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目的建立套式RT-PCR检测急性下呼吸道感染(ALRTI)患儿临床样本鼻病毒(HRV)基因的方法,了解上海地区ALRTI患儿HRV感染的现状。方法收集2005年1月至12月住院的342例确诊为ALRTI的患儿的深部鼻咽分泌物(NPS)标本。用套式RT-PCR方法检测NPS中的HRV基因,并进一步对扩增产物进行序列分析。对HRV在ALRTI患儿中所占比例、性别、年龄、季节分布及临床特点进行分析。结果在342例ALRTI患儿的NPS标本中,套式RT- PCR检测到46例HRV阳性,占13.5%;选取15份HRV阳性样本,用另一对引物扩增并测序,测得的核苷酸序列经BLAST比较,与基因库中已知HRV序列的同源性为83%～97%;15份样本序列之间核苷酸同源性在64.4%～98.4%,核苷酸变异在1.6%～48.3%;15份样本序列在进化树中聚类为两簇;15份样本序列变异度较大,可表现为核苷酸的点突变,也可是邻近几个碱基同时突变,甚至有单个或邻近几个碱基的核苷酸缺失及插入突变。46例阳性患儿中,男33例,女13例,男女之比为2.5:1。HRV阳性患儿中,3岁以下婴幼儿检出38例,占HRV总检出数的82.6%;1岁以下患儿检出27例,占58.7%。HRV所致ALRTI全年可见,在3～5月为最高峰。本组46例套式RT-PCR检测HRV阳性患儿中,诊断支气管肺炎45例,喘息性支气管炎1例;以中等度以下发热为主:39例患儿末梢血WBC<10×10~9/L,占84.8%;37例中性粒细胞<0.50,占80.4%;36例C反应蛋白<8 mg/L,占78.3%,均符合病毒性肺炎的特点。合并症较少,绝大部分病情较轻,所有患儿均治愈。结论套式RT-PCR检测HRV基因快速、简便。HRV基因组有高度变异的特点。HRV感染在本地区小儿ALRTI中占有一定比例。HRV所致ALRTI缺乏特异性,病情相对较轻且预后较好。
Objective To establish a method of nested RT-PCR for detection of clinical samples of rhinovirus (HRV) in children with acute lower respiratory tract infection (ALRTI) and to understand the status of HRV infection in children with ALRTI in Shanghai. METHODS: Deep nasopharyngeal secretions (NPS) samples from 342 children with ALRTI admitted to hospital from January 2005 to December 2005 were collected. The HRV gene in NPS was detected by nested RT-PCR and the amplified product was further sequenced. The proportion, gender, age, season distribution and clinical features of HRV in children with ALRTI were analyzed. Results Among 342 NRS samples of ALRTI children, 46 HRV positive samples were detected by nested RT-PCR, accounting for 13.5%; 15 HRV positive samples were selected and amplified with another pair of primers and sequenced. The measured nucleotide The BLAST results showed that the homology of the nucleotide sequence with the known HRV sequence in the gene library was 83% -97%. The nucleotide sequence homology of the 15 samples was 64.4% -98.4% and the nucleotide variation was 1.6 % ~ 48.3%; 15 sample sequences clustered into two clusters in the phylogenetic tree; 15 sample sequences with large variation can be expressed as nucleotide point mutations, but also adjacent several base mutation, and even Nucleotide deletion and insertion mutations of single or near several bases. 46 cases of positive children, 33 males and 13 females, male to female ratio of 2.5: 1. In HRV-positive children, 38 cases of infants under 3 years old, accounting for 82.6% of the total number of HRV detection; 27 cases of children under 1 year old accounted for 58.7%. HRV caused by ALRTI visible throughout the year, in March to May is the peak. The group of 46 cases of HRV positive cases detected in children with bronchial pneumonia, bronchial pneumonia in 45 cases, 1 case of asthmatic bronchitis; with moderate fever mainly: 39 cases of peripheral blood WBC <10 × 10 ~ 9 / L, accounting for 84.8%; 37 cases of neutrophils <0.50, accounting for 80.4%; 36 cases of C-reactive protein <8 mg / L, accounting for 78.3%, are in line with the characteristics of viral pneumonia. Complications less, most of the less serious, all children were cured. Conclusion The nested RT-PCR detection of HRV gene is rapid and simple. The HRV genome is highly variable. HRV infection accounts for a proportion of pediatric ALRTI in this region. ALRTI due to HRV lack of specificity, the condition is relatively mild and the prognosis is good.