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Dec 13, 2012 - Abstract Simple, cost-effective approach for routine sur- veillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly ...
Parasitol Res (2013) 112:825–828 DOI 10.1007/s00436-012-3212-3

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 (Sundar and Rai 2002; Murray 2010) and PKDL therapy (Ramesh et al. 2011). Routine surveillance of MIL susceptibility in prevailing field isolates is necessary to ascertain the clinical life of this drug. For many antileishmanial drugs such as antimonials, pentamidine and amphotericin B, in vitro amastigote susceptibility assays using macrophage infection model are

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required as extracellular promastigotes are intrinsically tolerant to the drug (Lira et al. 1999; Singh et al. 2006) or depict poor/partial correlation with amastigote drug susceptibility (Vermeersch et al. 2009). Unfortunately, the amastigote system is labor intensive, hereby hampering the close surveillance of drug susceptibility in the field. Additionally, lack of assay reproducibility and variations are also reported to occur due to use of different cell lines (Seifert et al. 2010). A simplified biological assay for MIL susceptibility monitoring is therefore a pressing need. In the current study, we validated a simplified colorimetric assay measuring parasite susceptibility to MIL using promastigotes for routine monitoring of MIL susceptibility. The assay depicted concordance with the standard intracellular amastigote assay, in two different labs in India. The performance of MIL susceptibility assays was assessed at distinct parasite stages in clinical isolates of VL and PKDL as well as in MIL-induced parasites with high MIL tolerance. The study establishes a simplified biological tool applicable for surveillance of MIL susceptibility in clinical isolates from MIL-treated cases (including failures and relapses) to track MIL resistance.

Parasitol Res (2013) 112:825–828

Preparation of drug stocks MIL (batch 1149149) stock solution (20 mM) was prepared in water, filter sterilized and stored at −20 °C until use. MIL stock solution was serially diluted freshly over six concentrations in respective media with 10 % FBS and penicillin (100 U/ml)/ streptomycin (100 μg/ml). Drug susceptibility assay at intracellular amastigote stage The drug susceptibility assays were performed in two different labs, at the National Institute of Pathology, ICMR, New Delhi, India [referred to as Lab 1] and the Indian Institute of Chemical Biology (IICB), CSIR, Kolkata, India [referred to as Lab 2]. The amastigote drug susceptibility was assessed in Lab 1 as described previously using murine macrophage cell line J774A.1 (Kumar et al. 2009). Mouse peritoneal macrophages (PECs) were used in Lab 2 for amastigote susceptibility monitoring as described previously (Mukhopadhyay et al. 2011). Drug susceptibility assay at promastigote stage

Materials and methods Materials M199 medium, RPMI-1640 medium, penicillin, streptomycin, soluble starch and resazurin were purchased from Sigma; L-glutamine was supplied by Invitrogen, USA. Fetal bovine serum (FBS) was purchased from Gibco, Grand Island, NY, USA, 16-well chamber slides from Nunc, Rochester, NY, USA, and Diff-Quik® stain solution obtained from Dade Behring, Newark, DE, USA. Parasite culture Clinical isolates of L. donovani were prepared from splenic aspirates of VL patients (n017) reporting to the Kala-azar Medical Research Center (KAMRC), Muzaffarpur, Bihar, or from dermal lesions of PKDL patients (n0 7) reporting to the Department of Dermatology, Safdarjung Hospital, New Delhi, following the guidelines of the ethical committee of the respective institute. The promastigotes were maintained in vitro in M199 medium with 10 % FBS at 25 °C, and clonal populations were generated as described previously (Mukhopadhyay et al. 2011). One VL isolate (BHU 573 clone-3) was made resistant to MIL (12, 37 and 49 μM MIL) as described elsewhere (Seifert et al. 2003). L. donovani BPK 206/0 cl-10 was employed as internal control between different experiments.

Resazurin dye/AlamarBlue® (7-Hydroxy-3H-phenoxazin3-one 10-oxide) was employed for promastigote susceptibility testing. Resazurin is a redox indicator that converts to fluorescent and colorimetric, resorufin dye by the metabolically active cells (Toté et al. 2009). Assays were performed in sterile 96-well plates using late log-phase promastigotes (105/well), containing MIL concentrations ranging from 0.4 μM to 390 μM. After 72-h incubation at 25 °C, 50 μl resazurin [0.0125 % (w/v in PBS)] was added, and plates were incubated for a further 24 h. In Lab 1, cell viability was measured fluorometrically (λex 550 nm; λem 590 nm) on Infinite M200 multimode reader (Tecan). The experiments were repeated at least twice in quadruplicates. In Lab 2, cells were incubated with AlamarBlue® for 4 h at 37 °C, and OD was measured at 570 nm. The assay was performed in triplicate, and the results were expressed as percentage reduction in the parasite viability compared to untreated control wells. Fifty percent inhibitory concentration (IC50) was calculated by sigmoidal regression analysis in both labs. Statistical analysis IC50 was calculated using Microcal Origin 6.0 software. Spearman’s rank correlation coefficient was calculated to determine the correlation between the in vitro sensitivities of promastigotes and amastigotes and to determine the correlation between the promastigote assays in two labs.

Parasitol Res (2013) 112:825–828

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Fig. 1 Inter-stage miltefosine susceptibility of Leishmania donovani parasites from VL cases. Scatter plot showing the IC50 values of promastigote vs amastigote susceptibility: a Lab 1 (n020), b Lab 2 (n017)

Correlation was considered significant if r≥0.70. Statistical significance was accepted as a P value of