# Application of Amplicon Metagenomics to Identify Fungal Pathogens in Formalin-Fixed Paraffin-Embedded Samples: Proof of Concept in Animals with Fungal Pathologies

## TL;DR

This study shows that amplicon metagenomics can identify fungal pathogens in preserved FFPE tissues, offering a new tool for diagnosis and research.

## Contribution

The study demonstrates a proof of concept for using amplicon metagenomics in FFPE samples to identify fungal pathogens.

## Key findings

- Amplicon metagenomics successfully identified fungal pathogens in 9 out of 11 FFPE samples.
- The ITS1 and 28S D1 rRNA gene markers were effective for fungal identification in FFPE tissues.
- The method is valuable for retrospective studies and polymicrobial community research with suboptimal DNA preservation.

## Abstract

The identification of fungal pathogens in formalin-fixed paraffin-embedded (FFPE) tissues is an unmet need in human and animal medicine, and sequence-agnostic approaches are needed to identify emerging pathogens. Eleven FFPE biopsy specimens with etiologic diagnoses of fungal disease based on standard testing of paired fresh tissue samples were utilized here to evaluate metabarcoding approaches. The cases included tissues from three dogs, three cats, one box turtle, one goat, one common loon, and one gray tree frog. The diagnoses from the fresh tissues in these cases were Microsporum canis, Penicillium sp., Exophiala sp. (likely E. jeanselmei), Verticillium sp., Rhizopus sp., atypical Cryptococcus neoformans, Conidiobolus spp., Aspergillus fumigatus, Cryptococcus neoformans var grubii, Batrachochytrium dendrobatidis, Fusarium solani, Blastomyces dermatitidis, Coccidiodes immitis, and Histoplasma capsulatum. We compared the ITS1 and 28S D1 rRNA gene genetic markers in combination with several bioinformatic strategies to identify fungal pathogens in the FFPE tissue samples, with a success rate of 9/11. These methods could allow diagnosticians who receive only FFPE tissues and see fungal pathogens to speciate the pathogens and could be of value in retrospective studies wherein FFPE tissue is the only archived tissue. Furthermore, these techniques could be of use to researchers investigating polymicrobial communities where DNA preservation is suboptimal.