Expert consensus on the standardization of broth microdilution method for drug susceptibility testing of Mycobacterium tuberculosis in China

doi:10.19982/j.issn.1000-6621.20250070

Abstract

Abstract:

Drug resistant tuberculosis has long been a serious challenge in the field of global public health. At present, it is difficult to provide accurate and effective treatment plans for patients based solely on binary phenotypic drug sensitivity and genotypic drug sensitivity tests. The microbroth dilution drug sensitivity test based on the microplate not only covers a variety of drugs and multiple concentrations at one time, but also provides quantitative drug resistance information for the clinic, so it is receiving more and more attention. In order to use the microbroth dilution drug sensitivity test in a reasonable, scientific, and standardized manner, the Tuberculosis Basic Professional Branch of Chinese Antituberculosis Association had organized experts in relevant fields to formulate this consensus on technical characteristics, application value, standardized operation, result interpretation, and precautions in actual operation of this drug sensitivity method, and proposed 12 recommendations, aiming to provide unified guiding principles for clinical and laboratory staff who are related to tuberculosis.

Key words: Mycobacterium tuberculosis, Microbial sensitivity tests, Broth microdilution, Consensus development conferences as topic

CLC Number:

R-331
R372

Tuberculosis Basic Professional Branch, Chinese Antituberculosis Association. Expert consensus on the standardization of broth microdilution method for drug susceptibility testing of Mycobacterium tuberculosis in China[J]. Chinese Journal of Antituberculosis, 2025, 47(5): 535-545. doi: 10.19982/j.issn.1000-6621.20250070

Figures/Tables 4

References 39

[1]	World Health Organization. Global tuberculosis report 2024. Geneva: World Health Organization, 2024.
[2]	World Health Organization. Technical manual for drug susceptibility testing of medicines used in the treatment of tuberculosis. Geneva: World Health Organization, 2018.
[3]	Tan Y, Hu Z, Zhao Y, et al. The beginning of the rpoB gene in addition to the rifampin resistance determination region might be needed for identifying rifampin/rifabutin cross-resis-tance in multidrug-resistant Mycobacterium tuberculosis isolates from Southern China. J Clin Microbiol, 2012, 50(1): 81-85. doi:10.1128/JCM.05092-11.
[4]	Xia H, Zheng Y, Zhao B, et al. Assessment of a 96-Well Plate Assay of Quantitative Drug Susceptibility Testing for Mycobacterium Tuberculosis Complex in China. PLoS One, 2017, 12(1): e0169413. doi:10.1371/journal.pone.0169413.
[5]	World Health Organization. Optimized broth microdilution plate methodology for drug susceptibility testing of Mycobacterium tuberculosis complex. Geneva: World Health Organization, 2022.
[6]	European Committee on Antimicrobial Susceptibility Testing. Area of Technical Uncertainty (ATU) in antimicrobial susceptibility testing[EB/OL].[2025-02-26]. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/Area_of_Technical_Uncertainty_-_guidance_2019-1.pdf.
[7]	Köser CU, Georghiou SB, Schön T, et al. On the Consequences of Poorly Defined Breakpoints for Rifampin Susceptibility Testing of Mycobacterium tuberculosis Complex. J Clin Microbiol, 2021, 59(4): e02328-20. doi:10.1128/JCM.02328-20.
[8]	Maurer FP, Courvalin P, Böttger EC, et al. Integrating forecast probabilities in antibiograms: a way to guide antimicrobial prescriptions more reliably?. J Clin Microbiol, 2014, 52(10):3674-3684. doi:10.1128/JCM.01645-14. pmid: 25100821
[9]	Valsesia G, Hombach M, Maurer FP, et al. The Resistant-Population Cutoff (RCOFF): a New Concept for Improved Characterization of Antimicrobial Susceptibility Patterns of Non-Wild-Type Bacterial Populations. J Clin Microbiol, 2015, 53(6):1806-1811. doi:10.1128/JCM.03505-14. pmid: 25762769
[10]	Valsesia G, Roos M, Böttger EC, et al. A statistical approach for determination of disk diffusion-based cutoff values for systematic characterization of wild-type and non-wild-type bacterial populations in antimicrobial susceptibility testing. J Clin Microbiol, 2015, 53(6):1812-1822. doi:10.1128/JCM.03506-14. pmid: 25762772
[11]	Blöchliger N, Keller PM, Böttger EC, et al. MASTER: a model to improve and standardize clinical breakpoints for antimicrobial susceptibility testing using forecast probabilities. J Antimicrob Chemother, 2017, 72(9):2553-2561. doi:10.1093/jac/dkx196. pmid: 28859448
[12]	Köser CU, Robledo J, Shubladze N, et al. Guidance is needed to mitigate the consequences of analytic errors during antimicrobial susceptibility testing for TB. Int J Tuberc Lung Dis, 2021, 25(10):791-794. doi:10.5588/ijtld.21.0428. pmid: 34615575
[13]	Kadura S, King N, Nakhoul M, et al. Systematic review of mutations associated with resistance to the new and repurposed Mycobacterium tuberculosis drugs bedaquiline, clofazimine, linezolid, delamanid and pretomanid. J Antimicrob Chemother, 2020, 75(8):2031-2043. doi:10.1093/jac/dkaa136. pmid: 32361756
[14]	CRyPTIC Consortium. Quantitative measurement of antibiotic resistance in Mycobacterium tuberculosis reveals genetic determinants of resistance and susceptibility in a target gene approach. Nat Commun, 2024, 15(1):488. doi:10.1038/s41467-023-44325-5.
[15]	Jouet A, Gaudin C, Badalato N, et al. Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs. Eur Respir J, 2021, 57(3): 2002338. doi:10.1183/13993003.02338-2020.
[16]	Vargas R Jr, Freschi L, Spitaleri A, et al. Role of Epistasis in Amikacin, Kanamycin, Bedaquiline, and Clofazimine Resis-tance in Mycobacterium tuberculosis Complex. Antimicrob Agents Chemother, 2021, 65(11): e0116421. doi:10.1128/AAC.01164-21.
[17]	Foongladda S, Banu S, Pholwat S, et al. Comparison of TaqMan Array Card and MYCOTB^TM with conventional phenotypic susceptibility testing in MDR-TB. Int J Tuberc Lung Dis, 2016, 20(8):1105-1112. doi:10.5588/ijtld.15.0896. pmid: 27393547
[18]	World Health Organization. Technical Report on critical concentrations for drug susceptibility testing of isoniazid and the rifamycins (rifampicin, rifabutin and rifapentine). Geneva: World Health Organization, 2021.
[19]	Ismail NA, Ismail F, Joseph L, et al. Epidemiological cut-offs for Sensititre susceptibility testing of Mycobacterium tuberculosis: interpretive criteria cross validated with whole genome sequencing. Sci Rep, 2020, 10(1):1013. doi:10.1038/s41598-020-57992-x. pmid: 31974497
[20]	Heysell SK, Moore JL, Peloquin CA, et al. Outcomes and use of therapeutic drug monitoring in multidrug-resistant tuberculosis patients treated in virginia, 2009—2014. Tuberc Respir Dis (Seoul), 2015, 78(2):78-84. doi:10.4046/trd.2015.78.2.78.
[21]	Makhado NA, Matabane E, Faccin M, et al. Outbreak of multidrug-resistant tuberculosis in South Africa undetected by WHO-endorsed commercial tests: an observational study. Lancet Infect Dis, 2018, 18(12):1350-1359. doi:10.1016/S1473-3099(18)30496-1. pmid: 30342828
[22]	Beckert P, Sanchez-Padilla E, Merker M, et al. MDR M.tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era. Genome Med, 2020, 12(1):104. doi:10.1186/s13073-020-00793-8.
[23]	夏辉, 郑扬, 宋媛媛. 世界卫生组织《优化肉汤微孔板法结核分枝杆菌复合群药物敏感性试验方法学》解读. 中国防痨杂志, 2022, 44(7): 641-645. doi:10.19982/j.issn.1000-6621.20220187.
[24]	World Health Organization. WHO consolidated guidelines on tuberculosis. Module 4: treatment-drug-resistant tuberculosis treatment. Geneva: World Health Organization, 2020.
[25]	Rancoita PMV, Cugnata F, Gibertoni Cruz AL, et al. Validating a 14-drug microtitre plate containing bedaquiline and delamanid for large-scale research susceptibility testing of Mycobacterium tuberculosis. Antimicrob Agents Chemother, 2018, 62(9): e00344-18. doi:10.1128/AAC.00344-18.
[26]	Clinical and Laboratory Standards Institute. Performance Standards for Susceptibility Testing of Mycobacteria, Nocardia spp., and Other Aerobic Actinomycetes. 2nd ed. Waynesboro, Virginia: Clinical and Laboratory Standards Institute, 2023.
[27]	Schön T, Köser CU, Werngren J, et al. What is the role of the EUCAST reference method for MIC testing of the Mycobacterium tuberculosis complex?. Clin Microbiol Infect, 2020, 26(11):1453-1455. doi:10.1016/j.cmi.2020.07.037.
[28]	CRyPTIC Consortium. Epidemiological cut-off values for a 96-well broth microdilution plate for high-throughput research antibiotic susceptibility testing of M.tuberculosis. Eur Respir J, 2022, 60(4): 2200239. doi:10.1183/13993003.00239-2022.
[29]	中华人民共和国国家卫生健康委员会. WS/T 807—2022 临床微生物培养、鉴定和药敏检测系统的性能验证. 2022-01-02.
[30]	Kaniga K, Cirillo DM, Hoffner S, et al. A Multilaboratory, Multicountry Study To Determine MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing of Selected First-Line Antituberculosis Drugs, Second-Line Injectables, Fluoroquinolones, Clofazimine, and Linezolid. J Clin Microbiol, 2016, 54(12):2963-2968. doi:10.1128/JCM.01138-16. pmid: 27654338
[31]	Kaniga K, Cirillo DM, Hoffner S, et al. A Multilaboratory, Multicountry Study To Determine Bedaquiline MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing. J Clin Microbiol, 2016, 54(12): 2956-2962. doi:10.1128/JCM.01123-16. pmid: 27654337
[32]	Schena E, Nedialkova L, Borroni E, et al. Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC^TM MGIT^TM 960 system. J Antimicrob Chemother, 2016, 71(6): 1532-1539. doi:10.1093/jac/dkw044. pmid: 27076101
[33]	Thermo Fisher Scientific. Thermo Scientific^TM Sensititre^TM MIC Susceptibility Plates for Mycobacterium tuberculosis (011-MYCOTB CID10470)[EB/OL].[2025-02-26]. https://www.thermofisher.cn/.
[34]	Thermo Fisher Scientific. Thermo Scientific^TM Sensititre^TM MIC Susceptibility Plates for Mycobacterium tuberculosis (034-MYCOTB CID10470)[EB/OL].[2025-02-26]. https://www.thermofisher.cn/.
[35]	World Health Organization. WHO Consolidated Guidelines on Drug-resistant Tuberculosis Treatment. Geneva: World Health Organization, 2019.
[36]	Clinical and Laboratory Standards Institute. CLSI M100:Performance Standards for Antimicrobial Susceptibility Testing (35th ed)[EB/OL]. [2025-02-26]. https://clsi.org/shop/standards/m100/.
[37]	中华人民共和国国家卫生健康委员会. 国家卫生健康委关于印发人间传染的病原微生物目录的通知. 国卫科教发〔2023〕24 号. 2023-08-18.
[38]	中华人民共和国国家卫生和计划生育委员会.WS 233—2017 病原微生物实验室生物安全通用准则. 2017-07-24.
[39]	中华人民共和国国家卫生健康委员会. 可感染人类的高致病性病原微生物菌(毒)种或样本运输管理规定. 卫生部令第45号. 2005-12-28.

药物中文名	药物英文名	药物英文缩写	推荐级别	推荐浓度范围(mg/L)
利福平	Rifampicin	RIF	最高	0.016~2
异烟肼	Isoniazid	INH	最高	0.016~2
左氧氟沙星	Levofloxacin	LFX	最高	0.06~8
莫西沙星	Moxifloxacin	MFX	最高^a	-
贝达喹啉	Bedaquiline	BDQ	最高	0.008~1
利奈唑胺	Linezolid	LZD	最高	0.06~4
氯法齐明	Clofazimine	CFZ	最高	0.008~1
德拉马尼	Delamanid	DLM	最高	0.002~0.25
普托马尼	Pretomanid	PMD	最高^b	-
吡嗪酰胺	Pyrazinamide	PZA	最高^c	-
乙胺丁醇	Ethambutol	EMB	较高	0.125~8
D-环丝氨酸	D-cycloserine	DCS	较高	1~64
特立齐酮	Terizidone	TZD	较高^d	-
乙硫异烟胺	Ethionamide	ETO	较高	0.125~16
丙硫异烟胺	Prothionamide	PTO	较高^e	-
卡那霉素	Kanamycin	KAN	一般^f	0.125~16
阿米卡星	Amikacin	AMK	一般^g	-
链霉素	Streptomycin	STR	不推荐	-
卷曲霉素	Capreomycin	CPM	不推荐	-
对氨基水杨酸	Para-aminosalicylic acid	PAS	不推荐^h	-
利福喷丁	Rifapentine	RFT	不推荐ⁱ	-
利福布汀	Rifabutin	RFB	不推荐	-
氧氟沙星	Ofloxacin	OFX	不推荐^j	-

药物名称 (缩写)	WHO推荐ECOFF 值(mg/L)^[5]	CLSI推荐折点 (mg/L)^[26]	EUCAST推荐折点(mg/L)^[27]	CRyPTIC Consortium 推荐ECOFF值 (UKMYC6/5)(mg/L)^[28]	本共识拟临时推荐折点(mg/L)
利福平(RIF)	0.5	0.5	-	0.5	0.5
异烟肼(INH)	0.125	0.12	-	0.1(0.2/0.4为临界MIC)	0.125
左氧氟沙星(LFX)	1	-	-	1	1
莫西沙星(MFX)	-	-	-	1	0.5
贝达喹啉(BDQ)	0.125(或0.25)	-	0.25	0.25	0.25
利奈唑胺(LZD)	1.0(或2.0)	-	-	1	1
氯法齐明(CFZ)	0.25(或0.5)	-	-	0.25	0.25
德拉马尼(DLM)	0.06(或0.125)	-	0.06	0.12	0.125
普托马尼(PTM)	-	-	2	-	2
乙胺丁醇(EMB)	4	≤2.0为敏感,4.0为不确定,≥8.0为耐药	-	4(4为临界MIC)	≤2.0为敏感,4.0为不确定结果,≥8.0为耐药
D-环丝氨酸(DCS)	32(或64)	-	-	-	32
乙硫异烟胺(ETO)	4	-	-	4(4为临界MIC)	4
卡那霉素(KAN)	4	-	-	4	4
阿米卡星(AMK)	-	-	-	1	1
链霉素(STR)	-	-	-	-	2
卷曲霉素(CPM)	-	-	-	-	-
对氨基水杨酸(PAS)	-	-	-	-	2
利福布汀(RFB)	-	-	-	0.12	-
利福喷丁(RFT)	-	-	-	-	-
氧氟沙星(OFX)	-	-	-	-	2

药物名称(缩写)	MIC值质控范围(mg/L)^a
药物名称(缩写)	CLSI-冻干药粉微孔板^[26]	CLSI-冷冻微孔板^[26]	其他研究
利福平(RIF)	≤0.12	0.06~0.25	0.03~0.25^[30]
异烟肼(INH)	≤0.12	0.03~0.12	0.03~0.25^[30]
左氧氟沙星(LFX)	-	0.12~1	0.12~1^[30]
莫西沙星(MFX)	≤0.5	0.06~0.5	0.06~0.5^[30]
贝达喹啉(BDQ)	-	0.015~0.06	0.015~0.06^[31]
利奈唑胺(LZD)	-	0.25~2	0.25~2^[30]
氯法齐明(CFZ)	-	0.03~0.25	0.03~0.25^[30]
德拉马尼(DLM)	-	-	0.002~0.016^[32]
普托马尼(PMD)	-	-	-
乙胺丁醇(EMB)	≤0.5~2	0.25~2	0.25~2^[30]
D-环丝氨酸(DCS)	-	-	4~16^[33-34]
特立齐酮(TZD)	-	-	-
乙硫异烟胺(ETO)	0.6~2.5	-	-
卡那霉素(KAN)	1.2~5	0.25~2	0.25~2^[30]
阿米卡星(AMK)	0.25~1	0.25~2	0.25~2^[30]
链霉素(STR)	0.5~2	-	-
卷曲霉素(CPM)	-	0.5~4	0.5~4^[30]
对氨基水杨酸(PAS)	≤0.5	-	-
利福喷丁(RFT)	-	-	-
利福布汀(RFB)	≤0.12	-	-
氧氟沙星(OFX)	0.5~2	0.25~2	0.25~2^[30]