Amplicon-Based Next-Generation Sequencing for Detection of Fungi in Formalin-Fixed, Paraffin-Embedded Tissues: Correlation with Histopathology and Clinical Applications

Abstract Invasive fungal infections are increasing in prevalence due to an expanding population of immunocompromised individuals. To reduce morbidity and mortality, it is critical to accurately identify fungal pathogens to guide treatment. Current methodologies rely on histopathology, fungal culture, and serology, which are often insufficient for diagnosis. Here we describe the use of a lab-developed internal transcribed spacer (ITS) targeted amplicon-based Next Generation Sequencing (NGS) assay for the identification of fungal etiology in fungal stain positive FFPE tissues by using Illumina MiSeq. A total of 44 specimens from 35 patients were included in this study with varying degrees of fungal burden from multiple anatomical sites. NGS identified 20 unique species across the 54 total organisms detected, including 40 molds, 10 yeasts, and 4 dimorphic fungi. The histopathological morphology and the organisms suspected by surgical pathologist were compared with the organisms identified by NGS, with 100% (44/44) and 93.2% (41/44) concordance, respectively. In contrast, fungal culture only provided an identification in 27.3% (12/44) of specimens. We demonstrated that NGS is a powerful method for accurate and unbiased fungal identification in FFPE tissues. A retrospective evaluation of the clinical utility of the NGS results also suggests this technology can potentially improve both the speed and the accuracy of diagnosis for invasive fungal infections.