应用基因芯片直接检测结核病患者各类标本中利福平和异烟肼耐药的价值

doi:10.3969/j.issn.1000-6621.2019.02.004

摘要/Abstract

摘要：

目的研究结核分枝杆菌(MTB)耐药基因芯片直接检测结核病患者临床标本中利福平和异烟肼耐药相关基因的临床价值。方法回顾性分析125例通过硝基苯甲酸(PNB)和噻吩-2-羧酸肼(TCH)鉴别培养为MTB的临床标本,其中痰标本90例、支气管灌洗液12例、脓液12例、胸腔积液6例、组织标本3例、腹腔积液和尿液标本各1例,应用绝对浓度法同时进行利福平(RFP)和异烟肼(INH)药物敏感性试验(简称“绝对浓度法药敏试验”),并用基因芯片直接检测临床标本中MTB rpoB、katG和inhA基因型。以绝对浓度法药敏试验为标准,评价基因芯片检测RFP、INH耐药和耐多药(MDR)的敏感度和特异度,并且比较两种检测结果的一致性。结果在125例结核病患者临床标本中,绝对浓度法药敏试验显示对RFP、INH的耐药率和MDR发生率分别为20.0%(25/125)[其中高耐占88.0%(22/25)、低耐占12.0%(3/25)]、17.6%(22/125)[其中高耐占18.2%(4/22)、低耐占81.8%(18/22)]、16.8%(21/125)。初治和复治结核病患者临床标本中MDR-MTB的检出率分别为7.6%(7/92)和39.4%(13/33)。以绝对浓度法药敏试验为标准,应用基因芯片直接检测临床标本中MTB rpoB、katG和inhA基因型,预示对RFP、INH耐药和MDR的敏感度分别为72.0%(18/25)、63.6%(14/22)和61.9%(13/21),特异度分别为91.0%(91/100)、86.4%(89/103)和89.4%(93/104),两种方法的一致率分别为87.2%(109/125)、82.4%(103/125)和84.8%(106/125)。结论应用耐药基因芯片直接检测结核病患者临床标本中MTB对 RFP和INH耐药的相关基因突变具有中等的敏感度和较高的特异度,可快速检出对RFP、INH耐药和MDR-TB患者,为临床早期开展有效化疗提供实验依据。

关键词: 标本制备, 结核,抗多种药物性, 核酸探针, 点突变, 微生物敏感性试验, 利福平, 异烟肼, 对比研究

Abstract:

Objective To study the clinical value of direct detection on Mycobacterium tuberculosis (MTB) rifampicin (RFP) and isoniazid (INH) resistance-related genes in clinical specimens from the patients with tuberculosis (TB) by gene chip. Methods A retrospective analysis of 125 clinical specimens identified as MTB by culture using the media with nitrobenzoic acid (PNB) and thiophene-carboxylic acid hydrazide (TCH) was carried out, including sputum specimens (n=90), bronchial lavage fluid (n=12), pus (n=12), pleural effusion (n=6), tissue specimens (n=3), peritoneal effusion (n=1) and urine specimen (n=1). RFP and INH susceptibility tests were performed simultaneously by absolute concentration method, and MTB rpoB, katG and inhA genotypes in clinical specimens were detected directly by gene chip. Using absolute concentration method as control, the sensitivities and specificities of gene chips for detecting RFP, INH resistance and multidrug resistance (MDR) were evaluated, and the consistency of the two methods was compared. Results Of 125 clinical specimens from TB patients, the results of absolute concentration method showed that RFP-, INH- and multidrug-resistant (MDR) rates were respectively 20.0% (25/125) (88.0% (22/25) high level resistance and 12.0% (3/25) low level resistance), 17.6% (22/125) (18.2% (4/22) high level resistance and 81.8% (18/22) low level resistance) and 16.8% (21/125). The detection rates of MDR-MTB were 7.6% (7/92) and 39.4% (13/33) in the clinical specimens from primary and recurrent TB patients, respectively. Compared with absolute concentration method, the sensitivities of detecting RFP-, INH-resistant and MDR-MTB were 72.0% (18/25), 63.6% (14/22) and 61.9% (13/21) by gene chip, respectively. The specificity was 91.0% (91/100), 86.4% (89/103) and 89.4% (93/104), respectively. The consistency rates of the two methods were 87.2% (109/125), 82.4% (103/125) and 84.8% (106/125), respectively. Conclusion MTB, RFP, and INH resistance-related gene mutation detection in clinical samples of TB patients have moderate sensitivities and high specificities by gene chip. It can be used to quickly detect RFP-, INH-resistant, and MDR-MTB to provide experimental basis for early effective chemotherapy.

Key words: Specimen handling, Tuberculosis,multidrug-resistant, Nucleic acid probes, Point mutation, Microbial sensitivity tests, Rifampin, Isoniazid, Comparative study

白雪娟,刘银萍,张俊仙,王杰,吴雪琼. 应用基因芯片直接检测结核病患者各类标本中利福平和异烟肼耐药的价值[J]. 中国防痨杂志, 2019, 41(2): 138-144. doi: 10.3969/j.issn.1000-6621.2019.02.004

Xue-juan BAI,Yin-ping LIU,Jun-xian ZHANG,Jie WANG,Xue-qiong WU. Direct detection of rifampicin and isoniazid resistance-associated genes in clinical specimens from the patients with tuberculosis using gene chip[J]. Chinese Journal of Antituberculosis, 2019, 41(2): 138-144. doi: 10.3969/j.issn.1000-6621.2019.02.004

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结核病患者	例数	绝对浓度法药敏试验[例数(构成比,%)]
结核病患者	例数	MDR	单耐RFP	单耐INH	对RFP、INH敏感
初治	92	7(7.6)	3(3.3)	1(1.1)	81(88.0)
复治	33	13(39.4)	2(6.1)	0(0.0)	18(54.5)

例数	RFP绝对浓度法药敏试验	RFP基因型(rpoB)	INH绝对浓度法药敏试验	INH基因型
例数	RFP绝对浓度法药敏试验	RFP基因型(rpoB)	INH绝对浓度法药敏试验	katG	inhA
87	敏感	野生型	敏感	野生型	野生型
1	敏感	野生型	敏感	315(AGC>ACC)	野生型
3	敏感	野生型	敏感	野生型	-15(C>T)
1	敏感	531(TCG>TTG)	敏感	野生型	野生型
5	敏感	531(TCG>TTG)	敏感	315(AGC>ACC)	野生型
1	敏感	526(CAC>TAC)	敏感	315(AGC>ACC)	野生型
1	敏感	513(CAA>CCA)	敏感	野生型	-15(C>T)
1	敏感	531(TCG>TTG)	高度耐药	315(AGC>ACC)	-15(C>T)
1	高度耐药	野生型	敏感	野生型	野生型
2	高度耐药	531(TCG>TTG)	敏感	野生型	-15(C>T)
1	高度耐药	516(GAC>TAC),531(TCG>TTG)	敏感	野生型	-15(C>T)
1	高度耐药	531(TCG>TTG)	高度耐药	315(AGC>ACC)	野生型
5	高度耐药	531(TCG>TTG)	低度耐药	315(AGC>ACC)	野生型
1	高度耐药	526(CAC>CGC)	低度耐药	315(AGC>ACC)	野生型
1	高度耐药	526(CAC>TAC)	低度耐药	315(AGC>ACC)	野生型
2	高度耐药	531(TCG>TTG)	低度耐药	野生型	-15(C>T)
1	高度耐药	526(CAC>TAC)	高度耐药	野生型	-15(C>T)
2	低度耐药	531(TCG>TTG)	低度耐药	315(AGC>ACC)	野生型
2	高度耐药	531(TCG>TTG)	低度耐药	野生型	野生型
1	高度耐药	野生型	高度耐药	野生型	野生型
4	高度耐药	野生型	低度耐药	野生型	野生型
1	低度耐药	野生型	低度耐药	野生型	野生型

检测技术	绝对浓度法药敏试验(例)						敏感度 (%)	特异度 (%)	阳性预测值(%)	阴性预测值(%)	一致率 (%)
检测技术	耐RFP (25)	不耐RFP (100)	耐INH (22)	不耐INH (103)	MDR (21)	非MDR (104)	敏感度 (%)	特异度 (%)	阳性预测值(%)	阴性预测值(%)	一致率 (%)
耐药基因芯片检测
耐RFP(27)	18^a	9^b	16	11	15	12	72.0	91.0	66.7	92.9	87.2
不耐RFP(98)	7^c	91^d	6	92	6	92
耐INH(28)	16	12	14^a	14^b	13	15	63.6	86.4	50.0	91.8	82.4
不耐INH(97)	9	88	8^c	89^d	8	89
耐多药(24)	16	8	14	10	13^a	11^b	61.9	89.4	54.2	92.1	84.8
非耐多药(101)	9	92	8	93	8^c	93^d