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qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue
Published on: July 9, 2014
Assessment of panfungal PCR performance with formalin-fixed paraffin-embedded tissue specimens†
Shawn T Clark1, Yvonne C W Yau1,2, Aaron Campigotto1,2
1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, M5S 1A8, Canada.
Abstract:
We reviewed the performance of a panfungal ITS-2 PCR and Sanger sequencing assay performed on 88 FFPE specimens at The Hospital for Sick Children (Toronto, Canada) in 2019. A potential fungal pathogen was identified by ITS PCR in 62.7 and 2.9% of positive and negative direct slide examination of tissue specimens, respectively. ITS amplicons were detected in 87/88 specimens, with 53/88 (60.2%) considered as 'positive-contaminants' and 34/88 (38.6%) as 'positive-potential pathogen' upon sequencing. Potential pathogens included Blastomyces dermatitidis (17.1%), Cryptococcus neoformans (17.1%), Histoplasma capsulatum (14.3%) and Mucormycetes (11.4%). Laboratories should only perform ITS PCR on FFPE tissues if fungal elements have been confirmed on histopathology slides.
Lay Summary:
In this study, we examined how well a DNA-based test could detect DNA from fungi in archived human biopsy tissues. The best performance was achieved if fungi were seen in the tissue under a microscope before being tested. Our results indicate that we should only use this test if these conditions are met.
Insights
A fungal DNA test on FFPE tissues showed high contamination rates. The internal transcribed spacer PCR (ITS PCR) assay is most reliable for detecting fungal pathogens when fungal elements are confirmed by histopathology first.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Histopathology
Background:
- Archived human biopsy tissues (FFPE) were analyzed for fungal DNA detection.
- A panfungal internal transcribed spacer 2 (ITS-2) PCR and Sanger sequencing assay was evaluated.
- Histopathology confirmation of fungal elements is crucial for accurate molecular testing.
Purpose of the Study:
- To assess the performance of an ITS-2 PCR and Sanger sequencing assay for detecting fungal DNA in FFPE specimens.
- To determine the reliability of the assay in identifying fungal pathogens versus contaminants.
Main Methods:
- Analysis of 88 FFPE specimens using a panfungal ITS-2 PCR and Sanger sequencing assay.
- Comparison of molecular results with direct slide examination (histopathology).
- Identification and categorization of detected fungal DNA as 'positive-contaminants' or 'positive-potential pathogen'.
Main Results:
- ITS amplicons were detected in 98.9% (87/88) of specimens.
- 60.2% (53/88) of positive results were identified as contaminants.
- 38.6% (34/88) of specimens yielded potential fungal pathogens, including Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum, and Mucormycetes.
Conclusions:
- The panfungal ITS-2 PCR and Sanger sequencing assay demonstrates a high rate of contaminant detection in FFPE tissues.
- The assay is most effective when fungal elements are pre-confirmed via histopathology.
- Laboratories should exercise caution and implement strict protocols when using ITS PCR on FFPE tissues.
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