Panfungal PCR for identification of fungi from histopathology-positive formalin-fixed, paraffin-embedded tissues and analysis of factors affecting performance

This study evaluated performance of pan-fungal polymerase chain reaction (PCR) for identification of fungi from histopathology-positive formalin-fixed, paraffin-embedded (FFPE) tissues.

We selected FFPE tissue biopsies with histopathologically proven fungal elements and simultaneous culture requests from 2020 to 2023. DNA extraction from blocks was performed using the QIAamp DNA FFPE Kit (QIAGEN). Conventional PCR with ITS1 and ITS4 primers was conducted, followed by gel electrophoresis and sequencing using the Basic Local Alignment Search Tool. Fisher exact and Mann-Whitney U tests were performed to determine associations of various factors with DNA amplification and concordant results.

From a total of 96 samples, 35 (36.5%) were amplified; of these 35 samples, 7 (20.0%) yielded sequencing results. Overall, 7 of 96 (7.3%) demonstrated agreement with histopathology and 4 of 96 (4.2%) exhibited agreement with both microbiology and histopathology. Analysis of factors influencing fungal DNA amplification revealed strong associations with 10% potassium hydroxide smear positivity and sterile vs nonsterile tissue. Factors found to be significant for concordant ITS sequencing results with histopathology were septate hyphae vs nonseptate hyphae, DNA concentration, and time elapsed between sample collection and PCR.

We found low positivity for pan-fungal PCR for fungal identification from FFPE tissues. Although the diagnostic yield from FFPE samples was low, optimizing conditions that influence DNA yield may improve results.