耐药MTB分离株对不同组合抗结核药品体外药物敏感性试验的结果分析

doi:10.3969/j.issn.1000-6621.2020.11.008

摘要/Abstract

摘要：

目的分析耐药MTB分离株对不同组合抗结核药品(简称“不同组合药品”)进行体外药物敏感性试验(简称“药敏试验”)的检测结果,评价不同组合药品的最低抑菌浓度(MIC)及对耐药菌株的抗结核协同作用。方法搜集同济大学附属上海市肺科医院“十二五”国家科技重大专项入组的148例首次复治肺结核患者,通过对患者痰标本进行BACTEC MGIT 960和改良罗氏法培养获取167株MTB分离株,对75例患者经上述两种方法进行药敏试验结果均为阳性的92株耐药菌株(包括36株MDR-MTB、54株XDR-MTB和2株单耐H菌株),采用MIC三维棋盘法分别以H-R、Mfx-Pa、Mfx-Pa-Rfb、Mfx-Pa-Rft等4种组合药品进行体外药敏试验,计算不同药品组合的协同率。结果药品未组合前的H、R、Rfb、Rft、Pa体外药敏试验MIC值的中位数(四分位数)[M(Q₁,Q₃)]分别为4(1,8)、24(2,64)、0.5(0.125,1)、4(4,4)、8(4,8)mg/L,均明显高于组合后的H[2(1,4)mg/L]、R[1(0.25,16)mg/L]、与Mfx-Pa组合后的Rfb[0.125(0.03125,0.125)mg/L]、与Mfx-Pa组合后的Rft[1(1,1)mg/L]、与Mfx/Mfx-Rfb/Mfx-Rft组合后的Pa[分别为0.0075(0.0075,0.875)、0.0075(0.0075,0.125)、0.0225(0.0075,0.5)mg/L](Z=-4.855,-6.908,-8.386,-8.632,-8.094,-8.335,-7.771,P值均<0.001);且H-R、Mfx-Pa、Mfx-Pa-Rfb、Mfx-Pa-Rft药品组合后的协同率分别为12.0%(11/92)、5.4%(5/92)、17.4%(16/92)、23.9%(22/92);其中H-R和Mfx-Pa组合分别与Mfx-Pa-Rfb和Mfx-Pa-Rft组合的协同率比较,差异均有统计学意义(χ²=12.670,P=0.002;χ²=19.420,P<0.001;χ²=18.640,P<0.001;χ²=28.500,P<0.001)。结论组合药品中H、R、Rfb、Rft、Pa的MIC值均低于未组合前该药品的MIC值,说明耐药菌株对组合后上述药品的敏感性增强。三药组合的协同率均优于两药组合,且Mfx-Pa-Rft较Mfx-Pa-Rfb组合表现出更好的协同作用。

关键词: 结核,抗多种药物性, 泛耐药结核病, 药物疗法,联合, 微生物敏感性试验, 体外研究, 药物协同作用, 对比研究

Abstract:

Objective The results of in vitro drug sensitivity test (drug sensitivity test) of drug-resistant MTB isolates against different combinations of anti-tuberculosis drugs (different combinations of drugs) were analyzed to evaluate the minimum inhibitory concentration (MIC) of different combinations of drugs and the anti-MTB synergistic effect on drug-resistant strains. Methods One hundred and forty-eight first retreatment tuberculosis patients participating in the “twelfth five-year” national science and technology key projects undertaken by the Shanghai Pulmonary Hospital affiliated to Tongji University were collected, and 167 MTB isolates were obtained via culturing of sputum samples from patients by BACTEC MGIT 960 and modified Roche separation method. Ninety-two resistant strains (including 36 of MDR-MTB, 54 of XDR-MTB, and 2 of single H resistant strains) from 75 patients identified by the above two methods of drug susceptibility test were used to perform the in vitro susceptibility tests of 4 types of drug combinations, including H-R, Mfx-Pa, Mfx-Pa-Rfb and Mfx-Pa-Rft, respectively, through three-dimensional chessboard MIC detection, and the synergistic rates of different drug combinations were calculated. Results The median (quartile) (M(Q₁,Q₃)) of the MIC values of H, R, Rfb, Rft, and Pa before drug combination were 4 (1,8), 24 (2,64), 0.5 (0.125,1),4 (4,4), and 8 (4,8) mg/L, respectively, which were significantly higher than that of H (2 (1,4) mg/L), R (1 (0.25,16) mg/L), and Rfb (0.125 (0.03125,0.125) mg/L) after Mfx-Pa combination, Rft (1 (1,1) mg/L) after Mfx-Pa combination, Pa (0.0075 (0.0075, 0.875) mg/L, 0.0075 (0.0075, 0.125) mg/L, and 0.0225 (0.0075,0.5) mg/L) combined with Mfx/Mfx-Rfb/Mfx-Rft, respectively (Z=-4.855, -6.908, -8.386, -8.632, -8.094, -8.335, -7.771, all P values <0.001).The synergic rates of H-R, Mfx-Pa, Mfx-Pa-Rfb and Mfx-Pa-Rft were 12.0% (11/92), 5.4% (5/92), 17.4% (16/92) and 23.9% (22/92), respectively. The difference of the synergic rates between H-R and Mfx-Pa combination to Mfx-Pa-Rfb and Mfx-Pa-Rft combination, respectively, were statistically significant (χ ²=12.670, P=0.002;χ ²=19.420, P<0.001;χ ²=18.640, P<0.001;χ ²=28.500, P<0.001). Conclusion The MIC values of H, R, Rfb, Rft and Pa in the combined drugs are lower than those before combination, indicating that the sensitivities to the combined drugs of drug-resistant strains are enhanced. The synergy rates of the three drug combinations are better than that of the two drug combinations, and Mfx-Pa-Rft showed better synergy than Mfx-Pa-Rfb combination.

Key words: Tuberculosis,multi-drug resistant, Extensively drug-resistant tuberculosis, Drug therapy,combination, Microbial sensitivity tests, In vitro, Drug synergism, Comparative study

应若嫣, 黄晓辰, 王洁, 刘一典, 沙巍, 杨华. 耐药MTB分离株对不同组合抗结核药品体外药物敏感性试验的结果分析[J]. 中国防痨杂志, 2020, 42(11): 1183-1182. doi: 10.3969/j.issn.1000-6621.2020.11.008

YING Ruo-yan, HUANG Xiao-chen, WANG Jie, LIU Yi-dian, SHA Wei, YANG Hua. Clinical analysis of drug sensitivity tests of various drug-resistant MTB isolates against different combinations of anti-tuberculosis drugs in vitro[J]. Chinese Journal of Antituberculosis, 2020, 42(11): 1183-1182. doi: 10.3969/j.issn.1000-6621.2020.11.008

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参考文献 13

[1]	全国第五次结核病流行病学抽样调查技术指导组, 全国第五次结核病流行病学抽样调查办公室. 2010年全国第五次结核病流行病学抽样调查报告. 中国防痨杂志, 2012,34(8):485-508.
[2]	Ragonnet R, Trauer JM, Denholm JT, et al. High rates of multidrug-resistant and rifampicin-resistant tuberculosis among re-treatment cases: where do they come from? BMC Infect Dis, 2017,17(1):36. doi: 10.1186/s12879-016-2171-1. URL pmid: 28061832
[3]	沙巍, 肖和平. 未来理想的抗结核药物与化学治疗方案. 中国防痨杂志, 2012,34(11):693-696.
[4]	张琳琳, 杨华, 肖和平, 等. 棋盘法检测复治新方案核心药物对耐多药和广泛耐药结核分枝杆菌体外的联合作用. 中华结核和呼吸杂志, 2016,39(6):464-468. doi: 10.3760/cma.j.issn.1001-0939.2016.06.013.
[5]	Rey-Jurado E Tudó G de la Bellacasa JP, et al. In vitro effect of three-drug combinations of antituberculous agents against multidrug-resistant Mycobacterium tuberculosis isolates. Int J Antimicrob Agents, 2013,41(3):278-280. doi: 10.1016/j.ijantimicag.2012.11.011. URL pmid: 23312604
[6]	Moody JA. Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods, In: Clinical Microbiology Procedures Handbook. Washington DC:American Society Microbiology, 1992: 22.
[7]	Daley CL, Caminero JA. Management of Multidrug-Resistant Tuberculosis. Semin Respir Crit Care Med, 2018,39(3):310-324. doi: 10.1055/s-0038-1661383. doi: 10.1055/s-0038-1661383 URL pmid: 30071546
[8]	López-Gavín A, Tudó G, Vergara A, et al. In vitro activity against Mycobacterium tuberculosis of levofloxacin, moxifloxacin and UB-8902 in combination with clofazimine and pretomanid. Int J Antimicrob Agents, 2015,46(5):582-585. doi: 10.1016/j.ijantimicag.2015.08.004. doi: 10.1016/j.ijantimicag.2015.08.004 URL pmid: 26421981
[9]	Li G, Zhang J, Jiang Y, et al. Cross-resistance of isoniazid, para-aminosalicylic acid and pasiniazid against isoniazid-resistant Mycobacterium tuberculosis isolates in China. J Glob Antimicrob Resist, 2019,20:275-281. doi: 10.1016/j.jgar.2019.08.005. doi: 10.1016/j.jgar.2019.08.005 URL pmid: 31425771
[10]	Rothstein DM. Rifamycins, Alone and in Combination. Cold Spring Harb Perspect Med, 2016,6(7):a027011. doi: 10.1101/cshperspect.a027011. doi: 10.1101/cshperspect.a027011 URL pmid: 27270559
[11]	Lee H, Ahn S, Hwang NY, et al. Treatment outcomes of rifabutin-containing regimens for rifabutin-sensitive multidrug-resistant pulmonary tuberculosis. Int J Infect Dis, 2017,65:135-141. doi: 10.1016/j.ijid.2017.10.013. doi: 10.1016/j.ijid.2017.10.013 URL pmid: 29224631
[12]	Yan L, Kan X, Zhu L, et al. Short-course Regimen for Subsequent Treatment of Pulmonary Tuberculosis: A Prospective, Randomized, Controlled Multicenter Clinical Trial in China. Clin Ther, 2018,40(3):440-449. doi: 10.1016/j.clinthera.2018.01.013. doi: 10.1016/j.clinthera.2018.01.013 URL pmid: 29519716
[13]	刘宇红, 高微微, 李亮, 等. 单耐药和多耐药复治菌阳肺结核个体化治疗效果探讨. 中华结核和呼吸杂志, 2018,41(1):25-31. doi: 10.3760/cma.j.issn.1001-0939.2018.01.008.

板孔编号	1	2	3	4	5	6	7	8	9	10	11	12
A	-	0.03	0.06	0.125	0.25	0.5	1	2	4	8	16	32
B	0.125	0.03+0.125	0.06+0.125	0.125+0.125	0.25+0.125	0.5+0.125	1+0.125	2+0.125	4+0.125	8+0.125	16+0.125	32+0.125
C	0.25	0.03+0.25	0.06+0.25	0.125+0.25	0.25+0.25	0.5+0.25	1+0.25	2+0.25	4+0.25	8+0.25	16+0.25	32+0.25
D	0.5	0.03+0.5	0.06+0.5	0.125+0.5	0.25+0.5	0.5+0.5	1+0.5	2+0.5	4+0.5	8+0.5	16+0.5	32+0.5
E	1	0.03+1	0.06+1	0.125+1	0.25+1	0.5+1	1+1	2+1	4+1	8+1	16+1	32+1
F	2	0.03+2	0.06+2	0.125+2	0.25+2	0.5+2	1+2	2+2	4+2	8+2	16+2	32+2
G	4	0.03+4	0.06+4	0.125+4	0.25+4	0.5+4	1+4	2+4	4+4	8+4	16+4	32+4
H	8	0.03+8	0.06+8	0.125+8	0.25+8	0.5+8	1+8	2+8	4+8	8+8	16+8	-

作用	H-R	Mfx-Pa	Mfx-Pa-Rfb	Mfx-Pa-Rft
协同作用	11(12.0)	5(5.4)	16(17.4)	22(23.9)
不相关	81(88.0)	87(94.6)	66(71.7)	58(63.0)
拮抗作用	0(0.0)	0(0.0)	10(10.9)	12(13.1)

组合药品	单耐H菌株	MDR-MTB	XDR-MTB	总耐药菌株
H-R	1.375(0.5,2.25)	1.5(1.038,2)	1.188(0.75,1.5)	1.25(1,2)
Mfx-Pa	2(2,2)	1.375(1,2)	1.5(1,2)	1.5(1,2)
Mfx-Pa-Rfb	1.813(1.5,2.125)	1.289(0.8988,2.125)	1.64(1.059,2.319)	1.505(1,2.25)
Mfx-Pa-Rft	1.465(0.56,2.37)	1.25(0.7425,2.25)	1.438(1.155,2.31)	1.31(0.8213,2.288)

不同耐药菌株	H-R	Mfx-Pa	Mfx-Pa-Rfb	Mfx-Pa-Rft
MDR-MTB(36株)
协同作用	4(11.1)	2(5.6)	7(19.4)	11(30.6)
无相关	32(88.9)	34(94.4)	27(75.0)	21(58.3)
拮抗	0(0.0)	0(0.0)	2(5.6)	4(11.1)
XDR-MTB(54株)
协同作用	6(11.1)	3(5.6)	9(16.7)	10(18.5)
无相关	48(88.9)	51(94.4)	37(68.5)	36(66.7)
拮抗	0(0.0)	0(0.0)	8(14.8)	8(14.8)
单耐H(2株)
协同作用	1(1/2)	0(0/2)	0(0/2)	1(1/2)
无相关	1(1/2)	2(2/2)	2(2/2)	1(1/2)
拮抗	0	0	0	0

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