The Detection and Identification of Yeasts from Formalin-Fixed Paraffin-Embedded (FFPE) Tissues Using the Broad Fungal Assay on the PLEX-ID PCR-Electrospray Ionization Mass Spectrometry (ESI-MS) System
Simner J Patricia, Seanne P Buckwalter, Jim Uhl, Pritt S Bobbi. Mayo Clinic, Rochester, MN
Background: Diagnosis of yeast infections is typically accomplished by fungal culture, morphologic examination of affected tissues, and/or serologic studies. Although histology or cytology analysis is often successful in providing a definitive identification, results may be indeterminate when only few yeast forms are present or morphologic features are ambiguous. This is problematic when cultures are not submitted or fail to yield a causative agent. Newer molecular-based assays may be useful in these situations for providing a diagnosis or confirming the morphologic impression. The purpose of this study was to evaluate the PLEX-ID Broad Fungal Assay to detect and identify yeasts directly from FFPE tissues.
Design: The accuracy of the PLEX-ID Broad Fungal assay was compared to culture and histopathology results for 80 tissue specimens containing a variety of yeasts. Histopathologic results were confirmed by 2 independent reviewers. For the PLEX-ID test, a 40 micron section of the FFPE tissue block was digested with proteinase K, followed by extraction and PCR amplification of DNA using broad-range fungal primers. Amplified products were identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA sequencing.
Results: PLEX-ID analysis resulted in 71.3% (57/80) agreement with culture results to the species level with 6.3% (5/80) discordant results and 22.5% (18/80) not detected by the system. Four of the discordant results yielded organisms that may be considered environmental contaminants while one clinically important discordant result occurred (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing was negative on the DNA extract from the discordant sample. Lack of detection by the PLEX-ID did not correlate with organism load by histopathology. The majority of histology results (93.7%, 75/80) correlated with culture. In the remaining 6.3% (5/80), the PLEX-ID was able to identify the organism that grew in culture.
Conclusions: The PLEX-ID Broad Fungal Assay is capable of identifying fungi directly from FFPE tissues and can help in the diagnosis of difficult cases or confirm the histologic impression where fungal culture may not have been performed.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 245, Wednesday Morning