Advertisement

Parasitology Research

, Volume 112, Issue 2, pp 825–828 | Cite as

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
Original Paper

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.

Keywords

Clinical Isolate Visceral Leishmaniasis Resazurin Miltefosine Visceral Leishmaniasis Patient 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We are grateful to Dr Robert Vinson from Paladin for kindly providing miltefosine. This work was funded by Kaladrug-R (grant number EC-FP7-222895, www.leishrisk.net/kaladrug).

References

  1. Boelaert M, Criel B, Leeuwenburg J, Van Damme W, Le Ray D, Van der Stuyft P (2000) Visceral leishmaniasis control: a public health perspective. Trans R Soc Trop Med Hyg 94:465–471PubMedCrossRefGoogle Scholar
  2. Kumar D, Kulshrestha A, Singh R, Salotra P (2009) In vitro susceptibility of field isolates of Leishmania donovani to MIL and amphotericin B: correlation with sodium antimony gluconate susceptibility and implications for treatment in areas of endemicity. Antimicrob Agents Chemother 53:835–838PubMedCrossRefGoogle Scholar
  3. Lira R, Sundar S, Makharia A, Kenney R, Gam A, Saraiva E, Sacks D (1999) Evidence that the high incidence of treatment failures in Indian kala-azar is due to the emergence of antimony-resistant strains of Leishmania donovani. J Infect Dis 180:564–567PubMedCrossRefGoogle Scholar
  4. Mikus J, Steverding D (2000) A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. ParasitolInt 48:265–269Google Scholar
  5. Mukhopadhyay R, Mukherjee S, Mukherjee B, Naskar K, Mondal D, Decuypere S, Ostyn B, Prajapati VK, Sundar S, Dujardin JC, Roy S (2011) Characterisation of antimony-resistant Leishmania donovani isolates: biochemical and biophysical studies and interaction with host cells. Int J Parasitol 41:1311–1321PubMedCrossRefGoogle Scholar
  6. Murray HW (2010) Treatment of visceral leishmaniasis in 2010: direction from Bihar State, India. Future Microbiol 5:1301–1303PubMedCrossRefGoogle Scholar
  7. Murray HW, Berman JD, Davies CR, Saravia NG (2005) Advances in leishmaniasis. Lancet 366:1561–1577PubMedCrossRefGoogle Scholar
  8. Pandey BD, Pandey K, Kaneko O, Yanagi T, Hirayama K (2009) Relapse of visceral leishmaniasis after miltefosine treatment in a Nepalese patient. AmJTrop Med Hyg 80:580–582Google Scholar
  9. Rakotomanga M, Blanc S, Gaudin K, Chaminade P, Loiseau PM (2007) Miltefosine affects lipid metabolism in Leishmania donovani promastigotes. Antimicrob Agents Chemother 51:1425–1430PubMedCrossRefGoogle Scholar
  10. Ramesh V, Katara GK, Verma S, Salotra P (2011) Miltefosine as an effective choice in the treatment of post-kala-azar dermal leishmaniasis. Br J Dermatol 165:411–414PubMedCrossRefGoogle Scholar
  11. Ramesh V, Mukherjee A (1995) Post-kala-azar dermal leishmaniasis. Int J Dermatol 34:85–91PubMedCrossRefGoogle Scholar
  12. Seifert K, Escobar P, Croft SL (2010) In vitro activity of anti-leishmanial drugs against Leishmania donovani is host cell dependent. J Antimicrob Chemother 65:508–511PubMedCrossRefGoogle Scholar
  13. Seifert K, Matu S, Javier Perez-Victoria F et al (2003) Characterization of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine). Int J Antimicrob Agents 22:380–387PubMedCrossRefGoogle Scholar
  14. Singh R, Kumar D, Ramesh V, Negi NS, Singh S, Salotra P (2006) Visceralleishmaniasis, or kala azar (KA): high incidence of refractoriness to antimony is contributed by anthroponotic transmission via post-KA dermal leishmaniasis. J Infect Dis 194:302–306PubMedCrossRefGoogle Scholar
  15. Sundar S, Rai M (2002) Advances in the treatment of leishmaniasis. CurrOpin Infect Dis 15:593–598CrossRefGoogle Scholar
  16. Toté K, VandenBerghe D, Levecque S, Bénéré E, Maes L, Cos P (2009) Evaluation of hydrogen peroxide-based disinfectants in a new resazurin microplate method for rapid efficacy testing of biocides. J Appl Microbiol 107:606–615PubMedCrossRefGoogle Scholar
  17. Vermeersch M, da Luz RI, Toté K, Timmermans JP, Cos P, Maes L (2009) In vitro susceptibilities of Leishmania donovani promastigote and amastigote stages to antileishmanial reference drugs: practical relevance of stage-specific differences. Antimicrob Agents Chemother 53:3855–3859PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Arpita Kulshrestha
    • 1
  • Vasundhra Bhandari
    • 1
  • Rupkatha Mukhopadhyay
    • 2
  • V. Ramesh
    • 3
  • Shyam Sundar
    • 4
  • Louis Maes
    • 5
  • Jean Claude Dujardin
    • 6
  • Syamal Roy
    • 2
  • Poonam Salotra
    • 1
  1. 1.National Institute of PathologyIndian Council of Medical ResearchNew DelhiIndia
  2. 2.Division of Infectious Diseases and Immunology, Indian Institute of Chemical BiologyCSIRKolkataIndia
  3. 3.Department of DermatologySafdarjung HospitalNew DelhiIndia
  4. 4.Institute of Medical SciencesBanaras Hindu UniversityVaranasiIndia
  5. 5.Lab. of Microbiology, Parasitology and Hygiene (LMPH)University of AntwerpAntwerpBelgium
  6. 6.Unit of Molecular Parasitology, Department of ParasitologyInstitute of Tropical MedicineAntwerpBelgium

Personalised recommendations