分枝杆菌的实验室诊断.ppt

2020 2 8 1 LaboratoryMethodsfortheDiagnosisofMycobacterium分枝杆菌的实验室诊断 高雄荣总病理检验部微生物科窦慧琴99 10 19 2020 2 8 2 实验室诊断方法 药敏试验 4 12ds 培养 42ds Negative 鉴定 7ds 染色镜检 24hs 分生 7ds 2020 2 8 3 课程目标 1 了解结核菌的培养检体收集的方法检体收集的容器检体的前处理接种的培养基了解抗酸性染色 原理及判读标准了解MTB的鉴定方法认识自动化培养仪器 MGIT960 鉴定仪器 ProbeTec 原理及操作方法TiBiliatest 2020 2 8 4 课程目标 2 了解NTM的鉴定方法PCR PFLPMethod了解分子生物学鉴定方法 PCRrestriction enzymeanalysis PRA 之原理 操作 及结果判读Biochemistryreactionsmethod了解药敏试验的方法及使用的药物 2020 2 8 5 GeneralCharacteristics GPB 0 2 0 4x2 10umStrictlyaerobic5 10 CO2enhancegrowthHighlipidsincellwallMycolicacidLongchainfattyacid cellwall60 HydrophobicAcidfastnessResistacidandalkali 2020 2 8 6 结核菌的培养 检体收集的方法检体收集的容器检体的前处理接种的培养基 2020 2 8 7 检体采集 采集时间Sputum以清晨第一口痰为佳 3 5mL Gastriclavage15mL清晨抽取 胃液 体液 采集次数取痰前应先以开水漱口 减少食物残渣为提高培养率 应连续采检3天检体保存及运送尽速送检 4oC保存 以避免杂菌生长 2020 2 8 8 分枝杆菌检体收集容器 50cc蓝头离心管Sputum40ccTissue pusStool2ccFungalmediumBonemarrow blood检体收集容器 宽口 螺旋盖 防摔 避免泄漏 胃液 痰液尿液 体液 2020 2 8 9 加等量的6 NaOH NALC Sodiumcitrate化痰液强力Vortex约15 20秒挑取约5ml痰液至50ml离心试管室温静置20分钟加无菌PhosphateBuffer至约45ml 盖紧盖子离心3 800rpm约15分钟 4 倒掉上清液 留下2ml接种 取0 5ml检体至MGITtube0 1ml检体至LJ培养基同时做一片抹片染AcidFastStain 检体处理 2020 2 8 10 2020 2 8 11 接种的培养基 MGIT MycobacterisGrowthIndicatorTube ModifiedMiddlebrook7H9BrothRutheniumfluorescentcomplexMGITOADCOleicAcid 供分枝杆菌生长时利用Albumin 键结干扰分枝杆菌生长的游离脂肪酸Dextrose 提供能量来源Catalase 破坏具毒性的过氧化物MGITPANTA抗菌剂及抗霉菌剂抑制非分枝杆菌生长包含下列5种抗生素 PolymyxinB AmphoterinB Nalidixicacid Trimethoprim AzlocillinLowenstein Jensenslants 2020 2 8 12 MGITTechnology NegativeCulture LittleorNoFluorescence PositiveCulture StrongFluorescence 2020 2 8 13 InoculationandIncubation MGIT960 Becton DickinsonDiagnosticInstrumentSystems Inthe35 incubator0 5cc culturefor6weeksLowenstein JensenslantsInthe35 incubator0 1cc culturefor8weeks IdentificationBDProbeTecETsystemCTBassay 2020 2 8 14 Incubation TemperatureMost 35 37 Skin softtissue 25 30 M marinum M haemophilaeBlood Bonemarrow 36 38 AtmosphereMiddlebrookagars 5 10 CO2L Jmedium only7daysafterinoculationBACTEC othercommerciallybrothsystem donotrequireincreasedCO2Timeupto6 8weeks 2020 2 8 15 SolidMacroscopeSmooth coarsePigmentMicroscopeNeedle MediumMorphology LiquidMacroscopeCotton like MTB M kansasiiHomogenous NTMTurbidform ContaminationMicroscopeShort ball dot ladder cording 2020 2 8 16 MGITBrothMedium MicroscopeMorphologyCording MTB M kansasii M abscessusLadder M kansasii M gordonaeDot MACBall M fortuitumShort M abscessus M fortuitumNeedle OtherMycobacteriasp 2020 2 8 17 BrothMorphology Cording MTB Ladder M kansasii Dot MAC Ball M fortuitum Short M abscessus Long M gordonae Needle OtherMycobacteriasp M xenopi Cording Zoster Rapid Cording MTB Ladder M kansasii Dot MAC Ball M fortuitum Short M abscessus Long M gordonae Cording Zoster Rapid 2020 2 8 18 Solid Liquid 35 MGIT ST 35 LJ Isolation CultureMedium 35 7H11 Sub Test 2020 2 8 19 Rough M tuberculosiscomplex 7H11SolidMedium Smooth M abscessus M kansasii M gordonae 2020 2 8 20 Acid FastStainMethod Ziehl Neelsenstain hot Kinyounacidfaststain cold Fluorochromestain 2020 2 8 21 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 2020 2 8 22 Kinyounacidfaststain cold Heatfixed driedsmearAddkinyoun scarbofuschin5minRinseslidewithwateranddrainDecolorizewithacidalcohol 2minRinseCounterstainwithmethyleneblue 30 1 RinseAirdry Donotblot 1000Xobs redbacilli http secure provlab ab ca afb stain znstain htm 2020 2 8 23 Interpretation Ziehl Neelsen Kinyoun CDC USANo0Doubtful1 2 300fieldActualNo1 1 9 100field2 1 9 10field3 1 9 field4 9 field Acid faststain4 2020 2 8 24 MethodofExamination左右3次来回 上下9次来回 2020 2 8 25 结核菌室鉴定试验工作流程图 Arylsulfatase3 d 14 dNaCltoleranceNitratePigmentproductionUreaseCatalase SQTelluritereductionTween80Niacin MGIT960 LJMedium 染色镜检 TiBiliatest M tuberculosiscomplex AntimicrobialSusceptibility 7天 7天 快速试验 NTM Growthrate Arylsulfatase3 d Rapidgrowers BDProbeTecDTBTiBiliaPRA NTM BiochemicalTest BiochemicalTest Arylsulfatase3 dNaCltoleranceNitrate Slowgrowers 2020 2 8 26 BDProbeTecTMET 临床意义 利用分子生物学的方法StrandDisplacementAmplification SDA 来侦测检体中微量致病菌之遗传物质 DNAorRNA 以快速的协助临床诊断 PrincipleAmplification StrandDisplacementAmplification SDA IsothermalamplificationsystemDetection FluorescentEnergytransferInternalamplificationcontrol 2020 2 8 27 BDProbeTec鉴定 反应原理 BD专利之发夹状 Hairpin 探针 附图1 两端标有两种不同萤光染剂 一为Fluorescin与Rhodamine 两者在近距离时不产生任何萤光 但在遇到检体中之TargetDNA时 经由primer和DNA探针发生之融合 hybridize 与延展 elongation 反应 而使两股DNA分子上探针标记之萤光染剂因切割而分离 能量的转移 EnergyTransfer 造成fluorescin产生萤光 附图2 此一连串DNA序列放大之过程产生萤光讯号经detector连续侦测之后 由仪器判定为阳性反应 附图1 附图2 2020 2 8 28 BDProbeTecTMET鉴定 2020 2 8 29 Mycobacterialantigen M tuberculoisiscomplex MTBC Secretemorethan33differentproteinsHighlyspecificforM tuberculosiscomplexPredominantproteinsisMPB64 1984 24 kDaprotein initiallyisolatedfromculturefiltratesofMycobacteriumbovisBCGTokyo MPB64antigenwasfoundintheculturefluidofonlystrainsoftheM tuberculosisH37RvandsomesubstrainsofM bovisBCG 2020 2 8 30 MPB64Proteinfeatures MycobacterialproteinfractionSecretoryproteinM tuberculosiscomplexTiBiliaTestDetectionProtein105CFU ml HangzhouGenesisBiodetctionandBiocontrolCo 结核杆菌抗原快速菌种鉴定检验试剂 2020 2 8 31 Immunochromatographicassay ICAslidetest TheteststripconsistsofsamplepadreagentpadnitrocellulosemembraneabsorbentpadSimplecultureconfirmationtestforMTBCDetectMPB64withanti MPB64monoclonalantibody 2020 2 8 32 MPB64 C Specimen Colloid Anti MPB64mAbA SpecimenFlow NCmembrane Anti mousepAb 100 lSpecimen Positive Anti MPB64mAbB TiBiliatestofprinciple 2020 2 8 33 TiBiliatestofprinciple MPB64 TC Specimen SpecimenFlow NCmembrane Anti mousepAb 100 lSpecimen Negative Colloid Anti MPB64mAbA 2020 2 8 34 Thepositivebanddevelopedwithin5to10minIntensityofthecolorwasmaximumat15min TiBiliaTBOperationandInterpretation 2020 2 8 35 SimpleandRapidIdentificationoftheMTBCbyICAUsingAnti MPB64MonoclonalAntibodies JournalofClinicalMicrobiology November1999 Tokyo Theintensityofapositivetestcolorbandvariedfroma3 darkreddishpurple to1 easilyvisiblepinkband 2020 2 8 36 BCGGlaxo donotproduceMPB64antigenBCGJapan aregoodsecretorsoftheantigen 2020 2 8 37 NTM的鉴定方法 PCR RFLPMethodBiochemistryreactionsmethod 2020 2 8 38 7天 7天 分子生物 快速试验 NTM的鉴定方法 NTM Isolate7H11 Rapidgrowers PCR PRA GrowthcharacteristicsBiochemicalTest Arylsulfatase3 dNaCltoleranceNitrate GrowthcharacteristicsBiochemicalTest Arylsulfatase3 14 dNaCltoleranceNitratePigmentproductionUreaseCatalase SQTelluritereductionTween80Niacin Slowgrowers 2020 2 8 39 2hrs 3days 2周 2周 2020 2 8 40 3周 CatalaseSemi quantitative 4周 2 3周 2020 2 8 41 BiochemistryReactions MycobacteriumaviumComplex 2020 2 8 42 PCRrestriction enzymeanalysis PRA method PCR RFLPanalysisofhsp65genePCR RFLPanalysisofthehsp65 65 KilodaltonHeatShockProteinGene genewasperformedforallstrainsA439 bpsegmentamplifiedbyusingprimersTb11 5 ACCAACGATGGTGTGTCCAT Tb12 5 CTTGTCGAACCGCATACCCT DigestionoftheamplifiedproductwithtworestrictionenzymesBstEIIandHaeIII Roche Restrictionfragmentswerethenseparatedbyelectrophoresisona4 agarosegel Pronadisa andvisualizedbystainingwithethidiumbromideandUVlight 2020 2 8 43 Amplificationofa439 bpfragmentofthehsp65geneDigestionoftheamplifiedproductwithtworestrictionenzymesBstEIIandHaeIII PCRrestriction enzymeanalysis PRA 2020 2 8 44 ProcedureforPRA CelllysateorgenomicDNA 2hr 439bpPCRproductamplified 4hr PCRproductcheck 1hr Restricyionenzymedigestion 4hr Gelelectrophoresis 1 5hr Imagecapturingandmatching 0 5hr 2020 2 8 45 PCR Mycobacteria 439bp 12345678910Maker111213PCNC 2020 2 8 46 TB BHMM intra B HM simiaeB HM aviumB HM Marker BstEII HaeIII PRApatternsofdifferentspeciesofmycobacteria 2020 2 8 47 鉴定 30S 50S 16SrDNA DNAsequencing MicroSeq500 PRA JFormosMedAssoc1996 95 530 5 DirectAmplification specifictocertainspecies ABIPrism310 2020 2 8 48 AntimicrobialSusceptibility 1 BACTECMGIT960SIRE 临床意义 利用BACTECMGIT960方法测定M tuberculosiscomplex对streptomycin isoniazid rifampin及ethambutol之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streptomycin1 0 g ml isoniazid0 1 g ml rifampin1 0 g ml ethambutol5 0 g ml原理 BACTECMGIT960SIREKit是一种需时4 12天之定性试验根据比较M tuberculosiscomplex菌株在含药试管及不含药试管 生长对照 GrowthControl 之生长情形 BACTECMGIT960仪器持续监控试管内萤光的增加 2020 2 8 49 AntimicrobialSusceptibility 2 Streptomycininhibitproteinsynthesisbybindingto30Sribosomesubunit justlikeamikacinandkanamycinIsoniazid INH inhibitmycolicacidsynthesis generateactiveoxygenradical asanti NADmetaboliteRifampinRNApolymeraseEthambutolinhibitD arabinoseintoarabiogalactan 2020 2 8 50 MechanismsofResistance RNApolymerase Rifampin Ribosome Streptomycin CellWallBiosynthesis Isoniazid mycolicacidsynthesis Ethambutol arabinogalactan arabinomannansynthesis Ethionamide mycolicacidsynthesis Cycloserine peptidoglycansynthesis DNAgyrase Quinolones Tuberculosis Pathogenesis Protection andControlASMpress WashingtonDC pp 560 Pyrazinamidase Pyrazinamide 2020 2 8 51 ScantheASTsetintotheinstrumentRemovethesetwhencompletedPrintthereportwithASTinterpretationsofeitherSusceptibleorResistant Loading Report 2020 2 8 52 2020 2 8 53 TB室考题 1 本院使用的Acid FastStain为何种方法 A Ziehl Neelsenmethod B Fluorochromemethod C Kinyounmethod2 处理sputum消化 去污染试剂为何 A NaOH NALC Sodiumcitrate B NaCl SodiumcitrateNALC C NaOH Na2CO3 Sodiumcitra