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.     

Identification of Old World Leishmania species by PCR–RFLP of the 7 spliced leader RNA gene and reverse dot blot assay

The aim of the study was to assess the 7SL RNA PCR followed by restriction fragment length polymorphism (RFLP) and reverse dot blot (RDB) assays for use in identification of Old World Leishmania species. Species‐specific RFLP patterns were obtained for Leishmania major, Leishmania tropica and the Leishmania donovani complex when the 7SL RNA PCR product was digested with the restriction enzyme BsuRI, an isoschizomer of HaeIII. For the RDB assay, biotin‐labelled 7SL RNA amplicons were hybridized to Leishmania genus‐specific and species‐specific oligonucleotide probes immobilized onto a membrane. The Old World Leishmania species could be distinguished by using five probes: one that was a genus‐specific probe and hybridized to all Leishmania species (Lc), two that were specific for L. major (Lm1 and Lm2), one that was specific for L. tropica (Lt) and one that detected both L. major and L. tropica (Lmt). The PCR–RDB was 10 times more sensitive than 7SL PCR and can detect <1 parasite. In addition, the identification of species was easier and more reliable than with 7SL PCR–RFLP. 7SL PCR–RFLP detected parasites in 50 of 57 clinical samples, whereas PCR–RDB detected 53 and 55 were detected by amplification of kinetoplast (k) DNA. The 7SL RNA PCR has proven useful for direct diagnosis of Old World leishmaniasis, especially when combined with the RBD assay for species identification.     

Leishmaniasis in Turkey: molecular characterization of Leishmania from human and canine clinical samples

Human leishmaniasis, both visceral and cutaneous, and canine leishmaniasis have been reported in Turkey for centuries. However, the advent of new diagnostic tools during the last 30 years has led to the recognition that leishmaniasis is an important public health problem throughout the country. In most disease foci both canine and human leishmaniases exist together and identification of parasite species causing these diseases is a pre-requisite for understanding disease epidemiology. A total of 109 samples obtained from human and canine leishmaniasis cases were examined using internal transcribed spacer 1 PCR followed by restriction fragment length polymorphism analysis. Our results indicate that two species, Leishmania tropica and Leishmania infantum, are primarily responsible for cutaneous and visceral leishmaniasis, respectively, in Turkey. However, a new focus of human cutaneous leishmaniasis caused by L. infantum in Hatay region is described. This finding further stresses the importance of Leishmania species molecular characterization in prescribing appropriate therapy and understanding the disease's transmission in different endemic foci.     

Methylenetetrahydrofolate reductase C677T gene polymorphism and the association with dyslipidemia in type 2 diabetic Palestinian patients

BackgroundDyslipidemia in diabetes is common and characterized by hypertriglyceridemia with decreased levels of high‐density lipoprotein. The objective of this study was to assess the prevalence of MTHFR C677T polymorphism in Palestinian T2DM patients and to investigate the association between this polymorphism and lipid profile in diabetic patients with and without dyslipidemia.MethodsA total of 208 T2DM patients including 98 with dyslipidemia and 110 without dyslipidemia were enrolled in this study. The MTHFR C677T genotyping was conducted by PCR‐RFLP followed by agarose gel electrophoresis.ResultsThere were no significant differences in either the genotype distribution or allele frequency in T2DM patients with or without dyslipidemia (37.8% CC, 54% CT, 8.2% TT vs. 48.2% CC, 41.8% CT, 11% TT; p = 0.209). However, among the dyslipidemic group, the TT carriers have a higher HDL level (46.8 ± 17.8) compared to (CC+CT) carriers (34.68 + 11.9) (p = 0.01). In the group without dyslipidemia, there was a significant elevation in diastolic blood pressure (DBP) among the CC carriers (83.6 ± 10.6) compared to those who carried at least one mutant allele (CT+TT) (78.1 ± 11.1) (p = 0.009).ConclusionsThe study shows that in our Palestinian population the MTHFR 677TT genotype lowers DBP significantly in patients without dyslipidemia and is related to increased level of HDL in diabetic dyslipidemia patients.     

Multifarious characterization of leishmania tropica from a Judean desert focus, exposing intraspecific diversity and incriminating phlebotomus sergenti as its vector

The predominant sand fly species collected inside houses in Kfar Adumim, an Israeli village in the Judean Desert that is a focus of cutaneous leishmaniasis, was Phlebotomus papatasi, which was also caught attempting to bite humans. Phlebotomus sergenti, which is rarely seen inside houses, constituted the predominant sand fly species in caves near the village. Leishmania isolates from Ph. sergenti and humans typed as Leishmania tropica. Sand fly and human isolates produced similar small nodular cutaneous lesions in hamsters. Isolates produced excreted factor (EF) of subserotypes A(9) or A(9)B(2), characteristic of L. tropica and reacted with L. tropica-specific monoclonal antibodies. Isoenzyme analysis consigned the strains to the L. tropica zymodemes MON-137 and MON-275. Molecular genetic analyses confirmed the strains were L. tropica and intraspecific microheterogeneity was observed. Genomic fingerprinting using a mini-satellite probe separated the L. tropica strains into two clusters that were not entirely congruent with geographic distribution. These results support the heterogeneous nature of L. tropica and incriminate Ph. sergenti as its vector in this Judean Desert focus.     

Palestinian infantile visceral leishmaniasis caused by a genetic variant of Leishmania infantum belonging to a new zymodeme

The parasites causing a Palestinian case of infantile visceral leishmaniasis (IVL) and those from four dogs from the Jenin District were identified serologically, biochemically and molecular biologically as Leishmania infantum, showing dogs act as a reservoir. The strain from the human case was distinct because of its unique 200-bp kDNA-polymerase chain reaction (PCR) component in its restriction fragment length polymorphism (RFLP) profile after digestion with the endonuclease RsaI, and by the electrophoretic mobility of its malate dehydrogenase (MDH(140)), designating it the reference strain of a new zymodeme of L. infantum, MON-281.     

Rapid Diagnosis of Old World Leishmaniasis by High-Resolution Melting Analysis of the 7SL RNA Gene

High-resolution melt analysis PCR (HRM PCR) for diagnosis of Old World Leishmania was developed using the 7SL RNA gene. Cutaneous leishmaniasis samples were analyzed. Sensitivity and specificity of HRM PCR were significantly better (P < 0.001) than those of internal transcribed spacer 1 PCR and similar to those of kinetoplast DNA PCR.Leishmania causes cutaneous (CL), mucocutaneous, and visceral leishmaniasis (VL), and the diagnosis, treatment, and prognosis are dependent on accurate identification of the infecting parasite species. Microscopic and culture-based diagnostic methods are being replaced by rapid, highly sensitive and specific molecular techniques based on PCR that target coding and noncoding regions of the Leishmania genome (8). At minimum, these assays require post-PCR examination by agarose gel electrophoresis (1), and species identification requires additional processing by restriction fragment length polymorphism (RFLP) (8), hybridization (5), DNA sequencing (12), etc.High-resolution melt (HRM) analysis is a relatively new technique that allows direct characterization of PCR amplicons in a closed system. It measures changes in the fluorescence intensity of a DNA intercalating dye during dissociation from double-stranded DNA to single-stranded DNA and can differentiate between single nucleotide polymorphisms (SNP). HRM has been used for analysis of genomic mutations (7), SNP genotyping (4), Mycobacterium tuberculosis rifampin susceptibility (3), Staphylococcus aureus typing (10), etc. HRM is simpler, less expensive, and faster than conventional PCR assays that require post-PCR processing.A previous study comparing Leishmania 7SL RNA gene DNA sequences suggested that the gene might be a good diagnostic target (12). An HRM PCR assay for Old World Leishmania based on this gene (7SL-HRM) is described here that can be used to differentiate between L. tropica, L. major, and the L. donovani complex. The sensitivity and specificity of this assay, as well as species identification using clinical samples, were compared with those of assays frequently used for parasite diagnosis, internal transcribed spacer 1 (ITS1) and kinetoplast DNA (kDNA) PCR.Cultured Leishmania strains (n = 77) were grown in M199 medium containing 10% fetal calf serum (FCS) and antibiotics. L. infantum (MHOM/PS/1999/LRC-L773), L. major (MHOM/IL/1980/Freidlin), L. tropica genotype I (MHOM/IL/2006/LRC-L1286), and L. tropica genotype IV (IARA/IL/2000/LRC-L810) were used as reference strains. Clinical samples on filter paper for routine diagnosis by ITS1 PCR were prepared as described previously (1), and DNA was extracted using the High Pure PCR template preparation kit (Roche, Germany). All samples were analyzed by ITS1 PCR, and if positive, species were determined by RFLP (8). Some samples were analyzed by kDNA PCR (1). The Helsinki Committee for Human Research of the Hadassah Hospital, Ein Kerem, Jerusalem, Israel, approved this study.7SL RNA gene sequences of L. donovani complex (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"AY722733","term_id":"57340412","term_text":"AY722733"}}AY722733, {"type":"entrez-nucleotide","attrs":{"text":"AY722734","term_id":"57340413","term_text":"AY722734"}}AY722734, {"type":"entrez-nucleotide","attrs":{"text":"AY722735","term_id":"57340414","term_text":"AY722735"}}AY722735, {"type":"entrez-nucleotide","attrs":{"text":"AY781795","term_id":"59800236","term_text":"AY781795"}}AY781795, and {"type":"entrez-nucleotide","attrs":{"text":"AY781792","term_id":"59800233","term_text":"AY781792"}}AY781792), L. tropica ({"type":"entrez-nucleotide","attrs":{"text":"AY722722","term_id":"57340401","term_text":"AY722722"}}AY722722, {"type":"entrez-nucleotide","attrs":{"text":"AY722723","term_id":"57340402","term_text":"AY722723"}}AY722723, and {"type":"entrez-nucleotide","attrs":{"text":"AY781789","term_id":"59800230","term_text":"AY781789"}}AY781789), and L. major ({"type":"entrez-nucleotide","attrs":{"text":"AY722713","term_id":"57340392","term_text":"AY722713"}}AY722713, {"type":"entrez-nucleotide","attrs":{"text":"AY722714","term_id":"57340393","term_text":"AY722714"}}AY722714, and {"type":"entrez-nucleotide","attrs":{"text":"AY722715","term_id":"57340394","term_text":"AY722715"}}AY722715) were compared by multialignment (http://bioinfo.genotoul.fr/multalin/multalin.html) (2). The PCR primers CJ7SLF (5′-ACG TGG ACC AGC GAG GGT-3′) and QRT7SLR (5′-CGG TTC CCT CGC TTC AAC-3′), in conserved regions flanking a 119-bp polymorphic internal region, were designed using the primer3 software program (http://frodo.wi.mit.edu/primer3/) (6).7SL RNA PCR was carried out as follows: DNA samples or controls (2 μl) were added to 2× Thermo-Start PCR master mix (10 μl; Thermo Scientific), Syto 9 (1.5 μM; Invitrogen), primers (0.5 μM [each] final concentration), and ultra-pure PCR-grade water (Fisher); final volume, 20 μl/PCR. Amplification conditions were as follows: 15 min of denaturation at 95°C, followed by 40 cycles of denaturation for 5 s at 95°C; annealing for 10 s at 55°C; and a final extension for 2 min at 72°C. A 119-bp amplicon was observed with the Leishmania reference strains. HRM ramping was carried out at 0.1°C/s from 75 to 95°C. HRM PCR and analysis were performed using a Rotor-Gene 6000 real-time thermal analyzer (Corbett Life Science). Positive-control (reference strain DNA, 20 ng/reaction) and negative-control reactions were included in each experiment. PCR efficiency was evaluated using the threshold cycle (C_T), and a normalized melt window, 88 to 92°C, was used in analyzing HRM curves. Statistical analysis was performed using the SPSS computer software program, v.15.0.The sensitivity, specificity, and discrimination between species endemic to the Middle East (L. infantum, L. major, and L. tropica) were examined using reference strains (Fig. ​(Fig.11 and data not shown). Sensitivity was determined using 10-fold dilutions of each DNA. Plots of C_T versus log DNA concentration were linear over 7 orders of magnitude (2 × 10⁴ to 2 × 10⁻² pg; r² = 0.995), and detection limits were identical, 0.02 pg DNA/species. The average melting point (T_m) ± standard deviation (SD) for 30 7SL-HRM runs was as follows: L. major, 89.27 ± 0.06°C; L. tropica genotype IV, 89.49 ± 0.09°C; L. tropica genotype I, 89.78 ± 0.04°C; and L. infantum, 90.58 ± 0.02°C (indicating that each T_m is highly reproducible). Melting curves and T_m were unaffected by the quantity of DNA used and varied by ±0.06°C over the concentration range examined.Open in a separate windowFIG. 1.Comparison of normalized high-resolution melting (HRM) curves of the 7SL PCR amplicon obtained for Leishmania references species (Leishmania tropica genotype I [LtropI], MHOM/IL/2006/LRC-L1286; L. tropica genotype IV [LtropIV], IARA/IL/2000/LRC-L810; L. major [LmajC], MHOM/IL/1980/Freidlin; and L. infantum [LinfC], MHOM/PS/1999/LRC-L773) and five random patient samples (no. 845, 846, 847, 848, and 853).Seventy-seven leishmanial strains (for L. tropica, n = 43; for L. major, n = 19; for L. infantum, n = 13; and for L. donovani, n = 2) from the Mediterranean Region (n = 45), Middle East (n = 10), Central Asia (n = 5), Africa (n = 13), and India (n = 4) were examined (see the supplemental material). HRM analysis correctly typed all the strains, regardless of parasite geographic or host origin. In addition, 7SL-HRM distinguished between L. tropica microsatellite genotypes I (n = 34) and IV (n = 9) found in Israel and the Palestinian Authority (9). DNA sequencing showed this was due to an SNP, C/T, at position 98 in the targeted gene sequence (data not shown). Nonleishmanial kinetoplastids, Sauroleishmania tarentolae (P10 strain), Trypanosoma brucei (BF427 strain), Crithidia fasiculata (ATCC 11745), Leptomonas seymouri (ATCC 30220), Phytomonas davidi (ATCC 50166), and Trypanosoma cruzi, were all negative (data not shown).ITS1 PCR and 7SL-HRM were used to screen clinical samples (n = 192) (Fig. ​(Fig.11 and data not shown), and the results are summarized in Table ​Table1.1. By ITS1 PCR, 49% (94/192) were positive, while the remaining 51% gave no PCR product, i.e., they were negative. Of the positive samples, 81/94 (86%) produced sufficient product for species determination by RFLP analysis: L. tropica (n = 37), L. major (n = 41), or L. donovani complex (n = 3). An additional 20 samples were positive when examined by 7SL-HRM (total number positive = 114/192 [59%]), all of which could be typed: L. tropica (n = 48), L. major (n = 61), or L. donovani complex (n = 4). Species identification of samples examined by both assays correlated 100%. The remaining samples (n = 78; 41%) were negative. Significant correlation (Fisher''s exact test, two-tailed P value < 0.0001) and good agreement (κ = 0.78) were found between the assays. In addition, using HRM it was possible to distinguish between L. tropica genotypes I (n = 28) and IV (n = 20).

TABLE 1.

Comparison of human leishmaniasis diagnosis by ITS1 and 7SL-HRM PCR