流式细胞术鉴定结核分枝杆菌利福平异质性耐药可行性分析

doi:10.3969/j.issn.1673-8640.2022.06.015

检验医学 ›› 2022, Vol. 37 ›› Issue (6): 577-582.DOI: 10.3969/j.issn.1673-8640.2022.06.015

• 技术研究与评价·论著 • 上一篇下一篇

流式细胞术鉴定结核分枝杆菌利福平异质性耐药可行性分析

李华¹, 王伟亮², 谢贝¹, 杨瑜¹, 孟繁荣¹, 王楠¹, 刘志辉¹, 张言斌³()

1.广州市胸科医院肺部疾病研究所,广东广州 510095
2.佛山市第四人民医院结核病防治科,广东佛山 528000
3.广州市胸科医院结核内科,广东广州 510095

收稿日期:2021-08-17 修回日期:2021-10-19 出版日期:2022-06-30 发布日期:2022-07-28
通讯作者: 张言斌
作者简介:张言斌,E-mail： zh-yb2002@163.com。
李华,女,1986年生,博士,主管技师,主要从事结核病检验相关研究。
基金资助:
国家科技重大专项(2018ZX10715004);广东省自然科学基金(2014A030313755);广东省自然科学基金(2018A030313550);广州市高水平临床重点专科和培育专科建设项目(穗卫函〔2019〕1555号);广州市医学重点学科(结核病学)建设项目

Feasibility of the determination of heterogeneous drug resistance to rifampicin in Mycobacterium tuberculosis by flow cytometry

LI Hua¹, WANG Weiliang², XIE Bei¹, YANG Yu¹, MENG Fanrong¹, WANG Nan¹, LIU Zhihui¹, ZHANG Yanbin³()

1. Institute of Pulmonary Diseases,Guangzhou Chest Hospital,Guangzhou 510095,Guangdong,China
2. Department of Tuberculosis Prevention and Treatment,Foshan Fourth People's Hospital,Foshan 528000,Guangdong,China
3. Department of Tuberculosis,Guangzhou Chest Hospital,Guangzhou 510095,Guangdong,China

Received:2021-08-17 Revised:2021-10-19 Online:2022-06-30 Published:2022-07-28
Contact: ZHANG Yanbin

摘要/Abstract

摘要：

目的探讨流式细胞术（FCM）检测结核分枝杆菌（MTB）利福平（RFP）异质性耐药的可行性。方法将MTB临床分离株分别用12.5 、25、50 和100 μg/mL RFP处理3、7和10 d后,用荧光素双醋酸酯（FDA）染色,采用FCM检测其平均荧光强度（MFI）,计算敏感指数（实验管MFI/对照管MFI）,并进行差异性分析。确定不影响RFP耐药MTB临床分离株生长、但抑制RFP敏感MTB临床分离株生长的RFP处理浓度和时间。将30株RFP敏感MTB临床分离株和30株RFP耐药MTB临床分离株菌液混合,得到RFP耐药MTB所占比例分别为0%、25%、50%、75%和100%的RFP异质性耐药MTB菌液,按照确定的RFP浓度和时间处理,计算FDA染色后的敏感指数。采用受试者工作特征（ROC）曲线评估不同比例RFP异质性耐药MTB菌液的敏感指数界值和检测性能。结果基于FDA的FCM检测MTB RFP异质性耐药的较优RFP处理浓度为50 μg/mL,作用时间为10 d;用50 μg/mL RFP处理MTB 10 d时,基于FDA的FCM鉴别含0%、25%和100%的RFP异质性耐药MTB菌液样本的敏感指数界值分别为0.48和0.83,敏感性分别为96.2%和86.7%,特异性分别为96.7%和83.3%;处理14 d时,该方法鉴别含0%、25%和100% RFP异质性耐药MTB菌液样本的敏感指数界值分别为0.37和0.77,敏感性分别为96.7%和96.7%,特异性分别为93.3%和86.7%。结论 FCM在MTB异质性耐药检测中具有较高的应用价值。

关键词: 流式细胞术, 结核分枝杆菌, 利福平异质性耐药, 荧光素双醋酸酯

Abstract:

Objective To evaluate the feasibility of flow cytometry（FCM） in determining the heterogeneous drug resistance of Mycobacterium tuberculosis（MTB） to rifampicin（RFP）. Methods MTB clinical isolates were isolated by fluorescein diacetate（FDA） after treating with RFP of 12.5,25,50 and 100 μg/mL for 3,7 and 10 d,respectively. The mean fluorescence intensity（MFI） of MTB clinical isolates was determined by FCM,and the sensitivity index（MFI of treated/MFI of control） was calculated. The concentration and time of RFP treatment were chosen according to significance test of difference,which inhibited the growth of MTB clinical isolates sensitive to RFP but not affected the growth of MTB clinical isolates resistant to RFP. A total of 30 isolates of RFP sensitive clinical MTB isolates and 30 isolates of RFP resistant clinical MTB isolates were mixed to obtain hetero-resistant MTB,in which the proportions of resistant bacteria were 0%,25%,50%,75% and 100%,respectively. Hetero-resistant MTB were treated according to the above optimal RFP treatment concentration and time. After stained by FDA,the sensitivity index of samples was calculated. According to receiver operating characteristic（ROC） curve,the sensitivity index of hetero-resistant MTB with different proportions of RFP resistant bacteria and the dsetermination performance of the method were investigated. Results The optimal RFP treatment concentration and time of FDA based FCM in the determination of MTB heterogeneous drug resistance to RFP were 50 μg/mL and 10 d,respectively. To identify hetero-resistant MTB samples which containing 0%,25% and 100% RFP resistant MTB,the samples were treated with 50 μg/mL RFP for 10 d,the cut-off values of this method were 0.48 and 0.83,the sensitivities were 96.2% and 86.7%,and the specificities were 96.7% and 83.3%,respectively. When treated for 14 d,the cut-off values of this method were 0.37 and 0.77,the sensitivities of this method were 96.7% and 96.7%,and the specificities of this method were 93.3% and 86.7%,respectively. Conclusions FCM has a broad application prospect in the determination of MTB heterogeneous resistance.

Key words: Flow cytometry, Mycobacterium tuberculosis, Heterogeneous drug resistance to rifampicin, Fluorescein diacetate

中图分类号:

R446.1

李华, 王伟亮, 谢贝, 杨瑜, 孟繁荣, 王楠, 刘志辉, 张言斌. 流式细胞术鉴定结核分枝杆菌利福平异质性耐药可行性分析[J]. 检验医学, 2022, 37(6): 577-582.

LI Hua, WANG Weiliang, XIE Bei, YANG Yu, MENG Fanrong, WANG Nan, LIU Zhihui, ZHANG Yanbin. Feasibility of the determination of heterogeneous drug resistance to rifampicin in Mycobacterium tuberculosis by flow cytometry[J]. Laboratory Medicine, 2022, 37(6): 577-582.

图/表 5

图1 基于FDA的FCM检测MTB RFP异质性耐药的药物处理浓度和作用时间分析注：（a）RFP敏感MTB临床分离株经RFP处理后的FDA敏感指数;*P<0.001、**P<0.000 1; 3 d; 7 d; 10 d;（b）MTB临床分离株经不同浓度RFP处理10 d后的FDA敏感指数; 12.5 μg/mL; 25 μg/mL; 50 μg/mL; 100 μg/mL。

表1 50 μg/mL RFP作用不同比例RFP耐药MTB异质菌液样本10、14 d后FDA染色敏感指数

RFP耐药MTB比例/%	作用10 d FDA染色敏感指数	作用14 d FDA染色敏感指数
100	0.907±0.103	0.916±0.082
75	0.809（0.733~0.884）	0.807（0.752~0.895）
50	0.789±0.166	0.760±0.164
25	0.689±0.133	0.653±0.141
0	0.267±0.118	0.198±0.107

表2 RFP异质性耐药MTB经50 μg/mL RFP作用后FDA染色敏感指数的ROC曲线分析结果

分组	曲线下面积		临界值		敏感性/%		特异性/%
分组	10 d	14 d	10 d	14 d	10 d	14 d	10 d	14 d
AB	0.991	0.992	0.48	0.37	96.70	96.70	96.70	93.30
AC	0.999	0.997	0.50	0.51	100.00	96.70	96.70	96.70
AD	1.000	1.000	0.55	0.56	100.00	100.00	100.00	100.00
AE	1.000	1.000	0.62	0.62	100.00	100.00	100.00	100.00
BC	0.684	0.710	0.72	0.74	73.30	60.00	63.30	40.00
BD	0.750	0.867	0.74	0.74	73.30	80.00	66.70	63.30
BE	0.912	0.954	0.83	0.77	86.70	96.70	83.30	86.70
CD	0.562	0.649	0.84	0.75	43.30	80.00	73.30	33.30
CE	0.781	0.806	0.83	0.85	86.70	86.70	66.70	63.40
DE	0.739	0.763	0.82	0.86	86.70	86.70	56.70	50.00

图2 RFP异质性耐药MTB经50 μg/mL RFP作用后FDA染色敏感指数的ROC曲线注：（a）经RFP处理10 d;（b）经RFP处理14 d;A、B、C、D、E分别表示含有0%、25%、50%、75%和100%RFP耐药MTB; AB; AC; AD; AE; BC; BD; BE; CD; CE; DE; 参考线。

图3 30组RFP异质性耐药MTB混悬液经50 μg/mL RFP处理后FDA染色敏感指数的分布注：（a）RFP处理10 d;0.48和0.83分别为含0%和25%RFP耐药MTB的FDA敏感性指数界值;（b）RFP处理14 d;0.37和0.77分别为含25%和100%RFP耐药MTB的FDA敏感性指数临界值;0%、25%和100%分别表示含有0%、25%和100% RFP耐药MTB; 0%; 25%; 100%。

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流式细胞术鉴定结核分枝杆菌利福平异质性耐药可行性分析

Feasibility of the determination of heterogeneous drug resistance to rifampicin in Mycobacterium tuberculosis by flow cytometry

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