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Optimized Diagnostics for Improved Treatment Stratification in Invasive Fungal Diseases

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

Executive 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. The majority of IFD events are still caused by Candida and Aspergillus species, but there is an increasing incidence of resistant or previously uncommon yeasts and moulds including particularly Zygomycetes. Timely pathogen detection is a prerequisite for effective therapy in patients with IFD.
FUNGITECT has therefore focused on this medical priority and facilitated the development and validation of a specific set of novel molecular diagnostic tests for IFD targeting fungal DNA and protein motifs, as well as the enzymatic activity of fungal pathogens. Additionally, FUNGITECT permitted the establishment and implementation of highly effective diagnostic assays supported by Next-Generation Sequencing and a bioinformatics service platform.
The tests established and validated within this project included different assays based on the polymerase chain reaction (PCR) targeting different gene regions, which can be coupled with the identification of fungal species by sequence analysis of the amplified DNA segments. The tests were designed to facilitate detection of an extremely broad range of fungal microorganisms, thus justifying the designation as panfungal assays. These tests are available for diagnostic testing in the clinical setting. In addition to the optimized PCR-based approaches, a Next Generation Sequencing (NGS) test system has been established and validated. It was demonstrated that unbiased sequencing by this approach, with appropriate control parameters, permits broad detection of pathogens including fungi, bacteria and viruses in clinical samples, providing a sensitivity of detection clearly exceeding the yield of culture-based techniques, which currently represent the standard of diagnostic care. Additional improvement steps are being performed to further reduce the turnaround time and the sequencing costs, in order to render the method broadly available in the clinical setting.
In addition to optimized molecular diagnostic platforms for fungal pathogens, research within the FUNGITECT project also focused on the detection of fungal species by specific monoclonal antibodies (MoAbs) by employing ELISA-based biochip assays. A number of specific MoAbs targeting particularly Candida and Aspergillus species have been generated, several of which were deemed eligible for exploitation in this detection format. Although further optimization steps are still required, the novel Candida and Aspergillus MoAbs provide an excellent starting point for the development of diagnostic tests and for exploitation as a valuable tool for research purposes.
Another complementary diagnostic approach for detection of fungal microorganisms investigated within FUNGITECT included the development of an enzymatic biochip enabling the screening for fungal lytic enzyme activity in patient samples. The test was designed to permit rapid and inexpensive screening for fungal pathogens in clinical specimens, including particularly peripheral blood. The patented biosensor is based on topical degradation of a specific bio-layer by fungal lytic enzymes facilitating sensitive optical read-out. Upon optimizing the composition of the degradable bio-layer, the sensor was demonstrated to facilitate fungus detection in serum samples after an incubation time of 16 hours, and the available prototype has considerable potential for exploitation as a complementary diagnostic tool.
The bioinformatics service platform established within FUNGITECT includes a large repository of microbial sequences facilitating automated analysis of NGS data and permitting rapid identification of the pathogens present in the sample analyzed. The software developed for complete genome sequence comparison also enables mutation profiling which can be used for strain typing based on sequence tag biomarkers. The platform permits comparative genomics and investigation of pathogenicity on the gene, transcript and protein levels, and offers multiple additional functionalities for the users including also meta-information in order to provide an integrated view for decision support in clinical diagnostics. The platform represents an important tool for future application in hospitals and the medical research area.
Project Context and Objectives:
Fungal infections constitute an ever-growing significant medical problem causing enormous costs for healthcare worldwide. Importantly, invasive fungal disease (IFD) is a leading cause of morbidity and mortality in severely immunocompromised individuals, particularly in patients with hematological malignancies, bone-marrow and organ transplant recipients, long-term intensive care unit (ICU) patients, preterm neonates, and patients with inborn or acquired immune deficiencies (AIDS). The vast majority of IFDs is caused by Candida and Aspergillus species. However, changes in the epidemiology have occurred over the last decades, with a number of newly emerging fungal pathogens affecting even immunocompetent patients. Additionally, rare but pronounced antifungal drug resistance of some major fungal pathogens (e.g. C. glabrata, C.krusei A.terreus) poses a serious challenge for treatment. The overall mortality of 35-40% just for candidemias exceeds that of all Gram-negative bacterial septicemias. Thus, IFD and the associated medicare costs, which exceed billions of euros per year in Europe alone, represent an enormous burden to healthcare systems. Moreover, the widespread use of prophylactic or empirical treatment without firm evidence of IFD leads to a high rate of overtreatment associated with considerable toxic side effects, and broad-spectrum antimycotic therapy administered against unidentified fungal pathogens can promote the evolution of clinical drug resistance.
Rapid, reliable and species-specific diagnosis of fungal pathogens causing IFD is a prerequisite for cost-effective and successful therapy, but remains one of the great challenges and an unmet clinical need. The existing diagnostic approaches do not adequately meet the requirements for personalized treatment strategies often required for optimal management of IFD. There is a consensus that the stunningly high mortality of candidemias and other invasive fungal diseases is largely attributable to the present lack of adequately fast and reliable diagnostic tests leading to delayed and untargeted antifungal therapies which may display limited efficacy. Hence, changing epidemiology, the increasing variety of fungal pathogens, and the rising number of affected patients demand for broad-range and cost-effective clinical diagnostic tests, permitting also the prediction of pathogenicity and resistance to antifungal agents. The knowledge of stratified treatment options facilitating timely onset of adequate antifungal therapy are critical for successful clinical management and outcome of IFD. FUNGITECT has therefore focused on this medical priority and facilitated the development and validation of a specific set of novel molecular diagnostic tests for IFD targeting fungal DNA and protein motifs, as well as the enzymatic activity of fungal pathogens. Additionally, FUNGITECT permitted the establishment and implementation of highly effective diagnostic assays supported by Next-Generation Sequencing and a bioinformatics service platform, facilitating optimized treatment strategies adapted to individual patient requirements.

The main objectives of project FUNGITECT were:

1. Establishment of Standard Operating Procedures (SOPs) for patient sample retrieval and biomarker discovery.

This comprised 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 included 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 has been adapted and tested 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 was 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 was intended to be exploited as a biomarker for the diagnosis of IFD, but the task has been abandoned based on the evaluation of intermediate results. (WP3).

4. Optimization and validation of molecular targets.

Prior to clinical implementation, molecular targets identified in work packages 2 and 3 had to 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 was 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 platform will 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 was developed 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) encompassed 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 was planned to include longitudinal monitoring of ~200 febrile neutropenic periods in high-risk patients, and evaluation of novel diagnostic PCR-based 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 will secure the resulting IP, if pertinent, and launch the novel diagnostic services. (WP7).

A detailed description of individual objectives is provided in the following section.
Project Results:
Main scientific and technological results achieved per objective in FUNGITECT:

Objective 1. Establishment of Standard Operating Procedures (SOPs) for patient sample retrieval and biomarker discovery
The type of sample specimens for molecular analyses was selected, and blood sampling was performed according to established protocols. Adequate transportation of samples has been implemented, and strategies minimizing the risk of fungal contaminations were established. Moreover, stabilization of clinical samples for further analyses has been secured. Standard operating procedures (SOPs) were developed for safe handling of clinical materials with regard to storage, isolation of DNA and analysis by PCR assays targeting the internally transcribed spacer region (ITS-PCR), the 16S and 18S rRNA genes (rDNA-PCR), and unbiased amplification of microbial sequences and analysis by Next Generation Sequencing (NGS). The corresponding SOPs and protocols have been harmonized, and contamination-free DNA extraction from clinical samples has been implemented.
Special attention was given to the automated DNA extraction in a contained laboratory environment in order to prevent contamination. This was achieved by using a robotic system that was developed by the consortium partner Molzym in cooperation with another company. The automated device, SelectNA™plus, was optimized to prevent microbial DNA contaminations by aerosols, pumping solution and foaming during vacuum filtration. This was achieved by adapting the script of the robot (pumping, UV irradiation of the interior surfaces) and re-design of plastic consumables. The SOPs for loading of samples and maintenance of the robot were established, and SOPs for all molecular assays were set up successfully.


Objective 2. Development of monoclonal antibodies (MoAbs) and enzymatic tests for low-cost point-of-care diagnostics of IFD
In this assay developed by the consortium partner MUV, the detection of fungal pathogens at the protein level is facilitated by specific monoclonal antibodies. The assays for detection of a wide range of fungal pathogens, permitting both broad pathogen detection by using cross-reacting antibodies and specific pathogen detection by using antibodies identifying a specific fungal microorganism, were successfully developed using artificially spiked samples. However, testing of patient samples was not yet successful, because the sensitivity of antigen detection was too low, possibly because the patient sera available for analysis came from unknown disease stages with insufficient levels of fungal antigens.
Hence, further methodological improvements are required, before the biochip could be considered for clinical applications. For this purpose, additional verified sera from patients with fully confirmed invasive fungal disease will also need to be obtained for validation of the tests, before producing pilot macrochips suitable for diagnostic testing. Nevertheless, the availability of 11 anti-Candida and 3 anti-Aspergillus monoclonal antibodies, which were generated during FUNGITECT and were deemed eligible for a chip-based assay, is a promising starting point for further development of the test system, which may also provide a very useful tool for research applications.
The enzymatic assay, developed and performed by the consortium partner Tuzla, detects the presence of biologically active fungal proteases using a patented biosensor platform. The biochip was developed to permit rapid and inexpensive screening for broad detection of fungal pathogens in clinical specimens, including particularly peripheral blood, for the identification of invasive fungal diseases (IFD).The biochip includes an optimized polymer matrix placed on top of a reflective metal layer. In the presence of fungal proteins in the sample tested, the polymeric bio-layer is degraded by lytic enzymes secreted by fungi, if present in the peripheral blood sample analyzed. Topical degradation of the matrix leads to a color change, which can be readily seen by visual inspection, without the need for optical instruments. The testing of clinical specimens revealed that fresh samples provide readily visible results, while analysis of samples after long-term storage at freezing temperatures may compromise the analysis. Samples tested after short-term storage were more commonly positive in the biochip assay, and the results correlated with other tests targeting fungal DNA in a high proportion of instances. These observations indicated that false-negative results might frequently occur in samples stored for extended time periods prior to analysis, and provided support for the use of fresh clinical samples to achieve optimal results. Nevertheless, careful assessment of the specificity and comparison with other technical approaches is still required, before clinical implementation of the biosensor can be considered.

Objective 3. Identification of molecular markers (DNA&RNA) for patient stratification and pathogenicity prediction of fungal species
The complete workflow for analyzing DNA from patient samples, with a focus on circulating cell-free DNA (cfDNA) isolated from blood plasma, has been successfully established by the consortium partner Fraunhofer and evaluated using primary patient samples. RNA-based pathogen identification and RNA-based patient stratification suffered from very low yields of RNA, possibly as a result of the sample composition in this particular patient cohort, and was therefore discontinued for technical reasons, in light of the more promising DNA-based data for pathogen identification.
A next generation sequencing (NGS)-based approach referred to as Whole Genome Shotgun Sequencing (WGS) or unbiased sequencing was performed using cfDNA isolated from peripheral blood, and the sequence analysis was designed to detect fungal, bacterial and viral pathogens. The entire workflow was successfully applied to clinical isolates from patients with hematooncologic disorders presenting with febrile neutropenia as an indication of strongly impaired immune response and an ongoing infection. In a high proportion of instances, clinically relevant bacterial, fungal and viral species could be identified, with good correlation to clinical microbiology results, where available. Furthermore, positive results were obtained by NGS analysis in instances in which blood cultures were negative, indicating a higher sensitivity of the sequencing approach. Additionally, a workflow for antimicrobial resistance prediction was established, but due to the limited number of fungus-positive specimens and inadequate genome coverage, no resistance testing could be performed within the current study. Although positive identifications of fungi were limited, the general workflow could be confirmed by NGS-based detection using primary patient samples. These findings indicate that NGS-based species identification in patient samples works successfully and robustly not only for bacteria and viruses but also for fungi, and provides a promising technology for future clinical application.

Objective 4. Optimization and validation of molecular targets.
The rDNA PCR assay established by the consortium partner Molzym facilitates the detection of fungal and bacterial pathogens by amplification of target sequences within the 18S and 16S rRNA genes, respectively. The starting material for this test was cell-bound DNA isolated from whole blood samples using the proprietary MolYsis system. The assay was used for specific amplification of fungal and bacterial DNA, and the products were subjected to Sanger sequencing facilitating the identification of the microorganisms detected. Molzym's solutions including manual (centrifugation-based), semi-automated (magnetic beads-based) and fully automated (vacuum-based) extraction of fungal and bacterial DNA permitted successful identification of pathogens at the species, genus or family level by all extraction methods. Based on encouraging results that emanated from initial testing, the rDNA PCR approach was compared and validated against other molecular approaches to fungus detection and species identification based on DNA analysis. These included the ITS2-PCR and the unbiased NGS assay, which were tested concomitantly using a series of clinical samples, while blood cultures served as a reference.
ITS2-PCR revealed the highest rates of potential fungal infections. For instance, in one series, 18.8% of episodes tested positive for fungal DNA sequences by ITS2-PCR, as compared to 9.1% by 18S-PCR and 9.4% by NGS, respectively. The results indicated occasional, successive or mixed infections of different fungi, fungi with bacteria and/or viruses, and thus uncovered a complex structure of transient or long-term microbial colonization of the neutropenic patients studied. The diversity of fungal strains regarded as potentially relevant was high, and again, the highest diversity with regard to discriminated species was observed with ITS2-PCR (12 species), while 18S-PCR and NGS identified 4 species, respectively, with a low level of overlap between the different methods. Hence, the methods contributed additively to the detection and identification of fungi rather than concordantly indicating fungemias in culture-negative episodes. As a general conclusion, all three molecular assays contributed to the finding of a diverse and abundant population of potentially relevant fungi in the febrile neutropenic episodes studied.

Objective 5. Development of a CDMS based diagnostic software platform
A Central Data Management System (CDMS) for the validation of data along with a database for comprehensive collection of patient data was developed and optimized in collaboration of the entire consortium with the consortium partner Genedata. The Patient Sample Management module used as a web-based solution by the consortium to register patient data, track data and sample information as well as the status, has been transferred into the most recent version of the CDMS.
The integration enabled and provided seamless collaboration and communication between the project members and results could be shared and stored in the according projects. Anonymized patient data as well as data collected from individual analyses were managed by a professional clinical research assistant (CRA), and were fed into the patient database developed by Genedata, in order to serve all partners as a basis for comprehensive evaluation of their results in relation to the clinical data available. Genedata also developed a broad database of genome sequences from fungal and other species, including about 200 public and proprietary fungal genomes permitting analysis of genome architecture and sequence homology, but also facilitating assessment at the gene, transcript, protein and pathway level as well as the mutational level for strains. This tool was employed for the analysis of sequencing data to facilitate rapid identification of microorganisms present in the sample analyzed. The sequence-focused approach was extended by the FUNGITECT patient sample database and its meta-information in order to provide an integrated view for decision support in diagnostics. The integrated communication and collaboration functionality by storing results, views and sharing these with the CDMS is essential for point-of-care decision support required for stratified treatment. Importantly, the prototyping of end-user analysis software has been successfully accomplished and a major transition step for future application in hospitals and the medical research area has been made.

Objective 6. Clinical validation of diagnostic methods
The methods developed within the preceding work packages were implemented within two clinical studies, using adult and pediatric samples, respectively. In each study, samples were collected in two patient centers, ensuring independent sources for the study. Methods developed within the project were primarily focused on the detection of fungal infections. However, fungal pathogens share their respective niches with other microorganisms, and their interactions can influence the response to treatment and disease outcome. To address this important aspect, screening for fungal, bacterial and specific viral pathogens was performed and, as an additional effort, interactions between selected microorganisms were analyzed by appropriate tools. The diagnostic assays employed were designed to address various aspects and components of the fungal pathogens including DNA (free and cell-bound), polysaccharides, proteins, and their enzymatic activity (fungal proteases).
Several technical approaches were systematically tested and validated by exploiting clinical samples collected within the present project:
1) Panfungal RQ-PCR assay
This two-reaction assay based on the amplification of highly conserved sequences within the 28S region was developed and patented (patent number 1960536) by P1 CCRI. During the process of development, the panfungal PCR assay showed an exceptionally high sensitivity, indicating its suitability as a primary diagnostic screening technique, followed by secondary techniques providing more specific information (for example the semi-nested ITS2-PCR, see below). Cell-bound DNA isolated from peripheral blood was used as starting material. The panfungal RQ-PCR assay was designed to only permit discrimination between yeast and mould fungal species.
This assay and several other methods listed below were tested by using close to one thousand clinical samples from immunocompromised pediatric and adult patients during febrile neutropenic episodes. The proportion of specimens revealing positive test results for fungal DNA sequences was in the range of 25 % (including 12.7 % for mould sequences, 10.6 % for yeast sequences, 3 % testing positive for both). The fact that none of the patients included in the study suffered from a clinically proven fungal disease prevented conclusions regarding the assay performance. Detection of fungal DNA in a considerable proportion of samples indicated a high sensitivity, but limited specificity of the method. Although in PCR-based tests for fungal sequences, the risk of exogenous contamination contributing to the positive results cannot be ruled out, the systematic employment of multiple negative controls and the low number of positive results may argue against this possibility. More likely, the observations could be attributable to the detection of low- or non-pathogenic fungal species and/or to the detection of fungal DNA derived from inactive fungal pathogens.
2) Semi-nested ITS2-PCR assay followed by Sanger sequencing
This assay was established by P1 CCRI as a complementary diagnostic assay to the panfungal PCR. The PCR amplification targets ITS regions of the fungal genome, in which variable sequences, highly specific for individual fungal species, are flanked by conserved regions. The ITS2 PCR assay is designed as a two-step reaction. In case of a positive finding by electrophoretic analysis, the final PCR product is sequenced in order to identify the fungal pathogen(s). Similar to the panfungal RQ-PCR assay, cell-bound DNA isolated from peripheral blood samples is used as starting material. As indicated for the panfungal PCR assay, close to one thousand specimens were analyzed by this assay, and 14.9 % tested positive for fungal DNA. In about half of the cases, the samples also showed positive results by panfungal PCR, indicating only a partial overlap of concordant findings. Positive specimens were subjected to Sanger sequence analysis and revealed several environmental fungal species in addition to fungal pathogens typically observed in this setting. As none of the patients included in the study suffered from a clinically proven fungal disease, the DNA detected might reflect the presence of low- or non-pathogenic species, as suggested by the detection of environmental (e.g. plant-associated) fungal DNA. As outlined above, the positive and negative predictive values of the ITS2 seminested PCR could not be determined. However, the assay coupled with Sanger- or NGS-based sequencing has potential for becoming a useful diagnostic tool, because it permits sequence-based identification of the fungal microorganism(s) involved in the infection.

3) rDNA PCR assay
In this assay, introduced by the consortium partner Molzym, targeting the 18S rDNA fungal sequences, identification of the microorganisms amplified from whole blood specimens is performed by Sanger sequencing, as described above. A somewhat smaller series of clinical specimens available from adult patients (n=234) was tested by this method, and was compared to other molecular approaches facilitating fungal identification at the species level, including ITS2-PCR and unbiased NGS analysis, as outlined under Objective 4. The proportion of positive test results by the rDNA assay was similar to NGS analysis (9.1% and 9.4%, respectively), but lower than the yield by the ITS2-PCR assay (18.8%). Apart from these differences, the overlap of the fungal species identified by sequencing was very small between different assays. This observation highlights the great influence of sample processing on preferential detection of microbial species in the clinical specimens tested. Based on the clinical studies performed, it appears unlikely that PCR-based testing of peripheral blood samples will become a dominant tool for fungal screening in patients at risk of invasive fungal infection. Nevertheless, in comparison to culture-based fungal diagnosis, molecular detection methods offer greater speed and considerably higher yields of detectable fungal microorganisms. In contrast to the critical assessment of PCR-based fungal screening in peripheral blood specimens emanating from results of the present study, real-time quantitative (RQ)-PCR is a well-established screening approach for the diagnosis of viral infections.

4) Shotgun NGS-based sequencing
Unbiased NGS sequencing was performed by the consortium partner Fraunhofer using cell-free DNA isolated from peripheral blood, as described under Objective 3. NGS-based sequencing was designed to detect fungal, bacterial and viral pathogens. The microorganisms detected were primarily bacteria (20 unique species), followed by viruses (9 unique species) and fungi (4 unique species). Although positive identifications of fungi were limited, the general workflow could be confirmed by NGS-based detection using primary patient samples. These findings indicate that NGS-based species identification in patient samples works successfully and robustly not only for bacteria and viruses but also for fungi, and provides a promising technology for future clinical application.
The testing of clinical specimens by the molecular approaches employed in the present study, including different PCR-based methods and sequencing analysis, revealed an additive contribution to the detection and identification of fungi rather than concordant indication of fungemia by specific species. As a general conclusion, all tested molecular assays contributed to the finding of a diverse and abundant population of potentially relevant fungi in the neutropenic episodes studied in specimens that tested negative by blood culture-based techniques.

5) Biosensor-based screening of fungal lytic enzyme activity
This assay, developed and performed by the consortium partner Tuzla, detects the presence of biologically active fungal proteases using a biochip, as outlined above under Objective 2. The testing of more than 200 clinical specimens revealed that fresh samples provide superior results, while analysis of liquid samples after long-term storage strongly affects the analysis. Blood specimens from patients in febrile neutropenia tested by the biochip revealed positive signals in 5.7% of samples, including particularly those which had been recently collected, with a considerably higher positivity rate in the most recent samples. The data in relatively fresh clinical samples look promising, but more extensive evaluation is still required to determine the potential of this approach for future diagnostic application.

6) Antibody-based detection of fungal species
This assay, developed by the consortium partner MUV, has been described above under Objective 2. Clinical validation of this test system could not be completed due to the limitations in sensitivity of antigen detection, requiring further optimization before the test can be subjected to evaluation using clinical specimens.

7) Serological testing and screening for viremia
Screening for fungal membrane-associated polysaccharides (Galactomannan and Beta-D-Glucan), a well-established approach representing the current standard of routine monitoring for invasive fungal infections, was performed in serum samples of all patients included in the studies performed. The correlation between serological testing and the molecular assays employed was determined. None of the samples tested positive for Galactomannan and 5.7 % of samples tested positive for Beta-D-Glucan. Only a subset of fungal species contains Beta-D-glucan in their cell wall. Nevertheless, fungal DNA was found in 15 – 20 % of samples by the other assays employed, and only about one third of the glucan-positive samples tested positive for fungal DNA, indicating a poor correlation between serological testing and the PCR-based assays. The discrepant results could be, at least in part, attributable to the fact that the DNA detected often resulted from environmental (e.g. plant-associated) fungi. These environmental fungi may not shed glucan targeted by the serological assays, and conceivably do not cause disease in humans, which is in agreement with the observation that none of the patients included in the study suffered from a clinically proven fungal disease.
The employment of RQ-PCR tests for detection of select viruses in peripheral blood in nearly one thousand clinical specimens that were also screened for fungal DNA sequences, revealed positivity for viremia in 11.4 % of the specimens analyzed, the most common viruses being HHV6 in pediatric and EBV in adult specimens. In about 20 % of cases, viral DNA was detected in association with fungal DNA, possibly arguing against a strong correlation between the two infections. However, a larger cohort and more detailed analysis would be needed to permit conclusions on possible correlations between the co-appearance of particular species. Although it is conceivable that febrile episodes in neutropenic patients might be, at least in some instances, caused by viruses, patients are not routinely screened for invasive viral infections. Moreover, mixed infections could contribute to the severity of the clinical course. The relatively common detection of viremia in the present study might raise the awareness of this issue and stimulate the implementation of viral screening in this high-risk patient population, in order to further assess the role of invasive viral infections in this setting.
Due to the unexpected absence of clinically proven fungal diseases in the patient cohorts investigated, it was not possible to determine the positive and negative predictive values of the assays employed and, consequently, their potential for implementation in routine diagnostics. Apparently, the broad antifungal prophylaxis provided at the clinical centers involved was highly effective, without any occurrence of clinically relevant breakthrough fungal infections. Possible interpretations of fungus-positive results by different molecular assays were addressed above. While the overwhelmingly successful antifungal prophylaxis in the cohorts studied certainly has to been seen as a great clinical achievement, it has precluded comprehensive clinical evaluation of the fungus detection assays established or developed for the present project.
Perhaps most importantly, however, the data might indicate that the detection of fungal DNAemia does not necessarily reflect the presence of live fungal pathogens, thus limiting the diagnostic value of PCR-based fungal screening in peripheral blood. Nevertheless, the methods tested may provide useful information if combined with other detection assays, and unbiased NGS-assisted sequencing could become the method of choice for comprehensive microbial screening in the clinical setting.

Objective 7. Commercial exploitation and dissemination
The Deliverables and Milestones pertaining to this Objective are manifested by the brand name (FUNGITECT) with logo, tag line and brochure that was created at the start of the project. They are presented on the website, and are used for dissemination and exploitation of the project results. The website (www.FUNGITECT.eu) supports dissemination and exploitation.
The PCR-based assays for broad fungus detection and, in part, species identification, established by the consortium partners CCRI and Molzym, including panfungal RQ-PCR, ITS2-PCR and rDNA-PCR, are ready for exploitation in the diagnostic setting. The unbiased NGS-based approach, developed by the consortium partner Fraunhofer, has been validated using clinical specimens, and is ready to enter clinical studies needed to corroborate the ability of the assay to serve as a basis for therapeutic patient management. The macrochip-based assay for identification of fungal pathogens by monoclonal antibodies, developed by the consortium partner MUV, requires further improvement of the detection limit, before its employment in clinical testing may be considered. The biosensor designed to facilitate the detection of fungi in liquid specimens by fungus-mediated enzymatic degradation of a specifically designed and optimized biolayer on a chip, which was developed by the consortium partner Tuzla, showed very promising results, but more extensive testing in comparison to other established methods is still required before the platform might be deemed eligible for clinical diagnostics. Finally, the Central Data Management System (CDMS), developed by the consortium partner Genedata, has been successfully tested within FUNGITECT, and is currently being prepared for the transition to clinical application in hospitals and in medical research.
Dissemination was pursued via publications in peer- reviewed journals, presentations at conferences (e.g. oral communications, posters, etc.), and exhibitions in trade fairs.
Potential Impact:
Potential project impact:

The activities of FUNGITECT were directed towards individualized antimicrobial treatment to ensure timely and targeted antifungal therapy, in order to decrease the use of unnecessary or inadequate treatment by improving current diagnostic approaches. The establishment of a first line of rapid and inexpensive diagnostic tools based on standardized and validated assays including panfungal RQ-PCR, ITS2-PCR, and rDNA-PCR was successfully achieved. The clinical studies performed indicated that these molecular assays offering extremely broad detection of fungal species may not be ideally suited for screening in peripheral blood specimens, because fungal DNAemia caused by fungal species presumably non-pathogenic in humans, such as plant-associated microorganisms, is rather commonly detected by these assays. It is necessary, therefore, to combine these highly sensitive molecular assays with specific identification of the fungal sequences detected, e.g. by Sanger or NGS-based sequencing, to permit judicious assessment of the expected pathogenicity and relevance in the immunocompromised patient setting. With this prerequisite in mind, the validated assays indicated are available for clinical employment. The monoclonal antibody arrays and biochips detecting fungal enzymatic activity are promising tools, but require further improvement and testing to assess their full potential for clinical exploitation.
Development of the unbiased NGS-based sequencing technology within FUNGITECT offers unprecedented diagnostic options for highly effective management of complex infectious diseases such as sepsis caused by various pathogens, because the platform permits simultaneous screening for fungal, bacterial, and viral pathogens, which may be implicated in in febrile infections in immunosuppressed patients. The preparation for implementing unbiased NGS analysis in the clinical setting is currently ongoing, and will undoubtedly have a major impact on future diagnostic approaches in a wide area of medical applications.
The Central Data Management System (CDMS) developed within FINGITECT is an advanced bioinformatics tool providing complex meta-information to permit an integrated view for decision support in diagnostics, offering enormous potential for the point-of-care decision support required for stratified treatment.
The efficiency in the management of invasive fungal diseases will expectedly 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 continue contributing to the achievement of durable integration in the European Research Area and to fostering European competitiveness and excellence. A selection of results from the FUNGITECT project and their expected 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 invasive fungal diseases.

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

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

8. The knowledge that has emanated from the project will improve the standing of the participating partners, and sharing of the expertise among FUNGITECT partners will be invaluable for future projects in related research areas.

Some of the novel technical approaches developed within FUNGITECT require further optimization prior to clinical exploitation, and more funds and time will be needed to further develop these technologies. Due to the steadily decreasing costs for advanced sequencing approaches, such as those based on different next generation platforms, it may be expected that molecular techniques relying on this technology will greatly advance the diagnostic field in the near future. The ability to detect and identify any type of pathogen in the clinical samples analyzed, including bacteria, fungi, and viruses, will facilitate comprehensive diagnostic work-up of septic infections, thereby providing the basis for optimized treatment strategies leading to improved outcome of invasive infectious diseases.

Main dissemination activities and the exploitation of results:

FUNGITECT participants have presented project results at international events focusing on the functionality and advantages of the newly developed fungal diagnostic tools. This includes advances in sample preparation, disseminated by P6 Molzym, advances in Bioinformatics tools by P5 Genedata, development of Next Generation Diagnostics processes by P3 Fraunhofer, and the biology of pathogenic fungi and diagnostics by P1 CCRI and P2 MUW.

Dissemination was performed by talks and poster presentations on scientific meetings and trade fairs, and presenting with further research consortia active in combatting fungal disease:

• Austrian Microbiome Initiative (AMICI) Meeting
• Austrian Association of Molecular Life Sciences and Biotechnology (ÖGMBT) Meeting
• 14th ASM Conference on Candida and Candidiasis
• Trends in Medical Mycology conference
• Stockholm University, Department of Molecular Biosciences Lab of Per Ljungdahl – Training event
• 7th FEBS Advanced Lecture Course on Human Fungal Pathogens
• 2nd Midwinter Conference, Advances in Immunobiology
• 7th FEBS Advanced Lecture Course on Human Fungal Pathogens Sepsis Update
• CNAPS X
• 28th ECCMID
• ICCMg3,
• 27th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID)
• CNAPS XI
• International Conference on Medical and Biological Engineering (CMBEBIH 2017)
• NGS field 5th conference in Sendai, Japan
• Bioprocessing Summit
• PDA Virus Forum in Florence
• Boehringer Ingelheim NGS workshop
• Biogen NGS workshop
• BioMerieux Meeting
• Shionogi Pharma – Anti-infective development and patient isolates
• Chugai –Biosafety Meeting
• Astellas – Biosafety Meeting
• MSD – Biosafety Presentation
• Roche – NGS and Biosafety Presentation
• Lonza – NGS and Biosafety Presentation
• 70th AACC Annual Scientific Meeting
• 28th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID)
• 36. Österreichische Gesellschaft für Hygiene, Mikrobiologie und Präventivmedizin (ÖGHMP)
• Infectious Diseases Diagnostics Conference
• 70. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM)
• 18. Klinisch-Mikrobiologisch Infektiologisches Symposium (KMIS)
• 10. European Meeting on Molecular Diagnostics (EMMD)
• ASM Microbe
• 6. Regensburger Meeting für angewandte Molekulare Diagnostik (REMMDI)
• 11. Österreichischer Infektionskongress (ÖIK)


Apart from the events listed above, FUNGITECT has performed very activate dissemination in the form of publication of results in peer-reviewed journals and books. The following publications have acknowledged FUNGITECT and funding from the European Union:

Book:

Lion, Thomas. Human Fungal Pathogen Identification: Methods and Protocols. New York, NY: Springer New York, 2017. Print.

Entire book, edited by Prof Thomas Lion, coordinator of FUNGITECT (P1 CCRI) has acknowledged the project and respective EU funding, and in particular the research published in chapters 6, 13, 14 and 20.

Articles:

Wirnsberger, Gerald, Florian Zwolanek, Tomoko Asaoka, Ivona Kozieradzki, Luigi Tortola, Reiner A. Wimmer, Anoop Kavirayani, Friedrich Fresser, Gottfried Baier, Wallace Y. Langdon, Fumiyo Ikeda, Karl Kuchler, and Josef M. Penninger. "Inhibition of CBLB protects from lethal Candida albicans sepsis." Nat Med. 2016 Aug;22(8):915-23. doi: 10.1038/nm.4134

Kuchler, Karl, Sabrina Jenull, Raju Shivarathri, and Neeraj Chauhan. "Fungal KATs/KDACs: A New Highway to Better Antifungal Drugs?" PLoS Pathog. 2016 Nov 10;12(11):e1005938.
doi: 10.1371/journal.ppat.1005938

Agustinho, Daniel Paiva, Marco Antônio De Oliveira, Aldo Henrique Tavares, Lorena Derengowski, Valentina Stolz, Fernanda Guilhelmelli, Márcia Renata Mortari, Karl Kuchler, and Ildinete Silva-Pereira. "Dectin-1 is required for miR155 upregulation in murine macrophages in response to Candida albicans." Virulence. 2017 Jan 2;8(1):41-52. doi: 10.1080/21505594.2016.1200215

Czurda, S., S. Smelik, S. Preuner-Stix, F. Nogueira, and T. Lion. "Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination." J Clin Microbiol. 2016 Jan;54(1):148-52. doi: 10.1128/JCM.02112-15

Istel, Fabian, Tobias SchwarzmüLler, Michael Tscherner, and Karl Kuchler. "Genetic Transformation of Candida glabrata by Electroporation." Bio Protoc. 2015 Jul 20;5(14)

Istel, Fabian, Tobias SchwarzmüLler, Michael Tscherner, and Karl Kuchler. "Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata." Bio Protoc. 2015 Jul 20;5(14)

Nogueira MF, Pereira L, Jenull S, Kuchler K, Lion T. Klebsiella pneumoniae prevents spore germination and hyphal development of Aspergillus species. Sci Rep. 2019 Jan 18;9(1):218. doi: 10.1038/s41598-018-36524-8.
Czurda S, Lion T. Broad-Spectrum Molecular Detection of Fungal Nucleic Acids by PCR-Based Amplification Techniques. Methods Mol Biol. 2017; 1508:257-266. DOI: 10.1007/978-1-4939-6515-1_14

Czurda S, Lion T. Prerequisites for Control of Contamination in Fungal Diagnosis. Methods Mol Biol. 2017; 1508:249-255. DOI: 10.1007/978-1-4939-6515-1_13

Nogueira F., Istel F., Pereira L., Tscherner M., Kuchler K. Immunological Identification of Fungal Species. In: Lion T. (eds) Human Fungal Pathogen Identification. Methods in Molecular Biology, vol 1508. (2017) Humana Press, New York, NY. doi: 10.1007/978-1-4939-6515-1_20

Czurda S, Smelik S, Preuner-Stix S, Nogueira F, Lion T. Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination. J Clin Microbiol. 2016 Jan; 54(1):148-52. doi: 10.1128/JCM.02112-15.

Schrevens S, Van Zeebroeck G, Riedelberger M, Tournu H, Kuchler K & Van Dijck P (2018). Methionine is required for cAMP-PKA mediated morphogenesis and virulence of Candida albicans. Mol Microbiol. 2018 Aug;109(3):415-416. doi: 10.1111/mmi.14065. Epub 2018 Jul 24.

Agustinho D, de Oliveira AM, Tavares AH, Derengowski L, Stolz V, Guilhelmelli F, Mortari MR, Kuchler K & Silva-Pereira I. Dectin-1 is required for miR155 upregulation in murine macrophages in response to Candida albicans. Virulence. (2017); 8(1): 41–52. doi:10.1080/21505594.2016.1200215

Grumaz C, Kirsthaler P, Sohn K. The Molecular Blueprint of a Fungus by Next-Generation Sequencing (NGS). Methods Mol Biol. 2017; 1508. doi: 10.1007/978-1-4939-6515-1_21.

Decker SO, Sigl A, Grumaz C, Stevens S, Vainshtein Y, Zimmermann S, Weigand MA, Hofer S, Sohn K, Brenner T. Immune-Response Patterns and Next Generation Sequencing Diagnostics for the Detection of Mycoses in Patients with Septic Shock – Results of a Combined Clinical and Experimental Investigation. Int. J. Mol. Sci. 2017 18(8), 1796. Doi: 10.3390/ijms18081796

Decker SO, Krüger A, Wilk H, Grumaz S, Vainshtein Y, Schmitt FCF, Uhle F, Bruckner F, Zimmermann S, Mehrabi A, Mieth M, Weiss KH, Weigand MA, Hofer S, Sohn K, Brenner T. New approaches for the detection of invasive fungal diseases in patients following liver transplantation—results of an observational clinical pilot study. Langenbecks Arch Surg (2019). doi:10.1007/s00423-019-01769-y

Ibrišimović M, Ibrišimović Mehmedinović N, Dedić J, Kesić A, Marić S, Šestan A. Effects of various metal and drug agents on excretion of enzyme aspartyl proteinase in Candida albicans and its role in human physiological processes. CMBEBIH 2017, IFMBE Proceedings of the International Conference on Medical and Biological Engineering 2017, Volume 62, 2017, Springer Singapore, DOI10.1007/978-981-10-4166-2_110 ISBN: 978-981-10-4165-5

Ibrišimović Mehmedinović N, Ibrišimović M, Kesić A, Marić S. Perspective potential of polymer-based biosensor chips in food industry and clinical diagnostics chapter 10. in book Biodegradable polymers: recent developments and new perspectives, IAPC Publishing, 2017 May, DOI 10.5599/obp.14.9 ISBN: 978-953-56942-5-0

Lorenz MG, Lustig M, Linow M Fungal-grade reagents and materials for molecular analysis. In: Lion T (ed.), Human Fungal Pathogen Identification: Methods and Protocols, Methods in Molecular Biology (2017), vol. 1508, p. 141-150. DOI 10.1007/978-1-4939-6515-1_4 © Springer Science+Business Media New York
List of Websites:
Coordinator:
Thomas Lion
The Children's Cancer Research Institute
St. Anna Kinderkrebsforschung
Zimmermannplatz 8
1090 Vienna, Austria
T +43 1 40077 4890
Email: thomas.lion@ccri.at

Project manager:
Nuno Andrade
The Children's Cancer Research Institute
St. Anna Kinderkrebsforschung
Zimmermannplatz 10
1090 Wien, Austria
T +43 1 40470 4441
Email: nuno.andrade@ccri.at

Beneficiaries:
Karl Kuchler
Medical University of Vienna
Austria
Email: karl.kuchler@meduniwien.ac.at

Steffen Rupp
Kai Sohn
The Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB
Email: steffen.rupp@igb.fraunhofer.de
Email: kai.sohn@igb.fraunhofer.de

Nadira Ibrišimović
University of Tuzla
Bosnia and Herzegovina
Email: nadira.ibrisimovic@untz.ba

Thomas Hartsch
GENEDATA AG
Switzerland
Email: Thomas.Hartsch@genedata.com

Michael Lorenz
Molzym GmbH & Co. KG
Germany
Email: lorenz@molzym.com