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COLORIMETRIC ASSAY FOR SCREENING COMPOUNDS AGAINST LEISHMANIA AMASTIGOTES GROWN IN MACROPHAGES

An estimated 12 million persons throughout the world suffer from the protozoan disease leishmaniasis. Current treatments have liabilities including poor activity against some forms of leishmaniasis, toxicity, or the need for parenteral administration. Higher throughput methods to screen chemical compounds are needed to facilitate the search for new antileishmania drugs. In the mammalian host, Leishmania parasites exist as amastigotes that replicate within macrophages. Therefore, an in vitro screening assay using intramacrophage amastigotes most closely represents the natural infection. We have transfected strains of Leishmania major and Leishmania amazonensis with the ß-lactamase gene, which catalyzes a colorimetric reaction with the substrate nitrocephin. The growth of these ß-lactamase–expressing Leishmania within macrophages was quantified in 96-well plates using an optical density plate reader, thus simplifying the methodology for scoring inhibitor assays. This simple and relatively inexpensive colorimetric assay helps improve throughput for screening compounds for antileishmania activity.

Received June 24, 2004. Accepted for publication November 11, 2004.

Acknowledgments: The authors thank Dr. Stephen Beverley for providing the Leishmania expression vector, pIR1SAT. The authors thank Kendra Aikenhead and Robert Gillespie for technical assistance.

Financial support: This work was supported by a grant from the Keck Center for Microbial Pathogens at University of Washington (funded by the Keck Foundation).

Authors’ addresses: Frederick S. Buckner and Aaron J. Wilson, Division of Allergy and Infectious Diseases, Health Sciences Building I104E, Box 357185, 1959 N.E. Pacific St., University of Washington, Seattle, WA 98195-7185. Telephone: 206-616-9214, Fax: 206-685-8681, E-mail: fbuckner{at}u.washington.edu.

Reprint requests: Frederick S. Buckner, Division of Allergy and Infectious Diseases, Box 357185, University of Washington, Seattle, WA 98195-7185.

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