We have developed a PCR assay that in a single reaction distinguishes between Leishmania infantum and Leishmania donovani strains on the basis of different size of the amplicon. The targeted intergenic region between putative biopterin transporter and nucleotide binding protein on chromosome 35 is highly variable, species-specific and can be amplified from clinical samples. Based on the assay, five tested Leishmania archibaldi and L. infantum strains from the Sudan and Ethiopia clearly belong to L. donovani, which is in accordance with a recent multifactorial analysis of these strains. The nucleotide sequence reported in this paper has been submitted to the GenBankTM with accession number EU068004.
Flagellates of the Leishmania donovani complex are causative agents of human cutaneous and visceral leishmaniasis. The complex is comprised of L. donovani, Leishmania infantum and Leishmania archibaldi, although the latter is not now considered to be a valid species. Morphological distinction of Leishmania species is impractical, so biochemical, immunological and DNA-based criteria were introduced. Multilocus enzyme electrophoresis (MLEE) is the present gold standard. We have sequenced the genes encoding five metabolic enzymes used for MLEE, both to resolve the DNA diversity underlying isoenzyme mobility differences and to explore the potential of these targets for higher resolution PCR-based multilocus sequence typing. The genes sequenced were isocitrate dehydrogenase, malic enzyme, mannose phosphate isomerase, glucose-6-phosphate dehydrogenase, and fumarate hydratase, for 17 strains of L. infantum, seven strains of L. donovani, and three strains of L. archibaldi. Protein mobilities predicted from amino acid sequences did not always accord precisely with reported MLEE profiles. A high number of heterozygous sites was detected. Heterozygosity was particularly frequent in some strains and indirectly supported the presence of genetic exchange in Leishmania. Phylogenetic analysis of a concatenated alignment based on a total of 263 kb protein-coding sequences showed strong correlation of genotype with geographical origin. Europe and Africa appear to represent independent evolutionary centres.
