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目的 探讨新生儿化脓性脑膜炎(以下简称化脑)新的快速诊断方法.方法 2003年8至2006年2月浙江大学医学院附属儿童医院采用脑脊液细菌16S rRNA基因芯片技术对24例临床上疑似化脑患儿脑脊液(CSF)的细菌DNA进行测定,同期进行与CSF细菌培养的对照.结果 (1)49株细菌PCR扩增产物基因芯片杂交结果显示,33株C+菌其G+探针和通用探针均阳性,G-探针阴性;16株G-菌中,G-探针和通用探针均阳性,G+探针阴性,均能与相应特异的探针发生杂交.(2)大肠埃希菌DNA的PCR产物其16S rRNA基因芯片的最小检出量为1pg,约等于10个拷贝教,相当于2个细菌.(3)16S rRNA基因芯片检测24份脑脊液标本发现11份阳性,阳性率为45.83%(11/24),明显高于脑脊液培养的阳性率12.50%(3/24),差异有统计学意义(P<0.01).结论 脑脊液细菌16S rRNA基因芯片检测技术特异性强、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.     

荧光定量PCR测定在小儿非细菌性脑膜炎病原学诊断中作用及其临床意义

目的 研究疱疹类病毒与肺炎支原体(MP)在潍坊地区小儿中枢神经系统(CNS)感染发病中的作用 及荧光定量PCR测定(FQ-PCR)与临床的关系。方法 利用FQ-PCR法检测小儿非细菌性脑膜炎(非菌脑)脑脊液 (CSF)中EBV、HSV、CMV和MP的核酸,并与CSF中其特异性抗体结果进行比较。结果 192例非菌脑患儿中84 例病原DNA阳性(阳性率43.8％),且年龄越小,阳性率越高,以0-3岁组最高,占50.0％;阳性者中,EBV-DNA阳 性率最高(46.4％),其次是HSV-DNA和MP-DNA(28.6％和14.3％),CMV-DNA阳性率最低(10.7％);重型患儿 CSF 4种病原DNA的拷贝量明显高于轻型患儿(P<0.05,0.01);CSF病原DNA阳性率与特异性IgM阳性率分 别为43.8％(47／192例)和24.5％(84／192例)(P<0.01)。结论 小儿CNS感染发病中上述4种病原不可忽视; CSF病原DNA拷贝量检测对判断非菌脑病情具有重要指导意义;MP的直接损伤是MP脑膜炎的发病机制之一; FQ-PCR特异性强、敏感性高,需标本量少,是早期快速诊断小儿非菌脑的可靠方法,值得推广使用。     

荧光定量PCR及血清IgM检测对儿童肺炎支原体中枢神经系统感染诊断价值的研究

目的研究荧光定量PCR及特异性抗体MP-IgM检测在儿童肺炎支原体(MP)中枢神经系统感染早期诊断及疗效评估中的作用。方法利用荧光定量PCR检测非化脓性中枢神经系统感染儿童血及脑脊液中MP-DNA量,并与血及脑脊液中MP特异性抗体(MP-IgM)检测阳性结果比较。结果(1)在262例非化脑患儿不同的年龄组中,各组脑脊液MP-DNA的阳性率均明显高于脑脊液MP-IgM的阳性率(P<0.05)。在3岁以下患儿组中,血MP-DNA阳性率显著高于血MP-IgM的阳性率(P<0.05),而3岁以上各组中两种方法阳性率无显著差异(P>0.05)。(2)在重症MP中枢神经系统感染患儿其脑脊液及血MP-DNA的拷贝数均较轻症者明显升高(P<0.01)。(3)MP中枢神经系统感染患儿急性期脑脊液及血MP-DNA的拷贝数明显高于恢复期(P<0.01)。结论荧光定量PCR法可作为早期快速诊断儿童(尤其3岁以下患儿)MP中枢神经系统感染的方法,并且为评估病情轻重及疗效提供可靠依据。     

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目的探讨荧光定量聚合酶链反应(FQ PCR)检测儿童病毒性脑炎脑脊液(CSF)中的病原体的价值。方法应用定性聚合酶链反应(PCR)对2002年1月至2004年10月保定市儿童医院收治的病毒性脑炎患儿78例的CSF标本筛选,检测最常见的引起病毒性脑炎的6种病毒,对单纯疱疹病毒(HSV),柯萨奇病毒(CV),埃可病毒(EV)阳性者做FQ PCR检测,其中HSV分别在3个时点(治疗前、治疗中、出院时)检测DNA拷贝数。结果定性聚合酶链反应阳性标本FQ PC检测100%阳性,HSV DNA拷贝数在3个时点有统计学差异,病毒核酸拷贝数与临床表现一致。结论FQ PCR可作为儿童病毒性脑炎早期诊断的有效方法之一,动态观察HSV DNA含量有助于指导治疗,估计疾病的发展和预后     

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     

小儿结核性脑膜炎不同检测方法诊断价值研究

目的探讨不同实验室检查方法对小儿结核性脑膜炎(TBM)的诊断价值,为其早期诊断寻求方法。方法收集2009年3月至2011年2月新乡医学院第一附属医院结核内科和小儿内科的TBM住院患儿30例,另将同期在该院小儿内科诊断为非结核菌感染的脑炎、脑膜炎或其他脑部病变的30例住院患儿设为对照组。对两组患儿运用荧光定量PCR(FQ-PCR)技术检测脑脊液(CSF)中结核DNA、结核抗体、抗酸杆菌涂片、结核菌培养、腺苷脱氨酶(ADA)活性5种方法进行检测,并将结果进行比较分析。结果 FQ-PCR阳性率与抗酸杆菌涂片、结核菌培养、结核抗体检测差异均有统计学意义(P<0.05);30例TBM患者ADA值升高14例,对照组有6例升高,敏感性与FQ-PCR差异无统计学意义,但特异性(80%)低于FQ-PCR(100%),差异有统计学意义。涂片法和培养法对脑脊液检出率差异无统计学意义。FQ-PCR和ADA两种方法的TBM组和对照组之间差异均有统计学意义。结论 FQ-PCR检测脑脊液结核DNA,简便、快速、敏感性高、特异性强,有助于早期诊断。     

荧光定量聚合酶链式反应法检测婴儿尿液人巨细胞病毒DNA对人巨细胞病毒感染的诊断意义

目的探讨荧光定量聚合酶链式反应（FQ-PCR）方法检测婴儿尿液人巨细胞病毒（HCMV）DNA水平对婴儿HCMV感染的诊断价值。方法采用FQ-PCR检测348例临床疑似HCMV感染婴儿尿液中HCMVDNA水平。同时采用化学发光免疫分析法（CLIA）检测婴儿血清HCMV-IgM抗体。在尿液HCMVDNA检测阳性患儿中比较血清HCMV-IgM抗体检测阳性与阴性患儿尿液中HCMVDNA拷贝数差异。结果FQ-PCR检测尿液HCMVDNA的阳性率为41.74%,CLIA检测婴儿血清HCMV-IgM抗体的阳性率为19.83%。2种方法对HCMV感染诊断的符合率为76.7%。前者诊断阳性率显著高于后者（χ^2=69.44P〈0.01）。在尿液HCMVDNA检测阳性患儿中,IgM抗体阳性组尿液HCMVDNA拷贝数显著高于阴性组（P〈0.01）。结论FQ-PCR检测尿液HCMVDNA是早期诊断婴儿HCMV感染的敏感有效的方法。HCMV感染婴儿尿液中高HCMVDNA拷贝数与活动性HCMV感染密切相关;FQ-PCR方法检测尿液HCMVDNA水平对于判断是否为HCMV活动性感染具有一定意义。     

人博卡病毒实时荧光定量PCR检测方法的建立

目的建立人博卡病毒(human Bocavirus,HBoV)的实时荧光定量PCR(real-time fluorescent quanti-tative PCR,FQ-PCR)检测方法,检测呼吸道感染患儿标本的HBoV。方法设计HBoV NP1基因的引物和Taqman探针,扩增NP1基因片段,并将其克隆到pGEM-T Easy载体上,构建质粒标准品,建立FQ-PCR检测方法,进行敏感性、特异性试验,检测195份临床标本。结果所建立的FQ-PCR方法对临床其他呼吸道病毒不出现特异性扩增曲线,特异性好,线性范围为10～108copies/μl。195份临床标本中HBoV阳性12份,阳性率为6.2%。结论成功建立了HBoV实时荧光定量PCR检测方法并检测临床标本,为人博卡病毒感染的临床治疗提供快速、可靠的诊断依据。     

先天性症状性巨细胞病毒感染脑脊液病毒载量与听力损伤相关性

目的 探讨症状性先天性巨细胞病毒(cytomegalovirus,CMV)感染新生儿脑脊液(cerebrospinal fluid,CSF) CMV DNA载量与感音性听力损伤(sensorineural hearing loss,SNHL)的相关性.方法 36例先天性症状性CMV感染患儿,PCR法检测CSF CMV DNA载量,并于出生1个月内、生后6个月及1年左右行脑干听觉诱发电位检测.结果 (1)36例患儿,其中CSF CMV DNA阳性15例,阳性率41.2％,SNHL17例,SNHL发生率47.2％.(2) CSF CMV DNA阳性组SNHL发生率60.0％(9/15);阴性组SNHL发生率38.1％ (8/21),两组发生率比较差异无统计学意义(P =0.194).(3)CSFCMVDNA阳性组中,SNHL组与听力正常组CSF CMV DNA载量为3.35 ±0.68和3.17±0.56,两组载量比较,差异无统计学意义(P=0.36).结论 先天性症状性CMV感染患儿CSF CMV DNA是否阳性及其载量不是预测SNHL的指标.     

细菌16S rRNA基因荧光定量PCR诊断新生儿败血症

目的探讨新生儿败血症的快速可靠诊断方法,以提高临床检测细菌的速度及准确性。方法以细菌16SrRNA基因为靶序列,设计通用引物及TaqMan探针,建立细菌16SrRNA基因实时荧光定量PCR反应体系;选择临床引起新生儿败血症的常见菌如金黄色葡萄球菌、表皮葡萄球菌和大肠埃希菌等进行浓度梯度实验;对临床疑为新生儿败血症的830例感染性疾病的新生儿抽取静脉血分别做细菌16SrRNA基因荧光定量PCR和血培养检测。结果临床常见分离株金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌荧光定量PCR检测结果均为阳性;巨细胞病毒、EB病毒、乙肝病毒,新型隐球菌及白色念珠菌,人基因组DNA及空白对照均为阴性。细菌荧光定量PCR最低能检测到3个细菌,其荧光定量CT值为37．90;对临床疑为感染性疾病的830例患儿标本中,荧光定量PCR检测血标本阳性率5．18％（43／830）,血培养阳性率2．41％（20／830）,前者明显高于后者,差异具有统计学意义,P〈0．01,30例非感染性疾病患儿血标本荧光定量PCR检测及细菌培养均为阴性。若以血培养作为对照,荧光定量PCR方法的诊断敏感性为100％,特异性为97．16％,正确诊断指数为0．972。结论建立了细菌16SrRNA基因荧光定量PCR诊断新生儿败血症方法。其检测快速、简便,为新生儿败血症提供早期、敏感的病原学诊断依据。     

Rapid diagnosis of bacterial meningitis in children with fluorescence quantitative polymerase chain reaction amplification in the bacterial 16S rRNA gene

Polymerase chain reaction (PCR) techniques have been increasingly used to detect microbial DNA in cerebrospinal fluid (CSF) for the diagnosis of bacterial meningitis. In order to determine the rapidity, sensitivity and specificity of 16S rRNA-based fluorescence quantitative polymerase chain reaction (FQ-PCR), 16S rRNA-based FQ-PCR, CSF bacterial culture and CSF routine analysis were compared in the diagnosis of bacterial meningitis in children. Twenty children who were clinically suspected of bacterial meningitis were included in this study. A total of 2.0 ml of CSF was collected from every child and was subjected to 16S rRNA-based FQ-PCR, CSF culture and CSF routine analysis. Bacterial DNA copies and the cycle threshold (CT) value of the 16S rRNA-based FQ-PCR was recorded, and the results were compared with CSF culture and CSF routine analysis. Seven children were found to be positive with a rate of 35% (7/20) when detected with 16S rRNA-based FQ-PCR and four children displayed a positive rate of 20% (4/20) with the CSF culture method. These two groups displayed a significant difference, with a p-value of 0.002. The method of 16S rRNA-based FQ-PCR demonstrated a high specificity when compared to the standard microbes. A negative correlation was noted between the CT value and the bacteria DNA copies, and the CT value was indicative of the seriousness of bacterial meningitis. 16S rRNA-based FQ-PCR was proved to be a more rapid, sensitive and specific method compared with CSF culture and it should have promising usage in the diagnosis of bacterial meningitis.     

神经元特异性烯醇化酶对脑膜炎患儿的诊断价值

目的探讨脑脊液神经元特异性烯醇化酶(CSF-NSE)对脑膜炎患儿的诊断价值。方法采用ELISA对18例化脓性脑膜炎(化脑)、13例结核性脑膜炎(结脑)及25例病毒性脑膜炎(病脑)及18例正常儿童CSF-NSE进行测定。结果与对照组比较,化脑及结脑患儿CSF-NSE水平均显著升高(P均<0.05);病脑患儿无显著性差异(P>0.05);化脑及结脑患儿CSF-NSE水平较病脑患儿显著升高(P<0.05);化脑、结脑、病脑患儿CSF-NSE与其CSF白细胞数及蛋白水平均无相关性(P>0.05)。结论CSF-NSE测定可作为鉴别化脑和病脑的重要参考指标之一,也可作为评价脑膜炎患儿病情严重程度及预后的生化指标。     

Test characteristics and interpretation of cerebrospinal fluid gram stain in children

BACKGROUND: Few data exist regarding the test characteristics of cerebrospinal fluid (CSF) Gram stain among children at risk for bacterial meningitis, especially the rate of false positive Gram stain. METHODS: We conducted a retrospective cohort study of children seen in the emergency department of Children's Hospital Boston who had CSF obtained between December 1992 and September 2005. Patients who had ventricular shunts, as well as those who received antibiotics before CSF was obtained were excluded. Test characteristics of CSF Gram stain were assessed using CSF culture as the criterion standard. Patients were considered to have bacterial meningitis if there was either: (1) growth of a pathogen, or (2) growth of a possible pathogen noted on the final CSF culture report and the patient was treated with a course of parenteral antibiotics for 7 days or more without other indication. RESULTS: A total of 17,569 eligible CSF specimens were collected among 16,036 patients during the 13-year study period. The median age of study subjects was 74 days. Seventy CSF specimens (0.4%) had organisms detected on Gram stain. The overall sensitivity of Gram stain to detect bacterial meningitis was 67% [42 of 63; 95% confidence interval (CI): 54-78] with a positive predictive value of 60% (42 of 70; 95% CI: 48-71). Most patients without bacterial meningitis have negative Gram stain [specificity 99.9% (17,478 of 17,506; 95% CI: 99.8-99.9)] with a negative predictive value of 99.9 (17,478 of 17,499; 95% CI: 99.8-99.9). CONCLUSIONS: CSF Gram stain is appropriately used by physicians in risk stratification for the diagnosis and empiric treatment of bacterial meningitis in children. Although a positive Gram stain result greatly increases the likelihood of bacterial meningitis; the result may be because of contamination or misinterpretation in 40% of cases and should not, by itself, result in a full treatment course for bacterial meningitis.     

Haemophilus influenzae type b still remains a leading cause of meningitis among unvaccinated children--a prospective CSF analysis study

A prospective, hospital-based cerebrospinal fluid (CSF) analysis study was undertaken in 65 children who had diagnostic lumbar puncture on admission for suspected central nervous system infections. Twenty-three children were clinically diagnosed to have had sepsis and/or meningitis. CSF bacterial culture grew Haemophilus influenzae type b (Hib) in four cases and Streptococcus pneumonia (SP) was cultured in another child. Bacterial antigen was detected in 13 other CSF specimens and the pathogens were Hib (n = 9), SP (n = 3) and Group B Streptococcus (n = 1). No etiologic cause was identified to explain the abnormal CSF pleocytosis and biochemistry in the remaining five cases. In contrast, the CSF analysis was normal in 42 other children with probable viral and non-infectious neurological condition, mostly febrile convulsions. The overall frequency rate for all types of meningitis and especially for Hib meningitis were 43 and 31 cases per 100,000 children < 5 years of age, respectively. These findings support our earlier observations that Hib meningitis still remains the leading cause of childhood meningitis in our region. Also it reaffirms the observation that bacterial meningitis may often be under-reported if CSF positive culture alone is considered for the diagnosis.     

小儿化脓性脑膜炎临床和病因学分析

目的探讨化脓性脑膜炎的病因,为诊治提供科学依据。方法收集住院化脓性脑膜炎患儿371例,男252例,女119例;平均年龄(2.67±3.32)岁。对患儿临床表现及血液和脑脊液(CSF)相关参数进行分析。结果 371例中≤1岁患儿占46.36%,<3岁占80.59%,以发热(90.29%)、抽搐(52.56%)等症状就诊。82.21%患儿白细胞计数(WBC)>10×109/L,74.42%患儿中性粒细胞比率>50%,85.44%患儿脑脊液WBC≥500×106/L。血培养革兰染色阳性(GSP)37例,革兰阳性菌(GPB)24例,革兰阴性菌(GNB)13例。脑脊液培养阳性34例,GPB 19例,GNB 15例。脑脊液检测出肺炎链球菌8例,流感嗜血杆菌3例,奈瑟菌1例。死亡7例(1.88%)中,脑脊液2例GNB阳性,5例化脓性/混浊,4例蛋白>150 mg/dl和葡萄糖<1 mg/dl。结论化脓性脑膜炎的发病年龄多在婴幼儿阶段,临床表现多种多样,血液和脑脊液相关参数分析,能较好的提供病因诊断依据,并为临床治疗及预后提供参考。     

Peritonitis as a first manifestation of Neisseria type C meningitis]

A 15-year-old boy was first referred for a clinical presentation of revealed peritonitis. Abdominal endoscopy showed normal appendix but the presence of purulent peritoneal fluid. Antibiotics were immediately administered. Ten hours later clinical signs of meningitis occurred, and lumbar puncture was performed. CSF bacterial meningitis characteristics were present but no bacteria was observed. However Neisseria meningitidis type C was detected by direct peritoneal fluid examination and by 24(th) hour blood culture. Peritoneal and the CSF fluid culture were negative, but DNA analysis from peritoneal fluid was positive.     

不同中枢神经系统感染患儿脑脊液肝细胞生长因子水平变化的意义

目的通过观察病毒性脑炎、细菌性脑膜炎及结核性脑膜炎患儿脑脊液肝细胞生长因子(HGF)水平变化,探讨其是否可以作为一个鉴别诊断的生化指标,为临床疾病的诊断提供依据。方法选择临床确诊病毒性脑炎30例、细菌性脑膜炎21例、结核性脑膜炎19例及对照组24例,采用酶联免疫吸附试验双抗体夹心法检测各组患儿脑脊液HGF水平。同时将HGF水平分别与脑脊液中白细胞计数和蛋白定量进行直线相关分析,观察脑脊液中HGF水平与脑脊液中白细胞计数和蛋白定量的关系。结果细菌性脑膜炎组、结核性脑膜炎组脑脊液HGF水平高于病毒性脑炎组和对照组,结核性脑膜炎组高于细菌性脑膜炎组患儿,细菌性脑膜炎组、结核性脑膜炎组及病毒性脑炎组与对照组患儿比较均有显著性差异(Pa<0.05),病毒性脑炎组与对照组患儿比较差异无统计学意义(P>0.05)。结论 HGF有可能成为鉴别细菌性脑膜炎、结核性脑膜炎及病毒性脑炎的指标之一。     

Rapid diagnosis of pneumococcal meningitis: implications for treatment and measuring disease burden

BACKGROUND: Streptococcus pneumoniae is the leading cause of childhood pneumonia and meningitis worldwide. Isolation of this organism, however, is uncommon in resource-poor countries, in part because of extensive use of prior antibiotics. A rapid, highly sensitive immunochromatographic test (ICT) for S. pneumoniae was evaluated for the diagnosis of meningitis. METHODS: Cerebrospinal fluid (CSF) from 450 children with suspected meningitis was tested with ICT, and results were compared with CSF culture, latex agglutination test (LAT) and/or polymerase chain reaction (PCR). Serial CSF specimens from 11 patients were also evaluated for duration of positive results during effective antimicrobial therapy. FINDINGS: All 122 cases of pyogenic pneumococcal meningitis positive either by culture (N = 87) or PCR (N = 35) were positive by ICT, yielding 100% (122 of 122) sensitivity. All purulent CSF specimens from patients with meningitis caused by other bacteria by culture (N = 149) or by LAT (N = 48) or those negative by culture, LAT and LytA and thus of unknown etiology (N = 20), and normal CSF specimens (N = 104) were negative by ICT. Thus the specificity of ICT also was 100% (321 of 321), although negativity of ICT was not confirmed by PCR, if it was positive for other organisms either by culture or LAT. Serotyping of S. pneumoniae strains revealed 28 different serotypes, indicating that outcome of ICT are independent of diverse capsular serotype of pneumococcus. Antigen was detected by ICT for at least 10 days after presentation, and 1 was still positive on day 20, which was longer than for either LAT or PCR. INTERPRETATION: ICT for pneumococcal antigen in CSF is 100% sensitive and specific in diagnosing pyogenic pneumococcal meningitis and can detect approximately 30% more pneumococcal meningitis cases than with culture alone. The simplicity of the test procedure and the longevity of CSF antigen detection suggest the potential utility of ICT to estimate the true burden of pneumococcal disease, as for Haemophilus influenzae type b using data from meningitis, and to guide selection of appropriate antibiotic treatment, especially in resource-poor countries with widespread prehospital antimicrobial use.