Background Herpes stromal keratitis (HSK) is a common infectious ocular surface disease,with a higher recurrent rate,especially in necrotizing HSK.The diagnosis of HSK primarily depends on signs and symptoms,and specific laboratory diagnostic is lack.Objective This study was to clarify the expression of herpes simplex virus (HSV) in the corneal epithelium scrapings and tears of necrotizing HSK patients.Methods Thirty eyes of 30 patients with necrotizing HSK were enrolled in Nanjing Hospital of Nanjing Medical Hospital from September 2012 to September 2013 under the patient's informed consent.The eyes were examined by slit lamp microscope and scored.HSK patients received local and systemic therapy for 8 weeks and then an oral maintenance dose for 6 months.Corneal epithelial scrapings and tears samples were collected for HSV DNA detection by real-time PCR before and the 1 st,2nd,4th,6th and 8th week after therapy respectively.The difference of HSV positive rate was compared between corneal epithelium scrapings and tears samples using Chi-square test.Multilevel mixed effective model was employed to evaluate HSV concentration change in the samples at various time points in the HSV-positive patients of initial visit.The correlation between HSV concentration and clinical score was analyzed by Spearman rank correlation.Results HSV-positive rate was 46.4％ (13/30) in the corneal epithelial scrapings and 13.3％(4/30) in the tear samples,showing a significant difference between them (P =0.006).HSV-positive rate was significantly lower in the corneal epithelial scrapings 1 week,2 weeks and 4 weeks after treatment than before (P =0.001,0.003,0.004),and no HSV was detected 6 weeks and 8 weeks after treatment.No significant change in HSV-positive rate in tear samples in 1 week and 2 weeks after treatment in comparison with before treatment (P =1.000,0.583),and no HSV was detected after 4 weeks following treatment.The HSV concentration was 2460 (2 165-636500)/ml in initial 13 HSV-positive eyes of corneal epithelial scrapings and 0 (0-1150)/ml in initial 13 HSV-positive eyes of tear samples.Multilevel mixed effective model determined that HSV concentration was significantly lower in corneal epithelial scraping than that in tear (P =0.005),and HSV concentration was reduced with the lapse of time (P =0.001),with the faster rate of decline in the corneal epithelial scrapings (P =0.049).A positive correlation was found between initial HSV concentration and clinical scores (rs =0.844,P =0.000).Conclusiors Real-time PCR appears to be a powerful molecular tool for the detection of HSV in the HSK,especially in corneal epithelial scrapings of lesion.The initial positive outcome of viral DNA in corneal epithelial scrapings predicts a severe clinical procedure.%背景 单纯疱疹病毒性角膜炎(HSK)是一种常见的感染性眼表疾病,复发率高,重者可致盲,尤其是坏死性基质型HSK.以往对坏死性基质型HSK的诊断主要依靠临床症状和体征,缺乏特异性诊断方法. 目的 检测坏死性基质型HSK角膜上皮组织和泪液中单纯疱疹病毒(HSV) DNA的表达,为坏死性基质型HSK的实验室诊断提供依据. 方法 纳入2012年9月至2013年9月于南京医科大学附属南京医院诊断为坏死性基质型HSK患者30例30眼,裂隙灯显微镜下对临床症状进行炎症评分,予以局部及全身药物治疗8周后,全身用药改为维持量治疗6个月,收集其治疗前和治疗后1、2、4、6和8周的病变区角膜上皮组织和泪液标本,应用实时荧光探针定量PCR(real-time PCR)分别检测角膜上皮组织和泪液标本中HSV的浓度和阳性率,2种组织中HSV阳性率的差异比较采用x2检验,采用多水平混合效应模型比较初诊时角膜上皮组织和泪液中HSK检测结果均为阳性的患眼在各随访时间点HSV的浓度,采用Spearman秩相关法分析患跟治疗前角膜上皮组织中病毒浓度与临床症状体征评分的相关性. 结果 本组30眼治疗前角膜上皮组织和泪液标本中real-time PCR诊断HSV阳性者分别为13例和4眼,阳性率分别为46.4％和13.3％,差异有统计学意义(P=0.006).治疗后1、2和4周,角膜上皮组织中HSV阳性率均明显低于治疗前,差异均有统计学意义(P=0.001、0.003、0.004),治疗后6周和8周,均未诊出HSV;而治疗后1周和2周泪液中HSV阳性率与术前比较差异均无统计学意义(P=1.000、0.583),治疗后4周均未检测出HSV.治疗前30例患者中13例角膜上皮组织和泪液标本HSV阳性者病毒浓度分别为2460(2165 ～ 636500)/ml和0(0～1150)/ml.多水平混合效应模型显示,泪液标本中病毒浓度显著低于角膜上皮组织(P=0.005),随着治疗的进行,病毒浓度显著下降(P=0.001),且角膜上皮组织与泪液中下降速率差异有统计学意义(P=0.049).秩相关分析显示,治疗前角膜上皮组织中病毒浓度与临床症状体征评分值之间呈正相关(rs=0.844,P=0.000). 结论 Real-timePCR可检测HSK患眼角膜上皮组织中和泪液中HSV的表达,但病变区角膜上皮组织的检测有较高的检出率.治疗前病变区角膜上皮组织较高的病毒浓度者病变更为严重.
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