Related Experiment Video
Updated: Apr 19, 2026
Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
Published on: August 21, 2016
Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are
B Babouee Flury1, M Weisser, S Savič Prince
1Division of Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Petersgraben 4, Basel, 4031, Switzerland. baharak.babouee@phe.gov.uk.
Background:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
Insights
Detecting fungal DNA in FFPE tissues is difficult due to DNA damage and PCR inhibitors. Panfungal PCR assays accurately identify molds in degraded samples, even after 7 years, despite some sensitivity limits.
Area of Science:
- Medical diagnostics
- Molecular biology
- Mycology
Background:
- Formalin-fixed, paraffin-embedded (FFPE) tissues pose challenges for fungal DNA detection.
- DNA degradation and PCR inhibitors are significant hurdles in FFPE sample analysis.
- Accurate fungal identification is crucial for diagnosing invasive fungal diseases (IFD).
Purpose of the Study:
- To evaluate the efficacy of panfungal PCR assays for detecting fungal DNA in FFPE tissues.
- To compare the performance of two panfungal PCR targets (ITS2 and ITS1-2).
- To assess the impact of DNA quality, degradation, and PCR inhibition on fungal detection in FFPE samples.
Main Methods:
- Two 45-cycle panfungal PCR assays targeting ITS2 and ITS1-2 regions were evaluated.
- FFPE specimens from 27 patients with suspected invasive fungal disease were analyzed.
- DNA quality was assessed using beta-globin gene amplification, and PCR inhibition was tested by spiking Candida krusei DNA.
Main Results:
- The ITS2 PCR assay demonstrated a higher detection rate (53.8%) compared to the ITS1-2 assay (38%).
- Time-dependent degradation of human DNA was observed but did not correlate with fungal PCR results.
- Both assays showed analytical sensitivity comparable to culture methods for Candida krusei.
Conclusions:
- Panfungal PCR assays can accurately identify molds in highly degraded FFPE tissue samples.
- These assays are effective for identifying fungi in samples stored for up to 7 years.
- Sensitivity limitations are primarily due to PCR inhibition and formalin-induced DNA degradation.
