Biomarker and antifungal resistance in Aspergillus

Invasive aspergillosis (IA) is the most common cause of infection-associated mortality in patients being treated for haematological malignancies and is an emerging disease in solid organ transplant recipients, critical care patients and in those receiving novel immunomodulatory therapies. The annual cost of treating fungal infections in Europe is approximately 200 million Euro. Currently there is no sensitive, rapid and accurate diagnostic assay for IA or for monitoring the success of antifungal therapy (1). Antifungal drug resistance accounts, at least partially, for the poor outcomes in IA and intrinsic and acquired resistance have been increasingly documented (2). With this consortium, we propose to develop and evaluate a battery of in vitro assays for a comprehensive multimodality analysis, combining the detection of Aspergillus elements (RNA, polysaccharides, proteins), host factors including cytokine profiles, together with the individual genetic susceptibility of the host. Existing diagnostic parameters will be included in the analysis to validate the improvement of the methods under development over the existing ones. 1. Definition of A. fumigatus biomarkers. This WP focuses on the detection of Aspergillus biomarkers indicating active growth during the early stages of fungal development. This will include the detection of DNA, mRNA, proteins, polysaccharides, and other molecules released. One central part is the development of a highly sensitive, commercially available, standardized diagnostic assay for nucleic acid quantification (bMx in collaboration with MUI and others). These efforts include the development of suitable protocols allowing the extraction and purification of the nucleic acids from A. fumigatus, specific amplification and detection tools (mRNA and DNA targets will be investigated, to determine which target type is the best option to diagnose IA and to monitor its evolution during the course of infection). Final aim is to test prototypes in biological samples (whole blood, sera, bronchoalveolar lavage) from patients suffering from IA. 2. Detection and identification of resistant Aspergillus strains. We will isolate and characterize laboratory- induced mutant strains (3,4) in Aspergillus spezies (resistance against polyene, candine and azole) to investigate the nature of resistance; we plan to perform genetic and biochemical studies, run antifungal susceptibility testing assays and compare in vitro and in vivo outcome in an animal mouse model. Azole and candin resistant strains will be also collected from the different clinical partners of the consortium or will be obtained from collaborators (E.Mellado, Instituto Carlos III, Madrid, Spain). The CYP51 and FKS1 genes that are the targets of azoles and candins will be sequenced to localize the mutations inducing resistance. Results obtained are important for partner IP, which designs a microarray to identify all mutations. Gene expression profiles of mutant and wild type strains with and without the presence of the specific drug will be analyzed. Producing resistant strains can be undertaken in vitro under different growth conditions to see if the selection of mutation hot spots is depending on the nutritional environment of the fungus. 3. Biobank: clinical and applied aspects. For the prospective evaluation of the developed diagnostic assays, clinical specimens (whole blood, sera, BAL, and PBMC) from high risk patients are required. We will collect and archive adequate specimens and apply new tests designed for clinical usefulness. Herein we run the maintenance of the specimen and data library, data management, ethical issues, integrative analysis of the results and the development of a commercial strategy for diagnostic kits of IA. a) Analysis of human biomarkers: Cytokines and chemokines quantification in patient sera during the course of IA. We will evaluate the new TruCultureTM system, which allows easy, fast and reproducible analysis of individual blood samples directly in the blood tube (without isolation of immune cell populations) to define changes of the immune status caused by Aspergillus infections. TruCultureTM is an in vitro model that preserves physiological cellular interactions and allows easy measurements of the immune system without stimulation in a closed system. The system is designed to capture immune cell activity without manipulation of any variables. b) Genetic predisposition and high-risk patients. This project part includes a genetic - epidemiological, multi- centre study combining epidemiological expertise with the most modern molecular-genetic and biocomputing techniques to identify genetic risk factors for IA. We apply samples for this analysis. (1) Lancet 2004, 10:1467-74; (2) Antimicrob. Agents Chemother. 2009,53:1645-7; (3) Immun.1997, 65:5110-17; (4) Infcet. Immun.1996, 64:3244-51.

The scientists of the project AspBIOmics have set themselves the aim to improve the diagnosis of invasive aspergillosis. Improved diagnosis is reached by detection of the pathogen or characteristic components of the pathogen in patients serum and the identification of new recognition molecules (e.g., surface components of the fungus, genetic material of the fungus). Invasive aspergillosis is a fungal lung infection caused by Aspergillus spp.; these infections affect especially people with weakened immunity. Not only is the detection of aspergillosis a problem, but also the treatment of this infection. Specifically, therapy-resistant Aspergillus isolates cause difficulties, so their occurrence, frequency and clinical relevance were examined.This project allowed us to generate the following major contributions to the understanding of these fungal infections. The preventive therapy against invasive aspergillosis led to a declining incidence, however, other fungi occur in their place. Furthermore, we have found that when patients receive an appropriate preventive therapy, the serum test methods are not suitable for the detection of pathogens. Knowledge on this false positivity is new and essential information to the attending physician as it will impact on treatment decision. The incidence of treatment-resistant pathogens is currently negligible at the Medical University Hospital Innsbruck, because therapy-resistant Aspergillus strains occur at very low frequencies. Cytokines and chemokine quantification defined 9 characteristic blood values, which could significantly improve the early diagnosis of aspergillosis in the future.The data collected and results gained provide important insights, which will have an impact on the prevention of infection and patient care.