254例急性呼吸道感染病例病原学调查分析

目的 分析急性呼吸道感染病例的病毒病原构成,掌握主要病原体活动水平及变化规律,了解丰台地区急性呼吸道感染病原谱.方法 以2010-2012年成人急性呼吸道感染病例为研究对象,收集病例基础信息,采集鼻咽拭子标本,使用多重PCR及RT-PCR对流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、偏肺病毒、冠状病毒、鼻病毒、博卡病毒和肺炎支原体等9种病原进行检测,数据分析使用SPSS 17.0软件.结果 254例标本中154例检测阳性,阳性率为60.63％,男女阳性检出率无统计学差异.冬春季致病病原体以流感病毒、副流感病毒及支原体为主,夏季仅检出副流感和支原体.流感样病例的主要病原体为流感病毒及副流感病毒;肺炎病例致病病原体主要为肺炎支原体.2010-2011年流感流行季甲型流感病毒为优势病原,2011-2012年流感流行季乙型流感病毒为优势病原.结论 2010-2012年间,北京丰台地区引起成人急性呼吸道感染的主要病原为流感病毒、支原体和副流感病毒.不同季节的优势病原不同.     

多重实时荧光定量PCR对9种常见呼吸道病原体检测

目的探讨多重实时荧光定量PCR(MRT-PCR)检测9种常见呼吸道病原体的临床应用价值。方法采用Primer Express3.0(ABI)和Primer3 Input 4.0设计引物和探针建立MRT-PCR法,通过构建质粒标准品分析该方法的最低检出限和特异性。并对204例临床标本中副流感病毒1、2、3型、肺炎支原体、肺炎衣原体、呼吸道合胞病毒、腺病毒、甲型流感病毒、乙型流感病毒进行回顾性检测。结果 MRT-PCR法检测病原体的最低检出限达103copies/m L,特异性达100%,无交叉反应。204例咽拭子标本中,MRT-PCR检出呼吸道合胞病毒23例,副流感病毒1、2、3型13例,腺病毒15例,肺炎支原体15例,肺炎衣原体3例,甲型流感病毒13例与乙型流感病毒6例。该结果与其他试剂报告结果完全一致。结论多重实时荧光定量PCR具有快速、准确、特异性强等特点,在呼吸道病原体检测方面有重要价值。     

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。     

多重核酸检测在儿童下呼吸道感染病原学诊断中的应用分析

目的对临床诊断为下呼吸道感染(支气管肺炎、大叶性肺炎、支气管炎、毛细支气管炎)的住院患儿采集深部痰液进行细菌培养及咽拭子进行多重RT-PCR和毛细电泳联用技术,检测呼吸道常见病原(呼吸道合胞病毒、甲型流感病毒、乙型流感病毒、副流感病毒、鼻病毒、偏肺病毒、冠状病毒和腺病毒、博卡病毒、肺炎支原体、衣原体),分析多重PCR检测技术在儿童下呼吸道感染病原学诊断的临床应用价值。方法收集2018年12月至2019年2月在成都儿童专科医院住院的155例下呼吸道感染患儿的鼻咽分泌物及深部痰液标本,年龄在1月～14岁,病程在1～60天。结果本组患者男93例(60%),女62例(40%),平均发病时间为7.2天,≤7天94例,7天61例,1岁组39例(25.2%),1～3岁组42例(27.1%),3～5岁组41例(26.5%),5岁33例(21.3%),平均年龄3.1岁。多重核酸检测结果发现,155例患儿的鼻咽分泌物标本中至少有一种病原检测阳性的共计143份标本(占92.3%),其中有40份标本(占27.97%)检测到两种或两种以上病原。排名前三位的病原依次为、呼吸道合胞病毒、甲型流感病毒和偏肺病毒。按照不同年龄组病原阳性率分析,除呼吸道合胞病毒外(χ2=29.241,P0.001),其余病毒组间差异无统计学意义。通过多重PCR检查阳性标本中混合细菌感染者占57例共计59株(36.77%),各年龄组混合感染差异无统计学意义,其中卡他布兰汉菌28株(47.46%),流感嗜血杆菌18株(30.51%),肺炎链球菌9株(15.25%),金黄色葡萄球菌3株(5.08%),大肠埃希菌1株(1.70%);根据发病时间分为≤7天和7天组,核酸检测阳性结果≤7天组91例(58.71%),与7天组阳性例数52例(33.54%)(χ2=5.4,P=0.020.05)差异具有统计学意义。结论呼吸道合胞病毒是本院冬春季3岁以下儿童下呼吸道感染主要病原,偏肺病毒、流感病毒是5岁以下儿童该时期下呼吸道感染的主要病原,支原体感染仍多见于5岁以上儿童;和痰培养比较,进行多重PCR病原检测有助于全面了解儿童下呼吸道感染的病原,发病早期(≤7天)检测阳性率更高。     

石家庄市不同人群呼吸道感染病原体调查分析

目的 了解本地区急性呼吸道感染患者8种呼吸道病原体的流行情况.方法 选取2014年1月1日至2015年12月31日在石家庄市第一人民医院诊治的7545例呼吸道感染患者,对血清进行8种常见呼吸道病原体的IgM抗体检测.结果 本地区2014年检出率较高的呼吸道病原体依次是流感病毒B(17.5％)、流感病毒A(12.9％)、肺炎衣原体(11.8％),2015年检出率较高的呼吸道病原体依次是流感病毒B(20.6％)、肺炎支原体(20.0％)、流感病毒A(11.0％).2015年较2014年流感病毒A、流感病毒B、肺炎衣原体、嗜肺军团菌4种病原体IgM抗体阳性率有所降低,肺炎支原体IgM抗体阳性率有所升高;各年龄组八种病原体抗体阳性率均存在差异,与0～3岁组比较4～15岁组肺炎衣原体、流感病毒A、流感病毒B、副流感病毒、嗜肺军团菌、呼吸道合胞病毒IgM抗体阳性率较高,15岁以上组腺病毒、嗜肺军团菌、呼吸道合胞病毒IgM抗体阳性率较高;与4～15岁组比较15岁以上组腺病毒、嗜肺军团菌IgM抗体阳性率较高;且秋冬季与春夏季比较流感病毒A、流感病毒B、副流感病毒IgM抗体阳性率较高,而肺炎支原体IgM抗体阳性率较低.结论 本地区呼吸道感染的病原体以流感病毒A(INFA)、流感病毒B、肺炎支原体为主,不同年份、不同季节、不同年龄组病原体的种类和感染率具有一定差异性.     

儿童社区获得性肺炎血清免疫学病毒特异性抗体检测与呼吸道病毒核酸检测结果一致性分析

目的:探讨儿童社区获得性肺炎的病毒血清特异性抗体检测与呼吸道病毒核酸检测结果的一致性,以指导其临床应用。方法:109例经鼻咽拭子标本实时荧光定量PCR检测诊断为病毒感染的社区获得性肺炎患儿,采集其急性期和恢复期血清标本,采用间接免疫荧光法检测呼吸道常见病毒(呼吸道合胞病毒、腺病毒、甲型流感病毒、乙型流感病毒和副流感病毒...     

自动巢式多重PCR系统在呼吸道感染病例中快速检测呼吸道病原体的应用

目的 探讨自动巢式多重PCR系统在呼吸道感染病原体快速诊断中的应用价值.方法 2016年11月至2017年3月收集120例呼吸道感染患者的呼吸道标本和临床流行病学资料,利用自动巢式多重PCR系统对120份鼻咽拭子标本进行病原检测,对检测结果和临床资料进行统计学分析.结果 120份标本中单一病原检出68份,2种及2种以上病原混合检出22份,总检出率为75.0％(90/120),检出结果中以甲型流感病毒、呼吸道合胞病毒、百日咳杆菌为主.结论 自动巢式多重PCR系统敏感度高、特异性强,对呼吸道感染患者的病原学快速诊断具有重要价值.     

九项呼吸道病原体IgM检测500例分析

上呼吸道病原体90％以上为病毒,其次为支原体、衣原体和细菌等.呼吸道感染起病较急,进展快,需及时明确病原微生物种类,以便临床进行针对性治疗.使用9项呼吸道病原体IgM方法可检测的病原体包括嗜肺军团菌(LP-TS)、肺炎支原体(MP-TS)、Q热立克次体(QF-TS)、肺炎衣原体(CP-TS)、腺病毒(AD-TS)、呼吸道合胞病毒(RS-TS)、甲型流感病毒(FA-TS)、乙型流感病毒(FB-TS)以及副流感病毒(PIV-TS).现将检测的以上呼吸道感染为主要症状就诊的500例患者血清学结果报道如下.     

呼吸道合胞病毒荧光纳米颗粒快速检测试纸条检测效能评价

目的评估呼吸道合胞病毒(RSV)荧光纳米颗粒快速检测试纸条的灵敏度和特异性等检测效能。方法以培养的RSV和其他呼吸道病原体以及280例具有上呼吸道感染症状的患者作为标本来源。分别anjiang采用RSV荧光纳米颗粒快速检测试纸条和胶体金快速检测试纸条检测同一份标本,分析两种检测试纸条的检测效能。结果荧光纳米颗粒快速检测试纸条的灵敏度高于胶体金快速检测试纸条;对甲型流感病毒、乙型流感病毒、副流感病毒、呼吸道腺病毒和肺炎支原体无特异性反应。结论 RSV荧光纳米颗粒快速检测试纸条在检测性能上高于胶体金快速检测试纸条。     

九项呼吸道联检试剂对多种呼吸道感染病原体检测的临床意义

目的 为临床提供一种快速诊断呼吸道感染病原体的方法.方法 对1318例呼吸道感染患者的血清标本应用九项呼吸道联检试剂(间接免疫荧光法)同时检测九项主要病原体的IgM抗体,包括嗜肺军团菌(LP)、肺炎支原体(MP)、Q热立克次体(COX)、肺炎衣原体(CP)、腺病毒(ADV)、呼吸道合胞病毒(RSV)、甲型流感病毒(IFA)、乙型流感病毒(IFB)和副流感病毒1、2和3型(PIVS).结果 非典型病原体感染率为33.3％(439/1318),其中以肺炎支原体最为多见,其次为呼吸道合胞病毒,流感病毒B、A次之;混合感染率达15.7％(207/1318);感染人群以儿童多见.结论 九项呼吸道联检试剂(间接免疫荧光法)检测快速、操作简便、准确性高,检测范围广、利于早期发现,费用不高,适合各大医院推广应用.     

High-throughput, sensitive, and accurate multiplex PCR-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses

Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.     

Non-invasive sample collection for respiratory virus testing by multiplex PCR

Background

Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods.

Objective

Evaluate the utility of non-invasive sample collection using anterior nare swabs and facial tissues for respiratory virus detection by multiplex PCR.

Study design

Children aged 1 month–17 years evaluated in a pediatric emergency department for respiratory symptoms had a swab, facial tissue, and NPA sample collected. All samples were tested for respiratory viruses by multiplex PCR. Viral detection rates were calculated for each collection method. Sensitivity and specificity of swabs and facial tissues were calculated using NPA as the gold standard.

Results

285 samples from 95 children were evaluated (92 swab-NPA pairs, 91 facial tissue-NPA pairs). 91% of NPA, 82% of swab, and 77% of tissue samples were positive for ≥ 1 virus. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were most common. Overall, swabs were positive for 74% of virus infections, and facial tissues were positive for 58%. Sensitivity ranged from 17 to 94% for swabs and 33 to 84% for tissues. Sensitivity was highest for RSV (94% swabs and 84% tissues). Specificity was ≥95% for all viruses except HRV for both collection methods.

Conclusions

Sensitivity of anterior nare swabs and facial tissues in the detection of respiratory viruses by multiplex PCR varied by virus type. Given its simplicity and specificity, non-invasive sampling for PCR testing may be useful for conducting epidemiologic or surveillance studies in settings where invasive testing is impractical or not feasible.     

Viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients

Syndromic diagnosis by multiplex nucleic acid amplification tests is the most practical approach to respiratory tract infections since the symptoms are rarely agent-specific. The aim of this study was to investigate the respiratory viruses in children admitted to a university hospital with acute respiratory tract infection during the last 8 years by a multiplex polymerase chain reaction (PCR) assay. A total of 3162 respiratory samples collected from children between April 2011 and April 2018 tested by a multiplex real-time PCR assay. Two different commercial assays were used during the study period, "AusDiagnostics/Respiratory Pathogens 12 (AusDiagnostics)" used between April 2011 and December 2015, which changed to "Fast Track Diagnostics/Respiratory Pathogens 21 (Fast Track Diagnostics)" after January 2016 to cover more viruses. Nucleic acid extraction was done by EZ1 Advanced XL platform (QIAGEN). Respiratory pathogens detected in 1857 of the 3162 (58.7%) samples. The most prevalent viruses during the 8-year period were rhinovirus/enterovirus (RV/EV; 36.2%), respiratory syncytial virus (RSV; 19%), and influenza virus A/B (14.7%). Rhinovirus was the main contributor to the RV/EV group as shown by the assay used during the 2016-2018 period. RV/EV and adenoviruses detected throughout the year. Influenza virus was most frequently detected during January to March when both RSV and metapneumovirus were also in circulation. The coinfection percentage was 10.2%. Rhinovirus was the most common virus in coinfections while RSV plus rhinovirus/enterovirus were the most frequent combination. RSV and metapneumovirus showed a similar seasonal distribution to the influenza virus, which made it necessary to use a virological diagnostic assay during the influenza season.     

High incidence of rhinovirus infection in children with community-acquired pneumonia from a city in the Brazilian pre-Amazon region

Community-acquired pneumonia (CAP) is the leading cause of child death worldwide. Viruses are the most common pathogens associated with CAP in children, but their incidence varies greatly. This study investigated the presence of respiratory syncytial virus (RSV), adenovirus, human rhinovirus (HRV), human metapneumovirus (HMPV), human coronavirus (HCoV-OC43 and HCoV-NL63), and influenza A virus (FluA) in children with CAP and the contributing risk factors. Here, children with acute respiratory infections were screened by pediatrics; and a total of 150 radiographically-confirmed CAP patients (aged 3 months to 10 years) from two clinical centers in Sao Luis, Brazil were recruited. Patient's clinical and epidemiological data were recorded. Nasopharyngeal swab and tracheal aspirate samples were collected to extract viral nucleic acid. RSV, adenovirus, rhinovirus, FluA, HMPV, HCoV-OC43, and HCoV-NL63 were detected by real-time polymerase chain reaction. The severe CAP was associated with ages between 3 and 12 months. Viruses were detected in 43% of CAP patients. Rhinovirus infections were the most frequently identified (68%). RSV, adenovirus, FluA, and coinfections were identified in 14%, 14%, 5%, and 15% of children with viral infection, respectively. Rhinovirus was associated with nonsevere CAP (P = .014); RSV, FluA, and coinfections were associated with severe CAP (P < .05). New strategies for prevention and treatment of viral respiratory infections, mainly rhinovirus and RSV infections, are necessary.     

Clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two common viral pathogens in acute lower respiratory tract infections (ALRTI). However, the association of viral load with clinical characteristics is not well‐defined in ALRTI. To explore the correlation between viral load and clinical characteristics of RSV and HMPV in children hospitalized for ALRTI in Lanzhou, China. Three hundred and eighty‐seven children hospitalized for ALRTI were enrolled. Nasopharyngeal aspirates (NPAs) were sampled from each children. Real‐time PCR was used to screen RSV, HMPV, and twelve additional respiratory viruses. Bronchiolitis was the leading diagnoses both in RSV and HMPV positive patients. A significantly greater frequency of wheezing (52% vs. 33.52%, P = 0.000) was noted in RSV positive and negative patients. The RSV viral load was significant higher in children aged <1 year (P = 0.003), children without fever and wheezing (P = 0.015 and P = 0.000), days of illness <14 days (P = 0.002), children with bronchiolitis (P = 0.012) and children with RSV single infections (P = 0.000). No difference was found in the clinical features of HMPV positive and negative patients. The HMPV viral load had no correlation with any clinical characteristics. The incidences of severe disease were similar between single infection and coinfection for the two viruses (RSV, P = 0.221; HMPV, P = 0.764) and there has no statistical significance between severity and viral load (P = 0.166 and P = 0.721). Bronchiolitis is the most common disease caused by RSV and HMPV. High viral load or co‐infection may be associated with some symptoms but neither has a significant impact on disease severity for the two viruses. J. Med. Virol. 89:589–597, 2017 . © 2016 Wiley Periodicals, Inc.

     

Human metapneumovirus in hospitalized children in Amman,Jordan

Human metapneumovirus (HMPV) has recently been identified as an important cause of acute respiratory infections (ARI) in children worldwide. However, there is little systematic data on its frequency and importance as a cause of ARI in the Middle East. We conducted a viral surveillance study in children <5 years of age admitted with respiratory symptoms and/or fever at two major tertiary care hospitals in Amman, Jordan from 1/18‐3/29/07. Nose and throat swabs were collected and tested for HMPV and other respiratory viruses by real‐time RT‐PCR. A total of 743 subjects were enrolled. Forty‐four (6%) subjects were positive for HMPV, 467 (64%) were positive for RSV and 13 (1.3%) had co‐infection with both HMPV and RSV. The frequency of HMPV in January, February, and March was 4.1%, 3.0%, and 11.9% respectively. Clinical features associated with HMPV infection were similar to those of other respiratory viruses, except children with HMPV were more likely to present with fever than children not infected with HMPV. Children with HMPV and RSV co‐infection were administered supplemental oxygen and were admitted to the ICU more frequently than children infected with HMPV alone or RSV alone, though these differences did not reach statistical significance. We conclude that HMPV is an important cause of acute respiratory infections in children in Amman, Jordan. Longer surveillance studies are needed to better understand the seasonal epidemiology of HMPV and to assess if co‐infection with HMPV and RSV leads to more severe illness. J. Med. Virol. 82:1012–1016, 2010. © 2010 Wiley‐Liss, Inc.     

Seasonality and prevalence of respiratory pathogens detected by multiplex PCR at a tertiary care medical center

Respiratory tract infections (RTIs) are a leading cause of mortality and morbidity. Seasonality has been reported for many viruses, including influenza virus, respiratory syncytial virus (RSV), and the recently described human metapneumovirus (hMPV). We hypothesize that the availability of rapid, multiplex PCR diagnostics will provide better clinical care and new insights into the etiology and clinical spectrum of RTIs. We conducted a retrospective analysis of the incidence of respiratory pathogens at a 500-bed adult and 154-bed pediatric hospital tertiary care center. A total of 939 specimens from patients with an age range of 5 days to 91 years (median, 2 years) were tested by a multiplex respiratory pathogen PCR from November 14, 2011 to November 13, 2012. Sixty-five percent of specimens were positive for at least one pathogen. As the age of the patient increased, the positivity rate for the PCR decreased proportionately. Rhinoviruses/enteroviruses (Rhino/Entero) were the most prevalent (34.3 %) followed by RSV (19.2 %) and hMPV (6.2 %). Twelve percent of the positive samples were positive for multiple analytes, with Rhino/Entero and RSV being the most common combination. The peak months were September and May for Rhino/Entero infections, January for RSV and February for coronavirus. hMPV peaked 2 months after RSV, as has been observed recently in other studies. Multiplex PCR provides rapid diagnostic information that can be used to make knowledgeable clinical decisions and potentially reduce the use of antibiotics. Active respiratory PCR surveillance could also predict seasonal respiratory epidemics to allow for adequate planning of additional infection control measures.