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 comorbidities 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 though 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
We have gathered a team of multidisciplinary researchers, clinicians, SMEs and a patient organization, each with a unique expertise, technical platform and/or model systems that together provide the means to successfully conduct the multifaceted research proposed and efficiently disseminate/exploit the knowledge obtained. The project builds on eight distinct, yet highly integrated, scientific work packages (WP), schematically illustrated in the figure above in which also major achievements during the first period is shown.
As illustrated, the project is based on material collected from NSTI patients (WP2) as well as experimental model systems (WP1 & 6). The workflow include a comprehensive set of analyses (WP3 and WP5) followed by integration of results in advanced computational platforms (WP4). The results will be translated into novel diagnostic tests (WP7) and improved patient management (WP2 & 8). 5
In WP1 the main focus is to establish an experimental mouse model of NSTIs (Headed by partner 7, now partner 15). The specific aims are: (a) to establish an optimized BXD mouse model for NSTI caused by different pathogens; (b) to map disease modulating quantitative trait loci (QTLs); (c) to identify relevant genes from the mapped QTL(s) and determine host response pathways engaged in pathogenesis and disease outcomes. The main results obtained thus far are establishment of an optimized mouse model of NSTI using a well characterized clinical strain of S. pyogenes bacteria. In addition significant disease confounding factors were identified by determining if variation in age and gender of mice contribute significantly to variations in disease onset and severity. Accordingly, we identified optimal host age range and optimal infection dose range that expose variation in infection severity across the BXD mouse panel, due mainly to variation in genetic context among the various BXD strains. A preliminary list of preliminary disease modulating QTLs was generated for further computational modelling (WP4).
A crucial component to achieve this is access to patient samples linked to clinical data, eg. a NSTI patient cohort and associated biobank (WP2). Accordingly, the primary objective and aim of The Scandinavian study group of NSTIs (partners 2-6) is to establish a prospective study of NSTI patients at the major referral centers in Denmark, Sweden and Norway in order to obtain clinical data together with “state of the art” collected blood and tissue samples. By means of extensive laboratory analysis (WP3, WP5), these patient samples will be linked to detailed clinical information collected in the clinical databases. Overall, the WP2 group has met all of the first years project milestones by securing ethical approvals for the enrolment of patients into the INFECT study; by establishing a fully operational clinical database linking all 5 clinical centers to data entry; by prospective enrolment of patients into the INFECT study; and finally by securing the collection of blood- and tissue samples to the joint biobanks. Standard Operating Procedures for the correct handling of tissue and blood samples have been developed and the quality of the samples taken by the respective teams has been verified with laboratory results (i.e WP5). As of 31st of December 2013, clinical data and biobank samples from 73 NSTI patients are available for analyses in the various INFECT WPs. This is a major advance in INFECT as it not only allows us to achieve the INFECT objectives but also creates the largest and best defined patient cohort of NSTI infections available to date.
The aim of WP3 (headed by partner 8) is to analyze the complex host-pathogen interactions during the acute phase of necrotizing soft tissue infections to identify host as well as pathogen traits associated with disease development. Therefore a comprehensive examination of the virulence repertoire of isolated bacterial pathogens will be combined with transcriptomic, proteomic, and metabolomic analyses of blood and tissue samples from selected NSTI cases. Within the first year, samples of NSTI cases were received and as expected there was great heterogeneity among the isolates causing NSTI. The applied epidemiologic typing verified that Streptococcus pyogenes serotypes M1 and M3 are dominating causes in NSTI. Due to the dominance of S. pyogenes, these were selected for the initial whole genome sequencing and sequence analyses are currently ongoing. To investigate the role of the adaptive immune system within the infection process, a serology screening approach targeting all known streptococcal superantigens will be performed. The methodology has been established and analyses of patient sera can now ensue.
WP4, headed by partner 9, aims at identifying major molecular interaction networks between human host and infectious bacteria in NSTI through (i) integration, analyses, and modeling of data from WP1-3 and WP 5-6; and (ii) model-driven design of experiments to determine mechanistic actions relevant to novel therapeutic strategies (WP6) and to further explore the pathogenesis at local tissue-specific infection sites (WP5). During this first period, the various workflows and 6 procedures for data management, analysis and integration were established (partners 9, 11, 14). A standardized data handling procedure has been developed to ensure that the various types of data of the project will be managed consistently and efficiently to facilitate data analyses, modelling, hypothesis generation, testing and subsequent model and hypothesis refinement. A central data management platform has also been established at the Laboratory of Systems and Synthetic Biology department at Wageningen University in collaboration with LifeGlimmer (partner 11) to store and integrate heterogeneous data, e.g. clinical and experimental, and to provide a user friendly environment for all INFECT partners to pose queries to the integrated database as shown in the small figure above. All different queries are possible and the fact that partners can actively and independently pose questions offers a powerful approach to fully utilize the full potential of INFECT. Ultimately, this work package will assist in identification of biomarker sets for development of novel diagnostics in WP7.
WP5 focuses on analyses of host-microbe interactions at the infected tissue site using NSTI patient tissue biopsies (Partner 1). Specifically WP5 aims to (a) provide unique in vivo data on bacterial and host factors expressed at the site of infection, b) obtain quantitative measurements of bacterial load at the tissue site, and c) provide insight into mechanistic action of different therapies (including HBO and IVIG). Work accomplish during this first period include testing for predefined bacterial and host components hypothesized to be involved in NSTIs. One of the first actions was to establish detailed experimental protocols for analyses of antigens and bacterial load in infected patient tissue (collected in WP2). This was successfully completed and the data indicated that a host factor called HMGB1 might be a valuable biomarker for tissue injury. To further test this, we analyzed biopsies from patients with varying severity of S. pyogenes tissue infections, and the results demonstrated a significant correlation between HMGB1 and severity of tissue infections. Thus, HMGB1 is a potential candidate for the diagnostic test developed in WP7, and will be further explored in this respect. We are currently analyzing a larger panel of tissue biopsies from NSTI patients enrolled in INFECT for HMGB1 as well as other bacterial and host factors that will together with clinical outcome of infection provide a comprehensive insight into the events contributing to tissue injury.
The over all aim of WP6 (partner 1) is to explore how host and bacterial derived components contribute to the tissue pathogenesis in NSTIs. In humans, however, it is difficult to capture early events of host-pathogen interactions important for NSTI progression. Therefore one essential part of this project is to apply tissue-engineering methodologies to reconstitute human skin suitable for infection with bacterial strains isolated from patients with NSTIs and generate data to be used for computational modelling and systems biology analyses. To obtain this, human skin fibroblasts and keratinocytes were cultured under specific conditions promoting the formation of a multi cellular tissue specific organization. Further, the established tissue model was used for infection with live clinical NSTI bacterial isolates, and protocols for infections and sample preparations were standardized. Following these standardized protocols differential bacterial and host response patterns were revealed over time. These results, together with future sample analyses will be integrated in the computational modelling platforms, and subsequently, investigations using the skin tissue model will be performed to explore the role of newly identified host and bacterial 7 components potentially contributing to NSTIs. Using this approach, will also allow evaluation of novel interventions, their efficacy as well as mechanistic actions in NSTIs.
In WP7 (headed by partner 12) the focus is to translate obtained results (WP1-6) into a clinical compliant diagnostics approach by applying multiplex diagnostic technologies in both compact sequencing (pathogen detection - DNA) and compact profiling (disease traits – antigens). The tools will be designed to support therapeutic decisions as for example antibiotics treatment, decisions for surgery or closing wounds, and/or adjunctive therapy. During this first year, specifications for the diagnostics kits has been defined including major technical goals, such as a first set of targets to be analyzed and “environmental factors” of clinical routine. The work to define these specifications consisted of a thorough literature search, discussion and knowhow from analogue diagnostic challenges and above all personal interviews of the clinicians and NSTI experts involved in the project. The first prototypes of two test kits (early pathogen detection – bacteria and fungi, as well as monitoring of inflammatory status of patients) have been made available for evaluation and further improvement in the upcoming years before going into the clinical validation phase. The assay technologies of compact sequencing (DNA) and compact profiling (protein biomarkers) have been further developed.
WP8 (headed by partner 6) focuses on dissemination and exploitation of the knowledge generated in INFECT. Clinical guidelines will be established and disseminated, scientific advances will be published, and information material will be prepared for patients, medical staff and society. Work accomplished so far include the production on an INFECT-Leaflet offering information on the INFECT project and its partners. The Leaflet has been distributed extensively by the INFECT partners in the research field and in partners’ respective counties, in particular by partner 13. To provide updated information to health care workers, researchers, patients, relatives, political decision makers, industry and society in large an external website (http://www.fp7infect.eu/) has been created and was launched as early as January 2013. This external website offers information on the specific objective of the program, i.e. Systems Medicine approach to understanding and handling of severe tissue infections. The page contains sections for news, and information targeted at different target populations; the public, health care workers and researchers. The INFECT external, open access webpage will be dynamically updated by the INFECT consortium as the project develops new insight. The webpage is today an easily accessible, easy-to-use tool for a wide range of interested parties. In addition, there have been several press releases, TV interviews, and presentations of INFECT at various national and international meetings.
WP9 focuses on providing an efficient project management for INFECT and its 14 partners. Main activities the first period has been to establish a functional management structure to ensure efficient communication between the participants in the project, and to ensure full compliance with all legal and EC requirements. To this end, partner 1 has created a user-friendly access restricted internal website where documents and data are uploaded and shared among all partners of INFECT. Furthermore, instruments such as data integration panel reports and templates for reports have been created and distributed among the partners.
In conclusion, the INFECT consortium has established and proved the operational efficacy of an outstanding and unique system for collection of NSTI patients, advanced experimental model systems, and state-of-the-art analyses of samples. The information gathered will significantly contribute to further understanding of complex human and pathogen interaction and future prognostic markers may be demonstrated and current treatment procedures changed, thereby improving survival and subsequent quality of life for those patients who become victims of severe life threatening NSTI.