宏基因组纳米孔测序在结核浆液性积液诊断及合并感染筛查中的应用价值

doi:10.19982/j.issn.1000-6621.20250386

摘要/Abstract

摘要：

目的: 探究宏基因组纳米孔测序(简称“纳米孔测序”)技术在结核浆液性积液诊断中的应用价值,并评估其在快速、精准鉴定结核分枝杆菌及筛查合并感染病原体方面的优势。方法: 纳入2021年10月至2025年5月在南宁市第四人民医院就诊的疑似结核浆液性积液患者,采集其积液样本进行纳米孔测序,以最终临床诊断结果为参考标准,与传统检测方法(包括抗酸染色、TB-DNA检测、GeneXpert MTB/RIF和分枝杆菌固体培养)进行对比分析。结果: 纳米孔测序技术的敏感度(73.1%,19/26)、特异度(100.0%,27/27)、阳性预测值(100.0%,19/19)、阴性预测值(79.4%,27/34)、Kappa值(0.734)的总体诊断效能优于抗酸染色[敏感度为11.5%(3/26)、特异度为100.0%(27/27)、阳性预测值为3/3、阴性预测值为54.0%(27/50)、Kappa值为0.117]、培养[敏感度为30.8%(8/26)、特异度为100.0%(27/27)、阳性预测值为8/8、阴性预测值为60.0%(27/45)、Kappa值为0.312]、TB-DNA检测[敏感度为34.6%(9/26)、特异度为100.0%(27/27)、阳性预测值为9/9、阴性预测值为61.4%(27/44)、Kappa值为0.350]、GeneXpert MTB/RIF[敏感度为34.6%(9/26)、特异度为100.0%(27/27)、阳性预测值为9/9、阴性预测值为61.4%(27/44)、Kappa值为0.350]、结核抗体检测[敏感度为46.2%(12/26)、特异度为81.5%(22/27)、阳性预测值为70.6%(12/17)、阴性预测值为61.1%(22/36)、Kappa值为0.278]。纳米孔测序技术还显示,结核病患者合并非结核分枝杆菌、病毒、真菌、细菌感染等的比例(42.1%,8/19)较高。结论: 纳米孔测序技术在结核浆液性积液的诊断中展现出优异的性能,其诊断效能优于传统病原学检测方法,能够有效提高结核病的早期诊断率,并为合并感染的筛查提供新的解决方案。

关键词: 纳米技术, 结核, 心包积液, 胸腔积液, 敏感性与特异性

Abstract:

Objective: To explore the application value of nanopore sequencing in the diagnosis of tuberculous serous effusion and to evaluate its advantages in rapidly and accurately identifying Mycobacterium tuberculosis and screening for coinfecting pathogens. Methods: From October 2021 to May 2025, patients with suspected tuberculous serous effusion who visited Nanning Fourth People’s Hospital were enrolled. Their effusion samples were collected for nanopore sequencing. Their final clinical diagnoses were used as reference standard for comparing sequencing results with conventional tests (acid-fast staining, TB-DNA PCR, GeneXpert MTB/RIF, and solid mycobacterial culture). Results: Nanopore sequencing achieved significantly better diagnostic performance than any conventional test: sensitivity 73.1% (19/26), specificity 100.0% (27/27), positive predictive value (PPV) 100.0% (19/19), negative predictive value (NPV) 79.4% (27/34), and Kappa 0.734, surpassing those of acid-fast staining (sensitivity 11.5% (3/26), specificity 100.0% (27/27), PPV 3/3, NPV 54.0% (27/50), Kappa 0.117), culture (sensitivity 30.8% (8/26), specificity 100.0% (27/27), PPV 8/8, NPV 60.0% (27/45), Kappa 0.312), TB-DNA PCR (sensitivity 34.6% (9/26), specificity 100.0% (27/27), PPV 9/9, NPV 61.4% (27/44), Kappa 0.350), GeneXpert MTB/RIF (sensitivity 34.6% (9/26), specificity 100.0% (27/27), PPV 9/9, NPV 61.4% (27/44), Kappa 0.350), and TB antibody detection (sensitivity 46.2% (12/26), specificity 81.5% (22/27), PPV 70.6% (12/17), NPV 61.1% (22/36), Kappa 0.278). Moreover, nanopore sequencing revealed that 42.1% (8/19) of tuberculosis cases co-infected with nontuberculous mycobacteria (NTM), viruses, fungi, or bacteria. Conclusion: Nanopore sequencing demonstrated excellent performance in the diagnosis of tuberculous serous effusion, with diagnostic efficacy significantly better than that of traditional pathogen detection methods. It can effectively improve the early diagnosis rate of tuberculosis and offers a new solution for screening coinfections, showing important clinical application value.

Key words: Nanotechnology, Tuberculosis, Pericardial effusion, Pleural effusion, Sensitivity and specificity

中图分类号:

R52
R446.5

朱庆东, 赵春艳, 黄雪雯, 黄爱春, 曾春梅, 许超艳, 兰艳群, 宋畅. 宏基因组纳米孔测序在结核浆液性积液诊断及合并感染筛查中的应用价值[J]. 中国防痨杂志, 2026, 48(3): 335-341. doi: 10.19982/j.issn.1000-6621.20250386

Zhu Qingdong, Zhao Chunyan, Huang Xuewen, Huang Aichun, Zeng Chunmei, Xu Chaoyan, Lan Yanqun, Song Chang. The application value of nanopore sequencing in the diagnosis of tuberculous serous effusion and screening for coinfections[J]. Chinese Journal of Antituberculosis, 2026, 48(3): 335-341. doi: 10.19982/j.issn.1000-6621.20250386

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参考文献 19

[1]	Mortazavi-Moghaddam SG, Sharifzadeh GR, Rezvani MR. Status of Exudative Pleural Effusion in Adults of South Khorasan Province, Northeast Iran: Pleural Tuberculosis Tending toward Elderly. Iran J Med Sci, 2016, 41(4): 322-327.
[2]	Isiguzo G, Du Bruyn E, Howlett P, et al. Diagnosis and Management of Tuberculous Pericarditis: What Is New? Curr Cardiol Rep, 2020, 22(1): 2. doi:10.1007/s11886-020-1254-1. URL pmid: 31940097
[3]	朱庆东, 王琦, 宋畅, 等. 高通量测序技术在结核病诊断和耐药检测中的应用进展. 新发传染病电子杂志, 2024, 9(5): 78-82. doi:10.19871/j.cnki.xfcrbzz.2024.05.015.
[4]	Chen Q, Yi J, Liu Y, et al. Clinical diagnostic value of targeted next-generation sequencing for infectious diseases (Review). Mol Med Rep, 2024, 30(3): 153. doi:10.3892/mmr.2024.13277.
[5]	van Dijk EL, Jaszczyszyn Y, Naquin D, et al. The Third Revolution in Sequencing Technology. Trends Genet, 2018, 34(9): 666-681. doi:10.1016/j.tig.2018.05.008. URL pmid: 29941292
[6]	Wang Y, Zhao Y, Bollas A, et al. Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol, 2021, 39(11): 1348-1365. doi:10.1038/s41587-021-01108-x. URL pmid: 34750572
[7]	中华医学会呼吸病学分会胸膜与纵隔疾病学组(筹). 胸腔积液诊断的中国专家共识. 中华结核和呼吸杂志, 2022, 45(11): 1080-1096. doi:10.3760/cma.j.cn112147-20220511-00403.
[8]	Chen Y, Ling Y, Xu X, et al. Diagnostic Utility of Nanopore Sequencing for Tuberculous Serous Effusions. Infect Drug Resist, 2025, 18: 3661-3670. doi:10.2147/IDR.S524986. URL pmid: 40718371
[9]	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. 结核与肺部疾病杂志, 2024, 5(4): 376-378. doi:10.19983/j.issn.2096-8493.2024022.
[10]	Ebrahimzadeh A, Pagheh AS, Mousavi T, et al. Serosal membrane tuberculosis in Iran: A comprehensive review of evidences. J Clin Tuberc Other Mycobact Dis, 2023, 31: 100354. doi:10.1016/j.jctube.2023.100354.
[11]	赵春艳, 宋畅, 宋树林, 等. 纳米孔测序检测痰液样本在肺结核诊断中的应用. 中国临床医生杂志, 2024, 52(4): 413-416. doi:10.3969/j.issn.2095-8552.2024.04.009.
[12]	朱庆东, 宋畅, 赵春艳, 等. 纳米孔测序检测肺部组织样本在痰涂片阴性肺结核中的诊断价值. 临床肺科杂志, 2025, 30(6): 857-861. doi:10.3969/j.issn.1009-6663.2025.06.008.
[13]	闫晓婧, 王宇津, 王隽, 等. 纳米孔测序技术对涂阴肺结核患者诊断价值的多中心临床研究. 中国防痨杂志, 2025, 47(2): 169-174. doi:10.19982/j.issn.1000-6621.20240481.
[14]	McIntyre S, Warner J, Rush C, et al. Antibodies as clinical tools for tuberculosis. Front Immunol, 2023, 14: 1278947. doi:10.3389/fimmu.2023.1278947.
[15]	Zhao CY, Song C, Lin YR, et al. The diagnostic value of third-generation nanopore sequencing in non-tuberculous mycobacterial infections. Front Cell Infect Microbiol, 2025, 15: 1557079. doi:10.3389/fcimb.2025.1557079.
[16]	Chen J, Xu F. Application of Nanopore Sequencing in the Diagnosis and Treatment of Pulmonary Infections. Mol Diagn Ther, 2023, 27(6): 685-701. doi:10.1007/s40291-023-00669-8. URL pmid: 37563539
[17]	Griffiths P, Reeves M. Pathogenesis of human cytomegalovirus in the immunocompromised host. Nat Rev Microbiol, 2021, 19(12): 759-773. doi:10.1038/s41579-021-00582-z. URL pmid: 34168328
[18]	Otto M. Staphylococcus epidermidis-the ‘accidental’ pathogen. Nat Rev Microbiol, 2009, 7(8): 555-567. doi:10.1038/nrmicro2182.
[19]	Hosseini M, Shakerimoghaddam A, Ghazalibina M, et al. Aspergillus coinfection among patients with pulmonary tuberculosis in Asia and Africa countries; A systematic review and meta-analysis of cross-sectional studies. Microb Pathog, 2020, 141: 104018. doi:10.1016/j.micpath.2020.104018.

特征	合计(53例)	结核组(26例)	非结核组(27例)	统计检验值	P值
性别[例(构成比,%)]				χ²=0.053	0.819
男性	39(73.6)	20(76.9)	19(70.4)
女性	14(26.4)	6(23.1)	8(29.6)
年龄(岁, $\bar{x}±s$)	56.21±18.24	51.08±19.33	61.15±15.94	t=2.065	0.044
体质量指数(kg/m², $\bar{x}±s$)	21.32±3.15	20.56±3.34	22.06±2.83	t=1.754	0.086
合并症[例(发生率,%)]
非结核分枝杆菌感染	2(3.8)	0(0.0)	2(7.4)	χ²=2.001	0.157
恶性肿瘤	8(15.1)	2(7.7)	6(22.2)	χ²=2.182	0.140
高血压	8(15.1)	5(19.2)	3(11.1)	χ²=0.681	0.409
糖尿病	3(5.7)	2(7.67)	1(3.7)	χ²=0.395	0.530
肝炎	1(1.9)	0(0.0)	1(3.7)	χ²=0.981	0.322
HIV感染	6(11.3)	3(11.5)	3(11.1)	χ²=0.002	0.961

实验室指标	合计(53例)	结核组(26例)	非结核组(27例)	统计检验值	P值
CD3⁺[个/μl, M(Q₁,Q₃)]	780(541,1186)	775(538,1187)	794(607,1174)	Z=-0.552	0.590
CD4⁺[个/μl, M(Q₁,Q₃)]	402(308,614)	372(288,619)	405(331,576)	Z=-0.552	0.587
CD8⁺[个/μl, M(Q₁,Q₃)]	279(182,455)	223(166,430)	318(190,521)	Z=-1.005	0.319
CD4⁺/CD8⁺[M(Q₁,Q₃)]	1.53(1.08,2.25)	1.51(1.30,2.63)	1.55(0.99,2.03)	Z=1.041	0.302
白细胞计数[10⁹/L, M(Q₁,Q₃)]	6.4(5.5,8.5)	6.3(5.7,8.9)	6.5(5.1,7.8)	Z=1.014	0.315
红细胞计数(10¹²/L, $\bar{x}±s$)	3.98±0.94	3.99±0.96	3.96±0.95	t=-0.146	0.884
血红蛋白(g/L, $\bar{x}±s$)	107.60±24.13	107.88±23.22	107.33±25.41	t=-0.083	0.935
血小板计数(10⁹/L, $\bar{x}±s$)	312.47±130.64	322.50±123.61	302.81±138.73	t=-0.546	0.588
中性粒细胞[10⁹/L, M(Q₁,Q₃)]	4.4(3.6,6.3)	4.6(3.7,6.4)	4.4(3.5,6.2)	Z=0.747	0.460
淋巴细胞[10⁹/L, M(Q₁,Q₃)]	1.1(0.8,1.4)	1.2(0.9,1.5)	1.0(0.8,1.4)	Z=1.210	0.229
单核细胞[10⁹/L, M(Q₁,Q₃)]	0.5(0.4,0.7)	0.5(0.4,0.7)	0.5(0.4,0.6)	Z=0.605	0.546
嗜酸性粒细胞[10⁹/L, M(Q₁,Q₃)]	0.08(0.05,0.23)	0.08(0.04,0.17)	0.08(0.06,0.24)	Z=-0.480	0.637
嗜碱性粒细胞[10⁹/L, M(Q₁,Q₃)]	0.03(0.02,0.04)	0.03(0.02,0.04)	0.04(0.02,0.05)	Z=0.080	0.942
C反应蛋白[mg/L, M(Q₁,Q₃)]	27.6(5.0,68.3)	31.9(8.4,69.4)	20.2(5.0,60.7)	Z=0.569	0.569
血红细胞沉降率[mm/1h, M(Q₁,Q₃)]	55.0(27.0,100.0)	55.0(33.0,72.0)	56.5(25.5,102.5)	Z=-0.044	0.972

检测方法	最终诊断(例)		敏感度 (%,95%CI)	特异度 (%,95%CI)	阳性预测值 (%,95%CI)	阴性预测值 (%,95%CI)	Kappa值 (95%CI)	AUC (95%CI)
检测方法	结核 (26例)	非结核 (27例)	敏感度 (%,95%CI)	特异度 (%,95%CI)	阳性预测值 (%,95%CI)	阴性预测值 (%,95%CI)	Kappa值 (95%CI)	AUC (95%CI)
纳米孔测序			73.1 (51.9~87.6)	100.0 (84.5~100.0)	100.0 (79.1~100.0)	79.4 (61.6~90.7)	0.734 (0.558~0.910)	0.860 (0.748~0.972)
阳性	19	0
阴性	7	27
抗酸染色			11.5 (3.0~31.3)	100.0 (84.5~100.0)	3/3 (31.0~100.0)	54.0 (39.5~67.9)	0.117 (0.010~0.244)	0.560 (0.402~0.718)
阳性	3	0
阴性	23	27
培养			30.8 (15.1~51.9)	100.0 (84.5~100.0)	8/8 (59.8~100.0)	60.0 (44.4~73.9)	0.312 (0.124~0.500)	0.660 (0.508~0.812)
阳性	8	0
阴性	18	27
DNA检测			34.6 (17.9~55.6)	100.0 (84.5~100.0)	9/9 (62.9~100.0)	61.4 (45.5~75.3)	0.350 (0.156~0.544)	0.660 (0.508~0.812)
阳性	9	0
阴性	17	27
Xpert			34.6 (17.9~55.6)	100.0 (84.5~100.0)	9/9 (62.9~100.0)	61.4 (45.5~75.3)	0.350 (0.156~0.544)	0.673 (0.525~0.821)
阳性	9	0
阴性	17	27
结核抗体检测			46.2 (27.1~66.3)	81.5 (61.3~93.0)	70.6 (44.0~88.6)	61.1 (43.5~76.4)	0.278 (0.035~0.521)	0.627 (0.474~0.781)
阳性	12	5
阴性	14	22

检测类别	例数	构成比 (%)
纳米孔测序未检出结核分枝杆菌(34例)
未检出病原菌	22	64.7
单纯病毒感染	6	17.6
单纯细菌感染	4	11.8
病毒合并真菌感染	1	2.9
多种病原菌合并感染	1	2.9
纳米孔测序检出结核分枝杆菌(19例)
单纯结核分枝杆菌感染	11	57.9
结核分枝杆菌合并非结核分枝杆菌感染	1	5.3
结核分枝杆菌合并病毒感染	3	15.8
结核分枝杆菌合并真菌感染	1	5.3
结核分枝杆菌合并细菌感染	2	10.5
结核分枝杆菌合并多种病原菌感染	1	5.3