Identification of the causative agent of cutaneous leishmaniasis in Chichaoua province, Morocco.

Cutaneous leishmaniasis (CL) in Morocco is caused by three species, Leishmania major, L. tropica and L. infantum. CL has been known in Chichaoua province since 2000. Using DNA extracted from microscopic slides and parasite cultures, collected in the years 2006 and 2009, we identified for the first time L. tropica as the causative agent of CL in this region. Species identification was achieved by performing the ITS1-PCR-RFLP approach. By using this method it was possible to identify parasites in Giemsa stained slides containing less than five parasites per oil-immersion field even they were conserved for up to four months.

Molecular approaches for a better understanding of the epidemiology and population genetics of Leishmania.

Molecular approaches are being used increasingly for epidemiological studies of visceral and cutaneous leishmaniases. Several molecular markers resolving genetic differences between Leishmania parasites at species and strain levels have been developed to address key epidemiological and population genetic questions. The current gold standard, multilocus enzyme typing (MLEE), needs cultured parasites and lacks discriminatory power. PCR assays identifying species directly with clinical samples have proven useful in numerous field studies. Multilocus sequence typing (MLST) is potentially the most powerful phylogenetic approach and will, most probably, replace MLEE in the future. Multilocus microsatellite typing (MLMT) is able to discriminate below the zymodeme level and seems to be the best candidate for becoming the gold standard for distinction of strains. Population genetic studies by MLMT revealed geographical and hierarchic population structure in L. tropica, L. major and the L. donovani complex. The existence of hybrids and gene flow between Leishmania populations suggests that sexual recombination is more frequent than previously thought. However, typing and analytical tools need to be further improved. Accessible databases should be created and sustained for integrating data obtained by different researchers. This would allow for global analyses and help to avoid biases in analyses due to small sample sizes.

A clinical isolate of Leishmania donovani with ITS1 sequence polymorphism as a cause of para-kala-azar dermal leishmaniasis in an Ethiopian human immunodeficiency virus-positive patient on highly active antiretroviral therapy.

The diagnosis of para-kala-azar dermal leishmaniasis (PKDL/VL), either as an immune reconstitution inflammatory syndrome (IRIS)-associated syndrome or as a complication of visceral leishmaniasis (VL) during human immunodeficiency virus (HIV) co-infection in patients with or without highly active antiretroviral therapy, represents a challenge for prompt treatment. The aim of this study was to identify the causative Leishmania species and to clarify whether the post-kala-azar dermal leishmaniasis (PKDL)-like lesions appeared as a result of IRIS or not. A 31-year-old Ethiopian male patient, with stage IV HIV/acquired immune deficiency syndrome (AIDS), was clinically diagnosed with PKDL/VL. He had developed a generalized maculopapular rash on his face after initiation of highly active antiretroviral therapy. The Leishmania isolate obtained from the skin lesions was analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing of the ribosomal DNA internal transcribed spacer 1 (ITS1) and partial coding sequences of the heat shock protein 70 gene (hsp70). Restriction analysis of the ITS1 PCR product gave a unique RFLP pattern not seen before for any Leishmania isolate. Sequencing of both the ITS1 and hsp70 PCR products identified the causative species as Leishmania donovani, and further revealed the existence of five different sequence variants of the ITS1 among the 10 clones sequenced. The results indicate that PKDL/VL resulted from an infection by L. donovani. The sequence variants of ITS1 might be due to the presence of multiple strains/clones or the existence of intragenomic variations in the multicopy ITS1, or a combination of both.

Two recent but temporally distinct outbreaks of cutaneous leishmaniasis among foreign workers in the Dead-Sea area of Jordan.

Two temporally distinct outbreaks of human cutaneous leishmaniasis (CL), as well as scattered cases of the disease, have recently been observed close to the Dead Sea, in Jordan. Each of the two outbreaks, which occurred in 2004/2005 and 2007/2008, involved a group of foreign workers who were deployed within otherwise uninhabited locations. During each outbreak, about 20% of the workers were found infected with the causative parasite. In the earlier outbreak, 61 workers were found to have skin lesions like those of CL and all but three were confirmed by culture and/or the examination of smears (40 cases) or, in the case of 18 (86%) of the 21 suspected cases found smear- and culture-negative, by PCR. In the second outbreak, the cases were only identified from their clinical manifestations and their response to antileishmanial treatment (cryotherapy). Leishmania major was identified as the cause of the 2004/2005 outbreak and some sporadic cases that occurred, in 2004, along the shores of the Dead Sea. The burrows of potential reservoir hosts were found close to the outbreak locations, frequently under the chenopod Seidlitzia rosmarinus. The two outbreaks emphasise the continuing problem posed by the CL focus in the Mid Jordan Valley and its impact on humans who move into the area. Curiously, an investigation on the socio-economic conditions of the workers during the outbreaks identified a group of 48 workers who were living in air-conditioned rooms during the 2007/2008 outbreak, among whom no CL cases were found. In contrast, 26 of a neighbouring group of 124 workers, who were all living in non-air-conditioned rooms, developed CL lesions. The role of air conditioning, and of other factors and measures, in the prevention of the transmission of the causative parasites of CL merits further investigation and the attention of the local health authorities.

Identification of the agent causing visceral leishmaniasis in Uzbeki and Tajiki foci by analysing parasite DNA extracted from patients' Giemsa-stained tissue preparations.

Our present study is the first attempt to characterize Leishmania parasites from foci in Uzbekistan and Tajikistan endemic for visceral leishmaniasis (VL). PCR-sequencing of the ribosomal internal transcribed spacer 1 and multilocus microsatellite typing (MLMT) were applied to DNA extracted from preparations of Giemsa-stained bone marrow aspirates from 13 cases of VL. L. infantum was shown to cause VL currently occurring in this area. MLMT applying 14 microsatellite markers, previously shown to be polymorphic for strains of the L. donovani complex, revealed that microsatellite profiles of parasites causing human VL in the Namangan and Jizzakh regions in Uzbekistan, and Penjikent region in Tajikistan, basically coincide with those of strains of L. infantum MON-1. Furthermore, these parasites were assigned to a distinct cluster genetically clearly separated from the populations of L. infantum MON-1 from Europe, the Middle East and North Africa. The existence of a genetically homogeneous but distinct group of L. infantum MON-1 indicates that the parasites circulating in the Uzbeki and Tajiki foci studied have been restricted there for a long time rather than having been recently introduced from elsewhere by human or animal reservoir migration.

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.

Evaluation of PCR-RFLP (based on ITS-1 and HaeIII) for the detection of Leishmania species, using Greek canine isolates and Jordanian clinical material.

It is possible to detect and distinguish Leishmania parasites using PCR-RLFP - a combination of PCR and analysis of the fragment-length polymorphism seen when the amplicons are digested with one or more restriction enzymes. In the present study, clinical samples from 24 Jordanians suspected to have cutaneous leishmaniasis and cultures set up using leishmanial parasites from five Greek dogs were investigated using PCR, in which the internal-transcribed-spacer-1 (ITS1) region of the parasites' ribosomal-RNA gene was amplified, followed by HaeIII digestion of the resulting amplicons. The cultures, which were all maintained in Leibowitz L-15 medium with 20% foetal calf serum, were each investigated as serial dilutions. Using the PCR-RLFP analysis, each culture was identified as L. donovani and each was found positive for this parasite with a mean sensitivity of 66%-100% (depending on the culture dilution tested), a specificity of 100%, a mean positive predictive value of 100%, and a negative predictive value of 74.6%-100%. When simulated clinical samples, created by mixing human blood with known numbers of L. donovani promastigotes, were investigated, the PCR-RFLP gave optimal results (with a value of 100% each for sensitivity, specificity and positive and negative predictive values). When the real clinical samples (25 lesion aspirates and 20 samples of peripheral blood from 24 Jordanian patients) were investigated using the molecular method, 20 (84%) of the patients were found to have lesion aspirates that were PCR-RFLP-positive for L. major (although, by microscopy, only six were found to have amastigote-positive lesion aspirates). None of the blood samples from the Jordanian patients, however, was found PCR-positive.

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.

[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.

Epidemiology of cutaneous leishmaniasis in the endemic area of Jericho, Palestine.

This study of cutaneous leishmaniasis in Jericho city and the adjacent Aqbat-Jaber refugee camp investigated the seroprevalance of Leishmania major and the risk factors associated with acquiring the disease. Clinical and parasitology identification of cases showed children and young men were more affected, with the head most affected in children. Enzyme-linked immunosorbent assay (ELISA) was used to test sera from 190 individuals. The overall seroprevalence of cutaneous leishmaniasis was 26.3%. A case-control study of 247 individual in 37 households showed that a higher level of education of the head of the household and having children sleep under bed nets were significantly related to a lower incidence of cutaneous leishmaniasis.

Epidemiology of cutaneous leishmaniasis in the endemic area of Jericho, Palestine

This study of cutaneous leishmaniasis in Jericho city and the adjacent Aqbat-Jaber refugee camp investigated the seroprevalance of Leishmania major and the risk factors associated with acquiring the disease. Clinical and parasitology identification of cases showed children and young men were more affected, with the head most affected in children. Enzyme-linked immunosorbent assay [ELISA] was used to test sera from 190 individuals. The overall seroprevalence of cutaneous leishmaniasis was 26.3%. A case-control study of 247 individual in 37 households showed that a higher level of education of the head of the household and having children sleep under bed nets were significantly related to a lower incidence of cutaneous leishmaniasis

Sequential genotyping of Pseudomonas aeruginosa from upper and lower airways of cystic fibrosis patients.

A controversy exists concerning the adequate specimen to characterise colonisation of cystic fibrosis (CF) airways by Pseudomonas aeruginosa. Oropharyngeal, sputum and bronchoalveolar lavage samples were evaluated from 38 stable CF patients for the detection of P. aeruginosa, genetically different isolates within the same host and longitudinal variations in the genotype during repeated examinations. Bacterial isolates were typed by pulsed-field gel electrophoresis of deoxyribonucleic acid macrorestriction fragments. Sensitivity, negative and positive predictive values and specificity to detect P. aeruginosa were 35.7, 73.5, 83.3 and 96.2% for oropharyngeal cultures in nonexpectorating patients and 91.7, 94.1, 100 and 100% for sputum cultures from expectorating patients, respectively. Genotypes of Pseudomonas isolates recovered from oropharyngeal swabs and sputum differed to the strains recovered by bronchoscopy in 55% and 40%, respectively. In 62% longitudinal variations in the genotype occurred. One-half of these alterations were detectable by bronchoscopy only. In conclusion, sputum samples were of equal value as specimens from bronchoalveolar lavage to detect Pseudomonas aeruginosa colonisation. Cultures from the oropharynx are not suitable for characterising bacterial conditions in the cystic fibrosis lung. Different genotypes within the same host and longitudinal genetic alterations are common and may be detectable in the bronchoalveolar lavage fluid exclusively.

Competitive polymerase chain reaction used to diagnose cutaneous leishmaniasis in German soldiers infected during military exercises in French Guiana.

A competitive polymerase chain reaction (PCR) followed by enzyme-linked-immunoassay-based verification of PCR products has been developed, which facilitated the diagnosis of leishmaniasis in two German soldiers who underwent survival training in the jungle of French Guiana and returned with therapy-resistant pyoderma-like lesions. After treatment with liposomal amphotericin B, the skin manifestations disappeared, and leishmania DNA could no longer be detected by PCR. In the context of growing military involvement in areas where leishmaniasis is prevalent, this assay may help detect or, due to its internal controls, exclude cases of infection with this parasite.

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.

Visceral leishmaniasis in a German child who had never entered a known endemic area: case report and review of the literature.

We describe a case of visceral leishmaniasis in a 15-month-old German child. Diagnosis was significantly delayed because the patient had no history of travel to known endemic areas. Congenital or blood transfusion-associated leishmaniasis was ruled out. Possible modes of transmission (including a potential new autochthonous focus of the disease in central Europe) are discussed.

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.

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.