Challenges for In Vitro Leishmania Exploratory Screening: Comparison
View Latest Version
Please note this is a comparison between Version 2 by Jessie Wu and Version 1 by Anita Cohen.

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. 

  • leishmaniases
  • challenges

 1. Background

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].

2. Challenges Involved in Implementing Exploratory Screening to Identify In Vitro Hit Compounds against Leishmania

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 [13,14,15,16,17,18][5][6][7][8][9][10].
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 [19,20,21,22,23,24][11][12][13][14][15][16]. 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 [25,26,27][17][18][19]. 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 [14][6]. Nevertheless, this strategy does not prevent false negatives represented by hit compounds specific to intracellular amastigotes without highlighted activity on extracellular forms [28][20]. 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 [29][21]. Although some consider that promastigotes may not be as relevant as axenic amastigotes for screening purposes [30][22], there is still a lack of correlation between axenic artificial forms and intracellular amastigotes [14][6]. As such, models using host cells currently remain the gold standard in determining compound sensitivity [31][23] 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 [32,33][24][25], the influence of the macrophage infection rate [34][26], and the variety of host cells used (primary cells or cell lines) [35][27]. Thus, although many studies show a correlation between the results obtained on in vitro promastigotes and (axenic) amastigotes [36,37[28][29][30][31][32][33],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 [42][34]. Thus, cell viability detection was extensively used in the exploratory screening of antileishmanial compounds, especially for primary screenings on extracellular forms [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][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]. Indeed, there are many colorimetric assays that are usable, simple, inexpensive, and suitable for large-scale screening [43,44][35][36]. Some of those are poorly illustrated in the existing literature, such as the trypanothione reductase assay [45][37] or acid phosphatase assay [46[38][39][40][41],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) [38,50,51,52,53,54,55,56,57,58,59][30][42][43][44][45][46][47][48][49][50][51]. 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) [27,60[19][52][53][54][55][56][57][58][59],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) [68[60][61][62],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) [71][63] and in amastigote (microscopic counting of infected macrophages and the number of parasites per macrophage after staining) assays [71][63]. 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 [25][17]. Different tools were developed to automate this tiresome microscopic counting [72,73][64][65]. As an example, can be cited an automated microscopic image analysis, which can be applied to the quantification of drug activity [74][66]. Of note is a methodology using a colorimetric β-lactamase assay described on intramacrophagic amastigotes of L. donovani [16][8]. Nevertheless, in the field of cell viability analysis, flow cytometry constitutes an interesting alternative [75[67][68],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 [77,78][69][70] such as green fluorescent protein (GFP) [32,79,80,81][24][71][72][73] and bioluminescent gene reporters such as luciferase [82[74][75][76][77][78],83,84,85,86], or a combination of both [87,88,89,90][79][80][81][82]. These methods are proven to be more sensitive and enable faster read-outs and higher throughput [91][83]. 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 [92][84]. 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 [93][85] 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 [29,94,95][21][86][87]. 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 [96][88]. 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.

References

  1. World Health Organization. Leishmaniasis, 2014. Global Health Observatory. Available online: https://www.who.int/data/gho/data/themes/topics/gho-ntd-leishmaniasis (accessed on 31 October 2021).
  2. World Health Organization. A Global Brief on Vector-Borne Diseases; WHO/DCO/WHD/2014.1; World Health Organization: Geneva, Switzerland, 2014; p. 21.
  3. World Health Organization. Health Topics/Leishmaniasis. Available online: https://www.who.int/health-topics/leishmaniasis#tab=tab_1 (accessed on 3 November 2021).
  4. World Health Organization. Weekly Epidemiological Record: No 25. 2020, 95, 265–280. Available online: https://apps.who.int/iris/handle/10665/239492 (accessed on 3 November 2021).
  5. Corman, H.N.; Shoue, D.A.; Norris-Mullins, B.; Melancon, B.J.; Morales, M.A.; McDowell, M.A. Development of a target-free high-throughput screening platform for the discovery of antileishmanial compounds. Int. J. Antimicrob. Agents 2019, 54, 496–501.
  6. De Rycker, M.; Hallyburton, I.; Thomas, J.; Campbell, L.; Wyllie, S.; Joshi, D.; Cameron, S.; Gilbert, I.H.; Wyatt, P.G.; Frearson, J.A.; et al. Comparison of a High-Throughput High-Content Intracellular Leishmania donovani assay with an axenic amastigote assay. Antimicrob. Agents Chemother. 2013, 57, 2913–2922.
  7. Siqueira-Neto, J.L.; Song, O.-R.; Oh, H.; Sohn, J.-H.; Yang, G.; Nam, J.; Jang, J.; Cechetto, J.; Lee, C.B.; Moon, S.; et al. Antileishmanial high-throughput drug screening reveals drug candidates with new scaffolds. PLoS Negl. Trop. Dis. 2011, 4, e675.
  8. Mandal, S.; Maharjan, M.; Ganguly, S.; Chatterjee, M.; Singh, S.; Buckner, F.S.; Madhubala, R. High-throughput screening of amastigotes of Leishmania donovani clinical isolates against drugs using a colorimetric β-lactamase assay. Indian J. Exp. Biol. 2009, 47, 475–479.
  9. Turcano, L.; Torrente, E.; Missineo, A.; Andreini, M.; Gramiccia, M.; Di Muccio, T.; Genovese, I.; Fiorillo, A.; Harper, S.; Bresciani, A.; et al. Identification and binding mode of a novel Leishmania trypanothione reductase inhibitor from high throughput screening. PloS Negl. Dis. 2018, 12, e0006969.
  10. Norcliffe, J.L.; Mina, J.G.; Alvarez, E.; Cantizani, J.; de Dios-Anton, F.; Colmenarejo, G.; Gonzalez-Del Valle, S.; Marco, M.; Fiandor, J.M.; Martin, J.J.; et al. Identifying inhibitors of the Leishmania inositol phosphorylceramide synthase with antiprotozoal activity using a yeast-based assay and ultra-high throughput screening platform. Sci. Rep. 2018, 8, 3938.
  11. Fialho Junior, L.; da Fonseca Pires, S.; Burchmore, R.; McGill, S.; Weidt, S.; Conceição Ruiz, J.; Goncalves Guimarães, F.; Chapeourouge, A.; Perales, J.; Monteiro de Andrade, H. Proteomic analysis reveals differentially abundant proteins probably involved in the virulence of amastigote and promastigote forms of Leishmania infantum. Parasitol. Res. 2021, 120, 679–692.
  12. Jara, M.; Berg, M.; Caljon, G.; de Muylder, G.; Cuypers, B.; Castillo, D.; Maes, I.; del Carmen Orozco, M.; Vanaerschot, M.; Dujardin, J.-C.; et al. Macromolecular biosynthetic parameters and metabolic profile in different life stages of Leishmania braziliensis: Amastigotes as a functionally less active stage. PLoS ONE 2017, 12, e0180532.
  13. Yao, C.; Wilson, M.E. Dynamics of sterol synthesis during development of Leishmania spp. Parasites to their virulent form. Parasit. Vectors 2016, 9, 200.
  14. Rodriguez, N.E.; Dixit, U.G.; Allen, L.-A.H.; Wilson, M.E. Stage-specific pathways of Leishmania infantum chagasi entry and phagosome maturation in macrophages. PloS ONE 2011, 6, e19000.
  15. Rosenzweig, D.; Smith, D.; Opperdoes, F.; Stern, S.; Olafson, R.W.; Zilberstein, D. Retooling Leishmania metabolism: From sand fly gut to human macrophage. FASEB J. 2008, 22, 590–602.
  16. Burge, R.J.; Damianou, A.; Wilkinson, A.J.; Rodenko, B.; Mottram, J.C. Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex. PLoS Pathog. 2020, 16, e1008784.
  17. Debrabant, A.; Joshi, M.B.; Pimenta, P.F.; Dwyer, D.M. Generation of Leishmania donovani axenic amastigotes: Their growth and biological characteristics. Int. J. Parasitol. 2004, 34, 205–217.
  18. Gupta, N.; Goyal, N.; Rastogi, A.K. In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol. 2001, 17, 150–153.
  19. Dias-Lopes, G.; Zabala-Penafiel, A.; de Albuquerque-Melo, B.C.; Souza-Silva, F.; Menaguali do Canto, L.; Cysne-Finkelstein, L.; Alves, C.R. Axenic amastigotes of Leishmania species as a suitable model for in vitro studies. Acta Trop. 2021, 220, 105956.
  20. Bhattacharya, A.; Corbeil, A.; do Monte-Neto, R.L.; Fernandez-Prada, C. Of drugs and Trypanosomatids: New tools and knowledge to reduce bottlenecks in drug discovery. Genes 2020, 11, 722.
  21. Gupta, S.; Nishi. Visceral leishmaniasis: Experimental models for drug discovery. Indian J. Med. Res. 2011, 133, 27–39.
  22. Sereno, D.; Cordeiro da Silva, A.; Mathieu-Daude, F.; Ouaissi, A. Advances and perspectives in Leishmania cell based drug-screening procedures. Parasitol. Int. 2007, 56, 3–7.
  23. Vermeersch, M.; da Luz, R.I.; Tote, K.; Timmermans, J.P.; Cos, P.; Maes, L. In vitro susceptibilities of Leishmania donovani promastigote and amastigote stages to antileishmanial reference drugs: Practical relevance of stage-specific differences. Antimicrob. Agents Chemother. 2009, 53, 3855–3859.
  24. Tegazzini, D.; Díaz, R.; Aguilar, F.; Peña, I.; Presa, J.L.; Yardley, V.; Martin, J.J.; Coteron, J.M.; Croft, S.L.; Cantizani, J. A replicative in vitro assay for drug discovery against Leishmania donovani. Antimicrob. Agents Chemother. 2016, 60, 3524–3532.
  25. Prada, C.F.; lvarez-Velilla, R.A.; Balana-Fouce, R.; Prieto, C.; Calvo-Alvarez, E.; Escudero-Martinez, J.M.; Requena, J.M.; Ordonez, C.; Desideri, A.; Perez-Pertejo, Y.; et al. Gimatecan and other camptothecin derivatives poison Leishmania DNA-topoisomerase IB leading to a strong leishmanicidal effect. Biochem. Pharm. 2013, 85, 1433–2440.
  26. Fernandez, O.; Diaz-Toro, Y.; Valderrama, L.; Ovalle, C.; Valderrama, M.; Castillo, H.; Perez, M.; Saraviaa, N.G. Novel approach to in vitro drug susceptibility assessment of clinical strains of Leishmania spp. J. Clin. Microbiol. 2012, 50, 2207–2211.
  27. Hendrickx, S.; van Bockstal, L.; Caljon, G.; Maes, L. In-depth comparison of cell-based methodological approaches to determine drug susceptibility of visceral Leishmania isolates. Plos Negl. Trop. Dis. 2019, 13, e0007885.
  28. Tadele, M.; Abay, S.M.; Asaga, P.; Makonnen, E.; Hailu, A. In vitro growth inhibitory activity of Medicines for Malaria Venture pathogen box compounds against Leishmania aethiopica. BMC Pharmacol. Toxicol. 2021, 22, 71.
  29. Mathur, T.; Kumar, M.; Barman, T.K.; Raj, V.S.; Upadhyay, D.J.; Verma, A.K. Novel azoles with potent antileishmanial activity. Future Microbiol. 2021, 16, 871–877.
  30. Al-Halbosiy, M.M.F.; Ali, H.Z.; Hassan, G.M.; Ghaffarifar, F. Artemisinin efficacy against old world Leishmania donovani: In Vitro and ex vivo study. Ann. Parasitol. 2020, 63, 295–302.
  31. Souza, J.M.; de Carvalho, E.A.A.; Candido, A.C.B.B.; de Mendonça, R.P.; Fernanda da Silva, M.; Parreira, R.L.T.; Dias, F.G.G.; Ambrosio, S.R.; Arantes, A.T.; da Silva Filho, A.A.; et al. Licochalcone a exhibits leishmanicidal activity in vitro and in experimental model of Leishmania (Leishmania) infantum. Front. Vet. Sci. 2020, 7, 527.
  32. Monzote, L.; Herrera, I.; Satyal, P.; Setzer, W.N. In-vitro evaluation of 52 commercially-available essential oils against Leishmania amazonensis. Molecules 2019, 24, 1248.
  33. Antwi, C.A.; Amisigo, C.M.; Adjimani, J.P.; Gwira, T.M. In vitro activity and mode of action of phenolic compounds on Leishmania donovani. PLoS Negl. Trop. Dis. 2019, 13, e0007206.
  34. Fumarola, L.; Spinelli, R.; Brandonisio, O. In vitro assays for evaluation of drug activity against Leishmania spp. Res. Microbiol. 2004, 155, 224–230.
  35. Tempone, A.G.; Martins de Oliveira, C.; Berlinck, R.G.S. Current approaches to discover marine antileishmanial natural products. Planta Med. 2011, 77, 572–585.
  36. Davies-Bolorunduro, O.F.; Osuolale, O.; Saibu, S.; Adeleye, I.A.; Aminah, N.S. Bioprospecting marine actinomycetes for antileishmanial drugs: Current perspectives and future prospects. Heliyon 2021, 7, e07710.
  37. van den Bogaart, E.; Schoone, G.J.; England, P.; Faber, D.; Orrling, K.M.; Dujardin, J.-C.; Sundar, S.; Schallig, H.D.F.H.; Adams, E.R. Simple colorimetric trypanothione reductase-based assay for high-throughput screening of drugs against Leishmania intracellular amastigotes. Antimicrob. Agents Chemother. 2014, 58, 527–535.
  38. Gonzalez-Fajardo, L.; Fernández, O.L.; McMahon-Pratt, D.; Saravia, N.G. Ex vivo host and parasite response to antileishmanial drugs and immunomodulators. PLoS Negl. Trop. Dis. 2015, 9, e0003820.
  39. Carrio, J.; Riera, C.; Gallego, M.; Portus, M. In vitro activity of pentavalent antimony derivatives on promastigotes and intracellular amastigotes of Leishmania infantum strains from humans and dogs in Spain. Acta Trop. 2001, 79, 179–183.
  40. Bodley, A.L.; McGarry, M.W.; Shapiro, T.A. Drug cytotoxicity assay for African trypanosomes and Leishmania species. J. Infect. Dis. 1995, 172, 1157–1159.
  41. Bodley, A.L.; Shapiro, T.A. Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania. Proc. Natl Acad. Sci. USA 1995, 92, 3726–3730.
  42. Tada, H.; Shiho, O.; Kuroshima, K.; Koyama, M.; Tsukamoto, K. An improved colorimetric assay for interleukin 2. J. Immunol. Methods 1986, 93, 157–165.
  43. Khazaei, M.; Rahnama, V.; Motazedian, M.H.; Samani, S.M.; Hatam, G. In vitro effect of artemether-loaded nanostructured lipid carrier (NLC) on Leishmania infantum. J. Parasit. Dis. 2021, 45, 964–971.
  44. Mondêgo-Oliveira, R.; de Sá Sousa, J.C.; Moragas-Tellis, C.J.; de Souza, P.V.R.; Dos Santos Chagas, M.D.S.; Behrens, M.D.; Jesús Hardoim, D.; Taniwaki, N.N.; Chometon, T.Q.; Bertho, A.L.; et al. Vernonia brasiliana (L.) Druce induces ultrastructural changes and apoptosis-like death of Leishmania infantum promastigotes. Biomed. Pharmacother. 2021, 133, 111025.
  45. Ghosh, J.; Chakraborty, S.; Dey, S.; Mukherjee, D.; Sarkar, B.; Mallick, S.; Dutta, A.; Dutta, T.; Bhattacharjee, S.; Ghorai, N.; et al. Potential anti-leishmanial activity of a semi-purified fraction isolated from the leaves of Parthenium hysterophorus. Acta Parasitol. 2021, 66, 1480–1489.
  46. Rathnakar, B.; Sinha, K.K.; Prasad, S.R.; Khan, M.I.; Narsaiah, C.; Rameshwar, N.; Satyanarayana, M. Design, synthesis of biaryl piperidine derivatives and their evaluation as potential antileishmanial agents against Leishmania donovani strain Ag83. Chem. Biodivers. 2021, 18, e2100105.
  47. Guillon, J.; Cohen, A.; Das, R.N.; Boudot, C.; Gueddouda, N.M.; Moreau, S.; Ronga, L.; Savrimoutou, S.; Basmaciyan, L.; Tisnerat, C.; et al. Design, synthesis, and antiprotozoal evaluation of new 2,9-bis-1,10-phenanthroline derivatives. Chem. Biol. Drug Des. 2018, 91, 974–995.
  48. Fersing, C.; Boudot, C.; Pedron, J.; Hutter, S.; Primas, N.; Castera-Ducros, C.; Bourgeade-Delmas, S.; Sournia-Saquet, A.; Moreau, A.; Cohen, A.; et al. 8-Aryl-6-chloro-3-nitro-2-(phenylsulfonylmethyl)imidazopyridines as potent antitrypanosomatid molecules bioactivated by type 1 nitroreductases. Eur. J. Med. Chem. 2018, 157, 115–126.
  49. Castera-Ducros, C.; Paloque, L.; Verhaeghe, P.; Casanova, M.; Cantelli, C.; Hutter, S.; Tanguy, F.; Laget, M.; Remusat, V.; Cohen, A.; et al. Targeting the human parasite Leishmania donovani: Discovery of a new anti-infectious pharmacophore in 3-nitroimidazopyridine series. Bioorg. Med. Chem. 2013, 21, 7155–7164.
  50. Dutta, A.; Bandyopadhyay, S.; Mandal, C.; Chatterjee, M. Development of a modified MTT assay for screening antimonial resistant field isolates of Indian visceral leishmaniasis. Parasitol. Int. 2005, 54, 119–122.
  51. Williams, C.; Espinosa, O.A.; Montenegro, H.; Cubilla, L.; Capson, T.L.; Ortega-Barría, E.; Romero, L.I. Hydrosoluble formazan XTT: Its application to natural products drug discovery for Leishmania. J. Microbiol. Methods 2003, 55, 813–816.
  52. Mikus, J.; Steverding, D. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol. Int. 2000, 48, 265–269.
  53. Njanpa, C.A.N.; Wouamba, S.C.N.; Yamthe, L.R.T.; Dize, D.; Tchatat, B.M.T.; Tsouh, P.V.F.; Pouofo, M.N.; Jouda, J.B.; Ndjakou, B.L.; Sewald, N.; et al. Bio-guided isolation of anti-leishmanial natural products from Diospyros gracilescens L. (Ebenaceae). BMC Complement. Med. Ther. 2021, 21, 106.
  54. Zahid, M.S.H.; Johnson, M.M.; Tokarski, R.J., II; Satoskarb, A.R.; Fuchs, J.R.; Bacheldera, E.M.; Ainslie, K.M. Evaluation of synergy between host and pathogen-directed therapies against intracellular Leishmania donovani. Int. J. Parasitol. Drugs Drug Resist. 2019, 10, 125–132.
  55. de Sousa Araùjo, P.S.; de Oliveira, S.S.C.; d’Avila-Levy, C.M.; Dos Santos, A.L.S.; Branquinha, M.H. Susceptibility of promastigotes and intracellular amastigotes from distinct Leishmania species to the calpain inhibitor MDL28170. Parasitol. Res. 2018, 117, 2085–2094.
  56. Carvalho, S.G.; Siqueira, L.A.; Zanini, M.S.; Dos Santos Matos, A.P.; Quaresma, C.H.; da Silva, L.M.; de Andrade, S.F.; Severi, J.A.; Villanova, J.C.O. Physicochemical and in vitro biological evaluations of furazolidone-based beta-cyclodextrin complexes in Leishmania amazonensis. Res. Vet. Sci. 2018, 119, 143–153.
  57. Grekov, I.; Pombinho, A.R.; Kobets, T.; Bartunek, P.; Lipoldová, M. Calcium ionophore, calcimycin, kills Leishmania promastigotes by activating parasite nitric oxide synthase. Biomed. Res. Int. 2017, 2017, 130948.
  58. Corral, M.J.; González, E.; Cuquerella, M.; Alunda, J.M. Improvement of 96-well microplate assay for estimation of cell growth and inhibition of Leishmania with Alamar Blue. J. Microbiol. Methods 2013, 94, 111–116.
  59. Kulshresta, A.; Bhandari, V.; Mukhopadhyay, R.; Ramesh, V.; Sundar, S.; Maes, L.; Dujardin, J.-C.; Roy, S.; Salotra, P. Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in clinical isolates of Leishmania donovani. Parasitol. Res. 2013, 112, 825–828.
  60. Rajabi, O.; Sazgarnia, A.; Abbasi, F.; Layegh, P. The activity of ozonated olive oil against Leishmania major promastigotes. Iran J. Basic Med. Sci. 2015, 18, 915–919.
  61. Khanna, V.G.; Kannabiran, K.; Getti, G. Leishmanicidal activity of saponins isolated from the leaves of Eclipta prostrata and Gymnema sylvestre. Indian J. Pharmacol. 2009, 41, 32–35.
  62. Ganguly, S.; Bandyopadhyay, S.; Sarkar, A.; Chatterjee, M. Development of a semi-automated colorimetric assay for screening anti-leishmanial agents. J. Microbiol. Methods 2006, 66, 79–86.
  63. Want, M.Y.; Yadav, P.; Khan, R.; Chouhan, G.; Islamuddin, M.; Aloyouni, S.Y.; Chattopadhyay, A.P.; Al Omar, S.Y.; Afrin, F. Critical antileishmanial in vitro effects of highly examined gold nanoparticles. Int. J. Nanomed. 2021, 16, 7285–7295.
  64. Gomes-Alves, A.G.; Maia, A.F.; Cruz, T.; Castro, H.; Tomás, A.M. Development of an automated image analysis protocol for quantification of intracellular forms of Leishmania spp. PLoS ONE 2018, 13, e0201747.
  65. Yazdanparast, E.; Dos Anjos, A.; Garcia, D.; Loeuillet, C.; Shahbazkia, H.R.; Vergnes, B. INsPECT, an open-source and versatile software for automated quantification of (Leishmania) intracellular parasites. Plos Negl. Trop. Dis. 2014, 8, e2850.
  66. Moraes, C.B.; Alcântara, L.M. Quantification of parasite loads by automated microscopic image analysis. Methods Mol. Biol. 2019, 1971, 279–288.
  67. Costa, A.F.P.; de Brito, R.C.F.; Carvalho, L.M.; Cardoso, J.M.O.; Vieira, P.M.A.; Reis, A.B.; Aguiar-Soares, R.D.O.; Roatt, B.M. Liver infusion tryptose (LIT): The best choice for growth, viability, and infectivity of Leishmania infantum parasites. Parasitol. Res. 2020, 119, 4185–4195.
  68. Gadelha, A.P.R.; Brigagao, C.M.; da Silva, M.B.; Rodrigues, A.B.M.; Guimarães, A.C.R.; Paiva, F.; de Souza, W.; Henriques, C. Insights about the structure of farnesyl diphosphate synthase (FPPS) and the activity of bisphosphonates on the proliferation and ultrastructure of Leishmania and Giardia. Parasit. Vectors 2020, 13, 168.
  69. García-Bustos, M.F.; Moya Álvarez, A.; Pérez Brandan, C.; Parodi, C.; Sosa, A.M.; Buttazzoni Zuñiga, V.C.; Pastrana, O.M.; Manghera, P.; Peñalva, P.A.; Marco, J.D.; et al. Development of a fluorescent assay to search new drugs using stable tdTomato-Leishmania, and the selection of galangin as a candidate with anti-leishmanial activity. Front Cell. Infect. Microbiol. 2021, 11, 666746.
  70. Dube, A.; Gupta, R.; Singh, N. Reporter genes facilitating discovery of drugs targeting protozoan parasites. Trends Parasitol. 2009, 25, 432–439.
  71. Ertabaklar, H.; Çaliskan, S.O.; Kolli, B.; Ertug, S.; Özbilgin, A.; Malatyali, E.; Chang, K.P. Transfection of Leishmania tropica with green fluorescent protein (gfp) gene and investigation of the in vitro drug effect. Mikrobiyol. Bul. 2019, 53, 213–223.
  72. Pulido, S.A.; Muñoz, D.L.; Restrepo, A.M.; Mesa, C.V.; Alzate, J.F.; Vélez, I.D.; Robledo, S.M. Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs. Acta Trop. 2012, 122, 36–45.
  73. Bolhassani, A.; Taheri, T.; Taslimi, Y.; Zamanilui, S.; Zahedifard, F.; Seyed, N.; Torkashvand, F.; Vaziri, B.; Rafati, S. Fluorescent Leishmania species: Development of stable GFP expression and its application for in vitro and in vivo studies. Exp. Parasitol. 2011, 127, 637–645.
  74. Bringmann, G.; Thomale, K.; Bischof, S.; Schneider, C.; Schultheis, M.; Schwarz, T.; Moll, H.; Schurigt, U. A novel Leishmania major amastigote assay in 96-well format for rapid drug screening and its use for discovery and evaluation of a new class of leishmanicidal quinolinium salts. Antimicrob. Agents Chemother. 2013, 57, 3003–3011.
  75. Paloque, L.; Vidal, N.; Casanova, M.; Dumètre, A.; Verhaeghe, P.; Parzy, D.; Azas, N. A new, rapid and sensitive bioluminescence assay for drug screening on Leishmania. J. Microbiol. Methods 2013, 95, 320–323.
  76. Michel, G.; Ferrua, B.; Lang, T.; Maddugoda, M.P.; Munro, P.; Pomares, C.; Lemichez, E.; Marty, P. Luciferase-expressing Leishmania infantum allows the monitoring of amastigote population size, in vivo, ex vivo and in vitro. PLoS Negl. Trop. Dis. 2011, 5, e1323.
  77. Ashutosh; Gupta, S.; Ramesh; Sundar, S.; Goyal, N. Use of Leishmania donovani field isolates expressing the luciferase reporter gene in in vitro drug screening. Antimicrob. Agents Chemother. 2005, 49, 3776–3783.
  78. Roy, G.; Dumas, C.; Sereno, D.; Wu, Y.; Singh, A.K.; Tremblay, M.J.; Ouellette, M.; Olivier, M.; Papadopoulou, B. Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models. Mol. Biochem. Parasitol. 2000, 110, 195–206.
  79. Sharma, R.; Silveira-Mattos, P.S.; Ferreira, V.C.; Rangel, F.A.; Oliveira, L.B.; Celes, F.S.; Viana, S.M.; Wilson, M.E.; de Oliveira, C.I. Generation and characterization of a dual-reporter transgenic Leishmania braziliensis line expressing eGFP and Luciferase. Front. Cell. Infect. Microbiol. 2020, 9, 468.
  80. Calvo-Alvarez, E.; Cren-Travaillé, C.; Crouzols, A.; Rotureau, B. A new chimeric triple reporter fusion protein as a tool for in vitro and in vivo multimodal imaging to monitor the development of African trypanosomes and Leishmania parasites. Infect. Genet. Evol. 2018, 63, 391–403.
  81. Taheri, T.; Saberi Nik, H.; Seyed, N.; Doustdari, F.; Etemadzadeh, M.H.; Torkashvand, F.; Rafati, S. Generation of stable L. major(+EGFP-LUC) and simultaneous comparison between EGFP and luciferase sensitivity. Exp. Parasitol. 2015, 150, 44–55.
  82. Sadeghi, S.; Seyed, N.; Etemadzadeh, M.-H.; Abediankenari, S.; Rafati, S.; Taheri, T. In vitro infectivity assessment by drug susceptibility comparison of recombinant Leishmania major expressing enhanced green fluorescent protein or EGFP-Luciferase fused genes with wild-type parasite. Korean J. Parasitol. 2015, 53, 385–394.
  83. Calvo-Alvarez, E.; Alvarez-Velilla, R.; Fernandez-Prada, C.; Balana-Fouce, R.; Reguera, R.M. Trypanosomatids see the light: Recent advances in bioimaging research. Drug Discov. Today 2015, 20, 114–121.
  84. Berry, S.L.; Hameed, H.; Thomason, A.; Maciej-Hulme, M.L.; Saif Abou-Akkada, S.; Horrocks, P.; Price, H.P. Development of NanoLuc-PEST expressing Leishmania mexicana as a new drug discovery tool for axenic- and intramacrophage-based assays. PLoS Negl. Trop. Dis. 2018, 12, e0006639.
  85. Reimao, J.Q.; Oliveira, J.C.; Trinconi, C.T.; Cotrim, P.C.; Coelho, A.C.; Uliana, S.R. Generation of luciferase-expressing Leishmania infantum chagasi and assessment of miltefosine efficacy in infected hamsters through bioimaging. PLoS Negl. Trop. Dis. 2015, 9, e0003556.
  86. Dan-Goor, M.; Nasereddin, A.; Jaber, H.; Jaffe, C.L. Identification of a secreted casein kinase 1 in Leishmania donovani: Effect of protein over expression on parasite growth and virulence. PLoS ONE 2013, 8, e79287.
  87. Price, H.P.; Paape, D.; Hodgkinson, M.R.; Farrant, K.; Doehl, J.; Stark, M.; Smith, D.F. The Leishmania major BBSome subunit BBS1 is essential for parasite virulence in the mammalian host. Mol. Microbiol. 2013, 90, 597–611.
  88. Agostino, V.S.; Trinconi, C.M.; Galuppo, M.K.; Price, H.; Uliana, S.R.B. Evaluation of NanoLuc, RedLuc and Luc2 as bioluminescent reporters in a cutaneous leishmaniasis model. Acta Trop. 2020, 206, 105444.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
Video Production Service
Feedback