Identification and Differentiation ofLeishmaniaSpecies in Clinical Samples by PCR Amplification of the Miniexon Sequence and Subsequent Restriction Fragment Length Polymorphism Analysis

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Abstract
We recently developed a new PCR-restriction fragment length polymorphism (RFLP)-based assay using the miniexon sequence from the genusLeishmania. Here we report the application of this new genotyping method to naturally infected clinical samples for the differentiation of New and Old WorldLeishmaniaspecies. Of the newly developed assay and four currently applied diagnostic tests (i.e., in vitro cultivation, serology, and two other molecular assays using either the small subunit-internal transcribed spacer sequence or a repetitive genomic sequence), the miniexon assay showed the highest sensitivity, 89.7%, compared to 70.6, 57.1, 51.7, and 79.3%, respectively. Species differentiation was robust and reliable compared with that by two otherLeishmaniagenotyping techniques. The assay provides a valuable tool for the identification ofLeishmaniadirectly from clinical samples and enables determination of the infecting species by a facile technique with high discrimination power. SinceLeishmaniacauses a broad spectrum of diseases distinguished by different parasite and host factors, detection and characterization of the infecting species is crucial for the confirmation of a diagnosis as well as the establishment of the clinical prognosis and the initiation of an adequate therapeutic approach. The miniexon PCR-RFLP assay will facilitate such determination and might improve diagnosis and treatment of leishmaniasis.