W. Presber

PCR diagnosis and characterization of Leishmania in local and imported clinical samples

Leishmaniasis diagnosis in regions where multiple species exist should identify each species directly in the clinical sample without parasite culturing. The sensitivity of two PCR approaches which amplify part of the ssu rRNA gene and the ribosomal internal transcribed spacer (ITS), respectively, was determined using human and dog blood seeded with Leishmania promastigotes. ssu-rDNA-PCR was more sensitive than ITS1-PCR, however species identification was not possible by the former approach. When a nested ITS1-PCR was used its sensitivity equaled the ssu-rDNA-PCR. Digestion of ITS1 amplicon with the restriction enzyme HaeIII distinguished all medically relevant Leishmania species. ITS1-PCR was used to diagnose 162 local and imported suspected cases of leishmaniasis in Israel, the Palestinian Authority and Germany. 113 cases (69.7%) were positive by PCR and species identification was possible in 110 samples. Leishmania DNA was also amplified and identified at the species level from archived non-stained and Giemsa stained microscope slides.

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.

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.

Origins of Microsatellite Diversity in the Trichophyton rubrum- T. violaceum Clade (Dermatophytes)

ABSTRACT We analyzed the population structure of the anthropophilic dermatophyte species Trichophyton violaceum, which mainly causes tinea capitis, and T. rubrum, the most frequently isolated agent of dermatophytosis worldwide. A microsatellite marker (T1) was developed by using the enrichment technique for microsatellites. The T1 marker containing a (GT)8-10 repeat was proven to specifically amplify both species, underlining their close kinship. Four polymorphic alleles were detected within a set of about 130 strains by using polyacrylamide gel electrophoresis with this marker. An association with geographic origin of the isolates was apparent. Given the close relatedness of both species, these data suggest an African origin of the entire T. rubrum complex, followed by the emergence of a new genotype (B) in Asia with subsequent spread of this genotype over Europe and the United States.

Molecular detection of Microsporum persicolor in soil suggesting widespread dispersal in central India.

A survey was conducted from 1999 to 2003 as part of a microbial biodiversity study on geophilic and keratinophilic fungi in central India. Among the keratinophilic fungi recovered were 82 isolates belonging to the dermatophyte genus Microsporum. Species were provisionally identified by morphology and confirmed by PCR-RFLP and sequencing of the ITS regions of rDNA. Microsporum persicolor appeared to be preponderant in central Indian soils, outnumbering the common geophilic species of Microsporum filvum and Microsporum gypseum. Three dinucleotide microsatellite markers were developed and their use revealed immense intraspecific variation among Indian populations of M. persicolor which would indicate that this species was not recently introduced into India. No correlation was established between the genotypes and the geographical location or the habitat of the isolates.