Comparative Assessment of colorimetric assays in evaluating intracellular drug susceptibility of Leishmania tropica against conventional antileishmanial drugs

This study aims to identify the most sensitive colorimetric test for assessing intracellular drug susceptibility of Leishmania tropica to conventional antileishmanial drugs. To this end, the efficacy of four colorimetric methods—MTT, XTT, MTS, and WST-8—was compared using reference L. tropica promas...

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Veröffentlicht in:Parasitology international 2025-06, Vol.106, p.103021, Article 103021
Hauptverfasser: Yıldırım, Ahmet, Aksoy, Tülay, Balcıoğlu, İbrahim Cüneyt
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description This study aims to identify the most sensitive colorimetric test for assessing intracellular drug susceptibility of Leishmania tropica to conventional antileishmanial drugs. To this end, the efficacy of four colorimetric methods—MTT, XTT, MTS, and WST-8—was compared using reference L. tropica promastigotes. The intracellular drug susceptibility was further evaluated using the test with the widest absorbance range on isolates from Türkiye CL patients: two responsive to a single course of meglumine antimoniate (MA) and two that showed no clinical improvement after two treatments. CL isolates were identified via real-time PCR targeting the ITS1 region. Promastigote suspensions at standardized densities (0.08 × 106 to 10 × 106 promastigotes/well) were prepared in both RPMI (phenol red-containing) and RPMIØRP (phenol red-free) media, then analyzed with ELISA-based MTT, XTT, MTS, and WST-8 to identify the method with the broadest specific absorbance range. Intracellular drug susceptibility of CL isolates was subsequently assessed in a macrophage/amastigote model by infecting THP-1 macrophages with promastigotes from both reference and patient isolates, followed by treatment with MA, sodium stibogluconate (SSG), miltefosine (MTF), pentamidine (PMD), and amphotericin B (AmB). Promastigotes obtained from parasite rescue and transformation assays were analyzed using the most sensitive colorimetric method to determine IC₅₀ values. Species identification confirmed all four CL isolates as L. tropica, and the XTT assay provided the widest absorbance range in RPMIØRP media. IC₅₀ values for both treatment-responsive and unresponsive isolates were similar to those of the reference isolate, showing susceptibility to all tested drugs without statistically significant differences. Expanding the isolate set is necessary to further evaluate the predictive value of SbV (pentavalent antimonials) susceptibility for treatment outcomes. The identification of XTT as the most sensitive method for intracellular antileishmanial susceptibility testing is expected to aid in standardizing laboratory models and provide valuable insights for researchers and clinicians managing treatment-unresponsive CL cases. [Display omitted] •This study focuses on identifying the most sensitive colorimetric test (MTT, XTT, MTS, and WST-8) for evaluating intracellular antileishmanial drug susceptibility.•Four Leishmania spp. isolates from cutaneous leishmaniasis patients in Türkiye were identified as Leishm
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To this end, the efficacy of four colorimetric methods—MTT, XTT, MTS, and WST-8—was compared using reference L. tropica promastigotes. The intracellular drug susceptibility was further evaluated using the test with the widest absorbance range on isolates from Türkiye CL patients: two responsive to a single course of meglumine antimoniate (MA) and two that showed no clinical improvement after two treatments. CL isolates were identified via real-time PCR targeting the ITS1 region. Promastigote suspensions at standardized densities (0.08 × 106 to 10 × 106 promastigotes/well) were prepared in both RPMI (phenol red-containing) and RPMIØRP (phenol red-free) media, then analyzed with ELISA-based MTT, XTT, MTS, and WST-8 to identify the method with the broadest specific absorbance range. Intracellular drug susceptibility of CL isolates was subsequently assessed in a macrophage/amastigote model by infecting THP-1 macrophages with promastigotes from both reference and patient isolates, followed by treatment with MA, sodium stibogluconate (SSG), miltefosine (MTF), pentamidine (PMD), and amphotericin B (AmB). Promastigotes obtained from parasite rescue and transformation assays were analyzed using the most sensitive colorimetric method to determine IC₅₀ values. Species identification confirmed all four CL isolates as L. tropica, and the XTT assay provided the widest absorbance range in RPMIØRP media. IC₅₀ values for both treatment-responsive and unresponsive isolates were similar to those of the reference isolate, showing susceptibility to all tested drugs without statistically significant differences. Expanding the isolate set is necessary to further evaluate the predictive value of SbV (pentavalent antimonials) susceptibility for treatment outcomes. The identification of XTT as the most sensitive method for intracellular antileishmanial susceptibility testing is expected to aid in standardizing laboratory models and provide valuable insights for researchers and clinicians managing treatment-unresponsive CL cases. 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To this end, the efficacy of four colorimetric methods—MTT, XTT, MTS, and WST-8—was compared using reference L. tropica promastigotes. The intracellular drug susceptibility was further evaluated using the test with the widest absorbance range on isolates from Türkiye CL patients: two responsive to a single course of meglumine antimoniate (MA) and two that showed no clinical improvement after two treatments. CL isolates were identified via real-time PCR targeting the ITS1 region. Promastigote suspensions at standardized densities (0.08 × 106 to 10 × 106 promastigotes/well) were prepared in both RPMI (phenol red-containing) and RPMIØRP (phenol red-free) media, then analyzed with ELISA-based MTT, XTT, MTS, and WST-8 to identify the method with the broadest specific absorbance range. Intracellular drug susceptibility of CL isolates was subsequently assessed in a macrophage/amastigote model by infecting THP-1 macrophages with promastigotes from both reference and patient isolates, followed by treatment with MA, sodium stibogluconate (SSG), miltefosine (MTF), pentamidine (PMD), and amphotericin B (AmB). Promastigotes obtained from parasite rescue and transformation assays were analyzed using the most sensitive colorimetric method to determine IC₅₀ values. Species identification confirmed all four CL isolates as L. tropica, and the XTT assay provided the widest absorbance range in RPMIØRP media. IC₅₀ values for both treatment-responsive and unresponsive isolates were similar to those of the reference isolate, showing susceptibility to all tested drugs without statistically significant differences. Expanding the isolate set is necessary to further evaluate the predictive value of SbV (pentavalent antimonials) susceptibility for treatment outcomes. The identification of XTT as the most sensitive method for intracellular antileishmanial susceptibility testing is expected to aid in standardizing laboratory models and provide valuable insights for researchers and clinicians managing treatment-unresponsive CL cases. [Display omitted] •This study focuses on identifying the most sensitive colorimetric test (MTT, XTT, MTS, and WST-8) for evaluating intracellular antileishmanial drug susceptibility.•Four Leishmania spp. isolates from cutaneous leishmaniasis patients in Türkiye were identified as Leishmania tropica through real-time PCR.•Compared to RPMI (phenol red-containing), RPMIØRP (phenol red-free) medium showed a wider specific absorbance range.•The XTT test demonstrated the broadest specific absorbance range with increasing concentrations of promastigote suspensions in RPMIØRP medium.•The XTT test was the most effective colorimetric assay for assessing intracellular antileishmanial drug susceptibility of L. tropica promastigotes recovered from macrophage/amastigote models using conventional antileishmanial drugs.</description><subject>Colorimetric assays</subject><subject>Cutaneous leishmaniasis</subject><subject>Drug susceptibility</subject><subject>In vitro</subject><subject>Leishmania tropica</subject><issn>1383-5769</issn><issn>1873-0329</issn><issn>1873-0329</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2025</creationdate><recordtype>article</recordtype><recordid>eNp9kU2P0zAQhiMEYpeFf4CQj1xS_JHG9gVpVS0fUiUucLZcZ1JcOXbwOJX6Z_ituGTZI6eZsZ55X43fpnnL6IZR1n84bWabfSwbTnlXnwTl7Flzy5QULRVcP6-9UKLdyl7fNK8QT5SyrZTsZXMjtGSad_K2-b1LU9WxxZ-B3CMC4gSxkDQSl0LKfoKSvSMW0V6Q-EjgbMNS-XisU8nWQQhLsJkMeTkSXNDBXPzBB18uV5k9ePw52egtKTnN3llij9ZHLNUhnquZT9EGYmsTntjwVw5fNy9GGxDePNa75senh--7L-3-2-evu_t963jHSst6obQQvR6ZUkIO1FE9bunIqD4oECPTgwK7VXTb0_HQdYPtBsU4F5ICV5KKu-b9qjvn9GsBLGbyeL3MRkgLGsE6TSXlXFe0W1GXE2KG0cz1l2y-GEbNNRlzMmsy5pqMWZOpa-8eHZbDBMPT0r8oKvBxBaDeefaQDToP0cHgM7hihuT_7_AHgOulag</recordid><startdate>20250601</startdate><enddate>20250601</enddate><creator>Yıldırım, Ahmet</creator><creator>Aksoy, Tülay</creator><creator>Balcıoğlu, İbrahim Cüneyt</creator><general>Elsevier B.V</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20250601</creationdate><title>Comparative Assessment of colorimetric assays in evaluating intracellular drug susceptibility of Leishmania tropica against conventional antileishmanial drugs</title><author>Yıldırım, Ahmet ; Aksoy, Tülay ; Balcıoğlu, İbrahim Cüneyt</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c241t-163893369f18837d0c09f50f109b8e3f19d8ea580560fb44da4d8122370e28703</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2025</creationdate><topic>Colorimetric assays</topic><topic>Cutaneous leishmaniasis</topic><topic>Drug susceptibility</topic><topic>In vitro</topic><topic>Leishmania tropica</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Yıldırım, Ahmet</creatorcontrib><creatorcontrib>Aksoy, Tülay</creatorcontrib><creatorcontrib>Balcıoğlu, İbrahim Cüneyt</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Parasitology international</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Yıldırım, Ahmet</au><au>Aksoy, Tülay</au><au>Balcıoğlu, İbrahim Cüneyt</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparative Assessment of colorimetric assays in evaluating intracellular drug susceptibility of Leishmania tropica against conventional antileishmanial drugs</atitle><jtitle>Parasitology international</jtitle><addtitle>Parasitol Int</addtitle><date>2025-06-01</date><risdate>2025</risdate><volume>106</volume><spage>103021</spage><pages>103021-</pages><artnum>103021</artnum><issn>1383-5769</issn><issn>1873-0329</issn><eissn>1873-0329</eissn><abstract>This study aims to identify the most sensitive colorimetric test for assessing intracellular drug susceptibility of Leishmania tropica to conventional antileishmanial drugs. To this end, the efficacy of four colorimetric methods—MTT, XTT, MTS, and WST-8—was compared using reference L. tropica promastigotes. The intracellular drug susceptibility was further evaluated using the test with the widest absorbance range on isolates from Türkiye CL patients: two responsive to a single course of meglumine antimoniate (MA) and two that showed no clinical improvement after two treatments. CL isolates were identified via real-time PCR targeting the ITS1 region. Promastigote suspensions at standardized densities (0.08 × 106 to 10 × 106 promastigotes/well) were prepared in both RPMI (phenol red-containing) and RPMIØRP (phenol red-free) media, then analyzed with ELISA-based MTT, XTT, MTS, and WST-8 to identify the method with the broadest specific absorbance range. Intracellular drug susceptibility of CL isolates was subsequently assessed in a macrophage/amastigote model by infecting THP-1 macrophages with promastigotes from both reference and patient isolates, followed by treatment with MA, sodium stibogluconate (SSG), miltefosine (MTF), pentamidine (PMD), and amphotericin B (AmB). Promastigotes obtained from parasite rescue and transformation assays were analyzed using the most sensitive colorimetric method to determine IC₅₀ values. Species identification confirmed all four CL isolates as L. tropica, and the XTT assay provided the widest absorbance range in RPMIØRP media. IC₅₀ values for both treatment-responsive and unresponsive isolates were similar to those of the reference isolate, showing susceptibility to all tested drugs without statistically significant differences. Expanding the isolate set is necessary to further evaluate the predictive value of SbV (pentavalent antimonials) susceptibility for treatment outcomes. The identification of XTT as the most sensitive method for intracellular antileishmanial susceptibility testing is expected to aid in standardizing laboratory models and provide valuable insights for researchers and clinicians managing treatment-unresponsive CL cases. [Display omitted] •This study focuses on identifying the most sensitive colorimetric test (MTT, XTT, MTS, and WST-8) for evaluating intracellular antileishmanial drug susceptibility.•Four Leishmania spp. isolates from cutaneous leishmaniasis patients in Türkiye were identified as Leishmania tropica through real-time PCR.•Compared to RPMI (phenol red-containing), RPMIØRP (phenol red-free) medium showed a wider specific absorbance range.•The XTT test demonstrated the broadest specific absorbance range with increasing concentrations of promastigote suspensions in RPMIØRP medium.•The XTT test was the most effective colorimetric assay for assessing intracellular antileishmanial drug susceptibility of L. tropica promastigotes recovered from macrophage/amastigote models using conventional antileishmanial drugs.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>39719247</pmid><doi>10.1016/j.parint.2024.103021</doi></addata></record>
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subjects Colorimetric assays
Cutaneous leishmaniasis
Drug susceptibility
In vitro
Leishmania tropica
title Comparative Assessment of colorimetric assays in evaluating intracellular drug susceptibility of Leishmania tropica against conventional antileishmanial drugs
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