Determination of anti-leishmanial drugs efficacy against Leishmania martiniquensis using a colorimetric assay

Article history:
Received 8 January 2019
Received in revised form 1 March 2019
Accepted 18 February 2020

Atchara Phumee
Narissara Jariyapan
Saranyou Chusri
Thanaporn Hortiwakul
Oussama Mouri
Frederick Gay
Wacharee Limpanasithikul
Padet Siriyasatien


Autochthonous leishmaniasis caused by Leishmania martiniquensis cases in Thailand have dramatically increased in the recent years. L. martiniquensis infection primarily occurs in immunocompromised patients, especially AIDS patients. In Thailand, amphotericin B is the only drug available for leishmaniasis treatment, and some patients relapse after amphotericin B therapy.

Moreover, the efficacy of anti-leishmanial drugs against L. martiniquensis has not been evaluated to date. In this study, we determined the efficacy of various anti-leishmanial drugs against the promastigote and intracellular amastigote stages of L. martiniquensis using a colorimetric assay. Two strains (CU1 and CU1R1) were isolated from leishmaniasis HIV co-infected patient from Songkhla province, southern Thailand. The CU1 strain was isolated from the patient in 2011, and CU1R1 was isolated from the same patient in 2013, when he was diagnosed as relapse leishmaniasis. The third strain (LSCM1) used in this study has been isolated from immunocompetent patient from Lamphun province, northern Thailand. All strains were identified as L. martiniquensis by sequencing of ribosomal RNA ITS-1 and large subunit of RNA polymerase II gene. Bioassays have been conducted both with promastigote and intracellular amastigote stages of the parasite. All L. martiniquensis strains have been tested against amphotericin B, miltefosine and pentamidine to determine the efficacy of the drugs against the parasite by using a PrestoBlue. The efficacy of miltefosine and pentamidine exhibit no significant difference between each stage of L. martiniquensis among all strains. Surprisingly, the promastigote and intracellular amastigote of the CU1R1 isolate, which was isolated from a relapsed patient after amphotericin B treatment, exhibited a two-fold increased inhibitory concentration(IC50) against amphotericin B compared with other strains, and the difference was statistically significant (p b 0.05). Moreover, intracellular amastigotes isolated from CU1R1 exhibited slightly increased susceptibility to amphotericin B compared with the promastigote (p b 0.05). The result of this experiment is a scientific evident to support that in case of relapsed leishmaniasis caused by L. martiniquensis, increasing dosage of amphotericin B is essential.

Moreover, this study also determined efficacy of other anti-leishmanial drugs for treatment the leishmaniasis in Thailand in case of these drugs are available in the country and the clinicians should have alternative drugs for treatment leishmaniasis in Thailand apart from amphotericin B.


Leishmania is protozoa belonging to the Family Trypanosomatidae of the Order Kinetoplastida (Lainson and Shaw, 1987). These protozoans are parasites responsible for leishmaniasis. The parasites are transmitted to vertebrate hosts by the bite of female sand flies (Bates, 2007). The parasites have dimorphic life cycle, including an intracellular amastigote form and a promastigote form. The amastigote is characterized by a round or ovoid shape. The immotile amastigote multiplies in the cytoplasm of macrophages of vertebrate hosts. The promastigote form is characterized by a spindle-shape. This form uses a flagellum for motility and is found in female sand fly (Bates, 2007; Kato et al., 2010). Leishmaniasis occurs in tropical and subtropical areas and southern Europe (Steverding, 2017). Leishmaniasis is classified into the Old World and New World leishmaniasis. Old World leishmaniasis is caused by the subgenus Leishmania, which is found in Africa, Asia, the Middle East, the Mediterranean, and India. New World leishmaniasis is found in Central and South America and is caused by the L. mexicana complex and all species of the subgenus Viannia (Maltezou, 2010). There are three main clinical forms of the disease, including visceral, cutaneous, and mucocutaneous leishmaniasis (Lainson and Shaw, 1987; Maltezou, 2010). Clinical forms and treatment depend on the Leishmania species (Mouri et al., 2014). In Thailand, indigenous leishmaniasis is an emerging disease described in recent years. The disease was reported in northern, central and southern regions of Thailand (Maharom et al., 2008). Two major Leishmania species L. martiniquensis and L. siamensis (L. orientalis n. sp.) (Sukmee et al., 2008; Suankratay et al., 2010; Bualert et al., 2012; Chusri et al., 2012; Phumee et al., 2013; Noppakun et al., 2014; Osatakul et al., 2014; Phumee et al., 2014; Pothirat et al., 2014; Chiewchanvit et al., 2015; Siriyasatien et al., 2016; Jariyapan et al., 2018) were reported in Thailand. Several drugs are effective for the treatment of leishmaniasis, including pentavalent antimonials, amphotericin B deoxycholate, lipid formulations of amphotericin B, miltefosine, paromomycin, pentamidine and sodium stibogluconate; however, these drugs exhibit different modes of action, adverse effects, costs, and routes of administration (Croft and Brazil, 1982; Singh and Sivakumar, 2004; Singh, 2006; Morizot et al., 2016). Currently, several reports described the increasing drug resistance in many Leishmania species (Croft et al., 2006; Mohapatra, 2014). However, the level of resistance varies according to the Leishmania species, strain, stages of Leishmania and the distribution and frequency of resistance to anti-leishmanial drugs are unknown. The variation in intrinsic sensitivity among some Leishmania species to several drugs depends on genetic, molecular, and biochemical variations. In Thailand, amphotericin B is the only effective drug available for the treatment of leishmaniasis; however, some patients developed relapsed leishmaniasis after receiving amphotericin B treatment (Suankratay et al., 2010; Chusri et al., 2012; Phumee et al., 2013; Phumee et al., 2014; Pothirat et al., 2014; Siriyasatien et al., 2016). Moreover, the efficacy of amphotericin B and other antileishmanial drugs against L. martiniquensis has never been evaluated. Therefore, in this study, we sought to determine the efficacy of anti-leishmanial drugs against various strains of L. martiniquensis isolated from Thai patients using colorimetric assays. The resazurin-based assay PrestoBlueCell Viability Reagent was used because it is a non-toxic, water soluble, redox-sensitive dye. Data obtained from this study may potentially improve the treatment of leishmaniasis caused by L. martiniquensis in Thailand.


Determination of anti-leishmanial drugs efficacy against Leishmania martiniquensis using a colorimetric assay