[Pathogen spectrum in enteroviral infections among children in Beijing from 2010 to 2016].

Objective: To understand the epidemiological and etiological characteristics of enterovirus (EV)-associated diseases among children in Beijing from 2010 to 2016. Methods: This was a repeated cross-sectional study. The throat swabs were collected from children with probable EV-associated diseases at the Children' s Hospital Affiliated to Capital Institute of Pediatrics from 2010 to 2016. The samples were sent for pan-EV, enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) detection by real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR) . The viral types of non-EV-A71 and non-CV-A16 EV-positive samples were identified using modified RT-PCR and sequencing with CV-A6, EV-A/B group and 5 'UTR universal primers. The constituent ratios of the prevalence of different EV types in different age and gender groups were compared. Results: Of the 2 703 throat swabs, 1 992 (73.7%) samples were positive for EV, including EV-A71 (19.1%, 516/2 703), CV-A16 (24.3%, 658/2 703), CV-A6 (22.2%, 600/2 703), CV-A10 (4.5%, 122/2 703) and other types of EV (3.5%, 95/2 703). There was 1 case of EV-A71 and CV-A16 co-infection. The positive detection rate of EV-A group (excluding EV-A71, CV-A16, CV-A6 and CV-A10) increased from 11.3% (7/62) to 95.2% (59/62) after using the modified VP1-specific primers and PCR amplification conditions. During the period between 2010 and 2012, CV-A16 and EV-A71 predominated in EV-positive samples. However, CV-A6 accounted for 60.7% (68/112) in 2013, much higher than CV-A16 (23.2%, 26/112) and EV-A71 (12.5%, 14/112). In 2014, EVs were mainly of CV-A16 and EV-A71, but CV-A6 was the predominant type in 2015 (68.2%, 232/340) and in 2016 (38.6%, 151/391). The epidemic season of EVs was mostly from April to August, but CV-A6 showed a small epidemic peak from October to November. The male-to-female ratio of EV-positive patients was 1.50∶1, and EV-associated diseases mostly occurred in children under 5 years of age. Younger children were more susceptible to CV-A6 than to EV-A71 and CV-A16. Conclusions: From 2010 to 2016, there was a significant change in the spectrum of EVs in children with EV-associated diseases in Beijing. Since 2013, non-EV-A71 and non-CV-A16 increased, and CV-A6 gradually became one of the major pathogens of EV-associated diseases. The modified PCR primers and amplification conditions can effectively improve the reliability of test results.目的： 分析北京地区2010至2016年引起儿童肠道病毒（EV）相关疾病的病原谱变化及其各型别的流行趋势。 方法： 采用重复横断面研究方法，对2010—2016年收集的首都儿科研究所附属儿童医院感染科门诊疑似EV相关疾病患儿的口咽拭子标本进行EV筛查。使用实时荧光反转录（RT）PCR反应检测EV、肠道病毒71型（EV-A71）和柯萨奇病毒A组16型（CV-A16）。经自行设计引物、改进引物及PCR扩增条件，通过RT-PCR方法对EV阳性而EV-A71和CV-A16阴性标本依次用CV-A6、EV-A和B组特异性引物以及5'UTR通用引物进行扩增、测序以确定型别。通过计算构成比的方式分析不同EV型别的流行特点以及在不同年龄组的分布情况，使用χ(2)检验对患儿性别的差异进行统计学分析。 结果： 收集的2 703份口咽拭子标本中EV阳性标本共1 992例，总阳性率为73.7%，其中EV-A71的阳性率为19.1%（516份），CV-A16为24.3%（658份），CV-A6为22.2%（600份），CV-A10为4.5%（122份），其他型别EV为3.5%（95份），还有1份标本为EV-A71和CV-A16同时阳性，提示共感染。通过改进EV-A组VP1特异性引物和PCR扩增条件后，EV-A组（除EV-A71、CV-A16、CV-A6和CV-A10）阳性检出率由11.3%（7/62）增加至95.2%（59/62）。2010—2012年，EV阳性标本中CV-A16和EV-A71所占比例最高；然而2013年CV-A6代替CV-A16和EV-A71成为主导病原，所占比例达60.7%（68/112），远远高于CV-A16（23.2%,26/112）和EV-A71（12.5%,14/112）；2014年，EV型别仍以CV-A16和EV-A71为主；2015和2016年均以CV-A6所占比例最高，分别为68.2%（232/340）和38.6%（151/391）。EV的流行季节多为4至8月份，但每年稍有不同，CV-A6在10和11月份会出现小的流行高峰。EV阳性标本中的男女性别比为1.50∶1，以5岁以下儿童多见。与EV-A71和CV-A16相比，CV-A6更易感染年龄偏小的儿童。 结论： 2010—2016年北京地区儿童中EV感染的病例中EV病原谱发生了明显变化，自2013年以来，非EV-A71和CV-A16的病原增多，CV-A6逐渐成为EV感染性疾病的主要病原体之一。对PCR引物和扩增条件的改进可以有效地提高检测结果的可靠程度。.