EURESFUN - Integrated post-genomic approaches for the understanding, detection and prevention of antifungal drug resistance in fungal pathogens

EC contribution: : € 2.755.000
Duration: : 36 months
Starting date: : 01/11/2005
Funding scheme: : Specific Targeted Research Project
Keywords: : fungal pathogens, antifungal drug, antifungal resistance, diagnostic
Contract/Grant agreement number: : LSHM-CT-2005-518199
Project web-site: : http://www.chuv.ch/imul/euresfun

Background:

The EURESFUN (EUropean RESistance FUNgal) network will utilize genomics-based integrated approaches to study antifungal resistance in relevant fungal pathogens (Candida, Aspergillus). This network will study distinct resistance mechanisms (target mutations, drug efflux, signal transduction, transcriptional activation, cell wall alterations, biofilm formation) all of which can contribute individually or in combination to antifungal resistance in clinical isolates. Using microarray strategies, systematic deletion/over-expression approaches, the network will also elucidate new resistance mechanisms. This approach will unravel potential novel targets for antifungal drug discovery, but also yield diagnostic tools and mutations suitable for the use in resistance monitoring and surveillance.

Problem:

According to the WHO data, infectious diseases represent the most common cause of death in the world (WHO). Among infectious diseases, fungal infections also take a prominent position, ranking for instance number 4 in infections of hospitalized patients acquiring microbial infections. The frequency of fungal infections has been steadily increasing in the human population worldwide over the past decades. Several fungal pathogens cause severe fungal infections in hospitals. Among them, the most important are Candida albicans (Fig. 1), C. glabrata and Aspergillus fumigatus (Fig.1). C. albicans accounts for more than 50% of all fungal infections, causing both superficial and disseminated infections, while C. glabrata infections account for 10 - 20% of the cases. Although A. fumigatus infections are less frequent, the clinical outcome is fatal in 80- 90% of the cases. C. albicans and C. glabrata infections are responsible for systemic infections in a significant proportion of cancer and transplant patients and over half of these infections are still fatal.

Fig. 1: Microscope images of Candida albicans and Aspergillus fumigatus

The fight against fungal infections necessitates the use of antifungal agents. Current available antifungal drugs belong to 4 major different classes: the polyenes, the azoles, pyrimidine analogues and the echinocandins. Each of these classes contains different compounds with specific range of activities against the major fungal pathogens. The exposure of fungal pathogens to antifungal agents has different outcomes, one of them being the development of resistance. Studies investigating mechanisms of resistance to specific antifungal agents could reveal the cellular and genetic basis of resistance. However, some of these studies were initiated with hypothesis-based approaches and thus only give a limited view on the diversity of possible resistance mechanisms to specific agents. The availability of collection of mutants with systematic gene deletions has opened the way to procedures for screening genes involved in susceptibility of resistance to a given agent. This genome-based procedure was implemented in the S. cerevisiae collection of mutants deleted in non-essential genes with compounds such as fluconazole and caspofungin. Many of the genes identified in these studies are not only candidates genes that might be altered in drug-resistant strains of other fungi, but they also provide valuable information on potential primary and secondary target genes for new agents.

Aim:

Microarray strategies and gene deletion approaches performed mainly with C. albicans will help the design of new therapeutic strategies to improve efficacy of existing antifungal therapy, including combination drug therapies to generate synergism. Novel intervention strategies to minimise or bypass resistance will be validated using animal models. Based on this knowledge, cell-based assays for drug target genes will be established and used for drug discovery by a SME.
To monitor progression of systemic infections and monitor resistance, the consortium will establish a collection of clinical strains representing a wide range of antifungal susceptibilities. SMEs will pursue the development and validation of reliable diagnostic tools with commercial potential for rapid clinical strain identification and resistance testing, employing microarray technologies in combination with PCR technologies.
Finally this network will establish data on resistance incidence and prevalence and will link clinical data on susceptibility to known antifungals with therapy outcome. Major European reference centers will jointly set up and recommend European standards for resistance definition, resistance testing/prediction/definition, as well as improved breakpoint identification

Expected and obtained results:

Considering the present status of knowledge on antifungal resistance mechanisms, the incidence of antifungal resistance and problems associated with the prediction, detection, and surveillance of resistance, the EURESFUN consortium was assembled to undertake in order to undertake the several actions with the following expected results:

A better understanding of the molecular basis of antifungal drug resistance is required in order to facilitate the development of diagnostic tools and effective intervention strategies to prevent resistance. Therefore specific mutations linked to antifungal resistance will be identified.
Current approaches to antifungal therapy are limited by a restricted number of available agents and by their mono-therapeutic use. New approaches using drug combinations could improve therapeutic outcome and overcome resistance. Cellular components involved in the response of fungal cells to antifungal drugs could provide alternative targets for compounds that are able to potentiate the action of known antifungal drugs. Therefore, these components will be identified and their inhibitors isolated in order to test their effects in susceptibility to known antifungal drugs in vitro and in animal models.
Only a few European data are available on the epidemiology of antifungal resistance. Moreover antifungal susceptibility tests are not fully standardized among European countries. Therefore, the network will build up a collection of strains displaying a wide range of susceptibility to known antifungals and associated with molecular epidemiology data. This strain collection will be used 1) to establish a distribution of susceptibility data in order to propose microbiological breakpoints of resistance and
2) to survey mutations in known resistance genes in the strain collection in order to develop optimal diagnostic tools for resistance surveillance
Current methodologies for strain identification and susceptibility testing are time consuming and not fully reliable. Molecular diagnostic tools especially those able to detect resistance are needed. The knowledge obtained from previous objectives will be exploited in order to generate novel diagnostic tools using post-genomic approaches (eg oligonucleotide arrays). These tools will enable genotyping, species identification and antifungal resistance monitoring.

Potential applications:

Fungal infections are a major source of nosocomial infections associated with a high mortality. They have steadily increased over the last two decades and this will continue because of the increasing number of immuno-suppressive treatments and the aging of the population. This together with the increasing treatment costs makes fungal infections a major concern to the European health system. In this context, resistance of human pathogenic fungi towards the currently available antifungal agents, whether intrinsic or acquired, has a strong impact on the length of hospitalization as well as the number of invasive procedures that are associated with patient treatment. Therefore, antifungal resistance contributes to an increase in the social and financial cost of fungal infections. Approaches developed by this Network are aimed at providing diagnostic tools to rapidly detect mechanisms of resistance. These tools will undoubtedly help tailor the treatments in such a way that resistance does not emerge and that their duration is at a minimum. Hence, they will have a strong impact on the costs associated with the treatment of fungal infections and reduce the social burden of fungal infections. Furthermore, development of these innovative tools will reinforce the competitiveness of the European industry in the field of diagnostics. This Network will develop original research programs on the molecular mechanisms involved in antifungal tolerance or antifungal resistance. The Network is organized to translate the knowledge gained on these molecular mechanisms into novel approaches to potentiate existing antifungals. establishment of screening assays and the identification of lead compounds will pave the way to innovative combined antifungal therapies.

Coordinator:

Prof. Dominique Sanglard
Centre Hospitalier Universitaire Vaudois Institut de Microbiologie
Rue du Bugnon 48
1011 Lausanne, Switzerland
Tel. +41 21 314 40 83
Fax +41 21 314 40 60
E-mail : Dominique.Sanglard@chuv.ch

European Commission