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AMPLIFICATION OF HUMAN DNA BY PRIMERS TARGETED TO LEISHMANIA KINETOPLAST DNA AND POST-GENOME CONSIDERATIONS IN THE DETECTION OF PARASITES BY A POLYMERASE CHAIN REACTION

We evaluated the Leishmania Viannia-specific primers B1-B2 to detect Leishmania in normal skin and peripheral blood monocytes of patients with active cutaneous leishmaniasis. Southern blotting and sequencing of polymerase chain reaction (PCR) products confirmed the specificity of kinetoplast DNA (kDNA) amplification from tissue fluid from healthy skin, whereas the PCR with monocytes also amplified a human sequence of a size similar (718 basepairs) to the expected kDNA product (750 basepairs), resulting in false-positive results. Although B1 was not homologous to any human DNA sequence, B2 showed homology to a human chromosome 2 intergenic region (AC010878) at positions 35,881-36,599, which are spaced 718 nucleotides apart. Amplification of the human art3fact from monocyte DNA was confirmed using the primer B2 alone. Examination of other primers reported for the PCR of kDNA from various species of Leishmania showed that six of seven were homologous to human DNA sequences. These findings underscore the importance of exploiting sequencing, bioinformatics, and DNA probes to refine molecular amplification techniques and to validate the performance of primers when used for new applications.

Received June 18, 2004. Accepted for publication October 24, 2004.

Acknowledgments: We thank M. Ximena Varona, Horacio Cadena and Dr. Claudia Posso (Centro Internacional de Entrenamiento e Investigaciones Médicas) for provision of samples and clinical data, and Dr. Gareth D. Westrop and Professor Graham H. Coombs (University of Glasgow, Glasgow, United Kingdom) and Carolina J. Ramirez (Centro Internacional de Entrenamiento e Investigaciones Médicas) for facilitating the sequencing of the PCR products. We are also grateful to Dr. Douglas C. Barker (Medical Research Council, University of Cambridge, Cambridge, United Kingdom) for his longstanding collaborative research association in the area of Leishmania molecular diagnostics.

Financial support: This work was supported by program grant no. 059056/Z/99/Z from the Wellcome Trust (United Kingdom) and grant no. 162-2002 from the Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnologia Francisco Jose de Caldas.

Authors’ address: Carolina Vergel, John Walker, and Nancy G. Saravia, Centro Internacional de Entrenamiento e Investigaciones Medicas, Avenida 1a Norte No. 3-03, Cali, Valle, Colombia, Telephone: 57-2-668-2164, Fax: 57-2-667-2989, E-mails: carover{at}cideim.org.co, john_walker{at}cideim.org.co, and saravian{at}cideim.org.co.