多重PCR技术检测儿童下呼吸道感染病毒和不典型病原体的价值

Detection of viral and atypical pathogens in children with lower respiratory tract infection by multiple PCR technique

目的:全面了解病毒和不典型病原体在儿童下呼吸道感染(LRTI)病原中的分布,探讨多重PCR技术检测呼吸道病原的临床应用价值。方法:收集2014年1月至2014年12月温州医科大学附属第二医院育英儿童医院992例因LRTI住院的5岁以下患儿的鼻咽分泌物,用直接免疫荧光法检测呼吸道合胞病毒、流感病毒A、流感病毒B、副流感病毒、腺病毒和衣原体抗原,并提取核酸,采用基于先进片段分析的多重PCR技术测定呼吸道合胞病毒、流感病毒A、流感病毒B、副流感病毒、腺病毒、鼻病毒、博卡病毒、偏肺病毒、冠状病毒和肺炎支原体、衣原体基因。结果:本组患者男662例,女330例,<1岁组587例,1~3岁组287例,3~5岁组118例。多重PCR技术检测结果发现,851例患儿的鼻咽分泌物标本至少存在一种病原阳性(占85.8%),224份标本(占22.6%)检测到两种或两种以上病原。呼吸道合胞病毒和鼻病毒占所有检测病原的前两位。不同年龄组的病原阳性率分别为84.8%、89.2%和82.2%,组间差异无统计学意义(χ~2=4.416,P=0.110)。但<1岁组和1~3岁组的混合感染率均明显高于3~5岁组(χ~2=3.963、9.871,P=0.047、0.002)。鼻病毒可见于各年龄段LRTI患儿,而呼吸道合胞病毒则主要见于婴幼儿。和直接免疫荧光法比较,相同病原检出的一致性较好(Kappa=0.615,P<0.01),但PCR技术病毒的检出率更高(58.37% vs. 41.53%,χ~2=56.23,P<0.001)。结论:呼吸道合胞病毒和鼻病毒占本地区5岁以下儿童LRTI主要病毒和不典型病原的前两位,博卡病毒、冠状病毒和偏肺病毒亦可见,肺炎支原体少见;和直接免疫荧光法比较,多重PCR技术有助于全面了解儿童LRTI的病原。

Objective： To understand the distribution of viruses and atypical pathogens in children’s lower respiratory tract infections （LRTI）, and to investigate the clinical value of multiplex PCR technique. Methods： Nasopharyngeal secretions of 992 children under 5 years old who were hospitalized for LRTI in the Second Affiliated Hospital ＆ Yuying Children’s Hospital of Wenzhou Medical University were collected, from January 2014 to December 2014. Direct immunofluorescence assay was used to detect respiratory syncytial virus （RSV）, influenza virus A, influenza virus B, parainfluenza virus, adenovirus and Chlamydia antigen, and extract nucleic acid. Use multiple PCR method based on advanced fragment analysis to detect RSV, influenza A virus, influenza virus B, parainfluenza virus, adenovirus, human rhinovirus （HRV）, bocavirus, human metapneumovirus, coronavirus, mycoplasma pneumonia and Chlamydia gene. Results： There were 662 males and 330 females, 587 were in 〈1 year old group, 287 were in 1-3 years old group, 118 were in 3-5 years old group, the median age was 10 month. By using multiple PCR method, nasopharyngeal secretions from 851 patients were detected at least one pathogen （85.8%）, 224 detected two or more than two pathogens （22.6%）. RSV and HRV accounted for the top two of all detected pathogens, and the positive rates of pathogens in different groups were 84.8%, 89.2% and 82.2%, there was no significant difference between the three groups （χ2=4.416, P=0.110）. However, the mixed infection rates in 〈1 year old group and 1-3 year old group were significantly higher than 3-5 year old group （χ2=3.963 and 9.871, P=0.047 and 0.002）. As for LRTI children, HRV can be detected of all ages, while RSV is mainly in infants. Compared with immunofluorescence, the consistency of the same pathogen detection is good （Kappa=0.615, P〈0.01）, but multiple PCR method was better for detecting other pathogens （58.37% vs. 41.53%, χ2=56.23, P〈0.001）. Conclusion： RSV and HRV are the first two of the major virus and atypical pathogens of LRTI in children under 5 years old in this region. Bocavirus, coronavirus, and human metapneumovirus can also be detected, mycoplasma was rare. Compared with the direct immunofluorescence method, multiple PCR is helpful in comprehensive understanding the etiology of LRTI in children.