Department of Ophthalmology, Faculty of Medicine, Cairo University, Egypt.
Curr Eye Res. 2012 Nov;37(11):1005-11. doi: 10.3109/02713683.2012.698357. Epub 2012 Jun 29.
To compare polymerase chain reaction (PCR) to microbial culture and smear for detection and identification of bacterial and fungal pathogens in suspected microbial keratitis.
Corneal scrapings from 88 patients with suspected infectious keratitis were subjected to routine bacterial culture and sensitivity, Gram's stain, fungal culture; potassium hydroxide (KOH) wet mount, and PCR. PCR was performed with primer pairs targeted to the 16S and 18S r RNA gene. The result of the PCR was compared with conventional culture and Gram staining method.
By broad-range PCR, 40 (45.45%) cases were positive for fungi (90.9% sensitivity), 26 (29.5%) were culture positive (59.09% sensitivity), 29 (33%) of all patients were positive for bacteria by broad-range PCR (87.9% sensitivity) and 19 (21.6%) were culture positive (57.58% sensitivity). The time taken for PCR assay was 4-8 h whereas positive fungal cultures took 2-10 days and bacterial culture from 2 to 4 days. Smears were positive for fungi in 29 eyes (33% of cases, 65.91% sensitivity) and for bacteria in 11 eyes (12.5% of cases, 33.33% sensitivity).
DNA amplification with universal primers is a promising diagnostic tool in cases of infectious keratitis where routine laboratory culture failed to identify the pathogen. PCR may be performed in cases where the results of corneal scraping stains are negative without waiting for the results of the culture.
比较聚合酶链反应（PCR）与微生物培养和涂片，以检测和鉴定疑似细菌性和真菌性角膜炎中的细菌和真菌病原体。
对 88 例疑似感染性角膜炎患者的角膜刮片进行常规细菌培养和药敏试验、革兰氏染色、真菌培养、氢氧化钾（KOH）湿片和 PCR。PCR 采用针对 16S 和 18S rRNA 基因的引物对进行。将 PCR 结果与传统培养和革兰氏染色方法进行比较。
通过广谱 PCR，40 例（45.45%）真菌阳性（90.9%敏感性），26 例（29.5%）培养阳性（59.09%敏感性），29 例（33%）所有患者广谱 PCR 阳性（87.9%敏感性），19 例（21.6%）培养阳性（57.58%敏感性）。PCR 检测时间为 4-8 小时，而阳性真菌培养需 2-10 天，细菌培养需 2-4 天。涂片在 29 只眼（33%的病例，65.91%的敏感性）中真菌阳性，在 11 只眼（12.5%的病例，33.33%的敏感性）中细菌阳性。
在常规实验室培养未能鉴定病原体的感染性角膜炎病例中，使用通用引物进行 DNA 扩增是一种很有前途的诊断工具。在角膜刮片染色结果为阴性的情况下，无需等待培养结果，即可进行 PCR 检测。
