title: Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in clinical isolates of Leishmania donovani.
abstract: Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases (n = 17) against standard amastigote assay, in two different labs in India. The inter-stage MIL susceptibility correlated strongly (r = 0.70, p = 0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation (r = 0.72, p = 0.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions (n = 7, r = 0.78, p = 0.046) and MIL-induced parasites (r = 0.92, p = 0.0001; n = 3) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patients.
authors.affiliation: National Institute of Pathology, Indian Council of Medical Research, Safdarjung Hospital Campus, New Delhi 110029, India.
authors.forename: Arpita
authors.initials: A
authors.lastname: Kulshrestha
authors.forename: Vasundhra
authors.initials: V
authors.lastname: Bhandari
authors.forename: Rupkatha
authors.initials: R
authors.lastname: Mukhopadhyay
authors.forename: V
authors.initials: V
authors.lastname: Ramesh
authors.forename: Shyam
authors.initials: S
authors.lastname: Sundar
authors.forename: Louis
authors.initials: L
authors.lastname: Maes
authors.forename: Jean Claude
authors.initials: JC
authors.lastname: Dujardin
authors.forename: Syamal
authors.initials: S
authors.lastname: Roy
authors.forename: Poonam
authors.initials: P
authors.lastname: Salotra
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002460:Cell Line; D005470:Fluorometry / Q000379:methods*; D007194:India; D007893:Leishmania donovani / Q000187:drug effects*; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D010078:Oxazines / Q000378:metabolism; D021261:Parasitic Sensitivity Tests / Q000191:economics / Q000379:methods; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000981:Antiprotozoal Agents; D010078:Oxazines; D014966:Xanthenes; D010767:Phosphorylcholine; C005843:resazurin; C039128:miltefosine