Application of metagenomic next-generation sequencing technology in pathogen detection in patients with burns and patients with acute or chronic wounds
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摘要:
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。 Abstract: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. -
Key words:
- Metagenome /
- High-throughput nucleotide sequencing /
- Infection /
- Burns
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参考文献
(36) [1] ManningJ. Sepsis in the burn patient[J]. Crit Care Nurs Clin North Am, 2018, 30(3):423-430. DOI: 10.1016/j.cnc.2018.05.010. [2] ZhangPJ, ZouBW, LiouYC, et al. The pathogenesis and diagnosis of sepsis post burn injury[J/OL]. Burns Trauma, 2021, 9:tkaa047[2021-05-30]. https://pubmed.ncbi.nlm.nih.gov/33654698/.DOI: 10.1093/burnst/tkaa047. [3] PierrakosC,VincentJL.Sepsis biomarkers: a review[J].Crit Care,2010,14(1):R15.DOI: 10.1186/cc8872. [4] FanSL, MillerNS, LeeJ, et al. Diagnosing sepsis - the role of laboratory medicine[J]. Clin Chim Acta, 2016, 460: 203-210. DOI: 10.1016/j.cca.2016.07.002. [5] LloydKG, SteenAD, LadauJ, et al. Phylogenetically novel uncultured microbial cells dominate earth microbiomes[J]. mSystems, 2018, 3(5): e00055-18. DOI: 10.1128/mSystems.00055-18. [6] 张敏,李伯安,邱广斌.下一代测序技术在感染性疾病检测上的临床应用[J].中华检验医学杂志,2017,40(7):492-494.DOI: 10.3760/cma.j.issn.1009-9158.2017.07.004. [7] 张坚磊,马小军.感染性疾病病原学诊断新技术与临床应用策略[J].协和医学杂志,2018,9(5):399-403.DOI: 10.3969/j.issn.1674-9081.2018.05.004. [8] GuW, MillerS, ChiuCY. Clinical metagenomic next-generation sequencing for pathogen detection[J]. Annu Rev Pathol, 2019, 14: 319-338. DOI: 10.1146/annurev-pathmechdis-012418-012751. [9] 邓呈亮, 詹日兴, 刘洋, 等.成年大面积烧伤死亡患者的细菌感染及耐药情况分析[J].中华烧伤杂志, 2016, 32(11): 688-691.DOI: 10.3760/cma.j.issn.1009-2587.2016.11.011. [10] 柴家科. 实用烧伤外科学[M]. 北京: 人民军医出版社, 2014: 218. [11] BloemsmaGC, DokterJ, BoxmaH, et al. Mortality and causes of death in a burn centre[J]. Burns, 2008, 34(8): 1103-1107. DOI: 10.1016/j.burns.2008.02.010. [12] LavrentievaA, VoutsasV, KonoglouM, et al. Determinants of outcome in burn ICU patients with septic shock[J]. J Burn Care Res, 2017, 38(1): e172-e179. DOI: 10.1097/BCR.0000000000000337. [13] SingerM, DeutschmanCS, SeymourCW, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3)[J]. JAMA, 2016, 315(8): 801-810. DOI: 10.1001/jama.2016.0287. [14] 中国医师协会急诊医师分会, 中国研究型医院学会休克与脓毒症专业委员会. 中国脓毒症/脓毒性休克急诊治疗指南(2018)[J]. 感染、炎症、修复, 2019, 20(1): 3-22. DOI: 10.3969/j.issn.1672-8521.2019.01.001. [15] DeurenbergRH,BathoornE,ChlebowiczMA,et al.Application of next generation sequencing in clinical microbiology and infection prevention[J].J Biotechnol,2017,243:16-24.DOI: 10.1016/j.jbiotec.2016.12.022. [16] MwaigwisyaS, AssiriRAM, O'GradyJ. Emerging commercial molecular tests for the diagnosis of bloodstream infection[J]. Expert Rev Mol Diagn, 2015, 15(5): 681-692. DOI: 10.1586/14737159.2015.1029459. [17] LongY, ZhangYX, GongYP, et al. Diagnosis of sepsis with cell-free DNA by next-generation sequencing technology in ICU patients[J]. Arch Med Res, 2016, 47(5): 365-371. DOI: 10.1016/j.arcmed.2016.08.004. [18] 王凯飞, 沈定霞, 刘朝军, 等. 血清降钙素原定量检测与血培养结果的比较[J]. 中华检验医学杂志, 2012, 35(3): 243-246. DOI: 10.3760/cma.j.issn.1009-9158.2012.03.011. [19] WarhurstG, DunnG, ChadwickP, et al. Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review[J]. Health Technol Assess, 2015, 19(35): 1-142. DOI: 10.3310/hta19350. [20] BrennerT, DeckerSO, GrumazS, et al. Next-generation sequencing diagnostics of bacteremia in sepsis (Next GeneSiS-Trial): study protocol of a prospective, observational, noninterventional, multicenter, clinical trial[J]. Medicine (Baltimore), 2018, 97(6): e9868. DOI: 10.1097/MD.0000000000009868. [21] HuHL, GuoLY, WuHL, et al. Evaluation of next-generation sequencing for the pathogenic diagnosis of children brain abscesses[J]. J Infect, 2019, 78(4):323-337. DOI: 10.1016/j.jinf.2019.01.003. [22] 张军民, 周贵民. 厌氧菌血培养仍是值得重视的问题[J]. 中华检验医学杂志, 2005, 28(10): 979-980. DOI: 10.3760/j:issn:1009-9158.2005.10.002. [23] 洪秀华. 病毒感染的实验诊断方法及其价值[J]. 诊断学理论与实践, 2007, 6(3): 180-184. DOI: 10.3969/j.issn.1671-2870.2007.03.002. [24] ComarM, D'AccoltiM, CasonC, et al. Introduction of NGS in environmental surveillance for healthcare-sssociated infection control[J]. Microorganisms, 2019, 7(12):708. DOI: 10.3390/microorganisms7120708. [25] 柯创宏, 韩焕钦, 杨天骄, 等. 高通量测序技术检测支气管肺泡灌洗液在婴幼儿肺炎病原学诊断中的价值[J]. 中华实用诊断与治疗杂志, 2020, 34(4): 398-400. DOI: 10.13507/j.issn.1674-3474.2020.04.020. [26] 胡晓熠, 汪明, 王惠明. 高通量测序技术在尿路感染及腹膜透析相关腹膜炎病原学诊断中的价值[J]. 中华实用诊断与治疗杂志, 2021, 35(3): 230-233. DOI: 10.13507/j.issn.1674-3474.2021.03.004. [27] ChenPX, SunWW, HeYY. Comparison of the next-generation sequencing (NGS) technology with culture methods in the diagnosis of bacterial and fungal infections[J]. J Thorac Dis, 2020, 12(9): 4924-4929. DOI: 10.21037/jtd-20-930. [28] YinH, XuDL, WangDW. Diagnostic value of next-generation sequencing to detect periprosthetic joint infection[J]. BMC Musculoskelet Disord, 2021, 22(1): 252. DOI: 10.1186/s12891-021-04116-9. [29] GalazzoG, van BestN, BenedikterBJ, et al. How to count our microbes? The effect of different quantitative microbiome profiling approaches[J]. Front Cell Infect Microbiol, 2020, 10: 403. DOI: 10.3389/fcimb.2020.00403. [30] 中华医学会检验医学分会临床微生物学组, 中华医学会微生物学与免疫学分会临床微生物学组, 中国医疗保健国际交流促进会临床微生物与感染分会. 宏基因组高通量测序技术应用于感染性疾病病原检测中国专家共识[J]. 中华检验医学杂志, 2021, 44(2):107-120. DOI: 10.3760/cma.j.cn114452-20201026-00794. [31] 《中华传染病杂志》编辑委员会.中国宏基因组学第二代测序技术检测感染病原体的临床应用专家共识[J].中华传染病杂志,2020,38(11):681-689.DOI: 10.3760/cma.j.cn311365-20200731-00732. [32] 于威, 孟冬梅. 微生物学及检验技术[M]. 哈尔滨: 黑龙江科学技术出版社, 2008: 138. [33] 梁振山, 曾令兵, 万腊根. 基因组测序技术及其临床应用研究进展[J]. 实验与检验医学, 2016, 34(1): 48-51. DOI: 10.3969/j.issn.1674-1129.2016.01.016. [34] 黄晶晶, 肖盟, 徐英春. 二代测序技术在微生物和感染性疾病中的应用[J]. 协和医学杂志, 2018, 9(5): 448-452. DOI: 10.3969/j.issn.1674-9081.2018.05.014. [35] 杨继勇. 高通量测序技术在感染病原检测中的应用与展望[J]. 中华检验医学杂志, 2020, 43(5): 533-539. DOI: 10.3760/cma.j.cn114452-20190719-00438. [36] WilsonMR, NaccacheSN, SamayoaE, et al.Actionable diagnosis of neuroleptospirosis by next-generation sequencing[J].N Engl J Med, 2014, 370(25): 2408-2417. DOI: 10.1056/NEJMoal401268. -
表1 微生物培养法和mNGS法在11例烧伤患者和急慢性创面患者的23份标本中检出的微生物株数比较（株）
检测方法 铜绿假单胞菌 金黄色葡萄球菌 肺炎克雷伯菌 鲍曼不动杆菌 奇异变形杆菌 嗜麦芽窄食单胞菌 纹带棒状杆菌 粪产碱杆菌 摩氏摩根菌 微生物培养法 6 5 7 5 5 2 1 1 1 mNGS法 8 8 7 8 6 2 3 1 1 注：mNGS为宏基因组学二代测序；其他包括海分枝杆菌、不解糖卟啉单胞菌、微小微单胞菌、化脓隐秘杆菌、咽峡炎链球菌、斯氏普鲁威登菌、鸟肠球菌、无乳链球菌、耐药棒状杆菌、威隆气单胞菌、纽氏放线菌、黏质沙雷菌、化脓性链球菌、肠道沙门菌、毛霉菌、人类疱疹病毒、单纯疱疹病毒、人细小病毒B19 
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