Abstract 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. Introduction Miltefosine was introduced as first-line therapy against visceral leishmaniasis (VL) in the Indian subcontinent in 2002 to combat the growing incidence of antimony resistance.1 Since then, the number of treatment failures has steadily increased and reports have emerged of the few first miltefosine-resistant isolates.2,3 Previous work using both experimentally derived resistant isolates and a miltefosine-resistant clinical Leishmania infantum isolate demonstrated the involvement of mutations in the inward miltefosine transporter (MT) and its subunit ROS3 affecting effective drug uptake.4,5 Although cross-resistance between amphotericin B and other anti-leishmanial drugs has been suggested,6 evidence for the association between miltefosine and amphotericin B resistance is still scarce and mutations in the MT complex have so far never been directly linked to amphotericin B resistance.3,7–11 A recent study did, however, associate mutations in the MT complex with alterations in the lipid composition of the plasma membrane with subsequent cross-resistance to amphotericin B.12 Given the recent shift towards a single dose of liposomal amphotericin B followed by a short course of miltefosine as first-line treatment in the Indian subcontinent,13 combined with the recent reports mentioning not only the emergence of miltefosine resistance,3 but also of clinical amphotericin B resistance,7 investigation into a possible link between miltefosine and amphotericin B resistance becomes highly relevant. Hence, this in vitro study evaluated the amphotericin B susceptibility of a laboratory-derived and a clinical miltefosine-resistant isolate characterized by having defective MT and/or ROS3, and investigated whether their reconstituted knock-in transfectants restored miltefosine susceptibility. Materials and methods Ethics Experiments using laboratory rodents were carried out in strict accordance with all mandatory guidelines (European Union directives, including Revised Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes that came into force on 01/01/2013, and the Declaration of Helsinki in its latest version) and was approved by the Ethics Committee of the University of Antwerp, Belgium (UA-ECD 2011–17). Chemical compounds Miltefosine (hexadecylphosphocholine, Carbosynth, Berkshire, UK) was dissolved in MilliQ water at 20 mM (compound stock solution) and stored at 4 °C. Amphotericin B deoxycholate (Fungizone®, Bristol-Myers Squibb, Braine-l’Alleud, Belgium) and liposomal amphotericin B (AmBisome®, Gilead Sciences, CA, USA) were freshly dissolved on the day of the experiment at 5.4 mM (Fungizone®) and 6.4 mM (AmBisome®) in a 5% dextrose solution in MilliQ water. Leishmania strains The potential cross-resistance between amphotericin B and miltefosine was evaluated on a previously described panel of L. infantum strains:4 (i) MHOM/FR/96/LEM3323 and the naturally miltefosine-resistant MHOM/FR/2005/LEM5159, which were both isolated from French HIV-positive patients who underwent several rounds of amphotericin B and/or miltefosine treatment,14 and were kindly provided by Dr Laurence Lachaud (Laboratoire de Parasitologie-Mycologie et Centre National de Référence des Leishmanioses, Montpellier, France); (ii) the laboratory-selected miltefosine-resistant LEM3323-MIL; and (iii) the episomally transfected LEM3323-MIL + LiMT and LEM5159 + LiROS3, which both show a restored miltefosine susceptibility. Promastigotes were grown in HOMEM (Gibco®, Life Technologies, Ghent, Belgium) supplemented with 200 mM l-glutamine, 16.5 mM NaHCO3, 10% heat-inactivated FCS, 40 mg/L adenine, 3 mg/L folic acid, 2 mg/L d-biotin and 2.5 mg/L haemin at 25 °C and were subcultured twice weekly. Transfectants were grown under hygromycin pressure. Drug susceptibility assays In vitro promastigote and amastigote susceptibility to miltefosine and amphotericin B was evaluated as described earlier.15 In brief, IC50 values of log-phase promastigotes were determined by exposing the parasites to 2-fold serial drug dilutions starting from 40 μM miltefosine or 8 μM amphotericin B. After 72 h incubation, susceptibility of promastigotes was determined by viability testing upon addition of resazurin and fluorescence reading (Tecan®, GENios). Promastigote susceptibility to AmBisome® was not determined as this formulation specifically targets the macrophage. For amastigote susceptibility, primary peritoneal mouse macrophages were seeded into a 96-well plate in RPMI-1640 culture medium (Gibco®, Life Technologies) at a concentration of 30000 cells/well, infected 24 h later with stationary-phase promastigotes (multiplicity of infection 10:1) and exposed to 2-fold serial drug dilutions. After 4 days, the infected macrophages were fixed with methanol and stained with Giemsa. Amastigote susceptibility was microscopically determined by comparing the intracellular amastigote burdens between treated and untreated cells. Cut-off values for resistance were set at 15 μM for miltefosine and 2 μM for amphotericin B.16 Results Despite their previously confirmed differences in miltefosine susceptibility resulting from characterized genetic alterations in the MT gene (LEM3323-MIL) or the ROS3 gene (LEM5159),4 all L. infantum strains maintained full susceptibility to amphotericin B both as promastigote (Table 1) and amastigote (Table 2). Episomal reconstitution with WT MT or ROS3 of the respective miltefosine-resistant strains restored miltefosine susceptibility, but did not affect the amphotericin B resistance profile (Tables 1 and 2). Table 1. Promastigote susceptibility of the selected strains against miltefosine and amphotericin B (Fungizone®) Miltefosine Fungizone® LEM3323 5.3±0.3 0.06±0.01 LEM3323-MIL >40.0 0.06±0.01 LEM3323-MIL + LiMT 2.4±1.2 0.06±0.01 LEM5159 >40.0 0.06±0.01 LEM5159 + LiROS3 3.8±1.3 0.06±0.01 Miltefosine Fungizone® LEM3323 5.3±0.3 0.06±0.01 LEM3323-MIL >40.0 0.06±0.01 LEM3323-MIL + LiMT 2.4±1.2 0.06±0.01 LEM5159 >40.0 0.06±0.01 LEM5159 + LiROS3 3.8±1.3 0.06±0.01 Results are expressed as mean IC50 value (μM) ± SEM and are based on three independent replicates run in triplicate. Table 2. Intracellular amastigote susceptibility of the selected strains against miltefosine and amphotericin B (Fungizone®, AmBisome®) Miltefosine Fungizone® AmBisome® LEM3323 2.3±0.5 0.03±0.01 0.13±0.02 LEM3323-MIL >20.0 0.03±0.01 0.04±0.01 LEM3323 + LiMT 3.0±1.0 0.02±0.01 0.05±0.16 LEM5159 >20.0 0.01±0.01 0.02±0.01 LEM5159 + LiROS3 0.97±0.17 0.03±0.01 0.08±0.01 Miltefosine Fungizone® AmBisome® LEM3323 2.3±0.5 0.03±0.01 0.13±0.02 LEM3323-MIL >20.0 0.03±0.01 0.04±0.01 LEM3323 + LiMT 3.0±1.0 0.02±0.01 0.05±0.16 LEM5159 >20.0 0.01±0.01 0.02±0.01 LEM5159 + LiROS3 0.97±0.17 0.03±0.01 0.08±0.01 Results are expressed as mean IC50 value (μM) ± SEM and are based on three independent replicates run in triplicate. Discussion Identifying potential cross-resistance mechanisms in VL treatment becomes increasingly essential given the current recommendations towards combination therapy.17 In the Indian subcontinent, a current option is based on combination of a single injection of liposomal amphotericin B and a short 5 day course of oral miltefosine.13 Although resistance to either one of the drugs alone might emerge upon its recurrent application in the field, past research on antimony-resistant clinical isolates already identified potential acquisition of resistance to both miltefosine and amphotericin B.6 While miltefosine resistance has been linked to mutations in the MT/ROS3 transporter machinery responsible for its inward transport,5 amphotericin B resistance has been associated with an altered sterol composition and membrane fluidity.7 No definite link between the miltefosine translocation machinery and amphotericin B has yet been identified, suggesting the involvement of other mechanisms.18 A recent experimental selection of amphotericin B and miltefosine resistance on promastigotes revealed a low-level cross-resistance between miltefosine and amphotericin B next to MT mutations, but a functional link between these MT mutations and amphotericin B resistance was not fully substantiated and the observed cross-resistance was attributed to changes in the membrane lipid composition.12 In that particular study, reconstitution of the amphotericin B-resistant strain with a functional MT only had a very moderate impact on restoration of amphotericin B susceptibility. Moreover, the amphotericin B-resistant line was not substantially hampered in miltefosine-uptake suggesting that the mutated MT was still functioning normally, casting doubt on the role of MT in the low-level amphotericin B cross-resistance feature described in that study.12 The objective of the present study was to evaluate two genotypically and phenotypically characterized miltefosine-resistant L. infantum strains for their susceptibility to amphotericin B, both at promastigote and intracellular amastigote level. In spite of the confirmed mutations in the MT/ROS3 transporter complex and their defective miltefosine uptake,4 no cross-resistance to amphotericin B could be demonstrated. This absence of cross-resistance to amphotericin B is reassuring and refutes the direct involvement of the MT/ROS3 transporter complex in amphotericin B cross-resistance. As point mutations in the MT gene do not necessarily lead to functional changes in the transporter complex,4 it is more likely that one of the ample non-synonymous mutations or ploidy changes that were observed upon miltefosine-resistance selection may in fact be responsible for the described amphotericin B/miltefosine cross-resistance. To further evaluate a potential synergistic involvement of MT mutations and to identify other key genetic alterations involved in amphotericin B/miltefosine cross-resistance, additional resistant clinical isolates should be evaluated. Routine testing for cross-resistance of clinical isolates may be the only way to obtain a definite answer. Acknowledgements We thank Dr Laurence Lachaud (Laboratoire de Parasitologie-Mycologie et Centre National de Référence des Leishmanioses, Montpellier, France) for providing the LEM5159 and LEM3323 patient isolates. Special thanks to Francisco Gamarro, Santiago Castanys and Maria P. Sanchez-Cañete of the Instituto de Parasitologia y Biomedicina ‘Lopez-Neyra’, Granada, Spain for their contribution in the generation of the episomally restored susceptible parasite lines. Funding This work was funded by the Fonds Wetenschappelijk Onderzoek Vlaanderen [FWO No. G051812N (L. M.), 12I0317N (S. H.) and 1136417N (L. V. B.)] and a research fund of the University of Antwerp [TT-ZAPBOF 33049 (G. C.)]. LMPH participates in COST Action CM1307 (Targeted chemotherapy towards diseases caused by endoparasites) and is partner of the Antwerp Drug Discovery Network (ADDN, www.addn.be) and the Excellence Centre ‘Infla-Med’ (www.uantwerpen.be/infla-med). Transparency declarations None to declare. References 1 Dhillon GP, Sharma SN, Nair B. Kala-azar elimination programme in India. J Indian Med Assoc 2008; 106: 664, 666–8. Google Scholar PubMed 2 Rijal S, Ostyn B, Uranw S et al. Increasing failure of miltefosine in the treatment of kala-azar in Nepal and the potential role of parasite drug resistance, reinfection, or noncompliance. Clin Infect Dis 2013; 56: 1530– 8. Google Scholar CrossRef Search ADS PubMed 3 Srivastava S, Mishra J, Gupta AK et al. Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India. Parasit Vectors 2017; 10: 49. Google Scholar CrossRef Search ADS PubMed 4 Mondelaers A, Sanchez-Canete MP, Hendrickx S et al. Genomic and molecular characterization of miltefosine resistance in Leishmania infantum strains with either natural or acquired resistance through experimental selection of intracellular amastigotes. PLoS One 2016; 11: e0154101. Google Scholar CrossRef Search ADS PubMed 5 Perez-Victoria FJ, Castanys S, Gamarro F. Leishmania donovani resistance to miltefosine involves a defective inward translocation of the drug. Antimicrob Agents Chemother 2003; 47: 2397– 403. Google Scholar CrossRef Search ADS PubMed 6 Kumar D, Kulshrestha A, Singh R et al. In vitro susceptibility of field isolates of Leishmania donovani to miltefosine and amphotericin B: correlation with sodium antimony gluconate susceptibility and implications for treatment in areas of endemicity. Antimicrob Agents Chemother 2009; 53: 835– 8. Google Scholar CrossRef Search ADS PubMed 7 Purkait B, Kumar A, Nandi N et al. Mechanism of amphotericin B resistance in clinical isolates of Leishmania donovani. Antimicrob Agents Chemother 2012; 56: 1031– 41. Google Scholar CrossRef Search ADS PubMed 8 Cojean S, Houze S, Haouchine D et al. Leishmania resistance to miltefosine associated with genetic marker. Emerg Infect Dis 2012; 18: 704– 6. Google Scholar CrossRef Search ADS PubMed 9 Croft SL, Sundar S, Fairlamb AH. Drug resistance in leishmaniasis. Clin Microbiol Rev 2006; 19: 111– 26. Google Scholar CrossRef Search ADS PubMed 10 Seifert K, Perez-Victoria FJ, Stettler M et al. Inactivation of the miltefosine transporter, LdMT, causes miltefosine resistance that is conferred to the amastigote stage of Leishmania donovani and persists in vivo. Int J Antimicrob Agents 2007; 30: 229– 35. Google Scholar CrossRef Search ADS PubMed 11 Khan I, Khan M, Umar MN et al. Attenuation and production of the amphotericin B-resistant Leishmania tropica strain. Jundishapur J Microbiol 2016; 9: e32159. Google Scholar PubMed 12 Fernandez-Prada C, Vincent IM, Brotherton MC et al. Different mutations in a P-type ATPase transporter in Leishmania parasites are associated with cross-resistance to two leading drugs by distinct mechanisms. PLoS Negl Tropl Dis 2016; 10: e0005171. Google Scholar CrossRef Search ADS 13 Sundar S, Rai M, Chakravarty J et al. New treatment approach in Indian visceral leishmaniasis: single-dose liposomal amphotericin B followed by short-course oral miltefosine. Clin Infect Dis 2008; 47: 1000– 6. Google Scholar CrossRef Search ADS PubMed 14 Lachaud L, Bourgeois N, Plourde M et al. Parasite susceptibility to amphotericin B in failures of treatment for visceral leishmaniasis in patients coinfected with HIV type 1 and Leishmania infantum. Clin Infect Dis 2009; 48: e16– 22. Google Scholar CrossRef Search ADS PubMed 15 Vermeersch M, da Luz RI, Tote K et al. In vitro susceptibilities of Leishmania donovani promastigote and amastigote stages to antileishmanial reference drugs: practical relevance of stage-specific differences. Antimicrob Agents Chemother 2009; 53: 3855– 9. Google Scholar CrossRef Search ADS PubMed 16 Maes L, Cos P, Croft SL, The relevance of susceptibility tests, breakpoints, and markers. In: Ponte-Sucre A, Diaz E, Padrón-Nieves M, eds. Drug Resistance in Leishmania Parasites: Consequences, Molecular Mechanisms and Possible Treatments . Vienna: Springer, 2013; 407– 29. Google Scholar CrossRef Search ADS 17 WHO. Control of the Leishmaniases: Report of a Meeting of the WHO Expert Committee on the Control of Leishmaniases, Geneva, 22-26 March 2010. WHO Technical Report Series, 2010. 18 Vergnes B, Gourbal B, Girard I et al. A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death. Mol Cell Proteomics 2007; 6: 88– 101. Google Scholar CrossRef Search ADS PubMed © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: email@example.com.
Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Feb 1, 2018
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