宏基因组学第二代测序技术在烧伤患者和急慢性创面患者病原体检测中的应用

Application of metagenomic next-generation sequencing technology in pathogen detection in patients with burns and patients with acute or chronic wounds

目的探讨采用宏基因组学第二代测序(mNGS)技术检测烧伤患者和急慢性创面患者病原体的价值。方法采用回顾性观察性研究方法。选择2019年3月—2020年6月解放军总医院第四医学中心的11例符合入选标准的烧伤患者和急慢性创面患者(男10例、女1例,年龄23~85岁),共采集标本23份,其中全血标本6份、皮肤组织块标本1份、引流的脓液标本1份、创面分泌物拭子标本15份。每份标本均分为2份,分别采用微生物培养法、mNGS法检测病原体。统计2种方法检测出的病原体数量和种类以及mNGS法检测的相对丰度,并比较2种检测方法的一致性。对数据行配对Wilcoxon秩和检验。结果经微生物培养法检测,在23份标本中,5份标本未检出病原体;其余18份标本共检出35株病原体,属于9种细菌和2种真菌。5份标本均各检出单一病原菌,9份标本均各检出2种病原菌,4份标本均各检出3种病原菌。经mNGS法检测,在23份标本中,1份标本未检出病原体;其余22份标本共检出75株病原体,分属于28种细菌、3种真菌和3种病毒。8份标本均各检出单一病原体,5份标本均各检出2种病原体,2份标本均各检出3种病原体,3份标本均各检出4种病原体,2份标本均各检出6种病原体,各1份标本检出7、20种病原体。微生物培养法在每份标本中检出的病原体为2(1,2)种,明显少于mNGS法的2(1,4)种(Z=3.359,P<0.01)。在微生物培养法未检出病原体的5份标本中,mNGS法在其中2份标本中检出细菌、另2份标本中检出病毒。mNGS法检出的存在2种及2种以上细菌的标本13份,每份标本中相对丰度占第1位的细菌其相对丰度范围为28.8%~95.9%。在23份标本中,有7份(30.4%)标本采用2种方法检测的结果完全一致,5份(21.7%)结果完全不一致,11份(47.8%)结果不完全一致。结论与传统的微生物培养法相比,mNGS法检测敏感性更高、对病原体的检出能力更强,并且可判断混合感染的病原体的相对丰度,作为培养法的补充,可对烧伤和急慢性创面感染病原体的诊断产生重要作用。

Objective To explore the value of using metagenomic next-generation sequencing(mNGS)technology to detect pathogens in patients with burns and patients with acute or chronic wounds.Methods A retrospective observational study was conducted.From March 2019 to June 2020,11 patients with burns and patients with acute or chronic wounds(including 10 males and 1 female,aged 23 to 85 years)in the Fourth Medical Center of PLA General Hospital met the inclusion criteria and were recruited.A total of 23 specimens were collected,including 6 whole blood specimens,1 skin tissue specimen,1 drained pus specimen,and 15 wound secretion swab specimens.Each specimen was divided into two parts,which were subjected for pathogen detection using microbial culture method and mNGS method,respectively.The number and types of pathogens detected by the 2 methods and the relative abundance detected by the mNGS method were recorded,and the consistency of the two methods were compared.Data were statistically analyzed with paired Wilcoxon rank sum test.Results With the microbial culture method,no pathogen was detected in 5 of the 23 specimens,while 35 pathogens were detected in the remaining 18 specimens,belonging to 9 species of bacteria and 2 species of fungi.Five specimens had one pathogen while 9 specimens had 2 pathogens and 4 specimens had 3 pathogens detected in each specimen.With the mNGS method,no pathogen was detected in one of the 23 specimens,while 75 pathogens were detected in the remaining 22 specimens,belonging to 28 species of bacteria,3 species of fungi,and 3 species of viruses.Eight specimens had one pathogen,5 specimens had 2 pathogens,2 specimens had 3 pathogens,3 specimens had 4 pathogens,2 specimens had 6 pathogens,and 1 specimen had 7 pathogens,and 1 specimen had 20 pathogens detected in each specimen.The number of pathogens detected in each specimen by microbial culture method was 2(1,2)types,which was significantly less than 2(1,4)types by mNGS method(Z=3.359,P<0.01).In 5 specimens,no bacteria were detected by microbial culture method but mNGS method detected bacteria in 2 specimens and virus in 2 different specimens.The mNGS method detected two or more types of bacteria in 13 specimens,the relative abundance of bacteria with the 1st relative abundance ranking ranged from 28.8%to 95.9%in each specimen.Of the 23 specimens detected by two detection methods,7 specimens(30.4%)showed identical detection results,5 specimens(21.7%)showed totally different detection results,and 11 specimens(47.8%)had partially consistent detection results.Conclusions Compared with the traditional microbial culture method,the mNGS method has higher detection sensitivity and stronger capacity to detect pathogens,and can determine the relative abundance of pathogens in mixed infections.As a supplement to the culture method,the mNGS method is expected to play an important role in the diagnosis of infectious pathogens in burns and acute or chronic wounds.