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

Abstract

Abstract. Leishmania (Viannia) braziliensis is the main causative species of tegumentary leishmaniasis in Brazil. In this study, we evaluated the susceptibility of 16 clinical isolates of L. (V.) braziliensis from different regions of Brazil to miltefosine in vitro. Half-maximal inhibitory concentrations of miltefosine varied from 22.9 to 144.2 μM against promastigotes and from 0.3 to 4.2 μM against intracellular amastigotes. No significant differences were found between isolates of different geographical origins. A clear correlation between the EC 50 against promastigotes and amastigotes within each isolate was found. These findings contribute to the evaluation of miltefosine's potential and limitations for the treatment of tegumentary leishmaniasis in Brazil.Tegumentary leishmaniasis is a disease of importance in Brazil, where it is mainly caused by Leishmania (Viannia) braziliensis. The efficacy of the first-line drug, meglumine antimoniate, for the treatment of cutaneous leishmaniasis in areas of L. (V.) braziliensis predominance in Brazil can be as low as 53%.1 New therapeutic alternatives are highly desirable. Miltefosine (MF) (hexadecylphosphatidylcholine) was approved for the treatment of visceral leishmaniasis in India in 2002, where pentavalent antimony was already considered as ineffective due to widespread parasite resistance. 2This oral drug has also been approved for the treatment of tegumentary leishmaniasis in Colombia, after the demonstration of equivalent efficacy to antimony, and in other countries in South America.3 However, the response is heterogeneous in areas of high prevalence of L. (V.) braziliensis: for example, a clinical trial showed 83% efficacy for MF in cutaneous leishmaniasis in Bolivia, whereas a 53% cure rate was observed in Guatemala. 3,4 In Brazil, 70% success rates were observed in two MF clinical trials of cutaneous leishmaniasis due to L. (V.) braziliensis and Leishmania (V.) guyanensis. 1,5The aim of this work was to characterize the MF susceptibility of L. (V.) braziliensis clinical isolates from Brazilian patients with tegumentary leishmaniasis from two geographically distinct regions.Eight clinical isolates were obtained from lesion biopsies of patients with tegumentary leishmaniasis attending the Anuar Auad Tropical Diseases Hospital, Goiânia, Goiás, Brazil (Leishbank), 6 and eight isolates were obtained through needle aspiration of skin lesions from patients attending the health post of Corte de Pedra, Bahia, Brazil. After the isolation, cultures were frozen and recovered to perform this study. The isolates were typed by polymerase chain reaction of internal transcribed spacer of ribosomal DNA and hsp70 gene followed by restriction analysis using Hae III, as described. 7,8 The M2903 reference strain and 16 clinical isolates produced the expected profile for L. (V.) braziliensis (data not shown).The activity of MF against promastigotes was evaluated by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay as previously described.9 Approximatel...

Investigation of the pathways related to intrinsic miltefosine tolerance in Leishmania (Viannia) braziliensis clinical isolates reveals differences in drug uptake

“…Parasites were grown in M199 medium supplemented with 10% heat inactivated fetal calf serum (FCS), 0,25% hemin, and 2% male urine at 25 °C. L. (V.) braziliensis reference strain (MHOM/BR/1975/M2903) and three clinical isolates previously characterized for susceptibility to MF by our group (Espada et al, 2017) were used in this study: MHOM/BR/2005/LTCP16012, which is more sensitive to MF, and MHOM/BR/2006/LTCP16907 and MHOM/BR/2009/LTCP19446, which are more tolerant to the drug (Table 1).…”

“…Resistant clinical isolates have already been described (Srivastava et al, 2017). Furthermore, Leishmania susceptibility to miltefosine was shown to vary, not only between different species but also in isolates of the same species (Bhandari et al, 2012; Espada et al, 2017; Hendrickx et al, 2015; Kumar et al, 2009; Prajapati et al, 2013; Utaile et al, 2013; Yardley et al, 2005).…”

“…The half-maximal effective concentrations (EC 50 ) against these isolates varied 6-fold and 15-fold for promastigotes and intracellular amastigotes, respectively. Interestingly, these isolates had not been exposed to miltefosine previously and were recovered from patients before treatment with other leishmanicidal drugs (Espada et al, 2017).…”

Miltefosine susceptibility of isolates of Leishmania (Leishmania) infantum from dogs of the municipality of Embu-Guaçu, Brazil

“…1A and 1B). The in vitro susceptibility of isolates and the reference strains of L. (L.) infantum to miltefosine in promastigotes were determined as previously described 3 . The EC50 values of miltefosine against promastigotes ranged from 6,5 ± 0,9 to 34,14 ± 1,56 µM (Fig.…”

Activity of paromomycin against Leishmania amazonensis: Direct correlation between susceptibility in vitro and the treatment outcome in vivo

“…The susceptibility of L. amazonensis promastigotes to PM was determined using the MTT colorimetric assay, after incubation of 2 × 10 6 parasites per well in a 96-well plate in M199 medium for 24 h at 25 °C in the presence of increasing concentrations of PM as previously described ( Espada et al, 2017 ). Promastigotes were counted in a Neubauer haemocytometer and experiments were carried out in triplicate.…”