曲霉病實(shí)驗(yàn)診斷技術(shù)課件

曲霉病實(shí)驗(yàn)診斷技術(shù)WHATIS

NEW1曲霉病感染曲霉菌所引起的一種疾病，可侵犯皮膚、黏膜、眼、外耳道、鼻、鼻竇、支氣管、肺、胃腸道、神經(jīng)系統(tǒng)或骨骼，嚴(yán)重者導(dǎo)致敗血癥。由各種曲霉，主要是煙曲霉引起的疾病。呈世界性分布。Fungalballremovedfrom

lung2呼吸系統(tǒng)曲霉病變應(yīng)性支氣管肺曲霉?。ˋBPA）侵襲性肺曲霉?。↖PA）慢性肺曲霉?。–PA）單純肺曲菌球慢性空洞性肺曲霉病（CCPA）慢性纖維化性肺曲霉?。–FPA）曲霉結(jié)節(jié)亞急性侵襲性肺曲霉?。⊿AIA）Chronicpulmonaryaspergillosis:rationaleandclinicalguidelinesfordiagnosisandmanagement.

2015DavidW.Denning.onbehalfoftheESCMIDandEuropeanRespiratory

Society3侵襲性肺曲霉病的診斷1，這個(gè)病人是否是IA？2，能確診嗎3，擬診/疑診(Proven?)(Possible/Probable?)ICU醫(yī)生最困擾的問題IPA

Proven組織活檢無菌部位組織和液體的培養(yǎng)獲取組織標(biāo)本沒那么簡(jiǎn)單！培養(yǎng) 72h以上！確診的風(fēng)險(xiǎn)、時(shí)間等，臨床的確診的病例很少，<10%6曲霉菌分類學(xué)煙曲霉黃曲霉黑曲霉土曲霉構(gòu)巢曲霉。。。。Aspergillussection

Fumigati（A.fumigatus

complex）7A.

fumigatusA.lentulusandA.

fumisynnematus,A.fumigatiaffinisand

A.novofumigatus,

A.viridinutans,A.udagawae,and

otheratypical

strainsA.hiratsukae,A.brevipes,A.duricaulis,andA.unilateralis.8曲霉病的非培養(yǎng)診斷技術(shù)9Whatifinvasivemould

disease…at

risk…beginswithcolonisation

…at

risk…thenprogressestoinfection

…at

riskinfectionNucleic

acidbeta-D-glucangalactomannan…andfinallyto

disease.at

riskinfectiondiseasebeta-D-glucangalactomannanNucleic

acidDecidingwhento

interveneatrisk infectiondiseaseTargetedbeta-D-glucangalactomannanNucleic

acidDecidingwhento

interveneat

risk infectiondiseaseProphylaxisbeta-D-glucangalactomannanNucleic

acidDecidingwhento

interveneatrisk infection diseasebeta-D-glucanPre-emptivegalactomannanNucleic

acidDecidingwhento

interveneat

riskinfectiondiseasebeta-D-glucanDiagnostic

drivengalactomannanNucleic

acid1819半乳甘露聚糖(GM)半乳甘露聚糖(Glactomannan，GM)廣泛存在于曲

霉和青霉細(xì)胞壁，菌生長(zhǎng)時(shí)GM從菌絲頂端釋放，是最早釋放的抗原。曲霉菌屬在生長(zhǎng)期間會(huì)釋放曲霉菌表面抗原，在患者體液中可以檢測(cè)到循環(huán)半乳甘露聚糖抗原。JPeter,etal.Lacectinfectious

disease.2004;350-358SpecificityofGM>85%.SensitivityofGM：29%

～100%IPA的血標(biāo)本GM檢測(cè)（敏感性波動(dòng)大）影響血標(biāo)本GM檢測(cè)敏感性因素眾多!!!!影響血標(biāo)本GM檢測(cè)敏感性因素眾多抗真菌治療可影響血標(biāo)本GM

敏感性No

antifungalsAntifungals0.511.5SensitivityTimetodiagnosis

(Days)KierenA.CID.

2005;35(6):1753-1758GM 比較BALF and Blood(最初96小時(shí) )Galactomannan

indexBALFBLOODTime(hours)Williamhope.ANTIMICROBIALAGENTSANDCHEMOTHERAPY,2010,

4879–IFIG檢驗(yàn)報(bào)告單檢測(cè)試劑盒：曲霉菌半乳甘露聚糖定量檢測(cè)試劑盒（ELISA法）檢測(cè)名稱：曲霉菌半乳甘露聚糖定量檢測(cè)試劑盒（ELISA法）_2016年4月8日樣本類型：血清樣本單位：ug/L上限：0.85下限：0.65建立時(shí)間：2016/4/8

18:20:16擬合方式：對(duì)數(shù)坐標(biāo)，對(duì)數(shù)直線擬合序

號(hào)樣

本

編

碼Y(反應(yīng)值）X(濃度/劑量）陰性/陽性樣本情況描述0空白0.008-空白正常1R2a1.2310.25標(biāo)準(zhǔn)曲線正常2R2b1.1520.5標(biāo)準(zhǔn)曲線正常3R2c0.9671標(biāo)準(zhǔn)曲線正常4R2d0.682.5標(biāo)準(zhǔn)曲線正常5R2e0.5055標(biāo)準(zhǔn)曲線正常6質(zhì)控品a0.6173.4174質(zhì)控品質(zhì)控在控7質(zhì)控品b1.140.4376質(zhì)控品質(zhì)控在控8臨床標(biāo)本11.1020.5081-陰性9臨床標(biāo)本21.1340.4481-陰性10臨床標(biāo)本31.1930.3554-陰性11仁濟(jì)醫(yī)院BALF10.47>5.0+陽性12仁濟(jì)醫(yī)院BALF20.502>5.0+陽性13仁濟(jì)醫(yī)院血清1.0750.465-陰性GM 比較BALF and Blood(我們的患者)28痰培養(yǎng)

VS GM序號(hào)GM姓名培養(yǎng)陽性次數(shù)/總培養(yǎng)次數(shù)性別年齡科室痰培養(yǎng)陽性診斷1三次陽性YLF2/5男68歲急診內(nèi)科重癥煙曲霉呼吸衰竭，肺曲霉病臨床診斷2未檢測(cè)GMJWC2/2男79歲呼吸內(nèi)科煙曲霉肺惡性腫瘤3單次檢測(cè)陽性LZL1/5女81歲急診內(nèi)科煙曲霉支氣管炎4單次檢測(cè)陽性QYX4/6男82歲中心監(jiān)護(hù)室(綜合)煙曲霉腸梗阻5未檢測(cè)GMZLZ1/5男74歲神經(jīng)內(nèi)科重癥煙曲霉腦梗死6單次檢測(cè)陽性WRX1/4男71歲急診內(nèi)科重癥煙曲霉慢性支氣管炎急性發(fā)作7單次檢測(cè)陽性SXM2/4女91歲急診內(nèi)科重癥煙曲霉消化道出血8單次檢測(cè)陽性LHL1/2男63歲呼吸內(nèi)科煙曲霉慢性支氣管炎9單次檢測(cè)陽性WXZ2/3女64歲急診內(nèi)科煙曲霉慢性阻塞性肺病伴急性加重10單次檢測(cè)陽性GQD1/4男81歲急診內(nèi)科重癥煙曲霉氣胸2015-7-3李立剛血清<0.65

陰性2015-7-3李立剛血清<0.65

陰性2015-9-6葉小英BALF≥0.85

陽性2015-9-6葉小英血清≥0.85

陽性2015-9-11馮來寶血清0.83

弱陽性2015-9-11汪德艷血清<0.65

陰性2015-9-29張菊芳血清<0.65

陰性2015-9-29張菊芳血清<0.65

陰性2015-10-16郁志法BALF1.2

陽性2015-10-16郁志法血清<0.65

陰性2015-10-23郁志法BALF0.76

弱陽性2015-10-23郁志法血清<0.65

陰性2015-10-30王開寬BALF>5.0

陽性2015-10-30王開寬血清<0.65

陰性2015-11-3王開寬BALF0.98

陽性2015-11-3王開寬血清<0.65

陰性2015-11-6王森云BALF<0.65

陰性2015-11-6王森云血清<0.65

陰性2015-12-11夏同檸血清<0.65

陰性2015-12-11夏同檸csf<0.65

陰性2015-12-18施平BALF<0.65

陰性2015-12-18施平血清<0.65

陰性2016-1-22蔣濤血清<0.65

陰性2016-2-14張英杰BALF2.59

陽性2016-2-15顧海云BALF<0.65

陰性2016-2-15顧海云血清<0.65

陰性2016-3-8沈梅珍血清<0.65

陰性2016-3-15趙海然BALF<0.65

陰性2016-3-15趙海然血清<0.65

陰性2016-4-1陳建霞BALF<0.65

陰性2016-4-1陳建霞血清<0.65

陰性2016-4-1徐士財(cái)血清<0.65

陰性2016-4-20華桂靜血清<0.65

陰性2016-12-25劉淑芳BALF<0.65

陰性2016-12-25劉淑芳血清<0.65

陰性35例擬診患者來自東方醫(yī)院本部、上海市第一人民醫(yī)院、仁濟(jì)醫(yī)院、肺科醫(yī)院等ICUBALF12例腦脊液1例血清22例合計(jì)：35例血清 VS BALBALF

GM對(duì)于IPA的診斷價(jià)值Zou

Ml.

Systematic

review

and

meta-analysis

of

detecting

galactomannanin

bronchoalveolar

lavage

fluid

for

diagnosing

invasive

aspergillosis.

PloS

one.BALF GM試驗(yàn)?zāi)芊裉岣咧匕Y患者IPA診斷的準(zhǔn)確性？GM試驗(yàn)?zāi)芊裉岣咧匕Y患者IPA診斷的準(zhǔn)確性？AspICU局限性:1，僅納入氣道分泌物曲霉菌培養(yǎng)陽性患者2，無曲霉菌特異性抗原檢測(cè)免疫層析LFDmonoclonalJF5

antibodyPrattesJ.Americanjournalofrespiratoryandcriticalcaremedicine.

2014;190(8):922-9.36UsingtheEORTC/MSGcriteria,thesensitivitiesofqPCRandLFDwere100%andthesensitivityoftheGMtestwas87.5%(GMtestindexcutoff,>0.8),withthetestshavingspecificitiesofbetween66.7and86.7%.TheagreementbetweentheresultsofqPCRandLFDwasalmostperfect(Cohen’skappacoefficient0.93,95%confidenceinterval,0.81to1.00).LFDandqPCRcombinedhadasensitivityof100%andaspecificityof85.7%.CalcofluorstainingandcultureofallBALfluidsampleswerenegativeforfungalinfection.Themediantimefromthestartofmold-activeantifungaltherapytothetimeofcollectionofBALfluidwas6days.ReversingrolesandusingdualtestingbyLFD

andqPCRtoclassifycases,theEORTC/MSGcriteriahadasensitivityof83.3%.AllthreetestsareusefulforthediagnosisofIPAinBALfluidsamples.Despitethesignificantdelaysbetweenthestartofantifungaltherapyandbronchoscopy,unlikemicroscopyandculture,thebiomarkersremainedinformative.Inparticular,thecombinationofLFDandqPCRallowsthesensitiveandspecificdetectionof

IPA.Aspergillusantibodydiagnosisof

CPAPopulationIntentionInterventionSoRQoEReferenceCommentCavitaryornodularpulmonaryinfiltrateinnon-immunocompromisedpatientsIncontextofasthma/ABPA/CFDiagnosis

orexclusionofCPAAspergillus

IgGantibodyAspergillusprecipitinsAspergillusIgM

antibodyAspergillus

IgAantibodyAspergillusIgEantibodyAADDBIIIIIIIIIIIIGuitard,

2012;Baxter,2012;VanToorenenbergen,2012BTS,1970;

Uffredi,2003;

Kitasato,2009;

Ohba,2012;

Baxter,2012Brouwer,

1988;Schonheyder1987;

Nimomiya,1990;Denning,

2003;Agarwal,

2012IgGandprecipitinsteststandardisationincompleteMostinhousetestspoorlyvalidated,withuncertainsensitivitythemajor

problem.Sensitivity

forAspergillusnoduleuncertainAspergillusIgG

serology0.99 0.972 0.902 0.918AreaunderROCcurveresultsComparisonof4commercialassaysusing250patientswithCPAinManchesterandnormal

controlsfrom

UgandaPageI,

unpublishedSiemensSerion

ELISAOmega

ELISADynamiker

ELISAAspergillusIgGafterTBin

NigeriaAspergillusIgG(Dynamiker)duringanti-TB

therapySmear/GeneXpertnegativepatients–39%AspergillusIgGpositive,comparedwith23%intheSmear/GeneXpertpositive

patients.OladelaR,

unpublished病區(qū)性別年齡診斷曲霉IgG曲霉IgM呼吸內(nèi)科男73歲肺炎--呼吸內(nèi)科女72歲慢性支氣管炎急性發(fā)作--呼吸內(nèi)科女24歲急性上呼吸道感染;++/-呼吸內(nèi)科女64歲肺炎-+呼吸內(nèi)科男82歲急性上呼吸道感染;--急診內(nèi)科女59歲肺部感染--急診內(nèi)科女60歲肺部感染-+急診內(nèi)科重癥女97歲肺部感染--胸部腫瘤?？?內(nèi))女71歲肺部陰影+-急診內(nèi)科重癥男70歲慢性支氣管炎急性發(fā)作--急診內(nèi)科病房女86歲慢性支氣管炎急性發(fā)作+-呼吸道感染組曲霉抗體檢測(cè)40病區(qū)性別年齡診斷曲霉IgG曲霉IgM胸部腫瘤?？颇?1歲肺腫瘤--腫瘤科男56歲肺惡性腫瘤;--腫瘤科男72歲肺惡性腫瘤;--甲狀腺腫瘤?？婆?1歲甲狀腺結(jié)節(jié)+-甲狀腺腫瘤?？婆?5歲甲狀腺腫+-血液科女58歲急性白血病--血液科女66歲非霍奇金淋巴瘤化療--血液科男27歲骨髓增生異常綜合征+-血液科女63歲貧血;+-血液科女58歲非霍奇金淋巴瘤--腫瘤科女45歲結(jié)腸惡性腫瘤++/-腫瘤科男90歲前列腺惡性腫瘤--腫瘤科男60歲結(jié)腸惡性腫瘤+-腫瘤科女66歲盆腔惡性腫瘤+-腫瘤科女60歲直腸惡性腫瘤--非感染組曲霉抗體檢測(cè)41分子生物學(xué)檢查42Somemolecular

methodsRestrictionfragmentlengthpolymorphism

(Martin,2000)PCR:18SrDNA(Wuetal.,

2003)MultiplexPCR:ITSregions(Changetal.,2001,

2001)PCR-EIA(Morrisonetal.,2001,

2004)Identificationofonlyalimitednumberof

species18S(SSU)5.8S28S

(LSU)18S5SITS1ITS2IGS1IGS2ITSD1/D2ITS:internaltranscribedspacerIGS:intergenic

spacerITSandD1/D2regionofthe28Ssubunit–goodforspecies

identification44MayoClinic:DepartmentofLaboratoryMedicineandPathologyMycology/Mycobacteriology

LaboratoryRochester,MN5590516S/D2MicroSeq?

AnalysisMycologyD2

sequencing:Candidakruseishouldagree≥99%withthereference

sequence.Sampleswithmismatchesshouldbereviewedandeditedifwarrantedand%match

adjusted.Thereshouldbe270-290basesofreadablesequence.Boththeforwardandreversestrandsmustbeanalyzedandmeettheacceptance

criteria.Thespecimenscoremustbe≥

30.4546AsperGenius,anewmultiplexreal-timePCRassayconsistingoftwomultiplexreal-timePCRs,onethatidentifiestheclinicallyrelevantAspergillusspecies,andonethatdetectstheTR34,L98H,T289A,andY121FmutationsinCYP51Aanddifferentiatessusceptiblefrom

resistantA.fumigatus

strains.37bronchoalveolarlavage(BAL)fluidsamplesfromhematologypatientsand40BALfluidsamplesfromintensivecareunit(ICU)patientsusing

aBALfluidgalactomannanlevelof>1.0orpositivecultureasthegoldstandard.

22BALfluidsamplesfrompatientswithinvasiveaspergillosis(IA)(2proven,9probable,and11

nonclassifiable).47Theoptimalcyclethresholdvalueforthepresenceof

Aspergilluswas<36.Sixteenofthe19BALfluidsampleshadapositivePCR(2

Aspergillusspeciesand14A.fumigatus

samples).Thisresultedinasensitivity,specificity,andpositiveandnegativepredictivevaluesof88.9%,89.3%,72.7%,and96.2%,respectively,forthehematologygroupand80.0%,93.3%,80.0%,and93.3%,respectively,intheICU

group.TheCYP51Areal-timePCRconfirmed12wild-typeand2resistantstrains(1TR34-L98Hand1TR46-Y121FT289Amutant).Voriconazoletherapyfailedforboth

patients.TheAsperGeniusmultiplexreal-timePCRassayallowsforsensitiveandfastdetectionofAspergillusspeciesdirectlyfromBAL

fluidsamples.Moreimportantly,thisassaydetectsanddifferentiateswild-typefromresistantstrains,evenifBALfluidculturesremainnegative.4849標(biāo)準(zhǔn)？時(shí)間技術(shù)折點(diǎn)BALF

GM檢測(cè)存在的問題BALF GM檢測(cè)存在的問題灌注量：灌洗量至少5×20

ml，少于100

ml的灌洗量可能增加灌洗回收液體中的支氣管腔分泌物混雜?；厥樟浚哼_(dá)到規(guī)定的回收比例，回收率＞40%，活細(xì)胞95%以上。當(dāng)BAL的回收率小于25％時(shí)，BALF結(jié)果通常不可靠。若影像學(xué)提示兩肺為彌漫性病變，則灌洗部位通常選擇右肺中葉或左肺舌葉。若影像學(xué)提示局灶性病變，則灌洗部位為病變肺葉。灌洗過程中嚴(yán)格遵循“堵管”操作，為防止灌洗液返流引起感染播散，建議灌洗部位為四～五級(jí)支氣管。灌洗操作應(yīng)在保護(hù)性毛刷或支氣管組織活檢之前，防止紅細(xì)胞混雜影響結(jié)果分析。52合適的BALF標(biāo)準(zhǔn)合適的BALF標(biāo)準(zhǔn)53標(biāo)本采集時(shí)間：行纖維支氣管鏡檢查時(shí)標(biāo)本接收時(shí)間；及時(shí)處理（室溫保存最好不要超過1 h）須嚴(yán)格遵守?zé)o菌操作不混有血液，紅細(xì)胞數(shù)小于10%不應(yīng)混有多量的鱗狀上皮細(xì)胞(小于1%)第一份回收的標(biāo)本往往混有支氣管內(nèi)成分，不宜用于微生物檢查。54Proteomic

IdentificationMALDI-TOFMS(Matrix-AssistedLaserDesorptionIonization–Timeof FlightMassSpectrometry):examinetheprotein

profilefromthe

microorganism55真菌數(shù)據(jù)庫四十個(gè)不同屬的110多種真菌菌種庫內(nèi)菌種數(shù)量和菌株數(shù)量仍在持續(xù)增長(zhǎng)5758Identificationofthe11clinicalisolatesandthreereferencestrains

bymatrix-assistedlaserdesorption

ionization–timeofflightmassspectrometry(MALDI-TOFMS)showedthatonlysixoftheninestrainsofA.flavuswereidentified

correctly.NoneofthestrainsofA.nomiusandA.tamariiwascorrectlyidentified.β-Tubulinorthecalmodulingeneshouldbethegenetargetofchoice

foridentifyingA.flavus,A.nomius,andA.tamarii.ToimprovetheusefulnessofMALDI-TOFMS,thenumberofstrainsforeachspeciesinMALDI-TOFMSdatabasesshouldbeexpandedtocover

intraspeciesvariability.59JClinMicrobiol.

2016Jun1.pii:JCM.00906-16.[Epubaheadof

print]Perform