**1. Introduction**

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Protozoan parasites of the genus *Leishmania* are responsible of a large variety of clinical manifestations ranging from self-healing cutaneous forms (CL), through mucocutaneous lesions (MCL), to lethal if untreated visceral disease (VL). Nevertheless, there is no absolute correlation between a particular clinical form and a causative species [1]. For instance, parasites of the *L. donovani* complex are generally responsible for VL cases in the Old and New World but can also cause CL. Another example is the *L. tropica* species, which causes a CL form but its association to VL cases was occasionally reported [2]. Identification of *Leishmania* parasites is a central issue to patients' management and to control. Leishmaniases have a worldwide distribution but only absent in the poles regions, and in Australia where in spite of presence of the parasites in Kangourous no human cases were described. According to the WHO, 350 million people are at risk, with a prevalence of 12 million and more than 98 countries affected [3]. More than twenty species are responsible for leishmaniasis in humans. However, *Leish‐ mania* species present very similar morphologies in their flagellated, promastigote forms and their intracellular, amastigote forms which renders necessary the use of molecular or bio‐ chemical assays for their identification and characterization (see for review [4]).

The current identification and classification of *Leishmania* is still based on isoenzyme typing, using multilocus enzyme electrophoresis (MLEE) (reviewed in [5]). This approach has been widely used for the identification of *Leishmania*, but several limitations were reported. Most importantly, differences in electrophoretic profiles were shown to be due to heterozygosity at a single nucleotide position [6–8]. Molecular studies showed also that zymodemes included distinct DNA genotypes [7,9]. Consequently, other molecular studies do not always agree with the classification of *Leishmania* by MLEE. The other limitations of MLEE are that it requires

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bulk cultures of parasites, it is time-consuming, and it can be performed only in specialized laboratories. Therefore, alternative DNA based tools and assays are increasingly developed and used for effective investigation and characterization of the parasites.

Indeed, the diversity of *Leishmania* species, their vectors and their reservoir hosts is a main feature of leishmaniasis, so consequently the transmission cycles are very much dependent on the environment and are very prone to changes. So not only parasite identification is needed to establish etiology of the disease and understand the pathogeny, but knowledge of parasite diversity and its population structure is also needed for a better understanding of ecoepidemiology and its changing trends. For this purpose, molecular tools have been developed to allow differentiation of *Leishmania* parasites at species and strain levels within environmen‐ tal or patients' samples.

Molecular tools are based mainly on the amplification and subsequent restriction fragment length polymorphism (PCR-RFLP) of several targets including repeated gene families and coding and non-coding regions, or the sequence analysis of the products. Recently multilocus sequence typing (MLST) and multilocus microsatellite typing (MLMT) were also developed for *Leishmania* DNA typing. Each of these molecular markers or tools has its specific discrim‐ inatory power, advantages and drawbacks.
