Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis

Abstract

We recently developed a new PCR-restriction fragment length polymorphism (RFLP)-based assay using the miniexon sequence from the genus Leishmania. Here we report the application of this new genotyping method to naturally infected clinical samples for the differentiation of New and Old World Leishmania species. 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 other Leishmania genotyping techniques. The assay provides a valuable tool for the identification of Leishmania directly from clinical samples and enables determination of the infecting species by a facile technique with high discrimination power. Since Leishmania causes 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.

FIG. 1.

Coamplification of Leishmania DNA from clinical samples and a positive control template. Black triangles denote the 540-bp band of the positive control template, and grey triangles correspond to the Leishmania-specific PCR signal. Lanes 1 and 2, positive samples. The PCR products of the target DNA and the positive control template are visible on the agarose gel. Lanes 3 and 4, positive samples. Target DNA outcompeted the positive control template. Lane 5, PCR inhibition is indicated because the positive control template is not visible. Lane 6, previously inhibited sample after DNA dilution. Lane 7, previously inhibited sample after subsequent purification. Lane 8, negative sample with only control template visible.