Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Jun 1.
Published in final edited form as: Exp Parasitol. 2023 Mar 31;249:108519. doi: 10.1016/j.exppara.2023.108519

A short-term method to evaluate anti-leishmania drugs by inhibition of stage differentiation in Leishmania mexicana using flow cytometry

Christian Florian Teh-Poot 1, Victor Manuel Dzul-Huchim 1, Jonathan M Mercado 2, Liliana Estefanía Villanueva-Lizama 1,3,4, Maria Elena Bottazzi 3,4, Kathryn M Jones 3,4, Francis TF Tsai 2,5, Julio Vladimir Cruz-Chan 1,3,4,*
PMCID: PMC10231665  NIHMSID: NIHMS1889348  PMID: 37004860

Abstract

Leishmaniasis is a vector-borne neglected tropical disease caused by the Leishmania spp. parasite. The disease is transmitted to humans and animals by the bite of infected female sandflies during the ingestion of bloodmeal. Because current drug treatments induce toxicity and parasite resistance, there is an urgent need to evaluate new drugs. Most therapeutics target the differentiation of promastigotes to amastigotes, which is necessary to maintain Leishmania infection. However, in vitro assays are laborious, time-consuming, and depend on the experience of the technician. In this study, we aimed to establish a short-term method to assess the differentiation status of Leishmania mexicana (L. mexicana) using flow cytometry. Here, we showed that flow cytometry provides a rapid means to quantify parasite differentiation in cell culture as reliably as light microscopy. Interestingly, we found using flow cytometry that miltefosine reduced promastigote-to-amastigote differentiation of L. mexicana. We conclude that flow cytometry provides a means to rapidly assay the efficacy of small molecules or natural compounds as potential anti-leishmanials.

Keywords: Leishmania Mexicana, parasite differentiation, immunology, flow cytometry, miltefosine

Graphical Abstract

graphic file with name nihms-1889348-f0001.jpg

Introduction

According to the World Health Organization (WHO), 350 million people are at risk for Leishmania infection and an estimated 0.7 million to 1 million new cases occur annually (Alvar et al. 2012). Leishmaniasis is prevalent in tropical and subtropical regions with disease manifestation ranging from apparent self-healing cutaneous lesions to visceral infections that can be lethal if untreated (Torres-Guerrero et al. 2017; Burza and Croft 2018). Leishmania spp., the causative agents of leishmaniasis, are dimorphic protozoan parasites that are transmitted to mammals during a bloodmeal of an infected female Phlebetomine sandfly. Following invasion of the mammalian host, the flagellated promastigotes are phagocyted by macrophages and the environmental differences in the phagosome trigger parasite differentiation into morphologically distinct non-motile amastigotes (Mittra et al. 2013; Séguin and Descoteaux 2016). Differentiation of promastigotes to amastigotes is necessary to maintain Leishmania infection (Rittig and Bogdan 2000; Engwerda et al. 2004), with the amastigote form being responsible for all clinical manifestations of leishmaniasis (Kevric et al. 2015; Akhoundi et al. 2016). Despite having common side effects, miltefosine has been used as a first-line drug in the treatment of leishmaniasis (Patel et al. 2014). The mechanism of miltefosine is not fully elucidated, but it has been suggested that it leads to apoptosis by altering lipid-dependent Leishmania signaling pathways (Verma and Dey 2004; Pinto-Martinez et al. 2018).

The conventional methods of screening for anti-leishmanial drugs are based on in vitro assays using amastigotes from axenic cultures or in mammalian cell line cultures, staining with Giemsa dye, and counting by light microscopy. These assays are time-consuming, laborious, and depend on the experience of the technician (Gupta 2011; Patel et al. 2014; Zulfiqar et al. 2017; Caridha et al. 2019; Corman et al. 2019). Assays to assess differentiation of promastigote to amastigote have been used for both Old and New World Leishmania species (Bates et al. 1992; Debrabant et al. 2003; Sousa et al. 2019; Tavares et al. 2019). However, to isolate amastigotes from old world Leishmania species, infected macrophage cultures or commercial cell lines are required (Glaser et al. 1990; Bates et al. 1992), and even these obtain low yields of amastigotes which are often contaminated with undesirable host cell factors, making them unsuitable for biochemical studies (Peters et al. 1995; Wheeler et al. 2011; Gluenz et al. 2015). Furthermore, obtaining amastigotes is costly and time-consuming, as not all Leishmania spp. can be easily differentiated in axenic culture, in contrast to Leishmania mexicana, which changes from promastigotes to amastigotes by transferring in Schneider’s Drosophila (SD) medium and decreasing the pH to 5.5 (Dias-Lopes et al. 2021) (Bates 1994; Sereno and Lemesre 1997; Gupta et al. 2001).

Flow cytometry has been used to monitor both life cycle and stage differentiation of Leishmania spp. (Nuñez et al. 2001; Saraiva et al. 2005; Dayakar et al. 2012). However, intracellular and surface markers such as Green Fluorescent Protein (GFP), firefly luciferase, and annexin are expensive and sometimes unavailable in developing countries such as Mexico (Thalhofer et al. 2010; Bolhassani et al. 2011; Sadeghi et al. 2015; Ebrahimisadr et al. 2018). Previously, stage differentiation of Leishmania donovani (L. donovani) was described using forward scatter (FSC) and side scatter (SSC) parameters which correspond to size and complexity, respectively (Dayakar et al. 2012). With this in mind, we hypothesized that flow cytometry may be exploited for anti-leishmanial drug screening. To test this, we monitored the differentiation stage of L. mexicana promastigotes to amastigotes in the presence of miltefosine. Our results demonstrated that flow cytometry is a robust technique to distinguish morphologically distinct parasites during stage differentiation. In this study, we established a short-term method to evaluate L. mexicana parasite differentiation in vitro in presence of anti-leishmanial drugs using flow cytometry.

Materials and methods

Parasite cultures

GFP-transfected Leishmania mexicana MNYC/BZ/62/M379 and WT-Leishmania mexicana MNYC/BZ/62/M379 parasites were obtained from the American Type Culture Collection (Maryland, USA). Parasites were cultured in medium 199 (Gibco) to characterize the exponential growth of promastigotes in T-25 flasks and as starter culture for further experiments. The medium 199 culture was supplemented with 10% fetal bovine serum (Gibco), 1% sodium-pyruvate (Sigma-Aldrich), 1% penicillin-streptomycin (Gibco), 0.1% β-mercaptoethanol (Gibco) and 15.5 µM hemin (Sigma-Aldrich) and adjusted to pH 7 at 27°C (Bates 1994). For induced stage differentiation of axenic amastigotes, 106 promastigotes obtained from the late logarithmic growth phase were incubated in SD medium, supplemented with 20% fetal bovine serum (Gibco) and 15.5 µM hemin (Sigma-Aldrich) and adjusting the culture to pH 5.5 (Bates 1994). All cultures were performed at least two times to ensure reproducibility.

Microscopic evaluation

GFP-expressing parasites cultured in medium 199 were examined by light microscopy with fluorescent filters and captured with an EVOS FL imaging system (Thermofisher, USA) at 40x magnification. For the short-term anti-leishmania drug assay, a culture sample of the parasites was diluted 1:100 with 10% formaldehyde solution and counted at each time-point (0, 4, 8, and 24 h) in a Neubauer chamber using an Olympus CKX41 microscope (Scientific Inc., USA) at 40x magnification. To observe changes in cellular morphology, thin smears of parasite cultures were made on glass slides, air-dried, and stained with Diff-Quik Staining Protocol. Images were captured using a microscope equipped with a miniVID LW camera (Scientific Inc., USA) at 100x magnification.

Flow cytometry analysis

A culture sample of 500µL from each time point was washed twice with 1x PBS, centrifugated at 200 x g, and fixed using 0.04% formaldehyde. Parasites were resuspended in FACS buffer (BD Biosciences) and dissociated using a syringe needle 25G x16 mm before data acquisition. A total of 10,000 events were acquired in the FACSVerse cell analyzer (BD, New Jersey, USA) using the FACSuite software (BD Biosciences). Data were analyzed using FlowJo software version V10.0.7r2. The GFP marker was used to unequivocally identify Leishmania parasites with FSC and SSC parameters in the flow cytometry analysis. Voltages setting used to identify GFP-expressing parasites were FSC (218), SSC (325). In the short-term assay, an aliquot (500 µL) was analyzed by flow cytometry corresponding to each time point of WT-Leishmania parasites cultures (non-fluorescent). The voltage settings were adjusted to correctly identify the parasites in the FSC (202) and SSC (320) parameters. Promastigotes and amastigotes were identified using non-fluorescence marker and forward scatter area (FSC-A) and side scatter area (SSC-A) parameters. To avoid recording aggregated parasites a singlet discrimination was included based on a dot-plot of SSC-A and side scatter height (SSC-H) parameters. The percentage of each population was estimated by histogram of frequencies.

Promastigote to amastigote differentiation assay.

Differentiation assay was performed by incubating 106 WT-Leishmania Mexicana promastigotes from the late logarithmic growth phase as described above. Parasites incubated with supplemented SD medium alone were used as control. To inhibit the promastigote to amastigote differentiation, 40 µM of miltefosine (Cayman Chemical Company, Michigan, USA) was added to the culture and incubated for 24 h. Promastigotes and amastigotes forms were monitored by both microscopy and flow cytometry techniques simultaneously at 0, 4, 8 and 24 hours time points. This short-term assay was performed three times in parallel by microscopy and flow cytometry.

Results

In this study, we established a short-term method to evaluate parasite differentiation in vitro in presence of anti-leishmanial drugs using flow cytometry. As observed in Fig. 1A, the parasite culture reaches the exponential growth phase at day 7 post-incubation. The L. mexicana promastigotes in supplemented medium 199 were elongated flagellated parasites with positive motility as we observed by fluorescent microscopy (Fig. 1B).

Figure 1. Exponential and stationary stage of L. mexicana parasites in 199 medium.

Figure 1.

Open in a new tab

A) Culture started with 5×105 promastigotes in medium 199. Promastigote and amastigote forms were counted by light microscopy up to 9 days post-incubation. Data are represented by the mean ± SD. Dotted line represents the number of promastigotes in the started culture. B) L. mexicana promastigotes visualized using the EVOS FL imaging system at 40x magnification.

To characterize promastigote and amastigote forms by using FSC-A and SSC-A parameters (Fig. 2A), a new culture of GFP-parasites was started from the late logarithmic growth phase to induce axenic amastigotes and was followed for seven days by both microscopy and flow cytometry simultaneously. Results of flow cytometry at 24 hours post-incubation in SD medium showed that 83.7 % of L. mexicana parasites expressed GFP marker (Fig. 2B). Similarly, using FSC-A vs. SSC-A parameters allowed identification of both promastigote and amastigote parasite populations (Fig. 2C). Our results showed a frequency of 16.5 % for promastigotes (left side of the panel) and 83.5 % for amastigote populations (right side of the panel) (Fig. 2D). Interestingly, both microscopy (Fig. 3) and flow cytometry showed that approximately 80% of the promastigotes have differentiated into the amastigote-like form as soon as 24 hours. The differentiation stage was captured (Fig. 3) from promastigotes at 0 h (immediately after placed in SD medium) to 24 h post-incubation. Parasites showed a gradual decrease in thickness of the flagellum and 24 h post-incubation an ovoid shape with a flimsy flagellum was observed. These findings suggested that evaluating L. mexicana differentiation should be performed during the first 24 hours post-incubation in SD medium.

Figure 2. Flow cytometry gating strategy to identify parasite populations using a GFP marker.

Figure 2.

Open in a new tab

A total of 106 promastigotes from stationary phase were shifted from medium 199 into medium SD. Parasites were recovered 24 hours post-incubation, and 10,000 events were acquired using a BD FACSVerse Cell Analyzer. A) Doublet exclusion based on SSC-A and SSC-H parameters. B) Identification of live parasites through blue laser at 488 nm corresponding to GFP-expressing parasites. C) Dot-plot gating of GFP-expressing L. mexicana parasites using FSC-A and SSC-A. D) Histogram of promastigote and amastigote percentages based on FSC-A.

Figure 3. Parasites in SD-medium stained with Diff-Quik.

Figure 3.

Open in a new tab

Parasites were recorded at 0, 4, 8 and 24h post-incubation showing a change in the morphology. Parasites were stained with commercial kit Diff-Quik and images were captured at 100x magnification using an miniVID camera (LW Scientific Inc. USA) attached to Olympus CX23 microscope.

Finally, to validate the differentiation assay, promastigote cultures were incubated with miltefosine. Measurements were performed immediately after the addition of SD medium, and then at 4, 8, and 24 hours post-incubation. The results showed that 17.6% of the parasites in SD medium (control cultures) started to differentiate four hours post-incubation into amastigote-like forms. The amastigote-like parasites estimated by flow cytometry when incubated for 24 hours in medium alone were 58.4 %. Conversely, we observed that miltefosine at 40 µM showed an inhibitory effect on the differentiation of L. mexicana promastigotes towards amastigote-like forms, and 24 hours post-incubation the amastigote-like population was 21.8 % (Fig. 4). In parallel, to confirm the findings observed in flow cytometry the parasites in presence of miltefosine were counted by microscopy and showed decreased amastigote population from 5×106 in SD medium alone to 1×106 24 h post-incubation (Fig. 5).

Figure 4. Promastigote-to-amastigotes differentiation assay by flow cytometry.

Figure 4.

Open in a new tab

A total of 106 promastigotes were transferred to SD medium and incubated with 40 µM miltefosine or medium alone. Parasites were recovered, diluted in FACS buffer and 10,000 events were acquired in a BD FACSVerse Cell Analyzer at different time points (0, 4, 8 and 24 hours) post-incubation. Data are presented as histogram of frequencies where amastigotes-L population represents amastigotes-like forms.

Figure 5. L. mexicana amastigotes parasites in SD medium.

Figure 5.

Open in a new tab

A) Culture started with 1×106 promastigotes in medium SD. Parasites were counted by light microscopy up to 24 h post-incubation. Data are represented by the mean ± SD. Dotted line represents the number of promastigotes in the started culture.

Discussion

The increase in drug-resistant strains of Leishmania has underscored the importance of identifying novel anti-leishmanial drugs (Croft and Coombs 2003). The targets for developing anti-leishmanial drugs involve elucidating antioxidant and metabolic functions, blocking parasite proliferation, or inhibiting the differentiation of promastigotes to amastigotes (Colotti and Ilari 2011; Ilari et al. 2017). Reverse-Transcriptase PCR has been used to amplify stage-related genes that could be used to explore the effect of compounds with activity on Leishmania (Dayakar et al. 2012). However, contrasting results have been observed for amastin transcript levels, probably due to the stability of mRNA in axenic amastigote cultures, which limits its use compared to other assays as flow cytometry or microscopic counting. For parasite differentiation, in vitro assays are needed to evaluate the amastigote forms that take days or weeks to observe in cell cultures (Dayakar et al. 2012). In addition, in many cases, a Giemsa dye is required to identify promastigote and amastigote forms of Leishmania spp. Thus, lengthy in vitro assays could be biased due to frequent changes in the culture such as replication parasites and apoptosis.

On the other hand, flow cytometry is a versatile technique to identify and characterize cellular parameters, including intracellular molecules. This technique could also provide a means to rapidly identify various types of cell populations, and to reliably quantify large numbers of cells in a short period of time. Although flow cytometry is not a novel technique, it has not been further exploited. Studies have demonstrated that flow cytometry is a feasible tool to screen herbal compounds and metabolites against Leishmania amazonensis (L. amazonensis) by measurement of GFP-expressing parasites (Plock et al. 2001). In addition, the annexin V marker expressed in Leishmania major (L. major) infected cells after exposure with anti-leishmania compounds allowed measurement of parameters such as apoptosis and necrosis by flow cytometry (Ebrahimisadr et al. 2018). However, the GFP-transfected parasite dependent-assay or used expensive markers such as annexin could be an obstacle. Although in this study we used GFP-expressing parasites to identify the voltages where parasites can be visualized in flow cytometry, we were able to identify the promastigote and amastigote-like forms in the differentiation assay with only FSC and SSC parameters. In addition, flow cytometry provided a reliable method to identify the parasite forms compared to visualization by light microscopy, reducing technician-caused errors. We suggest that these practices are time consuming and inaccurate.

Previously, L. donovani parasite was evaluated by flow cytometry using side scatter and forward scatter parameters. The results showed a change in parasite population size corresponding to the differentiation of promastigotes forms into amastigotes (Dayakar et al. 2012). In accordance with this study, we visualized small-rounded amastigotes 7 days post-incubation by microscopy. Interestingly, we also observed that parasite size increased over 24 hours post-incubation in SD medium, which corresponded to the amastigote-like population identified by others (Bates et al. 1992; Castilla et al. 1995; Abdelhaleem et al. 2019; Ashrafmansouri et al. 2020). Indeed, Dayakar et al. also showed these parasite forms in an image acquired by electron microscopy 24 hours post-incubation (Dayakar et al. 2012). The amastigote-like population represents an intermediate stage between promastigotes and intracellular amastigotes forms reported in L. major as well as in L. donovani (Gupta et al., 2001; Abdelhaleem et al., 2019). Conversely to Dayakar’s observations using L. donovani strain we found that L. mexicana parasite turns from left to right in FSC in flow cytometry, confirmed by microscopy at the 24 h post-incubation (Dayakar et al. 2012). Dayakar and collaborators noted this interesting difference as they observed amastigote-like populations. Therefore, for L. mexicana we developed a 24h-drug assay considering that parasites after 24 h post-incubation are no longer a homogeneous population and parasites undergo apoptosis or replication within 7 days post-incubation.

Drugs such as miltefosine have been used for the treatment of leishmaniasis since the 1940s with moderate efficacy (Goodwin 1945; Maegraith and Brundrett 1947). However, variable drug susceptibility associated with different Leishmania spp. has been recognized (Decuypere et al. 2005; Patel et al. 2014). Even though miltefosine can affect both the promastigote and amastigote forms, promastigotes are more susceptible than amastigotes (Croft et al. 2006; Dorlo et al. 2012). It is well-described that miltefosine can alter the lipid metabolism of promastigotes, leading to the accumulation of lipids and induction of apoptosis (Paris et al. 2004; Zulueta et al. 2020). Despite the different susceptibility of amastigotes, the drug is still effective in treating leishmaniasis in vivo and in vitro studies (Verma and Dey 2004). Similarly, L. mexicana is generally reported to be more resistant to anti-leishmanial drugs than other Leishmania spp. (Taylor and Williams 1991). A limitation in this study is that the effect induced by different concentrations of miltefosine was not evaluated, although the dose used here was sufficient to observe changes after 24 hours incubation period in the differentiation of promastigotes to amastigotes. At a dose of 40 µM, miltefosine showed an evident reduction of the differentiation stage compared to parasites in the presence of medium SD alone.

Differentiation of Leishmania parasites depends on various factors including the specie, culture conditions, and the stage of infection. The mechanism by which miltefosine affects differentiation is complex and not fully understood. It is suggested to involve the modulation of various cellular processes including lipid metabolism, protein kinase C signaling, and DNA fragmentation (Verma and Dey 2004). Miltefosine also has been shown to induce the synthesis of lipophosphoglycan (LPG) in Leishmania parasites, which plays a role in the differentiation of promastigotes into amastigotes. However, its effectiveness as an anti-leishmania drug is due to its ability to interfere with multiple cellular processes including interaction with macrophage receptors and modulate the host immune response by triggering IFNγ (Paris et al. 2004; Wadhone et al. 2009). According to our findings, miltefosine could be blocking the differentiation of promastigotes to amastigotes and reducing the sub-population of promastigotes incubated for 24h. An evaluation of GFP-fluorescence by flow cytometry revealed that miltefosine at dose 0.4 µM exhibited a trypanocidal activity in cultures with L. amazonensis-infected human macrophages (Mehta et al. 2011). In L. donovani strains, the use of miltefosine has been shown to elicit an apoptotic-like pathway that kills both amastigotes and promastigotes within 24 hours (Verma and Dey 2004). Other studies found that L. major and Leishmania tropica were susceptible to miltefosine with a 50% inhibitory concentration (IC50) at 22 µM and 11 µM doses, respectively (Khademvatan et al. 2011).

The evaluation of promastigote-to-amastigote differentiation by in vitro assays could be used to screen new drugs or compounds against parasites as Leishmania spp., however, this technique is not robust enough and complementary assays are required. Also, we are aware that drug toxicity assays are essential during the development of new drug candidates. In sum, our results provide a reliable, rapid method to distinguish morphologically distinct L. mexicana parasites during differentiation stage 24 hours post-incubation in medium SD, using simple flow cytometry based on FSC and SSC parameters alone.

HIGHLIGHTS.

  • Flow cytometry provides a rapid means to quantify L. mexicana parasite differentiation in 24 h post-incubation

  • FSC and SSC parameters are enough robust to measure the differentiation stage of L. mexicana in a 24h-drug assay.

  • Flow cytometry is reliable and rapid method compared to microscopy in drug assays with axenic L. mexicana parasites.

Funding

J.M.M. was supported by National Institutes of Health Molecular Biophysics Training grant T32-GM008280. F.T.F.T. received support by grants from the Welch Foundation (Q-1530-20190330) and the National Institutes of Health R01-GM111084 and R01-GM142143 and M.E.B. by Robert J. Kleberg Jr. and Helen C. Kleberg Foundation.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declarations of interest: none

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical statement

The authors declare that the work is original, and all authors made a significant contribution to the conception, design, execution, or interpretation of the study. This submitted manuscript has not been published elsewhere and is not under consideration by another journal. This work does not involve the use of animal or human subjects, and all procedures were performed in compliance with relevant laws and institutional guidelines. All the authors have read and approved the “Policies for authors” and “Ethics” information provided by Elsevier at the following address: https://www.elsevier.com/about/policies/publishing-ethics#Authors

References

  1. Abdelhaleem AA, Elamin EM, Bakheit SM, Mukhtar MM, 2019. Identification and characterization of Leishmania amastigote and axenic form antigens. Trop Biomed 1;36(4):866–873. [PubMed] [Google Scholar]
  2. Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P, Delaunay P, Sereno D, 2016. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl. Trop. Dis 3;10(3):e0004349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ashrafmansouri M, Amiri-Dashatan N, Ahmadi N, Rezaei-Tavirani M, Seyyed S, Haghighi A, 2020. Quantitative proteomic analysis to determine differentially expressed proteins in axenic amastigotes of Leishmania tropica and Leishmania major. IUBMB Life 72(8):1715–1724. [DOI] [PubMed] [Google Scholar]
  4. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M,WHO Leishmaniasis control team., 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7(5):e35671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bates PA., 1994. Complete developmental cycle of Leishmania mexicana in axenic culture. Parasitology 108:1–9. [DOI] [PubMed] [Google Scholar]
  6. Bates PA, Robertson CD, Tetley L, Coombs GH, 1992. Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms. Parasitology 105:193–202. [DOI] [PubMed] [Google Scholar]
  7. Bolhassani A, Taheri T, Taslimi Y, Zamanilui S, Zahedifard F, Seyed N, Torkashvand F, Vaziri B, Rafati S, 2011. Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies. Exp. Parasitol 127(3):637–45. [DOI] [PubMed] [Google Scholar]
  8. Burza S, Croft SL, Boelaer t M., 2018. Leishmaniasis. Lancet 15;392(10151):951–970. [DOI] [PubMed] [Google Scholar]
  9. Castilla JJ, Sanchez-Moreno M, Mesa C, Osuna A, 1995. Leishmania donovani: in vitro culture and [1H] NMR characterization of amastigote-like forms. Mol Cell Biochem 26;142(2):89–97. [DOI] [PubMed] [Google Scholar]
  10. Caridha D, Vesely B, van Bocxlaer K, Arana B, Mowbray CE, Rafati S, Uliana S, Reguera R, Kreishman-Deitrick M, Sciotti R, Buffet P, Croft SL., 2019. Route map for the discovery and pre-clinical development of new drugs and treatments for cutaneous leishmaniasis. Int. J. Parasitol. Drugs. Drug Resist 11:106–117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Colotti G, Ilari A, 2011. Polyamine metabolism in Leishmania: from arginine to trypanothione. Amino Acids 40(2):269–85. [DOI] [PubMed] [Google Scholar]
  12. Corman HN, Shoue DA, Norris-Mullins B, Melancon BJ, Morales MA, McDowell MA, 2019. Development of a target-free high-throughput screening platform for the discovery of antileishmanial compounds. Int. J. Antimicrob. Agents 54(4):496–501. [DOI] [PubMed] [Google Scholar]
  13. Croft SL, Coombs GH, 2013. Leishmaniasis-current chemotherapy and recent advances in the search for novel drugs. Trends Parasitol 19(11):502–8. [DOI] [PubMed] [Google Scholar]
  14. Croft SL, Sundar S, Fairlamb AH. 2006. Drug resistance in leishmaniasis. Clin Microbiol Rev 19(1):111–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dayakar A, Chandrasekaran S, Prajapati VK, Veronica J, Sundar S, Maurya R, 2012. A rapid method to assess the stage differentiation in Leishmania donovani by flow cytometry. Exp Parasitol 132(4):495–500. [DOI] [PubMed] [Google Scholar]
  16. Debrabant A, Joshi MB, Pimenta PF, Dwyer DM, 2003. Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics. Int. J. Parasitol 34(2):205–17. [DOI] [PubMed] [Google Scholar]
  17. Decuypere S, Rijal S, Yardley V, De Doncker S, Laurent T, Khanal B, Chappuis F, Dujardin JC., 2005. Gene expression analysis of the mechanism of natural Sb(V) resistance in Leishmania donovani isolates from Nepal. Antimicrob Agents Chemother 49(11):4616–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dias-Lopes G, Zabala-Peñafiel A, de Albuquerque-Melo BC, Souza-Silva F, Menaguali do Canto L, Cysne-Finkelstein L, Alves CR, 2021. Axenic amastigotes of Leishmania species as a suitable model for in vitro studies. Acta Trop 220:105956. [DOI] [PubMed] [Google Scholar]
  19. Dorlo TP, Balasegaram M, Beijnen JH, de Vries PJ. 2012. Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis. J Antimicrob Chemother Nov;67(11):2576–97. [DOI] [PubMed] [Google Scholar]
  20. Ebrahimisadr P, Ghaffarifar F, Horton J, Dalimi A, Sharifi Z, 2018. Apoptotic effect of morphine, imiquimod and nalmefene on promastigote, infected and uninfected macrophages with amastigote of Leishmania major by flow cytometry. Iran J. Pharm. Res 17(3):986–994. [PMC free article] [PubMed] [Google Scholar]
  21. Engwerda CR, Ato M, Kaye PM, 2004. Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis. Trends Parasitol 20(11):524–30. [DOI] [PubMed] [Google Scholar]
  22. Glaser TA, Wells SJ, Spithill TW, Pettitt JM, Humphris DC, Mukkada AJ, 1990. Leishmania major and L. donovani: a method for rapid purification of amastigotes. Exp. Parasitol 71(3):343–5. [DOI] [PubMed] [Google Scholar]
  23. Gluenz E, Wheeler RJ, Hughes L, Vaughan S, 2015. Scanning and three-dimensional electron microscopy methods for the study of Trypanosoma brucei and Leishmania mexicana flagella. Methods Cell Biol 127:509–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goodwin LG., 1945. The chemotherapy of experimental leishmaniasis; a dose response curve for the activity of sodium stibogluconate. Trans. R. Soc. Trop. Med. Hyg 39(2):133–45. [DOI] [PubMed] [Google Scholar]
  25. Gupta N, Goyal N, Rastogi AK., 2001. In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol 17(3):150–3. [DOI] [PubMed] [Google Scholar]
  26. Gupta N, 2011. Visceral leishmaniasis: experimental models for drug discovery. Indian J. Med. Res 133(1):27–39. [PMC free article] [PubMed] [Google Scholar]
  27. Ilari A, Fiorillo A, Genovese I, Colotti G, 2017. Polyamine-trypanothione pathway: an update. Future Med. Chem 9(1):61–77. [DOI] [PubMed] [Google Scholar]
  28. Kevric I, Cappel MA., Keeling, JH., 2015. New world and old world Leishmania infections: a practical review., 2015. Dermatol. Clin 33(3):579–93. [DOI] [PubMed] [Google Scholar]
  29. Khademvatan S, Gharavi MJ., Rahim, F., Saki, J., 2011. Miltefosine-induced apoptotic cell death on Leishmania major and L. tropica strains. Korean J. Parasitol 49(1):17–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Maegraith B, Brundrett JC., 1947. Sodium stibogluconate in the treatment of kalaazar; report on the treatment of eight cases and the appearance of probable drug reactions. Ann. Trop. Med. Parasitol 41(1):118–28. [DOI] [PubMed] [Google Scholar]
  31. Mehta SR, Zhang XQ, Badaro R, Spina C, Day J, Chang KP, Schooley RT, 2010. Flow cytometric screening for anti-leishmanials in a human macrophage cell line. Exp. Parasitol 126(4):617–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mittra B, Cortez M, Haydock A, Ramasamy G, Myler PJ, Andrews, 2013. Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels. J. Exp. Med 11;210(2):401–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nuñez R, Kamau S, Grimm F, 2001. Flow cytometric assessment of drug susceptibility in Leishmania infantum promastigotes. Curr Protoc Cytom 11:Unit 11.14. [DOI] [PubMed] [Google Scholar]
  34. Paris C, Loiseau PM, Bories C, Bréard J. 2004. Miltefosine induces apoptosis-like death in Leishmania donovani promastigotes. Antimicrob Agents Chemother Mar;48(3):852–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Patel AP, Deacon A, Getti G, 2014. Development and validation of four Leishmania species constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies. Parasitology 141(4):501–10. [DOI] [PubMed] [Google Scholar]
  36. Peters C, Aebischer T, Stierhof YD, Fuchs M, Overath P, 1995. The role of macrophage receptors in adhesion and uptake of Leishmania mexicana amastigotes. J. Cell Sci 108 (12):3715–24. [DOI] [PubMed] [Google Scholar]
  37. Pinto-Martinez AK, Rodriguez-Durán J, Serrano-Martin X, Hernandez-Rodriguez V, Benaim G, 2017. Mechanism of action of miltefosine on Leishmania donovani involves the impairment of acidocalcisome function and the activation of the sphingosine-dependent plasma membrane Ca2+ channel. Antimicrob Agents Chemother 21;62(1):e01614–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Plock A, Sokolowska-Köhler W, Presber W, 2001. Application of flow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania Spp. Exp Parasitol 97(3):141–53. [DOI] [PubMed] [Google Scholar]
  39. Rittig MG, Bogdan C, 2000. Leishmania-host-cell interaction: complexities and alternative views. Parasitol Today 16(7):292–7. [DOI] [PubMed] [Google Scholar]
  40. Sadeghi S, Seyed N, Etemadzadeh MH, Abediankenari S, Rafati S, Taheri T, 2015. 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 53(4):385–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Saraiva EM, Pinto-da-Silva LH, Wanderley JL, Bonomo AC, Barcinski MA, Moreira ME, 2005. Flow cytometric assessment of Leishmania spp metacyclic differentiation: validation by morphological features and specific markers. Exp. Parasitol 110(1):39–47. [DOI] [PubMed] [Google Scholar]
  42. Séguin O, Descoteaux A, 2016. Leishmania, the phagosome, and host responses: The journey of a parasite. Cell Immunol 309:1–6. [DOI] [PubMed] [Google Scholar]
  43. Sereno D, Lemesre JL., 1997. Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents. Antimicrob. Agents. Chemother 41(5):972–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sousa JKT, Antinarelli LMR, Mendonça DVC, Lage DP, Tavares GSV, Dias DS, Ribeiro PAF, Ludolf F, Coelho VTS, Oliveira-da-Silva JA, Perin L, Oliveira BA, Alvarenga DF, Chávez MA, Brandão GC, Nobre V, Pereira GR, Coimbra ES, Coelho EAF, 2019. A chloroquinoline derivate presents effective in vitro and in vivo antileishmanial activity against Leishmania species that cause tegumentary and visceral leishmaniasis. Parasitol. Int 73:101966. [DOI] [PubMed] [Google Scholar]
  45. Tavares GSV, Mendonça DVC, Lage DP, Antinarelli LMR, Soyer TG, Senna AJS, Matos GF, Dias DS, Ribeiro PAF, Batista JPT, Poletto JM, Brandão GC, Chávez-Fumagalli MA, Pereira GR, Coimbra ES, Coelho EAF, 2019. In vitro and in vivo antileishmanial activity of a fluoroquinoline derivate against Leishmania infantum and Leishmania amazonensis species. Acta Trop 191:29–37. [DOI] [PubMed] [Google Scholar]
  46. Taylor DR, Williams GT, 1991. Differentiation and limited proliferation of isolated Leishmania mexicana amastigotes at 27 degrees C. Acta Trop 50(2):141–50. [DOI] [PubMed] [Google Scholar]
  47. Thalhofer CJ, Graff JW, Love-Homan L, Hickerson SM, Craft N, Beverley SM, Wilson ME, 2010. In vivo imaging of transgenic Leishmania parasites in a live host. J Vis Exp 27;(41):1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Torres-Guerrero E, Quintanilla-Cedillo MR, Ruiz-Esmenjaud J, Arenas R, 2017. Leishmaniasis: a review. F1000 Research 26;6:750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Verma NK, Dey CS, 2004. Possible mechanism of miltefosine-mediated death of Leishmania donovani. Antimicrob. Agents Chemother 48(8):3010–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wadhone P, Maiti M, Agarwal R, Kamat V, Martin S, Saha B. 2009. Miltefosine promotes IFN-gamma-dominated anti-leishmanial immune response. J Immunol Jun 1;182(11):7146–54. [DOI] [PubMed] [Google Scholar]
  51. Wheeler RJ, Gluenz E, Gull K, 2011. The cell cycle of Leishmania: morphogenetic events and their implications for parasite biology. Mol Microbiol 79(3):647–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zulfiqar B, Jones AJ, Sykes ML, Shelper TB, Davis RA, Avery VM, 2017. Screening a natural product-based library against kinetoplastid parasites. Molecules 12;22(10):1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zulueta Díaz YLM, Ambroggio EE, Fanani ML. 2020. Miltefosine inhibits the membrane remodeling caused by phospholipase action by changing membrane physical properties. Biochim Biophys Acta Biomembr Oct 1;1862(10):183407. [DOI] [PubMed] [Google Scholar]

RESOURCES