A. Mondelaers

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

During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a ΔLiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL.

Experimental selection of paromomycin and miltefosine resistance in intracellular amastigotes of Leishmania donovani and L. infantum.

Although widespread resistance of Leishmania donovani and L. infantum against miltefosine (MIL) and paromomycin (PMM) has not yet been demonstrated, both run the risk of resistance selection. Unraveling the dynamics and mechanisms of resistance development is key to preserve drug efficacy in the field. In this study, resistance against PMM and MIL was experimentally selected in vitro in intracellular amastigotes of several strains of both species with different antimony susceptibility background. To monitor amastigote susceptibility, microscopic determination of IC50-values and promastigote back-transformation assays were performed. Both techniques were also used to evaluate the susceptibility of field isolates from MIL-relapse patients. PMM-resistance could readily be selected in all species/strains, although promastigotes remained fully PMM-susceptible. Successful MIL-resistance selection was demonstrated only by promastigote back-transformation at increasing MIL-concentrations upon successive selection cycles. Important to note is that amastigotes with the MIL-resistant phenotype could not be visualized after Giemsa staining; hence, MIL-IC50-values showed no shift. The same phenomenon was observed in a set of recent clinical isolates from MIL-relapse patients. This study clearly endorses the need to use intracellular amastigotes for PMM- and MIL-susceptibility testing. When monitoring MIL-resistance, promastigote back-transformation should be used instead of the standard Giemsa staining. In-depth exploration of the mechanistic background of this finding is warranted.

In Vivo Selection of Paromomycin and Miltefosine Resistance in Leishmania donovani and L. infantum in a Syrian Hamster Model

ABSTRACT In 2002 and 2006, respectively, miltefosine (MIL) and paromomycin (PMM) were licensed in the Indian subcontinent for treatment of visceral leishmaniasis; however, their future routine use might become jeopardized by the development of drug resistance. Although experimental selection of resistant strains in vitro has repeatedly been reported using the less relevant promastigote vector stage, the outcome of resistance selection on intracellular amastigotes was reported to be protocol and species dependent. To corroborate these in vitro findings, selection of resistance in Leishmania donovani and Leishmania infantum was achieved by successive treatment/relapse cycles in infected Syrian golden hamsters. For PMM, resistant amastigotes were already obtained within 3 treatment/relapse cycles, while their promastigotes retained full susceptibility, thereby sharing the same phenotypic characteristics as in vitro-generated PMM-resistant strains. For MIL, even five treatment/relapse cycles failed to induce significant susceptibility changes in either species, which also corresponds with the in vitro observations where selection of an MIL-resistant phenotype proved to be quite challenging. In conclusion, these results argue for cautious use of PMM in the field to avoid rapid emergence of primary resistance and highlight the need for additional research on the mechanisms and dynamics of MIL resistance selection.

Evidence of a drug-specific impact of experimentally selected paromomycin and miltefosine resistance on parasite fitness in Leishmania infantum.

OBJECTIVES Although miltefosine and paromomycin were only recently introduced to treat visceral leishmaniasis, increasing numbers of miltefosine treatment failures and occasional primary resistance to both drugs have been reported. Understanding alterations in parasite behaviour linked to drug resistance is essential to assess the propensity for emergence and spread of resistant strains, particularly since a positive effect on fitness has been reported for antimony-resistant parasites. This laboratory study compared the fitness of a drug-susceptible parent WT clinical Leishmania infantum isolate (MHOM/FR/96/LEM3323) and derived miltefosine and paromomycin drug-resistant lines that were experimentally selected at the intracellular amastigote level. METHODS Parasite fitness of WT, paromomycin-resistant and miltefosine-resistant strains, in vitro and in vivo parasite growth, metacyclogenesis, infectivity and macrophage stress responses were comparatively evaluated. RESULTS No significant differences in promastigote fitness were noted between the WT and paromomycin-resistant strain, while clear benefits could be demonstrated for paromomycin-resistant amastigotes in terms of enhanced in vitro and in vivo growth potential and intracellular stress response. The miltefosine-resistant phenotype showed incomplete promastigote metacyclogenesis, decreased intracellular growth and weakened stress response, revealing a reduced fitness compared with WT parent parasites. CONCLUSIONS The rapid selection and fitness advantages of paromomycin-resistant amastigotes endorse the current use of paromomycin in combination therapy. Although a reduced fitness of miltefosine-resistant strains may explain the difficulty of miltefosine resistance selection in vitro, the growing number of miltefosine treatment failures in the field still requires further exploratory research.

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

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.

Comparative fitness of a parent Leishmania donovani clinical isolate and its experimentally derived paromomycin-resistant strain

Paromomycin has recently been introduced for the treatment of visceral leishmaniasis and emergence of drug resistance can only be appropriately judged upon its long term routine use in the field. Understanding alterations in parasite behavior linked to paromomycin-resistance may be essential to assess the propensity for emergence and spread of resistant strains. A standardized and integrated laboratory approach was adopted to define and assess parasite fitness of both promastigotes and amastigotes using an experimentally induced paromomycin-resistant Leishmania donovani strain and its paromomycin-susceptible parent wild-type clinical isolate. Primary focus was placed on parasite growth and virulence, two major components of parasite fitness. The combination of in vitro and in vivo approaches enabled detailed comparison of wild-type and resistant strains for which no differences could be demonstrated with regard to promastigote growth, metacyclogenesis, in vitro infectivity, multiplication in primary peritoneal mouse macrophages and infectivity for Balb/c mice upon infection with 2 x 107 metacyclic promastigotes. Monitoring of in vitro intracellular amastigote multiplication revealed a consistent decrease in parasite burden over time for both wild-type and resistant parasites, an observation that was subsequently also confirmed in a larger set of L. donovani clinical isolates. Though the impact of these findings should be further explored, the study results suggest that the epidemiological implications of acquired paromomycin-resistance may remain minimal other than the loss of one of the last remaining drugs effective against visceral leishmaniasis.

In vitro ‘time-to-kill’ assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and Leishmania infantum

Objectives Despite a continued search for novel antileishmanial drugs, treatment options remain restricted to a few standard drugs, e.g. antimonials, miltefosine, amphotericin B and paromomycin. Although these drugs have now been used for several decades, their mechanism of action still remains partly hypothetical and their dynamics of cidal action and time-to-kill are still poorly documented. Methods An in vitro time-to-kill assay on intracellular amastigotes of the laboratory reference strains Leishmania donovani (MHOM/ET/67/L82) and Leishmania infantum [MHOM/MA(BE)/67/ITMAP263] evaluated the cidal action dynamics of the listed reference drugs at three different concentrations: at IC50, 2 × IC50 and the near cytotoxic dose level (CC90: determined on MRC-5 cells). This assay focused on identifying the minimal exposure time needed to completely eliminate viable intracellular amastigotes, using the standard microscopic Giemsa assay and the promastigote back-transformation assay. Results While 100% reduction was microscopically apparent for most drugs, the promastigote back-transformation assay clearly demonstrated a concentration- and time-dependent cidal mechanism. The time-to-kill at 2 × IC50 was ≥240 h for pentavalent antimony (77 &mgr;g eq./mL), 96 h for trivalent antimony (44 &mgr;g eq./mL), 168 to >240 h for miltefosine (10 &mgr;M), 168 h for paromomycin (100 &mgr;M) and >240 h for amphotericin B (2 &mgr;M). No differences were noted between both Leishmania species. Conclusions Evaluation of the concentration- and time-dependent cidal activity using the promastigote back-transformation assay revealed striking differences in efficacy of the different antileishmanial reference drugs. This assay should allow in-depth pharmacodynamic evaluation of novel drug leads in comparison with the existing antileishmanial drug repertoire.

Molecular detection of infection homogeneity and impact of miltefosine treatment in a Syrian golden hamster model of Leishmania donovani and L. infantum visceral leishmaniasis

Control of visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani primarily relies on chemotherapy using an increasingly compromised repertoire of antileishmanial compounds. For evaluation of novel drugs, the Syrian golden hamster is considered as a clinically relevant laboratory model. In this study, two molecular parasite detection assays were developed targeting cathepsin-like cysteine protease B (CPB) DNA and 18S rRNA to achieve absolute amastigote quantification in the major target organs liver and spleen. Both quantitative PCR (qPCR) techniques showed excellent agreement with a strong correlation with the conventional microscopic reading of Giemsa-stained tissue smears. Using multiple single tissue pieces and all three detection methods, we confirmed homogeneity of infection in liver and spleen and the robustness of extrapolating whole organ burdens from a small single tissue piece. Comparison of pre- and post-treatment burdens in infected hamsters using the three detection methods consistently revealed a stronger parasite reduction in the spleen compared to the liver, indicating an organ-dependent clearance efficacy for miltefosine. In conclusion, this study in the hamster demonstrated high homogeneity of infection in liver and spleen and advocates the use of molecular detection methods for assessment of low (post-treatment) tissue burdens.

Miltefosine-resistant Leishmania infantum strains with an impaired MT/ROS3 transporter complex retain amphotericin B susceptibility

Objectives Increasing numbers of miltefosine treatment failures in visceral leishmaniasis therapy and reports of miltefosine resistance in the Indian subcontinent resulted in the recommendation to use liposomal amphotericin B as first-line therapy. Cross-resistance between miltefosine and amphotericin B has recently been documented, suggesting a role of mutations in the miltefosine transporter, a complex encoded by the MT and ROS3 genes. This study aimed to further explore the putative role of MT/ROS3 defects in the molecular basis of amphotericin B cross-resistance. Methods The susceptibility profiles of different miltefosine-resistant Leishmania infantum strains with well-characterized mutations in the transporter complex and the corresponding episomally restored susceptible parasite lines were determined using both the routine extracellular promastigote assay and the intracellular amastigote assay. Results In vitro amastigote and promastigote susceptibility testing of the two miltefosine-resistant and the episomally reconstituted L. infantum lines revealed full susceptibility to amphotericin B, despite the variable miltefosine susceptibility profile. Conclusions Mutations present in either the MT and/or ROS3 gene are not sufficient to elicit higher tolerance to amphotericin B. Additional synergistic adaptations may be responsible for the miltefosine/amphotericin B cross-resistance described earlier.

Chapter 18:Molecular Basis of Drug Resistance in Leishmania

Leishmaniasis has been treated for decades with pentavalent antimony preparations until the emergence of antimony resistance has forced a switch in hyperendemic areas towards alternative therapeutics, such as miltefosine, amphotericin B and paromomycin. The use of miltefosine and amphotericin B has particularly been encouraged as first-line therapy for visceral leishmaniasis, however, all are increasingly confronted with treatment failures and/or the emergence of drug resistance. This chapter provides a concise overview of the mode-of-action of the current anti-leishmanial drugs and links this to the different resistance mechanisms that have been proposed over the past years. The pivotal importance of proactive drug-resistance research is highlighted with reference to the most commonly used laboratory methods.