聚合酶链反应结合地高辛标记探针诊断儿童单纯疱疹病毒感染

目的　评价聚合酶链反应 (PCR )结合地高辛分子杂交技术在单纯疱疹病毒 (HSV) DNA检测中的临床意义。方法　应用该方法检测 3 6例HSV感染患儿血白细胞和 3例重症病毒性脑炎患儿脑脊液中HSV DNA。结果　HSV DNA检测阳性率 61.54% (2 4/ 3 9) ,其中HSV Ⅰ型阳性率占 75% (18/ 2 4) ,HSV Ⅱ型占16.66% (4/ 2 4) ,Ⅰ Ⅱ型混合型占 8.3 3 % (2 / 2 4)。结论　HSV病原体的PCR检测结合地高辛分子杂交技术对早期诊断儿童HSV感染及提高HSV感染患儿的疗效具有重要意义     

单纯性疱疹脑炎病人脑脊液中单纯疱疹病毒DNA的快速检测

单纯性疱疹病毒(HSV)可引起进行性脑坏死和脑水肿,如不进行治疗,死亡率高达70%。未经治疗的单纯性疱疹脑炎幸存者中约2/3有神经缺损,有时甚至很严重。有效的抗病毒药,如阿糖腺苷和无环鸟苷可供使用,而早期使用效果最佳。然而,由于EB病毒脑炎、囊膜病毒脑炎(togavirus encephalitis)和结核性脑膜炎可能有相似的症状和体征,故诊断不能仅仅依据临床标准。因此,需要一种早期诊断单纯性疱疹脑炎的精确诊断试验,但目前脑活检标本检查仍是早期诊断本病没有的确定方法,而脑活检证实的本病病人脑脊液(CSF)病毒培养的阳性率仅约4%。尽管CSF中的HSV抗原和抗休试验已有了进展,但在感染早期阳性的罕见。聚合酶链反应(PCR)是一种可将极小的DNA进行酶扩增以便用常规手段如DNA杂交进行检查的方法(一种成功地用于检测数种传染因子包括乳头瘤病毒和免疫缺陷病     

儿童EB病毒脑炎27例

目的　探讨儿童EB病毒脑炎 (EBE)临床特点及诊断依据。方法　对 2 7例经荧光定量PCR(FQ PCR)及巢氏PCR方法检测脑脊液病毒阳性而确诊的EBE与 2 6例单纯疱疹病毒脑炎 (HSE)患儿临床及脑脊液资料进行回顾性分析。结果　 13例患儿FQ PCR检测EB DNA结果为 (2 .82± 2 .0 3)× 10 3 拷贝 ;EBE患儿脑脊液白细胞异常较HSE少 (P <0 .0 5 )。结论　在儿童病毒性脑炎中EBE并不少见 ,约占 10 % ,且多单独出现而非传染性单核细胞增多症 (IM)的一部分。EBE临床表现无特异性 ,脑脊液EB DNA检测是诊断EBE敏感而可靠的方法。早期治疗预后较好     

小儿单纯疱疹病毒性脑炎19例临床观察

单纯疱疹病毒性脑炎(HSE)是病毒性脑炎中常见的一种。国内有关小儿HSE的报道仍不多见。1993年8月～1995年8月我们应用聚合酶链反应(PCR)DNA扩增技术对病房中疑似为脑炎的124例患儿脑脊液进行单纯疱疹病毒(HSV)DNA检测,发现HSE19例(15.3％),现报告如下。     

逆转录多重套式聚合酶链反应在儿童病毒性脑炎病原学研究中的应用

目的探讨早期同步检测病毒性脑炎(VE)主要病原新方法。方法应用逆转录多重套式聚合酶链反应(RT-M-nPCR)对100例VE患儿及73例对照组脑脊液(CSF)标本进行单纯疱疹病毒(HSV)DNA与肠道病毒(EV)RNA同步检测。结果整个过程约需5 h。该法与ELISA及经典nPCR方法比较,符合率分别为96%及100%。100例VE患儿CSF标本中,HSV阳性18例,EV阳性17例,在100例VE中,有26例重症,其中HSV感染14例(53.85%),EV感染7例(26.92%),不明病原5例(19.23%)。不同病原检出率与年龄的关系:年龄~3岁、~7岁、~14岁HSV分别检出7.4%、11.9%、35.5%,EV分别检出25.9%、19.05%、6.5%。结论RT-M-nPCR是一种能在早期同步检测HSV及EV的具有巨大潜在应用价值的病原检测技术;在VE重症中HSV感染最常见;HSV感染在年长儿发病率高,而EV感染在学龄前及婴幼儿中多见。     

小儿病毒性脑炎的病原研究

目的　 探讨小儿病毒性脑炎的病原。 方法 　应用酶联免疫吸附方法 (ELISA)测定 5 1例病毒性脑炎患儿的脑脊液 (CSF)和血清中多种病毒特异性IgM抗体。 结果 　 5 1例病毒性脑炎患儿CSF中病毒IgM抗体阳性 2 1例 ( 4 1 2 %) ,血清中病毒IgM抗体阳性 18例 ( 35 3%)。 结论 　本地区病毒性脑炎多由肠道病毒和疱疹病毒引起 ,其中以CoxB和HSV为主要病原。     

儿童病毒性脑炎多中心诊断治疗研究

目的　探讨儿童病毒性脑炎的诊断与治疗问题。方法　以多中心、前瞻开放、随机对比方法观察儿童病毒性脑炎的临床症状、体征及脑脊液常规生化 ;以聚合酶链反应 (PCR)技术检测儿童病毒性脑炎脑脊液病原 ;在综合治疗的前提下 ,以单一抗病毒药物治疗儿童病毒性脑炎。结果　本组 14 3例病毒性脑炎中发热 134例(93 7% ) ,头痛 86例 (6 4 7% ) ,呕吐 87例 (6 0 8% ) ,呕吐伴头痛 5 9例 ,惊厥发作 4 7例 (32 9% ) ,以全身阵挛发作为主 ;有精神症状 87例 (6 0 8% ) ,意识障碍 2 5例 (17 5 % ) ,颈抵抗 4 6例 (32 2 % ) ,运动障碍 2 0例 ,锥体束征 9例 ;脑脊液常规生化检查异常 90例 ,其中 86例白细胞增多 ,16例蛋白定量增高。 14 3例均做了脑脊液病原学检测 ,病毒阳性 84例 (5 8 7% ) ,其中肠道病毒阳性 4 0例 ,DNA病毒阳性 4 4例 ,另外还检测出了 6例支原体。在症状、体征和脑脊液常规生化检查阳性的 4 6例中 ,脑脊液病毒学阳性 2 6例 (5 6 5 % ) ,在症状和脑脊液常规生化检查阳性者 4 4例中 ,脑脊液病毒学阳性 2 3例 (5 2 3% )。在脑脊液病原学不明的情况下 ,在综合治疗的基础上 ,三种抗病毒药物单药抗病毒治疗临床疗效无差别。结论　除常规诊断条件外 ,病毒病原学检查十分重要 ,抗病毒治疗以覆盖更广     

荧光定量PCR法检测呼吸系统感染儿童肺炎支原体DNA的分析

为探讨儿童肺炎支原体 (MP)感染的早期诊断 ,用荧光定量PCR(FQ -PCR)技术检测1010例疑似MP感染患儿血、痰中的DNA ,结果发现1010例患儿中MP -DNA阳性401例 ,占39.70 % ,其中血液标本阳性率23.60 % ,DNA平均拷贝数7.88×102;痰、咽拭子标本阳性率41.71 % ,DNA平均拷贝数2.71×103。血液与呼吸道标本之间MP -DNA量差异有显著性(P<0.005)。提示FQ -PCR可快速、敏感、准确地定量检测标本中MP -DNA ,了解MP在患儿体内感染和复制情况 ,有助于临床明确诊断与治疗方案的选择     

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目的探讨儿童化脓性脑膜炎(化脑)新的快速诊断方法。方法2003-08—2005-12采用16SrRNA荧光定量法对浙江大学儿童医院49例临床疑似化脑患儿脑脊液(CSF)的细菌DNA进行测定;监测化脑患儿脑脊液细菌DNA拷贝数,同期进行CSF细菌培养的对照。结果(1)荧光定量PCR(FQ-PCR)检测49份脑脊液标本发现17份阳性,阳性率为34.7%(17/49),明显高于脑脊液培养的阳性率10.2%(5/49),差异具有显著性(P<0.01)。(2)对17份FQ-PCR阳性标本进一步测定细菌DNA的拷贝数,发现患儿病情与其DNA拷贝数呈正相关,与其Ct值(指每个反应管内的荧光信号到达设定的阈值时所经历的循环数)呈负相关,Ct值越低,脑脊液细菌DNA拷贝数越高,患儿的预后越差。(3)FQ-PCR、CSF细菌培养同时阳性的仅为5例。(4)对2例脑脊液FQ-PCR的产物测序,Ct值17.9的测序提示为大肠埃希菌,符合CSF细菌培养结果;Ct值31.8的,测序未果。结论荧光定量PCR特异性强、敏感性高,需标本量少,是早期快速诊断儿童化脑的可靠方法,具有较大的应用价值。     

2002-2005年北京儿童医院住院患儿病毒性脑炎流行病学分析

目的 了解北京地区儿童病毒性脑炎的流行病学特点.方法 对2002-01-2005-12在北京儿童医院住院治疗、出院诊断为病毒性脑炎的1 016例进行回顾性总结分析,入选病例急性期血清和(或)脑脊液检测病毒IgM抗体,标本选择检测的病毒抗体种类按照临床医生的申请进行.结果 1 016例病毒性脑炎患儿占住院总人数的1.0%,男女比例为2.21,年龄(6.4±4.0)岁.血清和(或)脑脊液病毒抗体阳性的病例共380例(38.5%),其中肠道病毒抗体阳性病例最多(44.7%),其次是流行性腮腺炎病毒(35.3%),单纯疱疹病毒(15.5%),风疹病毒(4.5%),乙型脑炎病毒(2.9%).1 016例中,有3例死亡.结论 肠道病毒、流行性腮腺炎病毒和单纯疱疹病毒是北京地区儿童病毒性脑炎最常见的病原.该研究中62.6%的病例仍然缺乏病原学证据,因此需要建立针对更多病毒的快速敏感的检测方法从而为病毒性脑炎的诊断和治疗提供病原学证据.     

Rapid diagnosis of herpetic encephalitis in children by PCR-microarray technology for simultaneous detection of seven human herpes viruses

The aim of the study was to evaluate retrospectively the usefulness of polymerase chain reaction (PCR)-microarray technology, which can simultaneously detect seven human herpes viruses for rapid and accurate diagnosis of herpetic encephalitis in children. We simultaneously amplified herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2); varicella-zoster virus; Epstein–Barr virus (EBV); cytomegalovirus (CMV); and human herpes virus 6 (HHV-6A and HHV-6B) by multiplex PCR, and genotyped by DNA microarray technology. The multiplex primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. Two hundred ninety cerebrospinal fluid (CSF) specimens from children with clinical suspicion of viral encephalitis were screened by PCR-microarray technology. The results were compared with those of TaqMan PCR kits of common herpes virus. The PCR-microarray technology could detect as few as 10 copies of viral loads. There was no nonspecific hybridizing signal between probes and no cross-reaction to DNA extracted from the pathogens we used. Of 290 cases, 11 were tested positive by PCR-microarray technology. Among them, three were positive for HSV-1, two were positive for HSV-2, one was positive for EBV, two were positive for CMV, two were positive for HHV-6A, one was positive for HHV-6B, and one showed mixed infection of HSV-2 and CMV, and the positive rate was 3.8%. Compared with the results of TaqMan PCR, the sensitivity of PCR-microarray technology was 91.7%, the specificity was 100%, and the index of accurate diagnosis was 0.917. None of the 30 control CSF specimens was tested positive in both methods. Our study suggests that the simultaneous detection of seven human herpes viruses by PCR-microarray technology is the method of choice for rapid, accurate, and specific etiological diagnosis of herpetic encephalitis in children.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

目的 建立对人疱疹病毒6型(HHV-6)能同时进行定量和分型的荧光定量PCR检测新方法,运用该方法对临床疑似病毒性脑炎患儿进行检测.方法 以HHV-6聚合酶基因区(U38)为靶序列,设计通用引物和特异性分型探针,建立能同时检测HHV-6型A/B亚型的荧光定量PCR方法,进行敏感性和特异性实验.对临床445例疑似脑炎患儿的脑脊液标本进行HHV-6荧光定量分型检测,阳性结果测序验证.结果 HHV-6A和HHV-6B病毒株荧光定鼍分型检测结果均为阳性,两亚型之间无交叉.单纯疱疹病毒1型和2型、水痘-带状疱疹病毒、巨细胞病毒、爱泼斯坦.马尔病毒、乙肝病毒、金黄色葡萄球菌、肺炎支原体、人类基因组DNA及空白对照均为阴性.HHV-6荧光定量分型最低能检测到10拷贝/μl HHV-6A/B.在临床445例疑似脑炎患儿脑脊液标本中检出HHV-6阳性21例(4.72%),其中HHV-6A阳性4例,HHV-6B阳性16例,HHV-6A和HHV-6B混合感染1例.整个PCR操作过程2-3 h.结论 HHV-6荧光定量分型方法能对HHV-6同时进行定量和分型,具有特异、敏感、简便、快速的特点,可为临床HHV-6感染性脑炎提供早期、敏感的诊断依据.     

Pathology of Chronic Herpes Infection Associated with Seizure Disorder: A Report of Two Cases with Tissue Detection of Herpes Simplex Virus 1 by the Polymerase Chain Reaction

Although uncommon, the association of chronic encephalitis with epilepsy is well recognized. While a viral etiology has been suspected based on the morphology, to date no virus has been successfully cultured from the brain in patients with Rasmussen's encephalitis. We describe the pathologic findings and report the detection of herpes simplex virus 1 (HSV1) in the brain in two patients who presented primarily with intractable seizures. In the first patient, an intrauterine infection was suspected as the underlying basis for the seizure disorder and the extensive cerebral calcification and gliosis. The second patient (with presumed HSV1 encephalitis at age 7 months) underwent a temporal lobectomy for medically refractory seizures at the age of 3 years and pathologic examination revealed a chronic encephalitis. While immunohistochemical, ultrastructural, and culture studies were negative for viral pathogens, molecular analysis by the polymerase chain reaction (PCR) revealed HSV1 DNA sequences in both cases. Thus our cases represent two examples of chronic encephalitis associated with a seizure disorder, where a definitive viral etiology was documented by PCR.     

病毒性脑炎、脑膜炎多病原的探讨

为探讨北主地区病毒性脑炎及脑膜炎的病原学构成,采用微量细胞培养法,应用人胚肺二倍体细胞（ＨＥＦ）、人表此样癌细胞（Ｈｅｐ－２）、猴肾传代细胞（Ｖｅｒｏ）及狗肾传代细胞（ＭＤＣＫ）等４种细胞系对８６例脑炎、脑膜为患儿的脑脊液、粪便及咽试标本进行了病毒分离。结果３６例分离到了病毒,阳性率为４１．８６＾（３６／８６例）,其中肠道病毒３型（ＡｄＶ３）８例,单纯疱疹病毒１例（ＨＳＶ１）７例,阳性率分别为２０     

抗N-甲基-D-天门冬氨酸受体脑炎与病毒性脑炎的关系

抗N-甲基-D-天门冬氨酸受体(anti-N-methyl-d-aspartate receptor,NMDAR)脑炎是一种常见的自身免疫性脑炎,近年来逐渐被神经医学领域所认识.该病具有相对一致的临床表现,脑脊液具有特异性的抗NMDAR抗体,免疫治疗有较好的临床效果.抗NMDAR脑炎与病毒性脑炎关系密切,在认识该病之前,其常常被误诊为病毒性脑炎.近来大量研究发现病毒感染是抗NMDAR脑炎的重要诱发因素,而部分病毒性脑炎尤其是单纯疱疹病毒脑炎的复发,是由病毒感染继发中枢神经系统免疫性炎性反应所致,需要进行免疫治疗.因此认识抗NMDAR脑炎及其与病毒性脑炎的关系,对患者的诊治与预后有重要影响.     

Epidemiological Profile of Acute Viral Encephalitis

Objective

To study the etiology and clinico-epidemiological profile of acute viral encephalitis in children with acute encephalitis syndrome (AES).

Methods

An observational study including 100 patients fulfilling the criteria for AES was conducted in children of age group 1 mo – 16 y. Viral isolation was done on RD cells, HEp-2 cells and Vero cells from the cerebrospinal fluid samples of suspected viral encephalitis (VE) cases. An enzyme immunoassay for IgM antibodies was performed for measles, mumps, Varicella zoster virus (VZV), Herpes simplex virus 1 (HSV1) and Japanese encephalitis virus (JEV). Multiplex polymerase chain reaction (PCR) was done for Cytomegalovirus, Epstein Barr virus (EBV), HSV1 & 2, VZV, Enterovirus, Parecho virus, Human Herpes virus (HHV 6, 7) and Parvovirus B19. A micro neutralization test was performed for Enterovirus 71.

Results

Out of enrolled 100 patients, 73 were of probable viral encephalitis. HSV1 (31.50%) was the commonest virus followed by Adenovirus (10.95%), Parvovirus (2.73%), JE virus (1.36%), Enterovirus (1.36%), EBV (1.36%), and mixed infection with HSV & EBV (1.36%). HSV 1 caused significant morbidity in children. The common computed tomography (CT) findings were hypodensities in the fronto- parietal lobe followed by cerebral edema.

Conclusions

The landscape of AES in India has changed in the previous decade, and both outbreak investigations and surveillance studies have increasingly reported non-JEV etiologies; because of these findings there is a need to explore additional strategies to prevent AES beyond vector control and JEV vaccination.

     

Suboptimal management of central nervous system infections in children: a multi-centre retrospective study

ABSTRACT: OBJECTIVE: We aimed to audit the regional management of central nervous system (CNS) infection in children. METHODS: The study was undertaken in five district general hospitals and one tertiary paediatric hospital in the Mersey region of the UK. Children admitted to hospital with a suspected CNS infection over a three month period were identified. Children were aged between 4 weeks and 16 years old. Details were recorded from the case notes and electronic records. We measured the appropriateness of management pathways as outlined by national and local guidelines. RESULTS: Sixty-five children were identified with a median age of 6 months (range 1 month to 15 years). Ten had a CNS infection: 4 aseptic meningitis, 3 purulent meningitis, 3 encephalitis [2 with herpes simplex virus (HSV) type 1]. A lumbar puncture (LP) was attempted in 50 (77%) cases but only 43 had cerebrospinal fluid (CSF) available for analysis. Of these 24 (57%) had a complete standard set of tests performed. Fifty eight (89%) received a third generation cephalosporin. Seventeen (26%) also received aciclovir with no obvious indication in 9 (53%). Only 11 (65%) of those receiving aciclovir had CSF herpes virus PCR. Seventeen had cranial imaging and it was the first management step in 14. Treatment lengths of both antibiotics and aciclovir were highly variable: one child with HSV encephalitis was only treated with aciclovir for 7 days. CONCLUSIONS: The clinical management of children with suspected CNS infections across the Mersey region is heterogeneous and often sub-optimal, particularly for the investigation and treatment of viral encephalitis. National guidelines for the management of viral encephalitis are needed.     

Production of new T cells by thymus in children: effect of HIV infection and antiretroviral therapy

The decrease in the number of CD4(+) T cells during HIV infection is the result of both peripheral destruction of cells by the virus and inadequate replacement of these cells. Aging and HIV infection lead to lower production of new T cells by the thymus, and, therefore, a complete restoration of the immune system is not generally achieved in infected adults after antiretroviral therapy. Because children have a completely functional thymus, we addressed the effects of HIV-1 infection on the production of new T cells in vertically infected children and whether the decrease of viral load after therapy results in a restoration of thymic function. To analyze the thymic function, T-cell receptor rearrangement excision circles were measured by quantitative PCR. Our results indicate that HIV-infected children have lower T-cell receptor rearrangement excision circle levels than age-matched uninfected children, likely due to an inhibitory effect of HIV on thymic function. Additionally, in some patients, the decrease in viral load after retroviral therapy allows the generation of new T cells by the thymus, thus recovering the normal number of CD4 cells.