Molecular epidemiology and population genetics in Leishmania.

Polymorphic DNA sequences have been amplified using different PCR-based techniques and used for species identification, strain discrimination and population genetic studies in Leishmania. A PCR fingerprinting method that uses single non-specific primers generates species-specific banding patterns with some intraspecies variation. This approach can be used to identify Leishmania species and also to discriminate strains of different Leishmania species. Cultivation of the parasites is, however, mandatory. PCR-restriction fragment length polymorphism of the internal transcribed spacer (ITS) in the ribosomal operon differentiates all Leishmania species, except members of the L. donovani and L. brasiliensis complexes. ITS-single-strand conformation polymorphism or ITS sequencing can detect strain specific-variation (except in L. infantum); culturing is not required. Species of Leishmania exhibit different degrees of genetic variation (L. tropica > L. aethiopica > L. major > L. donovani). Population analysis using co-dominant DNA markers developed by sequence-confirmed amplified region analysis revealed a primarily clonal structure in a L. donovani population from Sudan and suggested that occasional recombination events may occur in this population.

Identification and determination of the relationships of species and strains within the genus Leishmania using single primers in the polymerase chain reaction.

DNA polymorphisms were assessed in different species and strains within the genus Leishmania by amplifying genomic DNA with single non-specific primers. This polymerase chain reaction (PCR) method employed non-random primers which anneal to mini- and microsatellite DNA sequences like the M13 core sequence and the simple repeat sequences (GTG)5 and (GACA)4, and the T3B primer derived from an intergenic spacer for tRNA genes. Distinctive and reproducible sets of amplified DNA fragments were obtained for all Leishmania isolates tested. The number and size of amplification products were found to be characteristic for a given taxon. Highly similar PCR profiles were observed when genomic DNA of representatives of the L. donovani, L. mexicana or L. braziliensis complexes was amplified. By comparing PCR patterns of unidentified Leishmania isolates with those obtained from reference strains it was possible to identify these isolates at the species level. The information of the amplification patterns was used for the construction of phylogenetic trees to measure the genetic relatedness within the genus Leishmania.

Rapid identification of Candida species by DNA fingerprinting with PCR.

DNA polymorphisms in different species and strains of the genus Candida were assessed by amplifying genomic DNA with single nonspecific primers. This PCR method employed an arbitrary primer (the 10-mer AP3), a primer derived from the intergenic spacer regions (T3B), and the microsatellite primers (GTG)5 and (AC)10. Distinctive and reproducible sets of amplification products were observed for 26 different Candida and 8 other fungal species. The numbers and sizes of the amplification products were characteristic for each species. All yeast species tested could be clearly distinguished by their amplification patterns. With all primers, PCR fingerprints also displayed intraspecies variability. However, PCR profiles obtained from different strains of the same species were far more similar than those derived from different Candida species. By comparing species-specific PCR fingerprints of clinical isolates with those of reference strains, clinical isolates could be identified to the species level even if they could not be identified by routine biochemical methods.