Department of Ophthalmology, Cantonal Hospital Lucerne, Lucerne, Switzerland.
Invest Ophthalmol Vis Sci. 2010 Apr;51(4):1905-10. doi: 10.1167/iovs.09-3737. Epub 2009 Oct 22.
Purpose. Polymerase chain reaction (PCR) is increasingly important for the rapid detection of fungal keratitis. However, techniques of specimen collection and DNA extraction before PCR may interfere with test sensitivity. The purpose of this study was to investigate the use of DNA-stabilizing FTA filter paper (Indicating FTA filter paper; Whatman International, Ltd., Maidstone, UK) for specimen collection without DNA extraction in a single-step, nonnested PCR for fungal keratitis. Methods. Specimens were collected from ocular surfaces with FTA filter discs, which automatically lyse collected cells and stabilize nucleic acids. Filter discs were directly used in single-step PCR reactions to detect fungal DNA. Test sensitivity was evaluated with serial dilutions of Candida albicans, Fusarium oxysporum, and Aspergillus fumigatus cultures. Test specificity was analyzed by comparing 196 and 155 healthy individuals from Switzerland and Egypt, respectively, with 15 patients with a diagnosis of microbial keratitis. Results. PCR with filter discs detected 3 C. albicans, 25 F. oxysporum, and 125 A. fumigatus organisms. In healthy volunteers, fungal PCR was positive in 1.0% and 8.4% of eyes from Switzerland and Egypt, respectively. Fungal PCR remained negative in 10 cases of culture-proven bacterial keratitis, became positive in 4 cases of fungal keratitis, but missed 1 case of culture-proven A. fumigatus keratitis. Conclusions. FTA filter paper for specimen collection together with direct PCR is a promising method of detecting fungal keratitis. The analytical sensitivity is high without the need for a semi-nested or nested second PCR, the clinical specificity is 91.7% to 99.0%, and the method is rapid and inexpensive.
目的。聚合酶链反应（PCR）在真菌性角膜炎的快速检测中越来越重要。然而，PCR 之前的标本采集和 DNA 提取技术可能会干扰测试的灵敏度。本研究的目的是探讨在真菌性角膜炎的一步法非嵌套 PCR 中，不进行 DNA 提取，使用 DNA 稳定的 FTA 滤纸片（Indicating FTA filter paper；Whatman International，Ltd.，Maidstone，UK）采集标本的效果。
方法。使用 FTA 滤纸片收集眼表面标本，滤纸片可自动裂解采集的细胞并稳定核酸。滤纸片直接用于一步法 PCR 反应以检测真菌 DNA。通过对白色念珠菌、尖孢镰刀菌和烟曲霉培养物的连续稀释来评估检测的灵敏度。通过比较来自瑞士和埃及的 196 名和 155 名健康个体与 15 名微生物性角膜炎患者，分析检测的特异性。
结果。用滤纸片进行 PCR 检测到 3 株白色念珠菌、25 株尖孢镰刀菌和 125 株烟曲霉。在健康志愿者中，真菌 PCR 在来自瑞士和埃及的眼睛中分别有 1.0%和 8.4%为阳性。在 10 例经培养证实的细菌性角膜炎中，真菌 PCR 均为阴性，在 4 例真菌性角膜炎中转为阳性，但漏诊了 1 例经培养证实的烟曲霉角膜炎。
结论。FTA 滤纸片采集标本联合直接 PCR 是一种有前途的检测真菌性角膜炎的方法。该方法无需进行半巢式或嵌套式二次 PCR，分析灵敏度高，临床特异性为 91.7%~99.0%，且方法快速、廉价。
