Wolfgang Presber

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

Abstract 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.

Strain typing in Leishmania donovani by using sequence-confirmed amplified region analysis.

To differentiate strains of Leishmania donovani, allelic markers at the DNA level were developed by sequence-confirmed amplified region analysis (SCAR). Homologous fragments from different strains of L. donovani were amplified by PCR using random primers and subsequently screened for single-strand conformation polymorphisms. Direct sequencing revealed 55 sequence polymorphisms in eight co-dominant DNA markers; 38 of them were single point mutations. Heterozygosity was evident for 69% and fixed heterozygosity for 25% of all polymorphisms. At most polymorphic sites one of the segregation genotypes was missing. Nineteen unique multilocus genotypes were identified among 29 strains of L. donovani. One genotype was represented by eight Sudanese strains; also two strains from Sudan as well as two strains from Kenya, respectively, shared identical genotypes. All other strains had individual multilocus genotypes. Calculation of genetic distances showed a correlation between multilocus genotypes and the geographical origin of these strains. African strains were found in one well-supported cluster with Kenyan and Sudanese strains clearly separated. SCAR markers seem to represent a random sample of neutral genetic variation present in natural populations. They are co-dominant because they can detect all possible allele combinations in a diploid organism and may, therefore, be very useful for population genetic analysis in Leishmania.

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

Abstract 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 diversity and structure in Leishmania infantum populations from southeastern Europe revealed by microsatellite analysis

BackgroundThe dynamic re-emergence of visceral leishmaniasis (VL) in south Europe and the northward shift to Leishmania-free European countries are well-documented. However, the epidemiology of VL due to Leishmania infantum in southeastern (SE) Europe and the Balkans is inadequately examined. Herein, we aim to re-evaluate and compare the population structure of L. infantum in SE and southwestern (SW) Europe.MethodsLeishmania strains collected from humans and canines in Turkey, Cyprus, Bulgaria, Greece, Albania and Croatia, were characterized by the K26-PCR assay and multilocus enzyme electrophoresis (MLEE). Genetic diversity was assessed by multilocus microsatellite typing (MLMT) and MLM Types were analyzed by model- and distance- based algorithms to infer the population structure of 128 L. infantum strains.ResultsL. infantum MON-1 was found predominant in SE Europe, whilst 16.8% of strains were MON-98. Distinct genetic populations revealed clear differentiation between SE and SW European strains. Interestingly, Cypriot canine isolates were genetically isolated and formed a monophyletic group, suggesting the constitution of a clonal MON-1 population circulating among dogs. In contrast, two highly heterogeneous populations enclosed all MON-1 and MON-98 strains from the other SE European countries. Structure sub-clustering, phylogenetic and Splitstree analysis also revealed two distinct Croatian subpopulations. A mosaic of evolutionary effects resulted in consecutive sub-structuring, which indicated substantial differentiation and gene flow among strains of both zymodemes.ConclusionsThis is the first population genetic study of L. infantum in SE Europe and the Balkans. Our findings demonstrate the differentiation between SE and SW European strains; revealing the partition of Croatian strains between these populations and the genetic isolation of Cypriot strains. This mirrors the geographic position of Croatia located in central Europe and the natural isolation of the island of Cyprus. We have analysed the largest number of MON-98 strains so far. Our results indicate extensive gene flow, recombination and no differentiation between MON-1 and MON-98 zymodemes. No correlation either to host specificity or place and year of strain isolation was identified. Our findings may be associated with intensive host migration and common eco-epidemiological characteristics in these countries and give valuable insight into the dynamics of VL.

Molecular markers for Phlebotomus papatasi (Diptera: Psychodidae) and their usefulness for population genetic analysis

Three molecular typing tools: multilocus microsatellite typing, cytochrome b sequence analysis and internal transcribed spacer 2 (ITS2) sequence analysis, were evaluated for their usefulness in inferring the population structure of Phlebotomus papatasi sand flies. ITS2 sequence analysis did not prove suitable for inferring phylogenetic and population genetic relationships across P. papatasi sand flies. Microsatellite markers showed high resolution in differentiating globally distributed P. papatasi populations, whereas cytochrome b sequence analysis provided insight into the relationships between closely related populations from the Mediterranean. Population structure, differentiation and demographic history among P. papatasi are important for understanding patterns of dispersal in this species and for planning appropriate control measures.

Accelerated active case detection of visceral leishmaniasis patients in endemic villages of Bangladesh.

Background The visceral leishmaniasis (VL) elimination program in Bangladesh is in its attack phase. The primary goal of this phase is to decrease the burden of VL as much as possible. Active case detection (ACD) by the fever camp method and an approach using past VL cases in the last 6–12 months have been found useful for detection of VL patients in the community. We aimed to explore the yield of Accelerated Active Case Detection (AACD) of non-self reporting VL as well as the factors that are associated with non-self reporting to hospitals in endemic communities of Bangladesh. Methods Our study was conducted in the Trishal sub-district of Mymensingh, a highly VL endemic region of Bangladesh. We used a two-stage sampling strategy from 12 VL endemic unions of Trishal. Two villages from each union were selected at random. We looked for VL patients who had self-reported to the hospital and were under treatment from these villages. Then we conducted AACD for VL cases in those villages using house-to-house visit. Suspected VL cases were referred to the Trishal hospital where diagnosis and treatment of VL was done following National Guidelines for VL case management. We collected socio-demographic information from patients or a patient guardian using a structured questionnaire. Results The total number of VL cases was 51. Nineteen of 51 (37.3%) were identified by AACD. Poverty, female gender and poor knowledge about VL were independent factors associated with non self-reporting to the hospital. Conclusion Our primary finding is that AACD is a useful method for early detection of VL cases that would otherwise go unreported to the hospital in later stage due to poverty, poor knowledge about VL and gender inequity. We recommend that the National VL Program should consider AACD to strengthen its early VL case detection strategy.

Evaluierung eines PCR-Testsystems für den Direktnachweis von Chlamydia trachomatis

Ziel der vorliegenden Studie war ein Vergleich der Ergebnisse, die mit einem PCR-Testkitbzw. einem Enzymimmunoassaybeim Direktnachweis von Chlamydia trachomatis erhalten wurden. 394 Proben von 98 weiblichen und männlichen STD-Patienten sowie von 236 schwangeren Frauen (Zervikalund Urethralabstrichen sowie Urinproben) wurden untersucht Von insgesamt 31 positiven Ergebnissen wurden 29 mit der PCR und 21 mit dem EIA gefunden, was einerSensitivität von 93,5% bzw. 68% entsprach. Die Spezifität lag bei beiden Testsystemen bei ca. 100%. Bei der Überprüfung der W PCRpositiven, jedoch EIA-negativen Befunde in einem zweiten PCR-Ansatz mit einem anderen Primerpaar konnten 9 PCR-Befunde bestätigt werden, ein Ergebnis erwies sich als negativ. Bei 2 falsch-negativen PCR-Resultaten wurden nach einer Phenol/Chloroform-Extraktion der Proben positive PCRBefunde erhalten. Bei 3 Patienten mit positivem PCR-Nachweis wurde die Testung 3-4 Wochen nach Therapiebeginn wiederholt. In allen Fällen konnten keine Chlamydien-DNA mehr nachgewiesen werden. Die Vorteile des Amplicor PCR-Testkits sind die hohe Empfindlichkeit und die geringe Zeitdauer des Tests. Für Probenvorbereitung, Amplifikation und Detektion des PCR-Produkts werden etwa 4 bis 5 Stunden benötigt.