Abstract
Ever since their discovery about 60 years ago as therapeutic agent for visceral leishmaniasis (VL) or kala-azar, pentavalent antimonials (Sbv) have remained the first line treatment of choice all over the world including India. But recently, the number of kala-azar patients unresponsive to sodium stibogluconate (SSG) therapy, is steadily increasing in India. In this study, three clinical isolates, of which two were from SSG unresponsive and one from SSG responsive patients were evaluated for their infectivity and for their chemotherapeutic responses in vitro (macrophage–amastigote system) and in vivo (in hamsters). Persistence of SSG resistance was also checked by repeated passages in vitro as well as in vivo. The drug resistant strains (2039 and 2041) did not respond to SSG therapy both in vitro as well as in vivo but strains 2001 and Dd8 showed full sensitivity to SSG treatment. All the four strains responded well to amphotericin B and miltefosine treatment both in macrophages and in hamsters. The specific chemotherapeutic responses of all the strains to SSG were consistently persistent after repeated passages in cultures and in vivo, which indicates that these isolates are truly refractory to SSG treatment in field conditions. Two isolates were also transfected with green fluorescent protein (GFP) for the development of in vitro assay for studying antileishmanial activities of new and reference drugs in macrophages by flow cytometry.
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References
Berman JD (1997) Human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years. Clin Infect Dis 24:684–703
Berman JD, Dwyer DM, Weyler DJ (1979) Multiplication of Leishmania in human macrophages in vitro. Infect Immun 26:375–379
Bhattacharyya A, Mukherjee M, Duttagupta S (2002) Studies on stibanate unresponsive isolates of Leishmania donovani. J Biosci 27:503–508
Bora D (1999) Epidemiology of visceral leishmaniasis in India. Natl Med J India 12:62–68
Chan MM, Bulinski JC, Chang KP, Fong D (2003) A microplate assay for Leishmania amazonensis promastigotes expressing multimeric green fluorescent protein. Parasitol Res 89:266–271
Croft SL (2001) Monitoring drug resistance in leishmaniasis. Trop Med Int Health 6:1–7
Kamau SW, Grimm F, Hehl AB (2001) Expression of green fluorescent protein as a marker for effects of antileishmanial compounds in vitro. Antimicrob Agents Chemother 45:3654–3656
Lira R, Sundar S, Makharia A, Keney R, Gam A, Saravia E, Sacks D (1999) Evidence that the high incidence of treatment failure in Indian kala-azar is due to the emergence of antimony resistant strains of Leishmania donovani. J Infect Dis 180:564–567
Melby PC, Chandrasekar B, Zhao W, Coe JE (2001) The hamster as a model of human visceral leishmaniasis: progressive disease and impaired generation of nitric oxide in the face of a prominent Th1-like cytokine response. J Immunol 166:1912–1920
Murray HW (2001) Clinical and experimental advances in treatment of visceral leishmaniasis. Antimicrob Agents Chemother 45:2185–2197
Neal RA, Croft SL (1984) An in vitro system for determining the activity of compounds against the intracellular amastigote form of Leishmania donovani. J Antimicrob Chemther 14:463–475
Plock A, Sokolowska-Kohler W, Presber W (2001) Application of flow cytometry and microscopical methods to characterize the effects of herbal drugs on Leishmania spp. Exp Parasitol 97:141–153
Seifert K, Matu S, Perez-Victoria FJ, Castanys S, Gamarro F, Croft SL (2003) Characterization of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine). Int J Antimicrob Agents 22:380–387
Sereno D, Holzmuller P, Lemesre JL (2000) Efficacy of second line drugs on antimonyl-resistant amastigotes of Leishmania infantum. Acta Trop 74:25–31
Singh N (2002) Is there true Sb(V) resistance in Indian Kala azar field isoslates? Current Sci 83:101–102
Singh N, Dube A (2004) Fluorescent Leishmania: applications to antileishmanial drug testing. Am J Trop Med Hyg 71:400–402
Singh N, Singh RT, Sundar S (2002) Identification of a gene linked to drug resistance in field isolates of Leishmania donovani. Ann Trop Med Parasitol 96:839–841
Sundar S (2001) Drug resistance in Indian visceral leishmaniasis. Trop Med Int Health 6:849–854
Sundar S, More DK, Singh MK, Singh VP, Sharma S, Makharia A, Kumar PC, Murray HW (2000) Failure of pentavalent antimony in visceral leishmaniasis in India: report from the center of Indian epidemic. Clin Infect Dis 31:1104–1107
Thakur CP, Pandey AK, Kumar N, Kumar P, Hassan S, Narain S, Roy RK (1998) Do the diminishing efficacy and increasing toxicity of sodium stibogluconate in the treatment of visceral leishmaniasis in Bihar, India, justify its continued use as a first-line drug? An observational study of 80 cases. Ann Trop Med Parasitol 92:561–569
Ullman B, Carrero-Valenzuela E, Coons T (1989) Leishmania donovani: isolation and characterization of sodium stibogluconate (Pentostam) resistant cell lines. Exp Parasitol 40:159–163
World Health organization, http://www.who.int/mc/diseases/leish/index.html
Acknowledgements
We are thankful to the patients and staff of Kala-azar Research Center, Muzaffarpur for their kind consent and cooperation. We express our gratefulness to Dr. Shailja Misra-Bhattacharya, Sh. A. L. Vishwakarma for extending fluorescence microscope and FACS facility, respectively. Financial assistance to NS by the Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged. This is CDRI communication No. 6677.
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Dube, A., Singh, N., Sundar, S. et al. Refractoriness to the treatment of sodium stibogluconate in Indian kala-azar field isolates persist in in vitro and in vivo experimental models. Parasitol Res 96, 216–223 (2005). https://doi.org/10.1007/s00436-005-1339-1
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DOI: https://doi.org/10.1007/s00436-005-1339-1