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 et al., 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., 2017).

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.