Turkiye parazitolojii dergisi · 2026
Quantitative Analysis of the Intracellular Efficacy of Conventional Antileishmanial Drugs Against Leishmania tropica: A Translational Approach Using the THP-1 Macrophage-Amastigote Model.
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Yıldırım Ahmet, Aksoy Tülay, Özbilgin Ahmet
Аннотация
OBJECTIVE: This study aimed to quantitatively assess the intracellular susceptibility of Leishmania tropica (L. tropica) to conventional antileishmanial drugs using the parasite rescue and transformation assay (PRTA), which involves controlled lysis of infected THP-1 macrophages, release of viable amastigotes, their transformation into promastigotes under defined in vitro conditions, and subsequent luminometric analysis.
METHODS: L. tropica (MHOM/TR/2007/CBU001) promastigotes were cultured in Novy-MacNeal-Nicolle and Schneider's insect medium; THP-1 cells were cultured in RPMI-1640. PMA-differentiated THP-1 macrophages were infected to establish the macrophage-amastigote model. Intracellular susceptibility to five conventional drugs (amphotericin B, miltefosine, pentamidine, sodium stibogluconate, and meglumine antimoniate) was evaluated by PRTA through luminometric quantification (CellTiter-Glo®) of transformed promastigotes, with microscopic enumeration of amastigotes in Giemsa-stained slides used for validation. Inhibitory concentration 50 (IC50) values were calculated from dose-response curves and compared using Student's t-test and Pearson's correlation coefficient.
RESULTS: Amphotericin B and pentamidine showed the highest efficacy; miltefosine demonstrated moderate activity; antimonials exhibited relatively low efficacy. IC50 values obtained by luminometric and microscopic methods were highly concordant: the paired t-test revealed no significant difference (t=0.2172, p=0.8387), and Pearson's correlation confirmed a strong positive relationship (r=0.9944, p=0.005).
CONCLUSION: Luminometric analysis of transformed promastigotes provides accuracy comparable to that of microscopic enumeration of intracellular amastigotes, while being more efficient in time, labor, and resources. By enabling functional assessment of parasite metabolism, supporting high-throughput and automation with low operator dependency, this standardized method represents a reliable, scalable, and translationally relevant platform with strong clinical predictive value, particularly for species-specific models such as L. tropica.
METHODS: L. tropica (MHOM/TR/2007/CBU001) promastigotes were cultured in Novy-MacNeal-Nicolle and Schneider's insect medium; THP-1 cells were cultured in RPMI-1640. PMA-differentiated THP-1 macrophages were infected to establish the macrophage-amastigote model. Intracellular susceptibility to five conventional drugs (amphotericin B, miltefosine, pentamidine, sodium stibogluconate, and meglumine antimoniate) was evaluated by PRTA through luminometric quantification (CellTiter-Glo®) of transformed promastigotes, with microscopic enumeration of amastigotes in Giemsa-stained slides used for validation. Inhibitory concentration 50 (IC50) values were calculated from dose-response curves and compared using Student's t-test and Pearson's correlation coefficient.
RESULTS: Amphotericin B and pentamidine showed the highest efficacy; miltefosine demonstrated moderate activity; antimonials exhibited relatively low efficacy. IC50 values obtained by luminometric and microscopic methods were highly concordant: the paired t-test revealed no significant difference (t=0.2172, p=0.8387), and Pearson's correlation confirmed a strong positive relationship (r=0.9944, p=0.005).
CONCLUSION: Luminometric analysis of transformed promastigotes provides accuracy comparable to that of microscopic enumeration of intracellular amastigotes, while being more efficient in time, labor, and resources. By enabling functional assessment of parasite metabolism, supporting high-throughput and automation with low operator dependency, this standardized method represents a reliable, scalable, and translationally relevant platform with strong clinical predictive value, particularly for species-specific models such as L. tropica.