Periodic Reporting for period 1 - FIT-SEP (Fully integrated technology for predictive diagnosis of sepsis)
Reporting period: 2021-09-01 to 2023-08-31
Biomarkers that predict of S. aureus and S. pyogenes-caused sepsis are bacterial toxins, superantigens. S. aureus causes most deaths from infectious diseases in high-income countries. This situation is exacerbated by spread of multiple antibiotic resistant S. aureus (MRSA) in the community and hospitals.
Superantigens are commonly found in the serum in the absence of bacteraemia. It is hence not appropriate to detect them by PCR of the toxin-coding DNA sequences. The key to our innovation is detection of superantigen protein using novel DNA-containing detector nanorods. Binding of the detector particles to the analyte will be quantified via the nanorod DNA. This strategy (immune-PCR) combines immunodetection with sensitivity of PCR to achieve ultrasensitive detection.
The system devised in this action be a prototype for a novel class of devices for ultrasensitive detection of wide array of molecules, including explosives, hormones, or chemical pollutants. The affordable all-in-one plug and play design will allow use in the general practitioner’s office (point of care), at home, or even in the war zones or disaster areas.
Development of fully integrated point-of-care lab-on-chip prototype will require multidisciplinary effort where ER’s novel detector nanorods will be combined with the Eden’s expertise in design and engineering of microfluidic systems.
Objective: development of novel diagnostic tools for detection of superantigens
To overcome the limitations of current diagnostic assays for sepsis in the terms of sensitivity, speed and ease of use, we propose to produce a fully integrated system for highly sensitive detection of superantigens. Major increase in sensitivity will be achieved by using immune-PCR (iPCR), a method that combines immune detection of proteins with sensitivity of quantitative PCR, while the ease of use and speed are based on the microfluidic assay design. Our fully integrated lab-on-chip point-of-care device will have several advantages over the existing systems. Firstly, it will use, as detector particles, Experienced Researcher (ER)-developed DNA-protein nanorods displaying TCR Vβ. The protein-DNA link in these detector particles is essential for iPCR and it is achieved by natural production of the particles, rather than more expensive chemical coupling. Secondly, the assay will use immobilised MHC II to capture the superantigen-nanorod complex. Given that all superantigens bind MHC II, this approach will allow capture of all superantigen-nanorod complexes. The assay will allow specific detection of each superantigen by using, for superantigen-specific detection, a mixture of bar-coded nanorods, each displaying a TCR Vβ domain which preferentially binds a specific superantigen. The bar-coding of the nanorod DNA will in turn permit multiplexing of the assay at the PCR stage.
Superantigens are commonly found in the serum in the absence of bacteraemia. It is hence not appropriate to detect them by PCR of the toxin-coding DNA sequences. The key to our innovation is detection of superantigen protein using novel DNA-containing detector nanorods. Binding of the detector particles to the analyte will be quantified via the nanorod DNA. This strategy (immune-PCR) combines immunodetection with sensitivity of PCR to achieve ultrasensitive detection.
The system devised in this action be a prototype for a novel class of devices for ultrasensitive detection of wide array of molecules, including explosives, hormones, or chemical pollutants. The affordable all-in-one plug and play design will allow use in the general practitioner’s office (point of care), at home, or even in the war zones or disaster areas.
Development of fully integrated point-of-care lab-on-chip prototype will require multidisciplinary effort where ER’s novel detector nanorods will be combined with the Eden’s expertise in design and engineering of microfluidic systems.
Objective: development of novel diagnostic tools for detection of superantigens
To overcome the limitations of current diagnostic assays for sepsis in the terms of sensitivity, speed and ease of use, we propose to produce a fully integrated system for highly sensitive detection of superantigens. Major increase in sensitivity will be achieved by using immune-PCR (iPCR), a method that combines immune detection of proteins with sensitivity of quantitative PCR, while the ease of use and speed are based on the microfluidic assay design. Our fully integrated lab-on-chip point-of-care device will have several advantages over the existing systems. Firstly, it will use, as detector particles, Experienced Researcher (ER)-developed DNA-protein nanorods displaying TCR Vβ. The protein-DNA link in these detector particles is essential for iPCR and it is achieved by natural production of the particles, rather than more expensive chemical coupling. Secondly, the assay will use immobilised MHC II to capture the superantigen-nanorod complex. Given that all superantigens bind MHC II, this approach will allow capture of all superantigen-nanorod complexes. The assay will allow specific detection of each superantigen by using, for superantigen-specific detection, a mixture of bar-coded nanorods, each displaying a TCR Vβ domain which preferentially binds a specific superantigen. The bar-coding of the nanorod DNA will in turn permit multiplexing of the assay at the PCR stage.
The FIT-SEP project planned to develop a sepsis assay using microfluidic technology from EDEN and the nanorod technology from the candidate. The project was structured in 4 Work Packages (WPs). The first three WPs were focused on technological development (development of superantigen-detecting nanorods, immune-PCR assay development and microfluidic integration), while WP4 dealt with impact actions and more specifically the business plan of the technology. The project started on the 1st of September 2021.
Due to health issues, the project had to be terminated early. The termination date was set at the 9th of January 2022, the effective termination date of the grant.
The work carried out:
1. re-evaluating diagnostic targets for detection of sepsis
2. market research of diagnostic devices detecting various targets (markers) of Sepsis
A. Clinical diagnostics for sepsis
There is no definitive biochemical test used for sepsis. Patient’s age and underlying conditions as well as vital physiological measurements are used for initial diagnosis according to practice guidelines “Surviving Sepsis” campaign. Recommendation is for a 1-hr “bundle” to make initial diagnosis. Rapid intervention is the key to prevent fatal outcome.
Initial tests are: Serum Lactate, procalcitonin (PCT) and C-reactive protein (CRP). Blood Culture (to establish bacteremia and identify the organism). PCT is a rapid indicator of infection, whereas CRP is an indicator of inflammation. Serum lactate is the indicator of poor blood circulation and inability of the liver and kidneys to process this metabolite released by the muscles etc. Lactate is detected by enzymatic test. PCT and CRP tests are done by ELISA. Approved point-of-care (POC) rapid test for CRP is available. Dipstick is under development (Merck-AnteoTech)
Blood bacterial culture and characterization of the organism is an obligatory clinical test to identify the pathogen and adjust the treatment if the default antibiotic prescription is not appropriate. This process requires culturing of bacteria, antibiogram assay etc. It is a very slow process (a couple of days). New PCR methods are available for rapid detection, however they are not yet adopted in the clinic.
Due to health issues, the project had to be terminated early. The termination date was set at the 9th of January 2022, the effective termination date of the grant.
The work carried out:
1. re-evaluating diagnostic targets for detection of sepsis
2. market research of diagnostic devices detecting various targets (markers) of Sepsis
A. Clinical diagnostics for sepsis
There is no definitive biochemical test used for sepsis. Patient’s age and underlying conditions as well as vital physiological measurements are used for initial diagnosis according to practice guidelines “Surviving Sepsis” campaign. Recommendation is for a 1-hr “bundle” to make initial diagnosis. Rapid intervention is the key to prevent fatal outcome.
Initial tests are: Serum Lactate, procalcitonin (PCT) and C-reactive protein (CRP). Blood Culture (to establish bacteremia and identify the organism). PCT is a rapid indicator of infection, whereas CRP is an indicator of inflammation. Serum lactate is the indicator of poor blood circulation and inability of the liver and kidneys to process this metabolite released by the muscles etc. Lactate is detected by enzymatic test. PCT and CRP tests are done by ELISA. Approved point-of-care (POC) rapid test for CRP is available. Dipstick is under development (Merck-AnteoTech)
Blood bacterial culture and characterization of the organism is an obligatory clinical test to identify the pathogen and adjust the treatment if the default antibiotic prescription is not appropriate. This process requires culturing of bacteria, antibiogram assay etc. It is a very slow process (a couple of days). New PCR methods are available for rapid detection, however they are not yet adopted in the clinic.
B. Market research for diagnostic assays.
Market is saturated with assays for IL-6 (1700), CRP (794) PCT (195), endotoxin (LPS; 103). As can be seen in Table 1, a large number of diagnostics’ kit exist, ranging in price, sensitivity. However, there are no assays on the market for bacterial superantigens. Superantigens are not only important in sepsis, but also in local rapid infection like flesh-eating disease. They are also key to the poisoning by food contaminated with Streptococci or Staphylococci because they can’t be eliminated by boiling.
Superantigens are extremely stable toxins; they can kill in the absence of bacterium at low concentration. A sensitive assay (together with IL-6, CRP, PCT and endotoxin) would help identify the cause of sepsis.
Endotoxin assays are mostly used for quality testing of pharmaceuticals, to ensure the absence of LPS contamination. The issue with the LPS detection is in that its key ingredient is derived the horseshoe crabs, leading to their overfishing. Recombinant ingredients are being used increasingly and could be used as a base for the immune-PCR assays proposed in this project.
In conclusion, large number of assays that can be potentially used in the clinical practice to improve diagnostics of sepsis are available, whereas there are no commercial assays for superantigens. This is a serious gap, given that superantigens are directly involved in complications of skin and other infections with Streptococci and Staphylococci, such as flesh-eating disease.
Most assays suitable for cytokine storm or sepsis are in ELISA format, which take hours. Microfluidic assays, which would provide a rapid and fully integrated solution for diagnostics are not commercially available.
In conclusion, market saturation of standard ELISA assays is proportional to the likelihood of an assay to be used in the clinic or quality control of pharmaceuticals, whereas fully integrated rapid plug-and-play microfluidic-based tests have not been seen in the clinical diagnostics market.
Market is saturated with assays for IL-6 (1700), CRP (794) PCT (195), endotoxin (LPS; 103). As can be seen in Table 1, a large number of diagnostics’ kit exist, ranging in price, sensitivity. However, there are no assays on the market for bacterial superantigens. Superantigens are not only important in sepsis, but also in local rapid infection like flesh-eating disease. They are also key to the poisoning by food contaminated with Streptococci or Staphylococci because they can’t be eliminated by boiling.
Superantigens are extremely stable toxins; they can kill in the absence of bacterium at low concentration. A sensitive assay (together with IL-6, CRP, PCT and endotoxin) would help identify the cause of sepsis.
Endotoxin assays are mostly used for quality testing of pharmaceuticals, to ensure the absence of LPS contamination. The issue with the LPS detection is in that its key ingredient is derived the horseshoe crabs, leading to their overfishing. Recombinant ingredients are being used increasingly and could be used as a base for the immune-PCR assays proposed in this project.
In conclusion, large number of assays that can be potentially used in the clinical practice to improve diagnostics of sepsis are available, whereas there are no commercial assays for superantigens. This is a serious gap, given that superantigens are directly involved in complications of skin and other infections with Streptococci and Staphylococci, such as flesh-eating disease.
Most assays suitable for cytokine storm or sepsis are in ELISA format, which take hours. Microfluidic assays, which would provide a rapid and fully integrated solution for diagnostics are not commercially available.
In conclusion, market saturation of standard ELISA assays is proportional to the likelihood of an assay to be used in the clinic or quality control of pharmaceuticals, whereas fully integrated rapid plug-and-play microfluidic-based tests have not been seen in the clinical diagnostics market.