EXPERIMENTAL PARASITOLOGY 90, 236–243 (1998) ARTICLE NO. PR984326 Leishmania braziliensis: Characterisation of a Complex Specific Subtelomeric Repeat Sequence and Its Use in the Detection of Parasites 1 Guoliang Fu, Georgia Perona-Wright, and Douglas C. Barker 2 MRC Outstation of NIMR, Molteno Laboratories, Department of Pathology, University of Cambridge, CB2 1QP, U.K. Fu, G., Perona-Wright, G., and Barker, D. C. 1998. Leishmania million people are currently infected and a further 350 mil- braziliensis: Characterisation of a complex specific subtelomeric repeat lion are estimated to be at risk (WHO 1997). There is a broad sequence and its use in the detection of parasites. Experimental Parasi- spectrum of clinical symptoms, largely dependent upon the tology 90, 236–243. A 1.6-kb tandem repeat sequence had previously infecting species. In the New World, Leishmania species of been identified in the subtelomeric region of mini- and megabase chromosomes from Leishmania braziliensis. Southern hybridisation the mexicana complex cause cutaneous or diffuse cutaneous was used to demonstrate that the repeat is complex specific. The leishmaniasis. L. braziliensis complex members are also re- sequence was characterised in strains representing four species of sponsible for cases of mucocutaneous leishmaniasis, a se- the L. braziliensis complex. This data allowed an assessment of the verely disfiguring spread of the parasite from the initial evolutionary relationship of the four species. PCR primers targeted to lesion to mucosal sites. The necrosis of tissue is progressive the repeat amplify only DNA from species of the L. braziliensis com- plex. Titration assays indicate that a minimum of 50 fg of parasite and potentially fatal if untreated. Therapy involves the use DNA can be detected by PCR alone. Southern hybridisation increases of toxic pentavalent antimonials. Rapid and specific diagno- the limit of detection to 5 fg. Interspecies variation in the repeat sis is therefore needed in order to determine appropriate sequence enabled restriction enzyme digestion of PCR products to medical action. distinguish individual species within the L. braziliensis complex. Direct diagnosis of leishmaniasis is achieved by visualising  1998 Academic Press Index Descriptors and Abbreviations: Leishmania braziliensis com- amastigotes in microscopic examination of tissue sample plex; subtelomeric repeat; diagnosis; restriction enzyme digestion; bp, smears or by histological staining of sections. The scarcity base pairs; ELISA, enzyme-linked immunosorbent assay; IFAT, immu- of parasites in cutaneous lesions causes limited sensitivity, nofluorescence antibody testing; kDNA, kinetoplast deoxyribonucleic however (Marsden 1985). Immunological tests such as the acid; PCR, polymerase chain reaction. Montenegro skin test, IFAT, or ELISA cannot distinguish be- tween previous and current infections and show cross-reactiv- ity with other pathogens, including some which resemble Leishmania in their clinical presentation (Manson-Bahr INTRODUCTION 1987). Specific diagnosis requires the culture and characteri- sation of patient isolates using monoclonal antibodies or iso- Leishmaniasis is a parasitic disease of global importance: enzyme analysis. The in vitro culture involved is time consum- it is considered endemic in 88 countries, approximately 12 ing, expensive, and difficult (Wiegle et al. 1987; de Brujin et al. 1993). More recently techniques have been developed 1 The sequence data reported herein has been submitted to GenBank which are based on PCR amplification of parasite DNA. These and assigned the Accession Numbers AF031224, AF043685, are proving highly sensitive and specific in the diagnosis of AF043686, and AF043687. Leishmania infections. Previously described assays involve 2 To whom correspondence should be addressed. Fax: 44 1223 333737. E-mail: dcb12@mole.bio.cam.ac.uk. kinetoplast DNA (Rodgers et al. 1990 and others), ribosomal 236 0014-4894/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.