Association of G22A polymorphism of the adenosine deaminase (ADA) gene with biochemical characteristics in type 2 diabetic Palestinians

The adenosine deaminase G22A polymorphism (20q.11.33) affects the level of adenosine deaminase (ADA) expression, which plays an important role in the regulation of intracellular and extracellular concentrations of adenosine. Recent studies reported greater ADA activity in diabetic patients and showed the role of ADA in the modulation of insulin activity and glucose homeostasis. We investigated whether the G22A polymorphism of the ADA gene is associated with type 2 diabetes mellitus (T2DM) in the Palestinian population and assessed the relationship between the G22A variant and fasting plasma glucose (FPG), glycated hemoglobin (HbA1c) and lipid profile among T2DM patients. A total of 231 individuals with T2DM and a control sample of 101 non-diabetic participants were randomly selected from those who were attending United Nations Relief and Works Agency (UNRWA) clinics for treatment and/or follow up. Genomic DNA was extracted from peripheral blood samples and PCRRFLP was performed to identify the TaqI polymorphism G22A of the ADA gene. No significant differences were observed in the genotype and allele frequencies between T2DM patients and the control group. Yet, among diabetic patients, the GG genotype was significantly associated with higher FPG and HbA1c when compared to the GA+AA genotype but had no influence on blood pressure, BMI or other metabolic parameters. In conclusion, we confirm that the GG genotype of the ADA gene is associated with poor glycemic control in T2DM Palestinians and points to the association of the G22A variant with decreased activity of the ADA enzyme, which is of paramount importance in the pathophysiology of T2DM.The authors thank the patients for participating in the study. This research was financially supported by the deanship of scientific research of Al-Quds University, Palestine

Genetic, serological and biochemical characterization of Leishmania tropica from foci in northern Palestine and discovery of zymodeme MON-307

Background Many cases of cutaneous leishmaniasis (CL) have been recorded in the Jenin District based on their clinical appearance. Here, their parasites have been characterized in depth. Methods Leishmanial parasites isolated from 12 human cases of CL from the Jenin District were cultured as promastigotes, whose DNA was extracted. The ITS1 sequence and the 7SL RNA gene were analysed as was the kinetoplast minicircle DNA (kDNA) sequence. Excreted factor (EF) serotyping and multilocus enzyme electrophoresis (MLEE) were also applied. Results This extensive characterization identified the strains as Leishmania tropica of two very distinct sub-types that parallel the two sub-groups discerned by multilocus microsatellite typing (MLMT) done previously. A high degree of congruity was displayed among the results generated by the different analytical methods that had examined various cellular components and exposed intra-specific heterogeneity among the 12 strains. Three of the ten strains subjected to MLEE constituted a new zymodeme, zymodeme MON-307, and seven belonged to the known zymodeme MON-137. Ten of the 15 enzymes in the profile of zymodeme MON-307 displayed different electrophoretic mobilities compared with the enzyme profile of the zymodeme MON-137. The closest profile to that of zymodeme MON-307 was that of the zymodeme MON-76 known from Syria. Strains of the zymodeme MON-307 were EF sub-serotype A2 and those of the zymodeme MON-137 were either A9 or A9B4. The sub-serotype B4 component appears, so far, to be unique to some strains of L. tropica of zymodeme MON-137. Strains of the zymodeme MON-137 displayed a distinctive fragment of 417 bp that was absent in those of zymodeme MON-307 when their kDNA was digested with the endonuclease RsaI. kDNA-RFLP after digestion with the endonuclease MboI facilitated a further level of differentiation that partially coincided with the geographical distribution of the human cases from which the strains came. Conclusions The Palestinian strains that were assigned to different genetic groups differed in their MLEE profiles and their EF types. A new zymodeme, zymodeme MON-307 was discovered that seems to be unique to the northern part of the Palestinian West Bank. What seemed to be a straight forward classical situation of L. tropica causing anthroponotic CL in the Jenin District might be a more complex situation, owing to the presence of two separate sub-types of L. tropica that, possibly, indicates two separate transmission cycles involving two separate types of phlebotomine sand fly vector

Complete genome sequencing of SARS-CoV-2 strains: A pilot survey in Palestine reveals spike mutation H245N

Objectives: SARS-CoV-2, severe respiratory syndrome coronavirus-2, is an RNA virus that emerged from China sweeping the globe in the form of a pandemic that became an international public health concern. This pilot study aimed to describe the genetic variation and molecular epidemiology of SARS-CoV-2 in Palestine in fall 2020. Results: To achieve these aims, whole genome sequencing of SARS-CoV-2, phylogenetic analysis, haplotype networking and genetic diversity analysis were performed. These analyses revealed a unique spike mutation H245N that has never been reported before. The phylogenetic analysis depicted that three clusters existed in Palestinian SARS-CoV-2 genome sequences, in which cluster-I comprised the majority of clusters by 90%. Congruently, the haplotype network analysis depicted the same three clusters with a total of 39 haplotypes. The genetic diversity analysis showed that Cluster-I is highly diverse as confirmed by statistically significant mutation rate indices, Tajima's D and Fu-Li's-F tests (- 2.11 and 2.74, respectively), highest number of mutations (Eta = 120), highest number of haplotypes (h = 17), and highest average number of nucleotide differences between any two sequences (S = 118). The study confirmed the high genetic diversity among the Palestinian of SARS-CoV-2 which possessed high number of mutations including one which was reported for the first time

Molecular characterization of Anaplasma and Ehrlichia in ixodid ticks and reservoir hosts from Palestine: a pilot survey

Tick-borne anaplasmosis and ehrlichiosis are clinically important emerging zoonoses usually overlooked by veterinarians and physicians alike. This study aimed at detecting and genetically characterizing Ehrlichia and Anaplasma species in ixodid ticks and their animal hosts from the West Bank, Palestine. A total of 723 ixodid ticks belonging to three genera (Rhipicephalus, Hyalomma, Haemaphysalis) were collected from dogs, sheep, goats and camels. In addition, 189 blood samples were collected from dogs, sheep, camels, horses and a goat from the West Bank, Palestine. All tick and blood samples were investigated for the presence of Anaplasma and Ehrlichia targeting a 345 bp fragment of the 16S rRNA gene followed by sequence analysis. The infection rate of Anaplasma spp. in ticks was 6.5% (47/723). Anaplasma platys was identified in 28% (13/47) of them. Whereas, based on a partial sequence (851 bp) of msp4 gene, 38% (18/47) were identified as A. ovis. The species of the remaining 16 positive samples (16/47, 34%) could not be identified. Simultaneously, the infection rate of Ehrlichia spp. in the ticks was 0.6% (4/723). Three of which were E. canis and one was Ehrlichia spp. The infection rate of A. platys in dogs' blood samples was 10% (13/135), while it was 1.5% (2/135) for E. canis. The infection rate of Anaplasma in sheep blood samples was 40% (19/47), out of which 26% (5/19) were caused by A. ovis as revealed by msp4-PCR. Implementation of purely-spatial analysis by saTScan for all cases of Anaplasma revealed two statistically significant clusters in two districts; Tubas town and Majdal-Bani-Fadil village on the western hills of the Jordan Valley. Most cases of Anaplasma (83%) were from rural areas where life cycle components (vector, host and reservoir) abundantly interact. This study is the first in Palestine to reveal the presence of Anaplasma and Ehrlichia in ticks, dogs and sheep providing crucial platform for future epidemiological surveys and control strategies in the country and regio

Estrogen receptor 1 gene polymorphisms (PvuII and XbaI) are associated with type 2 diabetes in Palestinian women

supplementary filesBackground Type 2 diabetes mellitus (T2DM) is a multifactorial disease where both genetic and environmental factors contribute to its pathogenesis. The PvuII and XbaI polymorphisms of the estrogen receptor 1 (ESR1) gene have been variably associated with T2DM in several populations. This association has not been studied in the Palestinian population. Therefore, the aim of this study was to investigate the association between the PvuII and XbaI variants in the ESR1 and T2DM and its related metabolic traits among Palestinian women. Methods This case–control study included 102 T2DM and 112 controls in which PvuII and XbaI variants of the ESR1 gene were genotyped using amplicon based next generation sequencing (NGS). Results Allele frequencies of both PvuII and XbaI variants were not significantly different between patients and control subjects (P > 0.05). In logestic regression analysis adjusted for age and BMI, the ESR1 PvuII variant was associated with risk of T2DM in three genotypic models (P 0.025). Among diabetic group, an inverse trend with risk of cardio vascular diseases was shown in carriers of CG haplotype compared to those with TA haplotype (OR = 0.28, CI [0.09–0.90]; adjusted P = 0.035). Further, stratified analyses based on ESR1 PvuII and XbaI genotypes revealed no evidence for association with lipid levels (TC, TG, HDL, LDL). Conclusions This is the first Palestinian study to conclude that ESR1 PuvII and XbaI variants may contribute to diabetes susceptibility in Palestinian women. Identification of genetic risk markers can be used in defining high risk subjects and in prevention trials

Tracking the geographical origin of Plasmodium falciparum causing a rare severe case of malaria imported into Palestine, a zero-indigenous case area

Background Malaria cases in non-endemic zero-indigenous case areas are most likely to have been imported whatever of the route of importation. In countries recently declared malaria-free and now without local transmission, imported cases remain a threat to re-introduction of the disease and a burden on the health system. Case presentation Three days after returning from a long trip to malaria- endemic countries; Abyei-Sudan, Chad and Uganda, a 41-year-old male resident from Jericho, Palestine, suffered paroxysms of fever, general fatigue, myalgia, arthralgia, headache, and a strong desire to vomit. Thin and thick Giemsa-stained blood smears were prepared and examined microscopically using oil immersion. Immature trophozoites (ring forms) were seen to parasitize approximately 10% of the erythrocytes revealing hyperparasitemia equivalent to > 100,000 parasites/ µl indicating severe malaria [1, 2]. The double chromatin configuration (headphones) and accolé (applique) position are both indicative of Plasmodium falciparum infection. The 18S rRNA- PCR targeting the rPLU6-rPLU5 region was used to confirm the diagnosis. The next-generation sequencing (NGS) method was carried out according to the manufacturer’s instructions (Illumina® DNA Prep, (M) Tagmentation kit (20060060), Illumina) to identify Plasmodium spp. Furthermore, NGS produced a whole-genome sequence of 22.8Mbp of the 14 chromosomes and 25Kbp of the apicoplast. A BLAST search of the apicoplast DNA and selected chromosomal DNA revealed that P. falciparum was the causative agent. The merozoite surface protein-1 (msp-1) was used to construct a phylogenetic tree of 26 P. falciparum, including the one isolated from the patient from Jericho, which clustered with the Sudanese isolate indicating genetic relatedness between the two. Conclusion The travel history together with signs and symptoms of malaria, followed by prompt diagnosis using conventional microscopic inspection of Giemsa-stained films together with molecular DNA tracking tools like msp-1 were key means in tracking the place of origin of infection in the case of travel to multiple destination

Metagenomic profiling of ticks: Identification of novel rickettsial genomes and detection of tick-borne canine parvovirus.

Across the world, ticks act as vectors of human and animal pathogens. Ticks rely on bacterial endosymbionts, which often share close and complex evolutionary links with tick-borne pathogens. As the prevalence, diversity and virulence potential of tick-borne agents remain poorly understood, there is a pressing need for microbial surveillance of ticks as potential disease vectors. METHODOLOGY/PRINCIPAL FINDINGS: We developed a two-stage protocol that includes 16S-amplicon screening of pooled samples of hard ticks collected from dogs, sheep and camels in Palestine, followed by shotgun metagenomics on individual ticks to detect and characterise tick-borne pathogens and endosymbionts. Two ticks isolated from sheep yielded an abundance of reads from the genus Rickettsia, which were assembled into draft genomes. One of the resulting genomes was highly similar to Rickettsia massiliae strain MTU5. Analysis of signature genes showed that the other represents the first genome sequence of the potential pathogen Candidatus Rickettsia barbariae. Ticks from a dog and a sheep yielded draft genome sequences of Coxiella strains. A sheep tick yielded sequences from the sheep pathogen Anaplasma ovis, while Hyalomma ticks from camels yielded sequences belonging to Francisella-like endosymbionts. From the metagenome of a dog tick from Jericho, we generated a genome sequence of a canine parvovirus. SIGNIFICANCE: Here, we have shown how a cost-effective two-stage protocol can be used to detect and characterise tick-borne pathogens and endosymbionts. In recovering genome sequences from an unexpected pathogen (canine parvovirus) and a previously unsequenced pathogen (Candidatus Rickettsia barbariae), we demonstrate the open-ended nature of metagenomics. We also provide evidence that ticks can carry canine parvovirus, raising the possibility that ticks might contribute to the spread of this troublesome virus

Molecular epidemiology of human cutaneous leishmaniasis in Jericho and its vicinity in Palestine from 1994 to 2015

Cutaneous leishmaniases (CL) are vector-borne parasitic diseases endemic inmany countries of the Middle East including Palestine. Between 1994 and 2015, 2160 clinically suspected human cases of CL from the Jericho District were examined. Stained skin tissue smears and aspirates were checked by microscopy and cultured for promastigotes, respectively. For leishmanial species identification, amplification products from a PCR-ITS1 followed by RFLP analysis using Hae III. Data were analyzed using Epi Info free-software. The overall infection rate was 41.4% (895/2160), 56.3% (504/895) of the cases were male, 43.7% (391/895) female, 60.5% (514/849) children under age 14, 41.3% (259/627) of the cases were caused by Leishmania major and 57.3% (359/627) by Leishmania tropica. The case numbers peaked in 1995, 2001, 2004, and 2012. Statistically-significant clusters of cases caused by L. major were restricted to the Jericho District; those caused by L. tropica were from the districts of Jericho, Bethlehem, Nablus and Tubas. CL is seasonal and trails the sand fly season. Distribution of cases was parabolicwith fewest in July. Themonthly total number of cases of CL and just those caused by L.major correlated significantly with temperature, rainfall, relative humidity, evaporation, wind speed and sunshine (P b 0.05, r2= 0.7–0.9 and P b 0.05, r2=0.5–0.8, respectively). Cases caused by L. tropica, significantly, had a single lesion compared to cases caused by L. major (P=0.0001), which, significantly, had multiple lesions (P=0.0001). This and previous studies showed that CL is present in all Palestinian districts. The surveillance of CL has increased public awareness and molecular biologicalmethodology for leishmanial species identification is an essential addition to classical diagnosis. The overall results are discussed, correlated to climatic and environmental changes and largescale human activities.This work received financial support from grants of the Deutsche Forschungsgemeinschaft (DFG), Scho 448/6-1-3, Scho 448/8-1, Scho 448/8-2 that extended from 1998 until 2015. It also received support fromEurNegVeg COST Action TD1303 (Cost 037/13). At one time during the study WHO Eastern Mediterranean Region (EMRO), Division of Communicable Diseases (DCD) and the WHO Special Programme for Research and Training in Tropical Diseases (TDR): the EMRO/DCD/TDR Small Grants Scheme for Operational Research in Tropical and Communicable Diseases financially supported this work. We thank Dr. L. F. Schnur for going over our manuscript

Genetic diversity and haplotype analysis of Leishmania tropica identified in sand fly vectors of the genera Phlebotomus and Sergentomyia using next-generation sequencing technology.

Next-generation sequencing (NGS) was used to investigate the genetic diversity of Leishmania tropica in the sand fly vector, targeting the internal transcribed spacer 1 (ITS1) of the genus Leishmania. Bioinformatics analyses were conducted using Galaxy, MEGA version X, DnaSP ver. 6.12.03, and PopART 1.7 software for NGS analysis, phylogenetic tree, genetic diversity, and haplotype networking, respectively. A total of 307 engorged sand flies were trapped, with an overall Leishmania infection rate of 9.4 (29/307) and 6.8% by NGS and ITS1-PCR, respectively. Two Leishmania-infected sand fly genera were identified: Phlebotomus (10.2%, 26/254) and Sergentomyia (5.7% (3/53). The phylogenetic tree showed two clusters, cluster I included the four study sequences along with 25 GenBank-retrieved DNA sequences. Cluster II consisted of three sequences from Iran and Pakistan. The genetic diversity analysis for the 29 L. tropica sequences showed high haplotype (gene) diversity index (Hd) (0.62 ± 0.07) but low nucleotide diversity index (π) (0.04 ± 0.01). Tajima's D, a neutrality test, is more negative in cluster I (D = - 2.0) than in total population (D = - 1.83), but both are equally significant (P < 0.001), indicating that observed variation in cluster I and whole population is less frequent than expected. The median-joining haplotype network produced a total of 11 active haplotypes. In conclusion, L. tropica from sand flies in Palestine is monophyletic that assembled in one main phylogroup and one haplotype

Effectiveness of insecticide thermal fogging in hyrax dens in the control of leishmaniasis vectors in rural Palestine: A prospective study

Background: Zoonotic cutaneous leishmaniasis (ZCL) is endemic in Palestine and transmitted by Phlebotomus sand flies. They inhabit dens of hyraxes, the reservoir animal. Control measures were implemented since 1996 but cases still occur. We estimated the effect of insecticide thermal fogging inside hyrax dens on sand fly density and leishmania infection. Methodology/principal findings: During July-September 2019, we conducted a 12-week controlled interrupted time series study in two control and one intervention sites containing three hyrax dens each. We implemented Permethrin thermal fogging in the intervention site at week 6. We measured weekly and 36hrs post-intervention sand fly abundance inside dens using CDC light traps. We performed Next-Generation Sequencing to identify sand fly Leishmania spp. infection. We calculated the abundance reduction (AR) using Mulla's formula and negative binomial regression. Among 11427 collected sand flies, 7339 (64%) were females and 1786 (16%) were Phlebotomus spp. comprising ten species; P. sergenti was the dominant (n = 773, 43%). We report P. arabicus (n = 6) for the first time in Palestine. After fogging, Phlebotomus spp. AR was 93% at 36hrs, 18% and 38% at two and five weeks respectively and 41% during the complete post-intervention period. In the regression models, Phlebotomus spp. density in the intervention site decreased by 74% (IRR: 0.26, 95%CI: 0.11-0.57) at two weeks, 34% (IRR: 0.66, 95%CI: 0.48-0.90) at five weeks and 74% (IRR: 0.26, 95%CI: 0.12-0.59) during the complete period. The density of Leishmania infected sand flies decreased by 65% (IRR: 0.35, 95%CI: 0.26-0.48) at five weeks and 82% (IRR: 0.18, 95%CI: 0.07-0.42) for the complete period (zero infections until week two). Leishmania infection prevalence in the intervention site was 14% pre-intervention and 3.9% post-intervention. Conclusions/significance: Fogging hyrax dens reduced sand fly abundance and leishmania infection during the 5-week post-intervention period and especially the first two weeks suggesting it could be an effective source-reduction measure for ZCL vectors. Future randomized controlled trials are needed to confirm the effectiveness of fogging hyrax dens on decreasing ZCL incidence