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

doi:10.3969/j.issn.1673-8640.2022.06.015

Abstract

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

CLC Number:

R446.1

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.

Figures/Tables 5

References 19

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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

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

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

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