Unbiased DNA Pathogen Detection in Tissues: Real-World Experience With Metagenomic Sequencing in Pathology

Area of Science:

• Clinical diagnostics
• Infectious disease pathology
• Molecular diagnostics

Background:

• Pathogen detection in formalin-fixed paraffin-embedded (FFPE) tissues presents significant diagnostic challenges.
• Conventional methods may have limitations in sensitivity and breadth of pathogen coverage.
• Metagenomic next-generation sequencing (mNGS) offers a potential solution for comprehensive pathogen identification.

Purpose of the Study:

• To evaluate the feasibility, diagnostic yield, and pathogen spectrum of mNGS in routine infectious pathology.
• To assess mNGS performance in formalin-fixed paraffin-embedded (FFPE) tissue samples.
• To compare mNGS with conventional diagnostic methods for pathogen detection.

Main Methods:

• A low-depth mNGS workflow was implemented using the Thermo Fisher Ion Torrent platform.
• A total of 623 FFPE tissue samples were analyzed between November 2021 and April 2025.
• Results were validated using orthogonal methods such as PCR and immunohistochemistry when possible.

Main Results:

• mNGS identified at least one potentially pathogenic microorganism in 36.8% of FFPE samples.
• Bacteria were the most common pathogens (63.3%), followed by viruses (16.2%), fungi (12.2%), and parasites (3.9%).
• Mycobacteriaceae was the most frequent bacterial family identified; novel viral and fungal pathogens were also detected.

Conclusions:

• Metagenomic next-generation sequencing (mNGS) is a feasible and valuable addition to routine infectious pathology diagnostics.
• The assay demonstrated improved diagnostic yield and expanded pathogen detection capabilities compared to conventional PCR.
• mNGS proved robust for FFPE samples, supporting its broader adoption in tissue-based pathogen diagnostics.