,aLaboratoire de Parasitologie, Hygiène et Zoologie, Faculté de Pharmacie, 27 Bd. Jean Moulin, 13385 Marseille Cedex 05; ,bLaboratoire de Parasitologie, Faculté de Medecine; ,cService de Pédiatrie Pr. Garnier, Hôpital Nord, Marseille, France
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pore-forming antibiotic amphotericin B, extensively used to treat serious systemic fungal infections, has been shown to have efficacy against leishmaniasis in spite of its toxic side effects. Since 1984, it has been applied successfully to the treatment of both cutaneous and visceral leishmaniasis. 4,5 In spite of therapeutic failures in immunocompromised patients, 6 no primary amphotericin B resistance has been reported in Leishmania sp.; moreover, recent studies have demonstrated that effective doses of amphotericin B were stable after long-term treatments in immunocompromised patients. 7 Nevertheless, the possibility of emergence of amphotericin B resistant isolates has been proven by the experiments of Mbongo et al., 8 who obtained amphotericin B resistant promastigotes after in-vitro drug pressure. We demonstrated in a previous study 9 that flow cytometric determination of membrane potential changes in L. infantum promastigotes constituted an easy and reliable method for studying the interactions between amphotericin B and the parasite membrane and evaluating the susceptibility of isolates to the antibiotic.
In the present study, we planned to explore the effect of amphotericin B on the parasite membrane in the course of amphotericin B treatment in wild strains of L. infantum. For this purpose, amphotericin B induced membrane potential changes were measured in 45 isolates from immunocompetent children and immunocompromised patients with visceral leishmaniasis during the first episode and eventually after relapses.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Forty-five isolates of L. infantum were recovered from 30 patients with visceral leishmaniasis living in the area of Marseilles: 11 immunocompetent children (0.5-3 years old) treated with liposomal amphotericin B (liposomal AmB (AmBisome, Nexstar Pharmaceuticals, Paris, France) 3 mg/kg/day for 10 days), and 19 young adults (30-35 years old) infected with the human immunodeficiency virus (HIV), treated with intralipid amphotericin B (intralipid AmB (intralipid 20% emulsion, Pharmacia SA, Paris, France) 1-2 mg/kg/day for 21 days). Leishmania sp. parasites were recovered during the first episode, and eventually after relapses, from bone marrow aspirates and blood samples. They were isolated in NNN medium (Gibco, Paisley, UK) and cultivated in RPMI medium (Eurobio) supplemented with 15% fetal calf serum, France). They were analysed by the isoenzymatic method: all were identified as L. infantum and three zymodems were represented (28 MON 1, one MON 11 and one MON 108). Two referenced isolates that had never been treated with amphotericin B were added to the study: L. infantum MON 1 (MHOM/ FR/78/LEM75) and L. infantum MON 11 (MHOM/FR/80/ LEM189).
Flow cytometric assessment of amphotericin B-membrane interactions
Susceptibility of isolates to amphotericin B (Fungizone, Bristol Myers Squibb, Paris, France) was assessed immediately after isolation and growth of promastigotes (no more than 2 weeks between isolation and test). The method used for evaluating the susceptibility of parasites to amphotericin B was based on a flow cytometric technique extensively used in clinical microbiology 10 and adapted for use with leishmania promastigotes. This technique was established on the basis that the main mechanism of action of polyene antibiotics such as amphotericin B was an interaction with membrane sterols, inducing membrane potential depletion. The method consisted of measuring antibiotic-induced membrane potential changes by using a carbocyanine dye, 3,3'-dipentyloxacarbocyanine iodide (DIOC 5(3)), 11 which is positively charged and accumulates inside the cell according to the Nernst equation: DIOC 5(3)-related fluorescence variations in parasites were proportional to membrane potential changes. Parasites maintained in RPMI medium supplemented with 15% heat-inactivated fetal calf serum were treated with a range of amphotericin B (Sigma, St Louis, MO, USA) concentrations (from 0 to 10 mg/L). After a 3 h incubation period at 25°C, DIOC 5(3) (Molecular Probes, Eugene, OR, USA) was incorporated into each cell culture, at the final concentration of 0.5 µM at room temperature. Cells were run immediately on a FacScan analytical flow cytometer (Becton Dickinson, Paris, France). Ten thousand cells were used for each analysis and their relative fluorescence was estimated by the arithmetic mean of their distribution. Amphotericin B induced fluorescence inhibition was expressed as the percentage of fluorescence observed in amphotericin B treated promastigotes compared with the control culture. Dose-response curves representing the percentage of fluorescence compared with the control culture according to the concentration of amphotericin B incorporated in duplicate parasite cultures were calculated by a non-linear regression. Amphotericin B susceptibility was expressed by the 90% inhibitory concentration (IC 90), representing the concentration of antibiotic that induced 90% of fluorescence decrease compared with the control culture.
Assessment of amphotericin B susceptibility on Leishmania intracellular amastigotes
The activity of amphotericin B on the amastigote form of the parasite was assessed in adherent human monocytes (THP1) infected by the two referenced Leishmania sp. isolates and the isolate from patient 10 during the first episode and the following relapses. The test was performed according to the methods previously described by Ogunkolade et al. 12 Briefly, adherent cells were obtained by treating THP1-monocytes with 1 µM phorbol myristate acetate in RPMI medium. After a 48 h incubation period at 37°C, cells were rinsed and infected by a suspension of promastigotes using an infection ratio of 10/1 parasites to macrophages. Adapted dilutions of amphotericin B were added in duplicate cultures and incubated for 96 h at 37°C. The plates were fixed with methanol and stained with 10% Giemsa stain. The percentage of infected macrophages in each assay was determined microscopically at 1000x magnification. The activity of amphotericin B on amastigotes was expressed by the 50% inhibitory concentrations (IC 50), illustrating the concentrations of drug that produced a 50% reduction of infected macrophage compared with the control culture.
Statistical analysis
Comparison between independent variables such as amphotericin B susceptibilities observed in isolates from immunocompromised and immunocompetent patients was performed by the Mann- Whitney U-test. The Wilcoxon test was used for comparing double variables such as amphotericin B sensitivities observed before and after treatment in immunocompromised patients.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Results observed in the present study confirmed the efficacy of liposomal amphotericin B 6,15,16 in the treatment of glucantime-resistant visceral leishmaniasis among immunocompetent patients: no case of treatment failure was seen in children; moreover, an early reduction of inflammatory signs illustrated by fever disappearance after 44 h was observed and no secondary effect was found. In HIV-Leishmania sp. coinfected patients, amphotericin B courses of treatment involving intralipid emulsions were not fully effective: the first relapses occurred 1-15 months following treatment and, after the first episode, additional courses of treatment were similarly ineffective. Nevertheless, during relapses, no pulmonary or cutaneous localizations 17 could be detected. Liposomal formulation of amphotericin B has been shown to be more effective than lipid emulsion in the treatment of murine visceral leishmaniasis; 18 moreover, it has been demonstrated to reduce the acute and chronic side effects of the parent drug, permitting higher dosages to be tolerated. However, its cost was high 19,20 compared with that of intralipid amphotericin B; that was the main reason why, in the present study, liposomal amphotericin B was preferentially used only for immunocompetent children. A comparison between the clinical efficacy of each amphotericin B formulation was therefore not possible in these two populations.
The susceptibility to amphotericin B of Leishmania sp. isolates from patients was assessed by a new flow cytometric method that consisted of measuring drug induced membrane potential changes. This technique has been well validated with fungi; however, its application to Leishmania sp. promastigotes is recent. Nevertheless, we demonstrated in a previous study 9 that the concentrations of amphotericin B inducing a 90% decrease of membrane potential (IC 90) produced a 90% drop in parasite growth. Moreover, a strong correlation could be established between results obtained by flow cytometry and susceptibility levels determined by the conventional assessment of parasite growth, suggesting that this new method could be a reliable tool for estimating the sensitivity of parasites to polyene antibiotics.
Amphotericin B susceptibility measured in promastigotes during the first episode exhibited a
weak range of variations in both immunocompromised and immunocompetent patients (0.065
IC
90
0.654 mg/L), and no significant difference could be observed between IC
90 determined in each population. No primary resistance to amphotericin B could be
detected, as no treatment failure could be observed in immunocompetent children. On the
contrary, in HIV-Leishmania sp. coinfected patients, relapses could be observed
whatever the susceptibility of isolates. In fact, all HIV-Leishmania sp. coinfected
patients exhibited weak levels of T4 cells (<100/mm
3) during relapses, suggesting that the success of the
antileishmanial treatment depended greatly on patient immunity status.
After an initial cure, amphotericin B susceptibility was shown to decline in Leishmania sp. promastigotes: in patient 10, more particularly, the IC 90 increased 10-fold after six relapses. This result indicated that after long-term treatment in vivo, interactions between amphotericin B and the parasite membrane could be modulated, leading to a significant decrease of amphotericin B susceptibility in the promastigote form of the parasite. Results observed in infected THP1-cells indicated also that sensitivity to amphotericin B was reduced in the intracellular amastigote form of the parasite: the IC 50 was multiplied by three after six relapses. The mechanisms involved in this adaptation could possibly be similar to those observed in fungi, such as stable qualitative and quantitative alterations in the lipid composition of the membrane 21 or resistance to oxidation-dependent damage. 22 On this basis, the assessment of membrane potential changes by flow cytometry could be useful for the study of resistance to amphotericin B, as it has been demonstrated that the antibiotic killed unicellular parasites by forming aqueous pores permeable to small anions and cations, 23 leading to membrane depolarization. 24 Thus, in the clinical trial, flow cytometric measurement of amphotericin B susceptibility has been shown to be a reliable way of evaluating the susceptibility of yeasts and bacteria to pore-forming antibiotics. 10 Concerning Leishmania sp. parasites, however, although results for patient 10 showed that a reduction of amphotericin B susceptibility in promastigotes could be followed by a similar decrease of amastigote susceptibility, the application of this flow cytometric technique could not have any predictive value concerning leishmaniasis clinical outcome: drug susceptibility in the intracellular amastigote form of the parasite was shown to differ from that in the promastigote form. 25 Amphotericin B activity appeared also to depend on interactions with macrophages 26 and the efficacy of treatment was demonstrated to vary greatly according to patient immunity status. 18 Nevertheless, this technique could be used for examining rapidly the evolution of interactions between amphotericin B and the parasite membrane in the course of amphotericin B treatment and detecting the emergence of amphotericin B resistant isolates.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Herwaldt, B. L. & Berman, J. D. (1992). Recommendations for treating leishmaniasis with sodium stibogluconate (Pentostam) and review of pertinent clinical studies. American Journal of Tropical Medicine and Hygiene 46, 296306.[ISI][Medline]
3 . Faraut-Gambarelli, F., Piarroux, R., Deniau, M., Giusiano, B., Marty, P., Michel, G. et al.(1997). In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: a study of 37 strains collected from patients with visceral leishmaniasis. Antimicrobial Agents and Chemotherapy 41, 82730.[Abstract]
4 . Croft, S. L., Davidson, R. N. & Thornton, E. A. (1991). Liposomal amphotericin B in the treatment of visceral leishmaniasis. Journal of Antimicrobial Chemotherapy 28, Suppl. B, 11118.[ISI][Medline]
5 . Davidson, R. N., Croft, S. L., Scott, A., Maini, M., Moody, A. H. & Bryceson, A. D. (1991). Liposomal amphotericin B in drug resistant visceral leishmaniasis. Lancet 337, 10612.[ISI][Medline]
6 . Davidson, R. N. & Russo, R. (1994). Relapse of visceral leishmaniasis in patients who were coinfected with human immunodeficiency virus and who received treatment with liposomal amphotericin B. Clinical Infectious Diseases 19, 560.[ISI][Medline]
7
.
Durand, R., Paul, M., Pratlong, F., Rivollet, D., Dubreuil-Lemaire, M. L., Houin, R. et al. (1998). Leishmania infantum: lack of parasite resistance to
amphotericin B in a clinically resistant visceral leishmaniasis. Antimicrobial Agents
and Chemotherapy 42, 21413.
8
.
Mbongo, N., Loiseau, P. M., Billion, M. A. & Robert-Gero, M. (1998).
Mechanism of amphotericin B resistance in Leishmania donovani promastigotes. Antimicrobial Agents and Chemotherapy 42, 3527.
9 . Azas, N., Di Giorgio, C., Delmas, F., Gasquet, M. & Timon-David, P. (1997). Assessment of amphotericin B susceptibility in Leishmania infantum promastigotes by flow cytometric membrane potential assay. Cytometry 28, 1658.[ISI][Medline]
10 . Peyron, F., Favel, A., Guiraud-Dauriac, H., El Mzibri, M., Chastin, C., Dumenil, G. et al. (1997). Evaluation of a flow cytofluorometric method for rapid determination of amphotericin B susceptibility of yeasts isolates. Antimicrobial Agents and Chemotherapy 41, 153740.[Abstract]
11 . Ordonez, J. V. & Wehman, N. M. (1993). Rapid flow cytometric antibiotic susceptibility assay for Staphylococcus aureus. Cytometry 14, 81118.[ISI][Medline]
12 . Ogunkolade, B. W., Colomb-Valet, I., Monjour, L., Rhodes-Feuillette, A., Abita, J. P. & Frommel, D. (1990). Interactions between the human monocytic leukaemia THP1 cell line and Old and New World species of Leishmania. Acta Tropica 47, 1716.[ISI][Medline]
13 . Lazanas, M. C., Tsekes, G. A., Papandreou, S., Harhalakis, N., Scandali, A., Nikiforakis, E. et al. (1991). Liposomal amphotericin B for leishmaniasis treatment of AIDS patients unresponsive to antimonium compounds. AIDS 7, 101819.
14 . Sen Gupta, P. C. (1953). Chemotherapy of leishmanial diseases: a resume of recent researches. Indian Medical Gazette 88, 2035.
15 . Cascio, A., Gradoni, L., Scarlata, F., Gamiccia, M., Giordano S., Russo, R. et al. (1997). Epidemiologic surveillance of visceral leishmaniasis in Sicily, Italy. American Journal of Tropical Medicine and Hygiene 57, 758.
16 . di Martino, L., Davidson, R. N., Giacchino, R., Scotti, S., Raimondi, F., Castagnola, E. et al. (1997). Treatment of visceral leishmaniasis in children with liposomal amphotericin B. Journal of Pediatrics 13, 2727.
17 . Matheron, S., Cabie, A., Parquin, F., Mayaud, C., Roux, P., Antoine, M. et al. (1992). Visceral leishmaniasis in HIV infection: unusual presentation with pleuropulmonary involvements, and effect of secondary prophylaxis. AIDS 6, 23840.[ISI][Medline]
18 . Paul, M., Durand, R., Fessi, H., Rivollet, D., Houin, R., Astier, A. et al. (1997). Activity of a new liposomal formulation of amphotericin B against two strains of Leishmania infantum in a murine model. Antimicrobial Agents and Chemotherapy 41, 17314.[Abstract]
19 . Mullen, A. B., Carter, K. C. & Baillie, A. J. (1997). Comparison of the efficacies of various formulations of amphotericin B against murine visceral leishmaniasis. Antimicrobial Agents and Chemotherapy 41, 208992.[Abstract]
20 . Coukell, A. J. & Brogden, R. N. (1998). Liposomal amphotericin B. Therapeutic use in the management of fungal infections and visceral leishmaniasis. Drugs 55, 585612.[ISI][Medline]
21 . Brajtburg, J., Powderly, W. G., Kobayashi, G. S. & Medoff, G. (1990). Amphotericin B: current understanding of mechanisms of action. Antimicrobial Agents and Chemotherapy 34, 1838.[ISI][Medline]
22 . Sokol-Anderson, M., Sligh, J. E., Elberg, S., Brajtburg, J., Kobayashi, G. S. & Medoff, G. (1988). Role of cell defense against oxidative damage in the resistance of Candida albicans to the killing effect of amphotericin B. Antimicrobial Agents and Chemotherapy 32, 7025.
23 . Beggs, W. H. (1994). Physicochemical cell damage in relation to lethal amphotericin B action. Antimicrobial Agents and Chemotherapy 38, 3634.[Abstract]
24 . Ramos, H., Valdivieso, E., Gamargo, M., Dagger, F. & Cohen, B. E. (1996). Amphotericin B kills unicellular leishmanias by forming aqueous pores permeable to small cations and anions. Journal of Membrane Biology 152, 6575.[ISI][Medline]
25 . Callahan, H. L., Portal, A. C., Devereaux, R. & Grogl, M. (1997). An axenic amastigote system for drug screening. Antimicrobial Agents and Chemotherapy 41, 81822.[Abstract]
26 . Mozaffarian, N., Berman, J. W. & Casadevall, A. (1997). Enhancement of nitric oxide synthesis by macrophages represents an additional mechanism of action for amphotericin B. Antimicrobial Agents and Chemotherapy 41, 182529.[Abstract]
Received 28 July 1998; returned 6 October 1998; revised 26 November 1998; accepted 5 March 1999