CAJ | 학술논문

背景棘阿米巴角膜炎的致盲率极高,发病初期易误诊为真菌性角膜炎或病毒性角膜炎,常规的临床诊断方法费时较长,特异性差,极易错过治疗的“时间窗”.常规PCR法检测简单、特异、有效,但由于病变标本取材量少,不易提取组织DNA,限制了其临床应用. 目的探讨非提取组织DNA的直接PCR法扩增棘阿米巴虫株18S rRNA保守序列在棘阿米巴角膜炎快速诊断中的可行性. 方法从山东省眼科研究所暨青岛眼科医院诊治的10例棘阿米巴角膜炎患者的病变角膜中分离10株棘阿米巴虫株,经鉴定均为T4基因型虫株.用直接PCR法分别扩增棘阿米巴原虫、白念珠菌、绿脓杆菌、Ⅰ型单纯疱疹病毒以及正常人角膜上皮细胞以确定该方法的特异性.将棘阿米巴原虫稀释至不同的浓度以确定直接PCR法的敏感性.采用SPF级6周龄健康雌性BALB/c小鼠构建棘阿米巴角膜炎动物模型,于感染后第1、3、5、7、10、15天获取角膜组织,分别进行直接PCR、实时定量PCR(real-time PCR)、KOH封片镜检和原虫培养鉴定,比较各种方法的有效性和可行性.结果直接PCR法仅能扩增棘阿米巴DNA,其他病原体DNA均未扩增出；在每个获取的棘阿米巴角膜炎样本中最少可检测到10个棘阿米巴原虫.在棘阿米巴角膜炎小鼠模型中,直接PCR法在感染后第1、3、5、7、10、15天的阳性检出率分别为80.0％、90.0％、80.0％、70.0％、70.0％和50.0％,总阳性率为73.3％,高于原虫培养法的31.7％,差异有统计学意义(P=0.005);KOH封片镜检的总阳性率为56.7％,real-time PCR法检测的总阳性率为61.7％,均略低于直接PCR法,差异均无统计学意义(P=0.056、0.172).结论直接PCR法操作简单,在上述4种方法中对棘阿米巴原虫检测的特异性和敏感性最高,能够快速诊断棘阿米巴角膜炎,尤其对于待检标本少而无法提取DNA的患者可首选.
배경 극아미파각막염적치맹솔겁고,발병초기역오진위진균성각막염혹병독성각막염,상규적림상진단방법비시교장,특이성차,겁역착과치료적“시간창”.상규PCR법검측간단、특이、유효,단유우병변표본취재량소,불역제취조직DNA,한제료기림상응용. 목적 탐토비제취조직DNA적직접PCR법확증극아미파충주18S rRNA보수서렬재극아미파각막염쾌속진단중적가행성. 방법 종산동성안과연구소기청도안과의원진치적10례극아미파각막염환자적병변각막중분리10주극아미파충주,경감정균위T4기인형충주.용직접PCR법분별확증극아미파원충、백념주균、록농간균、Ⅰ형단순포진병독이급정상인각막상피세포이학정해방법적특이성.장극아미파원충희석지불동적농도이학정직접PCR법적민감성.채용SPF급6주령건강자성BALB/c소서구건극아미파각막염동물모형,우감염후제1、3、5、7、10、15천획취각막조직,분별진행직접PCR、실시정량PCR(real-time PCR)、KOH봉편경검화원충배양감정,비교각충방법적유효성화가행성.결과 직접PCR법부능확증극아미파DNA,기타병원체DNA균미확증출；재매개획취적극아미파각막염양본중최소가검측도10개극아미파원충.재극아미파각막염소서모형중,직접PCR법재감염후제1、3、5、7、10、15천적양성검출솔분별위80.0％、90.0％、80.0％、70.0％、70.0％화50.0％,총양성솔위73.3％,고우원충배양법적31.7％,차이유통계학의의(P=0.005);KOH봉편경검적총양성솔위56.7％,real-time PCR법검측적총양성솔위61.7％,균략저우직접PCR법,차이균무통계학의의(P=0.056、0.172).결론 직접PCR법조작간단,재상술4충방법중대극아미파원충검측적특이성화민감성최고,능구쾌속진단극아미파각막염,우기대우대검표본소이무법제취DNA적환자가수선.
Background Acanthamoeba keratitis is a sort of serious infectious eye disease with high causing-blindness rate.Acanthamoeba keratitis often is misdiagnosed as fungal keratitis or viral keratitis in the early stage.Because conventional clinical diagnosis methods show a low specificity and take a long time,timely treatment often is delayed.Conventional PCR does not apply well because the lesion sample is not enough to extract DNA.However,direct PCR can amplify 18S rRNA conserved sequence of acanthamoeba keratitis without the extraction of DNA.Objective This study was to discuss the feasibility for rapid diagnosis of acanthamoeba keratitis using direct PCR to amplify the gene 18S rRNA fragment.Methods Ten acanthamoeba strains were isolated from 10 eyes with acanthamoeba keratitis in Qingdao Eye Hospital.The sensitivity of the direct PCR assay was tested using different numbers of amoebas.The specificity of the assay was tested using DNA extracted from acanthamoeba,candida albicans,pseudomonas aeruginosa,herpes simplex virus-1 (HSV-1) and normal human corneal epithelial cell.Acanthamoeba keratitis models were established using infected method in clean 6-week-old female BALB/c mice.Corneal lesion samples were obtained 1 day,3,5,7,10,15 days after modeled.The effectivity and feasibility of the direct PCR assay for rapid diagnosis of acanthamoeba keratitis were evaluated and compared with culture method,corneal smear examination and real-time PCR.Results Direct PCR primers could only amplify DNA of acanthamoeba rather than other pathogens,and 10 stains of acanthamoeba were detected at least in each sample.During the development of acanthamoeba keratitis in the mice,the diagnosis positive rate of direct PCR was 80.0％,90.0％,80.0％,70.0％,70.0％ and 50.0％ in 1 day,3,5,7,10,15 days after modeled with the total positive rate 73.3％,which was higher than 31.7％ of culture method,56.7％ of corneal smear examination and 61.7％ of realtime PCR,with a significant difference between the direct PCR and culture method (P =0.005),but no significant difference was seen in the total positive rate between the direct PCR and real-time PC R (P =0.172) or corneal smear examination (P =0.056).Conclusions The direct PCR assay is a simple,rapid,highly specific and sensitive method for the rapid diagnosis of acanthamoeba keratitis,especially for the limited lesion sample.