Babouee Flury B, Weisser M, Prince S Savič, Bubendorf L, Battegay M, Frei R, Goldenberger D
Division of Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Petersgraben 4, Basel, 4031, Switzerland.
BMC Infect Dis. 2014 Dec 18;14:692. doi: 10.1186/s12879-014-0692-z.
Detection of fungal DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is challenging due to degradation of DNA and presence of PCR inhibitors in these samples. We analyzed FFPE samples of 26 patients by panfungal PCR and compared the results to the composite diagnosis according to the European Organization for Research and Treatment of Cancer (EORTC) criteria. Additionally we analyzed the quality of human and fungal DNA and their level of age-dependent degradation, as well as the existence of PCR inhibition in these tissue samples.
We evaluated two 45-cycle panfungal PCR tests that target the internal transcribed spacer 2 (ITS2) as well as the ITS1-5.8S-ITS2 (ITS1-2) region. The PCRs were applied to 27 FFPE specimens from 26 patients with proven invasive fungal disease (IFD), and one patient with culture and histologically negative but PCR-positive fungal infection collected at our institution from 2003 to 2010. Quality of DNA in FFPE tissue samples was evaluated using fragments of the beta-globin gene for multiplex PCR, inhibition of PCR amplification was evaluated by spiking of C. krusei DNA to each PCR premix.
In 27 FFPE samples the ITS2 PCR targeting the shorter fragment showed a higher detection rate with a sensitivity of 53.8% compared to the ITS1-2 fragment (sensitivity 38%). Significant time-dependent degradation of human DNA in FFPE sample extracts was detected based on partial beta-globin gene amplification which was not in correlation to successful panfungal PCR identification of fungal organisms. The analytical sensitivity of both assays compared with culture was 60 CFU/ml of a Candida krusei reference strain. The performance of the two tests in an Aspergillus proficiency panel of an international external quality assessment programme showed considerable sensitivity.
Panfungal diagnostic PCR assays applied on FFPE specimens provide accurate identification of molds in highly degraded tissue samples and correct identification in samples stored up to 7 years despite sensitivity limitations, mainly caused by partial PCR inhibition and DNA degradation by formalin.
由于福尔马林固定、石蜡包埋（FFPE）组织中的DNA降解以及这些样本中存在PCR抑制剂，从FFPE组织中检测真菌DNA具有挑战性。我们通过泛真菌PCR分析了26例患者的FFPE样本，并根据欧洲癌症研究与治疗组织（EORTC）标准将结果与综合诊断结果进行了比较。此外，我们分析了人类和真菌DNA的质量及其随年龄的降解水平，以及这些组织样本中PCR抑制的存在情况。
我们评估了两种针对内部转录间隔区2（ITS2）以及ITS1-5.8S-ITS2（ITS1-2）区域的45个循环的泛真菌PCR检测。这些PCR应用于2003年至2010年在我们机构收集的26例确诊侵袭性真菌病（IFD）患者和1例培养及组织学检查阴性但PCR检测阳性的真菌感染患者的27个FFPE标本。使用β-珠蛋白基因片段进行多重PCR评估FFPE组织样本中的DNA质量，通过向每个PCR预混液中加入克鲁斯念珠菌DNA评估PCR扩增抑制情况。
在27个FFPE样本中，针对较短片段的ITS2 PCR检测率更高，灵敏度为53.8%，而针对ITS1-2片段的灵敏度为38%。基于部分β-珠蛋白基因扩增检测到FFPE样本提取物中人类DNA存在显著的时间依赖性降解，这与成功通过泛真菌PCR鉴定真菌生物无关。与培养相比，两种检测方法对克鲁斯念珠菌参考菌株的分析灵敏度均为60 CFU/ml。在国际外部质量评估计划的曲霉能力验证组中，这两种检测方法的性能显示出相当高的灵敏度。
应用于FFPE标本的泛真菌诊断PCR检测能够在高度降解的组织样本中准确鉴定霉菌，并且能够在保存长达7年的样本中正确鉴定，尽管存在灵敏度限制，主要是由部分PCR抑制和福尔马林导致的DNA降解引起的。
