[1943] In Situ Hybridization (ISH) for Determination of Fungal rRNA Preservation in Sinonasal Fungal Disease

KT Montone, J Palmer, AC Chiu, DW Kennedy, DC Lanza, VA Livolsi, MD Feldman, I Nachamkin. University of Pennsylvania, Philadelphia, PA

Background: Sinonasal fungal disease (SNFD) is becoming increasingly recognized and identification of specific pathogens may aid in treatment, particularly those patients with invasive disease. ISH for rRNA has become a means for detecting fungi in tissues. One pitfall of rRNA ISH is not knowing whether the rRNA is preserved following routine tissue processing which in the sinonasal tract may include decalcification or in examples of acute necrotizing invasive fungal rhinosinusitis (ANIFRS) may involve frozen section prior to routine formalin-fixation and paraffin-embedding (FFPE). This study set out to determine the preservation of fungal rRNA sequences in SNFD.
Design: 169 SNFD specimens (15 fungus ball (FB), 96 allergic fungal rhinosinusitis (AFRS), and 58 acute necrotizing fungal sinusitis (ANIFRS)) were studied by ISH using a biotin-labeled locked nucleic acid (LNA) probe targeting pan-fungal rRNA. The 169 tissue specimens included 134 routinely FFPE tissues, 19 that had undergone decalcification followed by FFPE and 16 that had been frozen during intraoperative consultation followed by FFPE.
Results: 124 specimens (73%) were positive for pan-fungal rRNA by ISH. 78% of FFPE specimens were ISH positive while only 42% of decalcified specimens were positive. 12 specimens (75%) which had been frozen prior to FFPE were positive by ISH. ISH was positive in 64% of specimens with positive cultures and 11% with negative cultures. ISH was negative in 23% of specimens with a positive culture. 3% of specimens were negative by culture and ISH although fungal organisms were seen by histology. Based on histopathology, ISH was positive in 79% AFRS, 80% FB and 62% of AFIRS.

DiagnosisISH Positive
FFPEDCALFSTotal
FB9/12 (75%)3/3 (100%)012/15 (80%)
AFRS73/84 (87%)3/12 (25%)076/96 (79%)
AFIRS22/38 (58%)2/4 (50%)12/16 (75%)36/58 (62%)



Conclusions: Most SNFD specimens have preserved fungal rRNA with preservation being greatest in non-invasive cases. Decalcification of sinonasal specimens may limit rRNA preservation in SNFD limiting ISH. In routine FFPE, preservation of rRNA is lowest in ANIFRS possibly due to the extensive necrosis encountered in these tissues which may relate to organism viability. Of note, >30% of ANFIRS patients showed no growth in cultures. Intraoperative consultation via frozen section allows for preservation of fungal rRNA in the tissues. Pathologists should be aware that there may be some limitations of using rRNA ISH in SNFD.
Category: Techniques

Monday, March 22, 2010 9:15 AM

Platform Session: Section G 1, Monday Morning

 

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