ECCMID 曲霉菌诊断手段和疾病定义VIP

,2014 欧洲感染学会曲霉菌病治疗指南-诊断部分,2014 ESCMID Aspergillus Guideline- Diagnostic Group,Katrien Lagrou, Dieter Buchheidt, Malcolm Richardson, ManuelCuenca-Estrella, Chris Kibbler, Claus Peter Heussel, MarkusRuhnke, Mauritio Sanguinetti, Jrgen Lffler, Catherine Beigelman-Aubry, Cornelia Lass-Flrl,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,治疗前临床分析标本采集Pre-analytical clinical sample treatment,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,获取符合要求的单纯粘液标本，如痰液,To achieve ahomogenoussample ofviscous samplessuch as sputum,通过真菌溶解液使其液化，如：胰腺液或淀粉酶,Liquifaction using a mycolyticagent, eg. Pancreatin, sputolysin,A,III,必要检查；大量的痰液培养（完整标本）表现出更高的阳性率,通过肺泡灌洗液或沉淀物的分析以获得曲霉培养的最佳标本,To achieve optimal recoveryof Aspergillus from BAL byCentrifugation and investigationof the sediment,肺泡灌洗液/支气管吸引（最少10分钟吸引超过1000克标本）,Centrifugation of BAL/bronchial aspirates(1000g for at least 10 minutes),A,III,联合PCR，能够显著增加培养的敏感率,所有患者,Any,通过真菌溶解液使其液化，如：超声手段或二硫代苏糖醇,Liquifaction using sonicationAnd dithiothreitol,必要检查；曲霉分离取决于培养的标本量；真菌浓缩,Fraczek et al. 2013, Baxter et al. 2011,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,直接镜检：荧光标定Direct microscopy: Fluorescence brighteners,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,通过新鲜的临床标本进行真菌鉴定,To identify fungalelements in freshclinical specimens(e.g. BALs),荧光染色剂的应用：Calcofluor white, Uvitex 2B,Blancophor,Application of fluorescent dyes:Calcofluor white, Uvitex 2B,Blancophor,A,III,必要检测；没有对曲霉特异的检测手段；高敏感率；结果快速反馈；广泛应用；无法与其他酵母菌进行区分；标本的质量影响最后的结果获得,Lass-Flrl C. 2007, Rchel R. 1999, Chander J. 1993,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,诊断工具：组织病理Diagnostic tools Histopathology,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,确定组织切片和病变组织中的真菌,To identify fungalelements in histological sections and stains,组织病理检查；HE 染色GMS染色PAS染色,Histological examination Haematoxylin and eosin (HE);Gomoris methenamine silver stain (GMS); Periodic acid-Schiff (PAS);,A,III,组织学检查是基础检测；无法与其他酵母菌进行区分；HE染色：困难；GMS染色：移除基地细胞；对丝状真菌更为敏感PAS染色：有利于细胞计数和细胞细节检查；对曲霉非特异但具有较高的敏感性；形态学可以确定真菌的种类（毛霉菌，菌株分枝间的夹角呈90度，芽孢.）;快速检测；应用广泛；在130个中枢感染案例中73例涉及曲霉菌病（56%）；在组织检查阳性的案例中只有25%的培养阳性率,Denning D. 2003, Vyzantiadis TA. 2012, Sundaram et al. 2006, Rchel R. 1999, Lass-Flrl C. 2007, Chai JK. 2004, Kaufman L. 1997, Verweij PE. 1996, Hayden RT. 2003,荧光染色：Calcofluor white, Uvitex 2B,Blancophor,免疫组化单克隆抗体WF-AF-1或EB-A1原位杂交,ImmunohistochemistryMonoclonal antibody WFAF-1 or EB-A1 In situ hybridisation,A,II,B,II,可应用于冷冻标本或石蜡包裹的组织,具有潜在的价值-提供菌株的特异性数据和基因数据；可获得的商品化单克隆抗体：WF-AF-1是对烟曲霉，黄曲霉和黑曲霉特异性的抗体,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,临床标本培养Culture of clinical samples,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,从深部感染部位分离菌株（活检组织，血液，脑脊液等）,Primary isolationfrom deep sites(biopsies, blood,CSF),A,III,必要检测；血液抑制孢子的繁殖；BHI能够帮助分离孢子再生；从标本中重复分离获得一些菌落或同一真菌具有更大的临床意义。定量培养不能有效区分感染和定植。无菌部位分离真菌：确定种属,Cuenca-Estrella 2011, Richardson HM 2000,从非无菌标本中分离菌株（痰液，呼吸道吸引物，皮肤等）,Primary isolationfrom non-sterilesamples (sputum,respiratoryaspirates, skin),在沙保弱葡萄糖琼脂、马铃薯琼脂、脑心浸膏琼脂中以at 30C 和37C 培养72小时（特殊培养基）,Culture on SDA, BHI agar, PDA at 30C and 37C for 72 (specific media),在沙保弱葡萄糖琼脂、马铃薯琼脂、脑心浸膏琼脂中+ 庆大霉素和氯霉素以at 30C 和37C 培养72小时（特殊培养基）,Culture on SDA, BHI agar, PDA with gentamicin PLUSchloramphenicol at 30C and 37C for 72 h,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,人群/检测：培养阳性- 质谱鉴定MALDI-TOFPopulation/Test: Positive culture - MALDI-TOF,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,复合种属的鉴定,Identification ofspecies complex,A,II,必要检测；血液抑制孢子的繁殖；BHI能够帮助分离孢子再生；从标本中重复分离获得一些定植菌株或同一真菌具有更大的临床意义。定量培养不能有效区分感染和定植。无菌部位分离真菌：确定种属,Cuenca-Estrella 2011,肉眼和显微镜对前期培养标本进行直接镜检,Macroscopic and microscopicexamination from primary cultures,在特定的培养基中以25-30和37进行培养（2% MEA培养基和察氏培养基）病在显微镜下进行镜检,Culture on identification media at 25-30C and 37C(2% MEA and Czapek- Dox Agar) and microscopicexamination,在45下进行培养,Culture at 45C,B,III,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,培养阳性- 真菌形态学判断 确定真菌分子IDPositive culture Morphology Molecular ID,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,种属鉴定,Identification atspecies level,建立实验室内数据库,MALDI-TOF 质谱,MALDI-TOF MS identification,B,II,Alanio 2011, Bille 2012, De Carolis 2012, Lau 2013,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,种属鉴定,Identification atspecies level,一些案例中是基础检测；确定真菌编码,对微管蛋白ITS和调节蛋白进行测序,Sequencing of ITS beta-tubulin and calmodulin,A,III,Balajee 2009, Samson 2007,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,人群/筛选：分类Population/Test: Typing,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,疾病爆发研究,To study outbreaks,疾病分布分析和CSP分析,Microsatellite and CSP analysis,C,IIu,Araujo 2012, Hurst 2009, Guinea 2011, Rougeron et al. 2014,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,人群/筛选：原始标本的贮存Population/Test: Storage of original samples,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,所有患者,Any,避免临床曲霉菌活性的丢失；反应原始的真菌感染信息,To prevent loss ofviability of Aspergillusin clinical samples,and to reflect theoriginal fungal content,培养阳性标本短期保存：存放在4下以避免孢子活性丢失保留真菌原始信息,Clinical samples for culture - short-term storage: 4C toprevent loss of viability and to reflect the originalfungal content,A,III,所有患者,Any,避免生物标记物降解，如血清中/支气管毛刷/肺泡灌洗液的半乳甘露聚糖（GM）,To preventdegradation ofbiomarkers, eg.Galactomannan (GM)in serum orBAL/bronchial washes,在运送至实验室后迅速完成酶联反应。避免长时间或短时间将血清标本存放在4,Complete assay soon after delivery to laboratory. Avoidshort or long-term storage of serum at 4C.,A,I,已经知晓血清标本的GM水平长时间或短时间存放在4中会产生降解；肺泡灌洗液的GM水平在4下仍然稳定,血清和肺泡灌洗液的GM水平在零下20条件下存放11个月相对稳定,ASM Manual of Clinical Microbiology, 10th Edition, Johnson et al. 2013, Oren et al. 2012, Furfano et al. 2012, Wheat et al. 2014,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,人群/筛选：分离标本的贮存/防腐Population/Test: Storage/preservation of isolates,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,曲霉菌短期保存,Short-term maintenance ofAspergillus isolates,重复传代培养,Repeated sub-culture,A,I,曲霉菌长期保存,Long-termpreservation ofAspergillus isolates,水中贮存/矿物油中贮存/冻干贮存/冷冻（-80）/硅胶贮存/液氮贮存,Water storage/storageunder mineral oil/silica gelstorage/freezedrying/freezing (-80C/ ceramic beads/liquid nitrogen,A,I,长期贮存可以达到5年甚至更长；在此期间不需要再次的接种,Stockdale et al. In: Medical Mycology: A Practical Approach. IRL Press at Oxford University Press, 1989,在数年间保持曲霉菌的活性以用于重复培养；每月接种1次；保存在环境室温下,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,血液半乳甘露聚糖（GM）Blood galactomannan,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,持续粒细胞缺乏患者和接受异基因造血干细胞移植患者（未接受预防治疗）,Prolonged neutropenicpatients and allogeneicstem cell transplantationrecipients not on moldactiveprophylaxis,曲霉菌感染的诊断,To diagnoseInvasive aspergillosis,血液半乳甘露聚糖检测,Galactomannan in blood,A,I,血液半乳甘露聚糖检测,Galactomannan in blood,A,II,敏感性显著低于中心粒细胞缺乏患者,Morrissey 2013, Springer 2013, Leeflang 2008, Maertens 2007, Maertens 2002, Maertens 2001, Pfeiffer 2006, Cordonnier 2008, Maertens 2010,血液恶性肿瘤患者*中心粒细胞缺乏患者,Patients with a hematologicalMalignancy Neutropenic patients,血液恶性肿瘤患者*非中心粒细胞缺乏患者,Patients with a hematologicalMalignancy Non-Neutropenic patients,To diagnoseInvasive aspergillosis,曲霉菌感染诊断,每3-4天采集一次标本,Draw samples every 3-4 days,C,III,连续两次标本GM值0.5 为准确的诊断标准；前瞻性的GM随访需要结合HRCT和临床评估,B,I,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,血液半乳甘露聚糖（GM）Blood galactomannan,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,ICU 患者,ICU patients,曲霉菌感染诊断,To diagnoseInvasive aspergillosis,血液半乳甘露聚糖检测,Galactomannan in blood,C,II,Meersseman 2008, Guinea 2008, Tabarski 2012, Husain 2004, Pfeiffer 2006, Guique 2013, Martin-Rabadan 2013, Petraitiene 2011, Mikulska 2013, Vergedis 2012, Nucci 2014; Huang 2007,敏感性显著低于中心粒细胞缺乏患者,实体器官移植患者,Solid organ recipients,曲霉菌感染诊断,To diagnoseInvasive aspergillosis,血液半乳甘露聚糖检测,Galactomannan in blood,C,II,低敏感性；高特异性；数据多来自肺部移植患者（其他实体器官移植患者IA感染相对较少）,其他 患者,Other,曲霉菌感染诊断,To diagnoseInvasive aspergillosis,血液半乳甘露聚糖检测,Galactomannan in blood,对于患者的诊断需要综合临床症状，影像学检查和微生物表现；摄入冰棒；输血；抗生素应用，肠外营养可导致假阳性的发生。近期的研究中显示他唑巴坦不再是假阳性的影响因素；在组织胞浆菌，镰刀菌和马尔尼菲青霉菌以及副求孢子菌存在交叉反应。,Diagnosis should be based on integration of clinical, radiological and microbiological signs; false positive results reported due to ingestion of icepops, transfusions, antibiotics, Plasma-Lyte infusion; TazocinTM no longer responsible for false positive results in recent studies; cross reactivity in case of histoplasmosis, fusariosis, penicillosis and trichosporonosis,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,人群/筛选：肺泡灌洗液和脑脊液GM检测Population/Test: GM - BAL,患者人群Population,目的Intention,SoR,QoE,备注Comment,所有患者,Any,肺曲霉菌病的诊断,To diagnose pulmonaryaspergillosis,肺泡灌洗液GM检测是肺曲霉菌病诊断的有效手段，阳性折点为 0.5-1.0 之间（1.0)加LFD可以将敏感率增加到94%， GM(1.0)加PCR可以将敏感率增加到100%(拟诊/确诊IPA患者中特异性为95-98%),Hnigl et al. 2012, Held et al. 2013, Hnigl et al. 2014,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,抗体：侵袭性曲霉菌病 曲霉血清Antibody: Aspergillus serology in invasive aspergillosis 1,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,备注Comment,肺曲霉菌病/侵袭性曲霉菌病患者,Patients with Pulmonary and invasive aspergillosis,诊断肺曲霉菌病/侵袭性曲霉菌病,To diagnose pulmonary andInvasive aspergillosis,通过曲霉特性抗体交叉酶联法（EIA）进行诊断：Serion (Germany), Omega(France), Bio-Rad (France),Bio-Enoch (China),Detection of spergillusspecificantibodies by EIA:Serion (Germany), Omega(France), Bio-Rad (France),Bio-Enoch (China),C,II,从发病到抗体产生中位天数约10.8天侵袭性曲霉菌病急性期的辨识率为29-100%,Ruhnke et al. 2012, Von Eiff et al. 1995, Kappe and Rimek 2004, Cornillet et al. 2006, Weig et al. 2001, Du et al. 2012, Holmberg et al 1980, Manso et al. 1994,Mishra et al 1983,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,肺泡灌洗液的曲霉PCR检测BAL Aspergillus PCR,患者人群Population,目的Intention,干预手段Intervention,SoR,QoE,异基因骨髓移植患者（未给予霉菌预防治疗）,Patients undergoingallogeneic stem cellTransplantation recipients not on mold-active prophylaxis,预测侵袭性曲霉菌病,To predictIA,肺泡灌洗液 PCR,BAL PCR,B,II,B,II,血液恶性肿瘤持续中性粒细胞缺乏患者；ICU患者肺移植患者,Patients with hematologicalmalignancies andProlonged neutropeniaICU pts (mixed ptsPopulations) Lung Tx pts,诊断侵袭性曲霉菌病,To diagnoseIA,肺泡灌洗液 PCR,BAL PCR,参考文献Reference,Sanguinetti, JCM, 2003Rantakokko, JCM, 2003Lass-Flrl, JCM, 2004Musher, JCM, 2004Khot, BMC Inf Dis, 2008Frealle,EJClinMicrobInfDis,2009Bergeron, JCM, 2011Luong, Transpl 2011Buess, BMC Inf Dis, 2012Reinwald, Eur J Hematol, 2012Reinwald, JAC, 2012Hnigl et al, JCM 2014,Einsele, Lancet, 1998,备注Comment,严格的说不同的实验检测间存在差异；在未给予抗真菌治疗患者中检测效果更好；PCR+GM能够增加特异性,实验室检测,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,血液PCR的应用指南:Aspergillus PCR from blood recommended:,使用标准化的方法（EAPCRI发布了标准，见see J Clin Microbiol, 2010 2013,use of standardized methods (- EAPCRI publications, see J Clin Microbiol, 2010 2013),联合诊断：两种不同的独立检测方法，如GM ELISA和PCR,combination of 2 independent detection methods, e. g. GM ELISA and PCR superior to PCR only,联合诊断：两种不同的标本，如血浆和高质量的细胞碎片,combination of 2 independent detection methods, e. g. GM ELISA and PCR superior to PCR only,单一的PCR IA阴性结果可作为确证和拟诊的剔除标准,single PCR-negative result sufficient to exclude a diagnosis of proven or probable,2次阳性结果可作为对 IA的诊断（因为其较高的特异性）,2 positive tests are required to confirm diagnosis of IA (because of significantly higher specificity),Present by Cornelia Lass-Florl Australia