广州地区婴幼儿非细菌性下呼吸道感染病原研究

目的探讨广州地区婴幼儿非细菌性下呼吸道感染病原学特征.方法用EILSA法检测824例急性下呼吸道感染病人的5种常见病原体的抗体.392份咽拭子用荧光PCR检测流感病毒(Flu V).结果 824例中病原抗体检测阳性为RSV 182例(22.1%),AdV 70例( 8.5%),CBV 37例(4.5%),MP 135例(16.8%),CP 60例(7.3%),混合感染34例,392例病人Flu V阳性35例(8.9%).RSV、AdV的感染<3岁组明显高于>3岁组,Flu V、MP的感染>3岁组明显高于<3岁组. RSV、AdV、Flu V病毒感染自11月至次年4月为流行季节,在3月达高峰,5～10月散发.CBV的感染则呈全年散发.结论病毒是广州地区婴幼儿非细菌性下呼吸道感染的最常见病原.肺炎支原体次之.其中RSV感染占首位,MP有上升的趋势,流感病毒感染无流行迹象.<3岁儿童感染以RSV、AdV为主,>3岁则以Flu V, MP占优.病毒感染多在冬春季节流行.     

慢性阻塞性肺疾病急性加重患者呼吸道病毒的检测分析

目的 探讨呼吸道病毒感染与慢性阻塞性肺疾病急性加重(AECOPD)的相关性.方法 随机选择140例慢性阻塞性肺疾病急性加重(AECOPD)患者,60例健康老年志愿者为对照组,分别检测呼吸道合胞病毒(RSV)、单纯疱疹病毒(HSV)、腺病毒(ADV)、巨细胞病毒(CMV)、副流感病毒(PIV)、流感病毒A/B(FluA/B)特异性抗体IgM水平,对组间阳性率进行比较.结果 AECOPD组患者中IgM阳性率依次为RSV＞PIV＞ FluA/B＞CMV＞ADV＞ HSV.AECOPD组与对照组各病毒抗体阳性率比较差异有统计学意义(P＜0.05).结论 病毒感染是AECOPD重要因素,病毒感染参与了AECOPD病情的进展过程,在呼吸道病毒流行的季节应做好预防工作.     

2021～2022年深圳市某医院常见呼吸道病毒流行病学特点

目的 分析2021年7月至2022年7月在我院就诊的急性呼吸道感染患者呼吸道病毒的感染情况，并比较多重荧光定量PCR法和胶体金法检测流感病毒的诊断效能。方法 收集我院1 263例急性呼吸道感染患者的鼻拭子标本，采用多重荧光定量PCR法进行检测，分析不同性别、年龄、季节6种常见呼吸道病毒的感染特点。随机抽取2022年1月至7月407例患者标本，采用胶体金法检测甲型流感病毒(influenza A virus, Flu A)和乙型流感病毒(influenza B virus, Flu B),并与多重荧光定量PCR法检测结果进行比较。结果 1 263例患者中，呼吸道病毒阳性检出率为21.54%(272/1 263),呼吸道合胞病毒(respiratory syncytial virus, RSV)阳性检出率最高，为8.55%(108/1 263)。不同性别患者阳性检出率差异无统计学意义(P>0.05)。秋季呼吸道病毒阳性检出率最高(P<0.001),其中RSV在秋季检出率最高，Flu A在夏季检出率最高、Flu B在春季检出率最高、副流感病毒Ⅰ型(parainfluenza vi...     

呼吸道合胞病毒黏附糖蛋白基因新变异形式的发现及其感染患儿临床特征分析

目的分析北京地区呼吸道合胞病毒(respiratory syncytial virus, RSV)流行特征, 监测黏附糖蛋白(G)基因序列变异及感染患儿临床特征。方法收集2023年1月1日—2023年12月31日首都儿科研究所附属儿童医院急性呼吸道感染患儿呼吸道标本, 对经呼吸道病原体多重核酸检测确定为RSV阳性的样本, 进一步通过PCR方法扩增得到RSV G蛋白基因全长, 测序后建立系统发育进化树确定RSV分型及G蛋白序列变异, 通过电子病历系统获得临床资料, 分析北京地区RSV感染患儿临床特征。结果共收集5 489份急性呼吸道感染患儿呼吸道标本, 其中男3 046例, 女2 443例, 平均年龄4.36岁。核酸检测确定为RSV阳性的589例(10.7%, 589/5 489), 其中男349例, 女240例, 平均年龄(2.51±2.78)岁, 中位年龄0.48岁。2023年3月开始RSV呈现持续流行趋势, 存在两个流行高峰, 分别为5月(24.6%, 122/496)和12月(18.2%, 126/693)。2023年7月前以A亚型为优势亚型, 8—10月为两个亚型博弈阶段, 1...     

Xpert Mtb/RIF检测与BACTEC MGIT960结核培养药敏联合检测在结核病诊治中的应用分析

目的评估Xpert Mtb/RIF检测与BACTEC MGIT 960结核培养药敏联合检测在结核病诊治中的应用价值。方法对临床结核病患者的标本进行Xpert Mtb/RIF检测,同时进行BACTEC MGIT 960结核培养、鉴定、药敏试验。以BACTEC MGIT 960结核培养及药敏试验为金标准,分析Xpert Mtb/RIF检测的敏感性和特异性。结果以BACTEC MGIT 960结核培养及药敏试验为金标准,Xpert Mtb/RIF检测结核分枝杆菌的敏感性为87.7%(100/114),Xpert Mtb/RIF检测利福平耐药的敏感性为90%(45/50)、特异性为91.3%(53/58)。结论 Xpert Mtb/RIF检测敏感性及特异性高, BACTEC MGIT 960结核培养及药敏试验联合应用有助于及早进行有效诊治。     

Xpert MTB/RIF系统在盆腔结核诊断中的应用价值分析

目的探讨Xpert MTB/RIF系统检测盆腔积液中结核分枝杆菌对于盆腔结核的诊断价值。方法采集30例临床确诊为盆腔结核患者及30例盆腔恶性肿瘤患者(对照组)的盆腔积液,对积液同时采用离心涂片法、荧光定量PCR法和Xpert MTB/RIF系统进行结核分枝杆菌检测,比较三种方法的敏感性及特异性。结果离心涂片法、荧光定量PCR法和Xpert MTB/RIF系统检测结核分枝杆菌的敏感性分别为6.67%(2/30)、53.33%(16/30)和73.33%(22/30),三种方法间差异均有统计学意义(P均<0.05)。三种方法特异度分别为100.0%,96.7%,100.0%,差异无统计学意义。结论采用Xpert MTB/RIF系统对盆腔积液进行结核分枝杆菌检测敏感性高,特异度好,具有较高的临床应用价值。     

液相芯片技术与实时荧光定量PCR在检测儿童感染性腹泻病原体的比较

目的研究液相芯片检测儿童感染性腹泻病原体的效率,为感染性腹泻病的临床快速诊断提供依据。方法收集96例婴幼儿及儿童腹泻患者粪便样本,运用Luminex液相芯片进行检测,并与实时荧光定量PCR检测结果比较。结果液相芯片技术共检测出7种病原体,阳性检出率为65.63%(63/96),其中沙门氏菌的检出率最高,为50.00%(48/96);单病原感染的阳性率为39.58%(38/96),混合感染的阳性率为26.04%(25/96)。与实时荧光定量PCR相比,液相芯片检测腹泻病原体的敏感度和特异度分别为100.00%和71.74%,阴性预测值和阳性预测值分别为100.00%和79.37%(P<0.05)。结论与实时荧光定量PCR相比,液相芯片技术具有高通量、快速、可靠等特点,有利于感染性腹泻病原体的临床检测。     

Xpert结核分枝杆菌/利福平试验对结核病及耐多药结核病诊断价值的Meta分析

目的 评估Xpert结核分枝杆菌/利福平（MTB/RIF）试验对结核病的诊断价值.方法 检索PubMed、Medline、中国知网、万方数据库等,收集Xpert MTB/RIF试验对结核病诊断价值的文献,检索起止时间均为建库至2012年6月.2名研究者独立进行资料提取和文献质量评估.采用Meta-Disc 1.4软件进行Meta分析.结果 共纳入26篇文献,其中2篇文献涉及儿童病例,包含了13 270例来自临床患者的检测标本.Meta分析结果显示,Xpert MTB/RIF试验诊断结核病的汇总敏感度为87%（95%CI：86%～88%）、特异度为97%（95%CI：97%～97%）.按照结核病的类型和患者年龄进行亚组分析,Xpert MTB/RIF试验诊断肺结核的敏感度高于肺外结核病,90%（95%CI：89%～91%） vs 76%（95%CI：72%～79%）;诊断涂阴菌阳性和涂阳菌阳性结核病的敏感度分别为74%（95%CI：71%～76%）和99%（95%CI：98%～99%）;对儿童肺结核的诊断敏感度比成人肺结核低,74%（95%CI：65%～83%） vs 90%（95%CI：89%～92%）.Xpert MTB/RIF试验诊断耐多药结核病的敏感度为96%（95%CI：94%～97%）,特异度为98%（95%CI：98%～99%）.结论 Xpert MTB/RIF试验诊断结核病的价值较高,尤其是成人结核病及耐多药结核病.Xpert MTB/RIF试验在儿童结核病中的诊断价值由于纳入文献较少,尚待进一步研究.     

乌鲁木齐地区冬春季小儿呼吸道感染者中呼吸道合胞病毒感染的研究

目的 了解乌鲁木齐地区冬春季节呼吸道感染患儿中呼吸道合胞病毒(RSV)的感染状况以及分子流行病学的基本情况.方法 研究对象为2006年11月-2007年4月于新疆维吾尔自治区人民医院儿科住院以及部分于门诊就诊,明确诊断为急性呼吸道感染的患儿,采集咽拭子280份,呼吸道分泌物112份.对全部标本采用反转录聚合酶链反应(RT-PCR)方法进行RSV及其亚型检测.随机取5份阳性标本测序,进行序列比较及同源性分析.结果 392份样本中共检出RSV阳性68份,阳性率17.3%(68/392),其中A基因型64份,占93.3%(64/68),B基因型4份,占6.7%(4/68).5株测序结果提示当地RSV与其他国家及地区代表株之间的同源性为63.1%～99.4%.进化树进一步显示存在A、B两个亚型.结论 RSV是引起2006-2007年冬春季该医院儿童急性呼吸道感染的重要病原体,以A亚型为主要流行株.

Abstract：

Objective To research the infections of respiratory syneytial virus(RSV)in children with respiratory tract inflammation and define its molecular epidemic features in Urumchi.Methods SamDles were collected from November 2006 to April 2007 in the People's General Hospital of Xinjiang Uygur Autonomous Region,including 112 respiratory secretions and 280 nasopharyngeal swabs. RSV and its subgroups were detected by nested PCR.The five positive amplicons selected randomly from all positive samples were sequenced and compared with other RSV in GenBank by BLAST and DNAStar.Results of all 392specimens.68 RSV G gene segments were tested.Among them,RSV lineage A occupied 93.3%,while B occuDied 6.7%.The identities between them were 63.1%-99.4%.Phylogenetic analysis defined that they belonged to two different clusters.Conclusion RSV was one of the important viruses leading to children's respiratory tract infections in the People's General Hospital of Xinjiang Uygur Autonomous Region during winter and spring from 2006 to 2007.RSV subtype A was the prevalent genotype in the hospital dunng this epidemics.     

A novel PCR-based point-of-care method facilitates rapid,efficient, and sensitive diagnosis of influenza virus infection

ObjectiveThe aim of this single-centre study was the comparative analysis of the GeneXpert (Cepheid Inc.) and the LIAT (Roche) system for the rapid polymerase chain reaction (PCR)-based detection of influenza A (IA) and influenza B (IB) viruses.Patients and methodsDuring the 2017–2018 flu season, 651 prospectively collected samples (throat and nasal swabs) of patients with symptoms of influenza-like illness or acute respiratory infection were tested for the presence of IA and IB viruses using the GeneXpert and LIAT systems. To evaluate the usefulness for near-patient testing, a LIAT system was installed at the Department of Emergency Medicine, and sample testing was performed on site. Reference testing of all samples was performed with the Xpert Flu assay and for 313 samples in addition with the Xpert Xpress Flu/RSV (respiratory syncytial virus) assay at the central laboratory. Analysis of all samples was carried out within 24 hr after collection.ResultsOverall, 267 of the 651 samples analysed were positive for influenza viruses in at least one of the three assays investigated (IA, 88; IB, 179). The overall rates of agreement between the LIAT assay and the Xpert Flu assay was 96.0% for the detection of IA and IB viruses. The sensitivity and specificity of the LIAT assay compared to the Xpert Flu assay for the detection of IA was 98.80% (95% confidence interval (CI) 93.47–99.97%) and 99.12% (95% CI, 97.96% to 99.71%) and for the detection of IB 98.76% (95% CI 95.58–99.85%), and 96.33% (95% CI 94.26–97.81%), respectively. The LIAT assay showed a statistically significant higher detection rate of IB virus than the Xpert Flu assay (p <0.01). No significant difference was found between the detection rate of the LIAT assay and the Xpert Xpress Flu/RSV assay. The mean time to the availability of a definite test result was significantly shorter with the on-site LIAT system than the GeneXpert system (mean 59 min saving time; p <0.01).ConclusionThe LIAT system represents a robust and highly sensitive point-of-care device for the rapid PCR-based detection of influenza A and influenza B viruses.     

北京甲3流感流行中合并其它呼吸道病毒感染的调查

为获得北京甲３流感暴发中其它主要呼吸道病毒介入和血清学依据。方法应用系列呼吸道病毒桥联酶标试剂盒对２０４例患者分泌物进行多病毒抗原检测;应用间接免疫荧光法对流行前人群５０例和流行后患者恢复期血清４１例,分别作呼吸道合胞病毒;副流感病毒３型以及腺病毒７型的血清学检测。     

Performance Characteristics of Xpert Flu/RSV XC Assay

The Xpert Flu/RSV XC assay was compared to laboratory-developed tests (LDTs) (n = 207) and the Xpert Flu assay (n = 147) using archived nasopharyngeal swabs. The percentages of positive agreements with LDTs were 97.8% for influenza A, 97.2% for influenza B, and 89.3% for RSV. The sensitivity of influenza detection was improved with the Xpert Flu/RSV XC assay compared to the Xpert Flu assay.     

Evaluation of the Cepheid Xpert Flu Assay for rapid identification and differentiation of influenza A, influenza A 2009 H1N1, and influenza B viruses

The Xpert Flu Assay cartridge is a next-generation nucleic acid amplification system that provides multiplexed PCR detection of the influenza A, influenza A 2009 H1N1, and influenza B viruses in approximately 70 min with minimal hands-on time. Six laboratories participated in a clinical trial comparing the results of the new Cepheid Xpert Flu Assay to those of culture or real-time PCR with archived and prospectively collected nasal aspirate-wash (NA-W) specimens and nasopharyngeal (NP) swabs from children and adults. Discrepant results were resolved by DNA sequence analysis. After discrepant-result analysis, the sensitivities of the Xpert Flu Assay for prospective NA-W specimens containing the influenza A, influenza A 2009 H1N1, and influenza B viruses compared to those of culture were 90.0%, 100%, and 100%, respectively, while the sensitivities of the assay for prospective NP swabs compared to those of culture were 100%, 100%, and 100%, respectively. The sensitivities of the Xpert Flu Assay for archived NA-W specimens compared to those of Gen-Probe ProFlu+ PCR for the influenza A, influenza A 2009 H1N1, and influenza B viruses were 99.4%, 98.4%, and 100%, respectively, while the sensitivities of the Xpert Flu Assay for archived NP swabs compared to those of ProFlu+ were 98.1%, 100%, and 93.8%, respectively. The sensitivities of the Xpert Flu Assay with archived NP specimens compared to those of culture for the three targets were 97.5%, 100%, and 93.8%, respectively. We conclude that the Cepheid Xpert Flu Assay is an accurate and rapid method that is suitable for on-demand testing for influenza viral infection.     

Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15

The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, n = 563 patients) and the Xpert Flu/RSV XC assay (cohort 3, n = 102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9 % (cohort 1), 95 % (cohort 2), and 98 % (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7 % and 100 % and specificities of 100 % and 94.1 %, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3 %. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary.     

Retrospective and prospective verification of the Cepheid Xpert influenza virus assay

We performed a retrospective (n = 121) and prospective (n = 305) verification of the Cepheid Xpert Flu assay to determine its performance characteristics. The overall sensitivity and specificity were 93% and 100%, respectively. Nasopharyngeal specimen sensitivities were 100% for seasonal influenza A/H1 virus and influenza A/H3 virus, 90% for influenza A/2009/H1N1 virus, and 95% for influenza B virus.     

Inhibition of influenza A virus replication by influenza B virus nucleoprotein: an insight into interference between influenza A and B viruses

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP_FluB) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP_FluB suggest that functional NP_FluB is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP_FluB. Further analysis of NP_FluB indicated that it does not affect nuclear import of NP_FluA. Taken together, these findings suggest a novel role of NP_FluB in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.     

Evaluation of the Xpert Flu test and comparison with in-house real-time RT-PCR assays for detection of influenza virus from 2008 to 2011 in Marseille,France

Rapid documentation of respiratory specimens can have an impact on the management of patients and their relatives in terms of preventive and curative measures. We compared the results of the Xpert® Flu assay (Cepheid) with three real-time RT-PCR assays using 127 nasopharyngeal samples, of which 75 were positive for influenza A (with 52 identified as A/HINI-2009) and 52 were positive for influenza B. The Xpert® Flu assay presented a quasi-absence of non-interpretable tests, and showed sensitivity and specificity of 100% and 100% for Flu A, 98.4% and 100% for A/HINI-2009, and 80.7% and 100% for Flu B.     

SARS-CoV-2 N gene dropout and N gene Ct value shift as indicator for the presence of B.1.1.7 lineage in a commercial multiplex PCR assay

ObjectivesDetection and surveillance of SARS-CoV-2 is of eminent importance, particularly due to the rapid emergence of variants of concern (VOCs). In this study we evaluated if a commercially available quantitative real-time PCR (qRT-PCR) assay can identify SARS-CoV-2 B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared with the S or RdRp gene.MethodsVOC B.1.1.7 and non-B.1.1.7 SARS-CoV-2-positive patient samples were identified via whole-genome sequencing and variant-specific PCR. Confirmed B.1.1.7 (n = 48) and non-B.1.1.7 samples (n = 58) were analysed using the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay for presence of SARS-CoV-2 S, RdRp and N genes. The N gene coding sequence of SARS-CoV-2 with and without the D3L mutation (specific for B.1.1.7) was cloned into pCR™II-TOPO™ vectors to validate polymorphism-dependent N gene dropout with the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay.ResultsAll studied B.1.1.7-positive patient samples showed significantly higher Ct values in qRT-PCR (Δ6–10, N gene dropout on Ct values > 29) of N gene than the corresponding values of S (p ≤ 0.0001) and RdRp (p ≤ 0.0001) genes. The assay reliably discriminated B.1.1.7 and non-B.1.1.7 positive samples (area under the curve = 1) in a receiver operating characteristic curve analysis. Identical Ct value shifts (Δ7–10) were detected in reverse genetic experiments, using isolated plasmids containing N gene coding sequences corresponding to D3 or 3L variants.DiscussionAn N gene dropout or Ct value shift is shown for B.1.1.7-positive samples in the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.