分枝杆菌的实验室诊断.ppt

Laboratory Methods for the Laboratory Methods for the Diagnosis of MycobacteriumDiagnosis of Mycobacterium分枝杆菌的实验室诊断分枝杆菌的实验室诊断高雄荣总高雄荣总 病理检验部病理检验部微生物科微生物科 窦慧琴窦慧琴99.10.1999.10.197/29/20247/29/20241 1实验室诊断方法药敏试验药敏试验(4-12ds)(4-12ds)培养培养(42ds-Negative)(42ds-Negative)鉴定鉴定(7ds)(7ds)染色镜检染色镜检(24hs)(24hs)分生分生(7ds)(7ds)7/29/20247/29/20242 2课程目标 (1)了解结核菌的培养检体收集的方法了解结核菌的培养检体收集的方法检体收集的容器检体收集的容器检体的前处理检体的前处理接种的培养基接种的培养基了解抗酸性染色、了解抗酸性染色、 原理及判读标准原理及判读标准了解了解MTBMTB的鉴定方法的鉴定方法认识自动化培养仪器认识自动化培养仪器(MGIT 960)(MGIT 960)鉴定仪器鉴定仪器( (ProbeTecProbeTec) )原理及操作方法原理及操作方法TiBiliaTiBilia test test 7/29/20247/29/20243 3课程目标 (2)了解了解NTMNTM的鉴定方法的鉴定方法PCR-PFLP MethodPCR-PFLP Method了解分子生物学鉴定方法了解分子生物学鉴定方法-PCR restriction--PCR restriction-enzyme analysis (PRA)enzyme analysis (PRA)之原理、操作、及结果判之原理、操作、及结果判读读Biochemistry reactions methodBiochemistry reactions method了解药敏试验的方法及使用的药物了解药敏试验的方法及使用的药物7/29/20247/29/20244 4General CharacteristicsGPB: 0.2-0.4x2-10 umGPB: 0.2-0.4x2-10 umStrictly aerobic Strictly aerobic 5-10% CO2 enhance growth5-10% CO2 enhance growthHigh lipids in cell wallHigh lipids in cell wallMycolicMycolic acid acidLong chain fatty acid ( cell wall 60%)Long chain fatty acid ( cell wall 60%)HydrophobicHydrophobicAcid fastnessAcid fastnessResist acid and alkali Resist acid and alkali 7/29/20247/29/20245 5结核菌的培养检体收集的方法检体收集的方法检体收集的方法检体收集的方法检体收集的容器检体收集的容器检体收集的容器检体收集的容器检体的前处理检体的前处理检体的前处理检体的前处理接种的培养基接种的培养基接种的培养基接种的培养基7/29/20247/29/20246 6检体采集采集时间采集时间采集时间采集时间SputumSputumSputumSputum以清晨第一口痰为佳以清晨第一口痰为佳以清晨第一口痰为佳以清晨第一口痰为佳(3-(3-(3-(3-5mL)5mL)5mL)5mL)Gastric Gastric Gastric Gastric lavagelavagelavagelavage 15 15 15 15 mLmLmLmL清晨抽取清晨抽取清晨抽取清晨抽取胃液体液体液采集次数采集次数取痰前应先以开水漱口，取痰前应先以开水漱口，减少食物残渣减少食物残渣为提高培养率，应连续采为提高培养率，应连续采检检3 3天天检体保存及运送检体保存及运送尽速送检，尽速送检，4oC 4oC 保存，以保存，以避免杂菌生长避免杂菌生长7/29/20247/29/20247 7分枝杆菌检体收集容器50cc 50cc 50cc 50cc 蓝头离心管蓝头离心管蓝头离心管蓝头离心管Sputum < 5ccSputum < 5ccSputum < 5ccSputum < 5ccGastric Gastric Gastric Gastric lavagelavagelavagelavage 15cc 15cc 15cc 15cc胃液用的容器需加入胃液用的容器需加入胃液用的容器需加入胃液用的容器需加入Na2CO3Na2CO3Na2CO3Na2CO3可中可中可中可中和胃酸和胃酸和胃酸和胃酸 Urine > 40ccUrine > 40ccUrine > 40ccUrine > 40ccTissue, pusTissue, pusTissue, pusTissue, pusStool < 5ccStool < 5ccStool < 5ccStool < 5cc15cc 15cc 15cc 15cc 绿头离心管绿头离心管绿头离心管绿头离心管Body fluids >2ccBody fluids >2ccBody fluids >2ccBody fluids >2ccFungal mediumFungal mediumFungal mediumFungal mediumBone marrow/bloodBone marrow/bloodBone marrow/bloodBone marrow/blood检体收集容器：宽口检体收集容器：宽口检体收集容器：宽口检体收集容器：宽口, , , , 螺旋盖螺旋盖螺旋盖螺旋盖, , , , 防摔防摔防摔防摔, , , , 避免泄漏避免泄漏避免泄漏避免泄漏胃液痰液尿液 体液7/29/20247/29/20248 8加等量的加等量的 6% 6% NaOHNaOH-NALC-Sodium citrate-NALC-Sodium citrate化痰液化痰液强力强力 Vortex Vortex 约约 15 - 20 15 - 20 秒秒挑取约挑取约 5 ml 5 ml 痰液至痰液至 50 ml 50 ml 离心试管离心试管室温静置室温静置 20 20 分钟分钟加无菌加无菌 Phosphate Buffer Phosphate Buffer 至约至约 45 ml, 45 ml, 盖紧盖子盖紧盖子离心离心 3,800 rpm 3,800 rpm 约约 15 15 分钟分钟( 4℃)( 4℃)倒掉上清液倒掉上清液, , 留下留下 2 ml2 ml接种接种: :取取 0.5 ml 0.5 ml 检体至检体至 MGIT tubeMGIT tube0.1ml0.1ml检体至检体至LJ LJ 培养基培养基同时做一片抹片染同时做一片抹片染 Acid Fast StainAcid Fast Stain检体处理7/29/20247/29/20249 97/29/20247/29/20241010接种的培养基MGIT (MGIT (MycobacterisMycobacteris Growth Indicator Tube) Growth Indicator Tube) Modified Modified MiddlebrookMiddlebrook 7H9 Broth 7H9 Broth Ruthenium fluorescent complex Ruthenium fluorescent complex MGIT OADCMGIT OADC  Oleic AcidOleic Acid：供分枝杆菌生长时利用：供分枝杆菌生长时利用AlbuminAlbumin：键结干扰分枝杆菌生长的游离脂肪酸：键结干扰分枝杆菌生长的游离脂肪酸 DextroseDextrose：提供能量来源：提供能量来源CatalaseCatalase：破坏具毒性的过氧化物：破坏具毒性的过氧化物MGIT PANTAMGIT PANTA抗菌剂及抗霉菌剂抑制非分枝杆菌生长抗菌剂及抗霉菌剂抑制非分枝杆菌生长包含下列包含下列5 5种抗生素：种抗生素：PolymyxinPolymyxin B B、、AmphoterinAmphoterin B B、、NalidixicNalidixic acid acid、、TrimethoprimTrimethoprim、、AzlocillinAzlocillin Lowenstein-Jensen slantsLowenstein-Jensen slants7/29/20247/29/20241111MGIT TechnologyMGIT TechnologyNegative CultureNegative CultureFFFO2FO2FO2FO2FO2FFO2FO2CO2O2O2O2O2O2O2Vial HeadspaceGrowth Medium SensorLittle or No FluorescenceLittle or No FluorescenceFFFFFO2FO2FFFFCO2O2O2O2 VialHeadspaceGrowth Medium SensorPositive CulturePositive CultureStrong FluorescenceStrong Fluorescence7/29/20247/29/20241212Inoculation and IncubationMGIT 960 (Becton-Dickinson MGIT 960 (Becton-Dickinson Diagnostic Instrument Diagnostic Instrument Systems) Systems) In the 35 ℃ incubatorIn the 35 ℃ incubator0.5cc-culture for 6 weeks 0.5cc-culture for 6 weeks Lowenstein-Jensen slants Lowenstein-Jensen slantsIn the 35 ℃ incubatorIn the 35 ℃ incubator0.1cc-culture for 8 weeks. 0.1cc-culture for 8 weeks. IdentificationIdentificationBD BD ProbeTecProbeTec ET system ET systemCTB assayCTB assay7/29/20247/29/20241313IncubationTemperatureTemperatureMost: 35-37 ℃ Most: 35-37 ℃ Skin & soft tissue: 25-30℃Skin & soft tissue: 25-30℃M. M. marinummarinum, M. , M. haemophilaehaemophilaeBlood & Bone marrow: 36-38 ℃ Blood & Bone marrow: 36-38 ℃ AtmosphereAtmosphereMiddlebrookMiddlebrook agars: 5-10% CO2 agars: 5-10% CO2L-J medium: only 7 days after inoculationL-J medium: only 7 days after inoculationBACTEC & other commercially broth system: do not BACTEC & other commercially broth system: do not require increased CO2 require increased CO2 TimeTimeup to 6-8 weeksup to 6-8 weeks7/29/20247/29/20241414SolidSolidSolidSolidMacroscopeMacroscopeMacroscopeMacroscopeSmoothSmoothSmoothSmooth、、、、coarsecoarsecoarsecoarsePigmentPigmentPigmentPigmentMicroscopeMicroscopeMicroscopeMicroscopeNeedleNeedleNeedleNeedleMedium Morphology LiquidLiquidLiquidLiquidMacroscopeMacroscopeMacroscopeMacroscopeCotton-like: MTB. Cotton-like: MTB. Cotton-like: MTB. Cotton-like: MTB. M.kansasiiM.kansasiiM.kansasiiM.kansasiiHomogenous: NTMHomogenous: NTMHomogenous: NTMHomogenous: NTMTurbid form: ContaminationTurbid form: ContaminationTurbid form: ContaminationTurbid form: Contamination MicroscopeMicroscopeMicroscopeMicroscopeShortShortShortShort、、、、 ballballballball、、、、 dot dot dot dot 、、、、ladderladderladderladder、、、、 cordingcordingcordingcording7/29/20247/29/20241515MGIT Broth MediumMicroscope Morphology Cording：MTB、 M. kansasii、 M. abscessusLadder： M. kansasii 、M. gordonaeDot：MACBall：M. fortuitumShort：M. abscessus、 M. fortuitumNeedle：Other Mycobacteria sp.7/29/20247/29/20241616Broth MorphologyCordingCording：：MTBMTBLadderLadder：： M. M. kansasiikansasiiDotDot：：MACMACBall: M. Ball: M. fortuitumfortuitumShortShort：：M. M. abscessusabscessusLong: M. Long: M. gordonaegordonaeNeedleNeedle：：Other Other MycobacteriaMycobacteria sp (M. sp (M. xenopixenopi) )Cording-ZosterCording-Zoster：：RapidRapidCordingCording：：MTBMTBLadderLadder：： M. M. kansasiikansasiiDotDot：：MACMACBall: M. Ball: M. fortuitumfortuitumShortShort：：M. M. abscessusabscessusLong: M. Long: M. gordonaegordonaeCording-ZosterCording-Zoster：：RapidRapid7/29/20247/29/20241717SolidSolidSolidSolidLiquidLiquidLiquidLiquid35℃ MGIT -ST35℃ LJ35℃ LJIsolation : Culture Medium35℃ 7H11Sub & TestSub & Test7/29/20247/29/20241818RoughM. tuberculosis complex7H11 Solid MediumSmoothM. abscessusM. kansasiiM. gordonae7/29/20247/29/20241919Acid-Fast Stain MethodZiehl-Neelsen stain (hot) Kinyoun acid fast stain (cold)Fluorochrome stain 7/29/20247/29/20242020厚缩抹片制作呼吸道检体经过消化厚缩处理接种培养基剩余检体涂成 2x1公分大小 抹片静置待乾燥7/29/20247/29/20242121Kinyoun acid fast stain (cold)Heat fixed, dried smearHeat fixed, dried smearAdd Add kinyounkinyoun＇＇s s carbofuschincarbofuschin 5 min 5 minRinse slide with water and drainRinse slide with water and drainDecolorize with acid alcohol, 2 minDecolorize with acid alcohol, 2 minRinseRinseCounterstainCounterstain with with methylenemethylene blue, 30 blue, 30＇＇＇＇-1-1＇＇RinseRinseAir dry (Do not blot)Air dry (Do not blot)1000X obs. (red bacilli)1000X obs. (red bacilli)http://secure.provlab.ab.ca/.../afb_stain/znstain.htm7/29/20247/29/20242222Interpretation (Ziehl-Neelsen/Kinyoun)CDC-USACDC-USANoNo0 0DoubtfulDoubtful1-2/300 field 1-2/300 field Actual NoActual No 1+1+1-9/100 field 1-9/100 field 2+2+1-9/10 field1-9/10 field3+3+1-9/field1-9/field4+4+>9/field >9/field Acid-fast stain 4+Acid-fast stain 4+7/29/20247/29/20242323Method of Examination左右3次来回，上下9次来回7/29/20247/29/20242424结核菌室鉴定试验工作流程图 ArylsulfataseArylsulfatase 3-d 3-d、、14-14-ddNaClNaCl tolerance toleranceNitrateNitratePigment productionPigment productionUreaseUreaseCatalaseCatalase-SQ-SQTelluriteTellurite reduction reductionTweenTween 80 80NiacinNiacinMGIT 960、LJ Medium染色镜检TiBilia test M. tuberculosis complexAntimicrobial Antimicrobial SusceptibilitySusceptibility＜7 天＞7 天快速试验NTMGrowth rate ＆ Arylsulfatase 3-dRapid growers•BD ProbeTec DTB•TiBilia•PRA-NTMBiochemical TestBiochemical TestArylsulfataseArylsulfatase 3-d3-dNaClNaCl tolerance toleranceNitrateNitrateSlow growers+-7/29/20247/29/20242525BDProbeTecTMETBDProbeTecTMET临床意义：利用分子生物学的方法临床意义：利用分子生物学的方法临床意义：利用分子生物学的方法临床意义：利用分子生物学的方法Strand Displacement Strand Displacement Strand Displacement Strand Displacement AmplificationAmplificationAmplificationAmplification（（（（SDASDASDASDA）来侦测检体中微量致病菌之遗传物质（）来侦测检体中微量致病菌之遗传物质（）来侦测检体中微量致病菌之遗传物质（）来侦测检体中微量致病菌之遗传物质（DNA DNA DNA DNA or RNAor RNAor RNAor RNA））））, , , ,以快速的协助临床诊断。

以快速的协助临床诊断以快速的协助临床诊断以快速的协助临床诊断 PrinciplePrinciplePrinciplePrincipleAmplification: Strand Displacement Amplification (SDA) Amplification: Strand Displacement Amplification (SDA) Amplification: Strand Displacement Amplification (SDA) Amplification: Strand Displacement Amplification (SDA) Isothermal amplification systemIsothermal amplification systemIsothermal amplification systemIsothermal amplification systemDetection: Detection: Detection: Detection: FluorescentFluorescentFluorescentFluorescentEnergy transferEnergy transferEnergy transferEnergy transferInternal amplification controlInternal amplification controlInternal amplification controlInternal amplification control7/29/20247/29/20242626BD ProbeTec 鉴定反反应应原原理理：：BDBD专专利利之之发发夹夹状状(Hairpin)(Hairpin)探探针针( (附附图图1)1)两两端端标标有有两两种种不不同同萤萤光光染染剂剂，，一一为为FluorescinFluorescin与与RhodamineRhodamine，，两两者者在在近近距距离离时时不不 产产 生生 任任 何何 萤萤 光光 ，， 但但 在在 遇遇 到到 检检 体体 中中 之之Target Target DNADNA时时，，经经由由primerprimer和和DNADNA探探针针发发生生之之融融合合(hybridize)(hybridize)与与延延展展(elongation)(elongation)反反应应，，而而使使两两股股DNADNA分分子子上上探探针针标标记记之之萤萤光光染染剂因切割而分离。

剂因切割而分离能能 量量 的的 转转 移移 (Energy (Energy Transfer)Transfer)造造 成成fluorescinfluorescin产产生生萤萤光光( (附附图图2)2)，，此此一一连连串串DNADNA序序 列列 放放 大大 之之 过过 程程 产产 生生 萤萤 光光 讯讯 号号 经经detectordetector连连续续侦侦测测之之后后，，由由仪仪器器判判定定为为阳阳性反应附图1)(附图2)7/29/20247/29/20242727BDProbeTecTMETBDProbeTecTMET鉴定鉴定7/29/20247/29/20242828Mycobacterial antigenM. M. tuberculoisistuberculoisis complex (MTBC) complex (MTBC)Secrete more than 33 different proteinsSecrete more than 33 different proteinsHighly specific for M. tuberculosis complexHighly specific for M. tuberculosis complexPredominant proteins is MPB64 (1984)Predominant proteins is MPB64 (1984)24-kDa protein, initially isolated from culture 24-kDa protein, initially isolated from culture filtrates of Mycobacterium filtrates of Mycobacterium bovisbovis BCG Tokyo. BCG Tokyo. MPB64 antigen was found in the culture fluid of MPB64 antigen was found in the culture fluid of only strains of the M. tuberculosis H37Rv and some only strains of the M. tuberculosis H37Rv and some substrainssubstrains of M. of M. bovisbovis BCG BCG7/29/20247/29/20242929MPB 64 Protein featuresMPB 64 Protein featuresMycobacterial protein fractionSecretory proteinM. tuberculosis complexTiBilia Test Detection Protein105 CFU/mlHangzhou Genesis Biodetction and Biocontrol Co.结核杆菌抗原快速菌种鉴定检验试剂 7/29/20247/29/20243030ImmunochromatographicImmunochromatographic assay assay (ICA slide test ) (ICA slide test ) The test strip consists The test strip consists of of sample padsample padreagent padreagent padnitrocellulose membranenitrocellulose membraneabsorbent padabsorbent padSimple culture Simple culture confirmation test for confirmation test for MTBCMTBCDetect MPB64 with anti-Detect MPB64 with anti-MPB64 monoclonal MPB64 monoclonal antibodyantibody7/29/20247/29/20243131MPB64MPB64C C SpecimenSpecimenColloid- Anti-MPB 64 Colloid- Anti-MPB 64 mAbmAb A ASpecimen FlowNC membraneAnti-mouse Anti-mouse pAbpAb100μl Specimen100μl SpecimenPositivePositiveAnti-MPB 64 Anti-MPB 64 mAbmAb B BTiBiliaTiBilia test of principle test of principle7/29/20247/29/20243232TiBiliaTiBilia test of principle test of principleMPB64MPB64 T T C C SpecimenSpecimenSpecimen FlowNC membraneAnti-mouse Anti-mouse pAbpAb100μl Specimen100μl SpecimenNegativeColloid- Anti-MPB 64 Colloid- Anti-MPB 64 mAbmAb A A7/29/20247/29/20243333•The positive band developed within 5 to 10 min• Intensity of the color was maximum at 15 minTiBilia TB Operation and Interpretation7/29/20247/29/20243434Simple and Rapid Identification of the MTBC by ICA Using Anti-MPB64 Monoclonal AntibodiesJournal of Clinical Microbiology, November 1999, TokyoThe intensity of a positive test color band varied from a 3+ (dark reddish purple) to 1+ (easily visible pink band).7/29/20247/29/20243535BCG Glaxo, do not produce MPB64 antigenBCG Japan, are good secretors of the antigen7/29/20247/29/20243636NTM的鉴定方法PCR-RFLP MethodBiochemistry reactions method7/29/20247/29/20243737＜7 天＞7 天分子生物-快速试验NTM NTM 的鉴定方法的鉴定方法NTMNTMIsolate 7H11 Rapid growers•PCR-PRAGrowth characteristics Growth characteristics Biochemical Test:Biochemical Test:ArylsulfataseArylsulfatase 3-d 3-dNaClNaCl tolerance toleranceNitrateNitrateGrowth characteristics Growth characteristics Biochemical Test:Biochemical Test:ArylsulfataseArylsulfatase 3 3、、14-d14-dNaClNaCl tolerance toleranceNitrateNitratePigment productionPigment productionUreaseUreaseCatalaseCatalase-SQ-SQTelluriteTellurite reduction reductionTweenTween 80 80NiacinNiacinSlow growers7/29/20247/29/202438382hrs3 days2周2周7/29/20247/29/202439393周CatalaseCatalase Semi-quantitative Semi-quantitative4周2-3周7/29/20247/29/20244040Results with testsResults with tests% Positive% PositiveNiacinNiacin0 0Nitrate reductionNitrate reduction4 4Heat-stable Heat-stable catalasecatalase6060CatalaseCatalase >45 mm of foam >45 mm of foam2 2Non-Non-chromogenicitychromogenicity8787TweenTween 80 hydrolysis (10 days) 80 hydrolysis (10 days)2 2Slow growth rateSlow growth rate100100Growth (25°C)Growth (25°C)100100Growth (45°C)Growth (45°C)8080MacConkeyMacConkey agar agar5050TelluriteTellurite reduction reduction8181ArylsulfataseArylsulfatase1 1UreaseUrease2 2Resistance to Resistance to NaClNaCl (5%) (5%)0 0Biochemistry Reactions Mycobacterium Mycobacterium aviumavium Complex Complex7/29/20247/29/20244141PCR restriction-enzyme analysis (PRA) methodPCR-RFLP analysis of hsp65 genePCR-RFLP analysis of hsp65 genePCR-RFLP analysis of the hsp65 (65-Kilodalton Heat PCR-RFLP analysis of the hsp65 (65-Kilodalton Heat Shock Protein Gene) gene was performed for all strains Shock Protein Gene) gene was performed for all strains A 439-bp segment amplified by using primers A 439-bp segment amplified by using primers Tb11 (5'-ACC AAC GAT GGT GTG TCC AT) Tb11 (5'-ACC AAC GAT GGT GTG TCC AT) Tb12 (5'-CTT GTC GAA CCG CAT ACC CT)Tb12 (5'-CTT GTC GAA CCG CAT ACC CT)Digestion of the amplified product with two restriction Digestion of the amplified product with two restriction enzymes enzymes BstEIIBstEII and and HaeIIIHaeIII (Roche). (Roche). Restriction fragments were then separated by Restriction fragments were then separated by electrophoresis on a 4% electrophoresis on a 4% agaroseagarose gel ( gel (PronadisaPronadisa) and ) and visualized by staining with visualized by staining with ethidiumethidium bromide and UV bromide and UV light light 7/29/20247/29/20244242Agarose Electrophoresis: 4% agarose, 50VPCRRegions common to mycobacteriaHae IIIHae IIIBstEIIHae IIIBstEIInAmplification of a 439-bp fragment of the hsp65 genenDigestion of the amplified product with two restriction enzymes BstEII and HaeIIIPCR restriction-enzyme analysis (PRA)PCR restriction-enzyme analysis (PRA)7/29/20247/29/20244343Procedure for PRACell Cell lysatelysate or genomic DNA (2hr) or genomic DNA (2hr)439 439 bpbp PCR product amplified(4hr) PCR product amplified(4hr)PCR product check(1hr)PCR product check(1hr)RestricyionRestricyion enzyme digestion(4hr) enzyme digestion(4hr)Gel electrophoresis(1.5hr)Gel electrophoresis(1.5hr)Image capturing and matching (0.5hr)Image capturing and matching (0.5hr)7/29/20247/29/20244444PCR-Mycobacteria (439bp) 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 Maker 11 12 13 PC NC10 Maker 11 12 13 PC NC7/29/20247/29/20244545LaneLaneNoNoNoNoMGITMGITMGITMGIT外观外观enzymeenzymeResultResultMarkerMarker　　　　　　　　BstBst II IIHaeIIIHaeIII　　1,21,2　　T T R R245+120+80245+120+80160+140+80160+140+80MTBMTB　　 4,5 4,5178-011178-011TdTdS/RS/R245+120+100245+120+100155+140+60155+140+60　　M. M. intracellulareintracellulare6,76,7178-030178-030　　TdTd S/R S/R245+220245+220　　200+135200+135　　M. M. simiaesimiae I I　　8,98,9177-15177-15　　TdTd S/R S/R245+220245+220　　140+105140+105　　M. M. aviumavium　　TB -B H M M. intra-B -H M. simiae B, H TB -B H M M. intra-B -H M. simiae B, H M. avium B,H MM. avium B,H MMarkerBstEIIHaeIIIPRA patterns of different species of mycobacteria7/29/20247/29/20244646鉴定30S30S50S50S16S 16S rDNArDNADNA sequencingDNA sequencing( (MicroSeqMicroSeq 500) 500)HHB BH65 kD hspPRAPRAJ Formos Med Assoc 1996; 95: 530-5 J Formos Med Assoc 1996; 95: 530-5 MorphologyAFSColonyBiochemical testDirect Amplification:Direct Amplification:specific to certain speciesspecific to certain speciesABI Prism 3107/29/20247/29/20244747Antimicrobial Susceptibility (1) BACTEC MGIT 960 SIRE临床意义：利用临床意义：利用BACTEC MGIT 960BACTEC MGIT 960方法测定方法测定M. tuberculosis M. tuberculosis complexcomplex对对streptomycinstreptomycin、、isoniazidisoniazid、、rifampinrifampin及及ethambutolethambutol之之药物感受性药物感受性使用之药物厚度为套组使用说明中所建议之低厚度，即使用之药物厚度为套组使用说明中所建议之低厚度，即streptomycin 1.0μg/mlstreptomycin 1.0μg/ml、、isoniazidisoniazid 0.1μg/ml 0.1μg/ml、、rifampinrifampin 1.0μg/ml1.0μg/ml、、ethambutolethambutol 5.0μg/ml 5.0μg/ml原理：原理： BACTEC MGIT 960 SIRE Kit BACTEC MGIT 960 SIRE Kit 是一种需时是一种需时4-124-12天之定性试天之定性试验验根据比较根据比较M. tuberculosis complexM. tuberculosis complex菌株在含药试管及不含药试管菌株在含药试管及不含药试管( (生长对照，生长对照，Growth Control)Growth Control)之生长情形。

之生长情形 BACTEC MGIT 960 BACTEC MGIT 960 仪仪器持续监控试管内萤光的增加器持续监控试管内萤光的增加7/29/20247/29/20244848Antimicrobial Susceptibility (2)Streptomycin inhibit protein synthesis Streptomycin inhibit protein synthesis by binding to 30S ribosome subunit, just by binding to 30S ribosome subunit, just like like amikacinamikacin and and kanamycinkanamycinIsoniazid(INHIsoniazid(INH) inhibit ) inhibit mycolicmycolic acid acid synthesis; generate active oxygen synthesis; generate active oxygen radical; as anti-NAD metaboliteradical; as anti-NAD metaboliteRifampinRifampin RNA polymerase RNA polymeraseEthambutolEthambutol inhibit D- inhibit D-arabinosearabinose into into arabiogalactanarabiogalactan7/29/20247/29/20244949Mechanisms of ResistanceRNA polymeraseRNA polymerase RifampinRifampinRibosomeRibosome Streptomycin StreptomycinCell Wall BiosynthesisCell Wall Biosynthesis IsoniazidIsoniazid: : mycolicmycolic acid synthesis? acid synthesis? EthambutolEthambutol: : arabinogalactanarabinogalactan/ / arabinomannanarabinomannan synthesis synthesis EthionamideEthionamide: : mycolicmycolic acid synthesis? acid synthesis? CycloserineCycloserine: : peptidoglycanpeptidoglycan synthesis synthesisDNA DNA gyrasegyraseQuinolonesQuinolonesTuberculosis: Pathogenesis, Protection, and ControlTuberculosis: Pathogenesis, Protection, and ControlASM press, Washington DC. pp. 560ASM press, Washington DC. pp. 560. .PyrazinamidasePyrazinamidasePyrazinamidePyrazinamide7/29/20247/29/20245050Scan the AST set into Scan the AST set into the instrumentthe instrumentRemove the set when Remove the set when completedcompleted Print the report with Print the report with AST interpretations of AST interpretations of either Susceptible or either Susceptible or ResistantResistantLoading & Report7/29/20247/29/202451517/29/20247/29/20245252TB TB 室考题室考题1.( )1.( )本院使用的本院使用的Acid-Fast Stain Acid-Fast Stain 为何种方法为何种方法( (A)Ziehl-NeelsenA)Ziehl-Neelsen method (B). method (B). FluorochromeFluorochrome method ( method (C)KinyounC)Kinyoun method method 2.( _) 2.( _) 处理处理sputumsputum消化、去污染试剂为何消化、去污染试剂为何? (? (A)NaOHA)NaOH-NALC-Sodium citrate (-NALC-Sodium citrate (B)NaClB)NaCl - -Sodium citrate NALC (C) Sodium citrate NALC (C) NaOHNaOH- Na2CO3 -Sodium citrate- Na2CO3 -Sodium citrate3.( ) CDC Acid-Fast Stain 2+ 3.( ) CDC Acid-Fast Stain 2+ 判读标准为判读标准为 (A) 1-9/100 F (B) 1-9/10 F (C ) 1-(A) 1-9/100 F (B) 1-9/10 F (C ) 1-9/ F9/ F4.( ) Acid-Fast Stain 4.( ) Acid-Fast Stain 脱色剂为何脱色剂为何? (A) 3% Acid (? (A) 3% Acid (HClHCl) alcohol (B ) 3% Acid( ) alcohol (B ) 3% Acid( H2SO4) alcohol (C) 3% Acid (Acetone) alcoholH2SO4) alcohol (C) 3% Acid (Acetone) alcohol5.( )5.( )本院所使用的本院所使用的TB TB 药物试验为何药物试验为何 ( A ) Streptomycin (B) ( A ) Streptomycin (B) IsoniazidIsoniazid (C ) (C ) RifampinRifampin ( D ) ( D ) EthambutolEthambutol (E ) Penicillin-G (F ) (E ) Penicillin-G (F ) GentamicinGentamicin (G) (G) AmpicillinAmpicillin (H) (H) SXT (I) SXT (I) VancomycinVancomycin ( (复选题复选题) )6.( )6.( )胃液检体必须加入胃液检体必须加入NaCO3,NaCO3,目的在目的在(A)(A)中和胃酸中和胃酸 (B)(B)破坏结晶物质破坏结晶物质(C)(C)提供分枝杆菌生提供分枝杆菌生长时利用长时利用(D)(D)溶解菜渣溶解菜渣7.( )MGIT7.( )MGIT（（MycobacteriaMycobacteria Growth Indicator Tube Growth Indicator Tube）是培养分枝杆菌之培养基）是培养分枝杆菌之培养基, , 其中加其中加入入 Oleic acidOleic acid目的在目的在 (A)(A)键结干扰分枝杆菌生长的游离脂肪酸键结干扰分枝杆菌生长的游离脂肪酸(B)(B)提供分枝杆菌生长时利提供分枝杆菌生长时利用用(C)(C)提供能量来源提供能量来源(D)(D)破坏具毒性的过氧化物破坏具毒性的过氧化物8.( )8.( )以下何者是结核分枝杆菌在以下何者是结核分枝杆菌在MGITMGIT培养基的形态培养基的形态? (? (A)MacroscopeA)Macroscope: Cotton-like: Cotton-like、、Microscope: Short (Microscope: Short (B)MacroscopeB)Macroscope: Cotton-like: Cotton-like、、Microscope: cording Microscope: cording ( (C)MacroscopeC)Macroscope: homogenous: homogenous、、Microscope: cording (D) Microscope: cording (D) Macroscope:turbidMacroscope:turbid form form、、Microscope: dotMicroscope: dot9.( )9.( )本室本室MycobacteriaMycobacteria 的鉴定使用的方法是的鉴定使用的方法是(A)(A)利用分子生物学的方法利用分子生物学的方法Strand Strand Displacement AmplificationDisplacement Amplification（（SDASDA）来侦测）来侦测 ( (B)NiacinB)Niacin strip method + Nitrate strip method + Nitrate test(Ctest(C) )脉冲电泳法脉冲电泳法(Pulsed-Field Gel Electrophoresis) (D)PRA(PCR Restriction (Pulsed-Field Gel Electrophoresis) (D)PRA(PCR Restriction enzyme Analysis) (E) enzyme Analysis) (E) ImunochromatographicImunochromatographic assay-ICA ( assay-ICA (复选题复选题) )10.( )NTM10.( )NTM鉴定的生化试验中鉴定的生化试验中 potassium potassium telluritetellurite reduction reduction阳性反应的颜色为阳性反应的颜色为: : ( (A)yellowA)yellow ( (B)PinkB)Pink ( (C)BlackC)Black ( (D)colorlessD)colorless7/29/20247/29/20245353答案:1.C 2.A 3.B 4.A5.ABCD 6.A 7.A 8.B 9.A 10.C7/29/20247/29/20245454。