Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in cl
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ORIGINAL PAPER
Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in clinical isolates of Leishmania donovani Arpita Kulshrestha & Vasundhra Bhandari & Rupkatha Mukhopadhyay & V. Ramesh & Shyam Sundar & Louis Maes & Jean Claude Dujardin & Syamal Roy & Poonam Salotra
Received: 27 April 2012 / Accepted: 15 November 2012 / Published online: 13 December 2012 # Springer-Verlag Berlin Heidelberg 2012
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 (n017) against standard amastigote assay, in two different labs in India. The interstage MIL susceptibility correlated strongly (r00.70, p0 A. Kulshrestha : V. Bhandari : P. Salotra (*) National Institute of Pathology, Indian Council of Medical Research, Safdarjung Hospital Campus, New Delhi 110029, India e-mail: [email protected] P. Salotra e-mail: [email protected] R. Mukhopadhyay : S. Roy Division of Infectious Diseases and Immunology, Indian Institute of Chemical Biology, CSIR, Kolkata, India V. Ramesh Department of Dermatology, Safdarjung Hospital, New Delhi, India S. Sundar Institute of Medical Sciences, Banaras Hindu University, Varanasi, India L. Maes Lab. of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp, Belgium J. C. Dujardin Unit of Molecular Parasitology, Department of Parasitology, Institute of Tropical Medicine, Antwerp, Belgium
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 00.72, p 00.0012) 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 (n07, r00.78, p00.046) and MIL-induced parasites (r00.92, p00.0001; n03) 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.
Introduction Leishmania donovani is a protozoal pathogen causing visceral leishmaniasis (VL) or kala-azar in the tropics and subtropics including the Indian subcontinent (Boelaert et al. 2000; Murray et al. 2005). Post-kala-azar dermal leishmaniasis (PKDL) is a dermal sequel developing in 5–15 % VL patients in India that constitutes an important parasite reservoir (Ramesh and Mukherjee 1995). With widespread resistance to antimonial therapy, oral antileishmanial drug miltefosine (MIL) has been introduced for VL (Sund
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