Population structure and geographical subdivision of the Leishmania major vector Phlebotomus papatasi as revealed by microsatellite variation.

Multi-locus microsatellite typing (MLMT) has been employed to infer the population structure of Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) sandflies and assign individuals to populations. Phlebotomus papatasi sandflies were collected from 35 sites in 15 countries. A total of 188 P. papatasi individuals were typed using five microsatellite loci, resulting in 113 different genotypes. Unique microsatellite signatures were observed for some of the populations analysed. Comparable results were obtained when the data were analysed with Bayesian model and distance-based methods. Bayesian statistic-based analyses split the dataset into two distinct genetic clusters, A and B, with further substructuring within each. Population A consisted of five subpopulations representing large numbers of alleles that were correlated with the geographical origins of the sandflies. Cluster B comprised individuals collected in the Middle East and the northern Mediterranean area. The subpopulations B1 and B2 did not, however, show any further correlation to geographical origin. The genetic differentiation between subpopulations was supported by F statistics showing statistically significant (Bonferroni-corrected P < 0.005) values of 0.221 between B2 and B1 and 0.816 between A5 and A4. Identification of the genetic structure of P. papatasi populations is important for understanding the patterns of dispersal of this species and to developing strategies for sandfly control.

Genetic structure of Mediterranean populations of the sandfly Phlebotomus papatasi by mitochondrial cytochrome b haplotype analysis.

Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) is the main vector of Leishmania major Yakimoff & Schokhor; which is the cause of self-limiting cutaneous leishmaniasis in the Old World. This sandfly is found in houses, animal shelters, caves and rodent burrows. It has a large geographical range, which includes the Middle East and the Mediterranean regions. A population analysis of colony and field specimens of P. papatasi was conducted on 25 populations originating from 10 countries. The distribution of haplotypes of the maternally inherited mitochondrial cytochrome b gene were analysed to assess the population differentiation of P. papatasi. Alignment of a 442-basepair region at the 3' end of the gene identified 21 haplotypes and 33 segregating sites from 131 sandflies. The pattern of sequence variations did not support the existence of a species complex. The median-joining network method was used to describe both the origin of the haplotypes and the population structure; haplotypes tended to cluster by geographical location, suggesting some level of genetic differentiation between populations. Our findings indicate the presence of significant population differentiation for populations derived from Syria, Turkey, Palestine, Israel, Jordan and Egypt. Knowledge of population differentiation among P. papatasi populations is important for understanding patterns of dispersal in this species and for planning appropriate control measures.

Clinical diagnosis of cutaneous leishmaniasis: a comparison study between standardized graded direct microscopy and ITS1-PCR of Giemsa-stained smears.

Parasitological diagnosis of cutaneous leishmaniasis is absolutely necessary before treatment. Direct microscopy of scrapings taken from the margins of skin lesions is the most commonly used method for clinical diagnosis of leishmaniasis. In this study to evaluate the usage of stained smears as samples for PCR and the possible advantage of PCR, we compared the sensitivity of the diagnosis of Giemsa-stained skin scrapings by standardized graded direct microscopy with that of ITS1-PCR with the material of the same area of the slide. Three 5mm x 5mm squares were marked on each of the 20 Giemsa-stained touch smears from 20 clinically diagnosed Palestinian patients. Out of the 60 squares scanned for amastigotes under 100x oil-immersion light microscopy, 45 (75%) gave usable results and 23 of these were positive for Leishmania. Fifteen (25%) squares could not be scanned microscopically, 12 because of staining that was too thick and 3 because of inadequate staining. DNA from each scanned square was extracted separately after microscopy and run through ITS1-PCR. Of the 23 microscopy-positive squares, 20 (87%) of these were positive by PCR. Of the three that were negative, one failed to extract for DNA, the second showed only one amastigote in the entire square, and the third was normally graded as +1 but was not amplified for unknown reasons. Of the 22 squares negative for microscopy, 18 (82%) were ITS1-PCR positive. Additionally, all three improperly stained squares were ITS1-PCR positive. Of the 12 darkly stained squares, 11 were positive. A negative control group of 15 German individuals from which Giemsa-stained slides containing three squares each was prepared and these slides were also microscopically scanned and tested by ITS1-PCR. Both tests were negative with both methods. Compared to microscopy (data in parenthesis), PCR showed a sensitivity of 87% (37%) and a specificity of 100% (100%). We have concluded that Giemsa-stained smears are a readily usable sampling method for PCR and that ITS1-PCR is far more sensitive than microscopy.

Origins of microsatellite diversity in the Trichophyton rubrum-T. violaceum clade (Dermatophytes).

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.

The recent emergence of Leishmania tropica in Jericho (A'riha) and its environs, a classical focus of L. major.

Between 1997 and 2002, 49 strains of Leishmania were isolated from the cutaneous lesions of Palestinians living in and around Jericho. A polymerase chain reaction (PCR) amplifying the ribosomal internal transcribed spacer 1 (ITS1-PCR) was applied to their cultured promastigotes and to 207 individuals' skin scrapings spotted on filter-papers, 107 of which proved positive for leishmanial DNA. Species identification was performed by restricting the ITS1-PCR amplification products from the cultured promastigotes and the amastigotes in the scrapings with the endonuclease HaeIII. Of the 49 cultures, 28 (57%) were L. major and 21 (43%) were L. tropica. Of the 107 dermal samples tested directly, 53 (49.5%) were infected with L. major, 52 (48.5%) with L. tropica and two remained unidentified. This is the first time L. tropica has been exposed in the population of the Jericho area and on such a large scale. The itinerant behaviour of some of this population precludes categorically declaring that L. tropica has recently become established in this classical focus of L. major. For this and although 88.2% of the cases of L. tropica claimed not to have travelled out of the vicinity of Jericho, local infected sand fly vectors of L. tropica must be caught, identified and, if possible, shown to harbour infections, and, if one exists, an animal reservoir host should also be exposed to endorse whether the cases caused by L. tropica were imported or autochthonous.

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.

Application of flow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania Spp.

The World Health Organization has identified leishmania sis as a major public health problem, particularly in Africa, Asia, and Latin America. About 1.5 to 2 million people are affected annually by this parasitic infection. As there is no vaccine, there is still a strong need for sufficient drugs. In a preliminary screening, extracts of 50 different plants were evaluated for their possible leishmanicidal activity against the promastigote form of Leishmania mexicana amazonensis. Eighteen extracts showed at least 50% inhibition at 100 microg/ml. The ethanolic extract from Yucca filamentosa L. showed the strongest leishmanicidal activity (100% inhibition at 5 microg/ml). The bioactivity-guided fractionation of this extract led to the isolation of three main components (Yuccasaponins MC 1--3). In further experiments, the effect of Yuccasaponin MC 3 on the promastigote form of L. mexicana amazonensis was quantified and characterized using flow cytometry and specific fluorescent dyes [propidium iodide, Syto 9, and DiBAC(4)(3)]. The data revealed that the membrane of the promastigote is attacked. The effect of Yuccasaponin MC 3 on intracellular forms (amastigote) was also characterized; green fluorescent protein-transfected Leishmania major were used. By this method, an inhibition of intracellular growth of L. major was demonstrated. This paper shows that, together, flow cytometry and microscopy are quick, sensitive, and easily reproducible methods to describe the effects of drugs on parasites.

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.

Assessment of genetic relatedness of vaginal isolates of Candida albicans from different geographical origins.

PCR fingerprinting with single non-specific primers was used to type vaginal isolates of C. albicans from Portugal, Angola, Madagascar, and two regions of Germany (Berlin and Munich). In addition to analysing isolates that exhibited the normal biotype of C. albicans, the study included atypical strains that failed to assimilate glucosamine and N-acetylglucosamine, which were isolated from women in Angola and Madagascar. A total of 212 strains of C. albicans were studied, representing 87 different multi-locus genotypes. The genotypes of strains from each geographical population were highly similar but not identical. There was one exception: a strain from Portugal grouped with the typical strains from Angola. The typical and especially the atypical populations from Africa displayed less genotype variation than the populations from Europe. The Portuguese samples exhibited the greatest genotypic heterogeneity. Distance analysis (UPGMA) revealed a statistically weak correlation between genotype and geographical origin of the C. albicans isolates.

Molecular taxonomy of the Trichophyton rubrum complex.

The validity of taxa around Trichophyton rubrum was evaluated by a combination of phenetic and molecular methods. Morphological and physiological features were compared to results of sequencing of the internal transcribed spacer region of the ribosomal operon, PCR fingerprinting, and amplified fragment length polymorphism analysis. The 15 species and varieties investigated (Trichophyton circonvolutum, Trichophyton fischeri, Trichophyton fluviomuniense, Trichophyton glabrum, Trichophyton gourvilii, Trichophyton kanei, Trichophyton kuryangei, Trichophyton megninii, Trichophyton pedis, Trichophyton raubitschekii, Trichophyton rodhaini, Trichophyton rubrum var. nigricans, Trichophyton soudanense, Trichophyton violaceum var. indicum, and Trichophyton yaoundei) were reclassified or synonymized as T. rubrum or T. violaceum.

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 variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis.

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.

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.

Strain specificity of yeasts isolated from different locations of women suffering from vaginal candidosis, and their partners.

Sexual partners often harbour identical yeast strains in the vagina, in the orointestinal tract and in semen in cases of recurrent vulvovaginal candidoses. Specimen were collected from vagina, oral cavity and faeces of the patients, and from semen, oral cavity and faeces of their male partners. Mycological cultures were grown on Sabouraud glucose-agar and, if positive, specified by Candida-ID-Agar (BioMérieux), by formation of chlamydospores on rice agar, and by biochemotyping with the System Walkaway (Dade) or the API-32C system (BioMérieux). A polymerase chain reaction finger-printing technique with the T3B oligonucleotide as single primer was used for strain typing. Candida albicans was isolated from the vagina of 18 out of 21 patients, the vagina of one patient harboured a strain of Candida glabrata. The cultures obtained from vagina, oral cavity and faeces were genetically identical in 12 patients. From the partners of 15 patients C. albicans was cultured in at least one of the clinical samples. Identical strains were observed for eight of 15 couples, whereas four of these identical strains were cultured from semen. Further prospective investigations will prove whether a consequent treatment of both partners will eradicate identical yeast strains and will be able to improve the results of treatment in such women.

[Genetic structure of geographically different populations of candida albicans]. / Untersuchungen zur genetischen Struktur von Candida-albicans-Populationen unterschiedlicher geographischer Herkunft.

Codominant single-locus markers were developed by amplifying genomic DNA of C. albicans with pairs of random primers. Monomorphic PCR products were screened for polymorphisms by the SSCP technique. Sequencing confirmed that SSCP's were mostly due to single nucleotide substitutions in the polymorphic fragments. A total of 85 polymorphic loci were observed within 13 PCR fragments. Populations from Africa displayed less genotype variation than the populations from Europe and USA. Two genetically similar African C. albicans populations exhibiting an atypical biotype were strictly clonal and perhaps represent a geographically distributed clone. Analyses of "typical" C. albicans populations of different geographical origin provided however evidence for both clonality and recombination. Evidence for clonality was supported by the absence of segregation genotypes, and by deviation of genotypic frequencies from Hardy-Weinberg expectations. Tests for nonrandom association of alleles across loci revealed less evidence for linkage disequilibrium than expected for strictly clonal populations. Although all C. albicans populations tested were primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

Molecular taxonomy of Trichophyton mentagrophytes and T. tonsurans.

Most members of the anamorph genus Trichophyton are anthropophilic and have evolved with the human host. Classical parameters for the identification of dermatophytes include clinical features, cultural characteristics, conidial morphology and physiological test results. Phenotypic variability and pleomorphism due to culturing on artificial media is common among this group of organisms and has led to the description of numerous species. The validity of taxa around T. mentagrophytes and T. tonsurans was verified. Morphological and physiological features were compared to results of three different molecular techniques (sequencing of the internal transcribed spacer (ITS) region of the ribosomal operon, PCR fingerprinting and amplified fragment length polymorphism (AFLP) analysis). Twenty-four species or varieties investigated could be reduced to five taxa and were reclassified or synonymized as Trichophyton tonsurans, T. interdigitale, T. mentagrophytes, T. simii and T. erinacei.

Molecular markers reveal exclusively clonal reproduction in Trichophyton rubrum.

Genotypic variability among 96 Trichophyton rubrum strains which displayed different colony morphologies and were collected from four continents was investigated. Twelve markers representing 57 loci were analyzed by PCR fingerprinting, amplified fragment length polymorphism, and random amplified monomorphic DNA markers. Interestingly, none of the methods used revealed any DNA polymorphism, indicating a strictly clonal mode of reproduction and a strong adaptation to human skin.