Quality of Life and Management of Living Resources Programme

" Diagnostic and epidemiological markers for tracking of endemic and resurgent European leishmaniasis " (Leishmania genotyping).
 

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Leishmaniasis

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OBJECTIVES AND EXPECTED ACHIEVEMENTS

Objectives

The present proposal aims to :
1. exploit recent advances in molecular and phylogenetic techniques, speciality information from genome sequencing efforts, for the development of Leishmania genotyping.
2. assess applicability of new Leishmania genotyping to address key epidemiological questions.
More specifically this proposal aims to :
1. optimise Leishmania taxonomy and identify genotypic markers:
1.1. amplify and sequence selected DNA targets (housekeeping genes, metabo1ic enzyme coding genes, antigen-coding genes, intergenic regions, microsatellites, anonymous PCR markers):
1.2. construct comparative phylogenies from DNA sequence and allozyme data;
1.3. identify genotypic markers.
2. produce new genotyping methods based on selected genotypic markers, developed for reliability, speed, simplicity and capability for direct Leishmania typing from biological samples.
3. evaluate typing methods:
3.1. assess sensitivities and specificities.
3.2. assess applicability to clinical and veterinary samples of different origin.
3.3. assess applicability of genotyping methods to addressing key epidemiological questions, such as:
3.3.1. the extent of genetic isolation / gene flow between Leishmania geographical populations;
3.3.2. the role of reservoir hosts in transmission and in sustaining genetic diversity;
3.3.3. comparative Leishmania genetic diversity among HIV positive and among HIV negative patient groups;
3.3.4. the origins of new Leishmania zymodemes in HIV co-infected patients;
3.3.5. differential identification of re-infection or relapse in treated patients, especially the immunocompromised ;
3.3.6. detection of genetic recombination in Leishmania.
4. Construct and fun an European and International database and website for genotypic markers

 

Expected achievements

1. DNA sequence data from selected targets for reference Leishmania strains.
2. High-resolution molecular phylogenies from sequence data.
3. Assessment of robustness of allozyme typing in comparison with DNA based molecular phylogenetic analysis for each enzyme, and for several enzymes.
4. Optimised Leishmania taxonomy following extensive statistical, meta, and consensus phylogenies.
5. New clade specific genotypic markers.
6. New highly variable genotypic markers: microsatellites, intergenic regions.
7. New, reliable genotyping methods, which will be rapid, specific, sensitive and an alternative to or complement allozyme analysis.
8. Data on suitability of genotyping methods for elucidation of key epidemiological questions of topical concern: extent of genetic diversity and gene flow, transmission between and from reservoirs or human hosts, genetic recombination in Leishmania.
9. Preliminary answers to the above epidemiological questions.
10. Establishment of an accessible European and International database and website for genotypic markers.

PROJECT WORK PLAN

The project will focus on multi-gene DNA sequence characterisation of Leishmania which will consider different levels of sensitivity according to clinical or epidemiological needs: species (with associated clinical risk), deme (associated clinical risk and eco-epidemiology), strain (epidemiological tracking). Extensive sequence data will be used to construct phylogenies of the genus Leishmania., with special emphasis on L. infantum and relationship to the close species., L.donovani. Phylogenies will be compared with those based on isoenzyme electrophoresis and will provide a benchmark for the development of rapid., specific and sensitive molecular methods for typing the aetiological agent of visceral leishmanisis in Europe., L. infantum. Markers for typing other Leishmania species, detected in Europe from travel associated cases., may also be identified. The use of highly polymorphic DNA regions to help elucidate key epidemiological questions of topical concern will be evaluated with small field studies. Along with other aspects of the project, a Leishmania typing database will be constructed as a major output of aIl the data collected here.

Stocks representative of the known genetic and phenetic diversity among L. infantum and representatives of other Leishmania species will be selected for sequencing of selected DNA targets.

DNA target have been chosen according to the potential level of taxonomic discrimination and biological characterisation. We thug have house-keeping genes (genus to species discrimination); metabolic enzymes (genus to subspecies discrimination); intergenic regions (species to subspecies discrimination); antigens (expected to give mainly subspecies discrimination due to immune pressure); microsatellites (subspecies and strain discrimination). Anonymous targets were also considered. An excessive initial number of DNA targets has been chosen but only the most informative will be selected for further studies after testing with a limited number of reference stocks. The main strategy for identification of target sequences for amplification will be searches of sequence databases and design of specific primers based on sequences flanking the targets.

DNA sequence data will be used to build Leishmania phylogenies, with special emphasis on L. infantum. Phylogenies will be used to identify genetic markers at subgenus, species, subspecies and zymodeme levels. Typing methods will be developed according to the characteristics of each genetic marker to maximise sensitivity, specificity and throughput.

Applicability of the most polymorphic markers will be tested for answering fundamental epidemiological questions by bath phylogenetic based methods and standard population genetics:
--- structure of foci: Can humans be reservoirs or are they end hosts ?
Do parasites undergo genetic recombination in nature ?
Sympatry between distant and adjacent foci ?
--- HIV co-infection: Are the newly found L. infantum zymodemes present in other groups than the immunocompromised ?
Are those zymodemes present only in asymptomatic immunocompetent hosts ?
Do new zymodemes arise in the immunocompromised host ?
Are intravenous drug users the reservoir in a vector independent cycle ?
What are the epidemiological implications of HIV/Leishmania cases in the community ?
--- clinical problems : are there specific drug resistance strains that can be identified by genetic markers ?
Are some cases of relapses, in fact re-infections ?
--- divergence between L. infantum strains and origin of diverse genotypes.
The large volume of information generated in this project will be managed by an Internet accessible database, which will be created to include several aspects of Leishmania strain typing. The Leishmania typing database will collaborate with other relevant organisations, such as the current Leishmania database, the MLST database and the Leishmania genome sequencing project.
Throughout the project, some tasks will be divided between some partners without redundancy. These tasks will, therefore, be more quickly completed: for example, sequencing of some regions, protocol development, quality control, epidemiological studies.

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Leishmaniasis

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