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Background: Miltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency., Methodology/principal Findings: To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production., Conclusions/significance: Differential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Miltefosine is currently the only oral drug for visceral leishmaniasis, and although deficiency in an aminophospholipid/miltefosine transporter (MT) is sufficient to elicit drug resistance, very few naturally miltefosine-resistant (MIL-R) strains have yet been isolated. This study aimed to make a detailed analysis of the impact of acquired miltefosine resistance and miltefosine treatment on in vivo infection.Methods: Bioluminescent versions of a MIL-R strain and its syngeneic parental line were generated by integration of the red-shifted firefly luciferase PpyRE9. The fitness of both lines was compared in vitro (growth rate, metacyclogenesis and macrophage infectivity) and in BALB/c mice through non-invasive bioluminescence imaging under conditions with and without drug pressure.Results: This study demonstrated a severe fitness loss of MT-deficient parasites, resulting in a complete inability to multiply and cause a typical visceral leishmaniasis infection pattern in BALB/c mice. The observed fitness loss could not be rescued by host immune suppression with cyclophosphamide, whereas episomal reconstitution with a wild-type MT restored parasite virulence, hence linking parasite fitness to MT mutation. Remarkably, in vivo miltefosine treatment or in vitro miltefosine pre-exposure significantly rescued MIL-R parasite virulence. The in vitro pre-exposed MIL-R promastigotes showed a longer and more slender morphology, suggesting an altered membrane composition.Conclusions: The profound fitness loss of MT-deficient parasites most likely explains the low frequency of MIL-R clinical isolates. The observation that miltefosine can reverse this phenotype indicates a drug dependency of the MT-deficient parasites and emphasizes the importance of resistance profiling prior to miltefosine administration. [ABSTRACT FROM AUTHOR]

Background: Since miltefosine monotherapy against visceral leishmaniasis (VL) caused by Leishmania donovani has been discontinued in the Indian subcontinent due to an increase in the number of treatment failures, single dose liposomal amphotericin B is now advocated as a treatment option of choice. Paromomycin-miltefosine combination therapy can be used as substitute first-line treatment in regions without cold-chain potential. Previous laboratory studies in the closely related species Leishmania infantum have demonstrated that paromomycin monotherapy fairly rapidly selects for resistance producing a phenotype with increased fitness. Given the possible clinical implications of these findings for the current field situation, the present study aimed to identify the potential hazards of paromomycin-miltefosine combination therapy. Principal findings: Drug interaction studies using the fixed-ratio isobologram method revealed an indifferent interaction between paromomycin and miltefosine. In hamsters infected with L. infantum, the combination resulted in cumulative efficacy in reducing parasite burdens in the liver, spleen and bone marrow. Selected resistant lines against the single drugs did not display cross-resistance. When the intracellular amastigote stage was repeatedly exposed to the paromomycin-miltefosine combination, either in vitro or in vivo, no significant susceptibility decrease towards either drug was noted. Conclusions: These results suggest that implementation of paromomycin-miltefosine combination therapy indeed could represent a safe and affordable treatment option for L. donovani VL as miltefosine appears to overrule the anticipated rapid development of PMM resistance. [ABSTRACT FROM AUTHOR]

Kinetoplastids are the causative agents of leishmaniasis, human African trypanosomiasis, and American trypanosomiasis. They are responsible for high mortality and morbidity in (sub)tropical regions. Adequate treatment options are limited and have several drawbacks, such as toxicity, need for parenteral administration, and occurrence of treatment failure and drug resistance. Therefore, there is an urgency for the development of new drugs. Phenotypic screening already allowed the identification of promising new chemical entities with anti-kinetoplastid activity potential, but knowledge on their mode-of-action (MoA) is lacking due to the generally applied whole-cell based approach. However, identification of the drug target is essential to steer further drug discovery and development. Multiple complementary techniques have indeed been used for MoA elucidation. In this review, the different 'omics' approaches employed to define the MoA or mode-of-resistance of current reference drugs and some new anti-kinetoplastid compounds are discussed. [ABSTRACT FROM AUTHOR]