SUMMARY: Cutaneous and visceral leishmaniasis are amongst the most devastating infectious diseases of our time, affecting millions of people worldwide. The treatment of these serious diseases rely on a few chemotherapeutic agents, most of which are of parenteral use and induce severe side-effects. Furthermore, rates of treatment failure are high and have been linked to drug resistance in some areas. Here, we reviewed data on current chemotherapy practice in leishmaniasis. Drug resistance and mechanisms of resistance are described as well as the prospects for applying drug combinations for leishmaniasis chemotherapy. It is clear that efforts for discovering new drugs applicable to leishmaniasis chemotherapy are essential. The main aspects on the various steps of drug discovery in the field are discussed.

Leishmaniasis is a neglected tropical disease with two main clinical forms: cutaneous and visceral leishmaniasis. Diagnosis of leishmaniasis is still a challenge, concerning the detection and correct identification of the species of the parasite, mainly in endemic areas where the absence of appropriate resources is still a problem. Most accessible methods for diagnosis, particularly in these areas, do not include the identification of each one of more than 20 species responsible for the disease. Here, we summarize the main methods used for the detection and identification of leishmaniasis that can be performed by demonstration of the parasite in biological samples from the patient through microscopic examination, by in vitro culture or animal inoculation; by molecular methods through the detection of parasite DNA; or by immunological methods through the detection of parasite antigens that may be present in urine or through the detection of specific antibodies against the parasite. Potential new methods that can be applied for laboratory diagnosis of leishmaniasis are also discussed.

Miltefosine (MF) is the first oral compound used in the chemotherapy against leishmaniasis. Since the mechanism of action of this drug and the targets of MF in Leishmania are unclear, we generated in a step-by-step manner Leishmania major promastigote mutants highly resistant to MF. Two of the mutants were submitted to a short-read whole genome sequencing for identifying potential genes associated with MF resistance. Analysis of the genome assemblies revealed several independent point mutations in a P-type ATPase involved in phospholipid translocation. Mutations in two other proteins—pyridoxal kinase and α-adaptin like protein—were also observed in independent mutants. The role of these proteins in the MF resistance was evaluated by gene transfection and gene disruption and both the P-type ATPase and pyridoxal kinase were implicated in MF susceptibility. The study also highlighted that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. Whole genome sequencing was used to pinpoint known and new resistance markers associated with MF resistance in the protozoan parasite Leishmania. The study also demonstrated the polyclonal nature of a resistant population with individual cells with varying susceptibilities and genotypes. Leishmania spp. are parasitic protozoa responsible for a spectrum of diseases known as leishmaniasis. There are few drugs available for the treatment of these diseases, and miltefosine is the first oral drug used in treatment of visceral leishmaniasis, a form of the disease that can be lethal if not treated. In this study, we seek to understand the mechanism of action and identify targets of the drug by generating promastigote mutants highly resistant to miltefosine. Two independent mutants were submitted to short read whole genome sequencing. Genome analysis of these mutants has permitted us to identify point mutations in three genes (P-type ATPase, pyridoxal kinase and α-adaptin like protein) that were also present in other independent miltefosine resistant mutants. Some of the new genes identified here could be useful as potential markers for miltefosine resistance in Leishmania. Moreover, our approach has permitted us to highlight that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. This may have practical applications while studying resistance.

Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis. We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated. Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers. Leishmania are unicellular microorganisms that can be transmitted to humans by the bite of sandflies. They cause a spectrum of diseases called leishmaniasis, which are classified as neglected tropical diseases by the World Health Organization. The treatment of leishmaniasis is based on the administration of antimony-containing drugs. These drugs have been used since 1947 and still constitute the mainstay for leishmaniasis treatment in several countries. One of the problems with these compounds is the emergence of resistance. Our work seeks to understand how these parasites become resistant to the drug. We studied antimony-resistant Leishmania amazonensis mutants. We analyzed gene expression at the whole genome level in antimony-resistant parasites and identified mechanisms used by Leishmania for resistance. This work could help us in developing new strategies for treatment in endemic countries where people are unresponsive to antimony-based chemotherapy. The identification of common mechanisms among different species of resistant parasites may also contribute to the development of diagnostic kits to identify and monitor the spread of resistance.