Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections

Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex II V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children’s Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system (Qiagen, Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA), FluB, parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), enteroviruses (EnV), human bocaviruses (hBoV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1), and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9–100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%–40.0%) were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H1N1-p and RSV (p=0.011?0.000). The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in NPS specimens in pediatric inpatients at the time of admission. The sensitivity of multiplex RT-PCR was influenced by viral loads, specimen process methods, primer and probe design and amplification condition.     

GeXP多重PCR技术同时检测12种常见呼吸道病毒

本研究建立了一种基于GeXP多重基因表达遗传分析系统的多重RT-PCR检测方法,该方法可以同时检测12种呼吸道病毒,包括流感病毒A型和B型、季节性H1N1、副流感病毒1～3型、人鼻病毒、人偏肺病毒、腺病毒、呼吸道合胞病毒A型和B型、人博卡病毒。针对病原体保守区序列设计12种病毒的特异性引物,分别用已验证的阳性标本为模板检验多重体系的特异性。多重检测体系在10~3拷贝/μL水平可同时检测到12种病毒。另检测24份临床标本,以real-time RT-PCR为参考标准,进一步验证检测体系。结果表明,这种基于GeXP系统的新方法灵敏度高、特异性强,可以快速同时检测12种常见呼吸道病毒。     

五种主要上呼吸道病毒多重RT-PCR 检测方法的建立

目的:建立甲型、乙型流感病毒、呼吸道合胞病毒A型、B型(RSV-A、RSV-B)和腺病毒(ADV)五种主要上呼吸道病毒的多重RT-PCR检测方法。方法:利用Primer premier5.0分别针对甲型流感病毒的M基因、乙型流感病毒的PB1基因、RSV-A和RSV-B的F基因及ADV的hexon基因设计五对特异性引物,对Mg2+、dNTP、引物浓度及退火温度等进行优化,建立同时检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV的多重RT-PCR方法,并验证该检测方法的灵敏性。结果:所建立的五种病毒的多重RT-PCR方法可以同时或者分别扩增甲型、乙型流感病毒、RSV-A、RSV-B及ADV的141bp、635bp、525bp、377b和283bp基因片段,敏感度分别达到770PFU/ml、800PFU/ml、680PFU/ml、970PFU/ml和850PFU/ml,且五种病毒间无交叉反应。结论:所建立的多重RT-PCR方法可以迅速准确地检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV,为五种病毒的检测提供了一种方便易行的方法。     

Detection of human bocavirus and human metapneumovirus by real-time PCR from patients with respiratory symptoms in Southern Brazil

The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5% of samples and hBoV in 13.2%. A unique causative viral agent was identified in 46.2% samples and the coinfection rate was 43.7%. For hBoV, 98.3% of all positive samples were from patients with mixed infections. Similarly, 84.8% of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.     

Viral and Atypical Bacterial Etiology of Acute Respiratory Infections in Children under 5 Years Old Living in a Rural Tropical Area of Madagascar

Background

In Madagascar, very little is known about the etiology and prevalence of acute respiratory infections (ARIs) in a rural tropical area. Recent data are needed to determine the viral and atypical bacterial etiologies in children with defined clinical manifestations of ARIs.

Methods

During one year, we conducted a prospective study on ARIs in children between 2 to 59 months in the community hospital of Ampasimanjeva, located in the south-east of Madagascar. Respiratory samples were analyzed by multiplex real-time RT-PCR, including 18 viruses and 2 atypical bacteria. The various episodes of ARI were grouped into four clinical manifestations with well-documented diagnosis: “Community Acquired Pneumonia”(CAP, group I), “Other acute lower respiratory infections (Other ALRIs, group II)”, “Upper respiratory tract infections with cough (URTIs with cough, group III)”and “Upper respiratory tract infections without cough (URTIs without cough, group IV)”.

Results

295 children were included in the study between February 2010 and February 2011. Viruses and/or atypical bacteria respiratory pathogens were detected in 74.6% of samples, the rate of co-infection was 27.3%. Human rhinovirus (HRV; 20.5%), metapneumovirus (HMPV A/B, 13.8%), coronaviruses (HCoV, 12.5%), parainfluenza virus (HPIV, 11.8%) and respiratory syncytial virus A and B (RSV A/B, 11.8%) were the most detected. HRV was predominantly single detected (23.8%) in all the clinical groups while HMPV A/B (23.9%) was mainly related to CAP (group I), HPIV (17.3%) to the “Other ALRIs” (group II), RSV A/B (19.5%) predominated in the group “URTIs with cough” (group III) and Adenovirus (HAdV, 17.8%) was mainly detected in the “without cough” (group IV).

Interpretation

This study describes for the first time the etiology of respiratory infections in febrile children under 5 years in a malaria rural area of Madagascar and highlights the role of respiratory viruses in a well clinically defined population of ARIs.     

Development and evaluation of multiplex real-time RT-PCR assays for seasonal, pandemic A/H1pdm09 and avian A/H5 influenza viruses detection

Since the pandemic influenza A (H1N1) 2009 ((H1N1)pdm09) virus spread all over the world, the (H1N1)pdm09 virus has been circulating with seasonal influenza viruses. We developed rapid and sensitive one-step multiplex real-time RT-PCR assays (rRT-PCR) for simultaneous detection of influenza viruses currently circulating in humans, and the avian A/H5 virus. The detection limit of each assay was 4.8 to 1 copies per reaction and no cross-reactivity with other major respiratory pathogens was found. Analytical positive predictive value (PPV), negative predictive value (NPV) sensitivity and specificity were 100%, 94.1%, 93.7% and 100%, respectively. Clinical evaluation revealed that 1,976 (16.5%) of 11,963 throat swabs from patients with respiratory symptoms were confirmed as 1,651 (83.6%) A/H1pdm09, 308 (15.6%) A/H3 and 17 (0.8%) B virus during the 2010–2011 influenza season. Collectively, the multiplex rRT-PCR assays described here provide a practical tool for reliable implementation of influenza surveillance and diagnosis.     

2009～2010年上海地区急性呼吸道感染病毒病原谱分析

调查2009~2010年上海地区人群急性呼吸道感染(ARTI)的病毒性病原,探讨2009甲型H1N1流感暴发背景下呼吸道感染病毒病原谱的构成。采用套式多重反转录-聚合酶链反应(RT-PCR)和实时荧光定量RT-PCR方法,对来自2 044例患者的2 044份标本(包括2 005份鼻咽拭子和39份肺泡灌洗液),同时检测腺病毒(ADV)、副流感病毒(PIV)、甲型流感病毒(FluA)、乙型流感病毒(FluB)、微小核糖核酸病毒、呼吸道合胞病毒(RSV)、人偏肺病毒(hMPV)、冠状病毒(CoV)和人博卡病毒(HBoV)。其中,656(32.09%)份标本经呼吸道病毒检测为阳性,52份标本为双重感染。FluA检出率最高(13.36%),其后依次为微小核糖核酸病毒(10.23%)、FluB(4.84%)、ADV(1.96%)、PIV(1.76%)、RSV(1.32%)、CoV(0.59%)、hMPV(0.39%)和HBoV(0.20%)。但各月病毒检出率分布不均,2009和2010年呼吸道病毒检出率高峰出现在当年11月(53.07%和65.59%),低谷都出现在当年5月,且2009年5~9月的病毒检出率高于2010年同期(32.02%vs15.38%,P<0.05)。其中,2009甲型H1N1流感暴发导致2009年10月~2010年1月2009甲型H1N1流感病毒占当月检出FluA的100%,2009年6~9月也占当月检出FluA的较高比率,依次为90.91%(20/22)、75.00%(15/20)、48.00%(12/25)和56.25%(18/32)。比较甲型H3N2流感病毒和2009甲型H1N1流感病毒分别在上呼吸道感染(URTI)和下呼吸道感染(LRTI)中的检出率,无统计学差异(URTI,85.29%vs76.61%;LRTI,14.71%vs23.39%;P>0.05)。呼吸道病毒检出率还与年龄相关,0~4岁组和5~14岁组病毒检出率高于其他年龄组。在0~4岁及≥65岁组中,微小核糖核酸病毒检出率最高,FluA次之;其余年龄组中FluA检出率最高。混合感染中15岁以下儿童占50%(26/52),微小核糖核酸病毒与其他病毒混合感染占84.62%(44/52)。本研究表明,上海地区2009~2010年FluA是最常见的急性呼吸道感染病原,2009甲型H1N1流感病毒成为2009年FluA的优势亚型。微小核糖核酸病毒是混合感染中最常见的病原。结果提示,应长期监测主要呼吸道病毒的活动水平,并加强对微小核糖核酸病毒流行病学和致病性的研究。     

Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID₅₀/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID₅₀/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported.     

The Clinical and Etiological Characteristics of Influenza-Like Illness (ILI) in Outpatients in Shanghai,China, 2011 to 2013

Introduction

Clinical and etiological characteristics of influenza-like illness (ILI) in outpatients is poorly understood in the southern temperate region of China. We conducted laboratory-based surveillance of viral etiology for ILI outpatients in Shanghai from January 2011 to December 2013.

Materials and Methods

Clinical and epidemiological data from ILI outpatients, both children and adults, were collected. A total of 1970 nasopharyngeal swabs were collected and tested for 12 respiratory viruses using multiplex RT-PCR, and the data were analyzed anonymously.

Results

All 12 respiratory viruses were detected in the specimens. At least one virus was detected in 32.4% of 1970 specimens analyzed, with 1.1% showing co-infections. The most frequently detected agents were influenza A (11.7%), influenza B (9.6%), and rhinoviruses (3.1%).Other viruses were present at a frequency less than 3.0%. We observed a winter peak in the detection rate in ILI patients during 3 years of surveillance and a summer peak in 2012. HCoV, HADV, and HMPV were detected more frequently in children than in adults. Patients infected with influenza virus experienced higher temperatures, more coughs, running noses, headaches and fatigue than patients infected with other viruses and virus-free patients (p<0.001).

Conclusions

The spectrum, seasonality, age distribution and clinical associations of respiratory virus infections in children and adults with influenza-like illness were analyzed in this study for the first time. To a certain extent, the findings can provide baseline data for evaluating the burden of respiratory virus infection in children and adults in Shanghai. It will also provide clinicians with helpful information about the etiological patterns of outpatients presenting with complaints of acute respiratory syndrome, but further studies should be conducted, and longer-term laboratory-based surveillance would give a better picture of the etiology of ILI.     

569例呼吸道感染住院儿童7种常见呼吸道病毒病原学分析

目的了解呼吸道感染住院患儿呼吸道病毒的分布情况。方法选取2015年7月至2016年6月呼吸道感染的住院患儿病例,抽取鼻咽分泌物,采用直接免疫荧光法检测常见的7种病毒,即呼吸道合胞病毒(RSV)、甲型流感病毒(IVA)、乙型流感病毒(IVB)、副流感病毒1型(PIV1)、副流感病毒2型(PIV2)、副流感病毒3型(PIV3)、腺病毒(IVD)。结果共有569例样本送检,193例病毒检测阳性(33.92%),其中有3例为2种病毒的混合感染。男性共369例,女性共200例。其中RSV、PIV3、ADV的感染率居前三位。呼吸道病毒的感染率随着患儿年龄的增长逐渐下降,除新生儿外,6个月内的婴幼儿呼吸道病毒检出率最高。RSV检测阳性率自11月开始呈增高趋势,12月最高,达55.86%。PIV3的检出高峰出现在7月,达13.64%。结论 RSV是本地区儿童呼吸道感染最常见的病毒。呼吸道病毒的检出率与年龄、季节等因素有关。     

503例儿童急性呼吸道感染常见病毒的检测结果分析

目的通过对503例急性呼吸道感染患儿进行7种常见病毒[流感病毒A、B型（IVA、IVB）,腺病毒（ADV）,副流感病毒1、2、3型（PIV1、PIV2、PIV3）及呼吸道合胞病毒（RSV）]的检测,了解本地区2013年急性呼吸道感染患儿的病毒感染状况。方法应用免疫荧光法对503例急性呼吸道感染患儿的咽拭子进行7种常见病毒的检测。结果 503例患儿中有55例检出病毒阳性,总阳性率为10.93%,均为单一病毒感染。7种常见病毒中,RSV的感染率最高,为76.36%。在各年龄组中,〈1岁组的病毒检出率最高,为29.41%,随着年龄的增长,检出率逐渐降低。从季节分布来看,春季的感染率最高,为23.08%,其次为冬季,感染率10.13%。结论 RSV是2013年本地区儿童急性呼吸道感染的主要病毒,〈1岁组患儿的病毒检出率最高,春季为感染的高发季节。     

The cost of community-managed viral respiratory illnesses in a cohort of healthy preschool-aged children

Background

Acute respiratory illnesses (ARIs) during childhood are often caused by respiratory viruses, result in significant morbidity, and have associated costs for families and society. Despite their ubiquity, there is a lack of interdisciplinary epidemiologic and economic research that has collected primary impact data, particularly associated with indirect costs, from families during ARIs in children.

Methods

We conducted a 12-month cohort study in 234 preschool children with impact diary recording and PCR testing of nose-throat swabs for viruses during an ARI. We used applied values to estimate a virus-specific mean cost of ARIs.

Results

Impact diaries were available for 72% (523/725) of community-managed illnesses between January 2003 and January 2004. The mean cost of ARIs was AU$309 (95% confidence interval $263 to $354). Influenza illnesses had a mean cost of $904, compared with RSV, $304, the next most expensive single-virus illness, although confidence intervals overlapped. Mean carer time away from usual activity per day was two hours for influenza ARIs and between 30 and 45 minutes for all other ARI categories.

Conclusion

From a societal perspective, community-managed ARIs are a significant cost burden on families and society. The point estimate of the mean cost of community-managed influenza illnesses in healthy preschool aged children is three times greater than those illnesses caused by RSV and other respiratory viruses. Indirect costs, particularly carer time away from usual activity, are the key cost drivers for ARIs in children. The use of parent-collected specimens may enhance ARI surveillance and reduce any potential Hawthorne effect caused by compliance with study procedures. These findings reinforce the need for further integrated epidemiologic and economic research of ARIs in children to allow for comprehensive cost-effectiveness assessments of preventive and therapeutic options.     

Viral Etiology of Respiratory Tract Infections in Children at the Pediatric Hospital in Ouagadougou (Burkina Faso)

Background

Acute respiratory infections (ARIs) are a major cause of morbidity and mortality in children in Africa. The circulation of viruses classically implicated in ARIs is poorly known in Burkina Faso. The aim of this study was to identify the respiratory viruses present in children admitted to or consulting at the pediatric hospital in Ouagadougou.

Methods

From July 2010 to July 2011, we tested nasal aspirates of 209 children with upper or lower respiratory infection for main respiratory viruses (respiratory syncytial virus (RSV), metapneumovirus, adenovirus, parainfluenza viruses 1, 2 and 3, influenza A, B and C, rhinovirus/enterovirus), by immunofluorescence locally in Ouagadougou, and by PCR in France. Bacteria have also been investigated in 97 samples.

Results

153 children (73.2%) carried at least one virus and 175 viruses were detected. Rhinoviruses/enteroviruses were most frequently detected (rhinovirus n = 88; enterovirus n = 38) and were found to circulate throughout the year. An epidemic of RSV infections (n = 25) was identified in September/October, followed by an epidemic of influenza virus (n = 13), mostly H1N1pdm09. This epidemic occurred during the period of the year in which nighttime temperatures and humidity were at their lowest. Other viruses tested were detected only sporadically. Twenty-two viral co-infections were observed. Bacteria were detected in 29/97 samples with 22 viral/bacterial co-infections.

Conclusions

This study, the first of its type in Burkina Faso, warrants further investigation to confirm the seasonality of RSV infection and to improve local diagnosis of influenza. The long-term objective is to optimize therapeutic management of infected children.     

Simultaneous Detection and Identification of Enteric Viruses by PCR-Mass Assay

Simultaneous detection of enteric viruses that cause similar symptoms (e.g. hand, foot and mouth disease) is essential to the prevention of outbreaks and control of infections. In this study, a novel PCR-Mass assay combining multiplex polymerase chain reaction (PCR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and used for simultaneous detection of eight distinct human enteric viruses. Enteric viral isolates and standard viral RNAs were examined to determine the sensitivity and specificity of the PCR-Mass assay. Clinical performance was evaluated with a total of 101 clinical specimens from patients suspected of having hand, foot and mouth disease (HFMD). The results were compared to those of previous analyses using real-time RT-PCR. The identification of specific viruses and clinical specimens shows that the PCR-Mass assay performed as well as or better than standard methods with respect to indicating the presence of multiplex pathogens in a single specimen.     

人冠状病毒HCoV-NL63和HCoV-HKU1常规RT-PCR与实时荧光定量RT-PCR检测方法的建立及应用比较

分别设计HCoV-NL63和HCoV-HKU1特异的引物与荧光标记探针,并合成含靶基因的模板RNA,建立常规RT-PCR方法与实时荧光定量RT-PCR方法,对其灵敏性、特异性和可重复性以及用于临床样本的适用性等进行平行比较评价.结果表明:这两种方法皆可对HCoV-NL63或HCoV-HKU1进行特异性诊断,其中荧光定量RT-PCR方法检测灵敏度均可达10拷贝/25μL反应体积,不同批次重复检测结果间的变异系数均小于5%.上述方法应用于158份临床鼻咽拭子标本,其中荧光定量RT-PCR方法检出6份HCoV-NL63阳性标本,5份HCoV-HKU1阳性标本,而常规RT-PCR方法则分别检出HCoV-NL63阳性与HCoV-HKU1阳性各3份.对常规RT-PCR方法获得的阳性样品进行序列分析证实上述方法的可靠性.本实验成功建立了可用于临床标本检测的人冠状病毒HCoV-NL63和HCoV-HKU1常规RT-PCR方法与实时荧光定量RT-PCR检测方法,并初步证实荧光定量RT-PCR检测方法检出率明显高于常规RT-PCR方法,这为开展HCoV-NL63和HCoV-HKU1的流行监测及临床早期诊断提供了有效技术手段.