Community Research and Development Information Service - CORDIS

Periodic Report Summary 2 - FUNGITECT (Optimized Diagnostics for Improved Treatment Stratification in Invasive Fungal Diseases)

Project Context and Objectives:
Project Summary
Invasive fungal disease (IFD) is a leading cause of morbidity and mortality in the growing number of immunocompromised individuals, including particularly cancer patients, and bone-marrow or organ transplant recipients. Timely pathogen detection is a prerequisite for effective therapy in patients with IFD. FUNGITECT focuses on this medical priority and the plan is to develop, validate and market a specific set of novel molecular diagnostic tests for IFD targeting fungal DNA-, RNA- and protein motifs, as well as the enzymatic activity of fungal pathogens. Additionally, FUNGITECT provides a unique opportunity to establish and implement highly effective diagnostic assays supported by Next-Generation Sequencing and a bioinformatics service platform, facilitating optimized treatment strategies adapted to individual patient requirements.

Main objectives for this reporting period

1. Establishment of Standard Operating Procedures (SOPs) for patient sample retrieval and biomarker discovery. This comprises the elaboration of clinical study protocols and SOPs for sample retrieval and identification of select biomarkers applicable in DNA, RNA and protein-based analysis (WP1).

2. Development of monoclonal antibodies (MoAbs) and enzymatic tests for low-cost point-of-care diagnostics of IFD. The task includes the establishment of serological detection as a tool for the detection of fungal cell wall components and cytoplasmic antigens or metabolites in serum or other body fluids. Additionally, a patented biosensor for detection of secreted proteolytic enzymes is to be adapted and optimized for rapid and broad detection of major fungal pathogens (WP2).

3. Identification of molecular markers (DNA&RNA) for patient stratification and pathogenicity prediction of fungal species. The objective is the development of an open diagnostic test system based on Next Generation Sequencing (NGS) and a PCR-based targeted diagnostic approach to facilitate rapid, reliable and broad speciation of fungal pathogens from patient samples. Moreover, RNA is intended to be exploited as a biomarker for the diagnosis of IFD. (WP3).

4. Optimization and validation of molecular targets. Prior to clinical implementation, molecular targets identified in work packages 2 and 3 must undergo validation and standardization by analyzing a variety of well-defined fungal clinical isolates and by retrospective and prospective testing of patient samples (WP4).

5. Development of a CDMS based diagnostic software platform. A Central Data Management System (CDMS) based on Genedata elector is intended to integrate and maintain relevant pathogen genomes for functional reconstruction and marker identification for host susceptibility. Coupled with NGS data processing and analysis workflows of re-sequencing data, this should permit high-throughput genome-wide profiling and genealogy assessment. Identified mutations and their profiles can then be exploited to elucidate mechanisms of pathogenicity and drug resistance. The platform is planned to support assay development, treatment options and decision processes for scientists and clinicians (WP5).

6. Clinical validation of diagnostic methods. Clinical validation of diagnostic assays (WP1-4) and functional effectiveness of the CDMS diagnostic software (WP5) encompasses carefully designed prospective clinical studies, including a national study in adult patients, and an international study in pediatric patients at high risk of IFD involving major cancer centers. Each study is planned to include longitudinal monitoring of ~200 febrile neutropenic periods in high-risk patients, and evaluation of novel diagnostic assays in comparison to existing standard diagnostics (WP6).

7. Commercial exploitation and dissemination. Upon availability of conclusive clinical data proving the applicability of the new assays in the diagnostic setting, the commercial partners plan to secure the resulting IP and launch the novel diagnostic services. (WP7).
Project Results:
WP 1 - The activities planned under work package 1 have been accomplished, namely the establishment of clinical sample collection guidelines for molecular IFD analysis; and the establishment of standards for analytical processing in molecular IFD diagnostics. Deliverable “D1.1 Guidelines/SOP for clinical specimen collection and pre-analytical handling conditions” has been reached.
WP 2 - Novel stable anti-Candida monoclonal antibody (MoAb)-producing hybridomas were obtained and cryopreserved, and 10 novel anti-Candida MoAbs were successfully purified. A classical ELISA was established using recombinant antigen and the novel corresponding anti-Candida MoAbs.
Another technology, the patented biosensor chip, is being adapted for topical degradation of a specific bio-layer by fungal lytic enzymes facilitating sensitive optical read-out. The biosensor´s matrix was modified by adding fungal nutritional components such as glucose and peptone, which were incorporated directly into the polymer layer of the biosensor.
WP 3 We have established a complete workflow based on Next Generation Sequencing for the identification of fungal species in plasma samples of septic patients. This approach is independent from cultures and takes approximately 24-28 hours from sample to the diagnostic report. Dedicated bioinformatics workflows for the unambiguous identification at the species level were established and evaluated. Protocols for the isolation and analyses of RNA from in vitro and in vivo samples were also established and applied for the expression analyses of in vitro cultures of Candida albicans as well as for clinical specimens.
WP4 The guidelines for diagnostic assays have been established. An automated extraction device with appropriate positive and negative controls was implemented. Validations of manual and automatic extraction procedures were performed that included diverse clinical material such as CSF aspirates, blood, sonication fluids from bones, various tissues and other samples from other sides. The validations show that the extraction of microbial DNA from diverse clinical material works reliably. The automated extraction combined with assays will be implemented in the upcoming clinical study.
WP 5 The implemented FUNGITECT Central Data Management System (CDMS) provides and integrates different modules each consisting of a relational database and a user interface for data submission, access, analysis and result storage. The modules include a genome reference compendia, a biomarker database, a patient sample data management as well as bioinformatics workflows for processing, analysis and decision support.
WP6 To date, clinical specimens from 87 febrile neutropenic episodes in pediatric patients and 17 in adult patients have been subjected to broad molecular screening of fungal pathogens. The data revealed common occurrence of commensal skin fungi in the samples, which would have escaped detection if assays with a narrower spectrum of coverage had been used. Although the current findings indicate external contamination during sample collection, and may therefore not reflect clinically relevant blood stream infections, they may impact on the pre-analytical processes. The clinical validation of diagnostic methods is ongoing, and will be expedited due to the recent recruitment of three additional clinical centers for adult patients and a very large center for pediatric patients.
WP7 A brand name (FUNGITECT) with logo, tag line, brochure, and website were already created in the previous reporting period. The project has so far produced a sound scientific output (one technical book with 25 chapters on methods and protocols for human fungal pathogen identification and six peer-reviewed articles, including one Nature Medicine). The various participants of FUNGITECT have represented the project or disseminated its results in 12 scientific meetings/conferences and 9 industry and healthcare events.
Potential Impact:
The activities of FUNGITECT are directed towards individualized antimicrobial treatment to ensure timely and targeted antifungal therapy, and are expected to decrease the use of unnecessary or inadequate treatment. This aim will be achieved by improving the current diagnostic approaches. The establishment of a first line of rapid and inexpensive diagnostic tools based on standardized and validated assays including a) panfungal RQ-PCR, b) monoclonal antibody arrays, c) biochips detecting fungal enzymatic activity, and/or d) optimized PCR-based sequencing will permit rapid clinical implementation due to the low-costs and point-of-care applicability. For a second line of advanced fungal diagnostics, FUNGITECT will place particular emphasis on the development and exploitation of NGS-based technologies offering unprecedented diagnostic options for highly effective management of complex infectious diseases such as IFD.
The economic impact of implementing effective fungal diagnostics, as proposed in this project, is expected to be very significant. With the accurate diagnostic procedures introduced by FUNGITECT, the efficiency in the management of invasive fungal diseases will provide an economic landmark. It could represent the beginning in the inversion of the cost escalation inherently related to inadequate diagnostic methods resulting in suboptimal patient management often leading to significant overtreatment. In addition to expected benefits regarding the future economic development of the participating SMEs, the entire field of fungal diagnostics can profit from the new diagnostic possibilities. The FUNGITECT consortium will contribute to achieving durable integration in the European Research Area and to fostering European competitiveness and excellence. A selection of expected results from the FUNGITECT project and their impacts is listed below:

1. The identification of novel fungal biomarkers will expectedly increase the quality and diversity of fungal diagnostics, and will provide valuable diagnostic information.

2. The establishment of novel test systems will facilitate timely diagnosis of IFDs.

3. Precise diagnostic tools for identification of fungal species will provide the basis for targeted therapy, potentially with fewer side effects.

4. Improved diagnostics facilitating improved selection of antifungal agents will help avoiding unnecessary therapy, thus reducing treatment costs.

5. Improved diagnostics will decrease IFD-related morbidity and mortality.

6. The identification of host factors will facilitate assessment risk factors for IFD.

7. Bioinformatics tool and database development will improve clinical research, and will facilitate the development of novel or improved diagnostic assays.

8. Commercial exploitation will allow broad availability of the novel test systems, which will improve the quality of diagnostics at many clinical centers.

9. New technologies developed within the project will provide a basis for exploitation in related clinical research and diagnostic areas (e.g. bacteriology and virology).

10. The knowledge emanating from the project will improve the standing of the participating partners. Moreover, sharing of the expertise among FUNGITECT partners will be invaluable for future projects in related research areas.
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