微孔板Alamar blue显色法检测结核分枝杆菌氧氟沙星耐药性的研究
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摘要
目的评价微孔板Alamar blue显色法检测结核分枝杆菌氧氟沙星耐药性的可行性。方法用微孔板Alamar blue显色法对78株结核分枝杆菌检测氧氟沙星的最小抑菌浓度,记录获得结果时间,利用ROC曲线确定氧氟沙星的最佳耐药阈值,并和传统的药物敏感性实验罗氏培养基比例法进行比较。结果微孔板Alamar blue显色法获得结果平均时间为8d,氧氟沙星的最佳耐药阈值为2μg/mL,以罗氏培养基比例法为金标准,氧氟沙星的灵敏度和特异度分别为88.6%和94.1%。结论微孔板Alamar blue显色法是一种简便、敏感、快速的药物敏感性检测方法,可用于结核分枝杆菌氧氟沙星耐药性的快速检测。
We assessed the feasibility of microplate alamar blue assay for determining the ofloxacin resistance of Mycobacterium tuberculosis(M.tuberculosis).MIC of ofloxacin of 78 M.tuberculosisisolates was determined by microplate alamar blue assay and the time of obtaining the results was recorded;receiver operating characteristic(ROC)curve was used to determine the resistant threshold value of ofloxacin,and compared to the conventional proportional method.Results showed that the resistant threshold value of ofloxacin was 2μg/mL for microplate alamar blue assay,and compared to the gold standard proportional method,the sensitivity and specificity were 88.6% and 94.1%,respectively.And the time obtaining the results for microplate alamar blue assay was 8days.Microplate alamar blue assay is simple,sensitive and rapid,and is a promising alternative method for rapidly determining the resistance of ofloxacin of M.tuberculosis.
We assessed the feasibility of microplate alamar blue assay for determining the ofloxacin resistance of Mycobacterium tuberculosis(M.tuberculosis).MIC of ofloxacin of 78 M.tuberculosisisolates was determined by microplate alamar blue assay and the time of obtaining the results was recorded;receiver operating characteristic(ROC)curve was used to determine the resistant threshold value of ofloxacin,and compared to the conventional proportional method.Results showed that the resistant threshold value of ofloxacin was 2μg/mL for microplate alamar blue assay,and compared to the gold standard proportional method,the sensitivity and specificity were 88.6% and 94.1%,respectively.And the time obtaining the results for microplate alamar blue assay was 8days.Microplate alamar blue assay is simple,sensitive and rapid,and is a promising alternative method for rapidly determining the resistance of ofloxacin of M.tuberculosis.
引文
[1]World Health Organization.Anti-tuberculosis drug resistance in the world:fourth global report,WHO/HTM/TB/2008.394[R].Geneva:World Health Organization,2008.
[2]Zhao Y,Xu S,Wang L,et al.National survey of drug-resistant tuberculosis in China[J].N Engl J Med,2012,366(23):2161-2170.DOI:10.1056/NEJMoa1108789
[3]Mokrousov I,Jiao WW,Sun GZ,et al.Evolution of drug resistance in different sublineages of Mycobacterium tuberculosis Beijing genotype[J].Antimicrob Agents Chemother,2006,50(8):2820-2823.DOI:10.1128/AAC.00324-06
[4]Takiff H,Guerrero E.Current prospects for the fluoroquinolones as first-line tuberculosis therapy[J].Antimicrob Agents Chemother,2011,55(12):5421-5429.DOI:10.1128/AAC.00695-11
[5]Wells WA,Boehme CC,Cobelens FG,et al.Alignment of new tuberculosis drug regimens and drug susceptibility testing:a framework for action[J].Lancet Infect Dis,2013,13(5):449-458.DOI:10.1016/S1473-3099(13)70025-2
[6]Li GL,Zhang JR,Zhao XQ,et al.Rapid detection on resistant of rifampicin and isoniazid for Mycobacterium tuberculosis clinical isolates by Alamar Blue asssay[J].Chin J Zoonoses,2012,28(4):319-326.(in Chinese)李桂莲,张敬蕊,赵秀芹,等.微孔板Alamar blue显色法检测结核分枝杆菌利福平和异烟肼耐药性的研究[J].中国人兽共患病学报,2012,28(4):319-322.
[7]Martin A,Morcillo N,Lemus D,et al.Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs[J].Int J Tuberc Lung Dis,2005,9(8):901-906.
[8]Chauca JA,Palomino JC,Guerra H.Evaluation of the accuracy of the microplate Alamar Blue assay for rapid detection of MDRTB in Peru[J].Int J Tuberc Lung Dis,2007,11(7):820-822.
[9]Leonard B,Coronel J,Siedner M,et al.Inter-and intra-assay reproducibility of microplate Alamar blue assay results for isoniazid,rifampicin,ethambutol,streptomycin,ciprofloxacin,and capreomycin drug susceptibility testing of Mycobacterium tuberculosis[J].J Clin Microbiol,2008,46(10):3526-3529.DOI:10.1128/JCM.02083-07
[10]Laszlo A,Rahman M,Espinal M,et al.Quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/IUATLD Supranational Reference Laboratory Network:five rounds of proficiency testing,1994-1998[J].Int J Tuberc Lung Dis,2002,6(9):748-756.
[11]Basic professional committee,Chinese antituberculosis association.Rules for the inspection of the laboratory diagnosis of tuberculosis[M].Beijing:China Education and Culture Publishing House,2006:52.(in Chinese)中国防痨协会基础专业委员会.结核病诊断实验室检验规程[M].北京:中国教育文化出版社,2006:52.
[12]WHO.Policy guidance on drug-susceptibility testing(DST)of second-line antituberculosis drugs(WHO/HTM/TB/2008.392)[M].Geneva:World Health Organization,2008:11.
[13]World Health Organization.Global tuberculosis report 2013[R].Geneva,Switzerland:World Health Organization,2013:45-58.
[14]Wang Z,Cui ZL,Ma XL,et al.Evaluation of the application of trace MIC on the examination of drug sensitivity of Mycobacterium tuberculosis[J].Mod Prevent Med,2014,141(8):1487-1489.(in Chinese)王芝,崔振玲,马晓莉,et al.微量MIC检测结核分枝杆菌药物敏感性的应用评价研究[J].现代预防医学,2014,41(8):1487-1489.
[15]Jiang J,Zhang J,Zhang H,et al.The practical evaluation of second-line anti-tuberculosis drug sesceptibility test using MGIT960system[J].Chin J Antituberc,2012,34(8):527-531.(in Chinese)蒋俊,张娟,张红,et al.MGIT960系统行二线抗结核药物敏感性试验的实用性评价[J].中国防痨杂志,2012,38(8):527-531.
[16]Chen ZN,Zhao LL,Yi SL,et al.Clinical trial of BACTEC MGIT 960for drug susceptibility testing on Mycobacterium tuberculosis[J].Chin J Zoonoses,2013,29(2):166-169.(in Chinese)陈忠南,赵丽丽,易松林,等.BACTEC MGIT960在结核分枝杆菌药物敏感性试验中的临床研究[J].中国人兽共患病学报,2013,29(2):166-169.
[2]Zhao Y,Xu S,Wang L,et al.National survey of drug-resistant tuberculosis in China[J].N Engl J Med,2012,366(23):2161-2170.DOI:10.1056/NEJMoa1108789
[3]Mokrousov I,Jiao WW,Sun GZ,et al.Evolution of drug resistance in different sublineages of Mycobacterium tuberculosis Beijing genotype[J].Antimicrob Agents Chemother,2006,50(8):2820-2823.DOI:10.1128/AAC.00324-06
[4]Takiff H,Guerrero E.Current prospects for the fluoroquinolones as first-line tuberculosis therapy[J].Antimicrob Agents Chemother,2011,55(12):5421-5429.DOI:10.1128/AAC.00695-11
[5]Wells WA,Boehme CC,Cobelens FG,et al.Alignment of new tuberculosis drug regimens and drug susceptibility testing:a framework for action[J].Lancet Infect Dis,2013,13(5):449-458.DOI:10.1016/S1473-3099(13)70025-2
[6]Li GL,Zhang JR,Zhao XQ,et al.Rapid detection on resistant of rifampicin and isoniazid for Mycobacterium tuberculosis clinical isolates by Alamar Blue asssay[J].Chin J Zoonoses,2012,28(4):319-326.(in Chinese)李桂莲,张敬蕊,赵秀芹,等.微孔板Alamar blue显色法检测结核分枝杆菌利福平和异烟肼耐药性的研究[J].中国人兽共患病学报,2012,28(4):319-322.
[7]Martin A,Morcillo N,Lemus D,et al.Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs[J].Int J Tuberc Lung Dis,2005,9(8):901-906.
[8]Chauca JA,Palomino JC,Guerra H.Evaluation of the accuracy of the microplate Alamar Blue assay for rapid detection of MDRTB in Peru[J].Int J Tuberc Lung Dis,2007,11(7):820-822.
[9]Leonard B,Coronel J,Siedner M,et al.Inter-and intra-assay reproducibility of microplate Alamar blue assay results for isoniazid,rifampicin,ethambutol,streptomycin,ciprofloxacin,and capreomycin drug susceptibility testing of Mycobacterium tuberculosis[J].J Clin Microbiol,2008,46(10):3526-3529.DOI:10.1128/JCM.02083-07
[10]Laszlo A,Rahman M,Espinal M,et al.Quality assurance programme for drug susceptibility testing of Mycobacterium tuberculosis in the WHO/IUATLD Supranational Reference Laboratory Network:five rounds of proficiency testing,1994-1998[J].Int J Tuberc Lung Dis,2002,6(9):748-756.
[11]Basic professional committee,Chinese antituberculosis association.Rules for the inspection of the laboratory diagnosis of tuberculosis[M].Beijing:China Education and Culture Publishing House,2006:52.(in Chinese)中国防痨协会基础专业委员会.结核病诊断实验室检验规程[M].北京:中国教育文化出版社,2006:52.
[12]WHO.Policy guidance on drug-susceptibility testing(DST)of second-line antituberculosis drugs(WHO/HTM/TB/2008.392)[M].Geneva:World Health Organization,2008:11.
[13]World Health Organization.Global tuberculosis report 2013[R].Geneva,Switzerland:World Health Organization,2013:45-58.
[14]Wang Z,Cui ZL,Ma XL,et al.Evaluation of the application of trace MIC on the examination of drug sensitivity of Mycobacterium tuberculosis[J].Mod Prevent Med,2014,141(8):1487-1489.(in Chinese)王芝,崔振玲,马晓莉,et al.微量MIC检测结核分枝杆菌药物敏感性的应用评价研究[J].现代预防医学,2014,41(8):1487-1489.
[15]Jiang J,Zhang J,Zhang H,et al.The practical evaluation of second-line anti-tuberculosis drug sesceptibility test using MGIT960system[J].Chin J Antituberc,2012,34(8):527-531.(in Chinese)蒋俊,张娟,张红,et al.MGIT960系统行二线抗结核药物敏感性试验的实用性评价[J].中国防痨杂志,2012,38(8):527-531.
[16]Chen ZN,Zhao LL,Yi SL,et al.Clinical trial of BACTEC MGIT 960for drug susceptibility testing on Mycobacterium tuberculosis[J].Chin J Zoonoses,2013,29(2):166-169.(in Chinese)陈忠南,赵丽丽,易松林,等.BACTEC MGIT960在结核分枝杆菌药物敏感性试验中的临床研究[J].中国人兽共患病学报,2013,29(2):166-169.