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.