title: Detection, differentiation, and quantitation of pathogenic leishmania organisms by a fluorescence resonance energy transfer-based real-time PCR assay.
abstract: 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 >or=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 degrees 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], <2%). However, human samples containing low levels of parasites (100 parasites per ml of blood) showed higher variation (CV, 60.89%). Therefore, despite its superior analytical performance, care must be taken when real-time PCR is utilized for therapy monitoring.
authors.affiliation: National Reference Center for Tropical Infections. Leishmaniasis Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schulz
authors.forename: Katja
authors.initials: K
authors.lastname: Mellenthin
authors.forename: Gabriele
authors.initials: G
authors.lastname: Schönian
authors.forename: Bernhard
authors.initials: B
authors.lastname: Fleischer
authors.forename: Christian
authors.initials: C
authors.lastname: Drosten
mesh_terms: D000818:Animals; D015342:DNA Probes; D016054:DNA, Protozoan / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005838:Genotype; D006801:Humans; D007891:Leishmania / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D007896:Leishmaniasis / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S