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DETECTION AND IDENTIFICATION OF LEISHMANIA SPECIES WITHIN NATURALLY INFECTED SAND FLIES IN THE ANDEAN AREAS OF ECUADOR BY A POLYMERASE CHAIN REACTION

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.

Received April 28, 2004. Accepted for publication August 26, 2004.

We are indebted to Roberto Sud for technical assistance throughout the study; Dr. Ernesto Gutierrez (Minister of Health, Guayaquil, Ecuador) and Dr. Luiggi Martini R. (Vice-Minister of Health, Guayaquil, Ecuador) for coordinating our field activities; and to Drs. Teresa Flor, Jenny Rodriguez, and Yim-Yan Wong Chum (National Institute of Health and Tropical Medicine, Guayaquil, Ecuador) for their support during the laboratory phase of the study.

Financial support: This study was supported by the Ministry of Education, Science, Culture and Sports of Japan (grant no. 10037385, 15590371).

Authors’ addresses: Hirotomo Kato and Hiroyuki Iwata, Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan. Hiroshi Uezato and Shigeo Nonaka, Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0215, Japan. Ken Katakura, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18 Nishi 9, Kita-ku, Sapporo 060-0818, Japan. Manuel Calvopiña, Jorge D. Marco, Paola A. Barroso, Masataka Korenaga, and Yoshihisa Hashiguchi, Department of Parasitology, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan. Eduardo A. Gomez, Departamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica de Guayaquil, PO Box 10833, Guayaquil, Ecuador. Tatsuyuki Mimori, Department of Tumor Genetics and Biology, Graduate School of Medical Science, Kumamoto University, Kumamoto 860-0811, Japan.

Reprint requests: Hirotomo Kato, Department of Veterinary Hygiene, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan Telephone: 81-83-933-5900, Fax: 81-83-933-5899, E-mail: katoh{at}yamaguchi-u.ac.jp.

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