In Situ Hybridization (ISH) for Species Specific Fungal rRNA in Acute Necrotizing Invasive Fungal Rhinosinusitis
KT Montone, J Palmer, AC Chiu, DW Kennedy, DC Lanza, VA Livolsi, MD Feldman, I Nachamkin. University of Pennsylvania, Philadelphia, PA
Background: Acute necrotizing invasive fungal rhinosinusitis (ANIFRS) is an angioinvasive fungal infection which almost alway occurs in immunosuppressed patients. ANIFRS is most commonly caused by Aspergillus or Zygomyces infections and rarely other pathogens. A diagnosis of ANIFRS requires rapid intervention, but cultures may be negative. Identification of particular fungal subtypes is important for patient management. ISH for rRNA may be able to identify fungal organisms ANIFRS.
Design: 25 patients (58 specimens) with ANIFRS were identified. These included culture proven cases of Rhizopus sp (6), Aspergillus sp (9), Fusarium sp (1), Alternaria sp (1) and Paecilomyces sp (1) and 7 patients with negative cultures. Rapid (<3 hours) ISH for fungal RNA was performed using species specific biotin-labeled oligonucleotide DNA probes targeting rRNA of Aspergillus sp, Fusarium sp, Rhizopus sp and a rRNA sequence seen in a variety of Dematiaceous fungi. Preservation of fungal rRNA was determined using a pan-fungal rRNA probe.
Results: ISH with the panfungal probe showed fungal rRNA preservation in 35/58 specimens (60%). Preserved rRNA was identified in at least one specimen from 20 patients (6/6 Rhizopus sp, 7/9 Aspergillus sp, 1/1 Fusarium sp, 1/1 Alternaria sp, 1/1 Paecilomyces sp and 4/7 negative). ISH confirmed specific fungal rRNA in all rRNA preserved cases with the exception of the Paecilomyces sp case which was negative with the Dematiaceous sp probe. Negative controls consisted of ISH with the specific probes in the other fungal infections which in all cases were negative with the exception of the Aspergillus sp probe which resulted in weak staining in 3 of the Rhizopus sp cases although signal was minimal compared to that seen with the Rhizopus sp probe. Only 4/7 patients with negative cultures had preserved rRNA. Of these, 2 cases of Aspergillus sp and 1 case of Dematiaceous sp was identified by ISH.
Conclusions: 60% of ANIFRS had preserved fungal rRNA most likely due to tissue necrosis and decalcification. ISH can identify Aspergillus sp, Rhizopus sp, Fusarium sp and some Dematiaceous sp in ANIFRS. Controls must be performed and care taken not to over interpret weak signals especially when the Aspergillus sp probe is used in Rhizopus sp cases. rRNA ISH may aid fungal species identification in specimens with negative cultures but in this study which included 7 culture negative cases, only 4 had preserved rRNA with 3 additional specific fungal infections identified.
Category: Head & Neck
Tuesday, March 23, 2010 11:45 AM
Platform Session: Section G, Tuesday Morning