結(jié)核分枝桿菌傳統(tǒng)藥敏試驗(yàn)方法

1、傳統(tǒng)的藥敏實(shí)驗(yàn)傳統(tǒng)的藥敏實(shí)驗(yàn) 藥敏試驗(yàn)的目的選擇有效的方案藥敏試驗(yàn)的校正藥敏試驗(yàn)的校正獲獲得最相得最相關(guān)關(guān)的的臨臨床耐床耐藥標(biāo)藥標(biāo)準(zhǔn)準(zhǔn) 可能的敏感株的敏感可能的敏感株的敏感級(jí)別級(jí)別在治在治療療反反應(yīng)變應(yīng)變化方面化方面測(cè)測(cè)定敏感性水平定敏感性水平從從未接受未接受過(guò)過(guò)治治療療的患者的患者(PS)與與至少接受至少接受5/6個(gè)個(gè)月治月治療療的患的患者者(PR)的的標(biāo)標(biāo)本菌株的敏感性本菌株的敏感性進(jìn)進(jìn)行比行比較較 PS = 100 菌株菌株; PR = 50 菌株菌株Cantti G, et al. Bull WHO 1969; 41:21-43.可能敏感菌株可能敏感菌株/ /（可能無(wú)耐藥性菌株）在不同

2、（可能無(wú)耐藥性菌株）在不同藥物濃度下的抑菌分布藥物濃度下的抑菌分布Minimal inhibitory concentrations (MIC)最小抑菌最小抑菌濃濃度度Frequency of strainsClinicallysusceptiblestrains (PS)Breakpoint1 SD3 SDFrequency of strainsMinimal inhibitory concentrations (MIC)Clinicallysusceptiblestrains (PS)Clinicallyresistantstrains (PR)不同藥物濃度下可能敏感菌株和可能耐藥菌株最低

3、抑菌濃度的分布Frequency of strainsMinimal inhibitory concentrations (MIC)PSPR2 mcg/mlBreakpoint13.1 as “R”75.811.1 as “S”可能敏感/可能耐藥菌株對(duì)利福平的最小抑菌濃度的累積分布（此試驗(yàn)采用 L-J 培養(yǎng)基用絕對(duì)濃度法）85.8Frequency of strainsMinimal inhibitory concentrations (MIC)Clinicallysusceptiblestrains (PS)Clinicallyresistantstrains (PR)采用采用 L-J 培培

4、養(yǎng)養(yǎng)基基Do not quote this data!(n=81) (n=111) 校正的乙胺丁醇的臨界濃度(此試驗(yàn)采用改良羅氏培養(yǎng)基的絕對(duì)濃度法)42%50%In L-J MediumIn 7H10 Medium67%67%抗抗結(jié)結(jié)核核藥藥物的最低抑菌物的最低抑菌濃濃度比平均峰度比平均峰值值EMBETHOFXCFXINHRMPKMCPMPASSM推薦的臨臨界濃濃度藥藥物L(fēng)J*7H107H11Bactec 460MGIT 960異異煙肼肼0.20.20.20.10.1利福平40.01.01.02.01.0乙胺胺丁醇2.05.07.52.55.0鏈鏈霉素4.02.02.02.01.0吡嗪酰胺吡嗪

5、酰胺NRNRNR100.0100.0卡那霉素30.05.06.04.0阿米卡星40.01.01.0卷曲霉素40.010.010.01.252.5環(huán)環(huán)丙沙星2.02.02.02.01.0氧氟氧氟沙星2.02.02.02.02.0莫西沙星0.5 0.25 加替沙星1.0乙硫異酰胺異酰胺5.010.02.55.0丙硫異異煙胺胺40.01.252.5PAS1.02.08.02.0環(huán)絲酰胺環(huán)絲酰胺40.0NRNRNRNR氨苯氨苯硫脲脲NRNRNRNRNR利奈唑酮唑酮1.01.0*Proportion WHO推薦藥敏試驗(yàn)的臨界濃度, 2007(4) 濃縮過(guò)程中利福平的穩(wěn)定性濃縮過(guò)程中利福平的穩(wěn)定性抗結(jié)核藥

6、物的儲(chǔ)存抗結(jié)核藥物的儲(chǔ)存在密封的硅在密封的硅膠膠容器容器內(nèi)儲(chǔ)內(nèi)儲(chǔ)存存(最好在最好在真真空下空下儲(chǔ)儲(chǔ)存存)比例法比例法 (by Middlebrook & Cohn)質(zhì)量較差應(yīng)觀察6周）Report報(bào)告the proportion of colonies grown at the critical concentration of the drug. 在藥物的臨界濃度報(bào)告結(jié)果 Drug free Isoniazid Ethambutol Inoculum media 0.2 mcg/ml 2.0 mcg/ml 10-3 + + 3 10-5 26 9 0 % resistant 34.6

7、 0.1Interpretation Resistant Susceptible藥藥敏敏試驗(yàn)結(jié)試驗(yàn)結(jié)果果-11 . If use nichrome wire (0.7 mm wire/ 3 mm of internal diameter), inoculate oneloopful of bacterial suspension of 10-2 / or if use pipette, inoculate 0.1 ml of 10-3 / of bacterial suspension2. If use nichrome wire, inoculate one loopful of 10-4

8、/ bacterial suspension or if use pipette, inoculate 0.1 ml of 10-5 /.DrugNoneINHRIFSMEMBPNBConcentrations(/)00.240.04.02.0500.0Tube NoC1H1R1S1E1PBInoculum (10-4 )13+2+11870Tube NoC2H2R2S2E2Inoculum (10-6 )2282000InterpretationR (7.1) (2/28x100)S (0.03)(1/2800 x100)S (0.6)(18/2800 x100)S (0.3)(7/2800

9、 x100)MTB藥藥敏敏試驗(yàn)結(jié)試驗(yàn)結(jié)果果-21 . If use nichrome wire (0.7 mm wire/ 3 mm of internal diameter), inoculate oneloopful of bacterial suspension of 10-2 / or if use pipette, inoculate 0.1 ml of 10-3 / of bacterial suspension2. If use nichrome wire, inoculate one loopful of 10-4 / bacterial suspension or if us

10、e pipette, inoculate 0.1 ml of 10-5 /.DrugNoneINHRIFSMEMBPNBConcentrations(/)00.240.04.02.0500.0Tube NoC1H1R1S1E1PBInoculum (10-4 )14+2+21+3+0Tube NoC2H2R2S2E2Inoculum (10-6 )22+50010Interpretation?S?MTB藥藥敏敏試驗(yàn)結(jié)試驗(yàn)結(jié)果果-31 . If use nichrome wire (0.7 mm wire/ 3 mm of internal diameter), inoculate oneloo

11、pful of bacterial suspension of 10-2 / or if use pipette, inoculate 0.1 ml of 10-3 / of bacterial suspension2. If use nichrome wire, inoculate one loopful of 10-4 / bacterial suspension or if use pipette, inoculate 0.1 ml of 10-5 /.DrugNoneINHRIFSMEMBPNBConcentrations(/)00.240.04.02.0500.0Tube NoC1H

12、1R1S1E1PBInoculum (10-4 )13515000Tube NoC2H2R2S2E2Inoculum (10-6 )200000InterpretationRR?MTB 17個(gè)跨國(guó)參比實(shí)驗(yàn)室網(wǎng)絡(luò)第14輪熟練度測(cè)試結(jié)果（數(shù)據(jù)來(lái)源于Armand Van Deun 2007年）敏感性敏感性特特異異性性效能效能可重可重復(fù)復(fù)性性Isoniazid異異煙煙肼肼S敏感敏感=13R耐耐藥藥=17Average (%)99999998100%1314111495%4313Rifampicin利福平利福平S敏感敏感=10R耐耐藥藥=17Average (%)99999998100%14141214

13、95%3323StreptomycinS敏感敏感=11R耐耐藥藥=16Average (%)97919594100%12117995%5688EthambutolS敏感敏感=17R耐耐藥藥=4Average (%)88979697100%111381195%6476藍(lán)色數(shù)字表示實(shí)驗(yàn)室數(shù)量藍(lán)色數(shù)字表示實(shí)驗(yàn)室數(shù)量 51個(gè)國(guó)家（或地區(qū)）參比實(shí)驗(yàn)室利福平和異煙肼藥敏試驗(yàn)結(jié)果的一致性（世界衛(wèi)生組織第四期全球耐藥報(bào)告，2008）% Agreement一致性一致性Isoniazid異異煙煙肼肼Rifampicin利福平利福平10038 (77.6)37 (72.5)95-996 (12.2)9 (17.6)

14、90-944 (8.2)5 (9.8)901 (2.0)0Total number of labs 合合計(jì)計(jì)49 (100.0)51 (100.0)TestDrug藥藥物敏感性(%)特異異性(%)患病情況況下的耐藥結(jié)藥結(jié)果預(yù)測(cè)值預(yù)測(cè)值(%)1%15%110%130%1KM2100909.234.552.681.1CPM21009620.056.873.591.5OFX21009725.064.178.793.5ETH279918.232.049.479.0二線抗結(jié)核藥物耐藥預(yù)測(cè)值1 Prevalence rates2 Results of SLDST proficiency tests don

15、e once in 2004 (coordinated by Dr. Nuria Martine Casabona) (participated SRL=9)3 Based on sensitivity/ specificity of 9 rounds proficiency tests of SRLs, 1994-2002Resistance predictive values (%)Resistance prevalence 敏感性敏感性= 99.6%特特異異性性= 97.9%Frequency of strainsMinimal inhibitory concentrations (MI

16、C)Clinicallysusceptiblestrains (PS)Clinicallyresistantstrains (PR)采用采用 L-J 培培養(yǎng)養(yǎng)基基Do not quote this data!(n=81) (n=111)抗抗結(jié)結(jié)核核藥藥物的最低抑菌物的最低抑菌濃濃度比平均峰度比平均峰值值EMBETHOFXCFXINHRMPKMCPMPASSM抗結(jié)核藥物的儲(chǔ)存抗結(jié)核藥物的儲(chǔ)存在密封的硅在密封的硅膠膠容器容器內(nèi)儲(chǔ)內(nèi)儲(chǔ)存存(最好在最好在真真空下空下儲(chǔ)儲(chǔ)存存) 17個(gè)跨國(guó)參比實(shí)驗(yàn)室網(wǎng)絡(luò)第14輪熟練度測(cè)試結(jié)果（數(shù)據(jù)來(lái)源于Armand Van Deun 2007年）敏感性敏感性特特異異性性效能效能可重可重復(fù)復(fù)性性Isoniazid異異煙煙肼肼S敏感敏感=13R耐耐藥藥=17Av