应用流式细胞术鉴定死活结核分枝杆菌的实验研究
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摘要
【目的】探讨碘化丙锭(PI)和6-羧基荧光素二乙酸酯(6-cFDA)鉴别死、活结核分枝杆菌的性能,为应用流式细胞术定量检测与分析结核分枝杆菌药物敏感性和异质性耐药方法的创建提供基础性实验依据。【方法】应用PI和6-cFDA分别对10株1麦氏浊度结核分枝杆菌纯培养活菌悬液和其相应的1麦氏浊度85℃加热30 min的死菌悬液进行染色,应用流式细胞术检测其平均荧光强度(MFI),依据受试者工作特征曲线(ROC)确定区分死菌与活菌的MFI界值,然后对另外10株活菌悬液和10株死菌悬液进行相同实验,利用前述MFI界值进行细菌死活判定。【结果】①在确定MFI界值实验中:10份死菌悬液样本PI染色的MFI值呈非正态分布,其中位值为2 459,10份活菌悬液样本PI染色的MFI值呈正态分布,其均值为426±180,PI染色鉴定死菌的MFI界值为1 329;10份死、活菌悬液样本6-cFDA染色的MFI值均呈正态分布,其均值分别为49±4和7 144±4 512,6-cFDA染色鉴定活菌的MFI界值为1 021。②应用PI染色鉴定死菌的MFI界值和6-cFDA染色鉴定活菌的MFI界值检测另外10株活菌悬液和10株死菌悬液,其准确度分别为95%和100%,灵敏度分别为1.00和1.00,特异度分别为0.90和1.00,Yonde指数分别为0.90和1.00,阳性预测值分别为0.91和1.00,阴性预测值分别为1.00和1.00。【结论】PI染色能够良好鉴定结核分枝杆菌死菌、6-cFDA染色能够良好鉴定结核分枝杆菌活菌,基于PI、6-cFDA染色鉴定结核分枝杆菌活性将具有广阔的应用前景。
【Objective】To provide basic experimental basis for quantitative detection and analysis of antibiotic susceptibility and heteroresistance in Mycobacterium tuberculosis by flow cytometry,we investigated the performance of propidium iodide(PI)and 6-carboxyfluorescein diacetae(6-cFDA)in identifying dead or living Mycobacterium tuberculosis.【Methods】PI and 6-cFDA were used to stain 10 strains of pure cultured living Mycobacterium tuberculosis suspension with turbidity of 1 McClell and the corresponding suspension with turbidity of 1 McClell heated at 85℃ for 30 minutes,respectively. The mean fluorescence intensity(MFI)was measured by flow cytometry. The MFI boundary value for distinguishing dead and living Mycobacterium tuberculosis were determined according to receiver operating characteristic(ROC)curves. The same experiments were carried out with other 10 strains of living Mycobacterium tuberculosis suspension and 10 dead Mycobacterium tuberculosis suspension,and the MFI threshold was used to determine the viability of bacteria.【Results】(1)In the experiment of determining MFI boundary value,the MFI of 10 strains of dead bacteria suspension stained with PI was non-normal distribution,the median value was 2 459,and the MFI of 10 strains of living bacteria suspension stained with PI was normal distribution,the mean value was 426±180. The MFI boundary value of dead bacteria stained with PI was 1 329. The MFI of 10 strains of dead bacteria and 10 strains of living bacteria suspension stained with 6-cFDA was normal distribution,and the mean value was 49±4 and 7 144±4 511,respectively. The MFI threshold of 6-cFDA staining for living bacteria was 1 021.(2)The MFI thresholds for identifying dead bacteria by PI staining and the MFI thresholds for identifying living bacteria by 6-cFDA staining were used to detect 10 other living bacterial suspensions and 10 dead bacterial suspensions. The accuracy,sensitivity,specificity,Yonde index,positive predictive value and negative predictive value were 95% and 100%,1.00 and 1.00,0.90 and 1.00,0.90 and 1.00,0.91 and 1.00,1.00 and 1.00,respectively.【Conclusions】PI staining can detect dead Mycobacterium tuberculosis and 6-cFDA staining can detect living Mycobacterium tuberculosis. Identification of Mycobacterium tuberculosis activity based on PI and6-cFDA staining will have broad application prospects.
【Objective】To provide basic experimental basis for quantitative detection and analysis of antibiotic susceptibility and heteroresistance in Mycobacterium tuberculosis by flow cytometry,we investigated the performance of propidium iodide(PI)and 6-carboxyfluorescein diacetae(6-cFDA)in identifying dead or living Mycobacterium tuberculosis.【Methods】PI and 6-cFDA were used to stain 10 strains of pure cultured living Mycobacterium tuberculosis suspension with turbidity of 1 McClell and the corresponding suspension with turbidity of 1 McClell heated at 85℃ for 30 minutes,respectively. The mean fluorescence intensity(MFI)was measured by flow cytometry. The MFI boundary value for distinguishing dead and living Mycobacterium tuberculosis were determined according to receiver operating characteristic(ROC)curves. The same experiments were carried out with other 10 strains of living Mycobacterium tuberculosis suspension and 10 dead Mycobacterium tuberculosis suspension,and the MFI threshold was used to determine the viability of bacteria.【Results】(1)In the experiment of determining MFI boundary value,the MFI of 10 strains of dead bacteria suspension stained with PI was non-normal distribution,the median value was 2 459,and the MFI of 10 strains of living bacteria suspension stained with PI was normal distribution,the mean value was 426±180. The MFI boundary value of dead bacteria stained with PI was 1 329. The MFI of 10 strains of dead bacteria and 10 strains of living bacteria suspension stained with 6-cFDA was normal distribution,and the mean value was 49±4 and 7 144±4 511,respectively. The MFI threshold of 6-cFDA staining for living bacteria was 1 021.(2)The MFI thresholds for identifying dead bacteria by PI staining and the MFI thresholds for identifying living bacteria by 6-cFDA staining were used to detect 10 other living bacterial suspensions and 10 dead bacterial suspensions. The accuracy,sensitivity,specificity,Yonde index,positive predictive value and negative predictive value were 95% and 100%,1.00 and 1.00,0.90 and 1.00,0.90 and 1.00,0.91 and 1.00,1.00 and 1.00,respectively.【Conclusions】PI staining can detect dead Mycobacterium tuberculosis and 6-cFDA staining can detect living Mycobacterium tuberculosis. Identification of Mycobacterium tuberculosis activity based on PI and6-cFDA staining will have broad application prospects.
引文
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[12]Decoster DJ,Vena RM,Callister SM,et al.Susceptibility testing of Mycobacterium tuberculosis:comparison of the BACTEC TB-460 method and flow cytometric assay with the proportion method[J].Clin Microbiol Infect,2005,11(5):372-378.
[13]吴迪.应用流式细胞术检测结核分枝杆菌异烟肼耐药性的实验研究[D].2015.Wu D.An experimental study on susceptibility testing of Mycobacterium tuberculosis to isoniazid by flow cytometry[D].2015.
[14]Doig C.The efficacy of the heat killing of Mycobacterium tuberculosis[J].J Clini Pathol,2002,55(10):778-779.
[15]Chan LY,Mcculley KJ,Kessel SL.Assessment of cell viability with single-,dual-,and multi-staining methods using image cytometry[M]//Methods Mol Biol.2017;1601:27-41.
[16]罗燕飞,范小斌,龚彩平,等.应用二醋酸酯荧光素与碘化丙啶的流式细胞术分析三种细菌的活性[J].广州医科大学学报,2015(2):29-31.Yanfei L,Xiaobin F,Caiping G,et al.Flow cytometry for viability analysis of three bacteria species with fluorescein diacetate vs propidium iodide staining[J].Academic J Guangzhou Med Univ,2015(2):29-31.
[17]Decoster DJ,Vena RM,Callister SM,et al.Susceptibility testing of Mycobacterium tuberculosis:comparison of the BACTEC TB-460 method and flow cytometric assay with the proportion method[J].Clin Microbiol Infect,2010,11(5):372-378.
[2]Zignol M,Dean AS,Falzon D,et al.Twenty years of global surveillance of antituberculosis-drug resistance[J].N Engl J Med,2016,375(11):1081-1089.
[3]Mariandyshev A,Eliseev P.Drug-resistant tuberculosis threatens WHO′s End-TB strategy[J].Lancet Infect Dis,2017,17(7):674-675.
[4]Gazi MA,Islam MR,Kibria MG,et al.General and advanced diagnostic tools to detect Mycobacterium tuberculosis and their drug susceptibility:a review[J].Eur J Clin Microbiol Infect Dis,2015,34(5):851-861.
[5]Vasava MS,Bhoi MN,Rathwa SK,et al.Drug development against tuberculosis:Past,present and future[J].Indian J Tuberc,2017,64(4):252-275.
[6]余丽,李海成,黎贞燕,等.结核菌及其耐药性不同检测方法的临床应用评价[J].广东医学,2017(2):190-193.Yu L,Li HC,Li ZY,et al.Evaluation of clinical application of different detection methods for tuberculosis and its drug resistance[J].J Guangdong Med,2017(2):190-193.
[7]Kamakoli MK,Sadegh HR,Farmanfarmaei G,et al.Evaluation of the impact of polyclonal infection and heteroresistance on treatment of tuberculosis patients[J].Sci Rep,2017,7:41410.
[8]高旭,李静,柳清云,等.异质性耐药对结核分枝杆菌表型和基因型耐药检测结果的影响[J].中华结核和呼吸杂志,2014,37(4):260-265.Gao X,Li J,Liu QY,et al.Heteroresistance in Mycobacteria tuberculosis is an important factor for the inconsistency between the results of phenotype and genotype drug susceptibility tests[J].China Med Abstracts(Int Med),2014,37(2):260-265.
[9]Davey HM,Kell DB.Flow cytometry and cell sorting of heterogeneous microbial populations:the importance of single-cell analyses[J].Microbiol Rev,1996,60(4):641-696.
[10]付亮,龙军,袁小澎.流式细胞术快速检测产超广谱β-内酰胺酶细菌的研究[J].广东医学,2011,32(4):416-418.Fu L,Long J,Yuan XP.Rapid detection of extended-spectrumβ-lactamase producing bateriae with flow cytometry[J].J Guangdong Med,2011,32(4):416-418.
[11]Kennedy D,Wilkinson MG.Application of flow cytometry to the detection of pathogenic bacteria[J].Curr Issues Mol Biol,2017,23:21.
[12]Decoster DJ,Vena RM,Callister SM,et al.Susceptibility testing of Mycobacterium tuberculosis:comparison of the BACTEC TB-460 method and flow cytometric assay with the proportion method[J].Clin Microbiol Infect,2005,11(5):372-378.
[13]吴迪.应用流式细胞术检测结核分枝杆菌异烟肼耐药性的实验研究[D].2015.Wu D.An experimental study on susceptibility testing of Mycobacterium tuberculosis to isoniazid by flow cytometry[D].2015.
[14]Doig C.The efficacy of the heat killing of Mycobacterium tuberculosis[J].J Clini Pathol,2002,55(10):778-779.
[15]Chan LY,Mcculley KJ,Kessel SL.Assessment of cell viability with single-,dual-,and multi-staining methods using image cytometry[M]//Methods Mol Biol.2017;1601:27-41.
[16]罗燕飞,范小斌,龚彩平,等.应用二醋酸酯荧光素与碘化丙啶的流式细胞术分析三种细菌的活性[J].广州医科大学学报,2015(2):29-31.Yanfei L,Xiaobin F,Caiping G,et al.Flow cytometry for viability analysis of three bacteria species with fluorescein diacetate vs propidium iodide staining[J].Academic J Guangzhou Med Univ,2015(2):29-31.
[17]Decoster DJ,Vena RM,Callister SM,et al.Susceptibility testing of Mycobacterium tuberculosis:comparison of the BACTEC TB-460 method and flow cytometric assay with the proportion method[J].Clin Microbiol Infect,2010,11(5):372-378.
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