1、侵襲性深部真菌病的實驗室診斷,倪語星 上海交通大學醫(yī)學院 附屬瑞金醫(yī)院 臨床微生物科,侵襲性真菌病的致病菌,條件致病菌 致病性雙相真菌 念珠菌 組織胞漿菌 曲霉 球孢子菌 隱球菌 芽生菌 接合菌 馬內菲青霉 鐮刀菌 孢子絲菌 暗色真菌 酵母菌 毛孢子菌 枝頂孢霉,侵襲性真菌病(IFD)主要包括： 念珠菌病 隱球菌病 侵襲性曲霉病,高危人群+高危因素=IFD,IFD的高危人群和高危因素,廣譜抗生素應用 入住ICU 血液系統(tǒng)腫瘤病人（粒缺、骨髓移植） 器官移植 HIV感染 應用皮質激素 糖尿病 靜脈插管,尸體解剖中侵襲性真菌感染的發(fā)生率,Aspergillus spp.,Candida spp.,

2、All other,Prevalence at Autopsy %,Prevalence of invasive aspergillosis at necropsy at JW Geothe University Hospital,Frankfurt,Germany(Lancet ,2000;335:2076),54.84%,12.9%,3.23%,9.68%,19.35%,國內西南醫(yī)院尸解資料，(1971-2000)，郝飛教授提供,Aspergillus,Cryptococcus,Mucor,Candida,All others,侵襲性真菌病的流行病學特點 危險因素不斷增多，發(fā)生率逐年增高趨

3、勢，確切資料有待收集整理 白念珠菌仍然是最常見臨床分離致病菌 非白念珠菌增加（帶來的問題） 曲霉已成為重要的致死真菌,真菌感染的實驗診斷方法及問題,形態(tài)學檢查：經驗？陽性率？ 培養(yǎng)+鑒定：時間長，敏感性？ 血清學檢查：敏感性？特異性？ 分子生物學檢查：標準化？,真菌抗原、細胞壁成分檢測,GM試驗：血漿、血清、BAL、胸水、CSF，用于曲霉檢測； G試驗: 用于曲霉、念珠菌檢測，對隱球菌、接合菌無意義； 乳膠凝集試驗: 檢測隱球菌；,新生隱球菌乳膠凝集試驗,血清GM作為診斷的早期標志物,Marr and Leisenring Clin Infect Dis 2005; 41:S381,在BAL中

4、檢測GM作為早期診斷標志,Musher et al. J Clin Microbiol 2004: 42(12): 5517-22,Becker et al. Br J Haematol 2003; 121: 448,關于GM試驗與G試驗,可作為推定診斷的標準; GM: 檢測半乳甘露聚糖，對曲霉感染診斷特異性強，假陽性反應可以在青霉菌屬中出現；部分含青霉烷砜衍生物的抗菌藥物可以誘發(fā)陽性反應； G試驗: 檢測(1,3)-D-葡聚糖，在很多真菌中都可以出現陽性反應，但在隱球菌、接合菌、毛霉、根霉呈陰性反應；,Prospective utility of (1-3)-B-D-Glucan (BG),

5、 galactomannan (GM) and anti-Candida albicans germ tube antibodies (CAGT) for the diagnosis of invasive fungal disease (IFD) in haemato-oncology adult patients A. Alhambra1, M.S. Cutara2, J.M. Moreno1, A. Del Palcio Perez-Medel1, I. Moragues3, J. Pontn3, A. Del Palacio1 1Hospital Universitario Doce

6、de Octubre, MADRID, Spain 2Hospital Universitario Severo Ochoa, LEGANES, Spain 3Universidad del Pais Vasco, BILBAO, Spain,Invasive Candidiasis S SP PPV NPV CAGT (%) 57 93 44 96 BG (%) 77 86 39 97 Invasive Aspergillosis S SP PPV NPV GM (%) 92 94 73 98 BG (%) 57 84 42 91,CONCLUSIONS The incidence of I

7、FD correlated directly and significantly (x2 p=0.0005) with risk stratification group: highest proportion in the high-risk group. Since all the biomarkers have inherent limitations, a better diagnosis yield is achieved combining the biomarkers. All three biomarkers share high negative predictive val

8、ue and can exclude reasonably IFD in haematology adult patients treated with wide spectrum antifungals.,Evaluation of two serologic test for diagnosis invasive Aspergillosis C. Castro, A. Romero, A. Aller, T. Gonzalez, A. Gonzlez, E. Martn-Mazuelos H. U. Valme, SEVILLA, Spain,A total of 236 sera fro

9、m 51 patients in risk of IA were tested for GM using Platelia Aspergillus kit (Bio Rad, France) which 36 sera (10 patients) were tested for BG also using Fungitell kit (Associates of Cape Cod., USA). Patients were attended at the University Hospital of Valme from Seville from January of 2008 to Dece

10、mber 2008. Patients with GM index 0.5 in two consecutive samples have been marked as GM positive and samples with results 80pg/ml were marked as BG positive. All GM positive patients were classified according to EORTC/MSG criteria (2008) for probability of IA.,GM test,From 51 patient studied, 16 of

11、them showed at least one positive specimen (33 sera). Only 6 patients showed two consecutive positive results (0.5 GM test) and they show clinical signs or microbiological criteria for AI proven (3 patients) and probable (3 patients).,BG assay,The BG assay were used in parallel with GM in 36 sera wh

12、ich 26 showed positive result from 9 patients, (3 with AI proven and 6 AI probable). 3 patients showed positive results before for BG test ( 3,5 days) and 6 patients presented simultaneously both antigens. Never the GM test was the first serological test to show a positive result. G試驗陽性的9名患者中，G試驗單獨陽

13、性的有3個病人，兩種抗原同時陽性有6個病人，未出現單獨GM試驗陽性的情況。,Conclusion Calculating significant sensitivity for both detection methods was not feasible due to a low number of proven/probable AI. BG detection showed positive results before GM test and present the great advantage to be a “panfungal”antigen. BG detection sho

14、uld be used with other techniques for detection of invasive Aspergillosis infections.,真菌細胞壁結構示意圖,深部真菌感染患者血漿1-3-D葡聚糖檢測,病例選擇 深部真菌感染患者35例，年 齡1288歲，來自我院2004年1月到5月住院患者，均經培養(yǎng)證實存在深部真菌感染，感染部位包括呼吸道、泌尿道、血液及靜脈插管引起的系統(tǒng)性感染。正常健康對照組30人，來自我院健康查體者。 第四軍醫(yī)大學,檢測結果,正常對照組血漿1-3-D葡聚糖含量最高為7.29 pg/ml ，最低為0.45 pg/ml，平均值為2.832.57

15、pg/ml； 深部真菌感染組血漿1-3-D葡聚糖含量最高為168.9 pg/ml，最低為14.93pg/ml，平均值為54.0636.13 pg/ml。 經SPSS統(tǒng)計軟件T-檢驗分析，對照組與深部真菌感染組1-3-D葡聚糖平均值差異非常顯著（t=7.741,P0.001）。,討論,入選的深部真菌感染患者均經細菌培養(yǎng)證實為念珠菌感染，包括白色念珠菌23株、熱帶念珠菌8株、季也蒙念珠菌1株、克柔念珠菌1株和光滑球擬假絲酵母菌2株，無隱球菌感染。 如以10 pg/ml為cutoff值，則陽性率為100%; 以20 pg/ml為cutoff值，則陽性率為91.4。 葡聚糖檢測可在擬診早期為臨床醫(yī)生提

16、供機體是否感染真菌的可靠信息，因此葡聚糖含量檢測不失為一種實用的真菌感染早期診斷方法。,注意,使用青霉素類 加酶抑制劑 香菇多糖等 會引起假陽性！,PCR,PCR技術用于診斷,種特異-PCR 非特異 PCR 雜交,Standart single nested PCR-EIA Real-time,標本 全血 血漿 血清 BAL,最低檢測范圍 4-10 cfu/ml 25-100 fg DNA,原位雜交,目的基因 多拷貝基因,122 patients 323 samples 33 proven cases,Time Axis of Methods for Detection of Pulmonary Aspergillo