Detection, Differentiation, and Quantitation of Pathogenic Leishmania ...

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2003, p. 1529–1535 0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.4.1529–1535.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 41, No. 4

Detection, Differentiation, and Quantitation of Pathogenic Leishmania Organisms by a Fluorescence Resonance Energy Transfer-Based Real-Time PCR Assay Alexandra Schulz,1 Katja Mellenthin,2 Gabriele Scho ¨nian,3 Bernhard Fleischer,1 1 and Christian Drosten * National Reference Center for Tropical Infections1 and Leishmaniasis Group,2 Bernhard Nocht Institute for Tropical Medicine, Hamburg, and Institute of Microbiology and Hygiene, Humboldt University, Charite´, Berlin,3 Germany Received 11 October 2002/Returned for modification 31 December 2002/Accepted 13 January 2003

Real-time technology eliminates many of the pitfalls of diagnostic PCR, but this method has not been applied to differentiation of Leishmania organisms so far. We have developed a real-time PCR that simultaneously detects, quantitates, and categorizes Leishmania organisms into three relevant groups causing distinct clinical pictures. The analytical sensitivity (detection rate of >95% at 94.1 parasites/ml of blood) was within a range that has been determined previously to facilitate the confirmation of visceral leishmaniasis from peripheral blood. Parasites were successfully detected in 12 different clinical samples (blood, bone marrow, skin, and liver). The Leishmania donovani complex, the Leishmania brasiliensis complex, and species other than these could be clearly discriminated by means of distinct melting temperatures obtained with fluorescence resonance energy transfer probes (melting points, 72.7, 67.1, and 65.0°C, respectively). All three groups could be quantified within equal ranges. As in other real-time PCRs, the variability in the quantification of DNA was small (coefficient of variation [CV],