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Journal of Clinical Microbiology, March 2001, p. 849-854, Vol. 39, No. 3
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.3.849-854.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Development of a Species-Specific PCR Assay for Detection of Leishmania donovani in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis

Poonam Salotra,1,* G. Sreenivas,1 Gregory P. Pogue,2,dagger Nancy Lee,2 Hira L. Nakhasi,2 V. Ramesh,3 and N. S. Negi4

Institute of Pathology (ICMR), Safdarjung Hospital Campus,1 and Departments of Dermatology3 and Medicine,4 Safdarjung Hospital, New Delhi 110 029, India, and Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 208522

Received 2 August 2000/Returned for modification 9 October 2000/Accepted 22 December 2000

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.

* Corresponding author. Mailing address: Molecular Biology Lab, Institute of Pathology (ICMR), Safdarjung Hospital Campus, Post Box #4909, New Delhi 110 029, India. Phone: 91-11-6198402. Fax: 91-11-6198401. E-mail: salotra{at}vsnl.com.

dagger Present address: Director, Large Scale Biology Corp., Vacaville, CA 95688.

Journal of Clinical Microbiology, March 2001, p. 849-854, Vol. 39, No. 3
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.3.849-854.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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