直接免疫荧光法在儿童呼吸道感染病毒病原学分析中的应用

<正>呼吸道病毒感染是儿科常见的疾病,引起的症状多变,给临床诊断和治疗造成很大的困难。近年来,分子生物学技术的发展使得越来越多的新病毒被发现,使得呼吸道病毒的检测对于儿童呼吸道感染的诊疗越发重要[1-2]。一般实验室常用的病毒检测方法包括免疫学方法和核酸扩增技术[3]。核酸扩增检测技术有聚合酶链反应(polymerase chain reaction,PCR)、实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)等,其中     

呼吸道病毒特异性IgM检测对儿童呼吸道感染的指导意义

目的:统计分析急性呼吸道感染患儿5种常见呼吸道病毒特异性Ig M的检出情况用以指导临床。方法:2012年1月~12月我院儿科住院急性呼吸道感染患儿1 609例,收集血清采用间接免疫荧光方法检测呼吸道合胞病毒(RSV)、腺病毒(ADV)、甲型流感病毒(IVA)、乙型流感病毒(IVB)、副流感病毒(PIV)特异性Ig M。结果:1 609例患儿检出阳性标本263例(16.35%),IVB检出率最多,共136例(8.45%),其余依次为IVA 63例(3.92%)、PIV 62例(3.85%)、ADV 44例(2.73%)、RSV 42例(2.61%)。不同年龄组患儿阳性标本检出率不同,差异具有统计学意义(χ2=134.822,P0.01),随年龄增长阳性标本检出率逐渐增高,学龄期儿童检查率最高(34.55%);IVB、PIV、IVA阳性标本检出率在不同年龄阶段的比较差异具有统计学意义(χ2分别为102.660、86.145、39.791,均P0.05),IVB和IVA以学龄期儿童检查率为最高,分别达20.00%和8.64%,PIV以学龄前儿童检出率最高(12.07%)。不同呼吸道病毒Ig M阳性检出率在不同月份有各自的特点,IVB在3月和10月出现两个检出高峰,检出率分别达20.15%和11.11%;IVA在3月和5月出现两个检出高峰,检出率分别达10.27%和11.81%;PIV、RSV检出率在1~6月份平缓上升,在3月份检出率最高,分别达7.98%和6.46%,随后检出率又平稳下降;ADV在3、4、5三个月份检出率为最高,检出率达6.19%。结论:5种呼吸道病毒检出阳性率达16.35%,尤以IVB检出率为最高;呼吸道病毒特异性Ig M阳性检出率随年龄增长而逐渐增高,IVB和IVA以学龄期儿童感染率最高,而PIV以学龄前儿童感染率最高;5种呼吸道病毒感染在2012年有各自的流行特点。     

541例呼吸道病毒和病毒抗体检测分析

本文应用ＡＰＡＡＰ方法对５４１例患者的流感病毒Ａ（ＦｌｕＡ）、流感病毒Ｂ（ＦｌｕＢ）、副流感病毒２（ＰＩＶ２）、副流感病毒１．３（ＰＩＶ１．３）、腺病毒３．７（ＡＤＶ３．７）以及合胞病毒（ＲＳＶ）进行了检测,用ＥＬＩＳＡ法检测了７１例患者血清中的抗ＡＤＶ和ＲＳＶ抗体。结果显示呼吸道感染阳性率为４６．６％（２０８／４４６）,非感染性疾病组阳性率为１３．７％（１３／９５）。ＡＤＶ和ＲＳＶ抗原与抗体检测     

儿童呼吸道感染疾病检测降钙素原的临床价值

儿童呼吸道感染是儿科常见的临床疾病,60%以上由病毒引起,呼吸系统病毒通过损伤呼吸道上皮促进气道继发细菌感染.虽然病毒感染是主要原因之一,但儿童感染性疾病鉴别诊断是常见而又棘手的难题,其确诊主要依赖于血培养.     

2018—2020年徐州市儿童流感样病例呼吸道病毒病原学分析

目的 了解徐州市儿童急性呼吸道感染（parainfluenza virus,ARTI）中常见呼吸道病毒包括流感病毒（influenza virus,IFV）、呼吸道合胞病毒（respiratory syncytial virus,RSV）、副流感病毒（parainfluenza virus,PIV）、鼻病毒（human rhinovirus,HRV）、肠道病毒（human enterovirus,HEV）、腺病毒（adenovirus,ADV）、博卡病毒（human bocavirus,HBoV）以及偏肺病毒（metapneumovirus,MPV）的流行病学和病原学特征,为临床诊治及科学防控提供依据。 方法 依托徐州市流感监测哨点医院（徐州市儿童医院）,采用PCR的方法对2018—2020年间共3 140例流感样病例患儿的咽或鼻咽拭子样本核酸进行常见呼吸道病毒检测。 结果 26.59%（835/3 140）急性呼吸道疾病的患儿拭子样本中检出呼吸道病毒核酸,检出率由高到低依次为IFV 9.68%、RSV 5.32%、PIV 5.29%、HRV 3.63%、HEV 1.91%、ADV 1.53%、HBoV 1.02%、MPV 0.29%;病毒混合感染率为7.43%（62/835）;不同年龄段患儿阳性率差异有统计学意义（x²=21.71,P<0.05）,1~<3岁组儿童病毒总检出率最高;12—3月为病毒检出率高峰期;IFV和HEV常作为单一的致病因子感染使儿童发病。 结论 徐州市儿童病毒性急性呼吸道感染主要以IFV、RSV、PIV和HRV为主,不同时间呼吸道病毒检出率不同,流行期也有较大差异,尤其在流感流行期间其他呼吸道病毒常呈低检出率。     

儿童呼吸道合胞病毒感染的研究

呼吸道合胞病毒 (ｒｅｓｐｉｒａｔｏｒｙｓｙｎｃｙｔｉａｌｖｉｒｕｓ ,ＲＳＶ)是婴儿和体弱成人下呼吸道感染的主要病原之一 ,每年在世界范围内流行。ＲＳＶＡ、Ｂ亚型和基因型划分的流行病学意义尚未明确。有关ＲＳＶＡ、Ｂ型在我国流行情况尚未见报道 ,本文对 10年来患ＲＳＶ感染住院儿童ＲＳＶ各亚型、基因型进行分析。材料与方法对象与鼻咽分泌物 (ＮＰＳ)标本 :ＮＰＳ标本取材于北京儿童医院① 1997年 11月～ 1998年 5月 145例ＲＳＶ感染住院患儿 ,年龄从 0 4至 146 8月龄 ,平均8 6月龄 ,男 10 1例 ,女 4 4例 ;② 1999年 1月～ 3…     

一种快速检测巨细胞病毒免疫荧光法

用间接免疫荧光法（IIF）和改良法（改良IIF—第一抗体作用后洗涤,再加一次第一抗体）检测细胞内巨细胞病毒（CMV）抗原。结果表明,改良IIF检出CMV抗原的时间比IIF早,且在病毒感染量较低时,也能查到。文中讨论了改良IIF提高敏感性的可能原因是患者血清中存在着亲和力高、低两类抗体。     

直接免疫荧光检查对SLE等疾病诊断的意义

直接免疫荧光检查对ＳＬＥ等疾病诊断的意义孙智才魏国红张桂芬刘嵘刘艳华（解放军第２５４医院病理科，天津３００１４２）皮肤直接免疫荧光（ＤＩＦ）检查，对红斑狼疮（ＬＥ）和天疱疮等自身免疫性疾病有重要的诊断价值。我们从１９８３年１９９６年，应用ＤＩＦ法对...     

痰热清注射液治疗小儿上呼吸道感染伴发烧的临床疗效观察

目的观察痰热清注射液治疗小儿上呼吸道感染的临床疗效。方法将上呼吸道感染患儿80例随机分为治疗组和对照组,治疗组用痰热清注射液以0.6-0.8ml/kg·d加入5%葡萄糖溶液100-250ml中静滴,对照组用双黄连注射液1-2ml/kg·d加入5%葡萄糖溶液100-250ml中静滴,评定疗效。结果治疗组40例病人中痊愈24例,显效9例,有效6例,总有效率97.5%。结论痰热清注射液治疗上呼吸道感染起效快、效果好、无不良反应、,值得临床推荐和应用。     

An Electrokinetically-Controlled Immunoassay for Simultaneous Detection of Multiple Microbial Antigens

An electrokinetically-controlled heterogeneous immunoassay microchip for multiple analyte detection was developed in this study. Numerical simulation was employed to study the transport process in a microfluidic network (μFN). The operation parameters obtained from numerical simulation was then applied to immunoassay experiment. The effectiveness of the automatic electrokinetic control was demonstrated in a separate experiment using fluorescein dye. The immunoassay microchip was made of poly(dimethylsiloxane)(PDMS)/PDMS-coated glass using soft lithography and replica molding. Multi-antigen immobilization was accomplished by adsorbing the antigen molecules onto a PDMS-coated glass slide and by using a μFN. Immobilized lysate antigen of Escherichia coli O157: H7 at different concentrations was assayed and the lower detect limit was 3 μg/mL. The assay also displayed very good specificity, when different microbial lysate antigens were immobilized, including Escherichia coli and Helicobacter pylori, and the primary and secondary antibodies were mixtures of different species. The time required for the immunoassay, from antigen coating to signal detection, was only one hour. While still an un-optimized prototype, this automatic-operating, high-throughput immunoassay microchip shows a great potential in detecting multiple pathogenic infections efficiently for clinical applications.     

Antibiotic Exposure Prior to Respiratory Viral Infection Is Associated with Progression to Lower Respiratory Tract Disease in Allogeneic Hematopoietic Cell Transplant Recipients

Recent publications note an association between antibiotic exposure and respiratory viral infections (RVIs). Antibiotics affect microbiota and impair immune response against RVIs in mice, and low microbiome diversity is associated with pulmonary complications including viral lower respiratory tract disease (LRTD) in hematopoietic cell transplantation (HCT) recipients. In this study, we examined whether antibiotic exposure was associated with increased risk of disease progression in RVIs post-transplantation. We analyzed patients who underwent allogeneic HCT (June 2008 to February 2016) and had their first RVI due to parainfluenza virus (PIV), respiratory syncytial virus (RSV), or human metapneumovirus (MPV) during the initial 100 days post-transplantation. Antibiotic exposure in the 3 weeks before RVI onset was defined as (1) use of specific antibiotics versus none of these antibiotics and (2) number of antibiotic-days. Cox proportional hazards models were used to examine associations between antibiotic exposures and risk of viral disease progression to proven/probable/possible LRTD. Ninety HCT recipients (84 adults, 6 children) fulfilled study criteria; 33 progressed to LRTD. The number of antibiotic-days was associated with progression to LRTD after adjusting for neutropenia, steroid use, and either lymphopenia (hazard ratio, 1.41 [95% confidence interval, 1.04 to 1.92], P?=?.027) or monocytopenia (hazard ratio, 1.46 [95% confidence interval, 1.11 to 1.91], P?=?.006). Specific antibiotic classes was not associated with the outcome. Cumulative antibiotic exposure immediately before RVI onset is a risk factor for disease progression following PIV, RSV, and MPV infections post-transplantation. Larger cohort studies are needed to determine the impact of specific antibiotics or antibiotic classes on disease severity.     

Comparison of the Luminex Respiratory Virus Panel Fast Assay with In-House Real-Time PCR for Respiratory Viral Infection Diagnosis

The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens.Viral infections of the respiratory tract have traditionally been diagnosed in the laboratory by culture of respiratory specimens and direct fluorescent assay (DFA). However, the availability of real-time PCR has allowed us to detect respiratory viruses with greater sensitivity and shorter turnaround times (12). In recent years, a number of new respiratory viruses have been identified, so we must now consider a wider range of viruses in our diagnoses (see, e.g., references 1 and 14). However, the number of fluorophores that can be differentiated in a multiplex real-time PCR assay limits the number of viral targets that can be detected.One solution is to screen each specimen with several different multiplex real-time PCRs to cover a large number of viruses (4). An alternative, the xTAG respiratory virus panel (RVP) assay (Luminex Molecular Diagnostics Inc., Toronto, Canada), is based on suspension microarray technology, which enables the detection of a large number of targets in a single reaction (6, 9). The xTAG RVP assay has been shown to offer results comparable or superior to those of culture/DFA and nucleic acid tests for the diagnosis of respiratory viral infections (7, 10). Recently, the RVP assay has been used successfully for the detection of etiological agents in outbreaks of respiratory illness (3, 15).The latest version of this test, the RVP Fast assay, has a simpler protocol and a shorter turnaround time than the original assay but still detects 19 different viral and subtype targets: influenza A virus (with additional subtyping: H1, H3, and H5), influenza B virus, respiratory syncytial virus A (RSV-A), RSV-B, parainfluenza virus 1 (PIV-1), PIV-2, PIV-3, PIV-4, adenovirus, human metapneumovirus, coronaviruses 229E, NL63, OC43, and HKU1, enterovirus/rhinovirus (EV/RhV), and human bocavirus. Here we compare the performance of the RVP Fast assay with those of culture/DFA and in-house real-time PCR assays, using respiratory specimens collected for routine viral testing.     

CRP和WBC联合检测在儿童感染性疾病中的应用

目的：探讨CRP和WBC联合检测在儿科感染中的应用。方法对870例儿科急性呼吸道感染儿童进行CRP检测和血细胞分析。结果870例患儿WBC和CRP均升高占17%,WBC升高CRP正常占25%,WBC正常CRP升高占11%,WBC和CRP均正常占47%。结论检测患儿CRP水平对鉴别急性细菌性呼吸道感染与急性病毒性呼吸道感染和抗生素疗效观察具有重要价值,值得临床参考应用[1]。     

儿童真菌感染的病原学研究

目的探讨广州地区儿童真菌感染的病原分布特点及其耐药状况,为防治儿童真菌感染提供实验室依据。方法对患儿感染部位的真菌进行分离培养和鉴定：以ATB^TM FUNGUS3酵母样真菌药敏试验条进行常用抗真菌药物的敏感性分析。结果从患儿标本中分离出558株真菌,主要来自呼吸道有299株,占53．58％;其次是消化道、伤口（创口）、泌尿系统和血液等,分别占28．14％、6．27％、4．66％、3．76％。其中白色假丝酵母菌367株,占65．77％;其次为热带假丝酵母菌、光滑假丝酵母菌、近平滑假丝酵母菌、克柔假丝酵母菌、季也蒙假丝酵母菌等,分别占15．28％、5．02％、4．48％、3．41％、2．69％。从骨髓中检出5株马尔尼菲青霉,从脑脊液中检出3株新型隐球菌。真菌对两性霉素B、5-氟胞嘧啶、氟康唑、伊曲康唑、伏立康唑等总耐药率分别为8．78％、4．84％、10．54％、1．36％、0．85％。结论引起儿童真菌感染的主要病原菌是白色假丝酵母菌。对真菌感染应该有针对性地使用高效的抗真菌药物进行早期治疗。     

Evaluation of the Speed-oligo Direct Mycobacterium tuberculosis Assay for Molecular Detection of Mycobacteria in Clinical Respiratory Specimens

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.     

下呼吸道感染病原菌构成及耐药性分析

目的了解下呼吸道感染病原菌的分布特点及耐药情况,以更好地为临床合理使用抗菌药物提供科学依据。方法对2009年从临床住院患者分离出来的下呼吸道感染病原菌的分布情况及其耐药性进行回顾性调查和分析。结果 2253株阳性菌株中,G-杆菌有1598株,占70.93%,以铜绿假单胞菌（21.88%）、鲍曼不动杆菌（12.16%）、肺炎克雷伯菌（9.19%）为主;G＋球菌有448株,占19.88%,以金黄色葡萄球菌（18.06%）为主;真菌有207株,占9.19%,以白色假丝酵母菌为主（7.81%）。几种主要病原菌对各类抗菌药物的耐药程度各不相同,但大部分菌株均呈现多重耐药的现象。结论应加强细菌的耐药性监测,做好多重耐药菌患者的隔离治疗,加强抗菌药物应用的管理和督查,以减少多重耐药菌的产生和传播。     

病毒感染与逃避免疫反应的研究进展

病毒和宿主共生过程中,病毒为了生存繁衍形成多种逃避宿主免疫反应的方式。其中病毒抗原变异逃逸抗体的中和和病毒干扰补体激活的关键环节来抑制宿主的抗病毒状态是病毒拮抗机体体液免疫反应的主要途径。此外,病毒还利用编码多种细胞因子的类似物干扰细胞因子的正常功能,造成有利于病毒生存复制的内环境,从而使得病毒能够持续感染机体。