Multi-drug resistance in malaria under combination therapy: Assessment of specific markers and development of innovative, rapid and simple diagnostics

Projektinformationen

MALACTRES

ID Finanzhilfevereinbarung: 201889

Abgeschlossenes Projekt

Startdatum 1 Januar 2008

Enddatum 30 Juni 2013

Finanziert unter

• Specific Programme "Cooperation": Health

Gesamtkosten € 3 738 056,04

EU-Beitrag € 2 800 358,00

Koordiniert durch

HET KONINKLIJK INSTITUUT VOOR DE TROPEN
Netherlands

Periodic Report Summary 4 - MALACTRES (Multi-drug resistance in malaria under combination therapy: Assessment of specific markers and development of innovative, rapid and simple diagnostics.)

Project Context and Objectives:

Malaria is by far the world’s most important tropical parasitic disease. An estimated 300 – 500 million people become ill each year. Malaria is a disease cause by an infection with the parasite Plasmodium falciparum, a small single cell organism. The disease kills an estimated one - two million people per year, mainly young children in developing countries. Approximately 1 child dies every 30 - 45 seconds due to malaria. Other important risk groups are pregnant women and non-immune travelers. Prompt and accurate diagnosis and effective treatment are the corner stones of effective malaria disease management. However, the clinical diagnosis is not always very easy, as the primary symptoms may resemble those of other diseases (like the flu). Furthermore, the malaria parasite is becoming more and more resistant to the action of anti-malaria drugs. This is due to small changes in the parasite DNA (point mutations). Over-prescription of anti-malarials (confusion with other febrile diseases) and the uncontrolled selling of poor quality drugs contribute to the increase in drug resistant parasites. Newer drugs, the so-called artemisinin-based combination therapies (ACTs), are available, but expensive and at risk of loosing their effectiveness too. The MALACTRES project aims to assess specific genetic markers for Artemisinin-based combination drugs resistance and development of innovative, rapid and simple diagnostics for malaria.

The specific project objectives are:

• To develop and evaluate in disease endemic countries accurate low-tech molecular diagnostic tests for malaria;
• To identify alleles of candidate resistance genes associated with increased transmission success of P. falciparum after ACT treatment in completed clinical trials with endpoints at gametocyte or infected mosquito level;
• To conduct ACT treatment trials with transmission endpoints, and measure impact of resistance-associated alleles of key genes on:

- gametocyte prevalence, density and longevity;
- infectiousness of gametocyte-positive treated individuals to mosquitoes;
- infectiousness of randomly-selected treated individuals to mosquitoes.

• To conduct ACT treatment trials with parasitological and clinical efficacy endpoints, including in vitro and in vivo resistant determination test, and measure abundance of parasites carrying candidate markers among participants with treatment failure
• To develop new low-tech diagnostic tools that are able to demonstrate the presence of mutations conferring drug resistance in the Plasmodium population
• To investigate commercial value aspects (patents?) of developed tests.

The MALACTRES project will move our knowledge of ACT resistance forward in 2 complementary ways: 1) the project consortium will use its unprecedented access to DNA and RNA from parasites isolated from ACT-treated individuals, and from mosquitoes fed on blood from ACT-treated individuals, to identify and validate genetic markers for selective changes induced by ACT action; 2) The MALACTRES project will result in the development and validation of simple tests in new formats not only for these markers, but also for already established markers of relevance for which current detection methods are slow, laborious and technically demanding.

The work is a balanced blend of clinical field work, laboratory research and test development. The work will be linked to ongoing EU initiatives like European Developing Countries Clinical Trial Partnership (EDCTP).

Project Results:

For the RTD component of the project work was performed in three main areas: preparation and execution of clinical trials, test development and research towards candidate resistant markers. Developed molecular diagnostic tests, based either on PCR or NASBA principle, have been tested in single laboratory evaluations and ring trials, and tested that passed sensitivity and specificity criteria have been moved to phase II evaluations. First prototype tests for drug resistance markers have been developed and are under evaluation. Identification of possible ACT resistance confirming markers is making progress.

The work performed in work package 2, in which clinical trials comparing the efficacy of ACTs has been compared, is completed and there are indications for the development of resistance. Blood samples were collected for in vitro tests and this work is approaching its completion. Interesting clinical samples, i.e. parasite isolates that seems to be less responding to the therapeutic efficacy of ACTs, will be further genetically analysed in the framework of WP3 that has identified some interesting possible candidate resistance markers. The preparations for a second trial assessing the impact of ACT resistance on transmission of P. falciparum to mosquitoes have started and a detailed study protocol has been submitted to the ethical committees of Kenya (KEMRI) and the London School of Hygiene and Tropical Medicine.

Significant progress was made in the development and evaluation of low-tech diagnostic tests for malaria infection and the detection of potential resistance markers (work package 4). Based on an early prototype the following diagnostic test formats have been delivered:

1) Direct blood PCR-NALFIA system for the detection of Pan- Plasmodium based on the conserved region of the 18s gene was made. In this system an internal control based on the GAPDH human household gene was developed.
2) Multiplex PCR-NALFIA system (identification of 4 different malaria causing species
3) Various NASBA formats

Laboratory and Phase 1 evaluations (ring trials) of these formats have been performed and on the basis of the outcome of these it was decided to drop the NASBA format due to technical difficulties. System 1 and 2 are now being pushed forward in phase II trials in Burkina Faso (a P. falciparum environment) and Thailand (in an area where P. vivax is the dominating species).

Initial laboratory work towards the development of diagnostic microarray tests has started..

Finally, the first steps were taken to come up with a strategy for SNP detection in combination with low tech detection. As a prototype target the chloroquine resistance transporter has been chosen. This SNP has been extensively studied and can still be found in many Plasmodium populations. Up to this moment the target has been amplified with PCR and the traditional protocols have been reduced to an amplification time of 2 hours. Detection of the SNP is at this stage still performed by digestion with a restriction enzyme. However with an alternative enzyme (information can not be disclosed at this stage) complete digestion can be achieved in 5 minutes. The work is now also being extended to other resistance markers.

Work towards the IP situation around the project has made significant progress. The current expected test methodology for diagnostics is based upon two technologies: Nucleic acid sequence based amplification (NASBA) technology and PCR with resolution of the specific amplification products obtained via NASBA or PCR using nucleic acid lateral flow immuno assay (NALFIA) detector Lateral flow devices (LFDs).

PCR is now off patent and may be worth still considering as an alternative to isothermal amplification, however it does require a PCR machine with thermo-cycling capabilities, however these are far more readily available these days and often relatively cheap and some can carry out the PCR reaction in as little as 20-30 minutes and be fully portable. Developments and results from previous work packages indicate that PCR may be the most viable approach for exploitation. All other isothermals, with the exception of NASBA, require licensing and associated costs.

Additionally, following a meeting with development partners, the need to make the PCR part robust was identified, which led to investigations with numerous companies on ability to dry down PCR primers and amplification reagents. This is common practice for PCR, enabling much simpler and robust use in the field by end users. However we were unable to find any third parties who have previously dried down NASBA reagents, and several had severe concerns on if this could be achieved, particularly in the multiplex format described in MALACTRES. Standard PCR is the cheapest PCR methodology to commercialize.

Regarding the resolution of the PCR result in a simple, robust cost effective manner will be through the route of NALFIA (Nucleic Acid Lateral Flow ImmunoAssay) also referred to as NALFD (Lateral Flow Device) for detection of Amplicons using incorporated ligands. There appears to be an expired patent, EP 0 198 662 Method for nucleic acid hybridization, Published in 1986 by Amersham, indicating that the incorporated ligands approach to detection is free to use.

To ensure the NALFIA device is robust for use in the field the assay was converted form a dipstick version to a housed version. As previously reported we still believe that as it is a sandwiched based assay therefore infringes a group of established patents, specifically when the label is housed on the test device prior to sample addition, producing a homogenous assay. These are extremely contested area of patents and generally accepted that the most important portfolio of patents belongs to Inverness Medical innovations. However from April 2009 these patents have expired worldwide with the exception of the United States, and therefore are not considered to adversely prevent the use of this technique for MALACTRES.

The management work undertaken so far comprised the preparation of P1, P2 and P3 reports, organization together with project partners of project progress meetings in Burkina Faso, Kenya, Belgium, Nigeria and the UK, transfer of the P1 and P2 financing to the project partners, the maintenance of the project website and production and distribution of the external newsletter (in close collaboration with partners in the UK and Tanzania) and the communication between the various project partners and the commission. Furthermore, several dissemination activities were initiated.

Main results thus far:

The contractual and financial arrangements for the project are well in place. Grant agreement and consortium agreement have been signed by all beneficiaries and the financial resources have been distributed amongst the project partners. Several interactive and very informative project progress meeting have taken place (in Burkina Faso, Kenya, Belgium, Nigeria and the UK). The consortium has been able to present its activities already at several major conferences and active participation in more is being planned. Other dissemination activities include the establishment of the project website (at www.malactres.eu) and the publication of 4 external newsletters that have been send around to approximately 150 malaria experts all around the world. The first scientific publications arising from the work in peer reviewed journals are anticipated to be published in 2012. In total, the consortium expects to publish between 10 v-20 papers in total.

The scientific work has made considerable progress. Several clinical field studies have been completed by the consortium and, together with valuable clinical samples already in hand and those that will result from associated trials funded by EDCTP, will provide an important source for studying possible ACT (associated) resistance markers.

Genetic work has identified potential markers for resistance in partner drugs also and this information will be used for future test development.

Significant progress has been made in the development of diagnostic tests. Two tests have reached extensive phase 2 evaluations: A multiplex PCR-NALFIA Plasmodium test and a direct blood PAN-Plasmodium PCR-NALFIA test. These evaluations are currently completed in Thailand and Burkina Faso. Next, these tests will be further trialed in a phase 3 evaluation in Africa. The NASBA-NALFIA approach has been abounded as the test format is not robust enough.

The first prototype tests for drug resistance markers have been developed. A MLPA-based system is currently undergoing a full laboratory evaluation that includes clinical samples from MALACTRES trials.

A IP evaluation indicated that the consortium has the freedom to operate with the available technologies. An interesting development is the establishment of a direct blood PCR (circumventing the extraction of DNA from blood). Several industrial partners have shown interest in taking this technology further.

The MALACTRES consortium is on track with its research programme and anticipates an exciting coming period in which we will further work on discovering molecular markers for ACT resistance and develop appropriate diagnostic tests.

Potential Impact:

Although significant progress has been made in malaria control during the last decades, according to the World Health Organization (WHO) malaria remains a serious problem, particularly in sub-Saharan Africa, where approximately 90% of clinical cases occur. Malaria, either alone or in combination with other diseases, is estimated to kill almost 1 million people worldwide each year, and over 2400 million remain at risk. The Roll Back Malaria Programme recommendation number 4 from the 2000 annual program report reads: “Access to early diagnosis and effective treatment are essential parts of any malaria control effort. The four basic technical elements of the WHO’s Global Malaria Control Strategy are:

• To provide early diagnosis and prompt effective treatment of malaria;
• To plan and implement selective and sustainable preventive measures, including vector control;
• To detect early, contain or prevent epidemics;
• To strengthen local capacities in basic and applied research to permit and promote the regular assessment of a country’s malaria situation, in particular the ecological, social and economic determinants of the disease.

The MALACTRES project concentrates on the first of these elements: Early Diagnosis and Effective treatment. As most cases of malaria are still treated on the basis of clinical diagnosis, it is inevitable that many patients without the disease receive anti-malarial drugs. This causes unwarranted costs for patients/health services, the risk of side effects and increase of potential drug resistance. Conversely, lack of sensitivity may cause some patients suffering from malaria not to be treated, thereby risking progression to severe disease and death. The MALACTRES project will provide new sensitive and specific diagnostic tools, which will enable the rapid screening of hundreds of patients a day, making each test cheap and easy to execute possibly even in resource poor laboratories as sophisticated equipment and a constant source of electrical power will probably not be needed. The employed methodologies will also offer advantages in terms of safety, as the tests will not require toxic chemicals for its execution. Adequate treatment requires the use of effective drugs. Affordable and effective drugs, such as chloroquine, have been lost for the communities, due to the emergence of resistance. Artemisinin-based combination therapies (ACTs) should not be lost due to drug resistance, as suitable alternative combinations drugs will not be available within the next decade. Many African countries, and in the near future probably most, have chosen ACTs as first and second line treatment. It is important to continuously monitor ACTs efficacy so that early signs of resistance are promptly detected. This consortium has chosen to do this by conducting large scale field trials and to link the work with ongoing trials, like for example those sponsored by the EU in the framework of EDCTP. The work conducted in the MALACTRES project will result in important information on the possible existence of ACT resistance in Africa and will allow proper action to be taken towards preserving these drugs for malaria treatment and to ensure that proper treatment can be given. The information will be disseminated to appropriate authorities involved in malaria control on national (malaria control programmes, ministry of health etc.) as well as international (WHO) level. Furthermore, information on the molecular background of ACT resistance will receive much interest from the scientific community and will have a high impact on malaria research focusing on drug resistance. It will also lead to development of novel instrumentation that can be used to monitor emerging drug resistance. The project as a whole will have a significant impact on society’s ability to manage, control and eventually eradicate malaria. For the first time, reliable means not requiring operator expertise and sophisticated equipment for the detection of an individual malaria infection and/or presence of mutations conferring possible drug resistance will become available. In those countries where malaria is endemic, introduction of this technology will offer an extremely rapid and safe means of screening a large numbers of individuals and/or blood samples, possibly to be used within the context of monitoring drug efficacy and shortening the patient’s follow up. Indeed, an extremely sensitive method could detect after treatment low parasite densities that could be predictive of drug failure. If this was confirmed, it could enormously simplify the monitoring of drug efficacy.

List of Websites:

www.malactres.eu

Other report summaries

• Periodic Report Summary 1 - MALACTRES (Multi-drug resistance in malaria under combination therapy: Assessment of specific markers and development of innovative, rapid and simple diagnostics)