Faculty of Medicine, Université de Paris, Paris, France
Hôpital Cochin, Laboratory of Parasitology-Mycology,Hôpitaux Universitaires Paris Centre, Paris, France.
Br J Ophthalmol. 2021 Sep;105(9):1302-1306. doi: 10.1136/bjophthalmol-2020-316730. Epub 2020 Oct 24.
BACKGROUND/AIMS: keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.
1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. PCR assays detected different regions of the nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1-JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of isolates were also characterised.
Estimated prevalence of AK was 1.32%. The sensitivity of diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant genotype (84.6%).
Culture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.
背景/目的：角膜炎（AK）是一种罕见但危及视力的感染。角膜刮片的分子诊断提高了 AK 的诊断率。迄今为止，不同的分子靶标和条件已用于诊断。在这项研究中，我们前瞻性地比较了五种聚合酶链反应（PCR）检测方法对疑似 AK 的角膜样本的诊断性能。
从疑似 AK 的患者中获取了 1217 例角膜刮片样本。样本处理包括分子诊断和培养。PCR 检测方法检测核小亚基 rRNA 基因的不同区域：使用 Nelson、ACARNA 和 JDP1-JDP2 引物对的三种终点 PCR 检测方法，以及使用 Acant 引物-探针的两种实时 PCR 检测方法。实时 PCR 检测方法同时扩增人 DNA 和内部对照，以确保刮片质量和无抑制剂。由于缺乏金标准，使用潜在类别分析评估每种检测方法的性能。还对分离株的基因型进行了特征描述。
AK 的估计患病率为 1.32%。诊断性 PCR 的敏感性（73.3%至 86.7%）与培养（66.7%）或目标序列或技术无显著差异。通过分别组合两种或三种检测方法，敏感性可提高至 93.8%或 100%。PCR 的特异性（99.3%至 100%）在不同检测方法之间存在差异。T4 是主要的基因型（84.6%）。
培养和单一 PCR 检测方法可能导致 AK 的误诊。不同 PCR 检测方法的组合和提高样本质量可以提高诊断的敏感性。
