Objective:Diagnosis of yeast infections is typically accomplished by fungal smear and culture, histopathologic examination and/or serologic studies. Newer mass-spectrometry based assays may be useful for providing an identification of the yeast present when histologic examination is inconclusive, fungal cultures were not ordered, or when cultures failed to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID Broad Fungal Assay to accurately detect and identify yeasts in formalin-fixed, paraffin-embedded (FFPE) tissues.Methods:Tissue from seventy-eight FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID Broad Fungal assay. A forty micron FFPE tissue section from each case was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing.Results:PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered environmental contaminants while 1 clinically important discrepancy was observed (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7%, 70/78) correlated with culture results.Conclusions:The PLEX-ID Broad Fungal Assay is capable of identifying fungi directly from FFPE tissues and can be a useful adjunctive test to traditional culture and histopathology.