ECCMID 曲霉菌诊断手段和疾病定义

1、,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, Manuel Cuenca-Estrella, Chris Kibbler, Claus Peter Heussel, Markus Ruhnke, Mauritio Sanguinetti, Jrgen Lffler, Catherine Beigelman- Aubry, Cornelia Lass-Flrl,Present by

2、 Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,1,.,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 achie

3、ve a homogenous sample of viscous samples such as sputum,通过真菌溶解液使其液化，如：胰腺液或淀粉酶,Liquifaction using a mycolytic agent, 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 recovery of Aspergillus from BAL by Centrifugation and investigation of the

4、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 sonication And 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. 2011,2,.,Present b

5、y 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 fungal elements in fresh clinical specimens (e.g. BALs),荧光染色剂的应用：Calcofluor

6、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. 1993,3,.,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,

7、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 fungal elements 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 sil

8、ver 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,

9、 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,Immunohistochemistry Monoclonal 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

10、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,4,.,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 isolation from deep sites (bi

11、opsies, 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 isolation from non-sterile samples (sputum, respiratory aspirates, 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 37

12、C 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 PLUS chloramphenicol at 30C and 37C for 72 h,5,.,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2

13、015 in Barcelona,人群/检测：培养阳性- 质谱鉴定MALDI-TOF Population/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 of species 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

14、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 microscopic examination 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 microscopic examination,在45下进行培养,Culture at 45C,B,III,6,.,Pre

15、sent 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ID Positive 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 at species level,建立实验室内数据库,MALDI-TOF 质谱,MALDI-TOF MS identification,

16、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 at species 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 2007,7,.,Present by Co

17、rnelia 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

18、. 2014,8,.,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 of viability of Aspergillus in clinica

19、l samples, and to reflect the original 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 to prevent loss of viability and to reflect the original fungal 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 prevent degradation of biomark

20、ers, eg. Galactomannan (GM) in serum or BAL/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. Avoid short 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 Clini

21、cal Microbiology, 10th Edition, Johnson et al. 2013, Oren et al. 2012, Furfano et al. 2012, Wheat et al. 2014,9,.,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 C

22、omment,曲霉菌短期保存,Short-term maintenance of Aspergillus isolates,重复传代培养,Repeated sub-culture,A,I,曲霉菌长期保存,Long-term preservation of Aspergillus 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/storage under mineral oil/silica gel storage/freezedrying/ freezing (-80C/ ceramic beads/liquid nitrog

23、en,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,10,.,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 Pop

24、ulation,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 neutropenic patients and allogeneic stem cell transplantation recipients not on moldactive prophylaxis,曲霉菌感染的诊断,To diagnose Invasive 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,Ga

25、lactomannan 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 hematological Malignancy Neutropenic patients,血液恶性肿瘤患者 *非中心粒细胞缺乏患者,Patients with a h

26、ematological Malignancy Non-Neutropenic patients,To diagnose Invasive 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,11,.,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,血液半乳甘露聚糖（GM） Blood galactomannan,

27、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 diagnose Invasive 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, Mikulsk

28、a 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 diagnose Invasive 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 diagnose Invasive aspergillosis,血液半乳甘露聚糖检测,Galactomannan i

29、n 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, antibiotic

30、s, Plasma-Lyte infusion; TazocinTM no longer responsible for false positive results in recent studies; cross reactivity in case of histoplasmosis, fusariosis, penicillosis and trichosporonosis,12,.,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 Popula

31、tion/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 pulmonary aspergillosis,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（0.5为阴性排除）,A,II,DHaese 2012, Heng 2013, Luong 2012, Zou 2012, Fisher 2013, Reinwald et al. 2012,患者人群 Population,目的 Intention,所有患者,Any,Visco

32、li 2002, Verweij 1999 May 10,肺曲霉菌病的诊断,To diagnose pulmonary aspergillosis,干预手段 Intervention,SoR,QoE,备注 Comment,无被证实的折点,脑脊液的半乳甘露聚糖检测,Galactomannan in CSF,B,II,13,.,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,组织活检标本的GM检测 GM - Biopsies,患者人群 Population,目的 Intention,干预手段 I

33、ntervention,SoR,QoE,备注 Comment,所有患者,Any,鉴定组织中的半乳甘露聚糖水平,To detect galactomannan in tissue,将GM检测应用到肺活检中,To apply GM-test on lung-biopsies,B,II,折点为0.5;高敏感性（90%）和特异性（90%）；标本需要切片；为了进行预处理需要充足的标本；在等渗盐水中稀释,Klont 2004, Lass-Flrl 2007,14,.,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barc

34、elona,诊断工具-Beta-D-甘露聚糖试验 Diagnostic Tools - -D-glucan Assay,患者人群 Population,目的 Intention,干预手段 Intervention,SoR,QoE,备注 Comment,多人群；成人ICU；血液疾病，实体器官移植,Mixed population: Adult ICU, Hematological disorders, SOT,诊断侵袭性真菌感染（非曲霉特异性检测）,To diagnose IFD (not specific for aspergillosis),诊断性试验,Diagnostic assay,C,

35、II,一共有4种不同的试验方法；Fungitell是唯一被美国批准病在欧洲和美国上市的产品；其他方法仅在日本上市；总体敏感性为77%，特异性为85% 特异性的问题限制了其在曲霉中的应用 2个或更多的连续标本阳性：敏感性=65% ；特异性=93%；包含临床研究 试验方法cutoff值见存在差异：Wako试验敏感性=40-97%；特异性=51-99%,Karageorgopoulos 2011, Lu 2011,监测性试验,Screening assays,C,II,15,.,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 201

36、5 in Barcelona,生物标记物/侧流型诊断设备 Biomarker/ Lateral-Flow Device,患者人群 Population,目的 Intention,干预手段 Intervention,SoR,QoE,备注 Comment,血液肿瘤和实体器官移植患者,Hematological malignancy and solid organ transplant,曲霉菌感染诊断,To diagnose Invasive aspergillosis,通过肺泡灌洗液评价LFD(回顾性研究),Evaluation of LFD using BAL samples (retrospe

37、ctive study),B,III,在拟诊患者（probable IPA）中肺泡灌洗液的敏感率为100%，特异性为81%（PPV 71% NPV100%），5位临床诊断患者获得LFD阳性诊断； 无确诊患者,造血干细胞移植,Hematopoietic stem cell Transplantation (HSCT),应用LFD通过血清标本诊断曲霉菌感染,Diagnose IA LFD using serum samples,前瞻性的对照LFD和血清GM试验对101造血干细胞移植患者的检测,Prospective screening in 101 Patients undergoing allo

38、- HSCT Comparison to Asp- GM to serum,B,III,IA：1确诊，9拟诊，20临床诊断 1血清阳性（LFD） vs 2血清标本(GM)： 敏感性（GM/LFD） 40%/20% 特异性（GM/LFD） 86.8%/97.8% 诊断指数（GM/LFD） 3.03/11.12,免疫抑制患者（血液疾病患者65%）,Immunocompromised Pts (hematological Malignancies 64%),曲霉菌感染诊断,To diagnose Invasive aspergillosis,通过肺泡灌洗液评价LFD(回顾性研究),Evaluatio

39、n of LFD using BAL samples (retrospective study),B,II,LFD,GM,BGD,PCR的敏感性介于70-88%；GM(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. 2014,16,.,Present by Cornelia Lass-Florl Australia ,ECCMID 10th May 2015 in Barcelona,抗体：侵袭性曲霉菌病

40、 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 and Invasive aspergillosis,通过曲霉特性抗体交叉酶联法（EIA）进行诊断：Serion (German

41、y), Omega(France), Bio-Rad (France),Bio-Enoch (China),Detection of spergillusspecific antibodies 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 a

42、l. 2006, Weig et al. 2001, Du et al. 2012, Holmberg et al 1980, Manso et al. 1994, Mishra et al 1983,17,.,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

43、 undergoing allogeneic stem cell Transplantation recipients not on mold-active prophylaxis,预测侵袭性曲霉菌病,To predict IA,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 hematological malignancies and Prolonged neutropenia ICU pts (mixed pts Populations) Lung Tx pts,诊断侵袭性曲霉菌病,To di

44、agnose IA,肺泡灌洗液 PCR,BAL PCR,参考文献 Reference,Sanguinetti, JCM, 2003 Rantakokko, JCM, 2003 Lass-Flrl, JCM, 2004 Musher, JCM, 2004 Khot, BMC Inf Dis, 2008 Frealle, EJClinMicrobInfDis,2009 Bergeron, JCM, 2011 Luong, Transpl 2011 Buess, BMC Inf Dis, 2012 Reinwald, Eur J Hematol, 2012 Reinwald, JAC, 2012 H

45、nigl 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,18,.,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,us

46、e 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 sufficien