荧光PCR探针熔解曲线法与微孔板法检测MTB耐药性的临床应用比较

doi:10.3969/j.issn.1000-6621.2021.02.006

摘要/Abstract

摘要：

目的分析荧光PCR探针熔解曲线法与微孔板法药物敏感性试验(简称“药敏试验”)检测结核分枝杆菌(MTB)对抗结核药品耐药性结果的一致性及MTB基因突变与耐药的相关性,为临床诊疗优化提供参考。方法搜集2019年1—12月分离自西安市胸科医院就诊患者并经鉴定确认的343株MTB临床分离株,菌株均进行了微孔板法药敏试验和荧光PCR探针熔解曲线法检测。以微孔板法药敏试验结果为参照,评价荧光PCR探针熔解曲线法检测MTB对异烟肼、利福平、链霉素、乙胺丁醇、莫西沙星和左氧氟沙星耐药性的检测效能,并分析荧光PCR探针熔解曲线法检测的MTB基因突变与微孔板法药敏试验最低抑菌浓度(minimum inhibitory concentration, MIC)的相关性。结果以微孔板法药敏试验结果为参照,荧光PCR探针熔解曲线法检测MTB对异烟肼、利福平、链霉素、乙胺丁醇、莫西沙星和左氧氟沙星耐药性的敏感度、特异度、Kappa值分别为:96.20%(76/79)、95.28%(242/254)、0.88;93.62%(44/47)、94.58%(279/295)、0.79; 96.88%(62/64)、94.96%(264/278)、0.86;93.33%(14/15)、95.37%(309/324)、0.61;92.31%(24/26)、97.16%(308/317)、0.80;91.18%(31/34)、99.35%(307/309)、0.92。荧光PCR探针熔解曲线法检测结果与微孔板法表型药敏试验检测结果不一致的菌株中,发生异烟肼AhpC启动子区(-44~-30及-15~3位点),利福平rpoB基因507~512位点,链霉素rrs基因905~908位点多位点突变和乙胺丁醇embB基因406位点突变的菌株,表型药敏试验耐药率较低,分别为1/5、1/5、1/10和1/5;而发生异烟肼KatG基因315位点、利福平rpoB基因529~533位点、链霉素rpsL基因43位点突变的菌株,表型药敏试验耐药率较高,分别为92.42%(61/66)、92.31%(36/39)和95.74%(45/47)。结论荧光PCR探针熔解曲线法与微孔板法药敏试验检测MTB对抗结核药品耐药性结果具有较好的一致性;MTB对异烟肼、利福平、链霉素的耐药与其部分基因突变具有一定相关性。

关键词: 分枝杆菌,结核, 聚合酶链反应, 分子探针技术, 抗药性,细菌, 对比研究

Abstract:

Objective To analyze the consistency between fluorescence PCR probe melting curve and Micropore-plate drug sensitivity tests (MicroDST) in determining the drug resistance of Mycobacterium tuberculosis (MTB), as well as the correlation between MTB gene mutation and drug resistance, in order to provide reference for optimization of clinical diagnosis and treatment. Methods From January to December, 2019, a total of 343 MTB clinical isolates from patients in Xi’an Chest Hospital were collected, all the specimens were tested using fluorescence PCR probe melting curve and MicroDST. Based on the results of MicroDST, the detection efficiency of fluorescence PCR probe melting curve in MTB resistance of isoniazid, rifampin, streptomycin, ethambutol, moxifloxacin and levofloxacin were evaluated. And the correlation between the gene mutation of MTB detected by fluorescence PCR probe melting curve and the minimum inhibitory concentration (MIC) of MicroDST was analyzed. Results With reference of the results of MicroDST, the sensitivity, specificity, and Kappa value of fluorescence PCR probe melting curve for isoniazid, rifampin, streptomycin, ethambutol, moxifloxacin and levofloxacin were 96.20% (76/79), 95.28% (242/254), 0.88; 93.62% (44/47), 94.58% (279/295), 0.79; 96.88% (62/64), 94.96% (264/278), 0.86; 93.33% (14/15), 95.37% (309/324), 0.61; 92.31% (24/26), 97.16% (308/317), 0.80; 91.18% (31/34), 99.35% (307/309), 0.92. Among the isolates, results of which by the fluorescence PCR probe melting curve were inconsistent with the phenotypic drug sensitivity, the lower phenotype drug sensitivity rates were found in those with the mutation of AhpC promoter region (-44--30 and -15-3) of isoniazid, the rpoB 507-512 of rifampin, the rrs 905-908 of streptomycin and the embB 406 of ethambutol, and the rates were 1/5, 1/5, 1/10 and 1/5, respectively. However, the phenotype drug sensitivity rates were higher in the isolates with mutation of the KatG 315 of isoniazid, the rpoB 529-533 of rifampin and the rpsL 43 of streptomycin, the rates were 92.42% (61/66), 92.31% (36/39) and 95.74% (45/47), respectively. Conclusion It has showed a good consistency in detection of drug resistance of MTB by fluorescence PCR probe melting curve and MicroDST. The drug resistance of MTB to isoniazid, rifampin and streptomycin was correlated with some gene mutations.

Key words: Mycobacterium tuberculosis, Polymerase chain reaction, Molecular probe techniques, Drug resistance,bacterial, Comparative Study

王佩, 赵国连, 雷倩, 郑丹, 崔晓利, 周俊. 荧光PCR探针熔解曲线法与微孔板法检测MTB耐药性的临床应用比较[J]. 中国防痨杂志, 2021, 43(2): 132-138. doi: 10.3969/j.issn.1000-6621.2021.02.006

WANG Pei, ZHAO Guo-lian, LEI Qian, ZHENG Dan, CUI Xiao-li, ZHOU Jun. Comparison of the clinical application between fluorescence PCR probe melting curve and Micropore-plate method in determining the drug resistance of Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2021, 43(2): 132-138. doi: 10.3969/j.issn.1000-6621.2021.02.006

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参考文献 8

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荧光PCR探针熔解曲线法	微孔板法药敏试验		敏感度[%, (95%CI)]	特异度[%, (95%CI)]	阳性预测值 [%,(95%CI)]	阴性预测值 [%,(95%CI)]	阳性似然比	阴性似然比	约登指数	Kappa 值
荧光PCR探针熔解曲线法	耐药(株)	敏感(株)	敏感度[%, (95%CI)]	特异度[%, (95%CI)]	阳性预测值 [%,(95%CI)]	阴性预测值 [%,(95%CI)]	阳性似然比	阴性似然比	约登指数	Kappa 值
异烟肼			96.20 (88.55~ 99.01)	95.28 (91.68~ 97.42)	86.36 (77.00~ 92.45)	98.78 (96.17~ 99.68)	20.36	0.04	0.91	0.88
耐药	76	12
敏感	3	242
利福平			93.62 (81.44~ 98.34)	94.58 (91.17~ 96.76)	73.33 (60.11~ 83.55)	98.94 (96.66~ 99.72)	17.26	0.07	0.88	0.79
耐药	44	16
敏感	3	279
链霉素			96.88 (88.19~ 99.46)	94.96 (91.51~ 97.11)	81.58 (70.69~ 89.21)	99.25 (97.02~ 99.87)	19.24	0.03	0.92	0.86
耐药	62	14
敏感	2	264
乙胺丁醇			93.33 (66.03~ 99.65)	95.37 (92.32~ 97.29)	48.28 (29.89~ 67.10)	99.68 (97.93~ 99.98)	20.16	0.07	0.89	0.61
耐药	14	15
敏感	1	309
莫西沙星			92.31 (73.40~ 98.66)	97.16 (94.49~ 98.61)	72.73 (54.21~ 86.06)	99.35 (97.43~ 99.98)	32.51	0.08	0.89	0.80
耐药	24	9
敏感	2	308
左氧氟沙星			91.18 (75.19~ 97.69)	99.35 (97.42~ 99.98)	93.94 (78.38~ 98.94)	99.03 (96.96~ 99.75)	140.87	0.09	0.91	0.92
耐药	31	2
敏感	3	307

突变位点	表型药敏试验MIC分级计数(株)					耐药率 (%)
突变位点	<0.2μg/ml (敏感)	0.2μg/ml (耐药)	0.4μg/ml (耐药)	0.8μg/ml (耐药)	1.6μg/ml (耐药)	耐药率 (%)
单一位点突变
AhpC启动子区(-44~-30及-15~3位点)	4	0	0	0	1	1/5
inhA94密码子	0	0	0	0	0	0.00
inhA启动子区(-17~-8位点)	3	1	1	5	2	75.00
KatG315密码子	5	0	0	6	55	92.42
多位点突变
inhA94密码子和inhA启动子区(-17~-8位点)	0	0	0	0	1	1/1
inhA94密码子和KatG315密码子	0	0	0	0	2	2/2
inhA启动子区(-17~-8位点)和KatG315密码子	0	0	0	0	2	2/2
未检测到突变	242	1	0	1	1	1.22

突变位点	表型药敏试验MIC分级计数(株)					耐药率 (%)
突变位点	<1μg/ml (敏感)	1μg/ml (敏感)	2μg/ml (敏感)	4μg/ml (中度敏感)	8μg/ml (耐药)	耐药率 (%)
单一位点突变
rpoB基因507~512位密码子	4	0	0	0	1	1/5
rpoB基因513~520位密码子	1	0	0	0	1	1/2
rpoB基因521~528位密码子	3	1	0	1	4	4/9
rpoB基因529~533位密码子	2	0	1	0	36	92.31
多位点突变
rpoB基因507~512和521~528位密码子	0	0	1	0	0	0.00
rpoB基因507~512和529~533位密码子	1	0	0	0	0	0.00
rpoB基因513~520和521~528位密码子	1	0	0	0	2	2/3
未检测到突变	278	1	0	0	3	1.06

突变位点	表型药敏试验MIC分级计数(株)					耐药率 (%)
突变位点	<1μg/ml (敏感)	1μg/ml (敏感)	2μg/ml (敏感)	4μg/ml (中度敏感)	8μg/ml (耐药)	耐药率 (%)
单一位点突变
rpsL基因43位密码子	2	0	0	0	45	95.74
rpsL基因88位密码子	0	0	0	0	8	8/8
rrs基因513~517位点	1	0	0	2	7	7/10
rrs基因905~908位点	8	0	1	0	1	1/10
多位点突变
rpsL基因43位密码子和rrs基因905~908位点	0	0	0	0	1	1/1
未检测到突变	259	1	4	0	2	0.75

突变位点	表型药敏试验MIC分级计数(株)					耐药率 (%)
突变位点	<2.5μg/ml (敏感)	2.5μg/ml (中度敏感)	5μg/ml (耐药)	10μg/ml (耐药)	20μg/ml (耐药)	耐药率 (%)
embB基因306位密码子	7	2	8	2	0	52.63
embB基因378位密码子	1	0	0	0	0	0.00
embB基因406位密码子	3	1	1	0	0	1/5
embB基因497位密码子	1	0	3	0	0	3/4
未检测到突变	309	0	0	0	1	0.32