Experimental studies about Leishmania resistance to metal and antifolates have pointed out that gene amplification is one of the main mechanisms of drug detoxification. Amplified genes code for adenosine triphosphate-dependent transporters (multidrug resistance and P-glycoproteins P), enzymes involved in trypanothione pathway, particularly gamma glutamyl cysteine synthase, and others involved in folates metabolism, such as dihydrofolate reductase and pterine reductase. The aim of this study was to detect and quantify the amplification of these genes in clinical strains of visceral leishmaniasis agents: Leishmania infantum, L. donovani, and L. archibaldi. Relative quantification experiments by means of real-time polymerase chain reaction showed that multidrug resistance gene amplification is the more frequent event. For P-glycoproteins P and dihydrofolate reductase genes, level of amplification was comparable to the level observed after in vitro selection of resistant clones. Gene amplification is therefore a common phenomenon in wild strains concurring to Leishmania genomic plasticity. This finding, which corroborates results of experimental studies, supports a better understanding of metal resistance selection and spreading in endemic areas. 1. Introduction Visceral leishmaniasis (VL) constitutes a public health problem in East Africa, Asia, Mediterranean basin, and Central and South America. It is estimated that about 500？000 new cases occur each year. In East Africa and India, VL cause large-scale and tenacious epidemics with high case-fatality rates [1]. In numerous countries that cannot afford the cost of liposomal amphotericin B, first line treatment still relies on sodium stibogluconate or N-methyl glucamine [2]. Cases refractory to antimony treatment have been described for a long time in humans [3] as well as in dogs [4]. During the last decades, incidence of resistance to antimony drugs in Leishmania spp. increased dramatically in some VL foci [5]. The mechanisms contributing to drug resistance in vivo are poorly understood. In vitro studies of strains selected for metal resistance showed that several independent pathways could concur to the resistance phenotype [6]. Among them, overexpression of energy dependent transporters seems to play a major role in resistance to antimonials (reviewed in [7]). Genes coding for “ATP binding cassette” (ABC) transporters [8] and multidrug resistance (MDR) genes [9] have been shown to be amplified as extrachromosomal elements in strains selected in vitro for resistance to heavy-metals. Some works showed that
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