-house AK-PCR method. The performance of AK-BD MAX PCR was then assessed for routine testing on 40 clinical samples, mostly corneal scrapings. Frozen, ready-to-use, in-house PCR premixes were stable over 8 months. Overall, 34 of the 40 clinical samples (85%) were considered to be true negatives; 4 (10%), probable AK; and 2 (5%), possible AK. This newly developed AK-BD MAX PCR is reliable, rapid, and efficient, and should facilitate Acanthamoeba keratitis diagnosis.
棘阿米巴角膜炎（AK）的报道病例数量不断增加。分子诊断已成为眼科医生识别和确认这一具有临床问题的诊断的首选方法。然而，内部的分子诊断程序耗时，可能不符合情况的紧迫性。在这项研究中，对之前的 AK-PCR 技术进行了改进，以适应 BD MAX（德国海德堡贝克顿·迪金森公司）的使用，BD MAX 是一个用于分子生物学的完全集成、自动化平台，用于快速常规诊断 AK。比较了不同的方案以优化从棘阿米巴包囊中提取 DNA。评估了 AK-BD MAX PCR 的分析参数。同时用 AK-BD MAX PCR 和其开发的原始 AK-PCR 对 32 个样本进行了测试。验证了热冲击预处理方案。BD MAX 获得的分析参数与之前内部 AK-PCR 方法获得的参数相似。然后在 40 个临床样本（主要是角膜刮片）上评估 AK-BD MAX PCR 的常规检测性能。冷冻即用型内部 PCR 预混液在 8 个月内保持稳定。总体而言，40 个临床样本中的 34 个（85%）被认为是真正的阴性；4 个（10%）可能是 AK；2 个（5%）可能是 AK。这种新开发的 AK-BD MAX PCR 可靠、快速且高效，应有助于棘阿米巴角膜炎的诊断。
