【作者】 沈军；

【导师】 朱启镕；

【作者基本信息】 复旦大学 ， 儿科学， 2009， 博士

【摘要】 研究目的：采用TaqMan-MGB实时荧光RT-PCR (real-time reverse transcriptase PCR, real-time RT-PCR)检测临床样本中人偏肺病毒(human metapneumovirus, hMPV)N基因的方法,并进一步用传统RT-PCR扩增阳性样本cDNA以进行基因序列分析；用直接免疫荧光(direct immunofluorescence assay, DFA)的方法对引起儿童急性下呼吸道感染(acute low respiratory tract infection, ALRTI)的呼吸道合胞病毒(RSV),腺病毒(ADV),A、B型流感病毒(IFV),1、2及3型副流感病毒(PIV)等7种常见呼吸道病毒抗原进行检测；以了解上海地区ALRTI患儿中呼吸道病毒感染状况及hMPV在上海地区流行株的分子流行病学特点；通过对同期标本中支原体、沙眼衣原体荧光定量PCR检测结果及细菌学培养结果的调查,较清晰的揭示2006年10月至2008年2月期间本地区儿童ALRTI的病原谱构成比与变化特点。对象及方法：1.收集2006年10月到2008年2月确诊为ALRTI住我院48小时内共1347例的患儿深部鼻咽分泌物(nasopharyngeal secretion, NPS)1347份标本。2.用TaqMan-MGB实时荧光RT-PCR方法检测随机挑选(每周二、四采集)的622份NPS标本中人偏肺病毒(hMPV) RNA,阳性标本进一步用传统PCR扩增产物并进行基因序列分析。荧光RT-PCR的上游引物为5’-CATCAGGTAATATCCCACAAAATCAG-3’,下游引物为5’-GTGAATATTAAGGCACCTACACATAATAA-3’,探针为FAM-5’-TCAGCACCAGACACAC-NFQMGB-3’;测序传统PCR的上游引物为5’-AACCGTGTACTAAGTGATGCACTC-3’,下游引物为5’-CATTGTTTGACCGGCCCCATAA-3’。3.用直接免疫荧光法方法检测1347份NPS中的呼吸道合胞病毒(RSV),腺病毒(ADV),A、B型流感病毒(IFV),1、2及3型副流感病毒(PIV)抗原。4.对各种病毒在小儿ALRTI中所占比例、性别、年龄、季节分布及临床特点进行临床流行病学分析,并对hMPV检测及测序结果进行分子流行病学分析。基因序列分析用BLAST和DNAMAN软件对hMPV核苷酸序列作比较,并绘制基因进化树。5.调查同期标本中支原体、衣原体荧光PCR检测结果及细菌学培养结果。结果：1. TaqMan-MGB实时荧光RT-PCR检测hMPV基因共检测出24份阳性标本,检出率为3.86%,阳性样本cDNA用另一对引物进一步用传统RT-PCR扩增,16份标本阳性；随机选取5份传统RT-PCR产物进行测序,测得的核苷酸序列经过BLAST比较,与GeneBank中已知hMPV序列的同源性在84%～100%之间；用DNAMAN软件进行基因分析发现,5份样本序列在进化树中聚类为2簇；5份样本基因序列变异性可表现为核苷酸的点突变,也可是临近几个碱基同时突变,甚至有单个或临近几个碱基的核苷酸缺失及插入突变。2.622例患儿中,3月龄以下儿童hMPV检出率0.94%(2/213),3月龄到6足月以下儿童检出率3.95%(3/76),6月龄到1足岁以下儿童检出率2.73%(3/110),1岁到2足岁以下儿童检出率9.52%(6/63),2岁到5足岁以下儿童检出率6.98%(9/129),5岁以上儿童检出率3.23%(1/31)。2岁以下儿童占58.33%(14/24),5岁以下儿童占95.83%(23/24)。hMPV致ALRTI绝大多数在11月份到次年1月份之间(21/24)。2006-2007年冬季hMPV检出率6.60%(14/212),2007-2008年冬季hMPV检出率1.11%(2/180)。男性323例患儿中检测出hMPV阳性10例(3.10%),女性237例患儿检出hMPV阳性14例(5.91%)，男女检出率无差异(X2=2.63,P>0.05)。3.1347份标本中7种常见病毒共检出率为33.56%(452/1347),hMPV检出率为3.86%(24/622),总的病毒检出率35.19%(474/1347)；细菌检出率为24.64%(329/1335),支原体和衣原体检出率分别为7.85%(103/1312)和2.97%(39/1312),1347份标本总的病原学检出率为55.23%(744/1347),其中混合感染率为15.59%(210/1347),2份标本hMPV与RSV同时阳性,9份标本存在3种病原体同时感染。有44.77%的临床标本病原未明。4.RSV检出率为28.36%(382/1347),为最常见的感染原。2006-2007年冬季RSV检出率31.16%(124/398),2007-2008年冬季RSV检出率38.59%(120/311)；2007年春季、夏季、秋季RSV检出率分别为21.05%(36/171)、8.64%(7/81)和31.40%(92/293)。2岁以下儿童为RSV最易感人群。3月龄以下儿童检出率36.36%(184/506),3月龄到6足月以下儿童检出率31.58%(72/228),2岁以下儿童占91.88%(351/382),5岁以下儿童占98.69%(377/382)。RSV阳性患儿多有喘息表现。5.PIV检出率为2.82%(38/1347),低于RSV和hMPV;ADV检出率为1.86%(25/1347)；IFV检出率为1.19%(16/1347)；少见混合性病毒感染。6.支原体和衣原体检出率分别为7.85%(103/1312)和2.97%(39/1312)。支原体在5岁以上儿童中的检出率为34.5%,衣原体在3月龄以下儿童中的检出率为6.8%。结论：1. TaqMan-MGB实时荧光RT-PCR检测hMPV基因具有快速、操作简便、敏感度高的特点,可应用于临床检测。2006年10月至2008年2月本地区存在两种基因型hMPV流行。2.2006年10月至2008年2月上海地区ALRTI儿童NPS中hMPV的检出率为3.86%,2006-2007年冬季较2007-2008年冬季检出率高。hMPV所致儿童ALRTI以5岁以下小年龄儿童多见。3.病毒病原在本地区儿童ALRTI中占有重要地位；2岁以下儿童ALRTI以病毒性感染为主,2岁以上到5岁以细菌性感染为主,到5岁以上则以支原体感染占优势。3月龄以下衣原体有6.28%的检出率,值得关注。4.2岁以下儿童为RSV最易感人群；在本地区,2007-2008年冬季较2006-2007年冬季RSV有较强的流行；RSV感染更易表现有喘息症状。5.2006年10月至2008年2月本地区儿童ALRTI中,8种病毒检出率为35.19%,细菌检出率为24.64%,支原体和衣原体检出率分别为7.85%和2.97%,仍有44.77%的临床标本病原未明。更多还原

【Abstract】 Objective:Establish a relible and stable diagnostic method of TaqMan-MGB real-time reverse transcription-polymerase chain reaction (RT-PCR) to detect human metapneumovirus (hMPV) in the clinical respiratory samples. The aim of this study was to understand the clinical and molecular epidemiology of hMPV and other common respiratory pathogens among children with acute low respiratory tract infection (ALRTI) from Oct2006to Feb2008in Shanghai. In the mean time, we tested other seven common respiratory virus by direct immunofluorescence assays (DFA). We also analyzed the detection results of respiratory bacteria, mycoplasma pneumonia and Chlamydia among the targeted samples and enrolled patients to display the spectrum of respiratory pathogens and the changing pattern of pathogens with the time.Subjects and Methods:1.1347nasopharyngeal secretion samples were collected from children clinically disgnosed with ALRTI from Oct2006to Feb2008. One sample was taken from each patient within48hours of admission.2. All specimens were routinely detected for seven common respiratory viruses, bacteria, mycoplasma pneumonia and Chlamydia. Seven common respiratory viruses including respiratory syncytial virus (RSV), influenza virus (IFV) A and B, parainfluenza virus (PIV) type1-3and adenovirus (ADV) were detected by direct immunofluorescence assay. Mycoplasma pneumonia and Chlamydia were detected by real-time PCR. Bacteria were identified by culture.3. We selected622samples collected on every Tuesday and Thursday to detect N gene of hMPV by real-time RT-PCR. A forward primer with a sequence of5’-CATCAGGTAATATCCCACAAAATCAG-3’and a reverse primer with a sequence of5’-GTGAATATTAAGGCACCTACACATAATAA-3’and a probe with a sequence of5’-TCAGCACCAGACACAC-3’were used for real-time RT-PCR. The probe was labeled with6-carboxyfluorescein (FAM) at the5’end and with a quencher (NFQMGB) at the3’end. Traditional PCR was performed with another primer set (forward:5’-AACCGTGTACTAAGTGATGCACTC-3’, reverse:5’-CATTGTTTGACCGGCCCCATAA-3’) and PCR products were sequenced directly.4. The clinical characteristic of hMPV-positive cases and the prevalence of hMPV were analyzed. The nucleotide fragment of PCR products of hMPV target gene were confirmed in the Genbank by blast. Nucleotide sequences alignment and phylogenetic analysis of hMPV were performed with DNAMAN software.5. The clinical picture of seven respiratory viruses were analyzed and compared.6. The detection rates of mycoplasma pneumonia, chlamydia and bacteria were evaluated.Results:1. Of622samples, hMPV was detected to be positive in24(3.86%) samples.5hMPV-positive PCR products were sequenced. Nucleotide sequences of5hMPV gene were aligned with the submitted sequences of hMPV in Genbank, which revealed similarity at nucleotides level varied from84%to100%, which suggested2distinct genetypes. Phylogenetic tree analysis further demonstrated2different hMPV lineages circulating during the study period. By comparision of nucleotide sequences, point mutation of nucleotide was observed.2. The detection rates of hMPV were0.94%(2/213),3.95%(3/76),9.52%(6/63),6.98%(9/129) and3.23%(1/31) respectively among children aged<3months old,3months to6months,>6months to<1year,1year to<2years,2years to <5years and>5years. Among24hMPV-positive cases,14(58.33%) were less than2years of age and23(95.83%) were younger than5years old. hMPV infection occurred commonly from Nov to Jan of next year. The prevalence of hMPV in the winter of2006to2007was6.60%(14/212) and the prevalence of hMPV in the winter of2007to2008was1.11%(2/180). Of323boys,10(3.10%) were hMPV-positive; of237girls.14(5.91%) were hMPV-positive. There was no significant difference in detection rate between boys and girls (X2=2.63, P>0.05)。3. Of1347respiratory samples, seven common respiratory viruses were detected in452(33.56%) samples. Bacteria were isolated in329(24.64%) of1335samples. Mycoplasma pneumonia and Chlamydia were detected in103(7.85%) and210(15.59%) of1312samples, respectively. Co-infection rate was15.59%in1347samples. No etiological agents was identified in44.77%of samples。4. Of1347samples, RSV was identified in382(28.36%) which was the most common pathogen. The detection rates of RSV were31.16%(124/398),38.59%(120/311),21.05%(36/171)、8.64%(7/81) and31.40%(92/293) respectively in the winter of2006, the spring, the summer and the autumn of2007and the winter of2007. Among382RSV-infected cases, children younger than2years old accounted for91.88%(351/382) and children under5years of age accounted for98.69%(377/382), which indicates children younger than2years old were the most susceptible population for RSV. RSV-infected children usually presented with wheezing.5. Of1347samples, the detection rates of PIV, ADV, IFV were2.82%(38/1347),1.86%(25/1347) and1.19%(16/1347), respectively. Virus co-infection was rarely found.6. The detection rates of Mycolasma pneumonia were7.85%(103/1312) and Chlamydia were2.97%(39/1312), respectively. Mycolasma pneumonia was identiifed most commoly in children>5years old with a detection rate of34.5%. Chlamydia was mainly detected in infants<3months of age with a detection rate of6.8%.Conclusions:1. TaqMan-MGB real-time RT-PCR is a rapid and sensitive assay for detecting hMPV, therefore, it can be used to screen hMPV in the clinical specimens. The N gene of field hMPV strains existed slight variation of nucleotide.2. The detection rate of hMPV was3.86%amongst children with ALRTI from Oct2006to Feb2008in Shanghai. However, the prevalence of hMPV in the winter season of2006to2007was higher than that in the winter season of2007to2008. The majority of children infected with hMPV were younger than5years old. 3. Viruses are the important respiratory pathogens among Shanghainese children with ALRTI. Most of virus infection occurred in children<2years old, bacteria infection mainly occurred in children<2years old, mycoplasma pneumonia infection occurred in children>5years old most frequently. Chlamydia infection commonly occurred in infants<3months.更多还原