Fatty Acid Profiles of Leishmania major Derived from Human and Rodent Hosts in Endemic Cutaneous Leishmaniasis Areas of Tunisia and Algeria.

Leishmaniasis is a protozoal vector-borne disease that affects both humans and animals. In the Mediterranean Basin, the primary reservoir hosts of Leishmania spp. are mainly rodents and canids. Lipidomic approaches have allowed scientists to establish Leishmania spp. lipid profiles for the identification of cell stage specific biomarkers, drug mechanisms of action, and host immune response. Using an in silico approach of global network interaction between genes involved in fatty acid (FA) synthesis followed by the GC-MS approach, we were able to characterize the fatty acid profiles of L. major derived from human and rodent hosts. Our results revealed that the lipid profile of L. major showed similarities and differences with those already reported for other Leishmania species. Phospholipids are the predominant lipid class. FA composition of rodent parasites was characterized by a lower abundance of the precursor C18:2(n-6). One of the rodent clones, which also expressed the lowest lipid abundance in PL and TAG, was the least sensitive clone to the miltefosine drug and has the lowest infection efficiency. Our findings suggest that the lipid composition variation may explain the response of the parasite toward treatment and their ability to infect their host.

Meta-analysis and discussion on challenges to translate Leishmania drug resistance phenotyping into the clinic.

Antimicrobial resistance (AMR) threatens the prevention and treatment of infections caused by a large range of microorganisms. Leishmania is not an exception and treatment failure due to drug-resistant organisms is increasingly reported. Currently, no molecular methods and marker are validated to track drug-resistant organism and antimicrobial susceptibility tests are roughly not amenable to a clinical setting. Taking these facts into account, it is essential to reflect on ways to translate basic knowledge into methodologies aimed to diagnose leishmania drug resistance. As a matter of fact, a meta-analysis of the literature discloses the reliability of the promastigotes antimicrobial susceptibility tests (AST) to predict intracellular amastigotes susceptibility status. Promastigote cultures that are easy to perform, typically inexpensive and amenable to standardization should represent a candidate to diagnose resistance. Using AST performed on promastigote, we propose a way to improve leishmania drug resistance diagnosis in the framework of guidance and guideline of the bacterial drug resistance diagnosis. In this review, we highlight challenges that remained and discuss the definition of clinical breakpoints, including the epidemiological cutoff (ECOFF), to track drug-resistant isolates. Our analysis paves the ways to standardize and analyze anti-leishmania susceptibility tests output in order to guide the characterization of drug-resistant isolates, the clinical decision during treatment and the search for new molecular markers.

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.

Antimony susceptibility of Leishmania isolates collected over a 30-year period in Algeria.

BACKGROUND: In Algeria, the treatment of visceral and cutaneous leishmanioses (VL and CL) has been and continues to be based on antimony-containing drugs. It is suspected that high drug selective pressure might favor the emergence of chemoresistant parasites. Although treatment failure is frequently reported during antimonial therapy of both CL and VL, antimonial resistance has never been thoroughly investigated in Algeria. Determining the level of antimonial susceptibility, amongst Leishmania transmitted in Algeria, is of great importance for the development of public health policies. METHODOLOGY/PRINCIPAL FINDINGS: Within the framework of the knowledge about the epidemiology of VL and CL amassed during the last 30 years, we sampled Leishmania isolates to determine their susceptibility to antimony. We analyzed a total of 106 isolates including 88 isolates collected between 1976 and 2013 in Algeria from humans, dogs, rodents, and phlebotomines and 18 collected from dogs in France. All the Algerian isolates were collected in 14 localities where leishmaniasis is endemic. The 50% inhibitory concentrations (IC50) of potassium antimony tartrate (the trivalent form of antimony, Sb(III)) and sodium stibogluconate (the pentavalent form of antimony, Sb(V)) were determined in promastigotes and intramacrophage amastigotes, respectively. The epidemiological cutoff (ECOFF) that allowed us to differentiate between Leishmania species causing cutaneous or visceral leishmaniases that were susceptible (S+) or insusceptible (S-) to the trivalent form of antimony was determined. The computed IC50 cutoff values were 23.83 µg/mL and 15.91 µg/mL for VL and CL, respectively. We report a trend of increasing antimony susceptibility in VL isolates during the 30-year period. In contrast, an increase in the frequency of S- phenotypes in isolates causing CL was observed during the same period. In our study, the emergence of S- phenotypes correlates with the inclusion of L. killicki (syn: L. tropica) isolates that cause cutaneous leishmaniasis and that have emerged in Algeria during the last decade. CONCLUSION/SIGNIFICANCE: Our results provide insight into the spatiotemporal dynamics of Leishmania antimony susceptibility in Algeria. We highlight the need for the future implementation of an effective methodology to determine the antimony susceptibility status of Leishmania isolates to detect the emergence of and prevent the dissemination of drug-resistant strains.

Development of a Murine Infection Model with Leishmania killicki, Responsible for Cutaneous Leishmaniosis in Algeria: Application in Pharmacology.

In Algeria, Leishmania infantum, Leishmania major, and Leishmania killicki (Leishmania tropica) are responsible for cutaneous leishmaniosis. We established a murine model of L. killicki infection to investigate its infective capacity, some immunophysiopathological aspects, and its suitability for pharmacological purposes. Following the injection of L. major or L. killicki metacyclic promastigotes in the ear dermis of BALB/c mice, the course of infection was followed. The infection with L. killicki caused slower lesion formation than with L. major. The presence of L. killicki or L. major DNA and parasites was detected in the ear dermis and in lymph nodes, spleen, and liver. Lesions induced by L. killicki were nonulcerative in their aspect, whereas those caused by L. major were highly ulcerative and necrotic, which matches well with the lesion phenotype reported in humans for L. killicki and L. major, respectively. The treatment of L. killicki lesions by injection of Glucantime® significantly reduced the lesion thickness and parasite burden. Ear dermal injection of BALB/c mice constitutes a model to study lesions physiopathology caused by L. killicki and presents interest for in vivo screening of new compounds against this pathogen, emerging in Algeria.

Transmission potential of antimony-resistant leishmania field isolates.

We studied the development of antimony-resistant Leishmania infantum in natural vectors Lutzomyia longipalpis and Phlebotomus perniciosus to ascertain the risk of parasite transmission by sand flies. All three resistant strains produced fully mature late-stage infections in sand flies; moreover, the resistant phenotype was maintained after the passage through the vector. These results highlight the risk of circulation of resistant Leishmania strains and question the use of human drugs for treatment of dogs as Leishmania reservoirs.