MeltPro两步法在结核病诊断及耐药筛查中的应用价值

doi:10.19982/j.issn.1000-6621.20250107

摘要/Abstract

摘要：

目的:评价MeltPro^?MTBC联合MeltPro^?MDR荧光PCR熔解曲线技术(简称“MeltPro两步法”)对疑似肺结核患者的诊断及耐药性检测的应用价值。方法:采用前瞻性研究方法,参照入组标准收集2024年4—12月北京市疾病预防控制中心结核病门诊部就诊的疑似肺结核患者痰液样本,进行抗酸杆菌痰涂片镜检、分枝杆菌培养、GeneXpert MTB/RIF(简称“Xpert”)及MeltPro^? MTBC和MeltPro^? MDR检测,计算涂片镜检、Xpert及MeltPro^? MTBC方法检测结核分枝杆菌(MTB)的阳性检出率。以临床诊断为参考,评价涂片镜检、Xpert及MeltPro^? MTBC的检测效能,比较Xpert与MeltPro两步法的耐药检测结果,并分析Xpert检测MTB不同等级与MeltPro^? MTBC阳性检出及MeltPro^? MDR检测成功率的关系。结果:共收集219例疑似肺结核患者,其中肺结核患者143例(65.3%,包括确诊患者87例、临床诊断患者56例)、非结核分枝杆菌(NTM)感染6例(2.7%)、其他肺部疾病患者70例(32.0%);涂片镜检、Xpert、MeltPro^? MTBC检测阳性率分别为24.7%(54/219)、35.6%(78/219)、37.4%(82/219),差异有统计学意义(χ²=9.536,P=0.008);Xpert与MeltPro^? MTBC检测阳性率差异无统计学意义(χ²=0.158,P=0.691)。以临床诊断为参考,涂片镜检、Xpert和MeltPro^? MTBC检测MTB的敏感度分别为35.0%(50/143)、54.5%(78/143)和57.3%(82/143),特异度分别为94.7%(72/76)、100.0%(76/76)和100.0%(76/76),Kappa值分别为0.233、0.454和0.483。Xpert检测利福平耐药率为5.1%(4/78),MeltPro^? MDR检出利福平耐药率为6.8%(5/74)、异烟肼耐药率为14.9%(11/74)。MeltPro^? MDR对Xpert检测MTB为高、中、低、极低、阴性标本的耐药检测成功率分别为8/8、100.0%(24/24)、100.0%(24/24)、54.5%(12/22)、9.2%(6/65)。结论:MeltPro两步法在结核病诊断与利福平耐药筛查中与Xpert检测效能高度一致,且可同时检测利福平和异烟肼耐药性,是一种可靠便捷的结核病快速诊断及耐药筛查方法。

关键词: 分枝杆菌,结核, 多重聚合酶链反应, 核酸扩增技术, 评价研究

Abstract:

Objective: To evaluate the diagnostic value of MeltPro^? MTBC combined with MeltPro^? MDR fluorescent PCR melting curve technique (referred to as the “MeltPro two-step method”) for tuberculosis (TB) diagnosis and drug resistance detection in suspected pulmonary tuberculosis (PTB) patients. Methods: Adopting prospective research methods, sputum samples from suspected PTB patients who met the enrollment criteria and were initially diagnosed at Tuberculosis Outpatient Department in Beijing Center for Disease Prevention and Control from April to December 2024 were collected. Several detection methods including smear microscopy, mycobacterial culture, GeneXpert MTB/RIF (Xpert), MeltPro^? MTBC, and MeltPro^? MDR assays were performed separately, and the positive detection rates of Mycobacterium tuberculosis (MTB) by smear microscopy, Xpert, and MeltPro^? MTBC were calculated. The diagnostic performance of smear microscopy, Xpert and MeltPro^? MTBC was evaluated based on clinical diagnosis. Drug resistance by Xpert and the MeltPro two-step method were compared. Consistency between Xpert semi-quantitative MTB levels and MeltPro^? MTBC results, as well as the success rate of MeltPro^? MDR detection, were analyzed. Results: A total of 219 sputum specimens from suspected patients were collected, and 143 cases (65.3%) were diagnosed with PTB (87 confirmed, 56 clinically diagnosed), 6 cases (2.7%) had non-tuberculous mycobacterial (NTM) infections, and 70 cases (32.0%) were patients with other pulmonary diseases. The MTB positive detection rates by smear microscopy, Xpert, and MeltPro^? MTBC were 24.7% (54/219), 35.6% (78/219), and 37.4% (82/219), respectively (χ²=9.536, P=0.008), with no significant difference between MeltPro^? MTBC and Xpert (χ²=0.158, P=0.691). Using clinical diagnosis as the reference, the sensitivities of smear microscopy, Xpert, and MeltPro^? MTBC were 35.0% (50/143), 54.5% (78/143), and 57.3% (82/143), with specificities of 94.7% (72/76), 100.0% (76/76), and 100.0% (76/76), and Kappa values of 0.233, 0.454, and 0.483, respectively. Rifampicin (RFP) resistance rates were 5.1% (4/78) by Xpert and 6.8% (5/74) by MeltPro^? MDR, while isoniazid (INH) resistance was 14.9% (11/74) by MeltPro^? MDR. The detection success rate of MeltPro^? MDR correlated with Xpert semi-quantitative MTB levels: 8/8 for high, 100.0% (24/24) for medium, 100.0% (24/24) for low, 54.5% (12/22) for very low, and 9.2% (6/65) for negative samples. Conclusion: The MeltPro two-step method demonstrates high consistency with Xpert in TB diagnosis and RFP resistance screening while enabling simultaneous detection of RFP and INH resistance. It is a reliable and convenient method for rapid diagnosis of TB and screening of drug resistance.

Key words: Mycobacterium tuberculosis, Multiplex polymerase chain reaction, Nucleic acid amplification techniques, Evaluation studies

中图分类号:

R378.91
R52

陈双双, 王嫩寒, 赵琰枫, 樊瑞芳, 田丽丽, 陈昊, 罗萍, 李洁, 李传友, 代小伟. MeltPro两步法在结核病诊断及耐药筛查中的应用价值[J]. 中国防痨杂志, 2025, 47(7): 893-900. doi: 10.19982/j.issn.1000-6621.20250107

Chen Shuangshuang, Wang Nenhan, Zhao Yanfeng, Fan Ruifang, Tian Lili, Chen Hao, Luo Ping, Li Jie, Li Chuanyou, Dai Xiaowei. Application value of MeltPro two-step method in tuberculosis diagnosis and drug resistance screening[J]. Chinese Journal of Antituberculosis, 2025, 47(7): 893-900. doi: 10.19982/j.issn.1000-6621.20250107

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药物检测通道	基因位点
利福平
A-ROX	rpoB 529~533位密码子
A-CY5	rpoB 513~519位密码子
B-FAM	rpoB 519~528位密码子
B-HEX	rpoB 507~513位密码子
异烟肼
A-FAM	KatG 315密码子
A-HEX	inhA启动子区(-17~-8位点)
B-ROX	ahpC启动子区(-44~-30位点)
B-CY5	ahpC启动子区(-15~4位点)

样本号	Xpert检测		MeltPro^?MDR
样本号	MTBC浓度	突变探针	MeltPro^?MDR
1	高	B	rpoB 507~513,rpoB513~519
2	低	C	rpoB 513~519
3	低	E	rpoB 529~533
4	中	D	rpoB 519~528
5	极低	未检出突变	rpoB 507~513