Intracellular amastigote replication may not be required for successful in vitro selection of miltefosine resistance in Leishmania infantum

Abstract

Although miltefosine (MIL) has only recently been positioned as a first-line therapeutic option for visceral leishmaniasis, field reports note an increasing trend in treatment failures. Study of laboratory selected MIL-resistant strains is needed in the absence of confirmed resistant clinical isolates. In contrast to promastigotes, experimental in vitro selection of MIL-resistance on intracellular amastigotes has not yet been documented. This study reports for the first time the selection of MIL-resistance in Leishmania infantum LEM3323, a strain which clearly shows active intracellular replication. Starting from the hypothesis that active multiplication may be essential in the resistance selection process; several other L. infantum strains were evaluated. Although strain LEM5269 showed only marginally lower intracellular multiplication, selection for resistance failed, as was also the case for several other strains showing poor or no intracellular replication. These results suggest that intracellular multiplication may not be an absolute prerequisite for the outcome of experimental in vitro MIL-resistance selection in clinical field isolates.

Genomic and Molecular Characterization of Miltefosine Resistance in Leishmania infantum Strains with Either Natural or Acquired Resistance through Experimental Selection of Intracellular Amastigotes

“…The parent clone of L . infantum (MHOM/FR/96/LEM3323 Cl-4) was subjected to resistance selection on intracellular amastigotes, as previously described [ 13 , 17 ]. The resistance selection cycles were repeated until the arbitrarily set cut-off value of 15 μM for MIL-resistance on amastigote level was achieved [ 23 ].…”

“…The selected resistant population was cloned again using the micro-drop method and one clone was randomly selected to perform all experiments [ 13 ]. A stable MIL-resistant phenotype (LEM3323-MIL) had already been experimentally selected on intracellular amastigotes of the parent LEM3323 strain [ 17 ].…”

“…In the present study, the experimentally selected MIL-resistant L . infantum strain LEM3323 [ 17 ] was subjected to an in-depth phenotypic and molecular characterization in direct comparison to its drug-susceptible wild-type parent counterpart. To further validate the in vitro intracellular amastigote resistance selection assay [ 13 ], the naturally MIL-resistant L .…”

Phenotypic adaptations of Leishmania donovani to recurrent miltefosine exposure and impact on sand fly infection

“…No significant differences could be observed between WT-and MIL-exposed strains. The represented data are combined from 2 independent experiments with a minimum of 10 flies for each group and time point amastigotes both in vitro and in vivo failed to result in a drug-resistant phenotype [15,34], creating the possibility to assess the impact of repeated MIL exposure without the alterations in parasite fitness as observed with full MIL resistance [13,14,29]. In the present study, recurrent in vitro and in vivo MIL exposure only resulted in a marginal decrease in promastigote susceptibility, which corroborates other studies describing the existence of an 'intermediate' resistant phenotype characterized by a partial decrease in susceptibility as a step towards full resistance [31].…”

Combined treatment of miltefosine and paromomycin delays the onset of experimental drug resistance in Leishmania infantum

“…donovani [35]. Although both a MIL- and a PMM resistant phenotype could be generated in vitro within 5 drug selection cycles [13, 21], no shift in susceptibility was observed upon in vitro drug combination exposure (Table 2). Similar to MIL resistance selection on other strains, a shift in promastigote back-transformation could be observed, indicating that parasites were able to resist elevated drug concentrations [11].…”