Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens
Anna Lau1, Sharon Chen, Tania Sorrell
1Faculty of Medicine, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sidney, Australia.
Abstract:
Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n=38) or solely histologically proven (n=24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida, Cryptococcus, Trichosporon, Aspergillus, Fusarium, Scedosporium, Exophiala, Exserohilum, Apophysomyces, Actinomucor, and Rhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n=10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.
Insights
A new panfungal PCR assay accurately identifies fungal pathogens in tissue samples. This molecular method aids in diagnosing invasive fungal infections (IFIs) when combined with traditional tests.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Invasive fungal infections (IFIs) are increasing, necessitating precise pathogen identification.
- Current diagnostic methods like culture and histology have limitations.
Purpose of the Study:
- To develop and evaluate a panfungal PCR assay for detecting fungal DNA in tissue specimens.
- To compare the PCR assay's performance against conventional diagnostic methods.
Main Methods:
- A panfungal PCR assay targeting the ITS1 region of ribosomal DNA was developed.
- The assay was used on fresh and formalin-fixed, paraffin-embedded (PE) tissue from patients with IFIs.
- PCR products were sequenced for pathogen identification and correlated with histology and culture.
Main Results:
- The assay detected fungal pathogens in 93.6% of culture-proven and 64.3% of solely histologically proven IFIs.
- A broad range of fungal genera were identified, including Candida, Aspergillus, and Cryptococcus.
- Molecular identification sometimes differed slightly from culture results; all PCR-negative cases showed fungal hyphae on histology.
Conclusions:
- The panfungal PCR assay is a valuable tool for identifying fungal pathogens in tissue specimens.
- Combining PCR with conventional methods enhances diagnostic accuracy for IFIs.
- This assay can detect a wide spectrum of fungi, improving IFI diagnosis and management.
