肺泡灌洗液宏基因二代测序在儿童重症肺炎中的应用

Application of metagenomics next generation sequencing of bronchoalveolar lavage fluid in children with severe pneumonia

目的分析重症肺炎患儿肺泡灌洗液宏基因组二代测序(mNGS)研究病原体及其在儿童重症肺炎中的应用价值。方法收集2019年5月至2022年6月于西安市儿童医院呼吸科确诊为重症肺炎患儿的血液及呼吸道样本,将样本同时进行传统病原学和mNGS方法检测,比较两种方法的病原检出率及与临床诊断的符合率,分析重症肺炎患儿肺泡灌洗液mNGS检出病原分布,计数资料(性别、年龄、临床症状、影像学和临床结局,病原体检出阳性率、临床符合率和真阳性率)以构成比或率表示,采用Pearsonχ^(2)检验进行分析。结果共纳入79例重症肺炎患儿,其中男52例、女27例,年龄为1月龄至14岁4月龄;其中肺泡灌洗液mNGS检出阳性率为84.8%(67/79),与临床诊断符合率为73.4%(58/79),真阳性率为86.5%(58/67);传统方法检测病原阳性率为40.5%(32/79),与临床符合率为26.6%(21/79),真阳性率为65.6%(21/32)。mNGS检出阳性率、与临床诊断符合率以及真阳性率均高于传统检测方法,差异具有统计学意义(χ^(2)=33.14、P<0.001,χ^(2)=34.66、P<0.001,χ^(2)=5.89、P=0.015)。细菌为各年龄段(<6个月、6个月~2岁、2~5岁和>5岁)重症肺炎最常见病原,差异具有统计学意义(χ^(2)=22.05、42.71、37.79、22.77,P均<0.001),其中以肺炎链球菌最为多见,在5岁以上患儿中肺炎支原体在常见病原体中居第2位。春季和冬季是儿童重症肺炎的高发季节,差异具有统计学意义(χ^(2)=12.65、27.24,P均<0.001)。结论与常规检测方法相比,重症肺炎患儿应用肺泡灌洗液mNGS检测病原体,可显著提高病原体检出率及临床诊断符合率。肺炎链球菌是儿童各年龄段重症肺炎最常见病原,肺炎支原体是5岁以上儿童重症肺炎的主要病原;儿童重症肺炎多出现在春季和冬季。

Objective To investigate the pathogen distribution of metagenomic next-generation sequencing(mNGS)for bronchoalveolar lavage fluid(BALF)in children with severe pneumonia and its application value in children with severe pneumonia.Methods Blood samples and respiratory samples of children diagnosed with severe pneumonia in the respiratory department of Xi’an Children’s Hospital from May 2019 to June 2022 were collected,the pathogens were simultaneously tested by traditional microbiological assays and mNGS,and the pathogen detection rates and the coincidence rate with the clinical diagnosis of the two methods were compared.The pathogenic distribution of mNGS in BALF of children with severe pneumonia was analyzed.Enumeration data(gender,age,clinical symptoms,imaging,clinical outcome;positive rate of pathogen detection,clinical coincidence rate and true positive rate)were represented by rates or composition ratios,and were conducted by Pearson Chi-square test.Results Total of 79 children with severe pneumonia were enrolled.Among them,52 were male and 27 were female,aged from 1 month to 14 years and 4 months;the positive rate of pathogens detected by mNGS was 84.8%(67/79),73.4%(58/79)were consistent with clinical diagnosis,the true positive rate was 86.5%(58/67).While,32 cases(40.5%)were positive by traditional detection assays,26.6%(21/79)cases were consistent with clinical diagnosis,and the true positive rate was 65.6%(21/32).These rates of mNGS were significantly higher than those of traditional detection,with significance differences(χ^(2)=33.14,P<0.001;χ^(2)=34.66,P<0.001;χ^(2)=5.89,P=0.015).Bacteria were the most common pathogens of severe pneumonia in all age groups(<6 months,6 months to 2 years,2 to 5 years and>5 years),with significance differences(χ^(2)=22.05,42.71,37.79,22.77;all P<0.001).Streptococcus pneumoniae(SP)was the most common pathogen,and mycoplasma pneumoniae(MP)was the second common pathogen in the patients over 5 years old.Spring and winter were the peak seasons for severe pneumonia in children,with significance differences(χ^(2)=12.65,27.24;both P<0.001).Conclusions Compared with traditional detection assays,mNGS detection of pathogenic microorganisms in BALF in children with severe pneumonia can significantly improve the pathogen detection rate and clinical judgment coincidence rate.SP was the most common pathogen of severe pneumonia in children of all ages,and MP was the main pathogen of severe pneumonia in children over 5 years old.Severe pneumonia in children was more common in spring and winter.