分子鉴别诊断(MDD)技术在呼吸道病原体检测中的应用

目的:以靶序列富集多重PCR(Tem-PCR)和液相芯片(xMAP)检测技术结合,建立常见呼吸道病原体的分子鉴别诊断(MDD)平台,对其可靠性进行评估,并用于临床样品的检测.方法:采集苏州大学附属儿童医院呼吸科住院病例呼吸道灌洗液样品22例和苏州大学附属第一医院呼吸内科门诊病例咽拭子样品20例,使用Tem-PCR技术对样品的核酸进行多重扩增,以液相芯片技术进行高通量的多重检测.结果:对已知样品的检测表明该平台具有高度的特异性和敏感性.对42例临床样品进行检测的结果显示,22例呼吸道灌洗液的阳性检出率为63.6%.呼吸道灌洗液阳性检出率高于门诊病例咽拭子样品的阳性检出率,RNA病原体(以病毒为主)的阳性率高于DNA病原体(以细菌为主)的阳性率.结论:建立了呼吸道病原体的MDD平台,具备了对呼吸道传染性疾病的高通量快速诊断能力.     

分子鉴别诊断领域的革命性技术——Tem-PCR

分子鉴别诊断由于其高效、快速的特点,在公共卫生领域和个体化医疗中将发挥越来越蓖要的作用.分子鉴别诊断需要敏感、特异、可靠的多重扩增技术作为支撑,靶序列富集多重PCR(Tem-PCR)正是这样一项技术,能在一个反应体系中同时对多种靶基因高度敏感和特异的扩增,为分子鉴别诊断在应对公共卫生危机、食品安全、病毒分型、细菌耐药性分析和个体独特型遗传背景检测等方而的应用带来革命性变化.     

呼吸道合胞病毒离心蚀斑技术的建立

建立了一种呼吸道合胞病毒蚀斑新技术。将细胞悬液与病毒混合离心能使蚀斑数量显著增多，出斑时间缩短。同时蚀斑数量和出斑时间与离心力和离心时间成正比，细胞浓度以２．５×１０￣８／Ｌ、甲基纤维素浓度以１．５％为宜。与快速法及常规蚀斑法相比，离心蚀斑技术的出斑时间平均比后两者快２天左右，出斑达高峰时间比后两者提前３天左右。且本法具有蚀斑明显，敏感，重复性好，简便易行等特点。     

细胞表面分子在软组织肿瘤鉴别诊断中的应用

软组织肿瘤鉴别诊断是临床病理难点之一,免疫组化技术的应用使诊断准确性大为提高。但近年文献显示,传统免疫组化标记物,如中间丝蛋白,特异性仍不够强。有的学者试用“细胞表面分子”作为新的标记物。细胞表面分子(Cell surface molecules CSM)是位于细胞表面或镶于细胞膜内的大分子糖蛋白,目前主要用于白血细胞及肿瘤的研究。多     

多重PCR检测儿童呼吸道感染常见病毒

目的 对深圳、粤东地区儿童呼吸道病毒感染进行监测、分析,探讨其流行规律.方法 2006年6月-2008年6月,于汕头大学附属医院、深圳市第四人民医院、揭阳市人民医院采集毛细支气管炎、支气管肺炎、喘息支气管炎等的患儿咽拭子或鼻咽分泌物标本686份,多重PCR进行9种呼吸道病毒检测.结果 在686例标本中病毒阳性362例(52.77%),其中呼吸道感染患儿中呼吸道合胞病毒(RSV)感染占首位,达到31.22%(113/362),其次是鼻病毒(RHV)为16.85%(61/362),最低是流感病毒B型(IVB)占0.83%(3/362),流感病毒A型(IVA)占14.36%(52/362),副流感病毒1型(PIV1)和副流感病毒3型(PIV3)分别占7.73%(28/362)和8.29%(30/362),腺病毒(AdV)达到9.67%(35/362),而人博卡病毒(hBOV)和人偏肺病毒(hMPV)分别占6.08%(22/362)和4.97%(18/362).结论 RSV、RHV及IVA是华南地区儿童呼吸道感染的主要病毒病原,混合感染以RSV、IVA联合其他病毒感染为主,诊治时应根据所感染病原体制定有针对性的措施.     

动态观察血清AFP含量鉴别诊断病毒性肝炎与原发性肝细胞癌

甲胎蛋白 (ＡｌｐｈａＦｅｔｏｐｒｏｔｅｉｎ ,ＡＦＰ)含量增高诊断原发性肝细胞癌患者具有相对特异性 ,但临床上也常可见到病毒性肝炎患者ＡＦＰ含量增高 ,虽然对于病毒性肝炎的诊断价值不大 ,可常引起病人心理紧张恐惧。因此 ,对于其鉴别诊断具有重要意义。本文以动态观察原发性肝细胞癌与病毒性肝炎患者血清ＡＦＰ含量变化 ,鉴别诊断此两种肝病。现报告如下。对象、方法和结果一、病例 :30例 (男 2 7,女 3) ,原发性肝细胞癌患者均经临床及其他辅助检查确诊 ,非手术治疗的。年龄 33～ 80岁 ,平均 5 1 8岁 ;30例 (男 2 8,女 2 )病毒性肝炎…     

成都市流感样病例常见呼吸道病毒流行特征分析

目的 了解成都市流感样病例常见呼吸道病毒感染状况.方法 抽取2014年5月-2015年11月间成都市3个流感监测点采集的流感样病例的临床标本1367份,使用PCR法进检测甲型流感病毒、乙型流感病毒、丙型流感病毒、Ⅰ型副流感病毒、Ⅱ型副流感病毒、Ⅲ型副流感病毒、Ⅳ型副流感病毒和鼻病毒8种病毒及其亚型.结果 1367份标本中共374份8种病毒检测呈阳性,其中19例为混合感染,总阳性检出率为27.36％.阳性检出率夏季最高(32.68％),秋季最低(23.76％),春季和冬季分别为28.01％和31.26％,四季检出率结果有统计学差异(x2=17.334,P＜0.001).19例混合感染的样本,以鼻病毒合并其他病毒感染为主.结论 成都市流感样病例以鼻病毒、甲型和乙型流感病毒感染为主,其次为丙型流感病毒感染,偶发副流感病毒感染.     

Currently used nucleic acid amplification tests for the detection of viruses and atypicals in acute respiratory infections

For the detection of respiratory viruses conventional culture techniques are still considered as the gold standard. However, results are mostly available too late to have an impact on patient management. The latest developments include appropriate DNA- and RNA-based amplification techniques (both NASBA and PCR) for the detection of an extended number of agents responsible for LRTI. Real time amplification, the latest technical progress, produces, within a considerable shorter time, results with a lower risk of false positives. As results can be obtained within the same day, patient management with appropriate therapy or reduction of unnecessary antibiotic therapy in LRTI will be possible. A number of technical aspects of these amplification assays, and their advantages are discussed.The availability and use of these new diagnostic tools in virology has contributed to a better understanding of the role of respiratory viruses in LRTI. The increasing importance of the viral agents, Mycoplasma pneumoniae and Chlamydophila pneumoniae in ARI is illustrated. A great proportion of ARI are caused by viruses, but their relative importance depends on the spectrum of agents covered by the diagnostic techniques and on the populations studied, the geographical location and the season. The discovery of new viruses is ongoing; examples are the hMPV and the increasing number of coronaviruses. Indications for the use of these rapid techniques in different clinical situations are discussed. Depending on the possibilities, the laboratory could optimize its diagnostic strategy by applying a combination of immunofluorescence for the detection of RSV an IFL, and a combination of real-time amplification tests for other respiratory viruses and the atypical agents. When implementing a strategy, a compromise between sensitivity, clinical utility, turn around time and cost will have to be found.     

泸州市流感样病例感染常见呼吸道病毒的流行特征分析

目的 分析泸州市流感样病例感染常见呼吸道病毒的流行特征,为临床鉴别和疾病防控提供参考.方法 选取2014年6月-2016年1月流感哨点流感样病例标本1628例为研究对象,采用荧光定量PCR检测常发呼吸道病毒等,分析其流行特征.结果 1628份标本中,共检测出病毒642份,检出率为39.4％,鼻病毒检出率最高,其次为甲、乙型流感病毒、呼吸道合胞病毒B、腺病毒.夏季呼吸道病毒检出率最高,为13.2％,其次是冬季、春季,秋季检出率最低,不同季节病院检出率差异明显(P＜0.05).不同年龄组呼吸道病毒检出率存在显著差异(P＜0.05),0～4岁儿童病毒检出率最高.共23分标本出现混合感染,其中鼻病毒与其他病毒混合感染19份,其他病毒混合感染4份.结论 泸州市流感样常见呼吸道病毒感染以鼻病毒、甲型和乙型流感病毒为主,不同流行季节病原谱也不一致,应加强呼吸道病毒检测和防治工作.     

Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses

Acute respiratory tract infections are caused by a large number of viruses. Diagnostic methods have until recently been available only for a limited number of these viruses. With the objective to achieve sensitive assays for all respiratory viruses, a rational workflow in the laboratory, and a short turn‐around time, a real‐time PCR diagnostic platform for daily rapid detection of 15 respiratory viruses was developed. The system was evaluated on 585 stored nasopharyngeal aspirates from hospitalized children. Previous analysis by immunofluorescence and virus isolation identified viruses in 37% of the samples while the new PCR diagnostic panel detected 57% virus positive samples. The new platform was introduced in the laboratory in October 2007 and has then fully replaced the standard immunofluorescence assay for rapid detection of viruses and virus isolation. J. Med. Virol. 81:167–175, 2009. © 2008 Wiley‐Liss, Inc.     

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.     

Evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children

This study compares the performance of four commercial multiplex PCR assays (Resplex II Panel v2.0, Seeplex RV15, xTAG RVP and xTAG RVP Fast) and direct fluorescent antibody (DFA) staining and viral isolation. Seven hundred and fifty nasopharyngeal swabs were tested for 17 viral agents. In each assay, the sensitivity and specificity for each target were determined against a composite reference standard. Two hundred and eighty-eight out of 750 (38.4%) specimens were positive by DFA or viral isolation, while an additional 214 (28.5%) were positive by multiplex PCR, for a total positivity rate of 66.9%. Of 502 positive specimens, one virus was detected in 420 specimens (83.7%), two in 77 (15.3%), three in four (0.8%) and four in one case (0.2%). Compared with a composite reference standard, the inter-assay accuracy of the multiplex PCR assays varied, but all were superior to conventional diagnostic methods in detecting a broad range of respiratory viral agents in children. In addition, the sensitivity of two commercial assays, Resplex II Plus PRE and Seeplex Influenza A/B Subtyping, was determined relative to the Astra influenza Screen & Type assay for detection of influenza A viruses, including seasonal influenzas and pandemic H1N1 2009 influenza A virus. Using 75 positive and 55 negative nasopharyngeal swabs for influenza A by the Astra assay, the sensitivity of Seeplex and Resplex was 95.9% and 91.8%, respectively, with a specificity of 100% for both.     

Saliva as a diagnostic specimen for testing respiratory virus by a point-of-care molecular assay: a diagnostic validity study

Objectives

Automated point-of-care molecular assays have greatly shortened the turnaround time of respiratory virus testing. One of the major bottlenecks now lies at the specimen collection step, especially in a busy clinical setting. Saliva is a convenient specimen type that can be provided easily by adult patients. This study assessed the diagnostic validity, specimen collection time and cost associated with the use of saliva.

三种呼吸道病毒实验室检测方法比较

目的在3种呼吸道多病原检测方法中筛选出敏感的实验室检测方法。方法以73份上呼道感染病例咽拭子标本作为检测对象,利用3种呼吸道多病原病毒检测方法,对17种呼吸道病毒检测指标进行平行检测,根据不同呼吸道病毒检测指标检出率,评价3种试剂检测效果。结果自建RT-PCR及PCR检测方法共检出56个阳性指标,市售多重RT-PCR检测试剂盒共检出41个阳性指标,市售多重实时荧光RT-PCR检测试剂盒共检出87个阳性指标。结论多重实时荧光RT-PCR检测试剂盒具有更高的检出率,可用于呼吸道多病原病毒的实验室检测。     

赛沛Xpert Xpress Flu/RSV Assay在儿童急性呼吸道感染病原学检测中的应用评价

目的:评价赛沛Xpert Xpress Flu/RSV Assay(后简称Xpert)在儿童流感病毒和呼吸道合胞病毒感染实验室检测的效果。方法:2018年10月—2019年2月,在北京儿童医院门诊采集急性呼吸道感染儿童的鼻咽拭子,所有样本均采用Xpert和测序方法进行检测,不一致结果样本使用第三方检测方法进行验证,比较...