Rapid determination of minimum inhibitory concentration and cut-off concentration of Mycobacterium tuberculosis by nitrate reductase assay using liquid medium

doi:10.3969/j.issn.1673-8640.2022.06.014

Abstract

Abstract:

Objective Through performing a multicenter study of nitrate reductase assay（NRA） using liquid medium for the determination of multidrug-resistant Mycobacterium tuberculosis（MDR-MTB） and extensively drug-resistant Mycobacterium tuberculosis（XDR-MTB）,to evaluate the performance of this method,and to identify the minimum inhibitory concentration（MIC） and cut-off concentrations of rifampicin,isoniazid,ofloxacin,amikacin,kanamycin and capreomycin. Methods The study was divided into 3 phases. In the Phase Ⅰ,the MIC of 6 drugs was determined. Phase Ⅱ established the cut-off concentrations of the 6 drugs. In the Phase Ⅲ,the cut-off concentrations for the 6 drugs used in NRA were verified by large numbers of clinical samples [730 isolates of Mycobacterium tuberculosis（MTB）] with agar proportion method（APM） or MGIT 960 system as reference method. The consistency and accuracy of the validation results from 4 participating laboratories were compared. Results The MIC for the 6 drugs were as follows：rifampicin 1 mg/L,isoniazid 0.2 mg/L,ofloxacin 2 mg/L,amikacin 4 mg/L,kanamycin 5 mg/L,capreomycin 5 mg/L. The cut-off concentrations for the 6 drugs were as follows：rifampicin 1 mg/L,isoniazid 0.2 mg/L,ofloxacin 2 mg/L,amikacin 2 mg/L,kanamycin 5 mg/L,capreomycin 2.5 mg/L. The accuracies for rifampicin,isoniazid,ofloxacin,amikacin,kanamycin and capreomycin were 96.4%-100.0%. The median determination time of NRA was 7 d. Conclusions NRA performed in liquid medium can provide a quick,economic and convenient method for the first-line and second-line drug susceptibility testing of MTB resource-limited settings.

Key words: Mycobacterium tuberculosis, Nitrate reductase assay, Minimum inhibitory concentration, Cut-off concentration

CLC Number:

R446.5

DU Jinghui, HUANG Zikun, LIU Min, GONG Jingjing, WANG Dan, LIU Jibo, LIU Xu. Rapid determination of minimum inhibitory concentration and cut-off concentration of Mycobacterium tuberculosis by nitrate reductase assay using liquid medium[J]. Laboratory Medicine, 2022, 37(6): 568-576.

Figures/Tables 7

References 21

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药物名称	NRA MIC/（mg/L）	参考方法		修正前结果			修正后结果
药物名称	NRA MIC/（mg/L）	耐药/株	敏感/株	敏感性/%	特异性/%	准确性/%	敏感性/%	特异性/%	准确性/%
利福平	≥4	82		98.1	100.0	98.5	98.7	100.0	99.0
	2	64
	1	8
	0.5	3	3
	0.25		18
	≤0.125		28
异烟肼	≥0.8	121		97.6	100.0	98.1	98.8	100.0	99.0
	0.4	34
	0.2	10
	0.1	4	3
	0.05		11
	≤0.025		23
氧氟沙星	≥8	60		100.0	100.0	100.0
	4	8
	2	3
	1		5
	0.5		35
	≤0.25		95
阿米卡星	≥8	34		95.2	100.0	99.0	95.2	100.0	99.0
	4	6
	2	2	5
	1		17
	0.5		42
	≤0.25		100
卡那霉素	≥20	13		97.0	100.0	99.5	100.0	100.0	100.0
	10	14
	5	5
	2.5	1	6
	1.25		25
	≤0.62		142
卷曲霉素	≥20	14		93.1	100.0	99.0	96.4	100.0	99.5
	10	8
	5	5
	2.5	2	4
	1.25		18
	≤0.62		155

药物名称	NRA MIC/（mg/L）	参考方法		修正前结果			修正后结果
药物名称	NRA MIC/（mg/L）	耐药/株	敏感/株	敏感性/%	特异性/%	准确性/%	敏感性/%	特异性/%	准确性/%
利福平	≥4	82		98.1	100.0	98.5	98.7	100.0	99.0
	2	64
	1	8
	0.5	3	3
	0.25		18
	≤0.125		28
异烟肼	≥0.8	121		97.6	100.0	98.1	98.8	100.0	99.0
	0.4	34
	0.2	10
	0.1	4	3
	0.05		11
	≤0.025		23
氧氟沙星	≥8	60		100.0	100.0	100.0
	4	8
	2	3
	1		5
	0.5		35
	≤0.25		95
阿米卡星	≥8	34		95.2	100.0	99.0	95.2	100.0	99.0
	4	6
	2	2	5
	1		17
	0.5		42
	≤0.25		100
卡那霉素	≥20	13		97.0	100.0	99.5	100.0	100.0	100.0
	10	14
	5	5
	2.5	1	6
	1.25		25
	≤0.62		142
卷曲霉素	≥20	14		93.1	100.0	99.0	96.4	100.0	99.5
	10	8
	5	5
	2.5	2	4
	1.25		18
	≤0.62		155

药物名称	药物浓度/（mg/L）	实验室1			实验室2			实验室3			实验室4
药物名称	药物浓度/（mg/L）	敏感性/ %	特异性/ %	准确性/ %	敏感性/ %	特异性/ %	准确性/ %	敏感性/ %	特异性/ %	准确性/ %	敏感性/ %	特异性/ %	准确性/ %
利福平	0.5	100.0	100.0	100.0	97.1	86.7	94.0	100.0	86.7	95.0	100.0	93.3	98.0
	1	100.0	100.0	100.0	97.1	100.0	98.0	100.0	100.0	100.0	94.3	93.3	94.0
	2	88.6	100.0	92.0	94.1	100.0	95.9	94.3	100.0	96.0	91.4	100.0	94.0
异烟肼	0.1	97.5	90.0	96.0	100.0	80.0	96.0	97.5	90.0	96.0	100.0	100.0	100.0
	0.2	97.5	100.0	98.0	100.0	100.0	100.0	97.5	90.0	96.0	100.0	100.0	100.0
	0.4	87.5	100.0	90.0	95.0	100.0	96.0	87.5	100.0	90.0	90.0	100.0	92.0
氧氟沙星	1	100.0	100.0	100.0	100.0	90.0	94.0	95.0	93.3	94.0	100.0	96.7	98.0
	2	100.0	100.0	100.0	100.0	100.0	100.0	95.0	96.7	96.0	95.0	100.0	98.0
	4	90.0	100.0	96.0	100.0	100.0	100.0	95.0	96.7	96.0	85.0	100.0	94.0
阿米卡星	2	100.0	100.0	100.0	94.4	93.8	94.0	100.0	96.9	98.0	100.0	96.9	98.0
	4	94.4	100.0	98.0	94.4	93.8	94.0	88.9	96.9	94.0	100.0	100.0	100.0
	8	83.3	100.0	94.0	77.8	100.0	92.0	83.3	100.0	94.0	88.9	100.0	96.0
卡那霉素	2.5	93.3	88.6	90.0	100.0	97.1	98.0	93.3	97.1	96.0	100.0	91.4	94.0
	5	86.7	97.1	94.0	100.0	100.0	100.0	93.3	100.0	98.0	100.0	97.1	98.0
	10	80.0	97.1	92.0	86.7	100.0	96.0	80.0	100.0	94.0	93.3	97.1	96.0
卷曲霉素	2.5	91.7	94.7	94.0	100.0	97.4	98.0	91.7	100.0	98.0	100.0	97.4	98.0
	5	91.7	97.4	96.0	100.0	100.0	100.0	83.3	100.0	96.0	83.3	100.0	96.0
	10	58.3	100.0	90.0	83.3	100.0	96.0	66.7	100.0	92.0	83.3	100.0	96.0

药物名称	药物浓度/（mg/L）	实验室1			实验室2			实验室3			实验室4
药物名称	药物浓度/（mg/L）	敏感性/ %	特异性/ %	准确性/ %	敏感性/ %	特异性/ %	准确性/ %	敏感性/ %	特异性/ %	准确性/ %	敏感性/ %	特异性/ %	准确性/ %
利福平	0.5	100.0	100.0	100.0	97.1	86.7	94.0	100.0	86.7	95.0	100.0	93.3	98.0
	1	100.0	100.0	100.0	97.1	100.0	98.0	100.0	100.0	100.0	94.3	93.3	94.0
	2	88.6	100.0	92.0	94.1	100.0	95.9	94.3	100.0	96.0	91.4	100.0	94.0
异烟肼	0.1	97.5	90.0	96.0	100.0	80.0	96.0	97.5	90.0	96.0	100.0	100.0	100.0
	0.2	97.5	100.0	98.0	100.0	100.0	100.0	97.5	90.0	96.0	100.0	100.0	100.0
	0.4	87.5	100.0	90.0	95.0	100.0	96.0	87.5	100.0	90.0	90.0	100.0	92.0
氧氟沙星	1	100.0	100.0	100.0	100.0	90.0	94.0	95.0	93.3	94.0	100.0	96.7	98.0
	2	100.0	100.0	100.0	100.0	100.0	100.0	95.0	96.7	96.0	95.0	100.0	98.0
	4	90.0	100.0	96.0	100.0	100.0	100.0	95.0	96.7	96.0	85.0	100.0	94.0
阿米卡星	2	100.0	100.0	100.0	94.4	93.8	94.0	100.0	96.9	98.0	100.0	96.9	98.0
	4	94.4	100.0	98.0	94.4	93.8	94.0	88.9	96.9	94.0	100.0	100.0	100.0
	8	83.3	100.0	94.0	77.8	100.0	92.0	83.3	100.0	94.0	88.9	100.0	96.0
卡那霉素	2.5	93.3	88.6	90.0	100.0	97.1	98.0	93.3	97.1	96.0	100.0	91.4	94.0
	5	86.7	97.1	94.0	100.0	100.0	100.0	93.3	100.0	98.0	100.0	97.1	98.0
	10	80.0	97.1	92.0	86.7	100.0	96.0	80.0	100.0	94.0	93.3	97.1	96.0
卷曲霉素	2.5	91.7	94.7	94.0	100.0	97.4	98.0	91.7	100.0	98.0	100.0	97.4	98.0
	5	91.7	97.4	96.0	100.0	100.0	100.0	83.3	100.0	96.0	83.3	100.0	96.0
	10	58.3	100.0	90.0	83.3	100.0	96.0	66.7	100.0	92.0	83.3	100.0	96.0

实验室编号	液体培养基NRA法结果	参考方法^结果
		利福平		异烟肼		氧氟沙星		阿米卡星		卡那霉素		卷曲霉素
		耐药	敏感	耐药	敏感	耐药	敏感	耐药	敏感	耐药	敏感	耐药	敏感
1	耐药	70	0	82	0	50	1	37	2	30	3	21	3
	敏感	2	86	3	73	1	106	3	116	2	123	2	132
2	耐药	58	0	60	1	28	0	20	0	17	1	15	0
	敏感	0	61	0	58	0	91	2	97	0	101	0	104
3	耐药	106	2	112	3	69	2	45	5	36	5	18	3
	敏感	3	142	5	133	3	179	0	203	4	208	3	229
4	耐药	109	1	126	0	65	3	49	1	31	0	36	1
	敏感	5	85	3	71	0	132	2	148	1	168	2	161