Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing

Abstract

A polymerase chain reaction and single-strand conformation polymorphism determination (PCR-SSCP) was used to detect deoxyribonucleic acid sequence polymorphisms in the transcribed non-coding regions between the small and large sub-unit ribosomal ribonucleic acid (rRNA) genes in Leishmania donovani from 63 clinical samples collected in eastern Sudan, between April 1997 and October 1998. Specific Leishmania primers were used to amplify the internal transcribed spacer (ITS) regions of L. donovani isolates directly from clinical samples spotted on filter papers. Amplification products were subsequently analysed by SSCP. Eleven polymorphic patterns were detected in the first part of the spacer, the ITS1 region, and were sequenced. Most of the changes were due to deletions of adenine bases and AT pairs within the first 192 nucleotides of the ITS region. This is the first application of PCR-linked SSCP analysis for the detection of population variation with direct display of sequence variation in parasitologically positive clinical samples spotted on filter paper. Culturing the parasite is thus not required, which is beneficial particularly in epidemiological studies based on field work where obtaining cultures can be extremely difficult.