Application value of MeltPro two-step method in tuberculosis diagnosis and drug resistance screening

doi:10.19982/j.issn.1000-6621.20250107

Abstract

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

CLC Number:

R378.91
R52

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

Figures/Tables 6

References 26

[1]	北京市卫生健康委员会. 2023年北京卫生健康事业发展统计公报[EB/OL]. [2024-04-30]. https://wjw.beijing.gov.cn/sjfb/bjstjgb/bjstjgb2023/202404/t20240430_3649880.html.
[2]	Wang M, Zhang Y, Huang C, et al. A Whole-Genome Sequencing-Based Study to Delineate the Risk and Characteristics of Tuberculosis Transmission in an Insular Population Over 10 Years in Shanghai. Front Microbiol, 2022, 12:768659. doi:10.3389/fmicb.2021.768659.
[3]	World Health Organization. Global tuberculosis report 2024. Geneva: World Health Organization, 2024.
[4]	World Health Organization. Global tuberculosis report 2020. Geneva: World Health Organization, 2020.
[5]	World Health Organization. Policy Statement: Automated Real-Time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifampicin Resistance: Xpert MTB/RIF System. Geneva: World Health Organization, 2011.
[6]	国家呼吸内科医疗质量控制中心, 中华医学会结核病学分会, 中国防痨协会结核病控制专业分会, 等. 综合医疗机构肺结核早期发现临床实践指南. 结核与肺部疾病杂志, 2024, 5(1):1-14. doi:10.19982/j.issn.1000-6621.20230428.
[7]	赵雁林, 逄宇. 结核病实验室检验规程. 北京: 人民卫生出版社, 2015.
[8]	World Health Organization. Technical Report on critical concentrations for drug susceptibility testing of isoniazid and the rifamycins (rifampicin, rifabutin and rifapentine). Geneva: World Health Organization, 2021.
[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]	World Health Organization. WHO consolidated guidelines on tuberculosis. Module 3: diagnosis-rapid diagnostics for tuberculosis detection, 2021 update. Geneva: World Health Organi-zation, 2021.
[11]	Zhang L, Weng TP, Wang HY, et al. Patient pathway analysis of tuberculosis diagnostic delay: a multicentre retrospective cohort study in China. Clin Microbiol Infect, 2021, 27(7):1000-1006. doi:10.1016/j.cmi.2020.12.031.
[12]	陈双双, 代小伟, 田丽丽, 等. 北京市肺结核病人耐药筛查实施效果的研究. 中国人兽共患病学报, 2022, 38(11):1018-1022. doi:10.3969/j.issn.1002-2694.2022.00.152.
[13]	Liang Q, Jiang X, Jia J, et al. An early and trustable indicator suggestive of non-tuberculosis mycobacteria isolation in a high tuberculosis burden setting. J Infect, 2024, 88(5):106149. doi:10.1016/j.jinf.2024.106149.
[14]	张子胜. 7种实验方法诊断结核病的临床应用价值. 中国卫生标准管理, 2016, 7(9):163-164. doi:10.3969/j.issn.1674-9316.2016.09.112.
[15]	陈纪飞, 黄丽花, 罗兰波, 等. 舌拭子-PCR荧光探针法对肺结核诊断效能的评价. 中国防痨杂志, 2025, 47(1):51-60. doi:10.19982/j.issn.1000-6621.20240324.
[16]	张汇征, 李桓, 张珍, 等. 荧光PCR熔解曲线法在检测临床标本结核分枝杆菌及其耐药性中的应用. 中国实验诊断学, 2021, 25(3): 354-356. doi:10.3969/j.issn.1007-4287.2021.03.011.
[17]	王嫩寒, 田丽丽, 陈双双, 等. 荧光PCR探针熔解曲线技术检测痰标本结核分枝杆菌利福平耐药性的研究. 中国防痨杂志, 2023, 45(6):583-588. doi:10.19982/j.issn.1000-6621.20230086.
[18]	李静, 姜琦, 江渊, 等. PCR荧光探针技术对结核分枝杆菌复合群及利福平耐药性检测性能的评价. 中国防痨杂志, 2024, 46(10):1250-1258. doi:10.19982/j.issn.1000-6621.20240166.
[19]	Pang Y, Dong H, Tan Y, et al. Rapid diagnosis of MDR and XDR tuberculosis with the MeltPro TB assay in China. Sci Rep, 2016, 6:25330. doi:10.1038/srep25330. pmid: 27149911
[20]	苏碧仪, 周德旺, 马品云, 等. 荧光PCR探针熔解曲线法检测结核分枝杆菌对利福平和异烟肼耐药情况的研究. 结核病与肺部健康杂志, 2020, 1(4):245-248. doi:10.3969/j.issn.2096-8493.2020.03.008.
[21]	Huo F, Ma Y, Liu R, et al. Interpretation of Discordant Rifampicin Susceptibility Test Results Obtained Using Gene-Xpert vs Phenotypic Drug Susceptibility Testing. Open Forum Infect Dis, 2020, 7(8):ofaa279. doi:10.1093/ofid/ofaa279.
[22]	Gegia M, Winters N, Benedetti A, et al. Treatment of isoniazid-resistant tuberculosis with first-line drugs: a systematic review and meta-analysis. Lancet Infect Dis, 2017, 17(2):223-234. doi:10.1016/S1473-3099(16)30407-8. pmid: 27865891
[23]	Lempens P, Meehan CJ, Vandelannoote K, et al. Isoniazid resistance levels of Mycobacterium tuberculosis can largely be predicted by high-confidence resistance-conferring mutations. Sci Rep, 2018, 8(1):3246. doi:10.1038/s41598-018-21378-x.
[24]	李桂莲, 万康林. 结核分枝杆菌异烟肼耐药性——一个不容忽视的问题. 中国人兽共患病学报, 2019, 35(6):475-479. doi:10.3969/j.issn.1002-2694.2019.00.100.
[25]	Villegas L, Otero L, Sterling TR, et al. Prevalence, Risk Factors, and Treatment Outcomes of Isoniazid- and Rifampicin-Mono-Resistant Pulmonary Tuberculosis in Lima, Peru. PLoS One, 2016, 11(4):e0152933. doi:10.1371/journal.pone.0152933.
[26]	Shen Y, Liu Y, Krafft T, et al. Progress and challenges in infectious disease surveillance and early warning. Medicine Plus, 2025, 2(1). doi:10.1016/j.medp.2025.100071.

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)

类别	例数	涂片镜检阳性		Xpert阳性		MeltPro^? MTBC阳性
类别	例数	例数	阳性率(%)	例数	阳性率(%)	例数	阳性率(%)
确诊病例	87	50	57.5	78	89.7	75	86.2
临床诊断病例	56	0	0.0	0	0.0	7	12.5
非结核分枝杆菌感染	6	4	66.7	0	0.0	0	0.0
其他肺部疾病	70	0	0.0	0	0.0	0	0.0
合计	219	54	24.7	78	35.6	82	37.4

MeltPro^? MTBC	Xpert(例)		合计 (例)
MeltPro^? MTBC	阳性	阴性	合计 (例)
阳性	69	13	82
阴性	9	128	137
合计	78	141	219

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
方法	肺结核	非肺结核	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
涂片镜检			35.0 (27.3~43.4)	94.7 (86.4~98.3)	92.6 (81.3~97.6)	43.6 (36.0~51.6)	0.233
阳性	50	4
阴性	93	72
Xpert			54.5 (46.0~62.8)	100.0 (94.0~100.0)	100.0 (94.2~100.0)	53.9 (45.3~62.3)	0.454
阳性	78	0
阴性	65	76
MeltPro^? MTBC			57.3 (48.8~65.5)	100.0 (94.0~100.0)	100.0 (94.4~100.0)	55.5 (46.8~63.9)	0.483
阳性	82	0
阴性	61	76

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