Leishmania resistance to miltefosine associated with genetic marker.
Article Type: Letter to the editor
Subject: Genetic markers (Research)
Leishmaniasis (Genetic aspects)
Leishmaniasis (Research)
Single nucleotide polymorphisms (Research)
Authors: Cojean, Sandrine
Houze, Sandrine
Haouchine, Djamel
Huteau, Francoise
Lariven, Sylvie
Hubert, Veronique
Michard, Florence
Bories, Christian
Pratlong, Francine
Le Bras, Jacques
Loiseau, Philippe Marie
Matheron, Sophie
Pub Date: 04/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: April, 2012 Source Volume: 18 Source Issue: 4
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: France Geographic Code: 4EUFR France
Accession Number: 286114940
Full Text: To the Editor: During 2000-2010, serial Leishmania isolates obtained from an HIV-infected patient who was not responding to treatment showed a gradual decrease in in vitro miltefosine susceptibility. We performed L. donovani miltefosine transporter (Ldmt) gene analysis to identify an association between miltefosine resistance of reference L. donovani lines and variability in miltefosine response of L. infantum isolates. A new single-nucleotide polymorphism (SNP), L832F, was identified, which might be a marker of miltefosine resistance in leishmaniasis.

The patient, a 46-year-old woman, had lived in France since 1994 but regularly returned to Algeria, her country of birth. HIV-1 infection was diagnosed in 1991. Antiretroviral therapy was initiated in 1993, leading to undetectable viral load and a CD4+ T-cell count of 185 cells/[mm.sup.3] (reference >450/[mm.sup.3]). Concurrent conditions were thoracic herpes zoster in 1996, hairy leukoplakia of tongue, oropharyngeal candidiasis, and chronic renal failure of unknown cause since 2000.

Visceral leishmaniasis was diagnosed in 1998 by culture of a bone marrow smear, which showed intracellular amastigotes. Use of meglumine antimonate (Glucantime; Sanofi, Paris, France), a drug of choice for the treatment of leishmaniasis, was contraindicated because of pancreatitis in the patient and in vitro isolate susceptibility variation; therefore, induction therapy consisted of liposomal amphotericin B (AmpB [AmBisome; Astellas Pharma US, Deerfield, IL, USA]) at a dose of 3 mg/kg/d for 5 consecutive days, then 1x week for 5 weeks (total dose 30 mg/kg) during 1998-2000 (Table). The same medication was administered for relapses at 4 mg/kg/d for 5 days, then 4 mg/kg 1x week for 5 weeks (total dose 40 mg/kg) during 2001-2010. Given the adverse effects of AmpB and the availability of oral miltefosine (Impavido; AEterna Zentaris Inc., Quebec City, Quebec, Canada), the latter drug was used for maintenance treatment during 2001-2007 at 50 mg 2x/d. Leishmaniasis was monitored by leukocytoconcentration and culture of blood samples on Novy-Nicolle-McNeal medium.

When signs of biological and clinical relapse appeared, bone marrow was aspirated for parasite detection. After culture of the aspirate and isoenzyme determination, the strain was identified as L. infantum, zymodeme MON-24. Eleven relapses were documented; all were confirmed by positive direct examination of bone marrow or blood, but cultures of only 7 samples yielded positive results (Table).

The susceptibility of 4 cryopreserved isolates ([S.sub.1], [S.sub.3], [S.sub.4], and [S.sub.6]; Table) to AmpB and to miltefosine was studied in the in vitro promastigote and axenic amastigote form by determining the concentrations inhibiting parasite growth by 50% (1,2). The 50% inhibitory concentration (IC50) was determined in parallel for the following reference L. donovani lines: a wild-type L. donovani LV9 (MHOM/ET/67/HU3) line (LV9 WT), a wild-type L. donovani DD8 (MHOM/IN/80/DD8) line (DD8 WT), a laboratory miltefosine-resistant line obtained from LV9 WT (LV9 miltefosine-R, resistant to 90 umol/L miltefosine), and the laboratory AmBresistant line obtained from DD8 WT (DD8 AmB-R, resistant to 1.4 [micro]mol/L AmB) on promastigote and axenic amastigote forms (3,4).

The AmB susceptibility of the isolates did not change notably over time; [IC.sub.50] values ranged from 0.09 [micro]mol/L to 0.24 [micro]mol/L, regardless of parasite form, similar to those of wild-type reference strains (Table). In contrast, the [IC.sub.50] values of miltefosine increased greatly over time, from 5.00 [micro]mol/L to 50.10 [micro]mol/L. During the 6 years of follow-up with miltefosine maintenance therapy, the susceptibility of the isolate ([S.sub.6]) obtained 6 months after miltefosine treatment withdrawal in 2008 was 6-fold higher than that of the first isolate ([S.sub.1]) obtained in 2000.

The L. donovani miltefosine transporter protein (LdMT) promotes miltefosine translocation (5), and LdMT inactivation in L. donovani promastigotes leads to miltefosine resistance at the promastigote and amastigote stages (6). In 2003 and 2006 studies, several mutations were linked to the inability of parasites to take up miltefosine and to miltefosine resistance (5,7). In a 2009 study, the weak expression of LdMT and its [beta] subunit LdROS3 in L. braziliensis isolates was linked to diminished sensitivity (8). We sequenced the entire Ldmt gene (3,294 bp) in the reference strains and the clinical isolates for SNP analysis (5,7). Only 1 new SNP, L832F, was found in the miltefosine-resistant reference strain (LV9 miltefosine-R) and in clinical isolate [S.sub.6]. The L832 wild-type allele was found in isolate S1 and in the miltefosine-sensitive reference lines (LV9, DD8, and DD8 AmpB-R), whereas both alleles were found in isolates [S.sub.3] and [S.sub.4], with a decrease in the wild-type allele (Table). The last isolate, which was obtained 3 years after miltefosine withdrawal and could not be subcultured, had reverted to the wild-type allele (L832).

These results point to a relation between the 832F allele and diminished susceptibility to miltefosine. Analysis of this case of miltefosine resistance in a patient co-infected with Leishmania sp. and HIV strongly suggests that an SNP (L832F) in the Ldmt gene could represent a molecular marker of miltefosine resistance in L. infantum and L. donovani.

Sandrine Cojean, Sandrine Houze, Djamel Haouchine, Francoise Huteau, Sylvie Lariven, Veronique Hubert, Florence Michard, Christian Bories, Francine Pratlong, Jacques Le Bras, Philippe Marie Loiseau, and Sophie Matheron

Author affiliations: University of Paris-Sud, Chatenay-Malabry, France (S. Cojean, F. Huteau, C. Bories, P.M. Loiseau); Paris Descartes University, Paris, France (S. Houze, D. Haouchine, V. Hubert, J. Le Bras); Paris Diderot University, Paris (S. Lariven, F. Michard, S. Matheron); and Montpellier University, Montpellier, France (F. Pratlong)

DOI: http://dx.doi.org/ 10.3201/eid1804.110841

References

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(2.) Vermeersch M, da Luz RI, Tote K, Timmermans JP, Cos P, Maes L. In vitro susceptibilities of Leishmania donovani promastigote and amastigote stages to antileishmanial reference drugs: practical relevance of stage-specific differences. Antimicrob Agents Che mother. 2009;53:3855-9. http://dx.doi. org/10.1128/AAC.00548-09

(3.) Mbongo N, Loiseau PM, Billion MA, Robert-Gero M. Mechanism of amphotericin B resistance in Leishmania donovani promastigotes. Antimicrob Agents Chemother. 1998;42:352-7.

(4.) Seifert K, Matu S, Perez-Victoria J, Castanys S, Gamarro F, Croft SL. Characterisation of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine). Int J Antimicrob Agents. 2003;22:380-7. http://dx.doi. org/10.1016/S0924-8579(03)00125-0

(5.) Perez-Victoria FJ, Gamarro F, Ouellette M, Castanys S. Functional cloning of the miltefosine transporter. A novel P-type phospholipid translocase from Leishmania involved in drug resistance. J Biol Chem. 2003;278:49965-71. http://dx.doi. org/10.1074/jbc.M308352200

(6.) Seifert K, Perez-Victoria FJ, Stettler M, Sanchez-Canete MP, Castanys S, Gamarro F, 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. http://dx.doi. org/10.1016/j.ijantimicag.2007.05.007

(7.) Perez-Victoria FJ, Sanchez-Canete MP, Seifert K, Croft SL, Sundar S, Castanys S, et al. Mechanisms of experimental resistance of Leishmania to miltefosine: implications for clinical use. Drug Resist Updat. 2006;9:26-39. http://dx.doi. org/10.1016/j.drup.2006.04.001

(8.) Sanchez-Canete MP, Carvalho L, Perez-Victoria FJ, Gamarro F, Castanys S. Low plasma membrane expression of the miltefosine transport complex renders Leishmania braziliensis refractory to the drug. Antimicrob Agents Che mother. 2009;53:1305-13. http://dx.doi. org/10.1128/AAC.01694-08

Address for correspondence: Sandrine Cojean, UMR 8076 CNRS BioCIS; Groupe Chimiotherapie Antiparasitaire; Univ Paris Sud; 5 Rue Jean-Baptiste Clement, 92290 Chatenay-Malabry, France; email: sandrine. cojean@u-psud.fr
Table. Comparions of [IC.sub.50] for AmpB and miltefosine against
promastigotes and axenic amastigotes and distribution of LdMT SNPs
in Leishmania infantum isolates and reference strains *

                                                       [IC.sub.50],
                                                       [micro]mol/L
                                                       [+ or -] SEM

                                                           AmpB

Isolate              Year             AmpB             Miltefosine

--                   1998    3 mg/kg/d x 5 d; then          --
                                 1 x/wk x 5 wk

[S.sub.1]            2000    3 mg/kg/d x 5 d; then          --
                                 1 x/wk x 5 wk

--                   2001    4 mg/kg/d x 5 d; then      50 mg 2x/d
                                 1 x/wk x 5 wk

[S.sub.3]            2005    4 mg/kg/d x 5 d; then
                                 1 x/wk x 5 wk
                               ([double dagger])
[S.sub.4]

--                   2007    4 mg/kg/d x 5 d; then
                                 1 x/wk x 5 wk

[S.sub.6]            2008    4 mg/kg/d x 5 d; then          --
                                 1 x/wk x 5 wk

[S.sub.7]            2010    4 mg/kg/d x 5 d; then          --
                                 1 x/wk x 5 wk

Reference strain

  LV9 WT

  LV9
    Miltefosine-R

  DD8 WT

  DD8 AmpB-R

                       [IC.sub.50], [micro]mol/L [+ or -] SEM

                                        AmpB

                                                  Axenic
Isolate                Promastigotes           amastigotes

--                           --                     --

[S.sub.1]            0.09 [+ or -] 0.04     0.10 [+ or -] 0.03
                         ([dagger])

--                           --                     --

[S.sub.3]            0.13 [+ or -] 0.03     0.20 [+ or -] 0.03

[S.sub.4]            0.24 [+ or -] 0.01     0.15 [+ or -] 0.02
                         ([dagger])

--                           --                     --

[S.sub.6]            0.16 [+ or -] 0.03     0.11 [+ or -] 0.03

[S.sub.7]                    --                     --

Reference strain

  LV9 WT             0.03 [+ or -] 0.02     0.02 [+ or -] 0.05

  LV9                0.22 [+ or -] 0.04     0.70 [+ or -] 0.09
    Miltefosine-R

  DD8 WT             0.06 [+ or -] 0.02     0.05 [+ or -] 0.03
                         ([dagger])             ([dagger])

  DD8 AmpB-R         1.42 [+ or -] 0.06     1.00 [+ or -] 0.07
                         ([dagger])             ([dagger])

                     [IC.sub.50], [micro]mol/L [+ or -] SEM

                                   Miltefosine

                                                Axenic           Ldmt
Isolate                Promastigotes          amastigotes        SNP

--                          --                    --              --

[S.sub.1]           7.14 [+ or -] 0.56     5.00 [+ or -] 0.7     L832
                        ([dagger])            ([dagger])

--                          --                    --              --

[S.sub.3]           25.93 [+ or -] 1.46   21.00 [+ or -] 1.50   832L/F
                        ([dagger])            ([dagger])

[S.sub.4]           27.89 [+ or -] 1.76   31.90 [+ or -] 1.60
                        ([dagger])            ([dagger])

--                          --                    --              --

[S.sub.6]           44.30 [+ or -] 3.70}  50.10 [+ or -] 1.00    832F
                                              ([dagger])

[S.sub.7]                   --                    --             L832

Reference strain

  LV9 WT            4.46 [+ or -] 0.29     6.20 [+ or -] 0.3     L832
                        ([dagger])

  LV9               45.84 [+ or -] 2.40   54.20 [+ or -] 2.20    832F
    Miltefosine-R       ([dagger])            ([dagger])

  DD8 WT            17.40 [+ or -] 1.70   12.40 [+ or -] 1.50    L832

  DD8 AmpB-R        15.20 [+ or -] 1.00   10.30 [+ or -] 1.20    L832

* [IC.sub.50], 50% inhibitory concentration; AmpB, amphotericin B;
Ldmt, Leishmania donovani miltefosine transporter gene; SNP, single-
nucleotide polymorphism; --, assay not performed because sample
unavailable or not culturable; WT, wild type; R, resistant.

([dagger]) Significance was analyzed by using the nonparametric
Mann/Whitney U test to compare the [IC.sub.50] of the isolates with
the [IC.sub.50] of reference strains; p<0.01 was considered
significant. [IC.sub.50] of AmpB and miltefosine was compared with
[IC.sub.50] of reference strains and [S.sub.1]/[S.sub.3], [S.sub.1]/
[S.sub.4], and [S.sub.1]/[S.sub.6]. Miltefosine: [S.sub.1]/[S.sub.3],
[S.sub.1]/[S.sub.4], [S.sub.1]/[S.sub.6], [S.sub.4]/[S.sub.6]; p<0.01.
AmpB: [S.sub.1]/[S.sub.4] significant p<0.01; [S.sub.1]/[S.sub.3],
[S.sub.1]/[S.sub.6] not significant.

([double dagger]) For each relapse.
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