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

A polymerase chain reaction and single-strand conformation polymorphism det ermination (PCR-SSCP) was used to detect deoxyribonucleic acid sequence pol ymorphisms in the transcribed non-coding regions between the small and larg e sub-unit ribosomal ribonucleic acid (rRNA) genes in Leishmania donovani f rom 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 cli nical samples spotted on filter papers. Amplification products were subsequ ently analysed by SSCP. Eleven polymorphic patterns were detected in the fi rst part of the spacer, the ITS1 region, and were sequenced. Most of the ch anges were due to deletions of adenine bases and AT pairs within the first 192 nucleotides of the ITS region. This is thr first application of PCR-lin ked SSCP analysis for the detection of population variation with direct dis play of sequence variation in parasitologically positive clinical samples s potted on filter paper. Culturing the parasite is thus not required, which is beneficial particularly in epidemiological studies based on field work w here obtaining cultures can be extremely difficult.