上海地区儿童急性下呼吸道感染的鼻病毒基因检测及临床特征

Gene detection and clinical study of rhinovirus isolated from children with acute low respiratory tract infection in Shanghai

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息儿的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〈8mg／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 understand human rhinovirus （HRV） etiology of acute lower respiratory tract infection （ALRTI） in children in Shanghai area and establish a nested reverse transcription-polymerase chain reaction （nested RT-PCR） assay. Methods Three hundred and forty-two nasopharyngeal secretion （NPS） samples from ALRTI cases who were hospitalized were collected during January 2005-December 2005. Nested RT-PCR techniques were used to detect HRV-specific RNA. The PCR products were sequenced and data of nucleotides were analyzed. The proportion of HRV infection in children with ALRTI, the distribution of gender, age and season, and clinical characteristics were also investigated. Results Forty-six （13.5 %） of 342 samples were HRV positive detected by nested RT-PCR. The sequences of 15 positive samples shared high homology of 83 % -97 % with HRV sequence in GenBank. Within the 15 positive samples, nucleotide homology varied from 64.4% to 98.4%, and the ratio of genetic variation was from 1.6% to 48.3%. These 15 amplicons attribute to the two branches of HRV cladogram. The sequences of 15 amplicons were highly varied, in which single nucleotide mutation and several nearby nucleotides mutations were found. Ribonucleotide deletion and insertion in the nucleotide sequence was also found. HRV positive samples were detected in 33 boys and 13 girls, respectively. The ratio of infection cases between boys and girls was 2.5 ： 1. Of 46 HRV infected cases, 27 （58.7%） were less than 12 months of age and 38 （82.6%） were less than 3 years old. HRV infected ALRTI occured all the year round and peaked from March to May. Of the patients whose NPS samples were HRV positive detected by nested RT-PCR, 45 patients were diagnosed with bronchopneumonia and 1 was diagnosed with asthmatic bronchitis. Fever of most patients was moderate. The peripheral blood leukocyte counts in thirty-nine （84.8%） patients were less than 10 × 10^9/L. Neutrophil percentages in thirty-seven （80. 4%） patients were less than 0. 50. C-reactive protein of thirty-six （78.3%） patients were less than 8 mg/L. All of these features were the characteristics of viral pneumonia. The complications were not common and conditions of most patients were not severe. All the children were cured. Conclusions This nested RT-PCR technique is highly specific, rapid and convenient for the detection of HRV RNA in NPS of patients with ALRTI and the genome of HRV viruses is highly variable. The incidence of HRV infection predominates in children in Shanghai area. ALRTI of HRV is short of specificity and conditions of most patients are not severe and their prognoses are fine.