抗生素治疗时限对化脓性脑膜患儿脑脊液细菌16s rRNA基因检测的影响

目的：研究抗生素治疗时限对化脓性脑膜患儿脑脊液细菌16s rRNA基因检测的影响。方法：选取2014年2月至2016年3月在我院收治的化脓性脑膜炎患儿60例。对所有患儿予以16s rRNA基因检测和脑脊液细菌培养检测，比较两种方法检测结果，观察抗生素使用对两种检测方法的影响。结果：脑脊液细菌培养阳性率显著低于16s rRNA基因芯片检测阳性率[15%(9/60) vs.35%(21/60)](P<0.05)。抗生素应用早期的16s rRNA基因检测阳性率和抗生素使用后期的阳性率比较差异不显著[36.67%(11/30) vs.33.33%(10/30)](P>0.05)；抗生素使用早期的脑脊液细菌培养阳性率显著高于抗生素应用后期的阳性率[13.33%(4/30) vs.0.00%(0/30)](P<0.05)。结论：和脑脊液培养相比，16s rRNA基因芯片法检测化脓性脑膜患儿脑脊液细菌受抗生素治疗时限的影响更小。     

基因芯片快速检测脑脊液常见病原菌的应用研究

目的 探讨采用基因芯片方法在疑似颅内细菌感染患者的脑脊液标本中直接检测常见致病菌的可行性和临床意义,以期提高微生物检测的速度,将检验结果快速回馈给临床.方法 收集2015年3月至2016年7月疑似颅内感染患者脑脊液标本,从中筛检出30例阴性标本和77例阳性标本,从原始脑脊液标本中提取DNA,经PCR扩增后产物在基因芯片上杂交,其结果与同份标本的临床培养结果进行比较.结果 阴性标本中芯片法与培养法符合率为96.7%,阳性标本中芯片法与培养法符合率为41.6%,而剔除凝固酶阴性葡萄球菌后芯片法与培养法的符合率为78.1%.结论 基因芯片检测原始脑脊液标本对早期筛查具有一定的临床意义,提示临床初步抗生素的用药方向,减少用药的盲目性,减少耐药菌株的出现,有很好的发展前景.     

无菌性脑膜炎和脑炎病人脑脊液肠道病毒RNA的检测及其临床意义

目的 检测肠道病毒（EV）在中枢神经系统感染中的致病情况,探讨检测EV感染的方法。方法 就用逆转录－聚合酶链反应（RT－PCR0和病毒培技术检测46例无菌性脑膜炎及脑炎病人脑脊液（CSF）标本。结果 RT－PCR方法敏感特异;46例无菌性脑膜炎和脑炎急性期CSF标本中,31例EV阳性（67．4％）,14例病毒培养阳性（26．1％）。统计结果显示,RT－PCR敏感性明显高于病毒培养。结论 EV是引起无菌性脑膜炎和脑炎的重要病原;RT－PCR快速敏感特异,简单易行,易于推广,是诊断EV感染的有效方法。     

35例新生儿化脓性脑膜炎与脐炎的探讨

目的探讨新生儿化脓性脑膜炎与脐炎的关系.方法检测35例新生儿化脓性脑膜炎的脑脊液、血常规、脐分泌物并进行培养.结果化脓性脑膜炎与脐炎的关系密切,重症脐炎细菌进入血循环,由脐源性途径引起化脓性脑膜炎.结论新生儿脐炎消毒处理极为重要,以预防化脓性脑膜炎的发生.     

脑脊液腺苷脱氨酶测定在小儿结核性脑膜炎诊断中的应用

目的:探讨脑脊液腺昔脱氨酶(ADA)活性测定在小儿结核性脑膜炎诊断中的应用价值。方法:选择结核性脑膜炎35例、病毒性脑膜炎36例、化脓性脑膜炎32例及非脑膜炎40例,测定并比较各组患者脑脊液ADA活性及阳性率。结果:结核组脑脊液ADA活性和阳性率明显高于非结核组,差异有显著性(P<0.05);结核性脑膜炎组治疗前后脑脊液ADA活性比较,差异有显著性(P<0.05)。结论:脑脊液腺苷脱氨酶可作为诊断结核性脑膜炎的重要参考指标。     

聚合酶链反应在结核性脑膜炎中的应用

采用聚合酶链反应(PCR)技术对268份脑脊液标本进行结核分枝杆菌检测,同时以分离培养和涂片镜检做对比.结果以PCR有较高的检出率(38.4%),优于分离培养(23.1%)和涂片镜检(19.4%).说明该技术具有灵敏度高、快速简便等优点,对结核性脑膜炎的早期诊断、有重要的意义.     

半套式逆转录聚合酶链反应技术诊断中枢神经系统肠道病毒感染

肠道病毒 (ＥＶ)感染常见 ,所致疾病轻重不一 ,可表现为无症状感染或严重的脑膜炎、脑炎和心肌炎等。长期以来缺乏有效的病原学诊断方法。组织培养分离病毒费时 ,阳性率低 ,且病合 1至 2周得到结果时已无益于临床诊断和治疗。聚合酶链反应 (ＰＣＲ)方法能快速、特异地检测出脑膜炎病人脑脊液 (ＣＳＦ)中ＥＶＲＮＡ ,但感染初期及回顾性研究的临床标本中病毒滴度较低 ,扩增结果多为阴性 ,病毒培养也常常失败。我们采用二步扩增半套式ＰＣＲ方法检测无菌性脑膜炎病人脑脊液 (ＣＳＦ)和血清标本中肠道病毒ＲＮＡ ,发现该方法较常规一步ＰＣＲ敏…     

单克隆抗体酶联免疫吸附试验在结核性脑膜炎诊断中的初步应用

应用单克隆抗体酶联免疫吸附试验检测38例结核性脑膜炎，33例其它脑膜炎在112例非脑膜炎患者脑脊液中的结核杆菌抗原和抗体。结果脑脊液结核抗原阳性率为81．6％，假阳性率为2．8％；结核抗体阳性率为86．6%，假阳性率为3．4％．提示本法可作为结核性脑膜炎有效的辅助诊断方法。动态观察尚可为疗效判断提供依据。     

实时荧光定量PCR检测泌尿生殖道患者尿液解脲脲原体

目的应用实时荧光核酸恒温扩增检测技术（SAT）检测泌尿生殖道患者尿液解脲脲原体（UU）,并评价其敏感性和特异性。方法对140例疑似泌尿生殖道感染患者的拭子和尿液样本,分别采用培养法、SAT进行解脲脲原体检测,检测结果有差异的标本用实时荧光定量PCR法对拭子进行复测,根据实验结果评估SAT检测尿液UU的敏感性和特异性。结果 140例疑似患者试子培养和尿液SAT检测阳性率均为60%,两者比较差异无统计学意义（P〉0.05）,其中16例培养结果与SAT检测结果不一致,8例培养法阳性、SAT阴性的拭子PCR结果复测阳性;8例培养法阴性、SAT阳性的拭子标本PCR结果 4例阴性、4例阳性,结合培养法结果和PCR结果作为＂扩大金标准＂,得出SAT对尿液检测的敏感性为95.5%、特异性为100%。结论 SAT检测泌尿生殖道患者尿液UU具有取样方便,检测快速、准确等优点,适用于临床实验室泌尿生殖道UU的检测。     

实时荧光PCR与BACTEC MGIT 960快速培养在初治痰涂片阴性未受抗结核治疗的肺结核诊断中的应用

目的:探讨实时荧光PCR与BACTEC MGIT 960分枝杆菌快速培养(快速培养法)准确诊断初治痰涂片阴性(涂阴)且未经受抗结核治疗的肺结核可能性。方法:实验组:368份临床诊断活动性肺结核患者的初治涂片阴性痰标本;对照组:55份非结核性肺部疾病患者的痰液标本。用FQ-PCR技术、快速培养法及改良罗氏培养参考法对两组标本进行分析,观察指标为TB-DNA和结核菌阳性率。结果:实验组和对照标本中FQPCR技术、快速培养法和改良罗氏培养参考法检测的TB-DNA或结核菌阳性率分别为:16.85%、23.37%、22.01%和1%、0%、0%。三种方法分别平均耗时3h、9.6d和28d。以改良罗氏培养参考法为参照,FQ-PCR法和快速培养法的敏感性分别为69.1%和100%;特异性分别为91.2%和98.6%。结论:FQ-PCR和快速培养法均能提高初治涂阴肺结核患者的早期确诊率,但FQ-PCR的敏感性尚需进一步提高。     

Comparison of broad-range bacterial PCR and culture of cerebrospinal fluid for diagnosis of community-acquired bacterial meningitis

Appropriate, rapid and reliable laboratory tests are essential for the diagnosis and optimal antibiotic therapy of acute bacterial meningitis. Broad-range bacterial PCR, combined with DNA sequencing, was compared with culture-based methods for examining cerebrospinal fluid (CSF) samples from patients with suspected meningitis. In total, 345 CSF specimens from 345 patients were analysed, with acute community-acquired bacterial meningitis being diagnosed in 74 patients. The CSF of 25 patients was positive by both PCR and culture; 26 patients had CSF specimens positive by PCR only, and 14 patients had specimens positive by culture only. The sensitivity of PCR and culture for clinically relevant meningitis was 59% (44/74) and 43% (32/74), respectively, while the specificity was 97% (264/271) and 97% (264/271), respectively. The commonest bacterial rRNA gene sequences detected by PCR only were those of Streptococcus pneumoniae and Neisseria meningitidis (n = 12). PCR failed to detect the bacterial rRNA gene in seven specimens from patients with symptoms compatible with acute bacterial meningitis. Overall, the results demonstrated that PCR in conjunction with sequencing may be a useful tool in the diagnosis of bacterial meningitis. PCR is particularly useful for analysing CSF from patients who have been treated with antibiotics before lumbar puncture.     

Utility of the polymerase chain reaction in the diagnosis of tuberculous meningitis

Due to inconsistent clinical presentations and the lack of a rapid, sensitive and specific test, tuberculous meningitis (TBM) is particularly difficult to diagnose. The present study was carried out to determine the utility of the polymerase chain reaction (PCR) using INS primers in the diagnosis of TBM and to compare the efficacy of two different DNA extraction protocols. Fifty-seven cerebrospinal fluid (CSF) samples from suspected cases of meningitis -- 30 definitive/possible TBM and 27 non-TBM -- were processed for microscopy, culture and PCR. Results of computer tomographic (CT) scan findings were noted. The results of smear, culture and PCR were compared using culture and/or clinical response to treatment as the gold standard. The sensitivity of microscopy, culture, CT scan and PCR was 3.3%, 26.7%, 60.0% and 66.7%, respectively. PCR following QIAmp DNA extraction had a sensitivity of 66.7% compared to PCR following a DNA extraction protocol based on the use of cetyl trimethyl ammonium bromide (CTAB) (50%). PCR was positive in all culture-positive CSF samples using either extraction method. PCR is a rapid and sensitive technique; above all, it can diagnose tuberculous meningitis at a very early stage.     

Rapid diagnosis of bacterial meningitis by real-time PCR and fluorescence in situ hybridization

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.     

Evaluation of the roche AMPLICOR enterovirus PCR assay in the diagnosis of enteroviral central nervous system infections.

BACKGROUND: Enteroviruses cause a substantial number of cases of aseptic meningitis annually in the USA. While culture has been useful in the detection of patients with viral meningitis it is time-consuming and lacks sensitivity. Detection of viral nucleic acid in patient specimens has been demonstrated to improve enteroviral detection. OBJECTIVES: A research use only commercial amplification assay, the Roche AMPLICOR EV test, was compared to culture for the diagnosis of enteroviral meningoencephalitis. STUDY DESIGN: Four-hundred and sixty-five consecutive CSF samples sent prospectively for suspicion of enteroviral infection were evaluated by PCR and shell-vial culture. Clinical information and CSF analysis were used to resolve PCR positive, culture negative samples. Sensitivity and specificity were calculated using resolved data. RESULTS: There were 138 samples which met the definition of a true positive. Of these culture detected 77 (sensitivity 55.8%) and PCR detected 136 (sensitivity 98.6%). PCR missed two culture positive samples. Upon repeat testing, these CSF samples were found to contain inhibitors. CONCLUSIONS: The Roche AMPLICOR EV-PCR test was statistically more sensitive than culture (P<0.001) in the detection of enteroviruses in CSF in patients suspected of having enteroviral meningitis. This assay also has the advantage of a rapid turnaround time of 5-6 h compared to 3-5 days for culture.     

Multicenter Evaluation of the Amplicor Enterovirus PCR Test with Cerebrospinal Fluid from Patients with Aseptic Meningitis

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the “gold standard” were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.     

Improved Detection of Bacterial Central Nervous System Infections by Use of a Broad-Range PCR Assay

A universal PCR assay for bacteria and fungi detected meningitis pathogens in 65% of 20 cerebrospinal fluid (CSF) samples from patients with suspected central nervous system (CNS) infections compared to a 35% detection rate by culture and/or microscopy methods. Thus, the PCR assay can improve the diagnosis rate of infective meningitis when standard methods provide a negative result.     

Evaluation of the BioFire® FilmArray® Meningitis/Encephalitis panel in an adult and pediatric Ugandan population

BackgroundMeningitis causes significant mortality in sub-Saharan Africa and limited diagnostics exist. We evaluated the utility of the BioFire® FilmArray® Meningitis/Encephalitis multiplex PCR panel (BioFire ME) in HIV-infected adults and HIV-infected and uninfected children presenting with suspected meningitis in Uganda.MethodsWe tested cerebrospinal fluid (CSF) using a stepwise meningitis diagnostic algorithm including BioFire ME. We determined the diagnostic performance of BioFire ME for cryptococcal meningitis, using cryptococcal antigen (CrAg) and CSF culture as reference standards, and assessed other central nervous system (CNS) pathogens identified by the panel.ResultsWe evaluated 328 adult and 42 pediatric CSF specimens using BioFire ME. Of the adult CSF samples tested, 258 were obtained at baseline, and 70 were obtained from repeat lumbar punctures in cryptococcal meningitis. For Cryptococcus, sensitivity was 82%, specificity was 98%, PPV was 98%, and NPV was 79% in baseline specimens using CSF CrAg as the reference standard. Among follow-up specimens, a negative BioFire ME for Cryptococcus predicted CSF culture sterility with 84% NPV. Overall sensitivity was decreased at low fungal burdens: 29% for 0–99 Cryptococcus CFU/mL compared to 94% for ≥100 CFU/mL in baseline specimens. Other pathogens detected included E. Coli, H. influenzae, S. pneumoniae, CMV, enterovirus, HSV, HHV-6, and VZV. Two specimens tested positive for S. pneumoniae and one for Cryptococcus in the pediatric population.ConclusionsMultiplex PCR is a promising rapid diagnostic test for meningitis in adults and children in resource-limited settings. Cryptococcus at low fungal burdens in CSF may be missed by BioFire ME.