1572. Secreted Protease Detection as a Diagnostic Test for Invasive Aspergillosis
Session: Poster Abstract Session: Mycology: Diagnostic
Friday, October 28, 2016
Room: Poster Hall
Background: We have established a novel molecular assay that can aid in the clinical diagnosis of invasive aspergillosis. The assay is based on the detection of an abundant protease released by Aspergillus fumigatus and other related fungi during pulmonary invasion. Our assay accelerates and detects the specific catalytic turnover of the Asp f2 protease.

Methods: The protease was enriched from clinical specimens using a bead-immobilized monoclonal antibody specific for native Asp f2. The beads were then washed, exposed to a specific fluorogenic peptide, and fluorescence intensity was measured with a fluorometer. Exploitation of Asp f2’s intrinsic metalloprotease activity made this assay not only very specific, but also much more sensitive than ELISA or Western blotting. We evaluated the performance of the Asp f2 activity assay by analyzing 144 samples of brochoalveolar lavage fluid (BALF) from hematopoietic cell transplant recipients suspected for having invasive aspergillosis.

Results: The clinical diagnosis of proven or probable aspergillosis, according to the EORTC and MSG guidelines, was compared to our assay results. The protease assay has a sensitivity of 77.8% and a specificity of 96.1% for invasive aspergillosis. Furthermore, we determined that BALF from patients with A. fumigatus, A. versicolor, or A. niger infections had detectable Asp f2 activity.

We developed the assay based on the observation, that native Asp f2 isolated from A. fumigatus cultures had metalloprotease activity. This activity was inhibited by metalloprotease inhibitors but not by serine protease inhibitors and many other inhibitors. Furthermore, we engineered an artificial Asp f2 proenzyme consisting of a fusion of thioredoxin, yeast Smt3p, and Asp f2 residues 32-310. Therein, the Smt3p replaces the signal peptide region of Asp f2. The recombinantly expressed fusion protein was purified and activated with yeast Ulp1, which cleaves the fusion at the C-terminus of Smt3p, yielding an active recombinant Asp f2. The latter was inhibited by the same metalloprotease inhibitors, as was the native Asp f2.

Conclusion: Our Asp f2 activity assay is founded on a solid biochemical rationale. It generates reproducible results and may therefore become a valuable addition to the currently underdeveloped repertoire of molecular clinical assays for invasive aspergillosis.

Karine Bagramyan, Ph.D.1, Teresa Hong, BS1, Sanjeet Dadwal, MD2, James Ito, MD3 and Markus Kalkum, PhD1, (1)Molecular Immunology, City of Hope, Beckman Research Institute, Duarte, CA, (2)City of Hope, Duarte, CA, (3)Division of Infectious Diseases, City of Hope, Duarte, CA

Disclosures:

K. Bagramyan, None

T. Hong, None

S. Dadwal, None

J. Ito, None

M. Kalkum, None

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