多重聚合酶链反应技术对判定住院儿童肺炎病原谱的初步研究

Assessment of multiple polymerase chain reaction in detecting pathogens of children hospitalized with pneumonia:A preliminary study

目的探讨多重聚合酶链反应(multiple polymerase chain reaction,mPCR)技术在住院儿童肺炎病原谱中的临床应用。方法前瞻性纳入2019年9月至2020年6月上海交通大学附属儿童医院呼吸科收治的230例肺炎患儿的临床资料,采用mPCR技术检测病原体,同时采用传统方法检测病原体做自身对照。入院24 h内,采集患儿的鼻咽拭子用mPCR方法行35项病原体核酸检测;采集痰液行细菌培养和真菌培养,并用直接免疫荧光法行8项呼吸道病毒抗原检测;采集静脉血用颗粒凝集法行血清肺炎支原体抗体检测。结果230例肺炎患儿中男童136例(59.1%),女童94例(40.9%),平均年龄为(3.6±3.0)岁。mPCR方法检出前10位病原体依次为肺炎链球菌(17.0%)、人腺病毒(13.9%)、肺炎支原体(13.9%)、人鼻病毒(10.0%)、巨细胞病毒(10.0%)、人呼吸道合胞病毒(9.6%)、人冠状病毒(9.6%)、人博卡病毒(8.3%)、人偏肺病毒(7.4%)以及大肠埃希菌(6.1%)。m PCR检测结果中,3岁以下儿童中巨细胞病毒和大肠埃希菌的检出率显著高于3岁及以上儿童[(19.8%vs.0,χ^(2)=25.115,P=0.000)、(10.3%vs.1.8%,χ^(2)=7.422,P=0.006)],而肺炎支原体检出率则显著低于3岁及以上儿童(5.2%vs.22.8%,χ^(2)=14.928,P=0.000)。m PCR方法的1种或1种以上病原体检出率、2种或2种以上病原体检出率、病毒检出率、细菌检出率及真菌检出率与传统方法比较显著增高,差异有统计学意义[(78.7%vs.65.2%,χ^(2)=10.353,P=0.001)、(49.1%vs.11.7%,χ^(2)=75.941,P=0.000)、(58.7%vs.8.3%,χ^(2)=131.350,P=0.000)、(43.5%vs.17.4%,χ^(2)=36.964,P=0.000)、(3.5%vs.0.4%,χ^(2)=4.080,P=0.043)];而m PCR方法的肺炎支原体检出率与传统方法比较显著降低,差异有统计学意义(13.9%vs.50.0%,χ^(2)=68.874,P=0.000)。mPCR与传统方法之间相同病原检出的一致性欠佳(Kappa<0.5,P<0.05)。结论上海交通大学附属儿童医院住院肺炎最常见的病原体呈多种病毒和细菌,混合感染占半数。与传统检测方法比较,mPCR有助于早期识别肺炎病原。

Objective To understand the distribution of pathogens in hospitalized children with pneumonia,and to investigate the clinical value of multiple polymerase chain reaction(m PCR).MethodsA total of 230 children who were hospitalized for pneumonia in Shanghai Children’s Hospital from September 2019 to June 2020 were prospectively enrolled.Both m PCR and routine microbiological methods were performed in children.Nasopharyngeal and oropharyngeal(NP-OP)specimens were tested with m PCR which could detect 35 kinds of respiratory pathogen at the same time.Sputum specimens were tested with routine microbiological methods(culture and direct immunofluorescence assay)which could detect bacteria,fungus and 8 kinds of respiratory virus.Blood specimens were tested with routine microbiological methods(particle agglutination)which could detect Mycoplasma pneumoniae antibody.ResultsWe enrolled 230 cases,136(59.1%)boys and 94(40.9%)girls,and the median age of the patients was(3.6±3.0)years old.The top 10 pathogens detected by mPCR were Streptococcus pneumoniae(17%),human adenovirus(13.9%),Mycoplasma pneumoniae(13.9%),human rhinovirus(10.0%),cytomegalovirus Cytomegalovirus and Escherichia coli were more common among children younger than 3 years of age than among the children at and over 3 years old[(19.8%vs.0,χ^(2)=25.115,P=0.000),(10.3%vs.1.8%,χ^(2)=7.422,P=0.006)],while Mycoplasma pneumoniae was less common among children younger than 3 years of age detected by m PCR(5.2%vs.22.8%,χ^(2)=14.928,P=0.000).The detection rate of one or more pathogens,two or more pathogens,viral pathogens,bacterial and fungus pathogens using m PCR were significantly higer than that using routine microbiological methods[(78.7%vs.65.2%,χ^(2)=10.353,P=0.001),(49.1%vs.11.7%,χ^(2)=75.941,P=0.000),(58.7%vs.8.3%,χ^(2)=131.350,P=0.000),(43.5%vs.17.4%,χ^(2)=36.964,P=0.000),(3.5%vs.0.4%,χ^(2)=4.080,P=0.043)];however,the detection rate of Mycoplasm pneumoniae using m PCR was significantly lower than that using routine microbiological methods(13.9%vs.50.0%,χ^(2)=68.874,P=0.000).The consistency of the same pathogen detection between m PCR and routine microbiological methods was not good(Kappa<0.5,P<0.05).ConclusionThe most common pathogens are various viruses and bacteria in hospitalized children with pneumonia in Shanghai Children’s Hospital and nearly half of all cases had two or more pathogens detected.Compared with the routine microbiological method,mPCR is helpful in early identification of pneumonia pathogen in children.