125株耐多药结核分枝杆菌的吡嗪酰胺耐药及pncA基因突变分析

doi:10.3969/j.issn.1000-6621.2021.10.018

摘要/Abstract

摘要：

目的了解125株耐多药结核分枝杆菌对吡嗪酰胺(PZA)的耐药水平和pncA基因的突变特征,为PZA耐药的快速诊断提供依据。方法通过简单随机抽样方法选取2010—2019年福建省9个耐药监测点的125株耐多药菌株,采用BACTEC MGIT 960液体培养进行PZA药物敏感性试验(简称“药敏试验”),通过PCR扩增pncA目的基因,分析耐多药菌株对PZA的耐药性及pncA基因突变情况。结果 50株耐多药菌株对PZA耐药,PZA耐药率为40.0%(50/125)。男性患者PZA耐药率为39.0%(37/95),女性患者PZA耐药率为43.3%(13/30),差异无统计学意义(χ²=0.183,P=0.669);15~25岁、26~45岁、46~60岁、61~80岁患者PZA耐药率分别为58.3%(7/12)、 32.7%(16/49)、39.5%(17/43)、 47.6%(10/21),PZA耐药率在年龄组之间的差异无统计学意义(χ²=3.294,P=0.348);初治患者PZA耐药率为41.5%(21/65),复治患者PZA耐药率为38.3%(23/60),差异无统计学意义(χ²=0.134,P=0.715)。30株PZA耐药菌株检出pncA基因突变,突变类型22种,突变检出率为60.0%(30/50);PZA耐药株的pncA基因突变检出率[60.0%(30/50)]高于PZA敏感株的pncA基因突变检出率[5.3%(4/75)],差异有统计学意义(χ²=45.276,P=0.000)。结论福建省耐多药结核分枝杆菌的PZA耐药率较高,且 pncA基因突变检出率较高,临床上应关注PZA的耐药问题。

关键词: 分枝杆菌, 结核, 吡嗪酰胺, 抗药性, 基因突变

Abstract:

Objective To investigate the pyrazinamide (PZA) resistance of 125 multidrug-resistant (MDR) Mycobacterium tuberculosis isolates and the characteristics of mutation in pncA gene, to provide references for the rapid diagnosis of PZA resistance. Methods A total of 125 MDR strains were simple randomly selected from 9 drug-resistant surveillance sites in Fujian, 2010—2019. The PZA susceptibility was tested using the MGIT 960 system, and the pncA target gene was amplified by PCR to analyze the resistance of MDR strains to PZA and the mutation of pncA gene. Results A total of 50 MDR strains were resistant to PZA, and the PZA resistance rate was 40.0% (50/125). There was no statistical difference of the PZA resistance rate between the male (39.0% (37/95)) and the female (43.3% (13/30);χ²=0.183, P=0.669). The PZA resistant rates in the age-group of 15-25, 26-45, 46-60 and 61-80 years were 58.3% (7/12), 32.7% (16/49), 39.5% (17/43)and 47.6% (10/21), respectively, the difference was no statistically significant (χ²=3.294, P=0.348). There was no statistical difference of the PZA resistance rates between the new cases (41.5% (21/65)) and the retreatment cases (38.3% (23/60);χ²=0.134, P=0.715). The mutation of pncA gene was found in 30 PZA resistant MDR strains, and the types of mutations was 22. The mutation rate of pncA gene in PZA resistant MDR strain was 60.0% (30/50), which was significantly higher than that of the PZA sensitive strains (5.3% (4/75), χ²=45.276, P=0.000). Conclusion The PZA resistance rate of MDR Mycobacterium tuberculosis is high in Fujian, and the mutation of pncA gene is also high, attention should be paid to the drug resistance of PZA in clinic.

Key words: Mycobacterium tuberculosis, Pyrazinamide, Drug resistance, Gene mutation

赵永, 林淑芳, 林建, 戴志松, 魏淑贞. 125株耐多药结核分枝杆菌的吡嗪酰胺耐药及pncA基因突变分析[J]. 中国防痨杂志, 2021, 43(10): 1079-1083. doi: 10.3969/j.issn.1000-6621.2021.10.018

ZHAO Yong, LIN Shu-fang, LIN Jian, DAI Zhi-song, WEI Shu-zhen. Analysis of pyrazinamide resistance and pncA mutation in 125 multidrug-resistant Mycobacterium tuberculosis isolates[J]. Chinese Journal of Antituberculosis, 2021, 43(10): 1079-1083. doi: 10.3969/j.issn.1000-6621.2021.10.018

图/表 2

参考文献 14

[1]	张贺秋, 赵雁林. 现代结核病诊断技术. 北京: 人民卫生出版社, 2013:32-33.
[2]	Zhang S, Chen J, Shi W, et al. Mutations in panD encoding aspartate decarboxylase are associated with pyrazinamide resistance in Mycobacterium tuberculosis. Emerg Microbes Infect, 2013, 2(6): e34. doi: 10.1038/emi.2013.38. doi: 10.1038/emi.2013.38
[3]	Li J, Ji L, Shi W, et al. Trans-translation mediates tolerance to multiple antibiotics and stresses in Escherichia coli. J Antimicrob Chemother, 2013, 68(11): 2477-2481. doi: 10.1093/jac/dkt231. doi: 10.1093/jac/dkt231 URL
[4]	Dillon NA, Peterson ND, Rosen BC, et al. Pantothenate and pantetheine antagonize the antitubercular activity of pyrazinamide. Antimicrob Agents Chemother, 2014, 58(12): 7258-7263. doi: 10.1128/AAC.04028-14. doi: 10.1128/AAC.04028-14 URL
[5]	罗振华, 钱雪琴, 范齐文, 等. 结核分枝杆菌吡嗪酰胺耐药相关分子特征分析. 中华微生物学和免疫学杂志, 2015, 35(9): 660-664. doi: 10.3760/cma.j.issn.0254-5101.2015.09.005. doi: 10.3760/cma.j.issn.0254-5101.2015.09.005
[6]	Sheen P, Requena D, Gushiken E, et al. A multiple genome analysis of Mycobacterium tuberculosis reveals specific novel genes and mutations associated with pyrazinamide resistance. BMC Genomics, 2017, 18(1): 769. doi: 10.1186/s12864-017-4146-z. doi: 10.1186/s12864-017-4146-z URL
[7]	关平. 广东地区结核分枝杆菌吡嗪酰胺耐药的pncA、rpsA、panD基因分型特征研究. 国际检验医学杂志, 2019, 40(17): 2066-2069. doi: 10.3969/j.issn.1673-4130.2019.17.004. doi: 10.3969/j.issn.1673-4130.2019.17.004
[8]	朱大冕, 胡代玉, 刘洁, 等. 重庆地区多药结核分枝杆菌吡嗪酰胺耐药基因突变的特征分析. 中国防痨杂志, 2018, 40(2): 177-182. doi: 10.3969/j.issn.1000-6621.2018.02.013. doi: 10.3969/j.issn.1000-6621.2018.02.013
[9]	曹志敏, 陈玲. 结核分枝杆菌吡嗪酰胺耐药相关基因研究进展. 中国人兽共患病杂志, 2017, 33(10): 923-925. doi: 10.3969/j.issn.1002-2694.2017.10.015. doi: 10.3969/j.issn.1002-2694.2017.10.015
[10]	林淑芳, 魏淑贞, 赵永, 等. 福建省耐药多药结核分枝杆菌对吡嗪酰胺的耐药水平与基因型特征分析. 中国防痨杂志, 2018, 40(12): 1330-1334. doi: 10.3969/j.issn.1000-6621.2018.12.017. doi: 10.3969/j.issn.1000-6621.2018.12.017
[11]	周银发, 张山鹰, 魏淑贞, 等. 福建省耐多药结核分枝杆菌对吡嗪酰胺耐药性的研究. 结核病与肺部健康杂志, 2018, 7(2): 123-127. doi: 10.3969/j.issn.2095-3755.2018.02.010. doi: 10.3969/j.issn.2095-3755.2018.02.010
[12]	赵雁林, 逄宇. 结核病实验室检验规程. 北京: 人民卫生出版社, 2015:45-65.
[13]	Ramirez-Busby SM, Valafar F. Systematic review of mutations in pyrazinamidase associated with pyrazinamide resistance in Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother, 2015, 59(9): 5267-5277. doi: 10.1128/AAC.00204-15. doi: 10.1128/AAC.00204-15 pmid: 26077261
[14]	Miotto P, Cabibbe AM, Feuerriegel S, et al. Mycobacterium tuberculosis pyrazinamide resistance determinants: a multicenter study. MBio, 2014, 5(5): e01819-e01814. doi: 10.1128/mBio.01819-14. doi: 10.1128/mBio.01819-14

基本特征	耐药	敏感	χ²值	P值
性别			0.183	0.669
男性	37(39.0)	58(61.0)
女性	13(43.3)	17(56.7)
年龄组(岁)			3.294	0.348
15~25	7(58.3)	5(41.7)
26~45	16(32.7)	33(67.3)
46~60	17(39.5)	26(60.5)
61~80	10(47.6)	11(52.4)
患者类型			0.134	0.715
初治	27(41.5)	38(58.5)
复治	23(38.3)	37(61.7)

突变类型	氨基酸改变	菌株数	突变检出率(%)
22G→A	D8N	2	4.0
29A→C	Q10P	1	2.0
42C→A	C14终止	1	2.0
137C→A	A46E	1	2.0
137C→T	A46V	1	2.0
152A→C	H51P	1	2.0
202T→C	W68R	3	6.0
212A→G	H71R	1	2.0
231插入G	231插入G	1	2.0
246A→C	H82P	1	2.0
311G→T	S104I	4	8.0
95G→A,331G→A,385G→A	S32N,E111K,D129N	1	2.0
347T→C	L116P	1	2.0
278T→G,319G→A,376G→A,	V93G,E107K,E112K,Q141P,D126N,E181K	1	2.0
422A→C,541G→A
389插入GG	移码突变	2	4.0
403A→C	T135P	1	2.0
421C→T	转录终止	2	4.0
422A→C	Q141P	1	2.0
464T→G	V155G	1	2.0
478A→G	T160A	1	2.0
490T→C	S164P	1	2.0
523A→G	M175V	1	2.0

[1]	首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所, 中国防痨协会《中国防痨杂志》编辑委员会. 耐药肺结核全口服化学治疗方案中国专家共识(2021年版)[J]. 中国防痨杂志, 2021, 43(9): 859-866.
[2]	周文强, 张爽, 初乃惠. 耐药肺结核全口服治疗方案研究的现状和展望[J]. 中国防痨杂志, 2021, 43(9): 879-882.
[3]	石文卉, 初乃惠. 耐药肺结核患者氟喹诺酮类药物耐药情况及影响因素[J]. 中国防痨杂志, 2021, 43(9): 905-909.
[4]	林建, 林淑芳, 戴志松, 赵永, 周银发, 张添林, 魏淑贞. 2016—2019年福建省结核病耐药监测结果分析[J]. 中国防痨杂志, 2021, 43(9): 924-928.
[5]	于霞, 任汝颜, 文舒安, 梁倩, 董玲玲, 黄海荣. 13种抗生素对快生长分枝杆菌的体外抑菌效果评价[J]. 中国防痨杂志, 2021, 43(9): 947-951.
[6]	马婷婷, 任斐, 马进宝, 杨翰. 异烟肼耐药肺结核患者对丙硫异烟胺和对氨基水杨酸的耐药情况分析[J]. 中国防痨杂志, 2021, 43(9): 961-964.
[7]	夏辉, 赵雁林. 世界卫生组织《关于更新使用结核分枝杆菌及其耐药性核酸扩增检测技术的建议》的启示[J]. 中国防痨杂志, 2021, 43(8): 761-765.
[8]	胡严杰, 龚世伟, 任易. 核酸内切酶Surveyor酶切法快速检测MTB对吡嗪酰胺耐药性方法的建立及评估[J]. 中国防痨杂志, 2021, 43(8): 813-816.
[9]	杨婷婷, 高谦. 构建基于全基因组数据的结核病耐药及传播监测网络[J]. 中国防痨杂志, 2021, 43(7): 645-648.
[10]	易俊莉, 杨新宇, 陈昊, 赵琰枫, 陈双双, 张洁, 丁北川, 代小伟, 孙闪华, 武文清, 李传友. 1156株结核分枝杆菌临床分离株耐药情况分析[J]. 中国防痨杂志, 2021, 43(7): 677-681.
[11]	曹盼, 梁矿立, 袁吉欣, 李永波, 陈瑞, 张志飞, 仵倩红. 初治与复治空洞性利福平耐药肺结核患者的CT表现分析[J]. 中国防痨杂志, 2021, 43(7): 694-701.
[12]	常俊丽, 张建宏, 章瑛, 张海芳, 凡鹏飞, 李秋华, 丁雪玲. 一起学校利福平耐药肺结核聚集性疫情的调查分析[J]. 中国防痨杂志, 2021, 43(7): 751-754.
[13]	罗丹, 覃慧芳, 叶婧, 赵锦明, 秦翊翔, 蓝如束. 广西壮族自治区结核分枝杆菌耐药基因突变特征及其与基因型的相关性分析[J]. 中国防痨杂志, 2021, 43(6): 596-601.
[14]	王璐, 薛仲探, 王玉峰, 尚媛媛, 任卫聪, 姚丛, 高飞, 逄宇. 结核分枝杆菌对贝达喹啉药物敏感性动态监测及耐药制研究[J]. 中国防痨杂志, 2021, 43(5): 482-486.
[15]	刘敏, 李明俊, 余庆, 何坤, 吴玉珊, 杨红红, 李同心. 重庆市HIV与NTM双重感染患者的菌种分布及耐药情况[J]. 中国防痨杂志, 2021, 43(5): 501-505.