[Ecology and the genetic structure of sympatric leishmania species circulating in the intra-continental deserts of the south Palaearctic region]. / Ekologia i geneticheskaia struktura populiatsii simpatrichnykh vidov leishmanii, tsirkuliruiushchikh vo vnutrikontinental'nykh pustyniakh iuzhnoi Palearktiki.

A PCR fingerprinting approach with single non-specific primers--(GTG)5 and T3B--was apply to investigate variations in the genotyping of three species of Leishmania species within the Rhombomys opimus area. Forty-three strains of Leishmania major, L. turanica, and L. gerbilli circulating among great gerbils in Turkmenistan, Uzbekistan, Kazakhstan, and Mongolia were examined. PCR fingerprint revealed a high genetic intraspecific heterogeneity among L. turanica strains. Three groups of strains were clearly identified. The strains from Mongolia greatly differed from other L. turanica ones. The second group was formed by strains from Kazakhstan, they also demonstrated rather different patterns. L. turanica strains from Turkmenistan and Uzbekistan showed only minor differences, but greatly different from those from Kazakhstan and Mongolia. The groups identified by the PCR fingerprint correlate with the conditions of circulation: the duration of a transmission season and as the result of different periods of retention of Leishmania in the skin of great gerbils, as well as the presence or absence of L. major as coexisting species. There were no differences between L. turanica strains isolated from different hosts in the same geographical region, as well as between L. turanica strains isolated in the hyper- or meso and hypoendemic foci. There was no correlation between the genotypic and phenotypic characteristics of L. turanica. No intraspecific polymorphism was found among L. major and L. gerbilli strains from different geographical regions within the great gerbil area.

The ecology of cutaneous leishmaniasis in Nizzana, Israel: infection patterns in the reservoir host, and epidemiological implications.

We conducted an extensive interdisciplinary study in an emerging focus of cutaneous leishmaniasis in the Western Negev Desert of Israel between July 1998 and February 2000. The aims of the this study were to determine (1) the reservoir hosts, (2) the distribution of the pathogen within the host range, (3) the associations of host, vector, and pathogen within defined habitats, (4) the demographic distribution of the pathogen within the host populations, and (5) to apply the newly acquired epizootiological data to explain morbidity patterns in humans. Fourteen square (60 m width) sampling plots were delimited in three types of habitats each with a different kind of substrate: loess, sand, and sand-loess ecotone. Rodents and sand flies were trapped and several environmental variables were measured. Leishmania infections in rodents were detected microscopically in stained smears of ear tissue and by a Leishmania-specific polymerase chain reaction. Results indicate that, contrary to previous reports, Psammomys obesus and not Meriones crassus is the main reservoir host in the region. Additional rodents (12 Gerbillus dasyurus and two M. crassus) were also found positive for Leishmania DNA. Prevalence of Leishmania infections amongst P. obesus was highest in loess habitats (65%), intermediate in the sandy-loess ecotone (20%), and 0% in the sandy habitats. Psammomys obesus individuals in the loess habitat of the Nizzana ruins were larger, on average (probably older), than those in the sandy habitat of the Mt. Keren junction. Sand fly density was positively correlated to soil moisture being higher in the relatively humid plots of Nizzana ruins and much lower in the drier sandy soil of Mt. Keren. Elucidation of fundamental ecological factors affecting this disease has helped explain an apparent discrepancy between the distribution of the disease in the zoonotic system and among humans.

Genetic heterogeneity in the species Leishmania tropica revealed by different PCR-based methods.

A PCR fingerprinting approach, using single non-specific primers, as well as restriction and single-strand conformation polymorphism (SSCP) analyses of the amplified ribosomal internal transcribed spacer, were used to investigate genetic variability in the species Leishmania tropica. Twenty-nine strains of the 'L. tropica complex' from different Old World geographical areas were studied including 4 from Namibia, and 1 strain of L. killicki. All techniques revealed a high degree of genetic heterogeneity among the strains of L. tropica. The PCR fingerprinting displayed the highest discriminatory power, but can be applied only to cultured parasites. The internal transcribed spacer (ITS) region can be amplified directly from infected clinical samples and analysed subsequently. No strict correlation was discerned between the genetic variants and either the geographical origin of the strains or the clinical manifestations associated with human disease, except for the Namibian strains. Also, genetic variation did not correlate well with characterization by enzyme variant electrophoretic analysis. The strain of L. killicki always clustered together with the strains of L. tropica, suggesting it, probably, should not be considered a separate species of Leishmania. However, the 4 Namibian strains formed a distinct, statistically well-supported group closely related to but different from the other strains of L. tropica.

Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing.

Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.

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.

An epidemic of tinea corporis caused by Trichophyton tonsurans among children (wrestlers) in Germany.

An outbreak of tinea corporis due to Trichophyton tonsurans among 46 children (aged 7-17 years) was investigated. Most of them were wrestlers. Thirty-one strains were identified by conventional methods, but proved to be problematic in 15 isolates due to colony variation and reduced sporulation. They were identified as Trichophyton tonsurans by the use of molecular methods, for example, sequence comparison of the ribosomal internal transcribed spacer (ITS) region and polymerase chain reaction fingerprinting. No DNA polymorphisms were detected with any of the techniques used, suggesting clonal reproduction of the populations of the species and providing evidence for spatial and temporal stability of the lineage.

Molecular and conventional taxonomy of the Microsporum canis complex.

The validity of taxa around Microsporum canis was evaluated by a combination of phenetic and molecular methods. Morphological and physiological features were compared with results of sequencing of the internal transcribed spacer (ITS) region of the ribosomal operon, polymerase chain reaction (PCR) fingerprinting and amplified fragment length polymorphism (AFLP) analysis. The seven species investigated seem to be infraspecific taxa and were reclassified or synonymized as M. canis (teleomorph: Arthroderma otae), M. ferrugineum, and M. audouinii.

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.

Development of an oligonucleotide probe specific for Trichophyton rubrum.

A species-specific DNA probe was developed to detect the dermatophyte species Trichophyton rubrum. The selected oligonucleotide sequence is derived from the highly variable internal transcribed spacer 2 region of the ribosomal DNA operon. The specificity of the non-radioactive labelled oligonucleotide probe was tested against related dermatophytes, other eukaryotic microorganisms and against human DNA. No cross-hybridization was found, and hybridization signals were invariably detected in all T. rubrum strains investigated. In addition, no homologous sequences were found searching the EMBL database. Experiments to establish a method for isolating DNA directly from clinical specimens gave successful amplification and hybridization products in about 30% of the samples.

Phylogeny and taxonomy of the family Arthrodermataceae (dermatophytes) using sequence analysis of the ribosomal ITS region.

The internal transcribed spacer (ITS) region, covering the ITS1, ITS2 and 5.8S ribosomal DNA was used to evaluate phylogenetic relationships within the fungal family Arthrodermataceae. Sequences of variable length, ranging between 522 and 684 base pairs were aligned. An unrooted consensus tree based on parsimony analysis showed Trichophyton to be polyphyletic, and Microsporum to be paraphyletic. Non-monophyly of these two genera is in conflict with traditional classification. But this relation is not strongly supported by bootstrap analysis. Phylogenetic analysis showed that the two known members of the genus Epidermophyton grouped widely apart from each other. Within Trichophyton, our results suggest a separation of human pathogenic species and primarily geophilic species. Bootstrap support for these two groups is fairly high and both groups are recognized by current taxonomy. Three lineages were revealed within the T. mentagrophytes species complex. Microsporum canis, M. audouinii and M. equinum were found to be closely related. The topology of the tree was robust to various methods of analysis (parsimony and distance) and a different weighting scheme. Weighting of transversions over transitions did not improve the status of poorly supported branches of the tree.

Identification of common dermatophytes (Trichophyton, Microsporum, Epidermophyton) using polymerase chain reactions.

Polymerase chain reaction (PCR) fingerprinting detected DNA polymorphisms among frequently isolated species and strains of the genera Trichophyton, Microsporum and Epidermophyton. The patterns generated by this DNA-based method permitted species and strains to be identified. The conventional methods to identify dermatophytes rely on the expression of characteristic morphological features, as well as several physiological properties. Identification is often delayed or problematic because isolates may be slow to form conidia or produce atypical microscopic structures or colony appearances. Using non-specific primers such as (AC)10, (GTG)5, M13 core sequence and AP3, characteristic PCR profiles were generated for 17 species. Intraspecies variables were also observed for four of six varieties of T. mentagrophytes, whereas no detectable DNA variability was found within the three varieties of T. tonsurans. Comparing species-specific PCR fingerprints of clinical isolates with those of type strains, species could be identified by their PCR fingerprints, even if they could not be identified by the accepted phenotypic characteristics.

Molecular phylogeny and taxonomy of medically important fungi.

Some recent advances in study of molecular evolution and taxonomy of human pathogens are discussed. In systemic Onygenales as well as in Chaetothyriales, pathogenic species are phylogenetically intermingled with non-pathogenic taxa. When a teleomorph of Coccidioides immitis is eventually found, it is predicted to resemble Uncinocarpus, a genus otherwise comprising environmental species. In the dermatophytes, Trichophyton and Microsporum are paraphyletic, whereas Epidermophyton is polyphyletic. On the basis of 18S and ITS rDNA sequencing data, Exophiala anamorphs (black yeasts) are confirmed to be closely related to the ascomycete genus Capronia. The related neurotropic species Cladophialophora bantiana is remarkable in consistently having introns in its 18S rDNA gene.