Background

Chemotherapy is critically important in reducing the burden of leishmaniasis and the first line treatment for all clinical forms is pentavalent antimonials (SbV) such as sodium stibogluconate (Pentostam) and meglumine antimonite (Glucantime) (ref. 1). The increasing unresponsiveness to SbV, is a major concern. In Bihar (district of India), the most serious documented case of chemotherapy failure, 60% of the visceral leishmaniasis (VL) patients do not not respond to SbV (ref. 2). In Nepal, we found 10% unresponsiveness (ref. 3). A similar dramatic situation was observed in mucocutaneous leishmaniasis (MCL) where 30-40% SbV unresponsiveness to first round of treatment was observed. These patients however responded to new series of treatment but with a curative rate of only 20% (ref. 4).

The geographical and temporal grouping of SbV treatment failures suggests the emergence of antimony resistant strains. Indeed in Muzaffarpur (Bihar) SbV resistant Leishmania isolates were identified and a correlation was found between clinical outcome of SbV treatment and in vitro SbV sensitivity of corresponding L. donovani isolates (ref. 2;5). Alternative drugs are amphotericin B, paronomycine and the oral drug Miltefosine (hexadecylphosphocholine). Amphotericin B is a poorly tolerated and unpractical drug, while the lipid formulation, AmBisome is less toxic but too expensive. Miltefosine and paromomycine are promising alternatives but in vitro resistance to Miltefosine has already been demonstrated (ref. 6). All other tried drugs like ketoconazole or allopurinol failed against both CL and VL. A report (ref. 7) showed that generic SbV made in developing countries (1/14th of cost) is just as good as the expensive patented product. Therefore, closely monitoring and optimizing the use of SbV is expected to have a positive impact on the control of leishmaniasis.

Apart from emerging SbV resistance, there are many other possible causes for therapeutic failure. A summary is given in the table below.

On the level of the parasite:	On the level of treatment:	On the level of the host:
intrinsic insensitivity (i.e. species) drug resistance of the parasite ...	drug quality treatment compliance dosage ...	clinical presentation characteristics of the patient immunological response host genetics ...
Table: Possible causes of treatment failure

In this project, we will attempt to verify most of the listed possible causes, with a particular focus on the study of the emergence of SbV resistant parasites.

Determination of SbV sensitivity of Leishmania is not an easy task. Using radiorespirometric microtechniques (ref. 8) or radiolabelled thymidine incorporation inhibition assays (ref.9), it was possible to correlate drug tolerance with chemotherapy failure (ref. 5;10). The current method of choice is a in vitro biological assay in which inhibitory SbV doses are determined on amastigotes within cultured macrophages; however this method is expensive, cumbersome and time-consuming (ref. 11). A rational alternative would be to identify molecular markers associated to the SbV resistant phenotype and use those markers for the development of molecular tools to detect emerging or spreading SbV resistant strains. The limiting step is to find genetic markers that are linked to the SbV resistant phenotype under natural conditions.

Up to date all genetic markers reported in scientific literature corresponded to non-natural drug resistant Leishmania parasites. They were grown in laboratory conditions and adapted by discrete steps of increasing drug concentrations (ref. 12-16). In many cases the resistant phenotype was stable after several generations, even in the absence of the drug. In theory, this situation would not be different from that found in humans or other mammals, where suboptimal doses are administrated. In domestic mammals (dogs, cattle and mice) there is strong evidence that trypanosomatid populations are able to acquire resistance after exposure to subtherapeutic drug doses (ref. 17-19). Nevertheless, it has not been verified yet whether the mechanisms described for laboratory strains are also present in natural resistant parasites.

Studies on laboratory induced SbIII(or related AsIII) resistant Leishmania have revealed the following mechanism:

1) Reduced uptake of SbIII It was shown that the route of uptake of SbIII is mediated by an aquaglyceroporin (AQP1), a protein that also catalyses SbIII uptake in yeast and mammals (ref. 23;24). A recent study has shown that induced SbIII resistance correlates with a lower expression of AQP1, and this was shown to be associated with a lower intracellular SbIII accumulation (ref. 25). 2) Increased detoxification of SbIII The metal detoxification pathway in Leishmania basically consists of two steps: (i) conjugation of metals to thiols and (ii) direct export and/or sequestration of the metal/thiol conjugate. Both steps were shown to be upregulated in induced SbIII resistant Leishmania:

• Increased thiols: An increase of intracellular thiol levels (trypanothione & glutatione) was found in induced SbIII (and related AsIII) resistant Leishmania, the increase was caused by an overexpression or amplification (as extrachromosomal elements) of GCS, GS & ODC, genes involved in the biosynthesis of thiols (ref. 26-31).
• Increased sequestration: The gene ABCC1 codes for MRPA (PGPA), and was found to be amplified or higher expressed in induced SbIII resistant strains. Its role in resistance was proven by gene transfection. MRPA is located in the membrane of an intracellular organelle and was shown to transport SbIII conjugated to thiols. It seems that MRPA confers resistance by sequestration of the SbIII-thiol conjugates (ref. 26;29;32).

All the genes reported to be involved in induced SbIII resistance are candidate markers for natural SbV Leishmania resistance and will be studied in the project.

Spreading of natural drug resistance is not understood. It may depend on several factors: disease transmission cycle, parasite reproduction mechanisms, stability of the resistant phenotype in drug absence and virulence of resistant parasites:

• Anthroponosis and zoonosis imply different transmission cycles that will be explored in this project. The drug selective pressure is different under these two conditions.
• Reproduction of Leishmania is essentially clonal, but sexual exchanges might occur rarely (ref. 33). These parasites have extrachromosomal genetic elements but horizontal transfer has never been encountered.
• Stability of the resistance phenotype is dependent on the level of resistance induced, and highly resistant parasites could be transmitted from host to host without alteration of the resistance level (ref. 34). The isolation of naturally resistant parasites from sylvatic vectors (ref. 35) might be explained by stability of resistance phenotype, or by the occurrence of primary resistance (without requiring of a progressive adaptation of parasites to high SbVs concentrations.
• Virulence may be influenced by the resistant genotype. This possibility is supported by the report of Chang et al. (ref. 36): tunicamycin-resistant lines of L. amazonensis showing amplification of N-acetyl glucosaminyl transferase gene are also more virulent (larger lesions in BALB/c mice and faster growth rate as well as more amastigotes in macrophage model). A population genetics approach can address these questions as has been demonstrated for P. falciparum, in which all isolates (out of 11) resistant to mefloquine/halofantrine showed amplification of pfmdr1, but a totally different genotype (ref. 37).

Objectives

A. General Objective

The main objective of this project is to design molecular tools for gathering reliable information about the emergence and spreading of drug resistance in visceral and tegumentary leishmaniases. The quality of information provided by these tools is essential for surveillance strategies and rational policy development concerning SbV treatment.

B. Specific Objectives

• To obtain a representative collection of biopsies and parasite isolates from patients susceptible or refractory to SbV treatment in areas endemic for anthroponotic visceral (AVL) and zoonotic mucocutaneous leishmaniases (ZML).
• To identify genomic and functional modifications associated with SbV resistance in field isolates.
• To develop molecular tools to diagnose natural drug resistance.
• To identify the population structure of sensitive and drug resistant parasites for understanding the epidemiological dynamics of drug resistance.

Workplan

The objectives will be achieved through a multidisciplinary approach involving a field and laboratory component interacting on biochemistry, molecular tools and population genetics.

Deliverables

The specific deliverables we want to attain through this project are listed below:

• Documented biopsies and isolates from susceptible and refractory patients
• Information on the current status of SbV resistance in Nepal, Bolivia and Peru
• Information on basic immunological parameters in susceptible and refractory patients
• Panel of SbV resistant isolates from Nepal, Bolivia and Peru and corresponding standard sensitive ones
• List of possible new genes involved in SbV drug resistance
• Description of functional mechanisms used by SbV resistant Leishmania
• Demonstration of qualitative and/or quantitative variations of genetic markers in natural SbV resistance, which might constitute targets to develop diagnostic tools
• Guidelines for diagnosis and surveillance of drug resistance like (i) determination of the stage at which drug resistance detection method should be performed, before treatment (if resistance is pre-existing) or in the course of it (if resistance is an adaptive answer of the parasite to the treatment, f.i. in case of gene amplification), and (ii) definition of surveillance strategies according to the role of transmission cycle in drug resistance spreading
• Reference cryobank of isolates from SbV refractory and susceptible patients
• Oligonucleotides and protocols capable to discriminate between sensitive and resistant natural isolates
• Assessment of the sensitivity, specificity, Predictive Positive Value and Negative Predictive Value of developed tools
• Simplified protocol for SbV resistance diagnosis
• Information on population structure of sensitive and resistant isolates in Nepal (AVL) and Bolivia/Peru (ZCL)

Role of Partners

ITMA is responsible for (i) co-ordination of activities and project management , (ii) central cryopreservation of all isolates and (iii) population genetics studies. ITMA also contributes to the search of molecular markers and the design of detection tools, brings epidemiological support to the consortium and will actively participate in the writing of guidelines for surveillance of drug resistance.

Four partners are responsible for the fieldwork: BPKIHS leads the follow-up study of SbV treated patients in anthroponotic conditions (Nepal). This occurs in close collaboration with HUG, who is responsible for coordinating the recruitment and standardizing the management of patients and data collection in the field. In addition, HUG is responsible for immunological studies. CUMETROP and IMTAvH lead the follow-up study of SbV treated patients in zoonotic conditions (Bolivia and Peru, respectively). The three field partners exchange their respective clinical expertise in management of drug resistance. They provide documented biopsies and isolates for laboratory work packages.

LSHTM is responsible for (i) the production of parasite batches for in vitro SbV sensitivity assays and molecular analyses, (ii) the assays themselves, (iii) studies of selected metabolic pathways, and (iv) production of revertants from naturally resistant parasites. LSHTM provides sensitive and resistant isolates for molecular analyses of drug resistance mechanisms for population genetics. IMTAvH interacts with LSHTM for the development of functional assays, and is in charge of (i) molecular analyses of reported drug resistance mechanisms, and (ii) discovery of new ones. IMTAvH leads the search for molecular tools and will participate to the evaluation of the tools on human biopsies, in collaboration with field partners.