细菌感染诊断新进展

AdvancesintheDiagnosisofBacterialInfection

细菌感染诊断新进展BudgetSpaceissuesLackoftechnicalknowledgeNointerestfromleadershipWhatisthemainbarriertoimplementing

newdiagnosticsinyourhospital??ESCAPEHighpositivityrate

高阳性率Fastresults

快速结果回报Nofalsepositiveresults

无假阳性Differentiateinfectionfromcolonization

区分定植与感染Accurateresultsfordesigningadequateanti-infectivetreatment

结果精准用于指导合适的抗感染治疗Theexpectationofclinicians

临床医师的期望

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

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

parapsilosissensustricto,C.

orthapsilosis,

C.metapsilosisC.glabratacomplex:

C.glabratasensustricto,

C.bracarensis,C.

nivariensisRarespeciesinroutineworkMolecularidentification“金标准”:DNA提取+PCR+测序+blast临床标本或培养菌的DNA细菌：16SrRNA

真菌：ITSrRNA，D1/D2区表型、常规方法不能鉴定到种近平滑念珠菌复合体：近平滑念珠菌、拟平滑念珠菌、似平滑念珠菌光滑念珠菌复合体：C.

glabratasensustricto,C.

bracarensis,C.

nivariensis临床常规工作中少见菌的鉴定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

病原学检查：A群链球菌Empirictherapy:+penicillin经验性治疗：+青霉素Clinicaldiagnosis:severeinvasiveinfection,necrotizingfasciitiscomplicatedwithtoxicshocksyndromecausedbyS.

pyogenes

临床诊断：侵袭性化脓链球菌致坏死性筋膜炎并发毒素休克综合征Infectionprogressesrapidly王澎杨启文徐英春等，中华医院感染学杂志，2016;26(10):2251.图1图2Microfluidicchip

微流控芯片疾病病原核酸检测平台以

“疾病”

为基础呼吸道感染、尿路感染、胃肠道感染、血流感染等整合细菌/真菌/病毒/非典型病原体/寄生虫/耐药敏感、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/纯化/检测全自动TAT<1h

检测时间<1hEmergingInfectiousDiseases:

e.g.Ebola

新发感染性疾病：例如埃博拉病毒SIMPLE2minutestooperate只需2分钟手工操作时间CONVENIENTNoprecisequantification加样时无需精确定量FASTDetectiontime:1h仪器检测过程仅需~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)

胃肠道疾病Encephalitisandmeningitis脑炎及脑膜炎PCR–multi-nestedPCR多重巢式

Detectionofpathogens检测病原体Seventeenviruses 17种病毒Threebacteria 3种细菌Nineteenbacteria 19种细菌

FiveCandida 5种念珠菌Threeresistancegenes 3种耐药基因mecA,vanA/B,KPC(blakpc)Thirteenbacteria 13种细菌Fourprotozoa 4种原虫Fivevirus 5种病毒Sixbacteria 6种细菌Twofungi 2种真菌

Sevenvirus 7种病毒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

与直接免疫荧光染色(DFA)方法对比2011年12月–2012年4月结果63%的样本病毒检测阳性DFA不能检测鼻病毒,冠状病毒(26%）Microfluidicchip

微流控芯片Buildamicroflowonchip

芯片上构建微流路进行反应Capillaryelectrophoresis

毛细管电泳PCRandDNAhybridization

PCR反应和DNA杂交Immunodetection

免疫检测MALDI-TOF

质谱技术Microfluidicchip

微流控芯片Advantages技术优势Verylowenergy,samplesandreagentconsumption

能量、样品、试剂极少Fastandhigh

throughput

分析速度快，通量高Highlyintegratedandsmall

volume

可高度集成，体积小Future

direction

of

development

未来发展方向Lab-on-a-chip芯片实验室Enterococcusfaecium屎肠球菌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

基于电导率差异Pathogen-specificPCR

病原-特异性PCRMicrofluidicchip

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

病原快速药敏测定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药敏测定4小时内完成检测与CLSI符合率>90%可实现单细胞敏感性测定可实现自动化ChoiJ,etal.SciTranslMed.2014;6(267):267ra174.

DiskChip进样后碟式芯片DiskChipNucleicacidanalyzer恒温扩增微流控芯片核酸分析仪序号细菌名称拉丁名1肺炎链球菌Streptococcuspneumoniae2金黄色葡萄球菌Staphylococcusaureus3大肠埃希氏菌Escherichiacoli4肺炎克雷伯菌Klebsiella

pneumoniae

5铜绿假单胞菌Pseudomonasaeruginosa

6鲍曼不动杆菌Acinetobacter

baumannii

7嗜麦芽窄食单胞菌Stenotrophomonas

maltophilia

8流感嗜血杆菌Haemophilus

influenzae

9嗜肺军团菌Legionella

pneumophila

10肺炎支原体Mycoplasma

pneumoniae

11肺炎衣原体Chlamydophila

pneumoniae

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

(MRS)13结核分枝杆菌复合群Mycobacteriumtuberculosis

complexIsothermalamplificationondiskchip

恒温扩增碟式芯片法Object:Mycobacteriumtuberculosisandother12

commonrespiratorypathogenssimultaneouslyTurn-aroundtime(TAT):1hSensitivity:500copiesperreactionClinicalcoincidencerate≥90%comparedwithsequencing≥90%检测对象:结核分枝杆菌

复合群及12种常见呼吸道

病原菌同时检测检测时间:1h敏感性:500拷贝/反应临床符合率≥90%;与测序

结果符合率≥90%Traditionalmoleculardiagnosis:3independentrooms

传统分子诊断:需要3个独立房间提取extraction扩增amplification检测detection++XpertMTB/RIF(WHOrecommended)BloodstreaminfectionSepsis血流感染/败血症Culture-basedmethod基于培养方法Diagnosisfromdirectbloodspecimens血液标本直接检测Biomarkers生物标志物

MALDI-TOFMS“Revolutionofmicrobiologylaboratory”

“微生物实验室的革命”MassSpectrometryBizziniA,GreubG.ClinMicrobiolInfect.2010;16(11):1614-9.

MALDI-TOFMSPrinciple:Proteinmassspectracomparison

原理:蛋白谱图比对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/电喷雾离子质谱Principle:Combinesbroad-rangePCRswithESI-MS

原理:广泛PCR与电喷雾离子质谱结合Upto800pathogens(BACassay;IbisBiosciences)

可检测多达800种病原菌(BAC数据库)Bacteria,Fungi

细菌，真菌Virus(inthefuture)

病毒(将来)PCR/ESI-MSanalysisWorkflow检测流程

BacconiA,etal.JClinMicrobiol.2014;52(9):3164-74.331patientswithsuspicionofBSIfromJohnsHopkinsHospitalComparedtobloodculture:Sensitivity:83%(91%*)Specificity:94%(99%*)LOD:16CFU/mlforbacteria;4CFU/mlforCandidaspp.Clinicalstudy临床研究约翰霍普金斯医院，

331位疑似血流感染患者与血培养相比:敏感性:83%(91%*)特异性:

94%(99%*)检测下限:细菌16CFU/ml;

念珠菌4CFU/ml*Ifconfirmeddetectionswereconsideredtruepositives.*如果确认结果认定为真阳性BSI,bloodstreaminfectionBacconiA,etal.JClinMicrobiol.2014;52(9):3164-74.CD64:OneoftheFcreceptorsforIgGRapidlyincreasesinthepresenceofmicrobialwallcomponents,andsomeproinflammatorycytokinesSubstantiallydecreaseswithin48handbacktonormalbaselinelevelsafter7

daysCD64andPCTarebetterdiagnosticbiomarkersforearlydiagnosisofneonatalsepsisascomparedtoCRPBiomarkers

生物标志物CD64:IgGFc受体之一有微生物细胞壁成分和一些前炎性因子时迅速增加上述成分一旦消失48h内迅速下降，7天内恢复到基线水平CD64和PCT对早期败血症的诊断价值优于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

液相芯片法直接检测标本:咽拭子、鼻咽吸取物、支气管肺泡灌洗液检测时间:5h节省人力成本，提高诊断效率18RespiratoryVirus

18种呼吸道病毒病毒及其亚型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