支气管肺泡灌洗液纳米孔测序对涂片阴性肺结核诊断价值的验证性研究

doi:10.19982/j.issn.1000-6621.20230036

摘要/Abstract

摘要： 目的验证纳米孔测序技术用于疑似肺结核患者支气管肺泡灌洗液样本的诊断价值。方法采集2021年11月至2022年4月首都医科大学附属北京胸科医院就诊的50例符合入组标准的涂片阴性疑似肺结核患者的支气管肺泡灌洗液作为研究样本,对其进行BACTEC MGIT 960液体培养(简称“MGIT 960培养”)、GeneXpert MTB/RIF(简称“Xpert”)和纳米孔测序。以临床最终诊断为参照标准,比较3种方法对诊断肺结核的检测效能。结果 50例疑似肺结核患者中,最终确诊肺结核22例(44.0%)、非结核分枝杆菌病10例(20.0%)和细菌性肺炎18例(36.0%)。以最终临床诊断为参照标准,纳米孔测序测定技术、MGIT 960培养和Xpert检测的敏感度分别为72.7%(16/22)、27.3%(6/22)和31.8%(7/22),特异度分别为78.6%(22/28)、75.0%(21/28)和96.4%(27/28),符合率分别为76.0%(38/50)、54.0%(27/50)和68.0%(34/50),约登指数分别为0.51、0.02和0.28。结论纳米孔测序检测BALF样本具有较Xpert和MGIT 960培养更好的诊断效能,可提升疑似肺结核患者BALF的阳性检出率,但不能完全排除肺结核。

关键词: 结核,肺, 支气管肺泡灌洗液, 纳米技术, 分子诊断技术, 对比研究

Abstract:

Objective: To determine the diagnostic accuracy of a nanopore sequencing assay for testing of bronchoalveolar lavage fluid (BALF) samples from suspected pulmonary tuberculosis (PTB) patients. Methods: Fifty cases with suspected PTB from Beijing Chest Hospital from November 2021 to April 2022 were collected. These cases were diagnosed based on results of MGIT 960 culture, GeneXpert MTB/RIF testing and nanopore sequencing of BALF samples collected during hospitalization. Taking the final clinical diagnosis as the reference standard, diagnostic accuracies of the three assays were compared. Results: Among the 50 cases analysed in this study, 22 (44.0%) were diagnosed as tuberculosis, 10 (20.0%) non-tuberculous mycobacteria and 18 (36.0%) bacterial pneumonia. Taking the clinic diagnosis as the reference standard, the sensitivity of nanopore sequencing assay technology, MGIT 960 culture and Xpert assay were 72.7% (16/22), 27.3% (6/22) and 31.8% (7/22), respectively, the specificity were 78.6% (22/28), 75.0% (21/28) and 96.4% (27/28), respectively, the accuracy were 76.0% (38/50), 54.0% (27/50) and 68.0% (34/50), respectively, and the Yoden index was 0.51, 0.02 and 0.28, respectively. Conclusion: Nanopore sequencing assay of BALF samples may have better diagnostic performance than Xpert and MGIT 960 cultures, and may improve the positive detection rate of BALF in suspected PTB patients. But this method cannot exclude pulmonary tuberculosis.

Key words: Tuberculosis, pulmonary, Bronchoalveolar lavage fluid, Nanotechnology, Molecular diagnostic techniques, Comparative study

中图分类号:

R521
R446

闫晓婧, 王庆枫, 杨扬, 初乃惠, 聂文娟. 支气管肺泡灌洗液纳米孔测序对涂片阴性肺结核诊断价值的验证性研究[J]. 中国防痨杂志, 2023, 45(5): 487-492. doi: 10.19982/j.issn.1000-6621.20230036

Yan Xiaojing, Wang Qingfeng, Yang Yang, Chu Naihui, Nie Wenjuan. Diagnostic value of a nanopore sequencing assay of bronchoalveolar lavage fluid in smear-negative pulmonary tuberculosis[J]. Chinese Journal of Antituberculosis, 2023, 45(5): 487-492. doi: 10.19982/j.issn.1000-6621.20230036

图/表 1

参考文献 26

[1]	World Health Organization.Global tuberculosis report 2021. Geneva: World Health Organization, 2021.
[2]	World Health Organization. Automated Real-Time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifampicin Resistance: Xpert MTB/RIF Assay for the Diagnosis of Pulmonary and Extrapulmonary TB in Adults and Children. Geneva:World Health Organization, 2013.
[3]	Gliddon HD, Frampton D, Munsamy V, et al. A Rapid Drug Resistance Genotyping Workflow for Mycobacterium tuberculosis, Using Targeted Isothermal Amplification and Nanopore Sequencing. Microbiol Spectr, 2021, 9(3): e0061021. doi:10.1128/Spectrum.00610-21. doi: 10.1128/Spectrum.00610-21 URL
[4]	Liu Z, Yang Y, Wang Q, et al. Diagnostic value of a nanopore sequencing assay of bronchoalveolar lavage fluid in pulmonary tuberculosis. BMC Pulm Med, 2023, 23(1):77. doi:10.1186/s12890-023-02337-3. doi: 10.1186/s12890-023-02337-3
[5]	中华人民共和国国家卫生和计划生育委员会.WS 288—2017肺结核诊断. 2017-11-09.
[6]	Votintseva AA, Bradley P, Pankhurst L, et al. Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples. J Clin Microbiol, 2017, 55(5): 1285-1298. doi:10.1128/JCM.02483-16. doi: 10.1128/JCM.02483-16 pmid: 28275074
[7]	De Coster W, D’Hert S, Schultz DT, et al. NanoPack: visua-lizing and processing long read sequencing data. Bioinforma-tics, 2018, 34(15): 2666-2669. doi:10.1093/bioinformatics/bty149. doi: 10.1093/bioinformatics/bty149
[8]	Hill JT, Demarest BL, Bisgrove BW, et al. Poly peak parser: Method and software for identifification of unknown indels using sanger sequencing of polymerase chain reaction products. Dev Dyn, 2014, 243(12):1632-1636. doi:10.1002/dvdy.24183. doi: 10.1002/dvdy.24183 URL
[9]	Chen L, Cai Y, Zhou G, et al. Rapid Sanger sequencing of the 16S rRNA gene for identifification of some common pathogens. PLoS One, 2014, 9(2): e88886. doi:10.1371/journal.pone.0088886. doi: 10.1371/journal.pone.0088886 URL
[10]	Tewari D, Cieply S, Livengood J. Identifification of bacteria recovered from animals using the 16S ribosomal RNA gene with pyrosequencing and Sanger sequencing. J Vet Diagn Invest, 2011, 23(6):1104-1108. doi:10.1177/1040638711425583. doi: 10.1177/1040638711425583 URL
[11]	Jarvie T. Next generation sequencing technologies. Drug Discov Today Technol, 2005, 2(3): 255-260. doi:10.1016/j.ddtec.2005.08.003. doi: 10.1016/j.ddtec.2005.08.003 URL
[12]	Voelkerding KV, Dames SA, Durtschi JD. Next-generation sequencing: from basic research to diagnostics. Clin Chem, 2009, 55(4): 641-658. doi:10.1373/clinchem.2008.112789. doi: 10.1373/clinchem.2008.112789 pmid: 19246620
[13]	Simner PJ, Miller S, Carroll KC. Understanding the Promises and Hurdles of Metagenomic Next-Generation Sequencing as a Diagnostic Tool for Infectious Diseases. Clin Infect Dis, 2018, 66(5):778-788. doi:10.1093/cid/cix881. doi: 10.1093/cid/cix881 pmid: 29040428
[14]	Wilson MR, Naccache SN, Samayoa E, et al. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med, 2014, 370(25): 2408-2417. doi:10.1056/NEJMoa1401268. doi: 10.1056/NEJMoa1401268 URL
[15]	Satta G, Lipman M, Smith GP, et al. Mycobacterium tuberculosis and whole-genome sequencing: how close are we to unleashing its full potential? Clin Microbiol Infect, 2018, 24(6): 604-609. doi:10.1016/j.cmi.2017.10.030. doi: 10.1016/j.cmi.2017.10.030 URL
[16]	van Ingen J, Kohl TA, Kranzer K, et al. Global outbreak of severe Mycobacterium chimaera disease after cardiac surgery: a molecular epidemiological study. Lancet Infect Dis, 2017, 17(10): 1033-1041. doi:10.1016/S1473-3099(17)30324-9. doi: S1473-3099(17)30324-9 pmid: 28711585
[17]	Wang L, Liu D, Yung L, et al. Co-Infection with 4 Species of Mycobacteria Identified by Using Next-Generation Sequencing. Emerg Infect Dis, 2021, 27(11): 2948-2950. doi:10.3201/eid2711.203458. doi: 10.3201/eid2711.203458 pmid: 34670649
[18]	Zhu H, Zhu M, Lei JH, et al. Metagenomic Next-Generation Sequencing Can Clinch Diagnosis of Non-Tuberculous Mycobacterial Infections: A Case Report. Front Med (Lausanne), 2021, 8:679755. doi:10.3389/fmed.2021.679755. doi: 10.3389/fmed.2021.679755
[19]	Hendrix J, Epperson LE, Durbin D, et al. Intraspecies plasmid and genomic variation of Mycobacterium kubicae revealed by the complete genome sequences of two clinical isolates. Microb Genom, 2021, 7(1): mgen000497. doi:10.1099/mgen.0.000497. doi: 10.1099/mgen.0.000497
[20]	Stefani MMA, Avanzi C, Bührer-Sékula S, et al. Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases. PLoS Negl Trop Dis, 2017, 11(6): e0005598. doi:10.1371/journal.pntd.0005598. doi: 10.1371/journal.pntd.0005598 URL
[21]	Laver T, Harrison J, O’Neill PA, et al. Assessing the performance of the Oxford Nanopore Technologies MinION. Biomol Detect Quantif, 2015, 3:1-8. doi:10.1016/j.bdq.2015.02.001. doi: 10.1016/j.bdq.2015.02.001 URL
[22]	Alvarez JR, Skachkov D, Massey SE, et al. DNA/RNA transverse current sequencing: intrinsic structural noise from neighboring bases. Front Genet, 2015, 6: 213. doi:10.3389/fgene.2015.00213. doi: 10.3389/fgene.2015.00213 pmid: 26150827
[23]	Rang FJ, Kloosterman WP, de Ridder J. From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy. Genome Biol, 2018, 19(1): 90. doi:10.1186/s13059-018-1462-9. doi: 10.1186/s13059-018-1462-9 pmid: 30005597
[24]	Serpa PH, Deng X, Abdelghany M, et al. Metagenomic prediction of antimicrobial resistance in critically ill patients with lower respiratory tract infections. Genome Med, 2022, 14(1):74. doi:10.1186/s13073-022-01072-4. doi: 10.1186/s13073-022-01072-4 pmid: 35818068
[25]	Wang D, Wang W, Ding Y, et al. Metagenomic Next-Generation Sequencing Successfully Detects PulmonaryInfectious Pathogens in Children With Hematologic Malignancy. Front Cell Infect Microbiol, 2022, 12: 899028. doi:10.3389/fcimb.2022.899028. doi: 10.3389/fcimb.2022.899028 URL
[26]	Wei J, Sun J, Shuai X, et al. Sensitivity of PCR analysis (melting curve method) in diagnosis of Pulmonary Tuberculosis (PTB) based on Bronchoalveolar Lavage (BAL) by bronchoscope. Pak J Med Sci, 2022, 38(5):1333-1337. doi:10.12669/pjms.38.5.5480. doi: 10.12669/pjms.38.5.5480 pmid: 35799751