Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis

AIM: To design, optimize and validate a rapid, internally controlled real-time polymerase chain reaction (RT-PCR) test for herpes simplex virus (HSV) in the diagnosis of necrotizing herpes stromal keratitis. METHODS: Tears alone or together with corneal epithelium scrapings from 30 patients (30 eyes) suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores. RESULTS: The positive rate (46.4%) in the corneal epithelium group before the therapy was significantly higher than that (13.3%) in the tears group (P=0.006). There were 13 positive HSV patients before the therapy, the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group (paired t-test, P=0.0397). Multilevel mixed-effects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant (P=0.0049). The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment (r=0.844, P<0.0001). CONCLUSION: RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.     

聚合酶链反应检测角膜内皮炎房水中单纯疱疹病毒Ⅰ型DNA及带状疱疹病毒DNA

目的 评价聚合酶链反应技术（PCR）对角膜内皮炎的诊断价值，并以此解释病因，指导临床治疗。方法对临床诊断为角膜内皮炎的患者12例（12眼）及临床诊断为年龄相关性白内障的患者15例（15眼）分别用PCR方法进行房水中单纯疱疹病毒Ⅰ型（HSV-Ⅰ）DNA及带状疱疹病毒（VZV）DNA的检测，结果采用分类变量资料两样本率比较的四格表确切概率比较。结果共扩增角膜内皮炎患者12例（12眼），HSV-Ⅰ阳性者5例（5眼），阳性率为41．67％，共扩增对照组15例（15眼），HSV-Ⅰ阳性者0例，阳性率为0％，二者差异有显著性。结论PCR技术检测角膜内皮炎患者房水中的HSV-Ⅰ，可在分子水平上建立一种快速准确的诊断方法，为临床治疗提供可靠的依据。     

Unusual clinical presentations of new‐onset herpetic eye disease after ocular surgery

Purpose: To report five cases of new‐onset herpetic eye disease with unusual presentation after ocular surgery. Methods: Herpetic eye disease was suspected in five cases, three after cataract surgery and two after lamellar corneal transplantation surgery. Of these, four cases presented within 2–6 weeks of surgery. The clinical presentation was in the form of an epithelial defect, suspected epithelial down growth, graft oedema with unexplained anterior chamber inflammation and graft–host interface infection. A swab for viral detection with real‐time polymerase chain reaction was performed in all the described cases. Results: Herpes simplex disease was detected in all cases. All cases responded to the antiherpetic medications. Conclusions: Our study shows that new‐onset herpetic eye disease may occur after cataract surgery and lamellar corneal transplantation, and a high index of suspicion may be necessary for the diagnosis in such cases.     

多重聚合酶链反应快速诊断HSK和真菌性角膜炎的研究

目的:探讨多重聚合酶链反应(multiplexPCR,MPCR)技术快速诊断单纯疱疹病毒性角膜炎(HSK)和真菌性角膜炎的价值。方法:建立单纯疱疹病毒(HSV)和致病性真菌菌株的MPCR检测方法,检测49例患者54眼角膜刮片或泪液标本,对怀疑为真菌和混合感染的与涂片镜检和真菌培养比较。结果:MPCR5h可检测出标本中微量HSV和真菌,54份标本MPCR检测阳性43份(80%),其中怀疑有真菌感染的25份标本中17份真菌检测阳性(68%),阳性率明显高于真菌培养(χ2=11.57,P<0.005)和涂片镜检(χ2=13.33,P<0.005)。结论:MPCR速度快、敏感性和特异性高,有助于HSK和真菌性角膜炎的快速明确诊断。     

Polymerase chain reaction for diagnosis of herpetic intraocular inflammation

The authors present a polymerase chain reaction method for rapid and direct diagnosis of herpetic intraocular infections using small volume samples of intraocular fluid from 29 patients with various intraocular inflammatory diseases and 24 controls with senile cataract. Of six patients with early acute retinal necrosis from whom aqueous humor was tested, four were found to be positive for the presence of varicella-zoster (VZV) DNA while the other two were positive for the presence of herpes simplex virus (HSV) DNA. One of the patients with HSV DNA had been tested at an extremely early stage, at which time the aqueous humor viral antibody ratio did not predict a specific viral infection. Among four patients with acute retinal necrosis in relatively late stages following treatment with acyclovir from whom vitreous was obtained and tested, only one was found to have the presence of any viral DNA (VZV). On the other hand, the vitreous viral antibody ratio was found to be predictive of VZV infection in all four cases. VZV DNA was also detected in aqueous humor samples from four patients with suspected herpes zoster anterior uveitis, while HSV DNA was found in the aqueous humor of one patient with nonspecific keratouveitis. Neither human cytomegalovirus DNA nor human herpesvirus-6 DNA was detected in any sample included in this study. Finally, Epstein-Barr virus DNA was detected in the aqueous humor of the majority of patients studied and identified in cataract patients as well, suggesting either low specificity of the authors' assay for this virus or ubiquity of this virus in human eyes. In summary, the PCR method proved to be a very useful tool in establishing an etiological diagnosis in patients in the early stages of acute retinal necrosis, and in patients with anterior uveitis due to suspected HSV or VZV infection.     

聚合酶链反应在感染性葡萄膜炎诊断中的应用

聚合酶链反应（polymerase chain reaction,PCR）是一种有力的分子生物学工具,检测所需标本量少,耗时短,敏感性高,对于某些不典型的感染性葡萄膜炎,PCR能够在感染早期从极少量眼内液中检测出病原体的复制数量,提高诊断的效率。与传统的血清学抗体检测、病原微生物培养相比,PCR在辅助诊断方面表现出更大的优势。     

单纯疱疹病毒性角膜炎角膜组织病毒潜伏感染的PCR检测分析

 目的  探讨通过穿透性角膜移植获取的单纯疱疹病毒性角膜炎病变角膜组织中1型单纯疱疹病毒（HSV-1）DNA的表达情况及意义。设计 实验研究。研究对象  2010年5-12月北京同仁医院因病毒性角膜炎角膜白斑行穿透性角膜移植术后角膜标本20例,圆锥角膜、大泡性角膜病变和角膜营养不良等非感染性角膜病变的角膜标本20例。方法  对角膜组织标本中HSV-1 DNA进行聚合酶链反应（PCR）检测。 主要指标  HSV-1 DNA的阳性率。结果  单纯疱疹病毒性角膜炎静止期患者角膜组织中12/20例（60%）检出HSV-1 DNA,非感染性角膜组织中6/20例（30%）检出HSV-1 DNA（χ2=3.64,P=0.057）。结论  单纯疱疹病毒性角膜炎静止期角膜组织多数表达HSV-1 DNA,角膜内潜伏病毒是引起单纯疱疹病毒性角膜炎的可能原因,正常人角膜也可能有HSV-1的DNA存在。（眼科,2013,22：45-48）     

聚合酶链反应快速诊断真菌性角膜炎

目的　探讨聚合酶链反应 (PCR)技术快速诊断真菌性角膜炎的价值。方法　建立常见致病性真菌菌株的PCR检测方法 ,对兔茄病镰刀菌角膜炎模型研究并应用于临床检测 33例角膜刮片标本 ,结果与涂片镜检和真菌培养做比较。结果　PCR 5h可检测出标本中微量真菌 ,细菌、HSV、人和兔正常角膜组织均为阴性。动物模型和临床标本PCR敏感性分别为 84 .4 %和 81.8% ,均明显高于涂片镜检和真菌培养 (P <0 .0 5 )。结论　PCR速度快、敏感性和特异性高 ,有助于真菌性角膜炎的快速明确诊断。     

通用引物PCR方法快速检测兔眼真菌性角膜溃疡

目的 应用真菌通用引物聚合酶链反应PCR，对兔眼真菌性角膜溃疡进行快速检测，方法 建立兔眼烟曲霉菌、白色念珠菌和镰刀菌真菌性角膜溃疡的模型，根据国外文献报道的基因序列选出真菌通用引物，一对寡核苷酸引物B2F(5’-ACTTTCGTAG—GATAG-3’)和引物B4R(5’-TGATCGTCTTCGATAAAATA-3’)进行聚合酶链反应。结果 在687bp处出现DNA扩增带者为阳性。动物造型3d后，通过PCR技术、常规刮片和培养3种实验室手段诊断为真菌性角膜炎的敏感性分别为90.0％、33．3％和40.0％；5d后，PCR技术、常规刮片和培养的敏感性分别为96.6％、50．0％和60.0％．结论 通用引物PCR技术检测兔眼真菌性角膜溃疡快速、敏感、可靠。     

PCR检测在结核性葡萄膜炎诊断中的应用

结核性葡萄膜炎因难以获得病原学诊断依据,至今仍很难明确诊断。聚合酶链式反应（polymerase chain reaction,PCR）技术可扩增痕量级核酸的特点对少菌性感染的结核性葡萄膜炎而言是一大优势。在临床实际应用中,PCR技术的类别、靶基因序列的选用、眼内液样本类型及患者的选择等在一定程度上可能影响PCR检测的敏感度。对结核性葡萄膜炎致病机理及抗结核药物治疗反应等的深入剖析有助于临床医生从不同角度更好地分析PCR检测结果及治疗结局。（国际眼科纵览,2022, 46：66-70）     

Evaluation of the effects of acyclovir and/or human amniotic membrane on herpes virus culture and quantitative virus inactivity by real-time polymerase chain reaction

AIM: To investigate the permeability of amniotic membrane in herpes virus cell culture to acyclovir with real time polymerase chain reaction (RT-PCR).METHODS: Madin-Darby Bovine Kidney (MDBK) cell culture and Bovine Herpes Virus (BHV1) type 1 were used in the study. Cell cultures were grouped into two on the basis of herpes virus inoculation. Each group was sub-grouped into three. Amniotic membrane (V-HAM), acyclovir (V-A), and amniotic membrane and acyclovir (V-HAM-A) were applied to these subgroup cultures, respectively. After the application of the membrane and the drug, the cultures were evaluated at 24 and 48h for cytopathic effect positive (CPE+) with a tissue culture microscope. In the CPE (+) samples, the DNA was extracted for viral DNA analysis by RT-PCR.RESULTS: In control cultures without herpes virus CPE was not detected. Besides, amniotic membrane and acyclovir did not have cytotoxic effect on cell cultures. CPE were detected in Bovine Herpesvirus type-1 inoculated cell cultures after amniotic membrane and/or acyclovir application. DNA analysis with RT-PCR indicated that Cycle threshold (Ct) values were lower in the BHV1 and membrane applied group (amniotic membrane group< acyclovir group< membrane and acyclovir group). This showed that membrane did not have antiviral effect. The membrane and acyclovir cell culture groups with high Ct values indicated that membrane was permeable and had a low barrier effect to drug,CONCLUSION: In our in-vitro study, we found that amniotic membrane, which can be used in the treatment of corneal diseases, did not have antiviral effect. Besides, we detected that amniotic membrane was permeable to acyclovir in BHV-1 inoculated MDBK cell culture. However, more studies are necessary to investigate the quantitative effects of amniotic membrane and acyclovir.     

聚合酶链反应快速诊断棘阿米巴角膜炎的研究

目的:探讨聚合酶链反应(PCR)技术快速诊断棘阿米巴角膜炎的价值。方法:建立棘阿米巴标准虫株的PCR检测方法,并应用于临床检测24例角膜刮片标本,结果与原虫培养及100g/L氢氧化钾湿封片镜检做比较。结果:PCR5h可检测出标本中微量棘阿米巴原虫,对照细菌、真菌、I型单纯疱疹病毒、正常人角膜均为阴性。临床标本PCR敏感性为46%,明显高于原虫培养与100g/L氢氧化钾湿封片镜检(P<0.05)。结论:PCR速度快、敏感性和特异性高,有助于棘阿米巴角膜炎的快速明确诊断。     

细胞分离培养在实验室诊断眼部单纯疱疹病毒感染疾病的误诊和漏诊情况

目的:对比聚合酶链反应(PCR)和细胞分离培养在实验室诊断眼部单纯疱疹病毒(HSV)感染疾病的作用。方法:我们对连续患者的实验室结果(PCR实验,细胞分离培养及临床诊断)及病历进行回顾性研究在诊断眼部HSV感染疾病过程中,PCR检测结果、细胞分离培养及首诊结果进行了统计比较。结果:患者581例经过检查后,520例PCR检查阴性,细胞培养阴性(89.6%);0例PCR检查阴性,细胞培养阳性(0%);27例PCR检查阳性,细胞培养阴性(4.6%);34例PCR检查阳性,细胞培养阳性(5.8%)。PCR检测阳性率高于细胞分离培养(McNemars,P=0.0001)。47例PCR检查为HSV初诊感染阳性患者中,19例患者中有14例为细胞培养阴性结果(74%),5例HSV患者因缺PCR检测结果将被误诊;28例患者中有25例患者细胞培养阳性(占89%),3例HSV患者因缺PCR检测结果将被漏诊。结论:PCR诊断眼部HSV感染优于细胞分离培养。细胞分离培养对于眼部HSV感染的非典型表现不易诊断。     

聚合酶链反应快速诊断细菌性眼内炎的研究

目的　探讨聚合酶链反应 (PCR)技术快速诊断外源性细菌性眼内炎的价值。方法　建立常见致病性细菌的PCR检测方法 ,检测 3 6例眼内液标本 ,与细菌培养做比较。结果　PCR 5小时就可检测出标本中微量细菌 ,3 6例眼内液标本 3 1份阳性 (86 1% ) ,培养阳性 2 3份 (63 9% ) ,PCR阳性率明显高于培养 (χ2 =4 90 ,P <0 0 5 )。结论　PCR速度快、敏感性高 ,有助于外源性细菌性眼内炎的快速明确诊断。     

Cell culture isolation can miss the laboratory diagnosis of HSV ocular infection

AIM: We compared polymerase chain reaction (PCR) to cell culture isolation for the laboratory diagnosis of ocular herpes simplex virus (HSV) disease. METHODS: Laboratory and medical records of consecutive patients were reviewed for results of 1) HSV PCR testing, 2) HSV cell culture isolation, and 3) clinical diagnosis. PCR results were statistically compared to cell culture isolation and patients initially diagnosed for ocular HSV infection. RESULTS: Of 581 cases submitted for laboratory testing, 520 were PCR negative, cell culture negative (89.6%); 0 were PCR negative, cell culture positive (0%); 27 were PCR positive, cell culture negative (4.6%); and 34 were PCR positive, cell culture positive (5.8%). PCR tested more positive than cell culture isolation (McNemar's,P=0.0001). Of 47 HSV PCR positive cases with complete medical records, 19 were cell culture negative for HSV and 28 were cell culture positive for HSV. Fourteen of 19 cell culture negative cases (74%) (Without PCR, 5 cases of HSV would be missed) and 25 of the 28 cell culture positive cases (89%) (Laboratory testing was necessary for diagnosing 3 cases) were clinically diagnosed with HSV at the initial examination. CONCLUSION: PCR was a more definitive test for diagnosing HSV ocular infection than cell culture isolation. Cell culture isolation alone can miss an atypical presentation of HSV ocular infection.     

聚合酶链反应技术对棘阿米巴角膜炎诊断的临床应用

目的 探讨聚合酶链反应(PCR)技术临床应用于棘阿米巴角膜炎诊断的可行性及优越性。方法 以一段特异性通用引物并配合热启动聚合酶链反应技术来检测临床标本中的棘阿米巴原虫。结果 在门诊初诊为棘阿米巴角膜炎的13例(13只眼)中，通过PCR检测法有11只眼证实为棘阿米巴原虫感染，同时角膜刮片染色后镜检有5例发现了棘阿米巴包裹。结论 聚合酶链反应技术对棘阿米巴角膜炎的诊断有重要的临床应用价值。     

Applications of the polymerase chain reaction in clinical ophthalmology

Molecular biology has become a valuable component in many areas of medicine, including ophthalmology. Polymerase chain reaction (PCR) is the most widely used tool. It has proven to be a powerful technique in diagnosis and quantification of microorganisms and antibiotic resistance screening. For a growing number of ophthalmic conditions PCR testing can be conducted. It is therefore important that clinicians be knowledgeable about the indications, strengths, and limitations of the technique. The purpose of this review is to explore the current role of PCR in the diagnosis and management of eye disease.     

Correlation of quantitative polymerase chain reaction with clinical characteristics of patients with viral retinitis

Purpose:To evaluate the correlation of quantitative real-time polymerase chain reaction (qRT-PCR) to the clinical characteristics of patients with viral retinitis.Methods:Retrospective case series.Results:Aqueous or vitreous samples of 20 out of 35 eyes showed qRT-PCR positivity for virus etiology (57.14%). Cytomegalovirus (CMV) was most commonly identified in nine eyes (45%). The mean DNA copy number was 2,68,339.65 copies/mL (range: 90–3205397). DNA copy number significantly correlated with the extent of clinical involvement (P = 0.013); however, there was no correlation between DNA copy number and presenting visual acuity (P = 0.31), macular involvement (P = 0.675), optic nerve involvement (P = 0.14), and development of retinal detachment (P = 0.73). There was a significant correlation between the number of DNA copies and the timing of sampling (P = 0.0005). Samples taken earlier in the course of the disease had higher viral copies than later ones.Conclusion:qRT-PCR is useful in confirming a viral etiology in over 50% of cases of suspected viral retinitis. It correlates well with the extent of clinical involvement and timing of sampling.     

Polymerase chain reaction in the diagnosis of bacterial endophthalmitis

BACKGROUND—Microbiological investigations of vitreous fluid (VF) and aqueous humour (AH) specimens have often failed to detect the infecting agent in infectious endophthalmitis, resulting in a clinical dilemma regarding therapy. In this study, the polymerase chain reaction (PCR) was evaluated in the diagnosis of bacterial and Propionibacterium acnes endophthalmitis.
METHODS—58 intraocular specimens (30 VF and 28 AH) from 55 cases of endophthalmitis and 20 specimens (14 VF and 6 AH) as controls from non-infective disorders were processed for microbiological investigations. Nested PCR directed at the 16S rDNA using universal primers for eubacterial genome was done. PCR for P acnes was performed on specimens microbiologically negative by conventional techniques but eubacterial genome positive.
RESULTS—Of the 20 controls from non-infective cases, one (5%) was positive using eubacterial primers and none with P acnes primers. PCR for eubacterial genome showed 100% correlation with 20 (34.5%) bacteriologically positive specimens. Eubacterial genome, was detected in 17 (44.7%) of 38 bacteriologically negative specimens and nine (52.9%) out of the 17 were positive for P acnes genome. Among the 21 eubacterial PCR negative specimens, seven were fungus positive. By inclusion of PCR, microbiologically positive specimens increased from 46.5% to 75.8%. PCR on AH was as sensitive as that on VF for the detection of both eubacterial and the P acnes genome.
CONCLUSION—PCR performed on AH and VF is a reliable tool for the diagnosis of bacterial and P acnes endophthalmitis particularly in smear and culture negative specimens.

Keywords: polymerase chain reaction; bacterial endophthalmitis; infectious endophthalmitis