Nanopore靶向测序在呼吸道样本中检测非结核分枝杆菌的诊断价值研究

doi:10.19982/j.issn.1000-6621.20250007

摘要/Abstract

摘要：

目的: 探讨Nanopore靶向测序在呼吸道样本中检测非结核分枝杆菌(non-tuberculous mycobacteria, NTM)的诊断价值。方法: 回顾性收集2021年12月至2022年9月在浙江省杭州市红十字会医院结核病诊疗中心行呼吸道样本Nanopore靶向测序的455例患者的资料。根据诊断标准,455例患者最终诊断为NTM-PD患者(NTM-PD组)65例,诊断为非NTM-PD患者(非NTM-PD组)390例,455例患者的肺泡灌洗液或痰液同时进行Nanopore靶向测序、BACTEC MGIT 960液体培养(简称“液体培养”)和(或)PCR荧光探针检测,比较各种方法诊断NTM-PD的效能。结果: 以临床诊断结果为标准,Nanopore靶向测序、液体培养、PCR荧光探针检测呼吸道样本NTM的敏感度分别为92.3%(60/65)、83.1%(54/65)和57.1%(36/63),特异度分别为96.2%(375/390)、98.7%(385/390)和99.5%(380/382),阳性预测值分别为80.0%(60/75)、91.5%(54/59)和94.7%(36/38),阴性预测值分别为98.7%(375/380)、97.2%(385/396)和93.4%(380/407);Nanopore靶向测序、液体培养和PCR荧光探针的Kappa值分别为0.831、0.851和0.679。以液体培养为标准,Nanopore靶向测序和PCR荧光探针检测的敏感度分别为86.4%(51/59)和73.5%(36/49),特异度分别为98.0%(388/396)和94.4%(374/396),阳性预测值分别为86.4%(51/59)和62.1%(36/58),阴性预测值分别为98.0%(388/396)和96.6%(374/387);Nanopore靶向测序和PCR荧光探针的Kappa值分别为0.844和0.629。在65例临床诊断为NTM-PD组的患者中,Nanopore靶向测序和液体培养+基因芯片均为阳性的样本为50例,菌种鉴定的一致性为96.0%(48/50)。结论: Nanopore靶向测序在诊断NTM-PD中表现出较高的敏感度和特异度,与常规鉴定方法的一致性良好。在菌种鉴定方面,Nanopore靶向测序与液体培养结合基因芯片的检测结果具有高度一致性。Nanopore靶向测序能够对疑似NTM-PD患者的临床呼吸道样本进行快速检测,并获取菌种鉴定结果,为后续抗NTM治疗方案的制定提供指导。

关键词: 非典型性细菌, 分枝杆菌感染, Nanopores, 诊断技术和方法, 敏感性与特异性

Abstract:

Objective: To investigate the diagnostic value of Nanopore targeted sequencing for detecting non-tuberculous mycobacteria (NTM) in respiratory samples. Methods: Data from 455 patients who underwent Nanopore targeted sequencing of respiratory samples from December 2021 to September 2022 at the Tuberculosis Treatment Center of Hangzhou Red Cross Hospital in Zhejiang Province was retrospectively collected. According to diagnostic criteria, these 455 patients were finally diagnosed as NTM-PD patients (NTM-PD group; 65 patients) and non-NTM-PD patients (non-NTM-PD group; 390 patients), their alveolar lavage fluid or sputum were subjected to simultaneous Nanopore targeted sequencing, BACTEC MGIT 960 liquid cultures (liquid culture) and/or PCR fluorescent probes to compare their efficacy in diagnosing NTM-PD. Results: Using clinical diagnostic results as standard, the sensitivities of Nanopore targeted sequencing, liquid culture, and PCR fluorescent probe for detecting NTM in respiratory samples were 92.3% (60/65), 83.1% (54/65), and 57.1% (36/63), respectively; the specificities were 96.2% (375/390), 98.7% (385/390), and 99.5% (380/382), respectively; and the positive predictive values were 80.0% (60/75), 91.5% (54/59), and 94.7% (36/38); negative predictive values were 98.7% (375/380), 97.2% (385/396), and 93.4% (380/407); Kappa values were 0.831, 0.851 and 0.679, respectively. Using liquid culture as standard, the sensitivities of Nanopore targeted sequencing, and PCR fluorescent probe were 86.4% (51/59), and 73.5% (36/49), respectively; the specificities were 98.0% (388/396) and 94.4% (374/396), respectively; the positive predictive values were 86.4% (51/59) and 62.1% (36/58), respectively; and the negative predictive values were 98.0% (388/396) and 96.6% (374/387), respectively; Kappa values were 0.844 and 0.629, respectively. Among the 65 cases clinically diagnosed as NTM-PD, 50 samples got positive results for both Nanopore targeted sequencing and liquid culture+gene chip, and their strain identification consistency was 96.0% (48/50). Conclusion: Nanopore targeted sequencing demonstrated high sensitivity and specificity in diagnosing NTM-PD and showed strong concordance with conventional identification methods. In terms of strain identification, this method also exhibited high agreement with results obtained from liquid culture combined with gene chips. Consequently, Nanopore targeted sequencing is well-suited for rapid detection of clinical respiratory samples from patients suspected of having NTM-PD, and it can simultaneously provide strain identification results for informing subsequent development of anti-NTM therapeutic regimen.

Key words: Atypical bacterial forms, Mycobacterium infections, Nanopores, Diagnostic techniques and procedures, Sensitivity and specificity

中图分类号:

R446.5

应广智, 蔡青山, 马晓卿, 陈凌燕, 陈园园. Nanopore靶向测序在呼吸道样本中检测非结核分枝杆菌的诊断价值研究[J]. 中国防痨杂志, 2025, 47(5): 589-596. doi: 10.19982/j.issn.1000-6621.20250007

Ying Guangzhi, Cai Qingshan, Ma Xiaoqing, Chen Lingyan, Chen Yuanyuan. Diagnostic value of Nanopore targeted sequencing for detecting nontuberculous mycobacteria in respiratory specimens[J]. Chinese Journal of Antituberculosis, 2025, 47(5): 589-596. doi: 10.19982/j.issn.1000-6621.20250007

图/表 6

表1

图1

表2

两组患者一般资料的比较

资料	NTM-PD组(65例)	非NTM-PD组(390例)	统计检验值	P值
年龄(岁, $x ˉ$ ±s)	62.29±12.17	47.98±19.60	t=5.703	<0.001
性别[例(构成比,%)]			χ²=4.386	0.036
男性	33(50.8)	251(64.4)
女性	32(49.2)	139(35.6)
BMI( $x ˉ$ ±s)	19.20±3.40	20.50±3.40	t=2.923	0.004
样本类型[例(构成比,%)]			χ²=0.016	0.900
肺泡灌洗液	58(89.2)	350(89.7)
痰液	7(10.8)	40(10.3)
病史/合并疾病[例(发生率,%)]
慢性阻塞性肺疾病	12(18.5)	29(7.4)	χ²=8.261	0.004
肺结核病史	12(18.5)	80(20.5)	χ²=0.145	0.703
实体肿瘤	3(4.6)	12(3.1)	χ²=0.072	0.789
自身免疫性疾病	2(3.1)	8(2.1)	χ²=0.004	0.948
糖尿病	2(3.1)	56(14.4)	χ²=5.402	0.020
尘肺	2(3.1)	10(2.6)	χ²=0.000	1.000

表2

表3

表4

表5

参考文献 18

[1]	孔娇, 陈园园, 蔡青山, 等. 宏基因二代测序技术对非结核分枝杆菌肺病的诊断价值. 中国防痨杂志, 2022, 44(11): 1135-1140. doi:10.19982/j.issn.1000-6621.20220298.
[2]	van Ingen J, Boeree MJ, van Soolingen D, et al. Are phylogenetic position, virulence, drug susceptibility and in vivo response to treatment in mycobacteria interrelated?. Infect Genet Evol, 2012, 12(4): 832-837. doi:10.1016/j.meegid.2011.10.006.
[3]	Cowman S, van Ingen J, Griffith DE, et al. Non-tuberculous mycobacterial pulmonary disease. Eur Respir J, 2019, 54(1):1900250. doi:10.1183/13993003.00250-2019.
[4]	金向红, 黄向韵, 陈鲜宝, 等. 宏基因靶向三代测序在下呼吸道感染病原体检测的诊断效能和临床价值. 浙江临床医学, 2024, 26(6): 807-809, 813.
[5]	陶锋, 李一荣, 周艳梅, 等. 靶向宏基因组测序技术在不明原因肺部感染病原学诊断中的价值. 中国病原生物学杂志, 2024, 19(11): 1290-1294. doi:10.13350/j.cjpb.241108.
[6]	刘盛盛, 唐神结. 《非结核分枝杆菌病诊断与治疗指南(2020年版)》解读. 结核与肺部疾病杂志, 2021, 2(2): 108-115. doi:10.3969/j.issn.2096-8493.2021.02.004.
[7]	中国疾病预防控制中心. 中国结核病防治规划:痰涂片镜检质量保证手册. 北京: 中国协和医科大学出版社, 2004.
[8]	赵雁林, 刘志敏. 结核病实验室标准化操作与网络建设. 北京: 人民卫生出版社, 2013.
[9]	Gopalaswamy R, Shanmugam S, Mondal R, et al. Of tuberculosis and non-tuberculous mycobacterial infections-a comparative analysis of epidemiology, diagnosis and treatment. J Biomed Sci, 2020, 27(1): 74. doi:10.1186/s12929-020-00667-6.
[10]	Bryant JM, Grogono DM, Greaves D, et al. Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study. Lancet, 2013, 381(9877): 1551-1560. doi:10.1016/S0140-6736(13)60632-7.
[11]	秦中华, 景晔, 杜岩青, 等. 339株非结核分枝杆菌临床分离株菌种鉴定及耐药性分析. 中国防痨杂志, 2020, 42(6): 630-633. doi:10.3969/j.issn.1000-6621.2020.06.017.
[12]	李爱芳, 谈小文, 崔晓利, 等. 荧光PCR熔解曲线法在非结核分枝杆菌菌种鉴定中的应用价值. 中国防痨杂志, 2021, 43(7): 664-669. doi:10.3969/j.issn.1000-6621.2021.07.005.
[13]	刘珑玲, 陈世玖. 非结核分枝杆菌感染的分子生物学快速诊断技术. 中华医学杂志, 2014, 94(38): 3039-3040. doi:10.3760/cma.j.issn.0376-2491.2014.38.022.
[14]	周秋菊, 於青峰, 杨阳, 等. 非结核分枝杆菌临床分离株的流行病学及临床特征分析. 中国现代医生, 2020, 58(31): 125-129.
[15]	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. pmid: 34750572
[16]	Petersen LM, Martin IW, Moschetti WE, et al. Third-Genera-tion Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing. J Clin Microbiol, 2019, 58(1): e01315-19. doi:10.1128/JCM.01315-19.
[17]	Wei W, Cao J, Wu XC, et al. Diagnostic performance of metagenomic next-generation sequencing in non-tuberculous mycobacterial pulmonary disease when applied to clinical practice. Infection, 2023, 51(2): 397-405. doi:10.1007/s15010-022-01890-z.
[18]	缪青, 姚雨濛, 潘珏, 等. 宏基因二代测序技术对非结核分枝杆菌感染病原学诊断的价值. 中国临床医学, 2020, 27(4): 559-562. doi:10.12025/j.issn.1008-6358.2020.20201289.

名称	序列	长度
Nano-Myco-IS6110-F	TTTCTGTTGGTGCTGATATTGCGTGGCCAACTCGACATCC	585
Nano-Myco-IS6110-R	ACTTGCCTGTCGCTCTATCTTCAGTGTGGCTAACCCTGAACC	585
Nano-Myco-HSP65-F	TTTCTGTTGGTGCTGATATTGCCCAAGGACGAGACCACCAT	485
Nano-Myco-HSP65-R	ACTTGCCTGTCGCTCTATCTTCCAGATCCTCGTAGACACCGG	485
Nano-Myco-rpoB-F	TTTCTGTTGGTGCTGATATTGCGCGAGCTGATCCAAAACCAG	404
Nano-Myco-rpoB-R	ACTTGCCTGTCGCTCTATCTTCGTACGGCGTTTCGATGAACC	404

检测方法	临床诊断(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)
检测方法	阳性(65例)	阴性(390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)
Nanopore靶向测序			92.3 (83.0~97.5)	96.2 (93.7~97.8)	80.0 (70.8~86.8)	98.7 (97.0~99.4)	0.831 (0.757~0.895)
阳性(例)	60	15
阴性(例)	5	375
液体培养			83.1 (71.7~91.2)	98.7 (97.0~99.6)	91.5 (81.8~96.3)	97.2 (95.3~98.4)	0.851 (0.765~0.916)
阳性(例)	54	5
阴性(例)	11	385
PCR荧光探针^a			57.1 (44.0~69.5)	99.5 (98.1~99.9)	94.7 (81.6~98.6)	93.4 (91.4~94.9)	0.679 (0.562~0.768)
阳性(例)	36	2
阴性(例)	27	380

检测方法	液体培养(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)
检测方法	阳性(59例)	阴性(396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)
Nanopore靶向测序			86.4 (75.0~94.0)	98.0 (96.1~99.1)	86.4 (76.1~92.7)	98.0 (96.2~98.9)	0.844 (0.762~0.909)
阳性(例)	51	8
阴性(例)	8	388
PCR荧光探针			73.5 (58.9~85.1)	94.4 (91.7~96.5)	62.1 (51.3~71.7)	96.6 (94.7~97.9)	0.629 (0.501~0.730)
阳性(例)	36	22
阴性(例)	13	374

菌种	基因芯片法(54例)		Nanopore靶向测序(60例)
菌种	例数	构成比(%)	例数	构成比(%)
胞内分枝杆菌	40	74.1	41	68.3
鸟分枝杆菌	8	14.8	5	8.3
龟/脓肿分枝杆菌复合群	5	9.3	8	13.3
堪萨斯分枝杆菌	1	1.8	1	1.6
马萨分枝杆菌	0	0.0	1	1.6
哥伦比亚分枝杆菌	0	0.0	1	1.6
马萨分枝杆菌+胞内分枝杆菌^a	0	0.0	1	1.6
脓肿分枝杆菌+胞内分枝杆菌^a	0	0.0	1	1.6
脓肿分枝杆菌+偶发分枝杆菌^a	0	0.0	1	1.6