Abstract

BackgroundDetection 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.MethodsWe 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.ResultsIn 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.ConclusionPanfungal 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.

Highlights

  • 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 noticed two differing results; detection of H. aspergillata in the FFPE sample from patient no. 1 and A. lentulus in the sample from patient no. 3 from which A. nidulans and A. fumigatus were grown on culture respectively

  • Extraction using a second aliquot from formalin-fixed tissue and could confirm the PCR result with a 100% identity to H. aspergillata reference strains

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Summary

Introduction

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. 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. An appealing alternative for culture-independent identification of the fungal agent might be panfungal PCR performed retrospectively from FFPE tissue. Isolating high-quality DNA from FFPE biopsies is difficult because of the DNA degrading activity of the routinely used fixative formalin. Efficiency of DNA amplification from FFPE material depends on the time period for fixation [4,5]

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