纳米孔测序技术在2019冠状病毒病重型患者 继发感染诊断中的应用

Objective: To explore the application value of nanopore sequencing technology in the diagnosis and treatment of secondary infections in patients with severe coronavirus disease 2019 (COVID - 19). Methods: A total of 77 clinical specimens from 3 patients with severe COVID - 19 were enrolled. All specimens were heat - inactivated and then total nucleic acid was extracted based on magnetic bead enrichment, and a DNA library was constructed. Pathogen detection was carried out by nanopore sequencing technology. The sequencing data were compared with the pathogen database by Centrifuge software and analyzed for differences by R language to obtain the identification results of pathogenic microorganisms. The nanopore sequencing results were compared with the results of multiplex fluorescence quantitative PCR for respiratory pathogen screening and the clinical microbiological test results in the same period to verify the effectiveness of the nanopore sequencing technology. Results: The nanopore sequencing results showed that pathogens were detected in 44 specimens (the positive rate was 57.1%), and the pathogens included Klebsiella pneumoniae, Staphylococcus epidermidis, Enterococcus faecium and Dolosigranulum spp., etc. Among them, Klebsiella pneumoniae, Staphylococcus epidermidis and Enterococcus faecium were all detected in the clinical microbiological test based on microbial culture; Klebsiella pneumoniae was also detected in the multiplex quantitative PCR test for respiratory pathogen screening; Dolosigranulum spp. was only detected in the nanopore sequencing. Considering the clinical symptoms of the patients, antibacterial drug treatment was carried out, and the infection situation of the patients was controlled. Conclusion: Nanopore sequencing can rapidly identify potential pathogens in clinical specimens and assist in the clinical diagnosis and formulation of treatment plans for patients with severe COVID - 19.

目的：探讨纳米孔测序技术在2019冠状病毒病（COVID-19）重型患者继.发感染诊疗中的应用价值。方法：共入组了来自3例COVID-19重型患者的77份临.床标本，所有标本均经过热灭活后进行基于磁珠富集的总核酸抽提，构建DNA文.库，通过纳米孔测序技术进行病原体检测。测序数据采用Centrifuge软件进行病原.体数据库比对和R语言差异分析，以获得病原微生物的鉴定结果。比较纳米孔测序.结果与呼吸道病原体筛查多重荧光定量PCR检测及同一时期临床微生物检验结.果，以验证纳米孔测序技术的有效性。结果：纳米孔测序结果显示，44份标本检出.病原体（阳性率为57.1%），病原体包括肺炎克雷伯菌、表皮葡萄球菌、屎肠球菌.和狡诈菌等。其中，肺炎克雷伯菌、表皮葡萄球菌和屎肠球菌均在基于微生物培.养的临床微生物检验中检出；肺炎克雷伯菌同时在呼吸道病原体筛查多重定量.PCR检测中检出；狡诈菌仅在纳米孔测序中检出，综合考虑患者的临床症状进行抗.菌药物治疗，患者的感染情况得到控制。结论：纳米孔测序可快速鉴定临床标本中.的潜在病原体，辅助COVID-19重型患者的临床诊断和治疗方案的制订。