Leishmania antimony resistance/susceptibility in Algerian foci

Algeria is one of the most endemic countries for cutaneous and visceral forms of leishmaniosis. Strikingly, with more than 21,000 annual cases of cutaneous leishmaniosis recorded in 2010, the disease has a major public health impact. For all forms of leishmaniosis, the fi rst line treatment relies on antimonial containing drug i.e., Glucantime®, developed during the 1950’s. As early as 1986, antimonial treatment failure was reported during cutaneous leishmaniosis treatment. Linked to this therapeutic failure, Leishmania strains displaying a low susceptibility towards antimonials were isolated. Nevertheless, in Algeria, antimonial formulations still remain the fi rst line drug for all clinical forms of leishmaniosis. Therefore, an urgent need of knowledge on the baseline antimony susceptibility status of parasites strains in Algeria is required. These pieces of knowledge will shed light not only on the prevalence of antimony resistance in this area but also on underlying factors triggering this drug resistance in natural populations. Here, we performed a review of the literature on what is known about epidemiology, treatment failure, and antimony-resistance, in Algeria. We bring information on underlying mechanisms acting in antimony resistant parasites and discuss their potential to be used for diagnostic purpose. This analysis will help to set up protocols aiming at detecting antimony resistant strains in Algeria and to test the risk of transmission, two steps that are essential to defi ne public health policy in Algeria. Review Article Leishmania antimony resistance/ susceptibility in Algerian foci Eddaikra N1-3*, Ait-Oudhia K4, Oury B2,5, Moulti-Mati Farida3, Harrat Z1,6 and Sereno D2,5 1Laboratory of Parasitary Eco-epidemiology and Genetics of Populations, Pasteur Institute of Algiers, Petit Staoueli Road Dely Brahim, Algiers, Algeria 2Joint Research Unit IRD 224 MiVegec (Infectious Diseases and Vectors: Ecology, Genetics, Evolution and Control), Research Institute for Development (IRD), PO Box 64501, 34394 Montpellier cedex 5, France 3Laboratory of Analytical Biochemistry and Biotechnologies, Mouloud Mammeri University of Tizi Ouzou, Algeria 4National Veterinary Superior School, PO Box 161, Hassan Badi El-Harrach, Algiers, Algeria 5IRT Research Unit 177 InterTryp (“Hosts-VectorsParasites-Environment Interactions in Neglected Tropical Diseases Caused by Trypanosomatids”), Research Institute for Development (IRD), BP 64501, 34394 Montpellier cedex 5, France 6Laboratory of Biodiversity and Environment: Interactions, Genomes (LBEIG) of the Faculty of Biological Sciences of the University of Science and Technology Houari Boumediene (USTHB) of Algiers, Algeria Dates: Received: 19 September, 2017; Accepted: 27 September, 2017; Published: 28 September, 2017 *Corresponding author: Eddaikra N, Laboratory of Parasitary Eco-epidemiology and Genetics of Populations, Pasteur Institute of Algiers, Petit Staoueli Road Dely Brahim, Algiers, Algeria, Tel/Fax: +213 21 341920; +213 772366699; E-mail: neddaikra@yahoo.fr


Introduction
Leishmania sp. are causative protozoal agents of various forms of leishmaniosis, which is a signifi cant cause of morbidity and mortality in more than 98 countries and territories [1]. The a major health problem. In several regions of the world, the incidence of leishmaniosis outbreaks has been associated with urbanization, travel, climatic change and social confl icts [2,3].
In the absence of effective vaccines, the only feasible way to treat and control leishmaniosis is through the use of affordable medications. The current chemotherapeutic arsenal consists of molecules developed in the 1950s', including pentavalent antimony (Sb(V)) compounds (e.g., Pentostam®, Glucantime®), pentamidine and various formulations of the antifungal Amphotericin B and, more recently, miltefosine [4][5][6][7]. The choice of the chemotherapeutic protocol is frequently dictated by economic considerations instead of being based on clinical or biological indications. Therefore, antimonial containing drugs, one of the most affordable molecules, are in clinical use to treat all forms of leishmaniosis in most part of the word [8]. Therapeutic failure during antimony treatment remains a well-known problem, but antimony resistance is not the only factor responsible for the therapeutic failure. Indeed, 024-032. factors that are linked to the host (e.g., immunosuppression or malnutrition), the drug itself (e.g., drug batch or counterfeit drugs), the Leishmania species, the practitioner or the patient (e.g., incomplete treatment follow-up) will also play a role in parasite drug resistance and treatment failure [4,9,10]. In most parts of the world, the frequency of parasite antimony resistance linked to treatment failure is unknown [4,9,10]. This information should be crucial to address the risk of selection and transmission of drug-resistant parasites, particularly in areas where antimony is the only chemotherapeutic alternative.
Here we review the current epidemiological situation of leishmanioses in Algeria, address available tools to diagnose antimony resistance and, then focus on ongoing researches about antimony resistance in this area.

Update on past and current epidemiological data
The fi rst case description of leishmaniosis in Algeria was carried out by the Sergent brothers almost a century ago [11].

Human visceral and cutaneous leishmanioses
In Algeria, three distinct clinical forms of leishmaniosis are encountered. In the Sahara and the highland regions, the zoonotic form of leishmaniosis, caused by L major (ZCL), is endemic ( Figure 1). Two variants of L. major, zymodeme MON-25, and zymodeme MON-269 are reported to be present in Algeria [12,13]. The insect vector for L. major is Phlebotomus papatasi [14], and the wild rodents Psamommys obesus and Meriones shawi are the reservoir hosts of the parasite [15,16].
Moreover, natural infection of hedgehogs with L. major was reported [17].
In the central part of the country and particularly in the oasis of Ghardaia, the chronic form of cutaneous leishmaniosis is due to L killicki. L. killicki belongs to the L tropica complex and generally occurs in sympatry with L. major [18,19]. The annual incidence of this peculiar form of cutaneous leishmaniosis is estimated to be less than 100 cases per year, in Algeria. The proven vector for L. killicki is Phlebotomus sergenti [20], and the suspected reservoir host is Massoutiera mzabi, a rodent close to Ctenodactylus gundi that has been found naturally infected with L. killicki in Tunisia [21]. In the northern part of the country, a zoonotic cutaneous form caused by L infantum is sporadic.
The proven vector is Phlebotomus perfi liewi [22], and the variant L. infantum MON-24 [23], responsible for this cutaneous form, is present in dogs, the animal reservoir of this species. The visceral form of leishmanioses caused by L. infantum occurs mainly in the northern part of the country. The active visceral leishmaniosis foci are located in the region of Kabylie in the north of the country as well in the east in the departments of Jijel and Constantine . Some cases are also reported in the south, in Tassili N'ajjer and the Hoggar mountains in Illizi and Tamanrasset respectively [24]. The proven vector of L. infantum in Algeria is Phlebotomus perniciosus and canids, dogs and jackals are the main reservoirs [12,25].
Currently, an emerging focus of cutaneous leishmaniosis in the northern part of Algeria is reported [26]. Parasitological

Canine leishmanioses in Algeria
Since 1908 it is recognized that the dog plays a major role in the life-cycle of Leishmania infantum [28]. Various sandfl y species belonging to the genus of Phlebotomus act as vectors of the parasite [29]. Canine leishmaniosis (CanL) is a cosmopolitan disease with a worldwide distribution, mainly in tropical areas, but also in the temperate zones of North Africa, Europe, and Asia [30]. The Mediterranean basin is particularly affected and the disease is reported in almost all the countries bordering the Mediterranean, with relatively variable prevalence. In Algeria, the general prevalence of CanL has been under 11% for many years, it has recently increased to 25.1% in 2006 [31]. This increase in the prevalence of canine infection was followed by an increase in human leishmaniosis cases. In the region of Algiers for example, 12.2% more cases of human visceral and cutaneous leishmaniosis were recorded between 1990 and 1997 [32,33]. Until the 1980s', the prevalence of infection amongst dogs of Algiers was always below 10%. It is only in the last decade that the prevalence markedly increased, to reach 30% in the 1990s' and 20%-25% in the 2000s'. This change may be mainly explained by the concurrent and dramatic environmental changes that have occurred in Algeria, particularly the uncontrolled urbanization that took place in recent years. This new urbanization has led to a proliferation of In Algeria, unresponsiveness to Sb(V) to the treatment of zoonotic cutaneous leishmanioses is well documented in the focus of M'sila. In 1986 a survey carried out on ninety-seven children who received 60 mg/kg/day of meglumine antimoniate for 15 days, reported no signifi cant response compared to those receiving placebo. In vitro tests performed on intracellular amastigotes confi rmed that all the strains of L. major isolated from these children displayed a low susceptibility towards Sb (V) containing drugs in the form of Glucantime ® [37]. More recently, 9% of unresponsiveness to antimonial treatment was recorded in children with visceral leishmaniosis [38,39].
For the veterinarian practitioners, treatment of canine leishmaniosis remains diffi cult, given the complex pathogenesis of the disease associated with pleiotropic clinical signs [40].
All antileishmanial compounds in use to treat dogs can lead to temporary or permanent remission of clinical signs but no established protocol is effective to eliminate the infection.
While therapeutic protocols have evolved the drugs used for treatment haven't changed. In case of a confi rmed diagnosis, the practitioner has fi rstly to worry about the risks of transmission to man and then about chances of curing of the animal [41]. Infected dogs are often treated with the same drug used for humans. The treatment with antimonials is often long and costly and the toxicity of antimony causes serious side effects [42,43]. Currently, the association of meglumine antimoniate with allopurinol is used [43,44]. In Algeria, dog suffering of leishmaniosis is usually not treated. Therefore, there are no available data on treatment effi ciency. So as in other countries endemic for the leishmaniosis, veterinarian practitioners euthanize dogs diagnosed as positive in order to prevent the spread of the disease to humans.
In the occidental part of the Mediterranean basin where dogs are commonly treated with meglumine antimoniate, the occurrence of resistant strains to antimony is documented [45]. We recently highlighted the occurrence of antimonyresistance in strains belonging to the L. infantum MON-281 [46] and further probed their potential of being transmitted by sandfl ies [47]. We also checked for cross-resistance with other molecules in clinical use to treat the visceral leishmaniosis [2,46,48]. To clarify the risk of transmission of antimonyresistant strains in Algeria, it will be crucial to determine the confer fi tness advantages [49,50]. This fi nding will likely have

Available tools to monitor parasite antimony susceptibility
Various hosts and antimony-derived molecules play a role in the antileishmanial activity of Sb (V) in vivo. Therefore, ideally, to refl ect the in vivo activity of antimonials, the in vitro tests must monitor the antileishmanial activity of compounds playing a role in the killing pathway of Sb (V). In addition, the origin of the antimonial batch has to be taken into account, because commercially available Sb (V) solutions are not pure [4,51]. In fi ne in all cases, the IC50 determined in vitro must always be compared with clinical observations. In most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes. However, the best approach is to test leishmanicidal compounds toward amastigotes residing in macrophages.

Cellular models for in vitro testing antimonial susceptibility
Obviously, the intracellular amastigotes represent the ideal model because both the indirect activity, through the host cell, and the direct one on the parasite can be assessed. Unfortunately, methods that involve intracellular amastigotes are labor intensive, diffi cult to standardize and timeconsuming. In addition, the results are dependent upon the nature of the host cell used to grow Leishmania amastigotes [52][53][54]. Duplex quantitative reverse-transcriptase PCR (qRT-PCR), aimed at assessing drug activity against Leishmania intracellular amastigotes and their host cells can be performed. The assay simultaneously quantifi es Leishmania 18S ribosomal RNA and the human b2-microglobulin (b-2M) mRNA, used for monitoring drug cytotoxicity and test performance [55]. Nevertheless, this approach is time-consuming, cost prohibitive and not amenable to large-scale analysis. The development of reporter gene technologies has enabled the quantifi cation of Leishmania parasites in host cells and whole mammalian hosts [56][57][58][59]. These technologies have been tested to determine the drug susceptibility of fi eld isolates [60,61].

Animal models for in vivo antimonial susceptibility testing
Animal models have been largely used to study the biological and pathological features specifi c to cutaneous and visceral leishmaniosis, even that none can reproduce the various syndromes encountered in humans. Current animal models aim at investigating (i) host-parasite interactions by the way of pathogenesis or biochemical and biological changes, (ii) in vivo maintenance of parasites and (iii) clinical evaluation of drug candidates.
The principal problem of using animal models to explore antimony activity is the route of administration and the distribution to different sites of infection. Several animal species served as experimental hosts especially for CL: BALB/c mice and Syrian golden hamster, dogs and monkeys [71,72]. The choice of convenient animal models depends on Leishmania species under study. For instance, the most suitable animal models are C57BL/6 mice (or vervet-monkeys, or rhesus-monkeys) for L. major and CsS-16 mice for L. tropica. CBA mice are convenient to study physiopathological effects caused by the infection with L. amazonensis and CBA and golden hamsters or rhesus-monkeys can be used for L. braziliensis 71 . We have recently established a murine model for Leishmania killicki cutaneous infections and probed its suitability for pharmacological purposes [73]. This new model will now enable us to investigate the in vivo behavior of this emerging pathogen, under antimony in vivo antimony pressure.

Biomarkers for antimony resistance
The search for resistance biomarkers is based on the indepth understanding of the antileishmanial mode of action of antimony ( Figure 4) and resistance associated with in vitro selection [74]. Studies on the intracellular mode of action of Sb(III) demonstrates that thiol buffer capacity of Leishmania is deeply affected by Sb(III), through the inhibition of the trypanothione reductase [75], and via a decrease in the intracellular thiol content through an effl ux mechanism yet unidentifi ed [76]. These two mechanisms combine to profoundly compromise the thiol redox potential in drugsensitive parasites and lead ultimately to the accumulation of reactive oxygen species ROS [76,77].
Many of the specifi c genes associated with resistance to antimony have been discovered. These mechanisms generally involved: The limiting antimony entry into the cell. The entry of Sb (III) occurs through an AQP1 transporter [78], but the route of entry of Sb (V) is currently not identifi ed.
The increase of the target expression. Sb (III) inhibits the Trypanothione reductase activity leading to an accumulation of the reduced form of trypanothione. Overexpression of the Trypanothione reductase has been characterized in most of the antimony-resistant fi eld isolates [79], with some exceptions [80].
The decrease of the reduction of the prodrug form of Sb, Sb (V) to the active form Sb(III). This function is assumed by two reductases, TDR1, a thiol-dependent reductase, belonging to the Glutathione S-transferase family that shares homologies with the T. cruzi Tc52 protein [81], and LmACR2 that shares homologies with arsenate reductases [82]. Nevertheless, variations in reductase capacity of fi eld Leishmania isolates resistant to antimony have never been investigated.
The conjugation of the drug with thiol-containing molecules and sequestration or effl ux of the thiol conjugated drug. In all Kinetoplastidae, the major low molecular mass thiol is trypanothione in contrast with most other prokaryotic and eukaryotic cells, which utilize glutathione to maintain the intracellular thiol redox potential and preserve cells from harmful attacks of oxidative compounds. Trypanothione is a glutathione-spermidine conjugate that is formed following several enzymatic steps, via the synthesis of reduced gluthatione (GSH) and its subsequent conjugation to spermidine. Steps in the synthesis of spermidine involve ornithine decarboxylase (ODC) and spermidine synthase. The fi rst step in the biosynthesis of GSH is catalyzed by the γ-glutamylcysteine synthetase (γ-GCS) and the glutathione synthase. In the following step, two enzymes specifi c to trypanosome are involved in the pathway. First, the glutathionylspermidine synthase ligates reduced glutathione (GSH) to spermidine in a conjugate, the glutathionylspermidine. A second Kinetoplastidae-specifi c ligase, the trypanothione synthase conjugates a second GSH to glutathionylspermidine to produce trypanothione. The pool of reduced trypanothione is maintained by the trypanothione reductase (TR) that ensures the thiol redox potential.
Consequently, the overexpression of genes that leads to an increase in the intracellular level of reduced trypanothione (T (SH) 2 ), involves the formation of the Sb-trypanothione conjugate (Sb(TS 2 )) and its sequestration within a vacuole by the intracellular ABC transporter MRPA [83], or its effl ux from the parasite. In that way, the conjugation of Sb with trypanothione induces antimony resistance by using up free Sb (III) [81], within the amastigote. However, enzyme that conjugate Sb with trypanothione or other molecules containing thiols, is unknown.
Mutations associated with resistance represent molecular signatures largely used in drug resistance surveillance for other parasitic diseases. For instance, single nucleotide polymorphisms (SNPs) associated with Plasmodium susceptibility to drugs are often good indicators of the clinical drug resistance [84]. Unfortunately, no SNP have been proved to be indicative of antimony resistance in fi eld isolates. But another main mechanism of resistance evidenced in Leishmania consists in genomic rearrangements, which lead to the amplifi cation of resistance genes through homologous recombination between repeated sequences [85]. Amplifi ed genes known to be involved in antimony susceptibility should, therefore, represent good biomarkers for addressing antimony resistance [86].
If some of the above mentioned mechanisms are effectively encountered in resistant strains from the fi eld, any rule could be applied mainly because of the presence of additional unrelated or less specifi c mechanisms [87][88][89][90], or because of other yet unidentifi ed mechanisms of resistance. Nevertheless, signifi cant DNA amplifi cation and/or higher RNA expression occur in a majority of strains having an antimony resistant [91].
Recent metabolomic approaches have been used to investigate parasites displaying different susceptibility to antimony. Interestingly, a hierarchical clustering approach (antimony resistant and susceptible) revealed differences in the metabolite abundance for the drug-resistant and -sensitive clones [92]. However, in this study the sample size was not large enough to conclusively identify drug-resistant parasites.
The extensive procedures involved in culturing the parasites, the high cost, the large amount of time required and the need for highly skilled technicians to perform the metabolomic analyses limit their usefulness for antimony susceptibility surveys. But metabolomic studies might provide information on new metabolic markers for monitoring antimony resistance.

Conclusions
In Algeria, Sb (V) resistant L. major and L. infantum have been isolated from unresponsive patients and animal reservoirs [37,46]. Therefore, the emergence of such resistant parasites stressed questions about factors that are acting to select them but also required more knowledge on the risk of their transmission. In order to compare the relative fi tness of antimony-resistant and susceptible parasites, it would be interesting to survey the prevalence of both phenotypes over a large period of time. We think that it would be an adequate approach to assess the relevant natural fi tness of both phenotypes. The former is essential to study each of the fi tness components in more details, in order to understand why the relative fi tness between phenotypes differs, (e.g. due to differences in survival, reproduction or transmission [49,50].
These studies are of importance for public health policy.