The leishmaniases are a group of vector-borne diseases common to humans and certain mammals, mainly the dog, for zoonotic visceral forms. They are caused by flagellated protozoan parasites belonging to the Leishmania genus. At least 20 species are encountered in human pathology [1]. These parasites are transmitted through the bite of an infected hematophagous female phlebotomine sand fly.

There was an endemic in 98 countries and territories in 2020 [1], and the geographic distribution of these diseases evolved according to the movements of the insect vector driven by climatic and environmental changes (such as deforestation and urbanization) [2]. These diseases mainly affect poor people in Africa, Asia, and South America and are associated with malnutrition, population displacement, poor housing, weak immune system, and a lack of resources ^[1][2][1,2]. Clinically, there are three main forms of leishmaniasis [1]: (i) Cutaneous leishmaniasis, which represents the most common form; (ii) Mucocutaneous leishmaniasis; and (iii) Visceral leishmaniasis, which is potentially fatal if left untreated. Two epidemiological types of visceral leishmaniasis coexist worldwide. Firstly, the main epidemiological type around the Mediterranean basin involves L. infantum and is represented by zoonotic visceral leishmaniasis. This type is clinically characterized by the triad of mad fever, anemia, and splenomegaly and is transmitted from dogs to humans. Secondly, anthroponotic visceral leishmaniasis is more commonly found in India. It is characterized by additional adenopathy and cutaneous signs and is transmitted from human to human. Today, more than 1 billion people live in areas endemic for leishmaniasis and are at risk of infection. An estimated 30,000 new cases of visceral leishmaniasis occur annually, according to the WHO [3]. In recent years, a downward trend has been observed in the number of reported visceral leishmaniasis cases, notably due to the effect of the WHO’s visceral leishmaniasis elimination program [4]. In 2020, about 87% of global visceral leishmaniasis cases were reported from eight countries (Brazil, Eritrea, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan) [1].

Exploratory pharmacological in vitro screening is a method of scientific experimentation requiring the use of technical and technological resources to study and select, in a chemical library, the active hit compounds on a biological target. The identified hit compounds are starting points for pharmacomodulation studies to design and develop potential drugs. Therefore, exploratory in vitro screening is the very first key step in the drug development process. Given this, the instrumentation used to perform such screening is crucial. Indeed, the choice of equipment can result in significant differences in costs, required handling time, and quality of data (reproducibility and repeatability). From manual testing to semi-automated or even fully automated testing, these criteria could vary widely and represent a challenge for harmonizing results obtained by different research teams. Nevertheless, more and more examples of efficient high throughput screening against Leishmania are described in the literature ^{[5][6][7][8][9][10]}[13,14,15,16,17,18].

Another challenge posed by these exploratory in vitro screening tests is the biological target itself: the Leishmania parasite. Indeed, the exploratory screening for Leishmania involves in vitro exploratory screening on a whole protozoan parasite that exists in two morphological forms (promastigote and amastigote), which means there is a multitude of potential molecular targets. The Leishmania’s lifecycle requires the presence of two entities: the sand fly vector and a mammalian host. Various developmental stages throughout this lifecycle are required, but these different stages (promastigote and amastigote forms) involve many variations in diverse metabolic, biochemical, and biological pathways, which were progressively detailed in the literature ^{[11][12][13][14][15][16]}[19,20,21,22,23,24]. Various Leishmania forms are used experimentally in vitro to develop exploratory screening tests, which are promastigotes, intracellular amastigotes, and axenic amastigotes. These latter forms represent amastigotes that were adapted to grow and develop outside their host cells in a growth medium that mimics the intracellular conditions ^[17][18][19][25,26,27]. Therefore, it appears complicated to consider only one parasite form in the framework of an exploratory in vitro screening. If rapid primary screening can be performed on extracellular promastigotes and axenic amastigotes, it would be essential to confirm and identify false positives during a secondary screening of identified hit compounds on clinically relevant intramacrophagic amastigote forms ^[6][14]. Nevertheless, this strategy does not prevent false negatives represented by hit compounds specific to intracellular amastigotes without highlighted activity on extracellular forms ^[20][28]. Thus, exploratory screening tests designed to facilitate the rapid testing of a large number of compounds are usually performed on the extracellular promastigotes or axenic amastigotes, which both enable the performance of high throughput screenings with high reproducibility ^[21][29]. Although some consider that promastigotes may not be as relevant as axenic amastigotes for screening purposes ^[22][30], there is still a lack of correlation between axenic artificial forms and intracellular amastigotes ^[6][14]. As such, models using host cells currently remain the gold standard in determining compound sensitivity ^[23][31] since they provide essential information about the tested compound’s activity in the parasite’s natural environment. Nevertheless, these models also reveal variation factors and potential biases, such as a low replication rate of amastigotes compared to promastigotes ^[24][25][32,33], the influence of the macrophage infection rate ^[26][34], and the variety of host cells used (primary cells or cell lines) ^[27][35]. Thus, although many studies show a correlation between the results obtained on in vitro promastigotes and (axenic) amastigotes ^{[28][29][30][31][32][33]}[36,37,38,39,40,41], it seems important to take all these various factors and potential biases into account before implementing secondary screenings and interpreting them.

Another challenge in terms of harmonizing work carried out by different research teams is the wide variety of existing options for detection, acquisition, and data processing systems ^[34][42]. Thus, cell viability detection was extensively used in the exploratory screening of antileishmanial compounds, especially for primary screenings on extracellular forms ^{[19][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62]}[27,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70]. Indeed, there are many colorimetric assays that are usable, simple, inexpensive, and suitable for large-scale screening ^[35][36][43,44]. Some of those are poorly illustrated in the existing literature, such as the trypanothione reductase assay ^[37][45] or acid phosphatase assay ^{[38][39][40][41]}[46,47,48,49], while others are widely proven, notably, those related to intracellular metabolizing salts such as the most famous MTT assay (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) ^{[30][42][43][44][45][46][47][48][49][50][51]}[38,50,51,52,53,54,55,56,57,58,59]. This yellow salt is reduced to purple formazan crystals in living cells, allowing for easy determination of parasite viability. Other analogous tests are also described, such as the one using Alamar blue (resazurin) ^{[19][52][53][54][55][56][57][58][59]}[27,60,61,62,63,64,65,66,67], an oxidation-reduction indicator that changes its color from blue to red in living cells. The use of an analog of MTT, the MTS (3-(4,5-dimethylthiazol-2-yl-5(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) ^[60][61][62][68,69,70], is also described. However, these tests are not very sensitive and are mainly used for low throughput screenings. Furthermore, direct counting could also be used to evaluate the leishmanicidal activity of tested compounds both in promastigote (motility of promastigotes and examination of non-viable parasites after staining) ^[63][71] and in amastigote (microscopic counting of infected macrophages and the number of parasites per macrophage after staining) assays ^[63][71]. This method has the advantage of not requiring expensive equipment, but it is time-consuming, laborious to perform, unsuitable for large-scale screening, and suffers from a lack of reproducibility. Moreover, the determination of inhibitory concentration 50% (IC₅₀) may be inaccurate since the determination of parasite viability through a staining procedure is obviously difficult ^[17][25]. Different tools were developed to automate this tiresome microscopic counting ^[64][65][72,73]. As an example, can be cited an automated microscopic image analysis, which can be applied to the quantification of drug activity ^[66][74]. Of note is a methodology using a colorimetric β-lactamase assay described on intramacrophagic amastigotes of L. donovani ^[8][16]. Nevertheless, in the field of cell viability analysis, flow cytometry constitutes an interesting alternative ^[67][68][75,76], which is accurate and largely used to automate the reading of results. Another approach uses the detection and quantification of engineered cells expressing fluorescent gene reporters ^[69][70][77,78] such as green fluorescent protein (GFP) ^{[24][71][72][73]}[32,79,80,81] and bioluminescent gene reporters such as luciferase ^{[74][75][76][77][78]}[82,83,84,85,86], or a combination of both ^{[79][80][81][82]}[87,88,89,90]. These methods are proven to be more sensitive and enable faster read-outs and higher throughput ^[83][91]. Moreover, reporter proteins bear or produce an easily detectable response that can be quantified even in intracellular conditions, leading to the development of many experimental models ^[84][92]. As an example, the use of a traceable bioluminescent marker, such as NanoLuc-PEST, that correlates specifically with parasite viability could provide a more relevant in vitro assay for use in both axenic and intramacrophage amastigote models. This system was already described in L. mexicana ^[85][93] and could be adapted to other Leishmania species since the employed reporter protein expression vector (pSSU-int) was already successfully used in the main species involved in pathology ^[21][86][87][29,94,95]. Furthermore, a recent comparison of several bioluminescent reporters in a cutaneous leishmaniasis model indicated that NanoLuc-parasites, despite high bioluminescence intensity in vitro, were shown to be inadequate in discriminating between live and dead parasites in drug screening protocols. Bioluminescence detection from intracellular amastigotes expressing NanoLuc-PEST, red luciferase (RedLuc), or conventional luciferase (Luc2) proved more reliable than microscopy to determine parasite killing ^[88][96]. Nevertheless, this technology also presents several limitations, including a potential antibiotic cross-resistance conferred by induced antibiotic resistance allowing the selection of recombinant parasites and the difficulty of adapting it to Leishmania clinical isolates. Furthermore, these genotypic modifications of parasites could result in phenotypic consequences, such as biological transformations.