Sarah Hendrickx

Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol.

Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC50 values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC50 = 199 µM) compared to the parent strain (IC50 = 45 µM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.

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.

Role of oxidative stress and apoptosis in the cellular response of murine macrophages upon Leishmania infection

Leishmania parasites are able to survive in the macrophage, one of the most hostile environments of the vertebrate host. The present study investigated how Leishmania infection influences these host cell defence mechanisms. Macrophages were infected with antimony-susceptible and -resistant Leishmania strains. Free radical production in Leishmania-infected macrophages was measured by electron paramagnetic resonance. Apoptosis was detected with fluorescence microscopy using Annexin-V FITC labelling and with Western blotting to detect caspase-3 cleavage. Independent of their drug susceptibility profile or species background, all studied Leishmania strains induced a similar increase in free radical production in macrophages. O2 ●- production was significantly elevated during phagocytosis of the stationary phase promastigotes. Conversely, NO levels increased later in the infection and none of the strains induced capsase-3 cleavage. Leishmania donovani infection led to phosphatidylserine externalization only in RAW 264.7 cells. After an initial burst of O2 ●- during phagocytosis of promastigotes, amastigotes protect themselves by decreasing the O2 ●- production to the basal level. An increased NO production was observed 6 h after infection. Finally, induction of cell death is probably not essential in the survival of the parasite within the macrophage.

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.

In vitro and in vivo prophylactic and curative activity of the triterpene saponin PX-6518 against cutaneous Leishmania species

OBJECTIVES The oleanane triterpene saponin PX-6518, with known potent in vitro and in vivo activity against Leishmania donovani, was investigated for its spectrum against the cutaneous species Leishmania mexicana, Leishmania panamensis and Leishmania major. METHODS In vitro activity was based on the reduction of amastigotes in primary peritoneal mouse macrophages. BALB/c mice were injected with 2 × 10(6) amastigotes in the base of the tail (L. panamensis and L. major) or the foot (L. mexicana) and subcutaneously treated with PX-6518 [1-10 mg/kg body weight (BW)] or Pentostam(®) (250 mg/kg BW Sb(V) eq). Evolution of skin lesions was monitored in a prophylactic dose-finding study, and early curative [6 weeks post-infection (pi)] and late curative (>8-10 weeks pi) studies. RESULTS While moderate susceptibility to PX-6518 was obtained in vitro (IC(50): 1-4 µg/mL), excellent in vivo activity was demonstrated. In the prophylactic study (six administrations on alternate days, starting at 1 day pi), PX-6518 was 100% effective at 1 mg/kg BW against L. mexicana and L. panamensis, whereas L. major lesions could be prevented at 2 mg/kg BW. In the early curative (1 mg/kg BW once a week for 4 weeks) and late curative (1 mg/kg BW twice a week for 4 weeks) studies, PX-6518 completely healed L. mexicana and L. panamensis lesions, whereas L. major lesions were reduced by ∼ 50%. CONCLUSIONS This study demonstrates that PX-6518 possesses potent and broad-spectrum prophylactic and curative efficacy against cutaneous leishmaniasis in the BALB/c mouse model. L. major was the least susceptible species tested and parasitological cure could not be obtained.

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.

Evaluating drug resistance in visceral leishmaniasis: the challenges

SUMMARY For decades antimonials were the drugs of choice for the treatment of visceral leishmaniasis (VL), but the recent emergence of resistance has made them redundant as first-line therapy in the endemic VL region in the Indian subcontinent. The application of other drugs has been limited due to adverse effects, perceived high cost, need for parenteral administration and increasing rate of treatment failures. Liposomal amphotericin B (AmB) and miltefosine (MIL) have been positioned as the effective first-line treatments; however, the number of monotherapy MIL-failures has increased after a decade of use. Since no validated molecular resistance markers are yet available, monitoring and surveillance of changes in drug sensitivity and resistance still depends on standard phenotypic in vitro promastigote or amastigote susceptibility assays. Clinical isolates displaying defined MIL- or AmB-resistance are still fairly scarce and fundamental and applied research on resistance mechanisms and dynamics remains largely dependent on laboratory-generated drug resistant strains. This review addresses the various challenges associated with drug susceptibility and -resistance monitoring in VL, with particular emphasis on the choice of strains, susceptibility model selection and standardization of procedures with specific read-out parameters and well-defined threshold criteria. The latter are essential to support surveillance systems and safeguard the limited number of currently available antileishmanial drugs.

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.