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Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue
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Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

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Application of Amplicon Metagenomics to Identify Fungal Pathogens in Formalin-Fixed Paraffin-Embedded Samples: Proof

David B Needle1, Guillaume Reboul2, Patrick K Mitchell3

  • 1New Hampshire Veterinary Diagnostic Laboratory, College of Life Sciences and Agriculture, University of New Hampshire, Durham, NH 03824, USA.

Microorganisms
|March 27, 2025
PubMed

Insights

Metabarcoding approaches successfully identified fungal pathogens in 9 of 11 formalin-fixed paraffin-embedded (FFPE) tissues. This advance aids in diagnosing emerging fungal diseases from FFPE samples, crucial for veterinary and human medicine.

Area of Science:

  • Veterinary Mycology
  • Molecular Diagnostics
  • Pathogen Identification

Background:

  • Identifying fungal pathogens in formalin-fixed paraffin-embedded (FFPE) tissues presents a significant diagnostic challenge.
  • Current methods often struggle with degraded DNA in FFPE samples, limiting pathogen identification.
  • There is a need for sequence-agnostic methods to detect emerging fungal threats in archived tissues.

Purpose of the Study:

  • To evaluate the efficacy of fungal metabarcoding approaches for identifying pathogens in FFPE biopsy specimens.
  • To compare different genetic markers (ITS1, 28S D1 rRNA) and bioinformatic strategies for pathogen detection.
  • To assess the utility of these methods for retrospective studies and challenging diagnostic cases.

Main Methods:

  • Eleven FFPE biopsy specimens from various animal species with confirmed fungal diagnoses were analyzed.
  • DNA was extracted from FFPE tissues, and targeted gene regions (ITS1 and 28S D1 rRNA) were amplified.
  • Sequence data were processed using multiple bioinformatic pipelines to identify fungal species.

Main Results:

  • Metabarcoding successfully identified fungal pathogens in 9 out of 11 FFPE specimens, achieving an 82% success rate.
  • The study demonstrated the ability to speciate fungal pathogens directly from FFPE tissues.
  • Specific fungal genera identified included *Microsporum*, *Penicillium*, *Exophiala*, *Verticillium*, *Rhizopus*, *Cryptococcus*, *Conidiobolus*, *Aspergillus*, *Batrachochytrium*, *Fusarium*, *Blastomyces*, *Coccidioides*, and *Histoplasma*.

Conclusions:

  • Fungal metabarcoding using ITS1 and 28S D1 rRNA markers is a viable method for identifying fungal pathogens in FFPE tissues.
  • These sequence-agnostic techniques offer a valuable tool for diagnosticians working with FFPE samples and for retrospective studies.
  • The approach holds promise for investigating polymicrobial infections and situations with suboptimal DNA preservation.
Keywords:
FFPE sequencingbioinformaticsfungal diagnosismetabarcodingmolecular diagnostics

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