宏基因二代测序技术对感染性疾病患者的诊断价值及其临床应用

目的利用宏基因二代测序(mNGS)技术,检测其和传统培养法的区别,探讨病原微生物及检出序列数与感染患者病情程度和预后的相关性。方法选取2018年5月至2019年9月在上海市第十人民医院就诊的94例患者样本。分别进行传统培养和mNGS检测,对2种技术的诊断效果、假阳性率、假阴性率进行了分析。然后收集感染组75例患者的血液标本和临床信息,根据mNGS是否检出病原体将患者分为检出组(51例)和未检出组(24例)。测定外周血白细胞、C反应蛋白、降钙素原和中性粒细胞计数,用酶联免疫吸附试验法测定血清中细胞因子的浓度。分析患者病原体检出率与病情严重程度、住院时间等相关性分析。结果纳入的94例样本中,感染组75例(7979%),非感染组16例(1702%),未知组3例(319%)。样本类型中血液占比最多(34/94),其次为肺泡灌洗液(28/94)、组织、痰液、胸水、脓液等。下呼吸道感染所占比例最高(60/94),其次是血流感染、胸水、中枢系统感染、心血管系统感染等。mNGS的敏感度显著高于培养法(χ^2=-223.107,P<005)。mNGS的特异度与培养法差异无统计学意义(χ^2=3.840,P=005)。感染未检出组患者住院时间也明显高于检出组(t=4.724,P<005)。单因素分析发现检出组患者外周血白细胞计数、中性粒细胞计数、C反应蛋白、降钙素原显著高于未检出组(t值分别为-8.848、-4.724、-4.335、-4.335,P值均<005);检出组中肿瘤坏死因子α、白细胞介素2R、白细胞介素8水平均高于未检出组(t值分别为-5.585、-7.258、-4.678,P值均<005)。logistic多因素回归分析去除混杂因素后,显示性别、年龄、白细胞介素2R与病原体检出阳性有关。结论mNGS在结核、真菌、病毒和厌氧菌诊断方面优势更明显,其阳性率高于传统方法3倍以上。感染患者的病原体检出率较高,其检出病原体核酸序列是患者预后不良的高危因素,有显著的临床价值。

Diagnostic value and clinical application of metagenomic next-generation sequencing technology in infected patients

Objective To detect the difference between metagenomic next generation sequencing(niNGS)technology and traditional culture methods,and explore the relationship between read number of virus and the severity,prognosis of infectious patients.Methods The samples of 94 patients in Shanghai Tenth People's Hospital of Tongji University from May 2018 to September 2019 were enrolled.The diagnostic results,false positive rate,false negative rate of traditional culture and mNGS tests were analyzed.Then,the blood samples and clinical information of 75 patients in the infected group were collected.According to whether mNGS detected pathogens,the patients were divided into detection group(51 eases)and undetected group(24 eases).Peripheral white blood cells,C-reactive protein,pro calcitonin and neutrophil counts were measured,and the concentration of cytokines in the scrum was determined by enzyme linked immunosorbent assay.The correlation between the detection rate of pathogens and the severity of disease,hospital stay was analyzed.Results In 94 samples,75 eases(79.79%)were infected,16 eases(17.02%)were non infected,and three eases(3.19%)were unknown.On the sample type,the proportion of blood was the highest(34 eases),followed by alveolar lavage fluid(28 eases),tissue,sputum,pleural effusion,pus and so on.The majority of patients were diagnosed with lower respiratory system infections(60/94),followed by bloodstream infections,pleural effusion,central nervous system infections,and cardiovascular system infections.The sensitivity of mN(jS was significantly higher than that of culture method(X^2=-223.107,P<0.05).There was no significant difference in the specificity between mNGS and culture method(X^2=3.840,P=0.05).The hospital stay in the undetected group was also significantly higher than that in the detected group(t=4.724,P<0.05),One way analysis of variance revealed that white blood cells,neutrophil count,preactive protein,and procalcitonin in the detected group were significantly higher than those in the undetected group(t=-8.848,-4.724,-4.335,-4.335,all P<0.05).Furthermore,the levels of pro inflammatory cytokines(tumor necrosis factor ct,interleukin 2R,interleukin 8)in the detected group were significantly higher than those in the undetected group(t=-5.585,-7.258,-4,678,all P<0.05).The multivariate logistic analysis showed that gender,age,interleukin 2R were related with positive detection of pathogens.Conclusions mNGS has more obvious advantages in the diagnosis of tuberculosis,fungi,viruses and anaerobic bacteria,and its positive rate is more than three times higher than the traditional method.The detection rate of pathogens in infected patients is high,and the detection of pathogen nucleic acid scqucnccs is a high risk factor for poor prognosis of patients and has significant clinical value.