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Journal of Clinical Microbiology, November 2004, p. 5249-5255, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5249-5255.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity

Charles Mary,* Françoise Faraut, Laurie Lascombe, and Henri Dumon

Laboratoire de Parasitologie, Hopital de la Timone, Marseille, France

Received 6 April 2004/ Returned for modification 8 June 2004/ Accepted 2 August 2004

A real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to reach a sensitivity of 0.0125 parasites/ml of blood. In order to analyze the incidence of heterogeneity and number of minicircles, we performed comparative PCR by using the Leishmania DNA polymerase gene as a reporter. Assays performed in both promastigote and amastigote stages showed variations among different L. infantum and Leishmania donovani strains and the stability of the minicircle numbers for a particular strain. Analysis of blood samples from a patient who presented with Mediterranean visceral leishmaniasis confirmed the reliability of such an assay for Leishmania quantification in biological samples and allowed an estimation of positivity thresholds of classical tests used for direct diagnosis of the disease; positivity thresholds were in the range of 18 to 42, 0.7 to 42, and 0.12 to 22.5 parasites/ml for microscopic examination, culture, and conventional PCR, respectively. At the time of diagnosis, parasitemia could vary by a wide range (32 to 188,700 parasites/ml, with a median of 837 parasites/ml), while in bone marrow, parasite load was more than 100 parasites per 106 nucleated human cells. After successful therapy, parasitemia levels remain lower than 1 parasite/ml. In the immunocompromised host, relapses correlate with an increase in the level of parasitemia, sometimes scanty, justifying the need for assays with high sensitivity. Such sensitivity allows the detection of Leishmania DNA in the blood of 21% of patients with no history of leishmaniasis living in the Marseilles area, where leishmaniasis is endemic. This technique may be useful for epidemiologic and diagnostic purposes, especially for the quantification of parasitemia at low levels during posttherapy follow-up.

* Corresponding author. Mailing address: Laboratoire de Parasitologie, Hopital de la Timone, 266 rue Saint Pierre, 13385 Marseille, France. Phone: 33 (0) 4 91 38 49 55. Fax: 33 (0) 4 91 38 49 58. E-mail: cmary{at}ap-hm.fr.

Journal of Clinical Microbiology, November 2004, p. 5249-5255, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5249-5255.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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