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Am. J. Trop. Med. Hyg., 73(6), 2005, pp. 999-1004
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

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RAPID IDENTIFICATION OF LEISHMANIA COMPLEXES BY A REAL-TIME PCR ASSAY

GLENN WORTMANN*, LISA HOCHBERG, HUO-HSU HOUNG, COLLEEN SWEENEY, MICHAEL ZAPOR, NAOMI ARONSON, PETER WEINA, AND CHRISTIAN F. OCKENHOUSE
Infectious Disease Service, Walter Reed Army Medical Center, Washington, DC; Department of Entomology, Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Viral Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland; Infectious Disease Division, Uniformed Services University of the Health Sciences, Bethesda, Maryland; Department of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland

A real-time PCR assay for the detection of four Leishmania complexes (L. Viannia, L. mexicana, L. donovani/infantum, and L. major) was developed and evaluated. The assay was developed to detect the glucosephosphate isomerase gene and capitalizes on DNA sequence variability within that gene for Leishmania complex identification. Primer/probe sets were created and tested against a panel of 21 known negative controls and on DNA extracted from cultured promastigotes or from tissue biopsies from patients with cutaneous leishmaniasis. The assay was highly specific, as no amplification products were detected in the negative control samples while simultaneously retaining a high degree of complex-specific diagnostic accuracy for cultured organisms and patient clinical samples. Real-time PCR offers rapid (within hours) identification of Leishmania to the complex level and provides a useful molecular tool to assist both epidemiologists and clinicians.

Received October 9, 2004. Accepted for publication August 17, 2005.

Acknowledgments: Byran A. Arana, Niddia R. Rizzo, Flora Arana, Medical Entomology Research and Training Unit, Universidad del Valle de Guatemala, Guatemala City, Guatemala, for providing the Guatemalan clinical samples. Ron Neafie, Armed Forces Institute of Pathology, Washington, DC, for providing the negative control samples. Jeff Ryan, Col (ret), U.S. Army, Department of Entomology, WRAIR, for support and guidance.

Disclaimer: The views expressed here are those of the authors and should not be construed to reflect the views of the United States government.

* Address correspondence to Glenn Wortmann, 6900 Georgia Ave., NW, Walter Reed Army Medical Center, Washington, DC 20307-5001. E-mail: glenn.wortmann{at}na.amedd.army.mil

Authors’ addresses: Lisa Hochberg, Huo-hsu Houng, Colleen Sweeney, Peter Weina, and Christian F. Ockenhouse, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910. Glenn Wortmann, Michael Zapor, and Naomi Aronson, Walter Reed Army Medical Center, 6900 Georgia Ave., NW, Washington, DC 20307-5001, E-mail: glenn.wortmann{at}na.amedd.army.mil.

Reprint requests: Glenn Wortmann, Walter Reed Army Medical Center, 6900 Georgia Ave., NW, Washington, DC 20307-5001, Telephone: 202-782-6740, E-mail: glenn.wortmann{at}na.amedd.army.mil.




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