This study also demonstrates that resistance gene overexpression occurred more frequently than gene amplification, as observed in 11 out of 24 in vitro resistant isolates and 2 out of 23 in vitro susceptible isolates. Gene overexpression has been reported to occur as a consequence of gene amplification, which was initially described as a major feature associated with resistance at least within in vitro-selected mutants (7). This finding was not confirmed in our series, as gene amplification was rarely concomitant with gene overexpression. Indeed, their association was observed in only 4 isolates. According to Guimond et al., gene overexpression without gene amplification is more likely the consequence of enhanced mRNA stability than transcriptional up regulation, as no transcription promoters have yet been identified in Leishmania (15). Curiously, we also observed gene amplification in the absence of gene overexpression in 3 isolates. Although the occurrence of gene amplification without gene overexpression has been reported (15, 47), these findings are uncommon, and the significance of this isolated gene amplification remains uncertain.