Trial Information

Open, Prospective, Multicenter Trial to Evaluate the Clinical Significance of Combined Serological (Galactomannan ELISA, 1->3-β-D Glucan Assay) and Molecular (Nested Aspergillus PCR Assay, Multifungal DNA Microarray) Diagnostic Assays to Detect and Characterize Fungal Pathogens in Bronchoalveolar Lavage (BAL) and Blood Samples of Hematological High Risk Patients and to Detect Point Mutations Conferring Azole Resistance

The major problem in managing life threatening invasive fungal infections in patients (pts)
with acute leukemia and pts after allogeneic hematopoietic stem cell transplantation is the
lack of sensitive and specific diagnostic tools to identify fungal pathogens reliably and
early in the course of the disease. Because of deep neutropenia and low platelet count,
invasive diagnostic procedures are rarely feasible in time, therefore bronchoscopy with BAL
is the method of choice in diagnosing pulmonary infections, as the sensitivity of
culture-based methods from blood is low, especially in mould infections. Indirect methods,
so-called surrogate markers, are becoming increasingly important in this clinical setting.
These serologic markers, mainly galactomannan (GM) and recently 1(1,3)-β-D-glucan (BDG),
have been validated in clinical trials for blood samples, however the clinical significance
of testing BAL samples is up to now only based on retrospective data for GM and has not been
reported yet for BDG.

The aim of our prospective and multicentre diagnostic study is therefore to elucidate on the
sensitivity and specificity rates of these serologic markers in combination with molecular
tools (both an Aspergillus specific and a multifungal PCR based assay), as serologic markers
are not pathogen-specific, and furthermore to define species-specific cut-off values for BDG
in BAL samples.

Additionally, if genomic material of Aspergillus fumigatus is detected by PCR in a clinical
sample, we investigate fungal DNA for point mutations in the cyp51A gene mediating
resistance against common mould-active triazoles with novel rapid, sensitive and specific,
non-culture-based PCR-assays and sequencing to optimize antifungal treatment as early as
possible.

This study aims to improve the early, sensitive and specific diagnosis of invasive pulmonary
fungal infections and detect azole resistance patterns, it might impact on the prognosis in
hematologic patients; therefore antifungal treatment data and clinical outcome will be
recorded.

Clinical samples (BAL and blood) from approximately 100 pts suffering from acute leukemia
and pts after allogeneic stem cell transplantation with febrile neutropenia and lung
infiltrates diagnosed in a chest CT scan suggestive for fungal infection will be
investigated after pts`s informed consent in a multicentre, prospective trial. Pts will
undergo standardized diagnostic imaging and microbiological procedures for identification of
the underlying infectious pathogen. BAL and blood samples will be tested additionally for
GM, BDG and with a diagnostic nested Aspergillus PCR assay, a multifungal DNA-Microarray and
an azole resistance PCR assay. The molecular assays were established by our group and
encompass the detection of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus,
Candida albicans, Candida dubliniensis, Candida glabrata, Candida lusitaniae, Candida
tropicalis, Fusarium oxysporum, Fusarium solani, Mucor racemosus, Rhizopus microsporus,
Scedosporium prolificans, Trichosporon asahii and identify the three most common point
mutations that confer resistance to triazoles like voriconazole or posaconazole.

Results of other diagnostic means including culture findings as well as patients` clinical
data (e.g. duration of neutropenia, underlying disease and outcome including follow-up data
concerning antifungal treatment and mortality attributable to fungal infections) will be
documented, pseudonomyzed and included in our data bank.

BAL and blood aliquots of 20 control pts without immunosuppression (suffering from lung
diseases) will be collected and tested identically.

Case definitions will be made according to 2008 EORTC/MSG criteria. The duration of the
study will be approximately 24 months