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Journal of Clinical Microbiology, August 1998, p. 2173-2177, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Diagnosis of Kala-Azar by Nested PCR Based on Amplification of the Leishmania Mini-Exon Gene

Ken Katakura,1,* Shin-Ichiro Kawazu,2 Toshimitsu Naya,3 Koichi Nagakura,4 Mamoru Ito,5 Masamichi Aikawa,6 Jing-Qi Qu,7 Li-Ren Guan,7 Xin-Pin Zuo,8 Jun-Jie Chai,8 K.-P. Chang,9 and Yoshitsugu Matsumoto3

Department of Tropical Medicine, Jikei University School of Medicine,1 and Department of Molecular Immunology, University of Tokyo,3, Tokyo, National Institute of Animal Health, Tsukuba,2 Department of Infectious Diseases, Tokai University School of Medicine,4 and Research Institute of Medical Sciences, Tokai University,6 Isehara, and Laboratory of Immunology, Central Institute for Experimental Animal, Kawasaki,5 Japan; Institute of Parasitic Diseases, Shanghai,7 and Xinjiang Institute for Endemic Diseases Control and Research, Urumqi,8 China; and Department of Microbiology/Immunology, University of Health Sciences/Chicago Medical School, North Chicago, Illinois9

Received 10 December 1997/Returned for modification 2 March 1998/Accepted 28 April 1998

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.

* Corresponding author. Mailing address: Department of Tropical Medicine, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105, Japan. Phone: 81-3-3433-1111, ext. 2286. Fax: 81-3-3431-4459. E-mail: j30714{at}m-unix.cc.u-tokyo.ac.jp.

Journal of Clinical Microbiology, August 1998, p. 2173-2177, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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