In vitro susceptibility of Leishmania infantum to meglumine antimoniate in isolates from repeated leishmaniasis episodes in HIV-coinfected patients

J. Carrióa, C. Rieraa, M. Gállegoa, E. Riberab and M. Portúsa

a Laboratory of Parasitology, Facultat de Farmàcia, Universitat de Barcelona, Barcelona; b Service of Infectious Diseases, Hospital Vall d'Hebrón, Universitat Autònoma de Barcelona, Barcelona, Spain

Sir,

Leishmaniasis in HIV-coinfected patients is characterized by its recurrence, and post-treatment relapses are frequent.1 Pentavalent antimony (SbV) derivatives and amphotericin B are the two most commonly used drugs for the treatment of visceral leishmaniasis (VL). Owing to the severity and frequency of relapses, secondary prophylaxis against Leishmania is recommended in HIV-infected patients. Allopurinol has been used because of its ease of administration and low toxicity, but results have been unsatisfactory.2,3 The administration of SbV once a month has proved to be an effective low-cost treatment for the prevention of recurrent VL in HIV-coinfected patients. It is well tolerated and produces virtually no adverse side effects.3

Studies have shown a highly variable in vitro response of Leishmania infantum to SbV and the SbV IC50 of Leishmania strains, from patients with treatment failure, to be higher than that of strains from patients in whom treatment is successful.4

We studied the in vitro activity of meglumine antimoniate on promastigotes and intracellular amastigotes of repeated L. infantum isolates from patients treated with SbV, with and without prophylaxis with this drug, and from patients treated with amphotericin B. This allowed us to observe the role of drug pressure in inducing resistance to SbV in Leishmania strains.

L. infantum isolates were obtained from HIV-coinfected patients in and around Barcelona. Seven patients were treated with meglumine antimoniate (Glucantime, Rhône-Mérieux, Lyon, France; SbV 850 mg/day for 28 days). Four received monthly prophylaxis with the same dosage of this drug. Three other patients were treated with a lipid complex of amphotericin B (Abelcet, Pen, Barcelona, Spain) at varying dosages. For all patients, one isolate was obtained during the first VL episode and before treatment. Other isolates were obtained during VL relapses. All isolates were stored in liquid nitrogen until used for drug testing.

The in vitro susceptibility tests to meglumine antimoniate were performed on promastigote as well as intracellular amastigote stages.

Assays on promastigotes were performed as described previously.5 Briefly, promastigotes were cultured in Schneider's insect medium with heat-inactivated fetal calf serum. Serial dilutions of the drug in promastigote culture medium were performed in 96-well microtitre plates (Costar 3595; Corning Costar, Cambridge, MA, USA). Promastigotes (105) in their logarithmic growth phase were then added to each well and incubated at 26°C for 48 h. Growth was measured through acid phosphatase activity by the addition of p-nitrophenyl phosphate (Sigma N-6260; Sigma, St Louis, MO, USA). All assays were performed in duplicate at least twice.

Intracellular amastigotes were cultured in the murine monocyte-macrophage cell line RAW 264.7 (American Type Culture Collection) in RPMI-1640 medium (Bio-Whittaker 12-115; Boehringer-Ingelheim, Bio-Whittaker, Verriers, Belgium) in 8 LabTek Chamber Slide System (Nalge Nunc, Hamburg, Germany), as described before.5 Serial dilutions of the drug were added to each well and cultured for 2 days at 37°C in a 5% CO2 atmosphere. Drug activity was evaluated by the calculation of the percentage of infected cells. Counting was performed at three distinct places in the well and each assay was performed twice.

The concentration of antimony that produced a 50% reduction in growth (promastigotes) or in infected cells (intracellular amastigotes) (IC50) was determined from a least-squares linear regression of growth rate or percentage of infected cells versus log antimony concentration.

Our results on the susceptibility of L. infantum promastigotes to meglumine antimoniate correlate with those obtained for intracellular amastigotes (Pearson's r = 0.869, P < 0.001). This corroborates the lack of intrinsic stage-specific susceptibility of L. infantum strains to SbV.5 The susceptibility to meglumine antimoniate decreased in isolates obtained after a first course of treatment with SbV in patients from groups (a) and (b) (Wilcoxon test P = 0.018 when assays were performed on intracellular amastigotes). For patient LH-259 the susceptibility seemed to decrease progressively with the occurrence of three successive relapses. No difference in susceptibility to meglumine antimoniate was observed between strains obtained in the first leishmaniasis episode and in relapses from individuals receiving amphotericin B (FigureGo).



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Figure. In vitro susceptibility to SbV of intracellular amastigotes of L. infantum strains from HIV-coinfected patients in consecutive leishmaniasis episodes (first episode, open bars; second episode, grey bars; third episode, striped bars; fourth episode, black bars). (a) Patients treated with meglumine antimoniate (850 mg/day for 28 days). Time between episodes: LH-82, 9 months; LH-368, 1 month and LH-409, 1 month. (b) Patients treated with meglumine antimoniate (850 mg/day for 28 days) and secondary prophylaxis with this drug (850 mg monthly). Time between episodes: LH-96, 25 months; LH-159, 67 months; LH-168, 6 months; LH-259, 1, 2 and 2 months, respectively. (c) Patients treated with different dosages of amphotericin B. Time between episodes: LH-367, 18 months; LH-400, 2 months; and LH-432, 3 and 1 months, respectively. LH-values are assigned according to the laboratory. Results are represented as means ± s.d.; n = 6 (three counts from two repeated cultures).

 
This study confirms previous observations that the susceptibility of L. infantum strains to SbV decreases in patients undergoing treatment with meglumine antimoniate.4 A recent study on human and canine L. infantum isolates in Catalonia (unpublished data) found that 11 of the 43 strains analysed from individuals without treatment had SbV IC50 values that exceeded those in strains from unresponsive patients in the south of France (meglumine antimoniate IC50 > 70 mg /L, approximately equal to SbV 23 mg/L).4 It is to be expected that the drug pressure, caused by treatment and secondary prophylaxis with SbV administered to HIV-coinfected patients will increase the number of resistant Leishmania strains. The dissemination of such resistant strains among HIV patients may be favoured by the direct transmission of Leishmania via iv drug use.6

Further prospective studies with larger cohorts of patients are required to establish the clinical significance of, and the impact of treatment and prophylaxis on, the development of Leishmania strains resistant to SbV. The results of such studies may reveal the need to re-examine current therapy policies in Leishmania–HIV-coinfected patients and/or the use of alternative drugs during relapses in such patients.

Acknowledgments

The authors are grateful to Drs C. Muñoz (Hospital Santa Creu i Sant Pau, Barcelona), I. Gasser (Hospital General Vall d'Hebrón, Barcelona) and L. Moner (Hospital Residència Sant Camil, Sant Pere de Ribes) for providing the strains. The authors thank Robin Rycroft for correcting the English manuscript. This work was supported by the Spanish Ministerio de Sanidad y Consumo, project FIS 97-2004, and Comissionat per Universitats i Recerca, Generalitat de Catalunya exp. 1997 SGR 00341.

Notes

J Antimicrob Chemother 2001; 47: 120–121

References

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2 . Gradoni, L., Scalone, A., Gramiccia, A. & Troiani, M. (1996). Epidemiological surveillance of leishmaniasis HIV-1 infected individuals in Italy. AIDS 10, 785–91.[ISI][Medline]

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