Periodic Reporting for period 2 - FreeATPOC (Towards an instrument-free future of molecular diagnostics at the point-of-care)
Periodo di rendicontazione: 2022-01-01 al 2023-12-31
Molecular diagnostics are still based primarily on lab-based methods. Emerging technologies for application at the point of care (POC) or need, i.e. where a patient or sample is, have started gaining a big part of the molecular diagnostics market. Interestingly, available methods are divided in two groups: sophisticated instruments for ultra-sensitive temperature regulated enzymatic amplification of nucleic acids and extremely simple paper-based immuno-diagnostic kits. Complexity, the main disadvantage of the former, results in their poor translation to several applications at the POC. Simplicity, often accompanied by failures, of the latter is the reason for their limited adoption even in resource limited areas. Our vision is an instrument-free approach that combines the advantages of both groups, i.e. a method that does not use heaters or modules that require electricity, infrastructure and maintenance, but, still, takes advantage of enzymatically-amplified nucleic acids detection. To achieve the above, novel diagnostic tools for genetic amplification based on ligases, polymerases and restriction enzymes operating at ambient temperature will be developed, combined with quantitative smartphone colorimetric/UV detection. The global applicability of the new approach will be demonstrated during infectious disease (Influenza and HIV) testing in human samples (blood and swabs) and plant pathogens (Xylella fastidiosa) in plant-tissues. In all cases, we aim for a sample-to-result of less than 60 min, demonstrated sensitivities down to the clinically/field relevant values and a final cost in the order of $1 per assay. Combination of FREE@POC concept with newly developed predictive models will expand its utility to monitoring disease outbreaks and their spreading. This new generation of instrument-free molecular diagnostics is expected to revolutionize nucleic acid analysis at the POC but also in applications beyond resulting in substantial societal as well as economic benefits.
The concept behind FREE@POC is the development of a point-of-care diagnostic platform that is simple to use and relies on minimal equipment for control and operation, such as a smartphone. Specifically, it is proposed to use an isothermal amplification method that can operate close to room/body temperature, i.e. in the range of 25-37oC. Such a system would be classified as instrument- or electricity-free and would be ideal for operation not only close to the patient or the sample, but also in remote areas and the developing countries. In addition to low cost and complexity, we envision a system that would have the necessary sensitivity for detection even in crude samples. Overall, there are several challenges one has to overcome to produce a solution that is efficient in terms of detection capability and can also operate at the POC. Towards this end, all partners have put considerable effort in progressing the state of the art and producing novel tools of high performance and low cost. To maximize the project outputs and potential innovations by the end of the project, we followed several routes; these included various attempts to develop assays operating optimally at 25-37oC in terms of limit of detection as well as platforms and devices relying on basic instrumentation, with low technology/electricity dependency but with a high performance and detection capability.
All assays were developed upon extensive optimization of relevant steps, especially the sample pre-treatment for lysis at room temperature and under conditions compatible with the subsequent isothermal amplification. Regarding the HIV test, we demonstrated the detection of as little as 10 viral copies per reaction (or <1000 copies per ml of plasma) after isothermal (RPA) amplification at 37oC using our newly developed molecular lateral flow strip (LFS)-based technology. Such a high performance assay, leading to semi-quantitative HIV detection in patients’ plasma at the point of care is unique and has never been reported before. The same protocol and methodology were also applied to influenza detection, reaching a detection limit of 50 copies per reaction. Moreover, the HIV and influenza assays were validated by end users in a hospital environment, both in Europe and S. Africa, demonstrating the feasibility of the method. Finally, the universal and generic nature of the LFS molecular assay has been demonstrated during Xylella pathogens detection in the field using crude plant tissue. It is noted that the two assays developed for HIV and influenza detection are already commercially available and can be obtained for research purposes in the form of two kits.
Our newly developed and patented methodology, based on a molecular Lateral Flow Strip assay for amplicons’ detection and a 3D-printed LFS reader and relevant software, is an attractive solution for diagnostics at the POC using minimal instrumentation. The low cost of the reader (<70 euros), together with the simplicity in operation and rapid and semi-quantitative nature of the detection make the above innovation an attractive solution for global diagnostics. The method, currently at TRL=4-5, will form the main result for future exploitation and is the basis of FREE@POC business plan. Further innovations also produced within FREE@POC include a portable instrumentation which relies on the combination of paper microfluidics with an acoustic (SAW) biochip. This method also forms a generic platform suitable for nucleic acids detection but also antibodies and antigens. Such a flexible system has not been reported so far and a new patent application has been filed for this purpose. Overall, the project has resulted in several new approaches and methods, leading to two patent applications, two small pilot productions of HIV and influenza kits and a robust and attractive methodology for Xylella detection in the field using crude plant tissue.
All assays were developed upon extensive optimization of relevant steps, especially the sample pre-treatment for lysis at room temperature and under conditions compatible with the subsequent isothermal amplification. Regarding the HIV test, we demonstrated the detection of as little as 10 viral copies per reaction (or <1000 copies per ml of plasma) after isothermal (RPA) amplification at 37oC using our newly developed molecular lateral flow strip (LFS)-based technology. Such a high performance assay, leading to semi-quantitative HIV detection in patients’ plasma at the point of care is unique and has never been reported before. The same protocol and methodology were also applied to influenza detection, reaching a detection limit of 50 copies per reaction. Moreover, the HIV and influenza assays were validated by end users in a hospital environment, both in Europe and S. Africa, demonstrating the feasibility of the method. Finally, the universal and generic nature of the LFS molecular assay has been demonstrated during Xylella pathogens detection in the field using crude plant tissue. It is noted that the two assays developed for HIV and influenza detection are already commercially available and can be obtained for research purposes in the form of two kits.
Our newly developed and patented methodology, based on a molecular Lateral Flow Strip assay for amplicons’ detection and a 3D-printed LFS reader and relevant software, is an attractive solution for diagnostics at the POC using minimal instrumentation. The low cost of the reader (<70 euros), together with the simplicity in operation and rapid and semi-quantitative nature of the detection make the above innovation an attractive solution for global diagnostics. The method, currently at TRL=4-5, will form the main result for future exploitation and is the basis of FREE@POC business plan. Further innovations also produced within FREE@POC include a portable instrumentation which relies on the combination of paper microfluidics with an acoustic (SAW) biochip. This method also forms a generic platform suitable for nucleic acids detection but also antibodies and antigens. Such a flexible system has not been reported so far and a new patent application has been filed for this purpose. Overall, the project has resulted in several new approaches and methods, leading to two patent applications, two small pilot productions of HIV and influenza kits and a robust and attractive methodology for Xylella detection in the field using crude plant tissue.
Our newly developed and patented LFS-based molecular assay and reader have never been reported before. In addition, their use in combination with the two assays we designed and extensively optimized for HIV and influenza detection at the POC are clear advancements. Especially for HIV, the ability to discriminate cases where the viral load is below or above the 1000 copies per ml of plasma is significant to assess the effectiveness of antiretroviral therapy. Currently, visiting a hospital and having a PCR test is the only reliable way to assess the HIV load. Our FREE@POC innovation would allow testing to happen at a decentralised lab, with results being delivered the same day. The universal and generic nature of our solution is also another advancement of the state-of-the art. The demonstration of the use of the LFS molecular assay in the field for the detection of Xylella f. is a proof that the method can be applied to the whole One-Health spectrum.
All the above are expected to contribute significantly in advancing of the state of the art in the field of diagnostics and produce useful tools for application in the field. The fact that we are targeting both human and plant-borne pathogens is enhancing the impact of the work; potential applications could be found in the healthcare, but also agro/food safety, and could be implemented in both the developing and developed countries.
All the above are expected to contribute significantly in advancing of the state of the art in the field of diagnostics and produce useful tools for application in the field. The fact that we are targeting both human and plant-borne pathogens is enhancing the impact of the work; potential applications could be found in the healthcare, but also agro/food safety, and could be implemented in both the developing and developed countries.