Final Report Summary - ACUSEP (Integrated whole blood acoustophoresis and homogeneous nucleic acid detection cartridge for rapid sepsis diagnostics)
Executive Summary:
The aim of the ACUSEP project was to provide a proof-of-concept of a new method for sepsis diagnostics that would enable the identification of sepsis causing bacterium directly from a blood sample within a couple of hours. Sepsis is a world-wide health concern with increasing prevalence and an annual death toll of 135 000 in Europe. As severe sepsis may rapidly lead to organ dysfunctions and even death, urgent treatment is very important. The current diagnostics of sepsis still relies on blood culture-based methods for which it takes days to identify the causing pathogen and to confirm which antibiotics would be effective for treatment. Therefore, the antibiotic treatment is started on an educated guess, which may be wrong and lead to even fatal delay in administration of effective medication. Thus, rapid and reliable tests are urgently needed for early detection and identification of pathogens causing sepsis.
In the ACUSEP project a system was developed that integrates acoustophoresis-based sample preparation with polymerase chain reaction (PCR)-based detection and identification of the bacterium by its DNA. In addition, a novel method for simultaneous detection of multiple different bacteria in one PCR-reaction was demonstrated. For sample preparation, acoustophoretic devices were developed which used ultrasonic sound waves to separate blood cells from bacteria and plasma and then to separate and enrich bacteria from plasma. Sensitive PCR assays were developed to universally detect sepsis causing bacteria and to identify the top five most important species of these bacteria. PCR assays were also developed for the most important antibiotic resistance genes. The PCR assays utilized a novel “switchable lanthanide luminescence” label technology and in this project also novel label molecules were studied for this application. An automated ACUSEP system was created that integrated the acoustophoretic sample preparation devices to the PCR assays. Feasibility of the ACUSEP system was evaluated with blood samples of sepsis patients in a clinical study. The ACUSEP system detected and identified bacteria within two hours in the blood of patients who were later confirmed septic by blood culture
A proof-of-concept was provided of a novel, fast and easy-to-use method for sepsis diagnostics that integrated acoustophoretic sample preparation to sensitive PCR-based bacteria detection and identification. This system has high potential and if processed to a commercial product, it could become important in the care of suspected sepsis patients in the hospitals by enabling faster identification of the sepsis causing bacteria and of the possible antibiotic resistance they carry. Therefore it would faster secure the effective antibiotic treatment of the sepsis patient and thus decrease mortality and morbidity.
Project Context and Objectives:
Sepsis is a world-wide health concern with increasing prevalence and an annual death toll of 135 000 in Europe. Sepsis is caused by bacteria invading the blood circulation. Generally infections are contained locally within the initial site of infection and dealt by the immune system on site. However, in some cases the infection spreads systemic and results in sepsis. Severe sepsis may rapidly lead to organ dysfunctions and even death. Therefore, in case of severe sepsis urgent treatment is very important.
Sepsis is treated by antibiotic medication. Many bacteria can cause sepsis but certain antibiotics are only effective against certain bacteria. Moreover, bacterial strains carrying resistance to certain important antibiotics are alarmingly spreading in the world today. Therefore it would be good to know as soon as possible 1) if the person with sepsis-like symptoms has bacteria in the circulation, 2) which bacterial species is concerned, and 3) does this bacterium carry certain antibiotic resistance. The current diagnostics of sepsis still relies on blood culture based methods. Blood samples are cultured for bacterial growth at least overnight. When bacterial growth is detected in a positive sample, subcultures and microbiological tests are performed to identify the bacterium. Further subcultures are grown to discover the antibiotic susceptibility profile of the bacterium and thus to detect the possible presence of antibiotic resistance and confirm effective medication. As this traditional process relies on bacterial growth, it takes time, at least a few days. Therefore, the treatment of the critically ill sepsis patient has to be empirically decided on at the time of sepsis suspicion. The empiric decision is based on the local trends of the most probable causes of sepsis originating from infection at a certain tissue. However, this guess may be wrong and therefore effective treatment may be delayed even by some days. This delay may be fatal for the patient. Therefore rapid and reliable tests are urgently needed for early detection and identification of pathogens enabling more targeted and more appropriate antimicrobial therapy and thus, resulting in improved clinical outcome in patients with sepsis. There have lately been some advances in this field including implementation of mass spectrometry or molecular methods for the detection and identification of the pathogen. However, these techniques require constant availability of highly skilled staff and/or very expensive instrumentation. A major challenge for the development of faster diagnostic methods for sepsis is the low numbers of bacteria present in blood of sepsis patients.
The aim of ACUSEP project was to provide a proof-of-concept of a new method for sepsis diagnostics that would enable the identification of sepsis causing bacterium directly from a blood sample within a couple of hours. In addition, the method would allow simultaneous detection of certain antibiotic resistance genes possibly carried by the bacteria in the blood sample. The unique feature of the approach was the integration of acoustophoresis-based sample preparation method with polymerase chain reaction (PCR) based bacterium detection and identification. In the acoustophoretic sample preparation bacteria were separated from blood cells and collected from blood using ultrasonic sound waves. The objective was to be able to enrich bacteria from a large volume of blood sample to a minute sample volume free of blood cells and plasma to be analyzed in the PCR reaction. The ACUSEP project also aimed to apply a new switchable lanthanide luminescence label technology with novel label structures to allow ultrasensitive multiplexed detection of the DNA molecules amplified in a PCR reaction. The ultimate objective of the project was to combine the acoustophoretic sample preparation and PCR-based bacterium identification in an integrated system to provide simple, safe and rapid sample analysis.
Project Results:
In the ACUSEP project research work was divided into five work packages (WP), all of them with a specific focus.
The work related to development acoustophoretic systems under WP1 was performed in Lund University. In this WP different approaches were studied to isolate bacteria from blood cells and plasma by ultrasonic sound waves to a small volume to be analyzed by PCR assays. A separation unit was developed that enabled effective removal of blood cells form bacteria containing plasma. A trapping unit for high efficiency bacteria enrichment from plasma was also demonstrated. Finally a system was created that integrated the acoustophoretic separator and the acoustic trapping resonator. This system was shown to work well with bacteria spiked to blood samples from healthy volunteers and patients with suspected bacteremia.
The work of WP2 at the Autonomous University of Madrid focused on design, synthesis and testing of novel label molecules for switchable lanthanide luminescence-based detection of bacterial DNA. In total, more than one hundred new molecules were synthesized and several of them showed promising results in tests. In addition, within this WP, a method for producing surface-attached oligonucleotide probe arrays for the detection of multiple bacteria in one sealed PCR reaction container was developed at the University of Turku.
In WP3, a sensitive real-time quantitative PCR assay was developed to universally detect at least the top twenty sepsis causing bacteria in one broad-range bacteria assay. In addition, similar PCR assays were developed to detect and identify the five most important sepsis causing bacteria, antibiotic resistance gene mecA and its variant mecC simultaneously but separately in one reaction and most of the known variants of antibiotic resistance gene blaCTX-M in one reaction. In this WP also a proof-of-principle was shown for closed-tube PCR amplification and array-based multiplexed switchable lanthanide luminescence detection of sepsis causing bacteria. Label molecules from WP2 were utilized in this work, which was mainly conducted at the University of Turku with support from Abacus Diagnostica Oy and Tykslab (part of the Hospital District of Southwest Finland).
Work packages 1, 2 and 3 formed the scientific foundation for the ambitious aim of WP4 to create an integrated “ACUSEP system” combining the acoustophoresis-based sample preparation with the PCR assays. For the integrated system, PCR assays were converted to dry-reagent chips by Abacus Diagnostica Oy and the University of Turku to allow automated processing of the PCR reaction. Four successive prototypes of the integrated system were fabricated and tested at Lund University in collaboration with the University of Turku and Abacus Diagnostica Oy. The fourth automated prototype was evaluated with clinical samples of suspected sepsis patients in the clinical study of WP5. A study was designed, ethical approval was applied, infrastructure was established and the study plans were executed to compare the performance of the developed ACUSEP system to traditional sepsis diagnostics relying on blood culture at Lund University and to state-of-art commercial PCR-based method for sepsis diagnostics at the University of Turku with support from Tykslab. Bacteria were detected within two hours by the ACUSEP system in the blood of patients who were later confirmed septic by blood culture. Thus, a proof-of-concept was provided of a novel, fast and easy-to-use method for sepsis diagnostics that integrated acoustophoretic sample preparation to sensitive PCR-based bacteria detection and identification. However, for diagnostic use, there is still work to be done with the system to improve the sensitivity of the system and to increase the number of bacteria species identified in one assay.
Potential Impact:
The ACUSEP system developed in the ACUSEP project has high potential. It is fast and simple, which is a substantial advantage compared to the other available molecular diagnostic tools for sepsis diagnostics, which are labor-intensive and require highly specialized constantly staffed microbiological laboratories to benefit from the potential advances of obtaining a faster result compared to conventional culture methods. Here, the ACUSEP system could be an easy-to-use alternative that could be developed towards a completely automated system, which could be operated in a standard clinical chemistry laboratory or even onsite, e.g. on the emergency ward. With the fully developed ACUSEP system one could therefore overcome the limitations of the current methods and achieve simpler and faster identification of the sepsis causing bacteria and of the possible antibiotic resistance they carry. Thus it would then faster secure the effective antibiotic treatment of the sepsis patient and decrease mortality and morbidity.
From the socio-economic point of view, the annual cost of severe sepsis to Europe as a whole has been estimated to be approximately 7.6 billion euros. Sepsis concerns the whole community regardless of age and therefore affects also productivity. By reducing the sample-to-result time of bacteremia diagnosis from days to less than a couple of hours, appropriate treatment can be started earlier and the costs of the treatment and mortality are reduced. Moreover, by early identification of the causative organism, treatment with unnecessary antibiotics and risk for development of chemotherapeutic resistance is reduced.
The results and new knowledge created in the ACUSEP project have been and will be presented in scientific conferences and in peer-reviewed scientific journals. In addition, information of the project has become available to the general audience in the project website, press releases and in an interactive digital presentation at Vattenhallen Science Center, Lund, Sweden. As the project has demonstrated a system with high potential, the results of the project should be exploited by companies of the diagnostic industry.
List of Websites:
Project public website address: www.acusep.fi
Coordinator contact:
University of Turku, Turku, Finland
Principal investigator: Saara Wittfooth
Department of Biochemistry / Biotechnology
saara.wittfooth(at)utu.fi
Partner contacts:
Lund University, Lund, Sweden
Department of Biomedical Engineering
Principal investigator (acoustophoresis research): Thomas Laurell
thomas.laurell(at)bme.lth.se
Lund University / Skåne University Hospital, Lund, Sweden
Department of Hematology
Principal investigator (clinical study): Stefan Scheding
Stefan.scheding(at)med.lu.se
Autonomous University of Madrid, Madrid, Spain
Department of Organic Chemistry
Principal investigator: Juan Carlos Rodríguez-Ubis
jrubis(at)uam.es
The Hospital District of Southwest Finland, Turku, Finland
Laboratory of Turku University Central Hospital / Tykslab
Principal investigator: Kaisu Rantakokko-Jalava
kaisu.rantakokko-jalava(at)tyks.fi
Abacus Diagnostica Oy, Turku, Finland
Principal investigator: Tom Palenius
tom.palenius(at)abacusdiagnostica.com
The aim of the ACUSEP project was to provide a proof-of-concept of a new method for sepsis diagnostics that would enable the identification of sepsis causing bacterium directly from a blood sample within a couple of hours. Sepsis is a world-wide health concern with increasing prevalence and an annual death toll of 135 000 in Europe. As severe sepsis may rapidly lead to organ dysfunctions and even death, urgent treatment is very important. The current diagnostics of sepsis still relies on blood culture-based methods for which it takes days to identify the causing pathogen and to confirm which antibiotics would be effective for treatment. Therefore, the antibiotic treatment is started on an educated guess, which may be wrong and lead to even fatal delay in administration of effective medication. Thus, rapid and reliable tests are urgently needed for early detection and identification of pathogens causing sepsis.
In the ACUSEP project a system was developed that integrates acoustophoresis-based sample preparation with polymerase chain reaction (PCR)-based detection and identification of the bacterium by its DNA. In addition, a novel method for simultaneous detection of multiple different bacteria in one PCR-reaction was demonstrated. For sample preparation, acoustophoretic devices were developed which used ultrasonic sound waves to separate blood cells from bacteria and plasma and then to separate and enrich bacteria from plasma. Sensitive PCR assays were developed to universally detect sepsis causing bacteria and to identify the top five most important species of these bacteria. PCR assays were also developed for the most important antibiotic resistance genes. The PCR assays utilized a novel “switchable lanthanide luminescence” label technology and in this project also novel label molecules were studied for this application. An automated ACUSEP system was created that integrated the acoustophoretic sample preparation devices to the PCR assays. Feasibility of the ACUSEP system was evaluated with blood samples of sepsis patients in a clinical study. The ACUSEP system detected and identified bacteria within two hours in the blood of patients who were later confirmed septic by blood culture
A proof-of-concept was provided of a novel, fast and easy-to-use method for sepsis diagnostics that integrated acoustophoretic sample preparation to sensitive PCR-based bacteria detection and identification. This system has high potential and if processed to a commercial product, it could become important in the care of suspected sepsis patients in the hospitals by enabling faster identification of the sepsis causing bacteria and of the possible antibiotic resistance they carry. Therefore it would faster secure the effective antibiotic treatment of the sepsis patient and thus decrease mortality and morbidity.
Project Context and Objectives:
Sepsis is a world-wide health concern with increasing prevalence and an annual death toll of 135 000 in Europe. Sepsis is caused by bacteria invading the blood circulation. Generally infections are contained locally within the initial site of infection and dealt by the immune system on site. However, in some cases the infection spreads systemic and results in sepsis. Severe sepsis may rapidly lead to organ dysfunctions and even death. Therefore, in case of severe sepsis urgent treatment is very important.
Sepsis is treated by antibiotic medication. Many bacteria can cause sepsis but certain antibiotics are only effective against certain bacteria. Moreover, bacterial strains carrying resistance to certain important antibiotics are alarmingly spreading in the world today. Therefore it would be good to know as soon as possible 1) if the person with sepsis-like symptoms has bacteria in the circulation, 2) which bacterial species is concerned, and 3) does this bacterium carry certain antibiotic resistance. The current diagnostics of sepsis still relies on blood culture based methods. Blood samples are cultured for bacterial growth at least overnight. When bacterial growth is detected in a positive sample, subcultures and microbiological tests are performed to identify the bacterium. Further subcultures are grown to discover the antibiotic susceptibility profile of the bacterium and thus to detect the possible presence of antibiotic resistance and confirm effective medication. As this traditional process relies on bacterial growth, it takes time, at least a few days. Therefore, the treatment of the critically ill sepsis patient has to be empirically decided on at the time of sepsis suspicion. The empiric decision is based on the local trends of the most probable causes of sepsis originating from infection at a certain tissue. However, this guess may be wrong and therefore effective treatment may be delayed even by some days. This delay may be fatal for the patient. Therefore rapid and reliable tests are urgently needed for early detection and identification of pathogens enabling more targeted and more appropriate antimicrobial therapy and thus, resulting in improved clinical outcome in patients with sepsis. There have lately been some advances in this field including implementation of mass spectrometry or molecular methods for the detection and identification of the pathogen. However, these techniques require constant availability of highly skilled staff and/or very expensive instrumentation. A major challenge for the development of faster diagnostic methods for sepsis is the low numbers of bacteria present in blood of sepsis patients.
The aim of ACUSEP project was to provide a proof-of-concept of a new method for sepsis diagnostics that would enable the identification of sepsis causing bacterium directly from a blood sample within a couple of hours. In addition, the method would allow simultaneous detection of certain antibiotic resistance genes possibly carried by the bacteria in the blood sample. The unique feature of the approach was the integration of acoustophoresis-based sample preparation method with polymerase chain reaction (PCR) based bacterium detection and identification. In the acoustophoretic sample preparation bacteria were separated from blood cells and collected from blood using ultrasonic sound waves. The objective was to be able to enrich bacteria from a large volume of blood sample to a minute sample volume free of blood cells and plasma to be analyzed in the PCR reaction. The ACUSEP project also aimed to apply a new switchable lanthanide luminescence label technology with novel label structures to allow ultrasensitive multiplexed detection of the DNA molecules amplified in a PCR reaction. The ultimate objective of the project was to combine the acoustophoretic sample preparation and PCR-based bacterium identification in an integrated system to provide simple, safe and rapid sample analysis.
Project Results:
In the ACUSEP project research work was divided into five work packages (WP), all of them with a specific focus.
The work related to development acoustophoretic systems under WP1 was performed in Lund University. In this WP different approaches were studied to isolate bacteria from blood cells and plasma by ultrasonic sound waves to a small volume to be analyzed by PCR assays. A separation unit was developed that enabled effective removal of blood cells form bacteria containing plasma. A trapping unit for high efficiency bacteria enrichment from plasma was also demonstrated. Finally a system was created that integrated the acoustophoretic separator and the acoustic trapping resonator. This system was shown to work well with bacteria spiked to blood samples from healthy volunteers and patients with suspected bacteremia.
The work of WP2 at the Autonomous University of Madrid focused on design, synthesis and testing of novel label molecules for switchable lanthanide luminescence-based detection of bacterial DNA. In total, more than one hundred new molecules were synthesized and several of them showed promising results in tests. In addition, within this WP, a method for producing surface-attached oligonucleotide probe arrays for the detection of multiple bacteria in one sealed PCR reaction container was developed at the University of Turku.
In WP3, a sensitive real-time quantitative PCR assay was developed to universally detect at least the top twenty sepsis causing bacteria in one broad-range bacteria assay. In addition, similar PCR assays were developed to detect and identify the five most important sepsis causing bacteria, antibiotic resistance gene mecA and its variant mecC simultaneously but separately in one reaction and most of the known variants of antibiotic resistance gene blaCTX-M in one reaction. In this WP also a proof-of-principle was shown for closed-tube PCR amplification and array-based multiplexed switchable lanthanide luminescence detection of sepsis causing bacteria. Label molecules from WP2 were utilized in this work, which was mainly conducted at the University of Turku with support from Abacus Diagnostica Oy and Tykslab (part of the Hospital District of Southwest Finland).
Work packages 1, 2 and 3 formed the scientific foundation for the ambitious aim of WP4 to create an integrated “ACUSEP system” combining the acoustophoresis-based sample preparation with the PCR assays. For the integrated system, PCR assays were converted to dry-reagent chips by Abacus Diagnostica Oy and the University of Turku to allow automated processing of the PCR reaction. Four successive prototypes of the integrated system were fabricated and tested at Lund University in collaboration with the University of Turku and Abacus Diagnostica Oy. The fourth automated prototype was evaluated with clinical samples of suspected sepsis patients in the clinical study of WP5. A study was designed, ethical approval was applied, infrastructure was established and the study plans were executed to compare the performance of the developed ACUSEP system to traditional sepsis diagnostics relying on blood culture at Lund University and to state-of-art commercial PCR-based method for sepsis diagnostics at the University of Turku with support from Tykslab. Bacteria were detected within two hours by the ACUSEP system in the blood of patients who were later confirmed septic by blood culture. Thus, a proof-of-concept was provided of a novel, fast and easy-to-use method for sepsis diagnostics that integrated acoustophoretic sample preparation to sensitive PCR-based bacteria detection and identification. However, for diagnostic use, there is still work to be done with the system to improve the sensitivity of the system and to increase the number of bacteria species identified in one assay.
Potential Impact:
The ACUSEP system developed in the ACUSEP project has high potential. It is fast and simple, which is a substantial advantage compared to the other available molecular diagnostic tools for sepsis diagnostics, which are labor-intensive and require highly specialized constantly staffed microbiological laboratories to benefit from the potential advances of obtaining a faster result compared to conventional culture methods. Here, the ACUSEP system could be an easy-to-use alternative that could be developed towards a completely automated system, which could be operated in a standard clinical chemistry laboratory or even onsite, e.g. on the emergency ward. With the fully developed ACUSEP system one could therefore overcome the limitations of the current methods and achieve simpler and faster identification of the sepsis causing bacteria and of the possible antibiotic resistance they carry. Thus it would then faster secure the effective antibiotic treatment of the sepsis patient and decrease mortality and morbidity.
From the socio-economic point of view, the annual cost of severe sepsis to Europe as a whole has been estimated to be approximately 7.6 billion euros. Sepsis concerns the whole community regardless of age and therefore affects also productivity. By reducing the sample-to-result time of bacteremia diagnosis from days to less than a couple of hours, appropriate treatment can be started earlier and the costs of the treatment and mortality are reduced. Moreover, by early identification of the causative organism, treatment with unnecessary antibiotics and risk for development of chemotherapeutic resistance is reduced.
The results and new knowledge created in the ACUSEP project have been and will be presented in scientific conferences and in peer-reviewed scientific journals. In addition, information of the project has become available to the general audience in the project website, press releases and in an interactive digital presentation at Vattenhallen Science Center, Lund, Sweden. As the project has demonstrated a system with high potential, the results of the project should be exploited by companies of the diagnostic industry.
List of Websites:
Project public website address: www.acusep.fi
Coordinator contact:
University of Turku, Turku, Finland
Principal investigator: Saara Wittfooth
Department of Biochemistry / Biotechnology
saara.wittfooth(at)utu.fi
Partner contacts:
Lund University, Lund, Sweden
Department of Biomedical Engineering
Principal investigator (acoustophoresis research): Thomas Laurell
thomas.laurell(at)bme.lth.se
Lund University / Skåne University Hospital, Lund, Sweden
Department of Hematology
Principal investigator (clinical study): Stefan Scheding
Stefan.scheding(at)med.lu.se
Autonomous University of Madrid, Madrid, Spain
Department of Organic Chemistry
Principal investigator: Juan Carlos Rodríguez-Ubis
jrubis(at)uam.es
The Hospital District of Southwest Finland, Turku, Finland
Laboratory of Turku University Central Hospital / Tykslab
Principal investigator: Kaisu Rantakokko-Jalava
kaisu.rantakokko-jalava(at)tyks.fi
Abacus Diagnostica Oy, Turku, Finland
Principal investigator: Tom Palenius
tom.palenius(at)abacusdiagnostica.com