Nested polymerase chain reaction in the diagnosis of congenital cytomegalovirus infection

Objective. Human cytomegalovirus infection is highly prevalent in Indian population. It is the commonest congenitally acquired infection causing various anomalies. The diagnosis of infection in neonates is difficult as IgM may not be detected in all cases. The polymerase chain reaction is reported as alternative and better option in these patients. However, there is lack of data to substantiate this preference in a resource poor country like India.Methods : Blood samples from 930 neonates/fetuses were first tested for specific anti-CMV IgM antibodies using μ-capture enzyme linked immunosorbent assay, Mac-ELISA. Nested PCR was first standardised on clinically and therapeutically confirmed cases of CMV disease. In the second phase blood samples randomly from 20 babies suspected of CMV infection were collected for serology and PCR and both tests were run independently. Twenty healthy controls were also included. IgM ELISA and PCR were performed on these samples and results of these 20 samples were compared to evaluate the sensitivity and specificity of each method.Results : Out of 930 serum samples of suspected congenital CMV infection 188 (20.2%) were found positive for CMV specific IgM antibodies. While comparing the results of 40 paired samples, PCR was found to be highly specific (100%) but less sensitive than Mac-ELISA (95%) with negative predictive value of 100% and positive predictive value of 95%. Thus in congenital CMV infection Mac-ELISA was less costly, less cumbersome and more userfriendly.Conclusion : The Mac-ELISA seem to have parallel sensitivity and specificity as PCR for diagnosing congenital CMV infection.     

Detection of adenovirus DNA in clinical samples by SYBR Green real-time polymerase chain reaction assay

BACKGROUND: Adenoviruses are associated with a variety of diseases including upper respiratory tract infections, acute conjunctivitis, cystitis and gastroenteritis. Adenoviruses can also cause fatal disseminated infections in patients undergoing stem cell transplantation. Measurement of adenovirus load in clinical samples from localized adenovirus infections or disseminated adenovirus infections may provide important information for analyzing the pathogenesis of various adenovirus infections. The purpose of the present study was to develop and optimize a highly sensitive real-time polymerase chain reaction (PCR) assay to detect a wide range of adenoviruses and to detect adenovirus DNA in clinical samples from immunocompetent children. METHODS: Clinical samples of throat swabs and blood were collected from 111 patients suspected of having adenovirus infection. The copy number of adenovirus DNA was measured by real-time PCR assay. RESULTS: SYBR Green real-time PCR assay is able to detect 10-10(6) copies of standard adenovirus DNA per run. Adenovirus DNA was detected in all culture-positive samples serotyped as 1, 2, 3, 4, 5, 6, 8 and 11. Viral loads on throat swabs from immunocompetent children with adenovirus infection ranged from 10(5) to 10(11) copies/mL. Adenovirus DNA was detected in 60% of blood samples and copy number ranged from 10(3) to 10(5) copies/mL. CONCLUSION: SYBR Green real-time PCR is a useful quantitative tool for analysis of adenovirus DNA. The present results for immunocompetent children with adenovirus infections provided basic data for comparison with data obtained from immunocompromised patients.     

Detection of congenital cytomegalovirus infection in Chinese newborn infants using polymerase chain reaction

Polymerase chain reaction (PCR) amplification was used to detect cytomegalovirus (CMV) in 1000 urine specimens from Chinese newborns for defining the incidence of congenital CMV infection in the Chinese population. The major immediate-early and the late antigen genes of CMV were amplified and detected by gel electrophoresis. There were 18 congenitally infected infants found when tests were performed with one or both primer pairs. Comparing with tissue culture, PCR of both primer sets provided a sensitivity of 94%, a specificity of 100% and a predictive value of positive result of 100%.     

PCR检测慢性胃炎患儿胃液中幽门螺杆菌DNA

目的检测慢性胃炎儿童胃液中幽门螺杆菌DNA，探讨其在临床诊断中的实用价值。方法应用PCR检测胃液中幽门螺旋菌DNA，应用Warthin-Starry银染色检测胃粘膜组织中的幽门螺旋菌。结果胃炎组与非胃炎组比较，敏感性为75.0％，特异性为96．2％，两组有显著性差异（P＜0．01）。PCR与Warthin－Starry银染色比较，PCR优于Warthin—Starry银染色法。结论PCR检测患儿胃液中的幽门螺旋菌DNA，具有敏感、快速、特异、高效等优点，是基因水平上检测幽门螺旋菌一项新技术，用于慢性胃炎的早期诊断，具有较好的实用价值，值得推广应用。     

Detection ofBorrelia burgdorferi by nested polymerase chain reaction in cerebrospinal fluid and urine of children with neuroborreliosis

Diagnosis of neuroborreliosis is often difficult since history and clinical presentation may be non-specific and serological tests may initially be negative. We therefore tested the polymerase chain reaction (PCR) for the detection of borrelial sequences in CSF and urine samples of consecutive children with neuroborreliosis seen in a single summer season. Four of eight children were negative in serum for antibodies toBorrelia burgdorferi. Two of eight children were PCR-positive in CSF and one other child was positive in urine. In two out of four children PCR was the only laboratory test confirming the clinical diagnosis. All children recovered after treatment with third generation cephalosporins. When seven of eight children were re-examined 6 months later all were healthy and antibodies toB. burgdorferi were detected in their serum. PCR may assist the paediatrician in establishing a diagnosis of neuroborreliosis; however, a negative result does not rule out neuroborreliosis. PCR is an adjunct, but no substitute for clinical judgement and serology.     

应用实时聚合酶链反应技术检测儿童呼吸道标本中的呼吸道合胞病毒

目的建立实时聚合酶链式反应（PCR）技术检测儿科呼吸道标本中呼吸道合胞病毒（RSV）的方法.方法（1）根据RSV N基因序列设计合成分别用于扩增RSV A亚型和B亚型的TaqMan探针和引物,建立实时PCR方法,进行灵敏度和特异性检测.（2）采用实时PCR检测61例RSV检测阳性的冻存标本和103例新收集的呼吸道标本,并与病毒分离、间接免疫荧光、巢式PCR结果相比较.结果（1）实时PCR对A亚型RSV检测的灵敏度为5.25 pfu,B亚型为3.75 pfu,与巢式PCR相同.（2）实时PCR检测其他呼吸道病毒阳性标本,结果均为阴性;A亚型和B亚型之间无交叉.（3）实时PCR检测其他方法检测过的RSV阳性的冻存标本61例,A亚型（30例）阳性为27例（90%）,B亚型（31）阳性为27例（87.1%）.（4）103例新鲜标本中,实时PCR检出RSV 35例（A亚型7,B亚型28）,阳性率34.0%,其中巢式PCR阳性31例,阳性率30.1%;间接免疫荧光阳性22例,阳性率21.4%;病毒分离阳性9例,阳性率8.7%.实时PCR阴性的68例,其他方法均为阴性.实时PCR与三种方法的一致性检验,一致率在74.76%～96.12%（P均＜0.01）;差异性检验结果说明,实时PCR阳性检出高于间接免疫荧光和病毒分离（P均＜0.01）,而与巢式PCR差异无统计学意义（P＞0.05）.结论实时PCR检测儿科鼻咽分泌物标本中RSV的方法,敏感性、特异性和可重复性好,可用于急性呼吸道感染患儿标本中的RSV检测.     

应用聚合酶链反应技术检测微量残留白血病

为早期诊断和检测白血病患儿体内微量残留白血病（MRD），应用聚合酶链反应（PCR）技术及生物素标记克隆特异性探针斑点杂交方法，检测45例初发或复发急性白血病患儿免疫球蛋白重链（IgH）基因重排产生的第3互补决定区（CDR-Ⅲ），并对6例完全缓解患儿进行随访。结果表明，28例B-系急性淋巴细胞白血病（ALL）中25例（89．3％），9例T-系ALL中2例（22．2％）及1例粒-淋双标记白血病检出克隆性CDR-Ⅲ重排片段，7例急性非淋巴细胞白血病（ANLL）未见有意义的扩增带。6例完全缓解中，1例PCR扩增产物电泳后检测为阳性，但斑点杂交6例均显示阳性结果，且彼此间无交叉反应，检测肿瘤细胞的敏感度达10－5。研究提示本方法对早期诊断和监测MRD有重要意义。     

Increased urinary excretion of tubular enzymes and proteins in children with epilepsy

Cross-sectional analysis of children undergoing treatment with anti-epileptic drugs has shown an increased urinary excretion of tubular enzymes and proteins. This has usually been interpreted as a consequence of subclinical renal-tubular damage or enzyme induction. We measured excretion of tubular enzymes and proteins in 29 children who suffered from epileptic seizures and in 27 control children. Investigations were undertaken at diagnosis before the start of treatment and 3-4 months later. At diagnosis we found a slightly, but statistically significant increased excretion of N-acetyl-beta-glucosaminidase and alpha1-microglobulin. There was no significant difference between patients with an idiopathic and symptomatic aetiology of seizures or between patients with different seizure types. At the second investigation, in children treated with carbamazepine or valproate, no further increase occurred. We conclude that the increased excretion of tubular enzymes and proteins in children with epilepsy is most probably not due to a side-effect of the anti-epileptic drugs, but to a physiological alteration associated with the epilepsy itself. While the cause is unknown, the influence of serotonin metabolism is discussed.     

原位聚合酶链反应检测在B细胞系非霍奇金病中的作用

目的 探讨用原位聚合酶镇反应(PCR)检测B细胞系非霍奇金病的意义。方法 用免疫球蛋白重链(IgH)第三互补决定区引物，生物素标记扩增产物直接原位PCR扩增15例非霍奇金病患儿外周血淋巴细胞。结果 15例非霍奇金病中，12例检测到特异性基因重排，4例呈现淋巴瘤性白血病，治疗后骨髓完全缓解，2例间期细胞原位PCR仍显示有IgH基因重排，分别于3个月及6个月复发。结论 原位聚合酶镇反应检测非霍奇金病不失为一种简单、快速、灵敏的方法，并能帮助预测复发。     

FQ-PCR检测法诊断先天性巨细胞病毒感染的卫生经济学评价

目的：探讨采用荧光定量聚合酶链式反应(FQ-PCR)检测法诊断新生儿先天性巨细胞病毒（CMV）感染的经济学成本。方法对610例日龄在14 d以内的新生儿采用酶联免疫吸附法检测血清CMV抗体,对于CMV-IgM或IgG阳性新生儿采用FQ-PCR检测其尿液CMV含量,并分析确诊1例CMV感染病例的平均费用。结果CMV-IgM阳性新生儿的FQ-PCR阳性率为42.9%(15/35);单纯IgG阳性者为2.9%(16/547)。CMV-IgM阳性组CMV DNA对数值的均值为5.79±1.24,明显高于单纯CMV-IgG阳性组（4.11±0.87）（P<0.01）。CMV-IgM 阳性病例确诊先天性CMV感染的平均费用为256元/例,单纯IgG阳性者为3 760元/例。结论(1)CMV-IgM阳性新生儿CMV DNA含量高于单纯CMV-IgG阳性者;(2)FQ-PCR确诊CMV-IgG阳性的新生儿为先天性CMV感染的成本-效益比远高于CMV-IgM阳性的患儿,对于CMV-IgG阳性新生儿,该法不适合于大规模流行病学诊断性研究。［中国当代儿科杂志,2010,12（10）：796-798］     

PCR技术在儿童幽门螺杆菌感染中的应用

目的 建立一套适合儿童幽门螺杆菌感染的PCR检查方法，了解儿童幽门螺杆菌感染的可能途径。方法 对37例4～14岁患儿采用聚合酶链反应检测胃粘膜、胃液和唾液中的幽门螺杆菌，并作胃粘膜快速尿素酶、病理W—S银染色及细菌培养。结果 37例患儿中尿毒酶、病理W—S银染色、细菌培养、胃粘膜PCR、胃液PCR和唾液PCR的检出率分别为35．14％、40．54％、35．14％、45．95％、35．14％和5．40％；敏感性为68．8％、81．2％、81.3％、93．8％、81.3％、12．5％；特异性为90．5％、90．5％、100％、90．5％、100％、100％；2项或2项以上检出率阳性判断为幽门螺杆菌感染，37例患儿中阳性16例。结论 胃粘膜、胃液PCR方法同其他幽门螺杆菌检测手段相比幽门螺杆菌阳性检出率相似，PCR方法具有操作简便，特异性、敏感性高的优点，是诊断儿童幽门螺杆菌感染、判断药物疗效的有效检查手段。唾液PCR阳性率低可能与口腔缺乏特定的幽门螺杆菌生长环境有关，但也提示口-口传播是幽门螺杆菌感染的可能途径。     

筑巢式聚合酶链反应动态检测儿童微量残留白血病的研究

为探讨Ｔ细胞受体Ｖδ２－Ｄδ３基因重排检测对儿童急性淋巴细胞白血病分型诊断及微量残留病动态监测的意义，采用筑巢式聚合酶链反应技术，对４０例ＡＬＬ患儿进行了ＴＣＲ Ｖδ２－Ｄδ３基因重排的检测，并对其中３０例在其完全缓解后进行了ＭＲＤ的动态监测。     

Hepatitis B virus DNA. Detection with polymerase chain reaction in liver tissue of children with chronic hepatitis B]

BACKGROUND: Detection of hepatitis B virus DNA is a reliable evidence of the presence of the viral agent and its replication. Conventional hybridization techniques are limited to detect about 30,000 virions. With the polymerase chain reaction it became possible to extend the sensitivity by amplification of viral sequences. In our study we intended to test whether viral sequences could be found in liver tissue specimens negative for hepatitis B virus DNA by conventional hybridization techniques. METHODS: Hepatitis B virus DNA was detected by PCR in liver tissue of 37 children with chronic hepatitis B, negative for hepatitis B virus DNA by Southern blot hybridization. PCR was performed in a thermal cycler using Taq-polymerase and oligonucleotide primers within the hepatitis B core region. Hepatitis B virus DNA was visualized by ethidium bromide staining and subsequent Southern blot hybridization. RESULTS: 20 patients were HBeAg- and 17 anti-HBe-seropositive. Viral sequences were present in each of the 20 HBeAg positive HBsAg carriers and in 10 patients with anti-HBe. No hepatitis B virus DNA could be found in 7 children, all of them positive for anti-HBe. CONCLUSIONS: Our results confirm polymerase chain reaction to be a more sensitive method to detect hepatitis B virus DNA in the liver compared with conventional hybridization techniques. Every HBeAg positive carrier as well as the majority of anti-HBe positive patients present viral DNA in their liver. Polymerase chain reaction will be suitable to monitor viral replication in spontaneous course and treated patients.     

套式聚合酶链反应加限制酶分析检测母婴巨细胞病毒感染

为评价套式聚合酶链反应（套式ＰＣＲ）加限制酶分析在孕妇巨细胞病毒感染及其母婴宫内传播检测中的应用，采用套式ＰＣＲ加限制酶分析，病毒分离、电镜观察和特异性抗体测定，对各孕期孕妇外周血，脐血及死胎组织进行人巨细胞病毒（ＨＣＭＶ）检测。结果：３６７名孕妇ＨＣＭＶ阳性检出率为５．５％，其中，套式ＰＣＲ检出率（４．９％）高于病毒分离（３．０％，Ｐ＜０．０５）。６份ＨＣＭＶＤＮＡ阳性母血中，３份配对脐血ＨＣＭＶＤＮＡ也阳性，母－脐传播率为３／６。３对被证实为母－婴宫内传播ＨＣＭＶ的标本中，２对套式ＰＣＲ，病毒分离及特异性ＩｇＭ、ＩｇＡ均阳性，１对套式ＰＣＲ、病毒分离、特异性ＩｇＡ阳性，ＩｇＭ阴性。提示：套式ＰＣＲ能提高诊断ＨＣＭＶ的特异性与敏感性，对孕妇及胎儿／新生儿ＨＣＭＶ感染的研究有重要意义。     

Detection of enteroviruses in the cerebrospinal fluid by polymerase chain reaction: prospective study of impact on the management of hospitalized children

A polymerase chain reaction kit (AMPLICOR EV) for the detection of enteroviruses (EV-PCR) in the cerebrospinal fluid (CSF) was evaluated in clinical conditions in a prospective blinded-intention study. Forty-three children (mean age 2.7 years) hospitalized for suspected meningitis or fever of unclear etiology were enrolled. EV-PCR was performed on a daily basis. Results were available in less than 2 days in 72% of cases. EV-PCR was positive in nine (21%) children, including three infants without CSF pleocytosis. Knowing their EV-PCR result would have allowed a saving of 18 hospital days and 12 days of antibiotic therapy. The EV-PCR in the CSF can thus be practically useful for children hospitalized for meningitis or fever if available on-site on a daily basis.     

Absence of human herpesvirus-6 genome by polymerase chain reaction in children with Hodgkin disease: a Children's Cancer Group Lymphoma Biology Study

BACKGROUND: Human herpesvirus-6 (HHV6) has been implicated in adult lymphomas but the role that HHV6 has in lymphomagenesis is unclear. Because primary infection occurs in children, a prospective study was undertaken to detect HHV6 in those with pediatric Hodgkin disease (HD). OBSERVATIONS: Tumor was obtained from children with HD entered on a Children's Cancer Group Biology Study. Polymerase chain reaction (PCR) was performed using HHV6 primers. All 47 specimens (40 nodular sclerosing; 3 lymphocyte predominant; 3 mixed cellularity; and 1 unclassified) were negative for HHV6. CONCLUSIONS: The results demonstrate no HHV6 sequence in childhood HD.     

培养与聚合酶链反应联合进行住院患儿肺炎细菌病原学检测

目的 探讨细菌培养与病原特异性DNA联合检测住院患儿肺炎细菌病原的应用价值.方法对187例肺炎患儿的深部呼吸道吸引物进行肺炎链球菌、流感嗜血杆菌选择性培养和普通培养,并且对同一标本采用靶序列富集多重PCR(Tem-PCB)扩增结合Luminex液态芯片检测平台进行定量测定,检测肺炎链球菌、流感嗜血杆菌、金黄色葡萄球菌、肺炎克雷伯杆菌、大肠杆菌、嗜肺军团菌、绿脓杆菌、鲍曼不动杆菌等14种病原菌的特异性DNA.结果细菌培养的总检出率为40.1%(75/187,含3例检出2种病原菌),病原菌依次为流感嗜血杆菌17.1%、大肠杆菌8.6%、肺炎克雷伯杆菌6.4%、金黄色葡萄球菌4.8%、肺炎链球菌3.7%、绿脓杆菌1.6%、鲍曼不动杆菌1.1%和阴沟肠杆菌1.1%.以细菌培养或Tem-PCR任一阳性为标准,联合检测的总检出率为78.6%(147/187),病原菌依次为流感嗜血杆菌28.9%、肺炎链球菌19.3%、大肠杆菌8.6%、肺炎克雷伯杆菌6.4%、金黄色葡萄球菌5.9%、鲍曼不动杆菌5.9%、绿脓杆菌2.7%和阴沟肠杆菌1.1%.结论 Tem-PCR能提高流感嗜血杆菌、肺炎链球菌、金黄色葡萄球菌、绿脓杆菌和鲍曼不动杆菌的检出例数.细菌培养与病原特异性DNA联合检测应用能显著提高肺炎病原的检出率,可能更真实地反映肺炎的细菌病原学情况.