Pilot study: Live real-time imaging of life-threatening invasive fungal infections
Lead Research Organisation:
University of Aberdeen
Department Name: School of Medical Sciences
Abstract
Candida albicans and other Candida species are fungi which can cause life-threatening bloodstream infections in severely ill patients. In those who develop a Candida bloodstream infection, over 40% will die. These high death rates are due to poor diagnosis of infection, associated with delays in drug treatment, and limited drug choices.
Mouse models of fungal bloodstream infection remain an important tool in increasing our understanding of how infections occur and are essential for testing new drugs and diagnostic tools. However, at present, to monitor disease in mice over time requires sampling of groups of mice at many time points. Because there are differences between individual animals, 3-6 mice per group may be sampled at each time point. The ability to visualize infection in living mice would significantly reduce the numbers of mice used, as the same animal group can be repeatedly assessed during the study.
Attempts have been made to develop reporters for C. albicans previously, but none have successfully imaged bloodstream infections. Problems with interference from tissues have limited their use, as well problems getting substrates into fungal cells. Development of new fluorescent reporters, which use chemicals found naturally in mammals as substrates, suggest that these may be more suitable for imaging fungal infection.
The goal of this study is to modify an existing near-infrared reporter gene to allow it to be expressed in C. albicans cells. A whole range of different fungal strains will be created where the reporter gene expression is controlled by different promoters. These strains will be analysed in the laboratory to identify the strain which is most suitable for use in the mouse infection model. In the final part of the project the reporter will be tested in a small number of mice to confirm its suitability for imaging in live animals.
This project has the potential to massively reduce animal usage in fungal research where infection is monitored over time. Analyses of our own previous studies and those published by other research groups suggest that development of the new reporter for C. albicans could reduce the numbers of mice used in experiments by 75-90%.
Mouse models of fungal bloodstream infection remain an important tool in increasing our understanding of how infections occur and are essential for testing new drugs and diagnostic tools. However, at present, to monitor disease in mice over time requires sampling of groups of mice at many time points. Because there are differences between individual animals, 3-6 mice per group may be sampled at each time point. The ability to visualize infection in living mice would significantly reduce the numbers of mice used, as the same animal group can be repeatedly assessed during the study.
Attempts have been made to develop reporters for C. albicans previously, but none have successfully imaged bloodstream infections. Problems with interference from tissues have limited their use, as well problems getting substrates into fungal cells. Development of new fluorescent reporters, which use chemicals found naturally in mammals as substrates, suggest that these may be more suitable for imaging fungal infection.
The goal of this study is to modify an existing near-infrared reporter gene to allow it to be expressed in C. albicans cells. A whole range of different fungal strains will be created where the reporter gene expression is controlled by different promoters. These strains will be analysed in the laboratory to identify the strain which is most suitable for use in the mouse infection model. In the final part of the project the reporter will be tested in a small number of mice to confirm its suitability for imaging in live animals.
This project has the potential to massively reduce animal usage in fungal research where infection is monitored over time. Analyses of our own previous studies and those published by other research groups suggest that development of the new reporter for C. albicans could reduce the numbers of mice used in experiments by 75-90%.
Technical Summary
Candida bloodstream infections remain a significant problem in severely ill patients, particularly those in intensive care units and surgical patients. Mortality rates remain high due to difficulties in diagnosis, delays in initiation of antifungal therapy and limited choices of antifungal drugs.
Animal models of Candida bloodstream infection remain a vital element in the development of new antifungal agents and diagnostic tests, as well as increasing our understanding of how infection initiates and progresses. However, in temporal studies there is potential for large numbers of animals to be used. Because of inherent biological variation, groups of 3-10 animals are sampled at the each time point, with analyses based upon the mean or median values for each group. In vivo imaging of fungi in a single group of animals over the same time period would significantly (75-90%) reduce the number of animals required for the same study. However, in vivo imaging of invasive fungal infection requires a reporter which can be detected in the internal organs of mice. To date, none of the reporters developed for C. albicans has been able to accurately reflect disease progression.
Publication of new near-infrared reporters for mammalian cells has demonstrated that the near-infrared fluorescence can be detected deep within mice. In addition, only an endogenous compound, biliverdin, is required for fluorescence.
The aim of this project is to develop a codon-optimized near-infrared reporter (iRFP) for use in in vivo imaging of C. albicans. A range of constructs will be produced, with the Candida optimized reporter (camiRFP) expression controlled by various Candida gene promoters. Strains expressing the various strains will be extensively analysed in laboratory tests to identify the most suitable reporter for in vivo imaging. Finally, the Candida reporter gene will be validated in small scale experimental infection studies.
Animal models of Candida bloodstream infection remain a vital element in the development of new antifungal agents and diagnostic tests, as well as increasing our understanding of how infection initiates and progresses. However, in temporal studies there is potential for large numbers of animals to be used. Because of inherent biological variation, groups of 3-10 animals are sampled at the each time point, with analyses based upon the mean or median values for each group. In vivo imaging of fungi in a single group of animals over the same time period would significantly (75-90%) reduce the number of animals required for the same study. However, in vivo imaging of invasive fungal infection requires a reporter which can be detected in the internal organs of mice. To date, none of the reporters developed for C. albicans has been able to accurately reflect disease progression.
Publication of new near-infrared reporters for mammalian cells has demonstrated that the near-infrared fluorescence can be detected deep within mice. In addition, only an endogenous compound, biliverdin, is required for fluorescence.
The aim of this project is to develop a codon-optimized near-infrared reporter (iRFP) for use in in vivo imaging of C. albicans. A range of constructs will be produced, with the Candida optimized reporter (camiRFP) expression controlled by various Candida gene promoters. Strains expressing the various strains will be extensively analysed in laboratory tests to identify the most suitable reporter for in vivo imaging. Finally, the Candida reporter gene will be validated in small scale experimental infection studies.
Planned Impact
Successful development of a reporter for in vivo imaging of systemic fungal infection will have a significant impact on the numbers of mice currently used in fungal research. In our own published work, temporal characterization of Candida albicans systemic infection required a total of 52 mice. However, in vivo imaging of a reporter strain could have generated the same data using 6-10 mice, an 80% reduction in animal usage. Other research groups use similar approaches to monitor infection over time. In another study, where infection progression was monitored over 7 days, in vivo imaging could have reduced mouse numbers by 85%. Antifungal efficacy testing is another area where fungal burdens are monitored over time, e.g. 5-10 mice may be sampled at 5 or more different time points. In this example, in vivo imaging would reduce mouse numbers by 75-90%.
At present calculations on the animal reduction through use of a reporter and in vivo imaging assumes that groups of 6 mice would be required to image infection progression. However, this is based on the variation levels seen when kidney fungal burdens are measured; this could be much lower when the same group of animals are measured over time. In addition, in some virulence studies, large groups of mice are infected with some sampled at various time points to monitor fungal burdens, whilst others are monitored for survival. In a study where fungal burdens are measured at two time points, in vivo imaging of the mice infected for survival at those two time points would reduce animal usage by 66%. From these examples alone, it is clear that development of a C. albicans reporter gene will greatly reduce the numbers of animals currently used in fungal infection research. The reduction in animal usage will be much greater when large scale antifungal drug studies are taken in account.
The development of a reporter for C. albicans also has the potential to further refine the current intravenous challenge model of systemic candidiasis. At present, mice are intravenously infected with C. albicans and, in some studies, infection is allowed to progress until the mice become severely ill and are culled due to ill health. In vivo imaging would allow a maximum level of fluorescence to be set as a cut-off for culling animals. Potentially, this level could be reached prior to development of severe disease as we know that death is due to a progressive sepsis in response to the fungus.
As the project licence holder in my laboratory, this research would have significant impact on animal research carried out locally. I would strongly encourage people working under my licence to move towards imaging infection whenever possible and would enforce use of in vivo imaging for all temporal studies. Introduction of in vivo imaging would be greatly welcomed in our research group, as we are unwilling to carry out large scale temporal analysis of infection or antifungal therapy due to the large numbers of animals required at present.
Development of a reporter for in vivo imaging of C. albicans infection would also have great impact on the wider research community. The Aberdeen Fungal Group is a world leader in fungal research and our expertise in modelling fungal infection is internationally recognised. Successful development of the reporter would immediately lead to requests for the reporter strain and/or the reporter construct. The reporter strain and the constructs would be made freely available as the Candida community are keen to share materials, strains and expertise. There would also be the potential for companies developing new antifungal agents to use the in vivo imaging system to streamline pre-clinical trials.
At present calculations on the animal reduction through use of a reporter and in vivo imaging assumes that groups of 6 mice would be required to image infection progression. However, this is based on the variation levels seen when kidney fungal burdens are measured; this could be much lower when the same group of animals are measured over time. In addition, in some virulence studies, large groups of mice are infected with some sampled at various time points to monitor fungal burdens, whilst others are monitored for survival. In a study where fungal burdens are measured at two time points, in vivo imaging of the mice infected for survival at those two time points would reduce animal usage by 66%. From these examples alone, it is clear that development of a C. albicans reporter gene will greatly reduce the numbers of animals currently used in fungal infection research. The reduction in animal usage will be much greater when large scale antifungal drug studies are taken in account.
The development of a reporter for C. albicans also has the potential to further refine the current intravenous challenge model of systemic candidiasis. At present, mice are intravenously infected with C. albicans and, in some studies, infection is allowed to progress until the mice become severely ill and are culled due to ill health. In vivo imaging would allow a maximum level of fluorescence to be set as a cut-off for culling animals. Potentially, this level could be reached prior to development of severe disease as we know that death is due to a progressive sepsis in response to the fungus.
As the project licence holder in my laboratory, this research would have significant impact on animal research carried out locally. I would strongly encourage people working under my licence to move towards imaging infection whenever possible and would enforce use of in vivo imaging for all temporal studies. Introduction of in vivo imaging would be greatly welcomed in our research group, as we are unwilling to carry out large scale temporal analysis of infection or antifungal therapy due to the large numbers of animals required at present.
Development of a reporter for in vivo imaging of C. albicans infection would also have great impact on the wider research community. The Aberdeen Fungal Group is a world leader in fungal research and our expertise in modelling fungal infection is internationally recognised. Successful development of the reporter would immediately lead to requests for the reporter strain and/or the reporter construct. The reporter strain and the constructs would be made freely available as the Candida community are keen to share materials, strains and expertise. There would also be the potential for companies developing new antifungal agents to use the in vivo imaging system to streamline pre-clinical trials.
