Analysis of the characteristics of infectious pathogens in burn patients with sepsis based on metagenomic next-generation sequencing technology
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摘要:
目的 基于宏基因组学第二代测序（mNGS）技术,分析烧伤脓毒症患者感染病原体的特征。 方法 该研究为回顾性观察性研究。2021年7月—2023年12月,郑州市第一人民医院烧伤科收治109例符合入选标准的烧伤脓毒症患者,其中男68例,年龄57~92岁；女41例,年龄48~83岁。采集患者住院期间的血液、支气管肺泡灌洗液、脑脊液、痰液或其他液体标本,分别进行微生物培养（86例患者）和mNGS技术检测（109例患者）。统计采用mNGS技术检测的送检标本类型及检出病原体的情况,同时将患者分为入住重症监护病房（ICU）的ICU组（78例）和未入住ICU的非ICU组（31例）,并对2组患者感染的病原体进行分析。另对同时进行mNGS技术检测和微生物培养检测的86例患者标本的病原体检出情况进行分析。 结果 采用mNGS技术检测的109份标本中,血液标本42份、支气管肺泡灌洗液标本17份、痰液标本4份、脑脊液标本6份、脓液标本16份、组织液标本24份；共检测出39种病原体,其中细菌13种、真菌12种、病毒10种、寄生虫2种、支原体2种；检出病原体的总体阳性率为88.99%（97/109）。检出率排前3位的革兰阴性菌依次是肺炎克雷伯菌、鲍曼不动杆菌、假单胞菌,排前3位的革兰阳性菌依次是肺炎链球菌、金黄色葡萄球菌、粪肠球菌；检出率排前3位的病毒依次是人类疱疹病毒、巨细胞病毒、细环病毒；检出率排前3位的真菌依次是烟曲霉菌、白色念珠菌、黄曲霉菌。27例患者感染1种病原体,45例患者感染2种病原体,25例患者感染≥3种病原体。与非ICU组相比,ICU组患者检出的肺炎克雷伯菌、鲍曼不动杆菌、假单胞菌、肺炎链球菌、烟曲霉菌、巨细胞病毒的占比均明显升高（χ2值分别为8.62、7.93、3.93、5.48、4.28、5.58,P<0.05）。mNGS技术和微生物培养法检出的病原体中,最常见细菌的是肺炎克雷伯菌和鲍曼不动杆菌,最常见的真菌是曲霉菌属菌株和念珠菌属菌株。只能通过mNGS技术检测到的病原体有19种,如分枝横梗霉菌、耶氏肺孢子菌、结核分枝杆菌、病毒等；无采用微生物培养法检测到而采用mNGS技术检测不到的病原体。与采用微生物培养法相比,采用mNGS技术检出病原体的总体阳性率、细菌阳性率、真菌阳性率均显著升高（χ2值分别为45.52、5.88、4.94,P<0.05）。采用2种检测方法同时报告阳性结果的患者占27.91%（24/86）,采用mNGS技术检测结果为阳性而采用微生物培养法检测结果为阴性的患者占72.09%（62/86）。2种检测方法所得结果的一致性检验显示,差异无统计学意义（κ=0.02,P>0.05）。 结论 采用mNGS技术检测标本中病原体的阳性率高于采用常规微生物培养法,且能检测到后者不能检出的病原体,如分枝横梗霉菌、耶氏肺孢子菌、结核分枝杆菌、病毒等。采用mNGS技术检测有助于明确烧伤脓毒症患者感染病原体的种类,为临床用药提供依据和指导。 Abstract:Objective To analyze the characteristics of infectious pathogens in burn patients with sepsis based on metagenomic next-generation sequencing (mNGS) technology. Methods This study was a retrospective observational study. From July 2021 to December 2023, 109 burn patients with sepsis who met the inclusion criteria were admitted to the Department of Burns of the First People's Hospital of Zhengzhou, including 68 males aged 57 to 92 years and 41 females aged 48 to 83 years. Blood, bronchoalveolar lavage fluid, cerebrospinal fluid, sputum, or other fluid specimens were collected from the patients during their hospital stay for microbiological culture (86 patients) and mNGS technology detection (109 patients). The types of specimens and pathogens detected by mNGS technology were counted. Patients were divided into intensive care unit (ICU) group (78 cases) who were admitted to the ICU and non-ICU group (31 cases) who were not admitted to the ICU, and the pathogens for infection in the two groups of patients were analyzed. In addition, the detection of pathogens in the specimens of 86 patients who underwent both mNGS technology detection and microbiological culture detection was analyzed. Results Among the 109 specimens detected by mNGS technology, there were 42 blood specimens, 17 bronchoalveolar lavage fluid specimens, 4 sputum specimens, 6 cerebrospinal fluid specimens, 16 pus specimens, and 24 tissue fluid specimens; a total of 39 pathogens were detected, including 13 bacteria, 12 fungi, 10 viruses, 2 parasites, and 2 mycoplasmas. The overall positive rate of pathogen detection was 88.99% (97/109). Ranked by the detection rate, the top three Gram-negative bacteria were Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas spp, the top three Gram-positive bacteria were Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis; the top three viruses were human herpesvirus, cytomegalovirus, and circovirus; the top three fungi were Aspergillus fumigatus, Candida albicans, and Aspergillus flavus. Twenty-seven patients were infected with one pathogen, 45 patients with two pathogens, and 25 patients with three or more pathogens. Compared with those in non-ICU group, the proportions of Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas spp, Streptococcus pneumoniae, Aspergillus fumigatus, and cytomegalovirus detected in the patients in ICU group were significantly higher (with χ2 values of 8.62, 7.93, 3.93, 5.48, 4.28, and 5.58, respectively, P<0.05). In the pathogens detected by mNGS technology and microbiological culture method, the most common bacteria were Klebsiellapneumoniae and Acinetobacter baumannii, and the most common fungi were strains of Aspergillus and Candida. There were 19 pathogens those could only be detected by mNGS technology, such as Lichtheimia ramosa, Pneumocystis jirovecii, Mycobacterium tuberculosis, viruses, etc.; there were no pathogens detected by microbiological culture method that couldn't be detected by mNGS technology. Compared with those detected by microbiological culture method, the overall positive rate, bacterial positive rate, and fungal positive rate detected by mNGS technology were significantly increased (with χ2 values of 45.52, 5.88, and 4.94, respectively, P<0.05). The 27.91% (24/86) of patients were detected positive by both methods, and 72.09% (62/86) of the patients were detected positive by mNGS technology but negative by microbiological culture method. The consistency test of the results obtained by the two detection methods showed that the difference was not statistically significant (κ=0.02, P>0.05). Conclusions The positive rate of pathogen detection in specimens using mNGS technology is higher than that detected by using conventional microbiological culture method, and it can detect pathogens those cannot be detected by the latter, such as Lichtheimia ramosa, Pneumocystis jirovidii, Mycobacterium tuberculosis, viruses, etc. Detection using mNGS technology can help clarify the types of infectious pathogens in burns patients with sepsis, and provide basis and guidance for clinical medication. -
Key words:
- Burns /
- Infection /
- Sepsis /
- Metagenomics /
- Pathogen
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参考文献
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Table 1. 采用宏基因组学第二代测序技术检测烧伤脓毒症患者的不同类型标本感染病原体的情况（株）
病原体类型与种类 血液 支气管肺泡灌洗液 痰液 脑脊液 脓液 组织液 合计 革兰阴性菌 肺炎克雷伯菌 15 6 2 0 2 0 25 鲍曼不动杆菌 13 0 0 3 0 6 22 假单胞菌 7 5 2 2 2 2 20 铜绿假单胞菌 7 0 0 0 2 3 12 大肠埃希菌 4 0 0 1 0 1 6 产气克雷伯菌 0 0 0 0 1 2 3 革兰阳性菌 肺炎链球菌 6 6 4 0 0 0 16 金黄色葡萄球菌 0 0 3 0 4 3 10 粪肠球菌 0 2 2 0 2 1 7 结核分枝杆菌 0 4 2 0 0 0 6 屎肠球菌 0 2 2 0 0 0 4 鸟肠球菌 0 0 2 0 0 0 2 溶血葡萄球菌 1 1 0 0 0 0 2 病毒 巨细胞病毒 12 2 1 0 2 0 17 人类疱疹病毒 11 0 0 0 7 0 18 细环病毒 7 0 0 0 0 2 9 乙型肝炎病毒 3 0 0 0 0 0 3 单纯疱疹病毒 4 0 0 0 2 0 6 小细环病毒6型 1 0 0 0 0 0 1 人呼吸道病毒 0 1 0 0 0 0 1 默克尔细胞多瘤病毒 0 0 0 0 0 1 1 庚型肝炎病毒 1 0 0 0 0 0 1 人多瘤病毒 0 0 0 1 0 0 1 真菌 烟曲霉菌 4 5 2 1 0 3 15 白色念珠菌 3 4 0 1 2 3 13 黄曲霉菌 7 3 0 1 1 0 12 卷枝毛霉菌 2 2 2 0 1 2 9 耶氏肺孢子菌 1 5 1 0 0 0 7 热带念珠菌 2 2 1 0 0 1 6 德氏根霉菌 2 1 0 0 0 0 3 近平滑念珠菌 2 1 0 0 0 0 3 分枝横梗霉菌 1 0 0 0 0 1 2 黑曲霉菌 0 0 0 1 0 1 2 匍枝根霉菌 0 0 0 0 0 1 1 阿萨希毛孢子菌 1 0 0 0 0 0 1 其他病原体 寄生虫/支原体 1 1 1 1 0 0 4 合计 118 53 27 12 28 33 271 
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Table 2. 采用宏基因组学第二代测序技术检测2组烧伤脓毒症患者的病原体情况（株）
组别 样本数 肺炎克雷伯菌 鲍曼不动杆菌 假单胞菌 铜绿假单胞菌 大肠埃希菌 肺炎链球菌 金黄色葡萄球菌 粪肠球菌 结核分枝杆菌 屎肠球菌 非ICU组 31 8 6 8 4 2 5 2 2 1 1 ICU组 78 17a 16a 12a 8 4 11a 8 4 5 2 注：ICU指重症监护病房；其他包括产气克雷伯菌、鸟肠球菌、溶血葡萄球菌、乙型肝炎病毒、单纯疱疹病毒、小细环病毒6型、人呼吸道病毒、默克尔细胞多瘤病毒、庚型肝炎病毒、人多瘤病毒、卷枝毛霉菌、热带念珠菌、德氏根霉菌、近平滑念珠菌、分枝横梗霉菌、匍枝根霉菌、阿萨希毛孢子菌、班氏血丝虫、疟原虫、人型支原体、解脲支原体；与非ICU组比较,aP<0.05
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Table 3. 采用2种方法检出烧伤脓毒症患者的病原体情况（株）
检测方法 样本数 肺炎克雷伯菌 鲍曼不动杆菌 假单胞菌 铜绿假单胞菌 大肠埃希菌 肺炎链球菌 金黄色葡萄球菌 粪肠球菌 结核分枝杆菌 屎肠球菌 溶血葡萄球菌 mNGS技术 86 25 22 20 12 6 16 10 7 6 4 2 微生物培养 86 4 4 2 1 3 3 2 1 0 2 1 注：mNGS指宏基因组学第二代测序；其他包括产气克雷伯菌、乙型肝炎病毒、单纯疱疹病毒、小细环病毒6型、人呼吸道病毒、默克尔细胞多瘤病毒、班氏血丝虫、疟原虫、人型支原体、解脲支原体等,曲霉菌属包括黄曲霉菌、烟曲霉菌、黑曲霉菌,念珠菌属包括白色念珠菌、热带念珠菌、近平滑念珠菌,毛霉属包括卷枝毛霉菌、分枝横梗霉菌,孢子菌属包括耶氏肺孢子菌、阿萨希毛孢子菌,根霉属包括匍枝根霉菌、德氏根霉菌
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