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J Zhejiang Univ (Med Sci)  2021, Vol. 50 Issue (6): 748-754    DOI: 10.3724/zdxbyxb-2021-0158
    
Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19
JIA Xiaofang1,ZHANG Xiaonan1,LING Yun2,ZHANG Xinyu1,TIAN Di1,LIAO Yixin1,YI Zhigang1,LU Hongzhou3,*()
1. Scientific Research Department, Shanghai Public Health Clinical Center, Shanghai 201508, China;
2. Infectious Disease Department, Shanghai Public Health Clinical Center, Shanghai 201508, China;
3. Department of Infection and Immunology, Shanghai Public Health Clinical Center, Shanghai 201508, China
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Abstract  

Objective:To explore the application value of nanopore sequencing technique 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 collected. After heat inactivation, all samples were subjected to total nucleic acid extraction based on magnetic bead enrichment. The extracted DNA was used for DNA library construction, then nanopore real-time sequencing detection was performed. The sequencing data were subjected to Centrifuge software database species matching and R program differential analysis to obtain potential pathogen identification. Nanopore sequencing results were compared with respiratory pathogen qPCR panel screening and conventional microbiological testing results to verify the effectiveness of nanopore sequencing detection. Results: Nanopore sequencing results showed that positive pathogen were obtained in 44 specimens (57.1%). The potential pathogens identified by nanopore sequencing included Klebsiella pneumoniae, Staphylococcus epidermidis, Enterococcus faecium and Dolosigranulum pigrum, et al. Klebsiella pneumoniae, Staphylococcus epidermidis, Enterococcus faecium were also detected in clinical microbiological culture-based detection; Klebsiella pneumoniae was detected in respiratory pathogen screening qPCR panel; Dolosigranulum pigrum was only detected by the nanopore sequencing technique. Comprehensive considerations with the clinical symptoms, the patient was treated with antibiotics against Dolosigranulum pigrum, and the infection was controlled. Conclusion:Nanopore sequencing may assist the diagnosis and treatment of severe COVID-19 patients through rapid identification of potential pathogens.



Key wordsCoronavirus disease 2019      Severe acute respiratory syndrome coronavirus 2      Nanopore sequencing      Metagenomics next-generation sequencing      Pathogen detection     
Received: 05 June 2021      Published: 22 March 2022
CLC:  R372  
Corresponding Authors: LU Hongzhou     E-mail: luhongzhou@fudan.edu.cn
Cite this article:

JIA Xiaofang,ZHANG Xiaonan,LING Yun,ZHANG Xinyu,TIAN Di,LIAO Yixin,YI Zhigang,LU Hongzhou. Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19. J Zhejiang Univ (Med Sci), 2021, 50(6): 748-754.

URL:

https://www.zjujournals.com/med/10.3724/zdxbyxb-2021-0158     OR     https://www.zjujournals.com/med/Y2021/V50/I6/748


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

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定量聚合酶链反应(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%),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。


关键词: 2019冠状病毒病,  严重急性呼吸综合征冠状病毒2,  纳米孔测序,  宏基因组测序,  病原体检测 
Figure 1 Workflow diagram of the metagenomics next-generation sequencing assay based on nanopore sequencing

标本编号

患者编号

采集日期

标本类型

测序检出的

病原体名称

匹配总序列数

log2FC

序列覆盖率(%)

平均

覆盖深度

最大

覆盖深度

1

P1

2021年1月31日

痰液

黏滑罗斯菌

720

7.87

67.56

2.73

49

殊异韦荣菌

1401

4.94

36.09

0.10

71

2

P1

2021年2月16日

血浆

表皮葡萄球菌

3251

6.58

1.74

5.01

419

3

P2

2021年1月8日

肺泡灌洗液

嗜麦芽窄食单胞菌

113

3.50

3.32

0.06

8

4

P2

2021年1月8日

血浆

洛菲不动杆菌

1137

8.60

14.47

159.01

1110

5

P2

2021年1月12日

血浆

黄体微球菌

1968

6.66

0.28

0.65

390

6

P2

2021年1月17日

血浆

狡诈菌

4216

5.25

1.29

10.20

1762

7

P2

2021年1月27日

肺泡灌洗液

肺炎克雷伯菌

20?019

3.54

3.01

13.20

1561

8

P2

2021年1月31日

血浆

奥斯陆莫拉菌

2241

6.63

4.60

2.34

221

小韦荣球菌

570

无穷大

0.70

0.38

122

9

P3

2021年2月1日

痰液

屎肠球菌

36?863

5.02

6.20

24.83

3422

10

P3

2021年2月4日

痰液

纤细弯曲杆菌

550

9.97

0.30

1.61

543

11

P3

2021年2月12日

血浆

抗辐射不动杆菌

256

无穷大

11.17

7.01

144

12

P3

2021年2月16日

痰液

人γ疱疹病毒4

137

无穷大

47.70

2.93

30

Table 1 Potential pathogens identified by nanopore sequencing in 77 clinical samples from 3 patients with severe COVID-19
Figure 2 qPCR panel and nanopore sequencing results of severe COVID-19 patient samples
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