人3型腺病毒荧光定量PCR检测方法的建立

目的通过建立人3型腺病毒荧光定量PCR检测方法,快速鉴定人3型腺病毒感染,为早期诊断提供依据。方法以人3型腺病毒感染的细胞和常见的呼吸道消化道感染的病原体为研究对象,根据病毒六邻体高度保守的序列,设计荧光定量PCR反应体系,并分析实验的敏感性和特异性。结果人3型腺病毒TCID50细胞培养液中病毒核酸最低检出稀释度是10^-6,对应的拷贝数分别是5．802×10^3copies／mL和6．968×10^2copies／mL．呼吸道和消化道感染常见的病原体未出现交叉反应。结论荧光定量PCR的应用,可有效缩短检测时间,提高检测的敏感度。     

多重PCR结合核酸侵入反应及纳米金显色技术检测呼吸道病原体

目的 建立一种呼吸道病原体多重PCR结合核酸侵入反应及纳米金显色的检测方法.方法 针对几种重要的呼吸道病原体(甲型流感病毒、乙型流感病毒、SARS冠状病毒、嗜肺军团菌、脑膜炎奈瑟菌以及腺病毒)保守区基因设计引物,进行多重PCR反应、核酸侵入反应及纳米金显色反应,对多种呼吸道病原体同时进行检测,以人偏肺病毒、呼吸道合胞病毒、人鼻病毒、肺炎链球菌4种呼吸道病原核酸评价其检测特异性,以体外转录的病毒RNA或扩增的PCR片段评价其检测敏感性.结果 成功建立了一种呼吸道病原体多重PCR结合核酸侵入反应及纳米金显色的检测技术.建立的检测方法可特异的检测目的病原体,且与人偏肺病毒、呼吸道合胞病毒、人鼻病毒、肺炎链球菌无交叉反应.该方法对不同靶标的检测灵敏度介0.5 ～50拷贝/μL.结论 建立的呼吸道病原体多重PCR结合核酸侵入反应及纳米金显色技术的检测方法,具有较高的检测特异性及灵敏度,检测通量高,肉眼即可观察结果,在传染病病原体检测方面具有广阔的应用前景.     

人偏肺病毒研究进展

急性呼吸道感染是人类发病和死亡的重要病因之一.2002年WHO调查估计有1830万人死于感染性疾病,其中396万死于呼吸道感染.95%以上是下呼吸道感染.在发达国家病毒性支气管炎是最常见的婴幼儿住院原因.2001年,荷兰学者Van den Hoogen等人[1]分离到一种新的呼吸道病原人偏肺病毒(human Metapneumovirus,hMPV).此后世界各地相继报道并证实人偏肺病毒广泛存在,确定其为小儿下呼吸道感染的病原体,其发病率在小儿下呼吸道感染中约占10%.本文就近五年来的研究报道对人偏肺病毒的病毒学特征及病毒蛋白特性、病毒流行病学、临床表现及诊断方法、动物模型和疫苗的相关研究作一综述.     

兰州地区急性呼吸道感染患儿冠状病毒NL63的检测与临床研究

目的 探讨兰州地区冠状病毒NL63(HCoV-NL63)在急性呼吸道感染患儿中的流行现状及临床特点.方法 收集2006年11月至2009年10月兰州大学第一附属医院急性呼吸道感染(ARTIs)患儿1169例鼻咽分泌物,应用RT-PCR方法检测HCoV-NL63以及其余7种常见呼吸道病毒:鼻病毒(HRV),呼吸道合胞病毒(RSV),偏肺病毒(hMPV),流感病毒(IFVA,IFVB)副流感病毒1-3(HPIV1-3)及PCR方法检测腺病毒(ADV),博卡病毒(HBoV).结果 检测出HCoV-NL63阳性标本35例,检出率2.99％,2007年8、9月,2009年7、8月检测阳性标本阳性率较高,分别为23.53％、17.65％,50％、33.33％.2007年12月至2009年2月未检出HCoV-NL63阳性标本.25(25/35)例混合其他病毒感染,混合感染率为71.43％,最常见的混合感染病毒是HRV.3岁及以下和3岁以上HCoV-NL63感染组感染率差异无统计学意义.HCoV-NL63阳性患儿主要的诊断是支气管肺炎和毛细支气管炎,主要的症状是发热和咳嗽.HCoV-NL63单独感染组和混合感染组除消化道症状外,在其余症状和临床诊断方面,差异均无统计学意义.结论 HCoV-NL63是兰州地区呼吸道感染患儿的重要病原,夏季是兰州地区HCoV-NL63感染高峰期,HCoV-NL63的流行存在年度差异.HCoV-NL63感染存在很高的混合感染率,混合感染并不加重HCoV-NL63感染的病情.     

呼吸道合胞病毒抗原酶联免疫吸附测定检测方法的建立及应用

目的建它呼吸道合胞病毒抗原酶联免疫吸附测定(ELISA)检测方法,应用其对来源于本院的临床样本进行检测.方法建立ELISA方法,检测标本中的呼吸道合胞病毒,与呼吸道合胞病毒细胞培养检测方法进行对照,并利用该方法进行该病毒2007年在广州市的流行性分析.结果本方法的灵敏度略高于呼吸道合胞病毒的细胞培养检测方法,对甲型流感病毒(H3N2亚型、H1N1亚型)、乙型流感病毒、副流感病毒(Ⅰ型、Ⅲ型)、呼吸道腺病毒(Ⅲ型、Ⅶ型)无特异性反应.结论本呼吸道合胞病毒抗原ELISA检测方法可用于呼吸道合胞病毒感染的临床诊断和鉴别诊断.     

多重PCR检测方法和液态芯片技术在呼吸道病毒检测中的应用研究

目的 评估澳大利亚维多利亚感染性疾病参比实验室（The Victorian Infectious Diseases Reference Laboratory,VIDRL）的呼吸道病毒多重PCR检测方法和Luminex公司多指标同步分析液态芯片技术-呼吸道病毒检测试剂盒（xTAG RVP）,应用于多种常见呼吸道病毒检测的优势和缺点,为更好地、有针对性地选择合适技术进行临床实验诊断或公共卫生应急检测提供依据.方法 使用VIDRL的呼吸道病毒多重PCR检测方法和Luminex公司xTAG RVP试剂盒对198份临床流感样症状患者的咽拭子标本进行常见呼吸道病毒的核酸检测.结果 198份临床标本经VIDRL多重PCR方法和xTAG RVP方法检测,阳性率分别为38.89％和45.45％,符合率为72.22％.单一病毒的符合率为87.88％～100％.结论 xTAG RVP方法与VIDRL多重PCR方法相比,两者在常见呼吸道病毒检测中的特异性和敏感性相近,xTAG RVP方法具有明显的快速和高通量的优势.     

Taqman荧光定量RT—PCR在呼吸道感染患者临床标本非SARS冠状病毒检测中的应用

目的建立检测四种常见人非SARS冠状病毒核酸特异的快速、敏感的TaqManqRT—PCR检测方法,应用于急性呼吸道感染患儿的感染分析。方法分别应用TaqManqRT—PCR与普通RT—PCR平行检测248份呼吸道标本,对方法的灵敏性、特异性和稳定性以及临床标本的适用性进行比较评价,阳性标本以体外转录RNA为标准品进行病毒载量定量。结果本方法可对HKU1、NL63、229E、OC43四种冠状病毒进行特异性诊断,与其他病毒无交叉反应,检测灵敏度可达10拷贝／μl,检测线性范围可达10^1～10^8拷贝／μl,248份标本中HKU1、NL63、229E、OC43阳性率依次为1．2％,0．8％,1．2％,1．6％,其中OC43荧光RT—PCR法检出率高于普通RT—PCR,其余三种病毒两种方法检测结果一致。非SARS冠状病毒阳性标本均检出于12月至次年5月。结论建立的TaqManRealtimeRT-PCR法具有特异性强、灵敏性高的特点,是开展非SARS冠状病毒的临床检测与疾病监测的有效技术手段。     

多重实时PCR检测三种呼吸道病毒

目的建立多重实时PCR检测体系可同时快速检测引起人呼吸道感染的甲型流感病毒（IAV）、呼吸道合胞病毒（RSV）和SARS冠状病毒（SARS-CoV）。方法通过对PCR反应条件的优化，建立了同时检测IAV、RSV和SARS-CoV的多重实时PCR方法。结果经熔解曲线分析，证实了该法可同时或分别检测3种重要呼吸道病毒。检测灵敏度分别为10^0.7 TCID50／ml、1 TCID50／ml和10^-0.5 TCID50／ml。采用相同的反应体系和条件，分别对其他7种呼吸道病毒（包括乙型流感病毒，副流感病毒2型，柯萨奇病毒B3和B5血清型及腺病毒2、3和7血清型）进行扩增，均未检测出扩增产物。此种方法可在4h内完成。结论建立的多重实时PCR检测方法适用于3种重要呼吸道病毒的快速检测。     

中国大陆首例人感染高致病性禽流感病毒(H5N1)病例的实验室诊断

目的 对2005年10月湖南省湘潭市湘潭县发生的一名不明原因肺炎病例进行实验室检测,以确定导致该病例的主要病因.方法 采集病例呼吸道标本以及血清标本,对呼吸道标本利用分子鉴别诊断技术以及RT-PCR和实时荧光定量RT-PCR检测病毒核酸;通过血凝抑制试验以及微量中和试验检测血清中的特异性抗体.结果 该病例所有的呼吸道标本H5N1病毒特异性核酸及病毒分离均为阴性.红细胞凝集抑制及微量中和实验显示,恢复期血清较急性期血清H5N1特异性抗体阳转并且有4倍以上增高.结论 通过实验室检测结果分析,该病例为中国大陆第一例人感染高致病性禽流感病毒（H5N1）实验室确诊病例.     

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.     

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.     

Study of influenza C virus infection in France

From November 2004 to April 2007, specimens were obtained from 2,281 patients with acute respiratory tract illness in Normandy, France. Eighteen strains of influenza C virus were detected in these samples using a combined tissue culture/RT-PCR diagnostic method. Most patients with influenza C virus infection (13/18) were infants or young children (<2 years of age). The most frequent symptoms were fever and cough, and the clinical presentation of influenza C virus infection was similar to that of other respiratory viruses. Thirteen of the 18 infected patients were hospitalized; 3 presented with a severe lower respiratory infection. The hemagglutinin-esterase (HE) gene of 10 isolates was sequenced to determine the lineages of the circulating influenza C viruses. Phylogenetic analysis revealed that most of the isolated strains had an HE gene belonging to the C/Yamagata/26/81-related lineage. These results show that influenza C virus regularly circulates in Normandy and generally causes a mild upper respiratory infection. Because the differential clinical diagnosis of influenza C virus infection is not always easy, it is important to identify viral strains for both patient management and epidemiological purposes.     

Differential detection of rhinoviruses and enteroviruses RNA sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis

To define the role of enteroviruses and human rhinoviruses as etiological agents in childhood bronchiolitis, clinical aspirates from 84 infants admitted to hospital with symptoms of obstructive bronchiolitis were tested by picornavirus RT-PCR assay, adenovirus PCR assay and classical immunofluorescence antigen detection of common respiratory viral agents. Respiratory syncytial viruses (A&B) were detectable in 45 of 84 (53.6%) nasopharyngeal aspirates from infants with bronchiolitis, whereas coronaviruses, influenza viruses, and parainfluenza viruses were not detectable in the same samples. Adenoviruses were detectable by PCR in 11 of 84 (13.1%) nasopharyngeal swabs. By using a picornavirus RT-PCR assay followed by a differential molecular hybridisation, rhinovirus and enterovirus RNA sequences were detected in 16 of 84 (19%) and in 10 of 84 (11.9%) of the nasopharyngeal swabs tested. Positive human rhinovirus or enterovirus RT-PCR assay, however, was the only evidence of respiratory infection in 8 of 84 (9.5%) and in 7 of 84 (8.33%) of the studied patients. Respiratory syncytial viruses, human rhinoviruses, adenoviruses, and enteroviruses occur in dual infections detected in 18 of 84 (21.4%) respiratory samples tested. The median duration of stay in hospital was not significantly different between the patients demonstrating a single viral infection and those with a dual viral infection (6.22 +/- 2.07 vs. 5. 04 +/- 0.95 days; P > 0.05). In summary, combination of molecular and classical detection assays of common viruses can be used to demonstrate enterovirus and human rhinovirus respiratory infection in childhood bronchiolitis, and provides an improved approach to obtain new insights into concomitant viral respiratory tract infection in infants.     

Evidence of human metapneumovirus in children in Argentina

Human metapneumovirus (hMPV) is a virus, which was first associated with acute lower respiratory infection in children but is detected currently in all age groups. Clinical symptoms are similar to those described for respiratory syncytial virus (RSV) infections, ranging from mild respiratory illness to severe bronchiolitis and pneumonia in children. To date, no cases of hMPV have been reported in Argentina. In this study, 440 respiratory samples obtained during the period 1998-2002 from children under 5 years old with acute respiratory infection were evaluated. Routine detection for RSV, adenovirus, influenza, and parainfluenza was undertaken by immunofluorescent assay. Of the samples negative for these viruses, only 100 were available. All these samples were tested for hMPV by RT-PCR using primers for the L gene. Eleven out of 100 (11%) respiratory samples were positive for hMPV by RT-PCR. A higher frequency of detection was observed in spring. hMPV was detected in all the years studied, except in 2001. Ten out of 11 children positive for hMPV were hospitalized. Median age was 5 months. Of seven patients, five (71%) required oxygen supplementation. The most frequent diagnosis was bronchiolitis (86%), sometimes accompanied by conjunctivitis and otitis media. The present study showed that hMPV was associated with acute lower respiratory infections in children in Buenos Aires, Argentina. This evidence strongly suggests that hMPV is a common pathogen with a wide geographical distribution, which should be included in the routine diagnosis of respiratory viruses in young children.     

Detection of respiratory viruses in gargle specimens of healthy children

BackgroundRespiratory tract viral infection is one of the most common and important diseases in children. Polymerase chain reaction (PCR) tests are often used to detect viruses in samples, it is difficult to interpret the clinical significance of PCR positivity, which may reflect a past, imminent or active asymptomatic infection due to their high sensitivity. Although single respiratory viruses have been detected in samples from children with symptoms, other respiratory viruses can also be detected simultaneously. However, the clinical importance of these findings for the symptoms is not known.ObjectivesTo investigate the prevalence of respiratory viruses among children without any symptoms such as acute respiratory illness and/or fever.Study designFrom week twenty-five 2013 to week twenty-six 2014, gargle samples were collected from children once a week and these samples were subjected to real-time PCR to detect respiratory viruses. On each sampling day, we asked the parents about their children’s health condition.ResultsAmong the 286 samples collected, 200 were from asymptomatic children. In the asymptomatic condition, human parechovirus, adenovirus, enterovirus, rhinovirus, coronavirus 229E and HKU1 were observed in 45 episodes. In samples from symptomatic children, parainfluenza viruses, respiratory syncytial virus and coronavirus OC43 were detected in addition to those mentioned above.ConclusionsVarious viruses of different species were detected in the specimens from the children regardless of their health status. It might be speculated that host factors such as the function of the immune system influence the clinical outcome of the infection. However, this needs to be studied further.     

High Rates of Detection of Respiratory Viruses in the Nasal Washes and Mucosae of Patients with Chronic Rhinosinusitis

Respiratory viral infections are often implicated as triggers of chronic rhinosinusitis (CRS) flare-ups. However, there is a paucity of respiratory viral surveillance studies in CRS patients, and such studies could elucidate the potential role of viruses in promoting symptoms and aggravating mucosal inflammation. Therefore, a prospective case-control study was conducted to determine the prevalence of respiratory viruses in CRS patients and non-CRS controls. Nasal lavage fluids and turbinate epithelial cells were collected prospectively from 111 CRS patients and 50 controls. Multiplex PCR was used to identify common respiratory viruses in both sample types and the infection rate was compared between groups. Respiratory viruses were detected in 50.5% of lavage samples and in 64.0% of scraping samples from CRS patients. The overall infection rate was significantly different in CRS patients and controls (odds ratio, 2.9 in lavage and 4.1 in scraping samples). Multiple viral infections were detected more frequently in lavage samples from CRS patients than those from controls (P < 0.01; odds ratio, 7.7). Rhinovirus was the most prevalent virus and the only virus with a significantly different infection rate in CRS patients and controls in both samples (odds ratio, 3.2 in lavage and 3.4 in scraping samples). This study detected a higher prevalence of respiratory viruses in CRS patients than controls, suggesting that there may be significant associations between inflammation of CRS and respiratory viruses, particularly rhinovirus. Further studies should investigate the exact role of highly prevalent respiratory viruses in CRS patients during symptomatic aggravation and ongoing mucosal inflammation.     

Clinical evaluation of multiplex real-time PCR panels for rapid detection of respiratory viral infections

Respiratory viral infections are one of the leading causes of morbidity and mortality, particularly in children, the elderly and immunocompromised persons. Rapid identification of viral etiology is critical in ruling out non-viral infections, initiating antiviral treatment and limiting the spread of the infection. Multiplex assays of more than one viral gene target in a single tube have the advantage of rapid screening of a large number of potential viral pathogens in a short time. A multiplex real-time PCR assay was used in this study for detection of respiratory RNA and DNA viral infections in 728 specimens received from 585 adult and pediatric patients comprised of symptomatic and asymptomatic organ transplant recipients and non-recipients for diagnosis of respiratory illnesses and for routine clinical monitoring. Multiplex PCR was more sensitive than the multiplex immunofluoresence culture assay (R-mix) and also detected additional respiratory viruses that were not covered by the R-mix panel. The number of respiratory viruses detected in symptomatic patients was significantly higher than asymptomatic patients in both adult and pediatric patients. Herpesviral infections were the predominant cause of lower respiratory tract infection in the organ transplant recipients, whereas respiratory syncytial virus was the most common pathogen in non-transplant patients particularly children. Multiplex real-time PCR for detection of respiratory viruses has the potential for rapid identification of viral pathogens. In this era of emerging viral infections, addition of newer viral targets to the multiplex PCR panels will be beneficial in determining both patient management and public health epidemiology.     

Seasonal variations of respiratory viruses and etiology of human rhinovirus infection in children

BackgroundUsing the polymerase chain reaction (PCR) method it is possible to detect uncultivable viruses and discover multiple viral infections. However, the clinical importance of these findings in relation to symptoms is not known.ObjectivesThe seasonal fluctuations of respiratory viruses and the clinical outcomes of single infections and dual infections were investigated.Study designNasal aspirate samples were obtained from outpatients and inpatients of a children’s hospital and these samples were subjected to real-time PCR to detect 16 respiratory viruses. Seasonal variations of the 16 viruses and the clinical outcomes such as wheezing, the need for oxygenation and prolonged hospitalization of patients with single viral infections and multiple infections were determined for the 5 most often detected viruses.ResultsAmong 512 specimens analyzed, one or more viruses were detected in 424 (83%) specimens. Two or more viruses were detected in 160 samples (31% of all samples). The epidemic peaks of the viruses did not coincide with each other. Rhinoviruses were the most frequently detected viruses and their coinfection rates were also higher. However, the disease severity in the lower respiratory tract did not differ in most respiratory viral infections regardless of whether there was single infection or dual infection with a rhinovirus and other respiratory virus.ConclusionsSeasonal distribution was seen for each virus. There were no significant differences in clinical symptoms in the children studied. Because the infection of rhinoviruses is the common occurrence in children, it is hypothesized that the factors related to disease severity are mainly the underlying conditions of the children.