Final Report Summary - HILYSENS II (Demonstration Activities for the clinical validation of the prototype HILYSENS Lab-on-a-Chip) Executive Summary:Current clinical diagnostic methods for Borrelia lack the necessary sensitivity and specificity to detect early cases, as well as late manifestations of the disease, such as chronic or autoimmune-related infections. This can lead to late, delayed, or inadequate treatment which can cause serious disabling, difficult to treat and long term problems, such as neuroborreliosis or arthritis.The HILYSENS II project comprised the demonstration activities for the HILYSENS Lab-on-a-Chip prototype developed during the 2-year HILYSENS research and development project, funded by the ‘Research for the Benefit of SMEs’ program of the European Commission's Seventh Framework Programme (FP7). The developed prototype included a biochip (aka Lyme Card) and a portable fluorescence reader (aka the Reader), which combined enable a more specific and sensitive detection of human serological responses to Borrelia species in patients with Lyme disease.The HILYSENS II demonstration activities, which involved SMEs from Portugal, Italy, Germany and Spain, and a clinical academic partner from Sweden, included prototype optimization, scale-up and validation in clinical environment.At beginning of project, both Reader and Lyme card chip have been re-designed to be more robust and allow an economically viable massive production. In parallel, an ELISA assay based on Lyme card antigen bouquet (aka Hilysens ELISA) was also developed for central validation of Lyme card system (Reader and Lyme card).At the end of project, the Lyme card system was validated at Borrelia testing centres in Germany and Sweden, using over 400 serum and cerebrospinal fluid (CSF) samples from both healthy and suspected lyme disease patients. In parallel, a blind confirmatory testing with random samples using the Hilysens ELISA assay was performed in Portugal.At the end of project, which lasted 24 months, the Reader and its software were successfully validated and certified (CE-marked and ISO62304) to operate in clinical laboratories. On the other hand, the validation performed with the Lyme card itself was not successful and needs further developments and testings to reach the market (estimated to second quarter of 2017).As contingency plan for Lyme card failure, an equivalent Lyme well assay - i.e a microplate well coated with same antigen bouquet array of Lyme Card – was also assessed at end of project. This equivalent Lyme well assay presented good preliminary results and was shown to be a promising alternative/complementary assay to the Lyme card biochip.Project Context and Objectives:Lyme disease is the most common tick-borne infection in Europe and North America. The disease is a multisystemic disorder that can affect a range of tissues, including the skin, heart, nervous system, and to a lesser extent the eyes, kidneys, and liver. The illness is caused by the spirochete (spiral-shaped bacteria) Borrelia burgdorferi, which is transmitted by the bite of infected ticks of the genus Ixodes. Lyme Disease is endemic in 63 countries across the world, including the EU27 countries. The Regional Office for Europe of the World Health Organisation (WHO) estimates about 85,000 cases of Lyme Disease annually in Europe (from available national data) but stresses that this number is largely underestimated as case reporting is highly inconsistent in Europe and many infections go undiagnosed. The same report highlights that in much of Europe, the number of reported cases of Lyme Borreliosis has increased from the early 1990s, and the geographic distribution of cases has also expanded due to climate change.Early symptoms of Lyme Disease include fever, headache, fatigue, depression and a characteristic circular skin rash called erythema migrans (EM). Left untreated, later symptoms may involve the joints, heart, and central nervous system. When detected and treated early, the infection and its symptoms are eliminated by antibiotics in most cases. Late, delayed, or inadequate treatment can lead to more serious symptoms, which can be disabling and difficult to treat. Occasionally, symptoms such as arthritis persist after the infection has been eliminated by antibiotics, which suggests that Borrelia can promote chronic infections and infection-induced autoimmunity.Given the shortcomings of current laboratory methods, diagnosis has to be based on the clinical findings. However, since the clinical findings are very diverse and may mimic those of many common diseases (e.g. arthritis, fibromyalgia, multiple sclerosis), patients who suffer from the symptoms of the chronic disease may go through several months or years of repeated misdiagnosis and inadequate treatment. Besides the obvious deleterious effect on patients’ health and quality of life, this represents a big economic burden for European Health Systems. Moreover, lack of diagnosis results in underestimation of disease prevalence and health risks, leading to critical delays in starting awareness and preventive policies. Therefore, there is a crucial need to develop a sensitive tool for laboratory diagnosis of Borrelia burgdorferi acute and chronic infections to provide novel treatment options for patients suffering from their harmful infestations.Based on the current market needs, two main required features have been clearly identified has needing improvement: 1. To improve on the current WB technique, which is very labor-intensive, time-consuming, and difficult to standardize, the new tool should be robust, easy-to-use, and enable unambiguous interpretation of the test results. 2. To avoid the need for two different tests to secure a diagnosis and allow chronic infections to be detected, new antigens that have been reported in the literature in the last years but have not yet reached clinical use should be included. With these premises in mind and with the input of all the partners, the HILYSENS consortium set the goal of developing: A highly-sensitive, specific and robust diagnostic tool for Lyme Disease that is easy to use and interpret by frontline physicians and clinical laboratories and that will become the standard detection tool, rising awareness of this disease among European clinicians and permitting accurate quantification of the increasing disease incidence in order to carry out prevention campaigns before the disease becomes epidemic. Namely, in a former HILYSENS research and development project, funded by the ‘Research for the Benefit of SMEs’ program of the European Commission's Seventh Framework Programme - FP7 (grant agreement 262411), a Lab-on-a-Chip prototype including a portable fluorescence reader (aka the Reader) and a biochip (aka Lyme Card), which combined enable a specific and sensitive detection of human serological responses to Borrelia species in patients with Lyme disease, as developed and its proof-of-concept successfully achieved.In this HILYSENS II project, we aimed to conduct demonstration activities for the optimization, scale-up and validation of the Lyme Card system in clinical environment. Namely, the HILYSENS II demonstration activities, which involved SMEs from Portugal, Italy, Germany and Spain, and a clinical academic partner from Sweden, The Lyme card system was optimized by SMEs from Portugal, Italy and Spain and validated at Borrelia testing centres in Germany and Sweden, and, in parallel, a confirmatory testing was also performed in Portugal. By the end of the 24-month Demonstration Activity it was expected that the HILYSENS diagnostic system would be submitted for approval by regulatory authorities in Europe and the US, in order to enter these markets shortly thereafter, provided that the system had passed validation. Only the Reader and software did pass validation and achieve CE-marking. Whereas, the remaining foreground still needs further developments to be qualified to reach the clinical market, which is estimated to happen within the first half of 2017.Project Results:The HILYSENS II project has started with the verification of Lyme Card prototype performance inherited from the former R&D HILYSENS project, to progress towards its scale-up production, clinical validation and regulatory approval. The activities included the verification of antigens on the Lyme card (including consistency of antigen coating and immunogenic/antigenic properties upon coating), the development of ELISA reference assay with Lyme card antigens, verification of Lyme card integrity, consistency and performance upon manufacturing, establishment of pooled sera for Reader calibration, verification of Reader performance, final optimizations and scale-up production of Lyme card and Reader, prototype quality assessment, validation in clinical environment and application to regulatory approvals.In particular, since HILYSENS project, the Lyme card biochips had to be considerably re-designed to improve their reliability and enable an affordable and effective scaled-up production. Consequently, the Reader had also to be profoundly revised to be compatible with the new design of Lyme cards. This was performed taking also into consideration more efficient strategies for Reader manufacturing and also considering quality specifications to comply with regulatory demands. All this resulted in improvements of both Lyme card and Reader, while enabled the start of mass production of Lyme cards and production of more robust Readers (to avoid damage which proved to be an issue with previous prototype versions).The Lyme card Reader has successfully progressed from TRL6 to TRL8, being currently complete and qualified to reach the EU market (CE-mark were successfully achieved for the Reader and other certifications, including ISO62304 for its software, are formally ready for submission). On the other hand, the Lyme card itself did not pass validation in clinical operational environment. A detailed assessment of the validation results, shown that some issues with Lyme card fluidics was the cause for validation failure. These issues are now being carefully addressed to proceed with additional validation of the Lyme card system and achieve its certification for clinical diagnostic.As contingency plan, a Lyme Well platform, equivalent to the Lyme Card biochip, was designed and tested at end of project. Preliminary data shown promising results towards a solution for Lyme system. A comparison made against current commercial immunological kits for Borrelia diagnostic, shown that the equivalent Lyme Well assay presented a good positive, negative and overall percent agreement (83.3 – 85.7%) with Borrelia IgM ELISA (Euroimmune). On the other hand, the positive percent agreement between Lyme well the Liaison Borrelia IgM assay (Diasorin) was lower (41.2%), but negative percent agreement with same assay was high (88.9%), with the overall percent agreement being 57.7%. When compared to Borrelia IgM blot assay (Euroimmune), the Lyme well assay presented a high positive percent agreement (81.5%), while negative percent agreement was a bit lower (59.5%). On the other hand, the positive percent agreement between Lyme well assay and recomBead Borrelia IgM 2.0 (Mikrogen Diagnostik) was considerably lower (40%) but the negative percent agreement was 100%. In summary, the overall percent agreement between Lyme well IgM assay and other commercial IgM kits varied between 53.8% and 84%, showing that there is a relative overall good agreement between Lyme well assay results and non-reference commercial assay kits. Nonetheless, it should be noted that to actually determine the sensitivity and specificity of the Lyme well assay for Borrelia, results needed to be correlated to clinical data, as none of the tested commercial kits available is a reference to estimated such performance. Therefore, results for Lyme well were compared against clinical record to estimate the current sensitivity and specificity of the method. Results shown that positive predictive value and negative predictive value were high (94% and 70%, respectively) for a healthy control group of patients, with a sensitivity of 83% and specificity of 87%. On the other hand, for a cohort of patients clinically suspicious for chronic Lyme disease the positive predictive value and negative predictive value were 79% and 60%, respectively, with a sensitivity of 83% and specificity of 60%. In overall, these results are promising having allowed the Lyme well platform to reach a TRL4, but further developments and validation of the platform should be performed to progress its TRL to level 8 and reach its proper certification for the clinical market.In parallel with Lyme Card prototype scale-up and validation, a reference ELISA test has been successfully developed incorporating the same antigens which are coated in the Lyme card. Results have been validated with control sera. In total, more than 300 patient samples have been collected and used for the testing and validation of Hilysens prototype, while properly taking into consideration all ethical issues and clinical data demands. At the end of the project, the Hilysens ELISA assay has reached a TRL7 but results from clinical validation shown that the assay still needs some minor modifications and further validation to be able to be qualified to reach a TRL8 and reach the market. Namely, when compared against current non-reference commercial immunological kits for Borrelia diagnostic, the Hilysens ELISA presented a quite high positive percentage agreement across all tested kits for IgG (79.6% - 100%) and a more broad positive agreement range for IgM (38.5% - 94.1%). On the other hand, the negative percent agreement across all assays was lower for both IgG (44.4% -77.8%) and IgM (44% - 88.9%) assays. This may either mean that Hilysens ELISA assay may have a lower specificity than other kits or that other non-reference assays provide more false negatives than the Hilysens ELISA assay, which is a known issue for current commercial Borrelia assays. In order to rule out which hypothesis is more valid, further studies need to be performed, such as running a resolver test for same samples and/or making a correlation with clinical data. Therefore, results for Hilysens ELISA were also compared against clinical records to estimate the current sensitivity and specificity of the method. In case of patient’s samples from Sweden clinical partner, the results shown a positive predictive value of 50% for IgG (5 out of 10 IgG-positive samples were true positives) and 27% for IgM (3 out of 11 IgM-positive samples were true positives). The negative predictive value was 81% for IgG (26 out of 32 negative IgG-results were true negative) and 82% for IgM (27 out of 33 negative IgM-results were from true negatives), with a sensitivity of 45% for IgG and 33% for IgM and a specificity of 84% for IgG and 77% for IgM. These results show that Hilysens ELISA performs well taking into account the facts that a) acute infection can be seronegative and b) the patients were samples in an endemic area with a high sero prevalence among healthy individuals. The Swedish partner’s samples were collected from patients with suspected neuro-Borrelia. To obtain this diagnosis in Sweden, the patients must fulfil two criteria. First, the patients must show clear clinical signs of neurological complications such as nerve root pain (adults) and/or facial paresis (children). Second, the patients must have pleocytosis, i.e. elevated levels of white blood cells in their cerebrospinal fluid (more than 5 million cells per litre). Thus, only patients with acute ongoing infection get a neuro-Borrelia diagnosis. It is well known that it takes up to 6 weeks from disease onset before patients with neuro-Borrelia seroconvert and produce intrathecal anti-Borrelia antibodies. Thus, a serum sensitivity of 45% for IgG and 33% for IgM could be expected when examining patients with acute neuroborreliosis, as the remaining patients have probably not yet developed any local intrathecal antibodies. Furthermore, the Swedish partner is located in the west region of Sweden where Borrelia is endemic and the seroprevalence in healthy adults is between 10-15%. Therefore, there will always be false-positive results in patients that are diagnosed NOT to have a Borrelia infection. A specificity of 85% for IgG and 77% for IgM may simply reflect this fact.In case of patient’s samples from German clinical partner, the sensitivity was determined to be 72% for both healthy control group and cohort of patients clinically suspicious for chronic Lyme disease, while specificity was 100% for healthy control group but lower drastically to 10% for the cohort of patients clinically suspicious for chronic Lyme disease. This poor specificity with the latest control group may be explained by the chronicity of the patients sera tested, which included only sera from chronic patients. The long term immunology is poorly understood, but one may hypothesize a low level B cell answer in such control group which patients were attending the clinic due to suspicion of chronic Lyme. In overall, the results also shown that the cut-off values for the Hilysens ELISA assay may still need to be reviewed, in particular the inclusion or interpretation of borderline results, especially for specific antigens (i.e. peptides and proteins), which is now being further addressed, in order to proceed with further validations and reach the market.Potential Impact:The Hilysens II project addresses the need to improve current diagnosis of Lyme disease, by demonstrating and validating a valuable novel diagnostic tool – the Lyme card system – which comprises a Lyme card biochip and a Reader, that together enables a user-friendly platform that delivers a quick and clear diagnostic report to medical doctors.The generated foreground of the project has a vast potential impact in several socio-economic groups, such as in the healthcare sector, including In Vitro Diagnostic (IVD) market, overall biotech and healthcare SMEs, hospitals/clinics, medical practitioners, Lyme disease patients and overall civil society.Impact on Health SystemLyme disease is the most common tick-borne infection in Europe and North America, with estimated 85,000 and 300,000 new cases per year, respectively. The disease is caused by bacteria from the complex Borrelia burgdorferi sl and transmitted by ticks of the genus Ixodes spp. First manifestations of the disease include the presence of Erithrema migrans and flu-like symptoms. In late manifestations it may occur Lyme arthritis, acrodermatitis and even neuroborreliosis. If the disease is left untreated it can affect heart, joint and central nervous system and might become a cause for death.There is an enormous long-term cost of Lyme disease to families, school systems, and healthcare systems. According to a study published in 1993 in Contingencies, an actuarial trade publication, the cost to society for Lyme disease was about one billion dollars per year. The average treatment and diagnosis and lost wages related to Lyme disease was €40,750 per year per patient. Cases have more than doubled since then, so current costs are probably €1000 million or more annually. Autoimmune and chronic conditions represent immense costs for European Health systems and their economic impact goes beyond the costs of healthcare treatments. Indirect costs, such as those from lost productivity, can match or sometimes exceed the direct costs.The Lyme card system will enable to better evaluate and monitor antimicrobials therapies and will help to medical practitioners to plan and guide therapeutic interventions. Moreover, the application of the Lyme card technology will contribute to quantifying accurately the actual cases of Lyme disease across Europe and worldwide, thereby solving the problem of underreporting and misdiagnosing of this disease. The action has to be taken at a European level, making sure that standard testing criteria are developed and disseminated so that effective control of this disease is achieved on time. Furthermore, the use of cost-effective, user-friendly diagnosis technology will play an important role in improving the quality of health care in all Member States by ensuring that practitioners and patients base their decisions on appropriate healthcare on systematically developed evidence.Advantages of Lyme card over existing diagnosticsProgress in diagnostics and therefore in treatment and control of diseases is mainly governed by technology. However, to ensure that the expected impact on the patients is achieved, any new-developed diagnostic technology must not only solve the technical problems of current methodology but also ensure that such technology will be successfully taken up by “lead-users” (technology manufacturers) and “end-users” (clinical laboratories in this case). Regarding the latter, a recent study on product usage of new molecular diagnostic tests for infectious diseases conducted in 11 European countries 41 identified the following factors restraining a more widespread use of new molecular techniques in infectious pathogen testing:• the current prices charged per test (mentioned in the top three of answers by 9/11 surveyed countries);• the lack of standardization;• the fact that the applied techniques are demanding in both the skills required of the user and the laboratory space in which the tests are carried out.HILYSENS II fully addresses the last two concerns by providing a compact and robust lab-on-chip system – the Lyme card system - designed to work with small volumes of complex fluids and without the need of expert operators. This tool together with a dedicated Reader and user-friendly software will enable more precise, accurate and reproducible testing making it possible to become the standard tool for the disease diagnosis. The use of microfluidics represents also a dramatic reduction in reagents costs compared to standard laboratory techniques. Moreover the Lyme card system goes further beyond to ensure that the expected impact on patient’s health will be achieved by solving a capital drawback of current lab-on-a-chip devices: their high production costs. The Lyme card will be the first lab-on-a-chip device that utilizes the capabilities of roll-to-roll hot embossing to the full, for making an entire lab-on-a-chip device and lay the ground for subsequent flexible low-cost mass production. The new approach addresses this issue by using direct mask-less technology, polymeric materials and allowing production by low-cost manufacturing technology such as moulding used for rubber and plastic components and hot embossing used for credit cards or CD and DVD production. Due to the combination of these features the target production price for the Lyme card microfluidic chip can be considerably low when compared to other current diagnostic platforms.Impact on consortium SMEsThe European market for molecular diagnostics represented 1.6 billion USD in 2014 and is estimated to reach 4.2 billion USD by 2023 at a CAGR of 11.4% from 2015 to 2023. In the particular case of bio-MEMS, where the Lyme card system fits in, its worldwide market was valued 2.5 billion USD in 2014 and is anticipated to grow at CAGR of over 25% from 2016 to 2023.Consequently, the exploitation of Hilysens II foreground will have a major impact on improving the competiveness of the consortium SMEs, which are the current sole owners of the foreground. Namely, it will permit the SMEs to increase sales and turnover, leading to growth and job creation, due to the manufacture and commercialization of the innovative Lyme card system to the healthcare sector. Direct economic benefits will accrue to the SMEs given that they will scale-up the production to supply the clinical market and have acquire additional skills in manufacturing the final products (Lyme card, including reagents, and Reader, including software). The HilysensII foreground will enable the consortium members to compete globally in a growing and highly competitive market, while benefiting from the commercialization of the Lyme card/Reader and its sales to hospitals and clinical laboratories across Europe and North America.In a broader sense, it is expected that the overall image of the European biotechnology sector will be raised and Lyme card system will contribute to providing impetus to the European Union in the global biotechnology innovation race. Furthermore, given that biotechnology is an enabling technology, the use and uptake of the Lyme card technology will have a broader economic impact to the competitiveness of European pharmaceutical companies, given that the information it provides will contribute to decreasing the costs and the associated risks of the antibiotic treatment by accurately diagnose patients suffering from Lyme disease.Impact on quality of life: benefit for patients Improvements in healthcare quality have an obvious impact on the quality of life of all EU and worldwide citizens. By enabling specific, highly-sensitive and cost-effective diagnostic of pleomorphic bacteria infections, the Lyme card system will greatly enhance the level of service offered to patients and ensure that healthcare providers become more effective. The Lyme card system hopes to solve the major problem faced by health professionals when treating Lyme disease patients: the lack of a reliable and standardized diagnostic test. Lyme card will be the first lab-on-a-chip tool that will allow specific and sensitive detection of acute, chronic and autoimmunity-associated to Lyme disease infections and will ensure non-invasive, fast, specific and easy diagnosing, prognosis and monitoring. Specific and sensitive detection of immunologic profiles will allow better point of care treatment and intervention of the disease. Still classified as a rare disease, Lyme disease is becoming a serious health problem in Europe. The disease is widespread and the prevalence is significantly higher than reported by health officials due to the failure in the sensitivity of the current diagnostic methods. The estimated 85,000 new cases reported annually in Europe, find a higher incidence in central and northern Europe, with focal areas of higher incidence occurring also in countries where the overall incidence is generally low. Moreover, the highest reported incidences of Lyme disease occur among children 5-14 years old and adults 45-54 years old.Due to its unique features, the Lyme card system will have a major impact on health and life quality of European citizens, with special emphasis on children, through: - Improvement of test sensitivity and specificity, which will permit early diagnosis of the disease before it progresses to the chronic phases. - Ease-of-use and result interpretation, together with the low production costs will allow decentralized testing and make sure that all patients are benefiting from the tool, with independence of their origin.- Public health preventive policies could be initiated together with educational measures for frontline physicians in European areas in which the disease incidence is increasing.Main dissemination activitiesIn order to maximize impact of Hilysens II foreground, the consortium has planned and implemented a series of events and actions to disseminate the project and its foreground across key target publics and a wide audience. Nonetheless, it should be noticed that such dissemination activities were restricted in terms of information, in order to protect the foreground for proper exploitation. Considering this, the consortium has drawn a common strategy for dissemination that: a) satisfies the need to publish and raise awareness of the technology; b) make sure that the technology is broadly disseminated so as to contribute to the commercial success of Lyme card, and; c) make available the project results to a broader scientific community (provided that exploitation is not risked). Namely, the consortium has undertaken the following dissemination activities: - Creation and maintenance of a project website (hilysensproject.eu) were information about the project and overall Lyme disease theme was published, having in mind different target publics and end-users (patients, physicians/clinicians, patients associations, etc.);- Creation of a promotional video about the project, made available to general public in YouTube;- Use of diverse marketing channels from consortium partners to disseminate project progresses and events, including the use of social networks (i.e. Facebook, LinkedIn, Twitter, etc), newsletters and MailChimp accounts, to mainly target scientific and clinical community which account for the majority of current consortium partners’ clients;- Participation in scientific and entrepreneurship meetings/conferences to disseminate the project across scientific community, industry and also assess potential investors for future sustainability and progression of Lyme Card system;- Published press-releases about project activities to target local media and, ultimately, wide civic society;- Prepared and executed info days about the project activities, which were held in a clinical and/or academic environment, such to mainly target clinicians, lab technicians, patients, healthcare administrators, scientific researchers, professors and biotech/healthcare students, among other publics that attended the events.(for more details on dissemination events, please consult Table in Template A2 – List of dissemination activities)In summary, 24 dissemination events have taken place across the 2-year project, having reached over 2.1 million (estimated) people from across the globe, including over 130 healthcare professionals. The Hilysens II consortium is committed to continue dissemination of Hilysens II‘s foreground even after closer of the current project. Namely, the project website will be kept online and active for at least 3 more years, gathering all crucial information about the project and new updates. Newsletter and Press releases will also be made as the Hilysens II’s foreground enters the market and is further optimized in future developments to further improve the Lyme Card System. Hilysens II consortium will also be committed to publish results in open-source peer-reviewed journals and also attend further conferences to further disseminate the project foreground, as well as seek for new investments for a prosperous exploitation of the Lyme Card System.All dissemination activities and materials will, as always, mention that “The research leading to these results has received funding from the European Union's Seventh Framework Program under grant agreement n. 606348.” and care will be taken to ensure that no confidential information is disclosed.Exploitation of ForegroundThe HILYSENS II’s foreground will be exploited by the consortium SMEs, either through their own commercial channels and/or through the creation of a spin-off company of the project. The Lyme card system and other exploitable products from the foreground are expected to reach the European market within the first half of 2017 and swiftly progress to other markets, such as the North American market.List of Websites:Project public website: http://hilysensproject.eu/Coordinator (STAB VIDA) contact: Mr. Orfeu Flores (orfeu@stabvida.com)Partner MicroBioDevices contact: Mr. Giovanni Ugo Altieri (gianugo.altieri@microbiodevices.com)Partner microLiquid contact: Mr. Borja Barreado (borjabarredo@microliquid.com)Partner BCA contact: Dr. Carsten Nicolaus (service@borreliosecentrum.de)Partner UGOT contact: Mrs. Kristina Eriksson (kristina.eriksson@microbio.gu.se)
