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侵袭性深部真菌病的实验室诊断

侵袭性深部真菌病的实验室诊断. 倪语星 上海交通大学医学院 附属瑞金医院 临床微生物科. 侵袭性真菌病的致病菌. — 条件致病菌 — 致病性双相真菌 念珠菌 组织胞浆菌 曲霉 球孢子菌 隐球菌 芽生菌 接合菌 马内菲青霉 镰刀菌 孢子丝菌

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侵袭性深部真菌病的实验室诊断

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  1. 侵袭性深部真菌病的实验室诊断 倪语星 上海交通大学医学院 附属瑞金医院 临床微生物科

  2. 侵袭性真菌病的致病菌 —条件致病菌 —致病性双相真菌 • 念珠菌 组织胞浆菌 • 曲霉 球孢子菌 • 隐球菌 芽生菌 • 接合菌 马内菲青霉 • 镰刀菌 孢子丝菌 • 暗色真菌 • 酵母菌 • 毛孢子菌 • 枝顶孢霉

  3. 侵袭性真菌病(IFD)主要包括： • 念珠菌病 • 隐球菌病 • 侵袭性曲霉病

  4. 高危人群+高危因素=IFD

  5. IFD的高危人群和高危因素 • 广谱抗生素应用 • 入住ICU • 血液系统肿瘤病人（粒缺、骨髓移植） • 器官移植 • HIV感染 • 应用皮质激素 • 糖尿病 • 静脉插管

  6. 8 6 4 2 0 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 尸体解剖中侵袭性真菌感染的发生率 All other Candidaspp. Aspergillus spp. Prevalence at Autopsy [%] Prevalence of invasive aspergillosis at necropsy at JW Geothe University Hospital,Frankfurt,Germany(Lancet ,2000;335:2076)

  7. All others Aspergillus Candida 12.9% 3.23% 9.68% Mucor 54.84% Cryptococcus 19.35% 国内西南医院尸解资料，(1971-2000)，郝飞教授提供

  8. 侵袭性真菌病的流行病学特点 危险因素不断增多，发生率逐年增高趋势，确切资料有待收集整理 白念珠菌仍然是最常见临床分离致病菌 非白念珠菌增加（带来的问题） 曲霉已成为重要的致死真菌

  9. 真菌感染的实验诊断方法及问题 • 形态学检查：经验？阳性率？ • 培养+鉴定：时间长，敏感性？ • 血清学检查：敏感性？特异性？ • 分子生物学检查：标准化？

  10. 真菌抗原、细胞壁成分检测 • GM试验：血浆、血清、BAL、胸水、CSF，用于曲霉检测； • G试验: 用于曲霉、念珠菌检测，对隐球菌、接合菌无意义； • 乳胶凝集试验: 检测隐球菌；

  11. 新生隐球菌乳胶凝集试验

  12. 血清GM作为诊断的早期标志物 Marr and Leisenring Clin Infect Dis 2005; 41:S381

  13. 在BAL中检测GM作为早期诊断标志 Becker et al. Br J Haematol 2003; 121: 448 Musher et al. J Clin Microbiol 2004: 42(12): 5517-22

  14. 关于GM试验与G试验 可作为推定诊断的标准; GM:检测半乳甘露聚糖，对曲霉感染诊断特异性强，假阳性反应可以在青霉菌属中出现；部分含青霉烷砜衍生物的抗菌药物可以诱发阳性反应； G试验:检测(1,3)-ß-D-葡聚糖，在很多真菌中都可以出现阳性反应，但在隐球菌、接合菌、毛霉、根霉呈阴性反应；

  15. Prospective utility of (1-3)-B-D-Glucan (BG), 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. Cuétara2, J.M. Moreno1, A. Del Palcio Perez-Medel1, I. Moragues3, J. Pontón3, A. Del Palacio1 1Hospital Universitario Doce de Octubre, MADRID, Spain 2Hospital Universitario Severo Ochoa, LEGANES, Spain 3Universidad del Pais Vasco, BILBAO, Spain

  16. 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

  17. CONCLUSIONS  The incidence of IFD 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 value and can exclude reasonably IFD in haematology adult patients treated with wide spectrum antifungals.

  18. Evaluation of two serologic test for diagnosis invasive Aspergillosis C. Castro, A. Romero, A. Aller, T. Gonzalez, A. González, E. Martín-Mazuelos H. U. Valme, SEVILLA, Spain

  19. A total of 236 sera from 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 December 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.

  20. GM test • From 51 patient studied, 16 of 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).

  21. BG assay • The BG assay were used in parallel with GM in 36 sera which 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试验阳性的9名患者中，G试验单独阳性的有3个病人，两种抗原同时阳性有6个病人，未出现单独GM试验阳性的情况。

  22. 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 should be used with other techniques for detection of invasive Aspergillosis infections.

  23. 真菌细胞壁结构示意图

  24. 深部真菌感染患者血浆1-3-β-D葡聚糖检测 病例选择 深部真菌感染患者35例，年 龄12－88岁，来自我院2004年1月到5月住院患者，均经培养证实存在深部真菌感染，感染部位包括呼吸道、泌尿道、血液及静脉插管引起的系统性感染。正常健康对照组30人，来自我院健康查体者。 第四军医大学

  25. 检测结果 • 正常对照组血浆1-3-β-D葡聚糖含量最高为7.29 pg/ml ，最低为0.45 pg/ml，平均值为2.83±2.57pg/ml； • 深部真菌感染组血浆1-3-β-D葡聚糖含量最高为168.9 pg/ml，最低为14.93pg/ml，平均值为54.06±36.13 pg/ml。 • 经SPSS统计软件T-检验分析，对照组与深部真菌感染组1-3-β-D葡聚糖平均值差异非常显著（t=7.741,P＜0.001）。

  26. 讨论 • 入选的深部真菌感染患者均经细菌培养证实为念珠菌感染，包括白色念珠菌23株、热带念珠菌8株、季也蒙念珠菌1株、克柔念珠菌1株和光滑球拟假丝酵母菌2株，无隐球菌感染。 • 如以10 pg/ml为cutoff值，则阳性率为100%; 以20 pg/ml为cutoff值，则阳性率为91.4％。 • 葡聚糖检测可在拟诊早期为临床医生提供机体是否感染真菌的可靠信息，因此葡聚糖含量检测不失为一种实用的真菌感染早期诊断方法。

  27. 注意 • 使用青霉素类 • 加酶抑制剂 • 香菇多糖等 会引起假阳性！

  28. PCR技术用于诊断 PCR • 种特异-PCR • 非特异 PCR • 杂交 • Standart "single" • "nested" • PCR-EIA • "Real-time" 原位杂交 • 标本 • 全血 • 血浆 • 血清 • BAL • 最低检测范围 • 4-10 cfu/ml • 25-100 fg DNA 目的基因 多拷贝基因

  29. CT positive chest X-ray PCR antigen culture histology ß-glucan 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time Axis of Methods for Detection of Pulmonary Aspergillosis 122 patients – 323 samples – 33 proven cases Kami M et al, Clin Infect Dis 2001;33:1504-12

  30. 结语 • IFD的实验室诊断及技术在不断地完善与发展，相信随着诊断技术的不断改进和创新，有关IFD的诊断将取得更大进展，一定能找到一些具备早期，快速、特异、具有独立诊断价值的检测方法，IFD的患病率和病死率不断上升的趋势一定会得到很好的控制。 • 临床微生物科应与临床密切配合, 加强IFD的检测和耐药性监测工作，帮助临床及时开始抗真菌治疗以及合理地选择用药，使患者得到及时而准确的医治，以免贻误治疗、影响预后。

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