Report of third project year (2015) - Summary

The overall goal of INFECT is to advance our understanding of the pathophysiological mechanisms, prognosis, and diagnosis of the multifactorial highly lethal necrotizing soft tissue infections (NSTIs). NSTI’s are rapidly spreading infections that may cause extensive soft tissue or limb loss, multiorgan failure and are associated with a considerable fatality rate. It is undisputed that rapid diagnosis and prompt intervention is directly related to survival. The initial presentation may be limited to unspecific symptoms such as tenderness, swelling, erythema and pain. Thus, diagnosis and management are difficult due to heterogeneity in clinical presentation, in co-morbidities and in microbiological aetiology. There is an urgent need for novel diagnostic and therapeutic strategies in order to improve outcome of NSTIs. To achieve this, a comprehensive and integrated knowledge of diagnostic features, causative microbial agent, treatment strategies, and pathogenic mechanisms (host and bacterial disease traits and their underlying interaction network) is required. INFECT is designed to obtain such insights through an integrated systems biology approach in patients and different clinically relevant experimental models.

Specific objectives of INFECT are to:

  • Unravel specific mechanisms underlying diseases responses through a bottom-up systems approach applied to clinically relevant experimental models.
  • Apply a top-down systems biology approach to NSTI patient samples to pin-point key host and pathogen factors involved in the onset and development of infection.
  • Identify and quantify disease signatures and underlying networks that contribute to disease outcome.
  • Exploit identified disease traits for the innovation of optimized diagnostic tools.
  • Translate the advanced knowledge generated into evidence-based guidelines for classification and management, and novel therapeutic strategies

Summary of periodic report for the third project year (2015):

The main objective of WP1 was to utilize BXD mice to identify disease associated genes, and to establish an experimental mouse model of NSTI caused by Streptococcus pyogenes using a panel of transgenic mice expressing human HLA-II alleles that are associated with predisposition or protection from NSTI (partner 1). Results from S. pyogenes infected BXD mice identified the IL-1b network as a susceptibility determinant. Using the HLA-II transgenic mice revealed that infected HLA-II mice expressing DR3 and DR4DQ8 were highly susceptible to large lesions and increased deaths due to NSTI caused by clinical isolate 5448 and to a lesser extent to INFECT clinical isolate 2006. Larger lesions seen in DR3 correlated with persistence of bacteria in skin and systemic dissemination to spleen.

During this reporting period, the main objective of WP2 was to continue clinical data registration and prepare data for export and analysis, patient enrolments and blood, tissue sampling to the INFECT biobank and to prepare draft manuscripts for analysis of clinical data with INFECT partners. Through action of all clinical partners 2-6 106 additional patients were enrolled during 2015 giving a total of 302 enrolled patients into the INFECT cohort. Fruitful interactions between clinical partners and computational partners (partners 9, 11) have generated a strong plan for an array of statistical analyses required to penetrate the comprehensive clinical dataset (illustrated in the figure below). This forms the basis for a number of scientific reports addressing key clinical challenges. Furthermore, partners 1, 7, 8, 10 and 14 have received clinical data information for analysis and subsequent scientific reports.

In WP3, the main objective is to identify specific pathogen and host disease signatures. During this reporting period further confirmed the dominant role of the species Streptococcus pyogenes and specifically of the serotype M1 as main causative of severe NSTIs but also identified members of the species Streptococcus dysgalactiae ssp. equisimilis as causing NSTIs (partner 8). In contrast, members of the Viridans group streptococci were observed predominantly in mixed infections. Studies aimed to identify pathogen traits associated with the outcome of NSTI mainly through the analysis of host-pathogen interactions on the cellular level were conducted for all three major groups of NSTI causing bacterial species, i.e. S. pyogenes, S. dysgalactiae and S. aureus, and factors crucial for tissue pathology could be identified (partners 1, 8-11). Extensive serology screening approach showed that a lack of protective antibodies against streptococcal exotoxins and against the bacteria represents a significant risk factor for the development of a severe NSTI (partner 8). Also the previously established transcriptomic workflow was applied, allowing detailed insights into both the host and the pathogen transcriptional in vivo response (partners 8-11).

In WP4, the data standardization protocols using well defined patient nomenclatures (id, day, type, severity) were further extended and integrated into the semantic data standardization and data management framework (T4.1) (partner 9, 11). This framework is further extended to handle the RNA sequencing data obtained from WP3. This allows for interconnection of expression profiles into the dynamic and metabolic models constructed during T4.3. To test, integrate and investigate streptococcus further 121 publicly available bacterial genomes were integrated to analyse metabolic diversity within this genus (partners 9, 11, 14).

During this reporting period, the main objectives of WP5 were to follow-up on results obtained in WP1-4 and to provide data to gain insight into pathogenic mechanisms as well as guide selection of samples for analyses in WP3 and WP4. Work achieved by partner 1 during the reporting period has included analyses of tissue biopsies from NSTI patients for bacterial and host factors. A large panel of tissue biopsies from NSTI patients infected with the main pathogens, i.e. S. pyogenes, S. aureus or group G streptococcus has been analyzed. The analyses provided novel insight including identification of biofilm in S. pyogenes infected NSTI into bacterial load, and important differences where found with respect to bacterial load and inflammation in biopsies with biofilm or without biofilm. Also important differences between patients infected with different bacteria were noted. The data have been used to phrase working hypothesis that will now be tested in WP4 and validated by further analyses of patient samples (WP3 and WP5) as well as in our experimental models (WP1 and WP6).

The over all aim of WP6 is to establish a clinically relevant, robust and reproducible multicellular system, a so called organotypic model, resembling normal human skin tissue that can be used for experimental infections and test of therapeutic strategies (partner 1). During the 1st and 2nd period, partner 1 focused the efforts on establishing the skin oragnotypic model and standardized protocols for infections of the skin model using INFECT bacterial isolates, including the two major pathogens Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis (SDSE). Withn the 3rd period infections of tissue models were also established with Staphylococcus aureus (partner 1 and 10). In tissue models infected with S. pyogenes analyses of gene and protein expression profiles revealed differential gene expression of both host (structural and inflammatory components) and bacterial (regulatory components and virulence factors), as well as altered host protein expression patterns. In addition, the skin tissue model was used to verify the capability of NSTI bacterial isolates to form biofilm as observed in patients (partner 1-6). Experiments with the INFECT 2006 isolate (S. pyogenes) was also performed to generate samples for dualRNA sequencing analysis (partner 8) from which data will be integrated in the data driven computational modelling platform (partners 9, 11, 14). Over all, the progress of work is well in line with the objectives and tasks of this work package and the work towards investiaging dosage and mechanistic action of therapeutic strategies was initiated. Results from the experiments performed so far were compiled into four separate manuscripts, out of which two were accepted for publication (Disease Models and Mechanisms, 2015 and Scientific Reports, 2015).

In WP7, Partner 16 (Cube Dx GmbH, former partner 12 Anagnostics Bioanalysis GmbH) works closely together with the clinical partners to develop and test diagnostic tools for early pathogen detection (based on compact sequencing) and inflammation and organ monitoring (based on compact profiling). As Anagnostics Bioanalysis GmbH had to file for insolvency in February 2015. Cube Dx has purchased the assets of Anagnostics with effect of early August 2015 and since then started to re-establish operations. Therefore, one of the main task for Cube Dx during its initial phase within the INFECT consortium was to re-establish all processes and SOPs concerning pathogen identification tests and inflammation tests as well. Above all the prototype production facility had to be set-up de novo, as a crucial production machine has been sold during insolvency and had to be repurchased (and set-up again). Partner 16 (former partner 12) has implemented improvements for the three (pathogen ID, resistances, inflammation) test prototypes as well as to the device (hyborg) related to the INFECT project – e.g. minor changes in production, quality assurance, the products (and its components) itself (e.g. revision of device), etc. Basis for those changes are e.g. the technical verifications, beta-tester feedback, general industry trends and other sources. Over all, the progress of work is again underway and is in line with expectations with respect to the objectives and tasks of this work package.

The main objective of WP8 is to disseminate knowledge generated within the INFECT project, directed at medical staff, professionals, patients, relatives, press and society in large (all partners). During the third reporting period main task have been dissemination of information of the INFECT consortium and project, and dissemination of knowledge generated in INFECT. This has been achieved by several means. In the third reporting period we have continued the work with preparing dissemination of clinical guidelines developed on basis of scientific progress made in the entire INFECT study period, and preparing an educational program for all target populations within the project period. By this year a total of more than 110 dissemination activities have been delivered, of which there have been about 40 during period 3. The partners have disseminated advances made through oral presentations nationally and internationally, in scientific communities as well as in health care settings (partner 1, 2, 5, 6, 13), for policy makers and patients and relatives, where the patient organization has been a key player (partner 13). Partner 13 organized the “Necrotising fasciitis – In depth” scientific conference in Blackpool UK attracting a wide range of targets for this work package. Furthermore, there have been five scientific meetings for various combinations of partners in Braunschweig (all partners), Copenhagen (partners 2, 3, 5, 6, 9, 11), Stockholm (basic sciences partners) and in Bergen (partners 1, 6). In this reporting period our external open access website, containing information of scientific advances, has been updated. Also, search strategies on the internet has been expanded and improved, and there have been almost twice the number of visitors to our webpage this year compared to the previous reporting period, including visits from more than 100 countries. The external website is used not only to provide information on the INFECT project, but also delivers updates on new insights obtained in the project. Finally, the study has received attention resulting in presentations in, and the project leader and the INFECT project has been presented by the European Commission Research and Innovation Magazine.

The aim of WP9 is to provide an efficient project management for INFECT and its 14 partners. Main activities during this period focused on ensuring efficient data generation as well as sharing and integration of data between the participants in the project. Beyond this the management included admistrative activities to ensure full compliance with all legal and EC requirements. Furthermore, data integration panel reports were composed and templates for reporting were updated and distributed among the partners. Also, an ethical review of the work conducted during the third period has been conducted by the external advisor. During this reporting period, the scientific contact within the consortium was boosted and several meetings were organised between partners. This included meetings between clinical and data modelling partners (2-6, 9,11), as well as meetings between the experimental/modelling partners (1, 8, 9 and 11). It also included bi-weekly skype conferences with a focus on data generation, standardization, sharing, integration, and interpretation between experimental partners (1, 8, 9, 11, 14 and 15). Overall, the project has reached a stimulating stage where several of the individual WP projects are reaching completion and thereby providing results that stimulate new joint initiatives advancing the research field.