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In Vitro Susceptibility to Miltefosine of Leishmania infantum (syn. L. chagasi) Isolates from Different Geographical Areas in Brazil.

ABSTRACT: Treatment of visceral leishmaniasis in Brazil still relies on meglumine antimoniate, with less than ideal efficacy and safety, making new therapeutic tools an urgent need. The oral drug miltefosine was assayed in a phase II clinical trial in Brazil with cure rates lower than previously demonstrated in India. The present study investigated the susceptibility to miltefosine in 73 Brazilian strains of Leishmania infantum from different geographic regions, using intracellular amastigote and promastigote assays. The EC₅₀ for miltefosine of 13 of these strains evaluated in intracellular amastigotes varied between 1.41 and 4.57 μM. The EC₅₀ of the 73 strains determined in promastigotes varied between 5.89 and 23.7 μM. No correlation between in vitro miltefosine susceptibility and the presence of the miltefosine sensitive locus was detected among the tested strains. The relatively low heterogeneity in miltefosine susceptibility observed for the 73 strains tested in this study suggests the absence of decreased susceptibility to miltefosine in Brazilian L. infantum and does not exclude future clinical evaluation of miltefosine for VL treatment in Brazil.

SUBMITTER: Espada CR

PROVIDER: S-EPMC8228039 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters.Methodology/principal findingsA transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival.Conclusions/significanceLuciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.

Project description:BACKGROUND:Visceral leishmaniasis (VL) is an infectious disease with a variety of clinical signs. The main form of parasite transmission to humans and other mammalian hosts is through the bite of infected arthropod females with Lutzomyia longipalpis serving as the main vector in the Americas. Dogs are the main urban domestic reservoirs of the parasite and the main source of vector infection due to their high prevalence in endemic areas and the large number of parasites in the skin of infected animals. Although miltefosine has been used in Europe since 2002 for treatment of VL infected dogs, in the Americas the treatment of dogs has not been recommended. Therefore, this study aimed to evaluate efficacy of miltefosine observing a reduction of clinical signs in infected dogs and the infectiveness to the vector by Leishmania (L.) infantum. METHODS:To our knowledge, this is the first controlled study using qPCR and xenodiagnosis to evaluate the efficacy of miltefosine (Milteforan®, Virbac) as a single treatment in Brazil. Thirty-five adult dogs with canine visceral leishmaniasis (CVL), confirmed by clinical and laboratory tests, were included in this study. They received miltefosine at a dose of 2 mg/kg every 24 h for 28 days. The dogs were observed over a three-month period, during which clinical evaluations based on a scoring system were conducted at pre-established times. Parasite load was assessed by cytology and real-time polymerase chain reaction (qPCR). Transmissibility to the vector was evaluated by xenodiagnosis. RESULTS:At the end of the period, the following were observed: (i) the remission of clinical signs with a reduction in clinical scores for 94.2% of the animals; (ii) a statistically significant reduction (98.7%) in parasitic load by qPCR; and (iii) a reduction in infectivity to sand flies. After treatment, 74.2% of the animals remained or had become non-infectious. CONCLUSIONS:Our study indicates that the use of miltefosine administered orally for 4 weeks contributes to a clinical improvement and reduction in infectivity of dogs to L. infantum.

Project description:BackgroundSuperoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids.ResultsWe obtained Leishmania infantum mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as FeSOD-A gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in FeSOD-A-/-/+ L. infantum mutant clones than in the wild-type (WT) parasite. The decrease in FeSOD-A expression in L. infantum did not interfere with the parasite growth or susceptibility to amphotericin B. Surprisingly, FeSOD-A-/-/+ L. infantum mutant clones were 1.5- to 2.0-fold more resistant to trivalent antimony and 2.4- to 2.7-fold more resistant to miltefosine. To investigate whether the decrease in FeSOD-A expression was compensated by other enzymes, the transcript levels of five FeSODs and six enzymes from the antioxidant defence system were assessed by RT-qPCR. The transcript level of the enzyme ascorbate peroxidase increased in both the FeSOD-A-/-/+ mutants tested. The FeSOD-A-/-/+ mutant parasites were 1.4- to 1.75-fold less tolerant to oxidative stress generated by menadione. Infection analysis using THP-1 macrophages showed that 72 h post-infection, the number of infected macrophages and their intracellular multiplication rate were lower in the FeSOD-A-/-/+ mutant clones than in the WT parasite.ConclusionsThe unsuccessful attempts to delete FeSOD-A suggest that this gene is essential in L. infantum. This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient L. infantum parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.

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In Vitro Susceptibility to Miltefosine of Leishmania infantum (syn. L. chagasi) Isolates from Different Geographical Areas in Brazil.

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