Drug Susceptibility in Leishmania Isolates Following Miltefosine Treatment in Cases of Visceral Leishmaniasis and Post Kala-Azar Dermal Leishmaniasis

Abstract

BackgroundWith widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.Methodology and Principal FindingsWe measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC50±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC50 = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC50 = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC50 = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC50 = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC50 = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC50 = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC50 = 7.05±2.24 µM and 6.18±1.51 µM.ConclusionThe in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.

Treatment Failure and Miltefosine Susceptibility in Dermal Leishmaniasis Caused by Leishmania Subgenus Viannia Species

“…Singlenucleotide polymorphisms (SNP) have been associated with functional variations of the LdMT/LdRos3 complex in L. donovani (9). However, our results and those of studies evaluating the relationship of these polymorphisms to drug susceptibility profiles of clinical strains have either not demonstrated an association or not provided consistent evidence thus far (19), substantiating the ra-…”

“…positions 1259 and 2567, previously reported to be associated with LbMT loss of function (9), and at position 630 analyzed in L. donovani strains isolated from patients with VL and PKDL who relapsed after treatment with miltefosine (19) revealed no association between these SNPs and the resistance phenotype of the clinical strains in our study or genetic variations between strains isolated before treatment and at treatment failure from the same patient (Fig. 5).…”

“…Strains of L. donovani isolated from visceral leishmaniasis and PKDL patients who relapsed following milte- fosine treatment were significantly more tolerant to miltefosine than the corresponding pretreatment strains (19). The selective potential of miltefosine treatment in visceral leishmaniasis was also substantiated by the incremental increase in tolerance of sequential isolates of Leishmania infantum obtained at relapse from an HIV-coinfected patient who received maintenance treatment with miltefosine for several years (22).…”

“…The presence of C1259A (T420N) and T2567C (L856P) polymorphisms in the LbMT gene, previously reported to be associated with experimentally derived miltefosine resistance (9), and G630A (W210*) analyzed in VL and post-kala-azar dermal leishmaniasis (PKDL) strains of L. donovani (19) were characterized by sequence analysis of PCR products. Total DNA was extracted from logarithmic-phase promastigotes using a DNeasy blood and tissue kit (Qiagen).…”

Susceptibility to Miltefosine in Brazilian Clinical Isolates of Leishmania (Viannia) braziliensis

“…[12][13][14][15][16] One of the limitations of the present study was to lack the evaluation of a relationship between in vitro susceptibility with in vivo response to MF. However, these isolates were not from patients who used MF.…”