<i>In Vitro</i> and <i>In Vivo</i> Miltefosine Susceptibility of a <i>Leishmania amazonensis</i> Isolate from a Patient with Diffuse Cutaneous Leishmaniasis

<div><p>Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against <i>Leishmania donovani</i> has been well documented. <i>Leishmania amazonensis</i> is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the <i>Leishmania amazonensis</i> line isolated from this patient, who had not been previously treated with miltefosine. <i>In vitro</i> tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than <i>L. amazonensis</i> type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the <i>miltefosine transporter</i> gene or in the <i>Ros3</i> or <i>pyridoxal kinase</i> genes. These analyses were conducted in parallel with the characterization of <i>L. amazonensis</i> mutant lines selected for miltefosine resistance using a conventional protocol to select resistance <i>in vitro</i>, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the <i>miltefosine transporter</i> gene. <i>In vivo</i> studies were also performed to evaluate the correlation between <i>in vitro</i> susceptibility and <i>in vivo</i> efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the <i>L. amazonensis</i> type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated <i>miltefosine transporter</i> gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between <i>in vitro</i> susceptibility determinations and response to chemotherapy <i>in vivo</i>. This study contributed to establish that the miltefosine transporter is essential for drug activity in <i>L. amazonensis</i> and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.</p>