615. Detection of Aspergillus Spp. in Human Tissues Samples by Immunohistochemistry and in Situ Hybridization
Session: Abstracts: Mycology
Friday, October 22, 2010
Background: The diagnosis of invasive asperigllosis (IA) is limited by poor sensitivity and specificity of microbiologic cultures, and the inability of conventional histopathologic tests to distinguish IA from invasive fungal infections (IFI) caused by non-Aspergillus moulds.  Aspergillus-specific immunohistochemistry (IHC) and in situ hybridization (ISH) could facilitate direct diagnosis of IA within tissue.

Methods: IHC and ISH were performed on deparaffinized, formalin-fixed sections from previously diagnosed cases of proven IFI, and slides interpreted by pathologist blinded to diagnosis.  IHC: Primary Aspergillus antibody (Abcam, MA) and secondary anti-rabbit antibody were detected with Avidin Biotin Complex and visualized using liquid DAB. ISH: Commercially synthesized Locked Nucleic Acid  probe for Aspergillus was detected using antiflourescein AP. Poly T stained tissue section was used to determine the area of tissue to be studied.

Results: 8 tissue sections were available from patients with IA (A. fumigatus=5; A. niger=1; non-speciated=2, and 12 sections were available from patients with IFI due to other fungi (Zygomyces= 6; Candida=5; Dactylaria gallopava=1). IA samples were obtained from 6 tissue biopsies (3 lungs, 2 upper airways, 1 vocal cord), and 2 autopsy samples. 2 serial sections from one patient with IA due to A. fumigatus were excluded from the study because positive control stains were negative.  The sensitivity of IHC and ISH for IA was 83% (5/6) and 100% (6/6), respectivelynb. IHC was falsely negative in 1 pt with IA due to A. fumigatus.  The specificity of IHC and ISH was 100% (12/12), as the tests were each negative in all sections associated with IFI due to non-Aspergillus fungi. IHC was associated with significant levels of non-specific background staining in 33% of the sections that could be analyzed, compared to 0% for ISH.

Conclusion: Data from this pilot study suggest that Aspergillus-specific IHC and ISH will facilitate diagnosis of IA and exclude IFI due to non-Aspergillus fungi.  ISH was superior to IHC by limiting false negative results and non-specific background staining. We are currently assessing IHC and ISH prospectively on non-fixed tissues.


Subject Category: M. Mycology including clinical and basic studies of fungal infections

Speakers:
Aniket Vadnerkar, MD , Pittsburgh University, Pittsburgh, PA
Cornelius Clancy, MD , Medicine, University of Pittsburgh, Pittsburgh, PA
Samuel Yousem , University of Pittsburgh, Pittsburgh, PA
Sanja Dacic , University of Pittsburgh, Pittsburgh, PA
Minh-Hong Nguyen, MD , University of Pittsburgh, Pittsburgh, PA

Disclosures:

A. Vadnerkar, None

C. Clancy, Yes
Pfizer: Grant Investigator, Research grant
Merck: Grant Investigator, Research grant
Ortho McNeil: Grant Investigator, Research support

S. Yousem, None

S. Dacic, None

M. H. Nguyen, Yes
Pfizer: Grant Investigator,
Merck: Grant Investigator,
ViraCor: Grant Investigator,

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