細菌感染診斷新進展

AdvancesintheDiagnosisofBacterialInfection

細菌感染診斷新進展BudgetSpaceissuesLackoftechnicalknowledgeNointerestfromleadershipWhatisthemainbarriertoimplementing

newdiagnosticsinyourhospital??ESCAPEHighpositivityrate

高陽性率Fastresults

快速結果回報Nofalsepositiveresults

無假陽性Differentiateinfectionfromcolonization

區(qū)分定植與感染Accurateresultsfordesigningadequateanti-infectivetreatment

結果精準用于指導合適的抗感染治療Theexpectationofclinicians

臨床醫(yī)師的期望

“Goldstandard”:extractionDNA+PCR+sequencing+blastDNAofclinicalsampleorculturedclonesBacteria:16SrRNA

Fungi:ITSrRNA,D1/D2regionConventionalmethodscannotidentifythespeciesC.parapsilosiscomplex:C.

parapsilosissensustricto,C.

orthapsilosis,

C.metapsilosisC.glabratacomplex:

C.glabratasensustricto,

C.bracarensis,C.

nivariensisRarespeciesinroutineworkMolecularidentification“金標準”:DNA提取+PCR+測序+blast臨床標本或培養(yǎng)菌的DNA細菌：16SrRNA

真菌：ITSrRNA，D1/D2區(qū)表型、常規(guī)方法不能鑒定到種近平滑念珠菌復合體：近平滑念珠菌、擬平滑念珠菌、似平滑念珠菌光滑念珠菌復合體：C.

glabratasensustricto,C.

bracarensis,C.

nivariensis臨床常規(guī)工作中少見菌的鑒定Rarespecies?CandidaquercitrusaXiaoM,etal.JClinMicrobiol.2014;52(8):3044-8.CHIF-NETIsolate10H16410H16710H176FluconazoleMIC16,R16,R16,RCHROMagarCandidadarkblue(C.tropicalis)darkblue(C.tropicalis)darkblue(C.tropicalis)BrillianceCandidadarkgreen(C.albicans)darkgreen(C.albicans)darkgreen(C.albicans)Vitek2YST(rate)C.pulcherrima(89%)C.pulcherrima(89%)C.pulcherrima(89%)API20CAUX(rate)C.lusitaniae(86.2%)C.lusitaniae(86.2%)C.lusitaniae(86.2%)Pathogen:StreptococcusgroupA

病原學檢查：A群鏈球菌Empirictherapy:+penicillin經驗性治療：+青霉素Clinicaldiagnosis:severeinvasiveinfection,necrotizingfasciitiscomplicatedwithtoxicshocksyndromecausedbyS.

pyogenes

臨床診斷：侵襲性化膿鏈球菌致壞死性筋膜炎并發(fā)毒素休克綜合征Infectionprogressesrapidly王澎楊啟文徐英春等，中華醫(yī)院感染學雜志，2016;26(10):2251.圖1圖2Microfluidicchip

微流控芯片疾病病原核酸檢測平臺以

“疾病”

為基礎呼吸道感染、尿路感染、胃腸道感染、血流感染等整合細菌/真菌/病毒/非典型病原體/寄生蟲/耐藥敏感、快速TAT,turn-aroundtimeDiseasepathogennucleicacidtestingplatformBasedonthe"disease“RTI,UTI,GI,BSI,etc.Bacteria/fungi/virus/atypicalpathogen/parasite/resistanceSensitiveandfastBiofire

FilmArray(bioMerieux)AutomaticDNAextraction/purification/detection

核酸提取/純化/檢測全自動TAT<1h

檢測時間<1hEmergingInfectiousDiseases:

e.g.Ebola

新發(fā)感染性疾病：例如埃博拉病毒SIMPLE2minutestooperate只需2分鐘手工操作時間CONVENIENTNoprecisequantification加樣時無需精確定量FASTDetectiontime:1h儀器檢測過程僅需~1hPCR–multi-nestedPCR多重巢式

Operationalprocess操作流程Upper

respiratory

infection(URP)

上呼吸道感染Bloodstreaminfection(BCID)

血流感染Gastrointestinaldisease(GI)

胃腸道疾病Encephalitisandmeningitis腦炎及腦膜炎PCR–multi-nestedPCR多重巢式

Detectionofpathogens檢測病原體Seventeenviruses 17種病毒Threebacteria 3種細菌Nineteenbacteria 19種細菌

FiveCandida 5種念珠菌Threeresistancegenes 3種耐藥基因mecA,vanA/B,KPC(blakpc)Thirteenbacteria 13種細菌Fourprotozoa 4種原蟲Fivevirus 5種病毒Sixbacteria 6種細菌Twofungi 2種真菌

Sevenvirus 7種病毒AstudyfromSeattleChildren'sHospitalTAT,turn-aroundtimeXuM,etal.AmJClinPathol.2013;139(1):118-23.MethodSampleno.AverageTAT(h)TAT<2h;<3h（%）FilmArray(12/2011–4/2012)25371.682;95DFA(12/2010–4/2011)139970;2FilmArraycomparedwithdirectfluorescenceassay(DFA)

Dec.14,2011toApr.19,2012Results:63%ofallsamplestestedpositiveforviralagentsDFAdoesnotdetectrhinovirusandcoronavirus(26%)FilmArray

與直接免疫熒光染色(DFA)方法對比2011年12月–2012年4月結果63%的樣本病毒檢測陽性DFA不能檢測鼻病毒,冠狀病毒(26%）Microfluidicchip

微流控芯片Buildamicroflowonchip

芯片上構建微流路進行反應Capillaryelectrophoresis

毛細管電泳PCRandDNAhybridization

PCR反應和DNA雜交Immunodetection

免疫檢測MALDI-TOF

質譜技術Microfluidicchip

微流控芯片Advantages技術優(yōu)勢Verylowenergy,samplesandreagentconsumption

能量、樣品、試劑極少Fastandhigh

throughput

分析速度快，通量高Highlyintegratedandsmall

volume

可高度集成，體積小Future

direction

of

development

未來發(fā)展方向Lab-on-a-chip芯片實驗室Enterococcusfaecium屎腸球菌Klebsiella

pneumoniae肺炎克雷伯菌Canddia

albicans

白念珠菌Microfluidicchip

微流控芯片CaiD,etal.LabChip.2014;14(20):3917-24.Enrichmentofpathogensinblood+PCR

富集血液中病原菌+PCRBasedonconductivitydifferences

基于電導率差異Pathogen-specificPCR

病原-特異性PCRMicrofluidicchip

微流控芯片CaiD,etal.LabChip.2014;14(20):3917-24.Rapidpathogensusceptibilitydetermination

病原快速藥敏測定Microfluidicchip

微流控芯片ChoiJ,etal.SciTranslMed.2014;6(267):267ra174.

RapidpathogensusceptibilitydeterminationIn4hoursCoincidencerate>90%comparedwithCLSISingle-celledsensitivitymeasurementAutomationMicrofluidicchip

微流控芯片病原快速藥敏測定4小時內完成檢測與CLSI符合率>90%可實現(xiàn)單細胞敏感性測定可實現(xiàn)自動化ChoiJ,etal.SciTranslMed.2014;6(267):267ra174.

DiskChip進樣后碟式芯片DiskChipNucleicacidanalyzer恒溫擴增微流控芯片核酸分析儀序號細菌名稱拉丁名1肺炎鏈球菌Streptococcuspneumoniae2金黃色葡萄球菌Staphylococcusaureus3大腸埃希氏菌Escherichiacoli4肺炎克雷伯菌Klebsiella

pneumoniae

5銅綠假單胞菌Pseudomonasaeruginosa

6鮑曼不動桿菌Acinetobacter

baumannii

7嗜麥芽窄食單胞菌Stenotrophomonas

maltophilia

8流感嗜血桿菌Haemophilus

influenzae

9嗜肺軍團菌Legionella

pneumophila

10肺炎支原體Mycoplasma

pneumoniae

11肺炎衣原體Chlamydophila

pneumoniae

12耐甲氧西林葡萄球菌methicillin-resistantStaphylococci

(MRS)13結核分枝桿菌復合群Mycobacteriumtuberculosis

complexIsothermalamplificationondiskchip

恒溫擴增碟式芯片法Object:Mycobacteriumtuberculosisandother12

commonrespiratorypathogenssimultaneouslyTurn-aroundtime(TAT):1hSensitivity:500copiesperreactionClinicalcoincidencerate≥90%comparedwithsequencing≥90%檢測對象:結核分枝桿菌

復合群及12種常見呼吸道

病原菌同時檢測檢測時間:1h敏感性:500拷貝/反應臨床符合率≥90%;與測序

結果符合率≥90%Traditionalmoleculardiagnosis:3independentrooms

傳統(tǒng)分子診斷:需要3個獨立房間提取extraction擴增amplification檢測detection++XpertMTB/RIF(WHOrecommended)BloodstreaminfectionSepsis血流感染/敗血癥Culture-basedmethod基于培養(yǎng)方法Diagnosisfromdirectbloodspecimens血液標本直接檢測Biomarkers生物標志物

MALDI-TOFMS“Revolutionofmicrobiologylaboratory”

“微生物實驗室的革命”MassSpectrometryBizziniA,GreubG.ClinMicrobiolInfect.2010;16(11):1614-9.

MALDI-TOFMSPrinciple:Proteinmassspectracomparison

原理:蛋白譜圖比對MassSpectrometryMALDI-TOFMSBactria/fungiidentificationAdvantage:ConvenientFastLimitation:EquipmentcostDatabase:

Shigellavs.E.coli;S.pneumoniaevs.S.mitis/oralisSamplepreparation:TB,moldsSpecies(No.)-16SrRNAsequencingNo.GenuslevelSpecieslevelMisidentificationNoIdentificationLactobacillusgasseri88800Lactobacillusvaginalis*1800018Bacteroidesfragilis15151500Bacteroidesvulgatus33300Bacteroidesovatus33300Bacteroidesnordii*22000Bacteroidesdorei*22000Peptoniphilusasaccharolyticus15141401Peptoniphiluscoxii*40013Peptoniphiluslacrimalis*21001Peptoniphilusduerdenii*10001Peptoniphilusmassiliensis*10001Prevotellabivia88800Prevotellacorporis*62013Prevotelladisiens11100Peptostreptococcusanaerobius14141400Finegoldiamagna99900Anaerococcustetradius*40013Anaerococcushydrogenalis*20002Anaerococcusvaginalis*10001Anaerococcuslactolyticus*10010Propionibacteriumacnes88800Clostridiumsporogenes22200Clostridiumorbiscindens*20002Clostridiumperfringens11100Clostridiumramosum11100Clostridiumbotulinum10010Parvimonasmicra44400Eggerthellahongkongensis*31002Eggerthellalenta11000Veillonellaparvula44400Odoribacterplanchnicus*30012Varibaculumcambriense*20002Total172118108747Mobiluncuscurtisii85503Mobiluncusmulieris22200Porphyromonasasaccharolytica54410Porphyromonasuenonis*32001Porphyromonasgingivalis*11100Porphyromonasendodontalis*10001Species(No.)-16SrRNAsequencingNo.GenuslevelSpecieslevelMisidentificationNoIdentificationMALDI-TOFMS(Vitek):

AnaerobicbacteriaPekingUnionMedicalCollegeHospital.Dataunpublished.MALDI-TOFMS(Bruker):

MolecularepidemiologystudyXiaoM,etal.JClinMicrobiol.2014;52(8):3044-8.PCR/Electrosprayionizationmassspectrometry

PCR/電噴霧離子質譜Principle:Combinesbroad-rangePCRswithESI-MS

原理:廣泛PCR與電噴霧離子質譜結合Upto800pathogens(BACassay;IbisBiosciences)

可檢測多達800種病原菌(BAC數(shù)據(jù)庫)Bacteria,Fungi

細菌，真菌Virus(inthefuture)

病毒(將來)PCR/ESI-MSanalysisWorkflow檢測流程

BacconiA,etal.JClinMicrobiol.2014;52(9):3164-74.331patientswithsuspicionofBSIfromJohnsHopkinsHospitalComparedtobloodculture:Sensitivity:83%(91%*)Specificity:94%(99%*)LOD:16CFU/mlforbacteria;4CFU/mlforCandidaspp.Clinicalstudy臨床研究約翰霍普金斯醫(yī)院，

331位疑似血流感染患者與血培養(yǎng)相比:敏感性:83%(91%*)特異性:

94%(99%*)檢測下限:細菌16CFU/ml;

念珠菌4CFU/ml*Ifconfirmeddetectionswereconsideredtruepositives.*如果確認結果認定為真陽性BSI,bloodstreaminfectionBacconiA,etal.JClinMicrobiol.2014;52(9):3164-74.CD64:OneoftheFcreceptorsforIgGRapidlyincreasesinthepresenceofmicrobialwallcomponents,andsomeproinflammatorycytokinesSubstantiallydecreaseswithin48handbacktonormalbaselinelevelsafter7

daysCD64andPCTarebetterdiagnosticbiomarkersforearlydiagnosisofneonatalsepsisascomparedtoCRPBiomarkers

生物標志物CD64:IgGFc受體之一有微生物細胞壁成分和一些前炎性因子時迅速增加上述成分一旦消失48h內迅速下降，7天內恢復到基線水平CD64和PCT對早期敗血癥的診斷價值優(yōu)于CRPYangAP,etal.ClinChemLabMed.2016;54(2):345-51.TanTL.PLoSOne.2016Mar22;11(3):e0152065.IncorporatesPLA2-IIAandCD64intothediagnosticalgorithmofsepsisGroupIISecretoryPhospholipaseA2(sPLA2-IIA)

2型分泌型磷脂酶A2M53,abroad,T37.7,muscularstiffness,T38.5,pharyngalgia,prosopalgiaFever5dWBC4.7×109/L,N63.4%,ALT81U/L,cTnI0.16ug/LDay2afteradmission：WBC6.5×109/L,N78.6%,CK229U/L,CKMB17.9ug/L,cTnI

3.72ug/L140/100mmHg,HR:123/minCT:patchyshadowofinferiorlingularsegmentonleftupperlobe,

smallnodularshadowonthelowerrightpleuralCryptococcusantigen,HIV

antibody,TORCH,EBV-IgM,CMV-pp65,influenzaAantibody,PCT,Widalreaction,SPOTTB,CMV-DNA,legionella,mycoplasma,chlamydia-Ab,bloodculture×3Thyroidfunction,ANAHR:100–110bpmDay4afteradmission：

CK46U/L,CKMB0.5ug/L,cTnI0.4ug/LCaseDirectlydetectsfromsamples:

throatswab,nasopharyngealaspirates,BALDetectiontime,5hSaveslaborcost,improvesdiagnosticefficiencyLiquidchip

液相芯片法直接檢測標本:咽拭子、鼻咽吸取物、支氣管肺泡灌洗液檢測時間:5h節(jié)省人力成本，提高診斷效率18RespiratoryVirus

18種呼吸道病毒病毒及其亞型ViralSubtypes呼吸道合胞病毒（RSV）RespiratorySyncytialVirus(RSV)

呼吸道合胞病毒A

RSVA

呼吸道合胞病毒

B

RSVB甲型流感病毒InfluenzaA

甲型流感病毒通用型

InfluenzaAmatrix

H1型

H1subtype

H3型

H3subtype

2009H1N1型

2009H1N1乙型流感病毒InfluenzaB副流感病毒

1Parainfluenza1副流感病毒

2Parainfluenza2副流感病毒

3Parainfluenza3副流感病毒

4Parainfluenza4人偏肺病毒

(hMPV)Metapneum