Final Report Summary - DIAGMAL (Translation of the direct-on-blood PCR-NALFIA system into an innovative near point-of-care diagnostic for malaria) Executive Summary:Accurate diagnosis of malaria is essential to timely install proper treatment. Treatment on the basis of clinical symptoms only, i.e. fever = malaria, has nowadays almost everywhere been abandoned in accordance with World Health Organisation guideline to de treatment on the basis of diagnostic testing (also to circumvent improper use of antimalarials and thereby increasing the risk of drug resistance). However, current diagnostic methods for malaria have limitations such as detection limit, antigen persistence after successful treatment or requiring technical expertise and significant infrastructure. During the last decade molecular diagnostic tests have conquered an important place in the diagnostic landscape as they are highly sensitive and allow for high throughput. However, these tests are incompatible with the point-of-care concept as they require sample processing (DNA extraction), have a contamination risk and often complicated read-out systems. Building on the successful outcome of the EU FP7 project MALACTRES, the current project (DIAGMAL) aimed to develop and evaluate a simple molecular diagnostic test for malaria that has the following advantages:- Direct on blood (no DNA isolation)- Simple read-out system (black/white), no need for gel electrophoresis or computer.- Closed system: reduced risk of contamination- Stabilizing reagents included: increased shelf life, no cold storage- Cheap, quick, sensitive and specific.Today we have an integrated direct-on-blood PCR-NAFIA system in place that can detect a malaria infection and a very high sensitivity (<1 parasite/µl blood). The diagnostic platform has been rigorously tested in different European laboratories. In addition, field evaluations have been performed in Kenya and Viet Nam and demonstrate ease of operation, sufficient sensitivity and specificity and confirmed product stability. Importantly, the DIAGMAL platform has the capacity to perform species discrimination (P. falciparum, P. vivax or other Plasmoidium species), which is important to make treatment decisions and has an internal amplification control to assess test performance. The main results of the project can be listed as follows:- Multiplex protocol for the direct on blood amplification of DNA of relevant malaria causing Plasmodium species has been established (WP 2);- Diagnostic evaluations in different EU laboratories of the diagnostic platform are good in terms of high sensitivity and specificity (WP 2);- Prototypes of the closed transfer unit have been produced and tested (WP 3);- Integration of the platform (i.e. multiplex amplification protocol and NALFIA read-out) has been established (WP4)- Data on stabilization of the individual assay components are available and demonstrate sufficient stability of the platform (WP 2 and 4)- Field evaluations of the DIAGMAL product have been completed in Kenya and Vietnam and demonstrate sufficient sensitivity and specificity of the test (WP 5)- Exploitation strategy is being developed and contacts have been made with several interested parties who in the end of the day could bring the DIAGMAL product to market (WP 6)- Dissemination activities have been numerous, with presentations at international conferences, articles in general newspapers and several scientific papers are currently being written (WP 6)The DIAGMAL platform is now ready for the final steps towards commercialization. Data for CE marking are available of the different test components and these is sufficient (independent) data available demonstrating the diagnostic performance of the test. The consortium is committed to make this final step and to bring the DIAGMAL test to market, preferably in collaboration with an expert diagnostic company with a large sales network in Europe and malaria endemic countries. Project Context and Objectives:Accurate diagnosis of malaria is essential to timely install proper treatment. Treatment on the basis of clinical symptoms only, i.e. fever = malaria, has nowadays almost everywhere been abandoned in accordance with World Health Organisation guideline to de treatment on the basis of diagnostic testing (also to circumvent improper use of antimalarials and thereby increasing the risk of drug resistance). However, current diagnostic methods for malaria have limitations such as detection limit, antigen persistence after successful treatment or requiring technical expertise and significant infrastructure. During the last decade molecular diagnostic tests have conquered an important place in the diagnostic landscape as they are highly sensitive and allow for high throughput. However, these tests are incompatible with the point-of-care concept as they require sample processing (DNA extraction), have a contamination risk and often complicated read-out systems. Building on the successful outcome of the EU FP7 project MALACTRES, the current project (DIAGMAL) aimed to develop and evaluate a simple molecular diagnostic test for malaria that has the following advantages:- Direct on blood (no DNA isolation)- Simple read-out system (black/white), no need for gel electrophoresis or computer.- Closed system: reduced risk of contamination- Stabilizing reagents included: increased shelf life, no cold storage- Cheap, quick, sensitive and specific.The specific project objectives are:1) Development and evaluation of a direct-on-blood multiplex PCR to detect and discriminate between Plasmodium falciparum, Plasmodium vivax and the other relevant Plasmodium species with an internal amplification control.2) Development of stabilization protocols for PCR and NALFIA reagents (dry-chemistry approach) to decrease heat-sensitivity of the end product during transport and storage.3) Development and production of a closed contamination-free sample transfer system that will be used to transfer the PCR amplicons to the NALFIA membrane.4) Final integration, evaluation and validation of a rapid PoC integrated system consisting of a direct-on-blood PCR, a closed transfer unit and a Nucleic Acid Lateral Flow ImmunoAssay (NALFIA) to detect malaria and to discriminate between P. falciparum, P. vivax and other relevant Plasmodium species with an internal amplification control.5) Evaluation and validation of the developed technologies in different laboratory, clinical and field settings with the aim to meet WHO requirements for Prequalification of Diagnostics.6) Dissemination and exploitation of the developed products/technologies by the SME partners.Project Results:The project has now been completed. The work was performed over a period of 42 months and involved three SME and one academic partner. Furthermore, the consortium was able to work closely together with other European laboratories for the evaluation of the test, several other companies who contributed to the stabilization experiments and finally also with two research entities in malaria endemic countries (i.e. Vietnam and Kenya) who significantly contributed to the field evaluations of the diagnostic platform. The consortium received frequent advise of a project advisory committee (PAC), comprising experts in the field of molecular test development, diagnosis of tropical infections in a European setting and implementation of diagnostic tests and setting up laboratories in resource limited countries. For the work in the endemic countries a Data Safety and Monitoring (DSMB) was established, and trial insurance was secured to comply with international regulations. A project website (www.diagmal.eu) was established at the start of the project. During the project, several dissemination activities were undertaken, frequent project meetings held (formal meetings every 6 months at one of the partner institutes and as much as needed through teleconferencing) and contacts have been made with interested diagnostic companies who could bring the DIAGMAL platform to market.During the first year of the project research was much focused on establishing a multiplex direct on blood amplification protocol, including an internal amplification. This multiplex amplification protocol has been established and is able to detect all different Plasmodium species (pan-Plasmodium), and specifically P. falciparum and P. vivax. Laboratory evaluations in Amsterdam (at the Royal Tropical Institute) and the London School for Hygiene and Tropical Medicine demonstrated that this protocol had significant sensitivity and specificity to be incorporated in the final test format. In parallel to the development of the multiplex protocol, work was done to finalize the simple read-out format of the test, the so-called nucleic acid lateral flow (NALFIA) device. These two components were in year 2 integrated into a single platform that was further evaluated in the laboratory. Importantly, much work was dedicated to the development and testing of prototypes of the closed transfer unit (CTU) as device to transfer the amplicons (the products of the DNA amplification process) to the NALFIA read-out strip. It is important to incorporate the CTU in the DIAGMAL platform as this will circumvent opening the reaction tube after the amplification process and is thereby reducing the risk of contamination. In order to circumvent a cold chain, and thereby making the DIAGMAL platform more suitable for use under harsh conditions, work towards the stabilization (freeze-drying) of the reagents and components has been done. Part of this work was sub-contracted to specialized companies in Europe. In the last phase of the project field evaluations in malaria endemic countries were performed. These were planned in Kenya (P. falciparum environment) and Vietnam (P. vivax) and in collaboration with partner institutes in these countries. Study protocols for the anticipated field evaluations were prepared and ethical clearance for these studies was sought and obtained. The field work was delayed due to the fact that in both countries malaria transmission was much lower than expected. There, the consortium applied for a 6 month non-cost extension, in order to be able to complete the field work. This non-cost extension was obtained and the work was successfully completed. Currently the data from these studies are being reported as part of a WHO pre-qualification dossier for nucleic acid amplification tests.During the project there were several formal and informal project meetings that have significantly directed the research programme of the project. An important role in this process was for the PAC whose advice is very important in particular to meet potential end-user’s requirements of the test. Dissemination activities were in integral part of the work performed and the DIAGMAL project has been highlighted at several scientific and marketing meetings and is also prominently visual in the Diagnostic Landscape series of UNITAID. Furthermore, promotional posters have been made and a dedicated project website has been established and maintained.The consortium is planning to continue its effort also after the end of the project. We have a validated diagnostic platform for malaria in hand. The DIAGMAL platform can be brought to market and discussions are ongoing with interested parties. The DIAGMAL test can significantly contribute to malaria elimination efforts as it is able to detect in a simple yet sensitive way low density infections. The consortium also expects further scientific output in the form of research publication and is planning to demonstrate the DIAGMAL platform a the MIM meeting in Dakar (2018), as primary venue for malaria research.Potential Impact:The final outcome of the project is to have available the DIAGMAL™ platform, a multiplex direct-on-blood PCR (dbPCR; maximum assay time 45 minutes) for human malaria-causing Plasmodium species. This multiplex assay will be able to detect and to discriminate between P. falciparum (worldwide the most important species and due to increasing drug resistance requiring specific treatment measures), P. vivax (although concerned to be benign, increasingly associated to morbidity and possibly also prone to develop drug resistance) and the other three species, P. malariae, P. ovale and P. knowlesi, who are causing less severe disease and are more easily treatable. The assay will include an internal amplification control based on a human gene and stabilization reagents to increase the shelf life of the test. A closed contamination-free transfer system will be developed and integrated into the system to transfer the resulting amplicons to a lateral flow membrane on which the recognition of specific amplicons will take place (NALFIA read-out; maximum assay time 15 minutes). The lateral flow test will have a 4 line-format (P. falciparum-specific, P. vivax-specific, “other species”-specific and an internal amplification control). This has now been achieved.The DIAGMAL test is designed as a almost near Point-of-care (PoC) molecular diagnostic test for malaria that is able to bypass the need for sophisticated laboratory systems by leveraging new technologies to diagnose infectious diseases almost near the bedside, or “point-of-care”. Diagnostics account for 2% of the cost of health care, but can influence >60% of treatment decisions. In resource limited areas where advanced laboratory services or adequate expertise are not available, access to PoCs may be the tool to be able to make a treatment decision. The prototype dbPCR-NALFIA already developed in the MALACTRES project (the predecessor of DIAGMAL) has shown to be an easy to use test and has been successfully used by local technicians without any specific training on the protocol. This has now also been confirmed for the dbPCR-NALFIA test. The platform has been independently been tested in several European laboratories and all operators reported ease of performance of the easy and simple read-out of test results. Moreover, the DIAGMAL test was evaluated by local research staff in two malaria endemic counties and also there it performed well and there were no majpr obstacles encountered in the execution of the test. Therefore, it is expected the DIAGNMAL will increase access to an accurate malaria diagnostic tool and thereby has impact on health care in both the developed as well as the developing world. The results of the project will have an impact on the healthcare system in the developed world as follows. It will allow for decentralized multi-analyte Point-of-Care diagnosis of a relevant import, and in certain regions in or at the borders of the EU emerging, disease (malaria) and therefore ensure timely and accurate diagnosis. It will bring molecular diagnosis near the patient and reduce healthcare costs. This will result in more appropriate patient management in the clinical setting and, therefore, will increase the well-being of the European citizens. Tests could be deployed at point of entry (borders, refugee camps), remote settings (outbreaks on cruise chips or troops stationed in disease endemic regions) and in the hands of general practitioners. The latter reduces the work load of specialized health centres and reduces costs encountered in tertiary health care as many suspected are already adequately seen at the primary health care level. For the developing countries it will help in reducing the spread of malaria and may contribute to the elimination in certain regions of the disease. Several malaria endemic countries are on the edge of eliminating the disease. The final push requires identification of all carriers (of asymptomatic) of the malaria parasites. In these cases however, the number of parasites are below the detection limit of conventional applied diagnostics (such as microscopy or rapid diagnostic tests). The availability of a diagnostic device that can detect at a very sensitive level the last remaining parasites would be a great tool in the hands of National Malaria Control Programmes in support of their elimination efforts. Next to health gains, malaria elimination will increase economic development, including productivity gains such as increased human capital and increased productivity of factors of production, such as land or capital. Furthermore, the project will also have an impact on the spread of antibiotic resistance; as a result of the proposed work proper treatment is offered based on diagnosis instead of presumptive treatment which is currently frequently being done ("fever"="malaria"). Finally, it will aid the diagnosis of malaria in pregnant women, which is often cumbersome due to sequestration of the parasite in the placental and as a consequence it is often hidden for less sensitive diagnostic tools such as microscopy, but can be picked-up by molecular diagnostics.The project will give rise to a number of outcomes suitable for commercial exploitation in the following areas:1) Through exploitation of the DIAGMAL platform the SMEs achieve direct and profitable business growth, further enabling investments in human capital and resources. This will lead to an expansion of the SMEs skill base, technology portfolio and production capacity2) Through development of the applied technologies, each SME will advance their own knowledge, product offering and services, allowing them to differentiate from competitors. This includes:- The closed transfer unit can be marketed as a stand-alone product and will be a new business field;- The NALFIA detection can be easily adapted to other DNA based tests capable of opening up the complete market of clinical microbiology with PoC tests for the involved SMEs which are now either not using DNA based approaches for their product or are more focused on RT-PCR in other diagnostic markets (QB);- The direct on blood concept can be further worked out for other infectious diseases or even other clinical conditions or disorders.List of Websites:www.diagmal.euH.D.F.H. Schalligh.d.schallig@amc.uva.nl+31.20.5665454
