Shandong Provincial Key Laboratory of Ophthalmology, Shandong Provincial Excellent Innovation Team Programme, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China.
Clin Microbiol Infect. 2014 Oct;20(10):O776-82. doi: 10.1111/1469-0691.12571. Epub 2014 Mar 5.
This study was aimed at developing a direct PCR assay without template DNA extraction for the rapid and sensitive diagnosis of infectious keratitis. Eighty corneal scrapings from 67 consecutive patients with clinically suspected infectious keratitis were analysed prospectively. Direct PCR was performed with all scrapings, with specific primers for fungi, bacteria, herpes simplex virus-1 (HSV-1) and Acanthamoeba simultaneously. The results were compared with those obtained from culture, smear, and confocal microscopy. Discrepant results were resolved according to the therapeutic effects of the corresponding antimicrobial drugs. The lowest detection limit of direct PCR was ten copies of each pathogen. Sixty-six scrapings yielded positive results with direct PCR, giving a total positive detection rate of 82.5% (66/80). For 34 patients with high suspicion of fungal keratitis, the positive detection rate of direct PCR was 84.8% (39/46). This rate increased to 91.2% (31/34) when repeated scrapings were excluded, and was significantly higher than the rates obtained with culture (35.3%, 12/34) and smear (64.7%, 22/34) (p <0.001), and was also higher than the rate obtained with confocal microscopy (74.1%, 20/27). The sensitivities for the diagnosis of infectious keratitis with direct PCR and culture were 98.0% and 47.1% (p <0.001), whereas the specificities were 81.8% and 100%, respectively. The time required to complete the entire direct PCR procedure was only 3 h. The direct PCR assay is a rapid diagnostic technique with high sensitivity and specificity for infectious keratitis, and it is expected to have an impact on the diagnosis and treatment of infectious keratitis in the future.
本研究旨在开发一种无需模板 DNA 提取的直接 PCR 检测方法，用于快速、灵敏地诊断感染性角膜炎。对 67 例连续临床疑似感染性角膜炎患者的 80 个角膜刮片进行前瞻性分析。直接 PCR 采用真菌、细菌、单纯疱疹病毒 1(HSV-1)和棘阿米巴的特异性引物同时进行。将结果与培养、涂片和共聚焦显微镜检查结果进行比较。根据相应抗菌药物的治疗效果解决不一致的结果。直接 PCR 的最低检测限为每种病原体的 10 个拷贝。直接 PCR 检测 66 个刮片呈阳性，总阳性检出率为 82.5%（66/80）。对于 34 例高度怀疑真菌性角膜炎的患者，直接 PCR 的阳性检出率为 84.8%（39/46）。排除重复刮片后，该比率增加到 91.2%（31/34），明显高于培养（35.3%，12/34）和涂片（64.7%，22/34）（p<0.001），也高于共聚焦显微镜（74.1%，20/27）。直接 PCR 和培养诊断感染性角膜炎的敏感性分别为 98.0%和 47.1%（p<0.001），特异性分别为 81.8%和 100%。完成整个直接 PCR 程序仅需 3 小时。直接 PCR 检测法是一种快速、敏感、特异的诊断技术，有望对感染性角膜炎的诊断和治疗产生影响。
