多色探针熔解曲线法在结核分枝杆菌耐药性检测中的应用

doi:10.3969/j.issn.2095-3755.2018.04.009

摘要/Abstract

摘要：

目的评价多色探针熔解曲线法(multicolor melting curve analysis,MMCA)在MTB耐药性检测中的临床应用价值。方法收集235例浓集抗酸杆菌检测阳性和(或)MTB分子检测阳性患者的晨痰,同时用MMCA和分枝杆菌液体培养及比例法药物敏感性试验(简称“药敏试验”)进行耐药性检测,采用Kappa检验比较两方法的一致性。两方法有差别的标本用基因测序的方法进行比较。结果 MMCA和比例法药敏试验检测利福平(RFP)、异烟肼(INH)、乙胺丁醇(EMB)、左氧氟沙星(Lfx)、莫西沙星(Mfx)及耐多药结核(MDR-TB)的符合率分别为94.04(221/235)、90.64%(213/235)、80.00%(188/235)、94.89%(223/235)、77.87%(183/235)、89.36%(210/235); RFP、INH、Lfx及MDR-TB两方法比较Kappa值分别为0.88、0.81、0.88、0.77,具有极好的一致性;以上不一致的标本经基因测序技术验证,痰液及培养分离株两种标本类型的MMCA与测序结果总符合率分别为89.80%(132/147)、98.64%(145/147)。结论采用MMCA法直接、快速检测临床患者痰液MTB耐药情况,与金标准比例法药敏试验及参考方法基因测序技术一致性好,且操作简便、快速、准确,在结核病诊断及耐药结核病快速筛查中将发挥重要作用。

关键词: 分枝杆菌,结核, 结核,抗多种药物性, 多种药物,耐药, 荧光免疫测定, 分子探针技术, 药物敏感性试验, 比对研究

Abstract:

Objective Evaluation of multicolor melting curve analysis (MMCA) in the detection of mycobacterium tuberculosis resistance in sputum Samples.Methods 235 morning sputum specimen, which were concentrated acid-fast bacterium-positive and/or M.tuberculosis molecular test positive, were collected. Then, the MMCA drug-resistant detection, mycobacterium fluid culture and proportion drug-susceptibility testing (DST) were conducted simultaneously. The results of the MMCA and DST were compared using Kappa test. Sanger sequencing was conducted for those samples that had discordant results between MMCA and DST.Results Concordance rates between MMCA and DST for rifampicin (RFP), isoniazid (INH), ethambutol (EMB), levofloxacin (Lfx), moxifloxacin (Mfx), and multidrug-resistant tuberculosis (MDR-TB) were 94.04%(221/235), 90.63% (213/235), 80.00% (188/235), 94.89% (223/235), 77.87% (183/235), and 89.36% (210/235), respectively. The Kappa value between these two methods for RFP, INH, Lfx and MDR-TB were 0.88, 0.81, 0.88, and 0.77, respectively, indicating an excellent consistency. The discordant samples were verified by Sanger sequencing. The total coincidence rates of sequencing results and MMCA for sputum and culture isolates were 89.80%(132/147) and 98.64%(145/147), respectively.Conclusion Direct and rapid detection of drug resistance of mycobacterium tuberculosis in sputum samples by MMCA has good concordance rates with DST or Sanger Gene sequencing. MMCA is easy to operate, rapid and accurate, and it will play an important role in the diagnosis of tuberculosis and rapid screening for drug-resistant tuberculosis.

Key words: Tuberculosis, multidrug-resistant, Fluoroimmunoassay, Molecular probe techniques, drug-susceptibility testing, Comparative study

蔚鸣,许鑫鑫,闫朋,张宝庆,刘春涛,吴景秋,刘玉琴,王开利,侯艳杰. 多色探针熔解曲线法在结核分枝杆菌耐药性检测中的应用[J]. 结核病与肺部健康杂志, 2018, 7(4): 268-274. doi: 10.3969/j.issn.2095-3755.2018.04.009

Ming YU,Xin-xin XU,Peng YAN,Bao-qing ZHANG,Chun-tao LIU,Jing-qiu WU,Yu-qin LIU,Kai-li WANG,Yan-jie HOU. Clinical application of multicolor melting curve analysis in detection of drug resistance of Mycobacterium tuberculosis in sputum samples[J]. Journal of Tuberculosis and Lung Health, 2018, 7(4): 268-274. doi: 10.3969/j.issn.2095-3755.2018.04.009

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药物	MMCA 检测	比例法药敏试验(份)			敏感度 (%)	特异度 (%)	准确度 (%)	阳性预测值 (%)	阴性预测值 (%)	约登指数	K值
药物	MMCA 检测	耐药	敏感	合计	敏感度 (%)	特异度 (%)	准确度 (%)	阳性预测值 (%)	阴性预测值 (%)	约登指数	K值
RFP	耐药	106^a	8^b	114	94.64	93.50	94.04	92.98	95.04	0.8814	0.88
	敏感	6^c	115^d	121
INH	耐药	99^a	9^b	108	88.39	92.68	90.64	91.67	89.76	0.8107	0.81
	敏感	13^c	114^d	127
EMB	耐药	19^a	40^b	59	73.08	80.86	80.00	32.20	96.02	0.5394	0.35
	敏感	7^c	169^d	176
Lfx	耐药	63^a	7^b	70	92.65	95.81	94.89	90.00	96.97	0.8846	0.88
	敏感	5^c	160^d	165
Mfx	耐药	21^a	49^b	70	87.50	76.78	77.87	30.00	98.18	0.6428	0.35
	敏感	3^c	162^d	165
INH+RFP	耐药	72^a	12^b	84	84.71	92.00	89.36	85.71	91.39	0.7670	0.77
	敏感	13^c	138^d	151

药物	MMCA与比例法药敏试验结果不一致(份)	比例法药敏试验敏感而MMCA 检测耐药[份(率,%)]	比例法药敏试验耐药而MMCA 检测敏感[份(率,%)]
RFP	14	8(57.14)	6(42.86)
INH	22	9(40.91)	13(59.09)
EMB	47	40(85.11)	7(14.89)^a
Lfx	12	7(58.33)	5(41.67)
Mfx	52	49(94.23)	3(5.77)^b
合计	147	113(76.87)	34(23.13)