HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Castilho, T. M. Articles by Floeter-Winter, L. M. Search for Related Content PubMed PubMed Citation Articles by Castilho, T. M. Articles by Floeter-Winter, L. M. Pubmed/NCBI databases Nucleotide Protein Substance via MeSH Medline Plus Health Information Leishmaniasis Related Collections Leishmaniasis

A Real-Time Polymerase Chain Reaction Assay for the Identification and Quantification of American Leishmania Species on the Basis of Glucose-6-Phosphate Dehydrogenase

A real-time polymerase chain reaction (PCR) test was developed on the basis of the Leishmania glucose-6-phosphate dehydrogenase locus that enables identification and quantification of parasites. Using two independent pairs of primers in SYBR-Green assays, the test identified etiologic agents of cutaneous leishmaniasis belonging to both subgenera, Leishmania (Viannia) and Leishmania (Leishmania) in the Americas. Furthermore, use of TaqMan probes enables distinction between L. (V.) braziliensis or L. (V.) peruviania from the other L. (Viannia) species. All assays were negative with DNA of related trypanosomatids, humans, and mice. The parasite burden was estimated by normalizing the number of organisms per total amount of DNA in the sample or per host glyceraldehyde-3-phosphate dehydrogenase copies. The real-time PCR assay for L. (Leishmania) subgenus showed a good linear correlation with quantification on the basis of a limiting dilution assay in experimentally infected mice. The test successfully identifies and quantifies Leishmania in human biopsy specimens and represents a new tool to study leishmaniasis.

Received June 21, 2007. Accepted for publication October 17, 2007.

Acknowledgments: We thank Professor L. Alexander for use of the ABI PRISM^® 7000 Sequence Detection System, Professor A. M. Castrucci and M. A. Visconti for use of the iCycler iQ Real-Time PCR Detection System, Professor R. Bucala for providing human DNA from healthy donors, and Ricardo A. Zampieri and Karen J. Goldsmith-Pestana for technical assistance.

Financial support: This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, Fundaçao de Amparo à Pesquisa do Estado de São Paulo, and National Institutes of Health grants AI-27811 and U19 AI065866.

^* Address correspondence to Lucile M. Floeter-Winter, Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo; Rua do Matão, Travessa 14, no. 101, Cidade Universitária, São Paulo, São Paulo, Brazil, CEP 05508-900. E-mail: lucile{at}ib.usp.br

Authors’ addresses: Tiago M. Castilho and Diane McMahon-Pratt, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06510-8034, E-mail: tiago.castilho{at}yale.edu. Luís Marcelo Aranha Camargo and Jeffrey Jon Shaw, Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av.Professor Lineu Prestes 1374, 005508-000 São Paulo, São Paulo, Brazil, E-mails: spider{at}icb5usp.med.br and jeffreyj{at}usp.br. Lucile M. Floeter-Winter, Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo; Rua do Matão, Travessa 14, no. 101, Cidade Universitária, São Paulo, São Paulo, Brazil, CEP 05508-900. Telephone and Fax: 55-11-3091-7503, E-mail: lucile{at}ib.usp.br.