高通量二代测序技术在耐药结核病诊断中的应用

doi:10.3969/j.issn.1000-6621.2020.11.011

摘要/Abstract

摘要：

目的探索新一代高通量二代测序(next-generation sequencing,NGS)技术在耐药结核病诊断中的应用价值。方法搜集2016—2017年甘肃省结核病耐药基线调查150株临床分离株,排除非结核分枝杆菌(non-tuberculous mycobacteria,NTM)菌株2株,进行比例法药物敏感性试验(简称“药敏试验”)及结核分枝杆菌耐药相关基因(包括katG、inhA、embB、rpoB、rpsL、rrs、gyrA和gyrB)的NGS技术检测,剔除测序失败的13株,最后纳入分析菌株135株。以比例法药敏试验结果为参考标准评价二代测序在耐药结核病中的检测效能。结果以药敏试验结果为参考标准,NGS技术检测异烟肼、乙胺丁醇、利福平、链霉素、氧氟沙星耐药的敏感度分别为:88.75%(71/80)、85.71%(18/21)、84.72%(61/72)、73.91%(51/69)、68.97%(20/29),特异度分别为100.00%(55/55)、86.84%(99/114)、96.83%(61/63)、 96.97%(64/66)、99.06%(105/106),Kappa值分别为0.87、0.59、0.81、0.71、0.76。异烟肼耐药基因突变主要在katG 315位点上,突变频率为97.18%(69/71);乙胺丁醇耐药基因突变主要在embB 306位点上,突变频率为88.89%(16/18);利福平耐药基因突变主要在耐药决定区,突变频率为93.65%(59/63);链霉素耐药基因突变主要在rpsL 43位点上,突变频率为79.25%(42/53);氧氟沙星耐药基因突变主要在gyrA 94位点上,突变频率为76.19%(16/21)。结论抗结核药物相关耐药基因NGS利福平、异烟肼、氧氟沙星耐药检测效能较好,能够满足临床结核病诊断需要。乙胺丁醇、链霉素已知耐药位点NGS耐药检测效能一般,需要对其耐药机制进行进一步的研究。

关键词: 分枝杆菌, 结核, 微生物敏感性试验, 结核, 抗多种药物性, 点突变, 诊断, 评价研究

Abstract:

Objective To explore the application value of next-generation sequencing (NGS) in the diagnosis of drug-resistant tuberculosis. Methods A total of 150 specimens of bacterial strain were selected from the Year 2016-2017 baseline survey on drug-resistant tuberculosis in Gansu Province, and tested by proportional drug sensitivity test (drug sensitivity test) and NGS of drug resistance-related genes (including katG, inhA, embB, rpoB, rpsL, rrs, gyrA, and gyrB). Two non-tuberculous mycobacteria (NTM) strains were excluded, 13 strains were failed to be sequenced, therefore, 135 strains were finally included. Results of drug sensitivity test were used as standard to evaluate the efficacy of NGS in the diagnosis of drug-resistant tuberculosis. Results Based on drug sensitivity results, sensitivities NGS in detecting isoniazid (INH), ethambutol (EMB), rifampicin (RFP), streptomycin (Sm), and ofloxacin (Ofx) were 88.75% (71/80), 85.71% (18/21), 84.72% (61/72), 73.91% (51/69) and 68.97% (20/29), respectively; and the specificities of the above drugs were 100.00% (55/55), 86.84% (99/114), 96.83% (61/63), 96.97% (64/66), and 99.06% (105/106), respectively; Kappa values were 0.87, 0.59, 0.81, 0.71, and 0.76, respectively. Gene mutations of INH-resistant were mainly in katG 315, with a mutation frequency of 97.18% (69/71); those in EMB) were in embB 306, with a mutation frequency of 88.89% (16/18); those in RFP were in the resistance-determining area, with a mutation frequency of 93.65% (59/63); those of Sm were in rpsL 43, with a mutation frequency of 79.25% (42/53); and those in Ofx were in gyrA 94, with a mutation frequency of 76.19% (16/21). Conclusion The efficacies of NGS in detecting RFP, INH and Ofx were high and could meet the needs of clinical diagnosis of tuberculosis; while the efficacies of NGS in detecting EMB and Sm were low, and further investigation of the mechanism of drug resistance of the two drugs was needed.

Key words: Mycobacterium tuberculosis, Microbial sensitivity tests, Tuberculosis, multidrug-resistant, Point mutation, Diagnosis, Evaluation studies

辜吉秀, 李晴, 马玲, 李银花, 王冬冬, 司红艳. 高通量二代测序技术在耐药结核病诊断中的应用[J]. 中国防痨杂志, 2020, 42(11): 1203-1208. doi: 10.3969/j.issn.1000-6621.2020.11.011

GU Ji-xiu, LI Qing, MA ling, LI Yin-hua, WANG Dong-dong, SI Hong-yan. Application of next-generation sequencing technology in the diagnosis of drug-resistant tuberculosis[J]. Chinese Journal of Antituberculosis, 2020, 42(11): 1203-1208. doi: 10.3969/j.issn.1000-6621.2020.11.011

图/表 3

表1

表2

表3

136株结核分枝杆菌对5种药物耐药的相关基因突变类型及频率分析

药品名称	相关基因及位点			突变类型		突变菌株数		突变频率(%)
异烟肼	katG 315			AGC-ACC		60		84.51
	katG 315			AGC-ATC		4		5.63
	katG 297			GGC-GTC		1		1.41
	inhA+katG 315			AGC-TGC+AGC-ACC		1		1.41
	inhA+katG 315			GGC-GCC+AGC-ACC		1		1.41
	inhA+katG 315			GAG-GGG+AGC-ACC		1		1.41
	inhA+katG 315			ACA-GCA+AGC-ACC		2		2.82
	ahpC+katG 142			GGC-GAC+GAC-GCC		1		1.41
合计						71		100.00
乙胺丁醇	embB 306			ATG-GTG		20		60.61
	embB 306+embB 807			ATG-GTG+AAC-AAG		1		3.03
	embB 306+embB 563			ATG-GTG+ATT-CTT		1		3.03
	embB 306			ATG-ATC		5		15.15
	embB 406			GGC-GCC		3		9.09
	embB 406			GGC-TGC		2		6.06
	embB 1024			GAC-AAC		1		3.03
合计						33		100.00
利福平	ropB 531			TCG-TTG		26		41.27
	ropB 531+ropB 645			TCG-TTG+TAC-CAC		2		3.17
	ropB 531+ropB 367			TCG-TTG+GCG-GTG		1		1.59
	ropB 531+ropB 531			TCG-TTG+TCG-TCT		1		1.59
	ropB 531+ropB 561			TCG-TTG+ATC-GTC		3		4.76
	ropB531+ropB472+ropB915			TCG-TTG+GAG-CAG+CCG-TCG		1		1.59
	ropB 531+ropB 584			TCG-TTG+TTC-TCC		1		1.59
	ropB 531+ropB 126			TCG-TTG+CCG-TCG		1		1.59
	ropB 531+ropB 126			TCG-TTG+CCG-CTG		1		1.59
	ropB 522			TCG-TGG		3		4.76
	ropB 533			CTG-CCG		3		4.76
	ropB 516			GAC-TAC		2		3.17
	ropB 126			CCG-TCG		1		1.59
	ropB 251			GTC-TTC		3		4.76
	ropB 526			CAC-CGC		2		3.17
	ropB 526			CAC-GAC		4		6.35
	ropB 511			CTG-CCG		1		1.59
	ropB 511+ropB 249			CTG-CCG+GTG-TTG		2		3.17
	ropB 511+ropB 665			CTG-CCG+GCC-GGC		1		1.59
	ropB 511+ropB 516			CTG-CCG+GAC-GCC		1		1.59
	ropB 511+ropB 526			CTG-CCG+CAC-GAC		1		1.59
	ropB 526+ropB 526			CAC-CGC+CAC-GAC		1		1.59
	ropB526+ropB526+ropB526			CAC-CGC+CAC-GAC+CAC-CAG		1		1.59
合计						63		100.00
药品名称		相关基因及位点	突变类型		突变菌株数		突变频率(%)
链霉素		rpsL 43	AAG-AGG		42		79.25
		rpsL 88	AAG-AGG		11		20.75
合计					53		100.00
氧氟沙星		gyrA 94	GAC-GGC		16		76.19
		gyrA 90	GCG-GTG		2		9.52
		gyrA 91	TCG-CCG		2		9.52
		gyrB	GTC-GCC		1		4.77
合计					21		100.00

表3

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