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The Leishmania genotyping project was born from the realisation that a molecular approach would give very detailed insight into the epidemiology of leishmaniasis in Europe. The project was initiated from the London School of Hygiene and Tropical Medicine (LSHTM), where a PhD programme had explored some of the genetic diversity in the Leishmania donovani and Leishmania infantum complex. As part of that research we became aware of others with similar interests and preliminary collaborative contacts were made with European investigators.

Accordingly, the broad aim of the project was defined as devising “diagnostic and epidemiological markers for tracking of endemic and resurgent European leishmaniasis”. This would be done through exploiting recent advances in molecular and phylogenetic methods. Several European partners were invited to join the project, according to their experience and expertise, and eight enthusiastically agreed to participate.

Briefly, the objectives of the project were established as :

1. Amplification and sequencing of selected Leishmania DNA targets that might provide epidemiological tools.
2. Construction of comparative phylogenies based on the sequence data.
3. Identification of new genotypic markers.
4. Evaluation of the specificities and sensitivities of the markers with different assay procedures and on clinical and veterinary samples.
4. Pilot application of the genotypic markers to epidemiological questions, such as: the extent of genetic isolation of Leishmania populations in European endemic foci; the role of dogs in sustaining genetic diversity; the comparative genetic diversity of Leishmania among HIV positive and HIV negative patient groups; the distinction of re-infection and relapse infections in the immunocompromised.
5. Construction of a database and website to carry outputs of the project.

The partnership provided a complementary and synergistic profile. Broadly, yet not exclusively, the UK and Belgian partners brought prior experience in PCR-based and DNA sequencing methods, the German partner provided crucial expertise in microsatellites, the Czech Republic had a strong track record in molecular taxonomy and phylogenetics, the Spanish, Portuguese and Greek partners had a variety of strengths and interesting epidemiologies to explore, and the French partner had extensive experience of previous European collaborations in the context of characterising zymodemes, the current gold standard for identification of Leishmania strains.

The project was divided into 10 workpackages and four principal phases, which overlapped. In the preparatory phase panels of Leishmania reference strains and isolates were assembled, grown in bulk, DNA extracted and distributed to relevant partners. The second phase focused firstly on the sequencing of the DNA targets, including housekeeping genes encoding enzymes used in multilocus enzyme electrophoresis (MLEE), a few antigen coding genes, intergenic regions, and markers detected through random amplification of polymorphic DNA (RAPD), and secondly, on the development of microsatellite methods. The sequence data from the enzyme genes were used to examine the genetic basis of isoenzyme mobility differences seen by MLEE, and sequences from all targets were incorporated into phylogenetics analysis. In the third phase the most promising genotypic markers were assessed as epidemiological tools, and procedures for using the markers were compared. In the fourth phase pilot epidemiological studies were conducted in Spain, Portugal and Greece.

The partners were privileged to meet together, approximately every 6 months, for a series of stimulating workshops, in Crete, Berlin, Madrid, London, Ceske Budjovice, Antwerp and Lisbon. In addition there has been a series of bilateral visits and working exchanges, particularly involving the PhD students who have undertaken their training within the framework of the project. The successful midterm review of the progress took place in conjunction with the workshop in London. The final meeting is due to take place in September 2005 in Montpellier.

The project has so far achieved the vast majority of its objectives. This is concisely illustrated by referring to the titles of the partners’ many presentations at Worldleish 3 in Sicily, in April 2005. Further details have been or will be published elsewhere. Here is a summary of some of the outputs of the project:

1. The provision of DNA from a reference panel of Leishmania DNA extracts.

2. Amplification and sequencing of 10 single copy housekeeping genes, encoding enzymes used in MLEE, for a panel of European and some non-European L. donovani and L. infantum isolates. This has revealed genetic polymorphisms that are not detected by MLEE, explained many mobility differences on MLEE, provided characteristics that split the zymodeme MON1, which predominates in Europe, and provided the basis for high resolution alternatives to MLEE.

3. Provision of a panel of microsatellite markers for L. infantum and L. donovani, including panel the splits the zymodeme MON1, for high resolution epidemiological analysis. Microsatellite markers have been applied to more than 200 isolates.

4. In parallel with enzyme encoding genes and microsatellites, comparative analyses of several intra and intergenic regions of antigen encoding genes, with an assessment of the research and field applications of the corresponding genotypic markers.

5. Detection of heterozygosity and mosaic genes, suggesting that genetic exchange might occur among some L. donovani populations.

6. Development and comparison of several procedures for applying genotypic markers, either in the research laboratory, or in a diagnostic/field context.

7. PCR-based specific and sensitive diagnostic tests.

8. Development of real-time PCR-melting curve analysis (MCA) for L. infantum and L. donovani.

9. Multiple phylogenetics analyses, with DNA sequence, restriction fragment polymorphism (RFLP), microsatellite and RAPD data, revealing robust genetic groups within the L. donovani and L. infantum complex, giving insight into relationships between the groups, and demonstrating the need for some taxonomic revision.

10. Most importantly, understanding of the molecular epidemiology of leishmaniasis in Spain, including the Balearic Islands, in Portugal and in Greece, encompassing canine leishmaniasis, and HIV-associated leishmaniasis.

11. Training of several PhD students.

12. Construction of the database and website.

In addition, the project has played a significant role in motivating other collaborative research proposals and networks that are relevant to the leishmaniases. These new enterprises do not only concern Europe but also the wider Mediterranean region (Leishmed), Africa (TryLeiDiag) and South America (LeishEpiNetSA).

Above all, a most rewarding characteristic of this European Commission Leishmania genotyping project has been the generous, spirit of true and open collaboration and synergy between the partners, without confrontation and without competition. Long lasting friendships and working relationships have been forged. This bodes well for projects that follow a similar model. Furthermore, it strengthens the view that such international networks can have a genuine impact on scientific progress and the improvement of public health and, through their strong consensus, can have a realistic influence on policy development, both within and beyond Europe.