Association of IFNAR2 rs2236757 and OAS3 rs10735079 Polymorphisms with Susceptibility to COVID-19 Infection and Severity in Palestine.

The clinical course and severity of COVID-19 vary among patients. This study aimed to investigate the potential correlation between the gene polymorphisms of the interferon receptor (IFNAR2) rs2236757 and oligoadenylate synthetase 3 (OAS3) rs10735079 with the risk of COVID-19 infection and its severity among Palestinian patients. The study was conducted between April and May 2021 on 154 participants who were divided into three groups: the control group (RT-PCR-negative, n = 52), the community cases group (RT-PCR-positive, n = 70), and the critically ill cases (ICU group; n = 32). The genotyping of the investigated polymorphisms was performed using amplicon-based next-generation sequencing. The genotypes distribution for the IFNAR2 rs2236757 was significantly different among the study groups (P = 0.001), while no statistically significant differences were found in the distribution of genotypes for the OAS3 rs10735079 (P = 0.091). Logistic regression analysis adjusted for possible confounding factors revealed a significant association between the risk allele rs2236757A and critical COVID-19 illness (P < 0.025). Among all patients, those who carried the rs2236757GA were more likely to have a sore throat (OR, 2.52 (95% CI 1.02-6.24); P = 0.011); the presence of the risk allele rs2236757A was associated with an increased risk to dyspnea (OR, 4.70 (95% CI 1.80-12.27); P < 0.001), while the rs10735079A carriers were less likely to develop muscle aches (OR, 0.34 (95% CI 0.13-0.88); P = 0.0248) and sore throat (OR, 0.17 (95% CI 0.05-0.55); P < 0.001). In conclusion, our results revealed that the rs2236757A variant was associated with critical COVID-19 illness and dyspnea, whereas the rs10735079A variant was protective for muscle aches and sore throat.

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.

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.

Concurrent molecular characterization of sand flies and Leishmania parasites by amplicon-based next-generation sequencing.

BACKGROUND: Phlebotomine sand flies are vectors of Leishmania parasites, which are the causative agents of leishmaniasis. Herein, we developed an amplicon-based next-generation sequencing (Amp-NGS) to characterize sand flies and Leishmania parasites simultaneously targeting partial fragments of 18S rDNA and ITS1 genes, respectively. METHODS: Our assay was optimized using reference sand fly (n = 8) and Leishmania spp. (n = 9) samples and validated using wild-caught sand flies from Palestine. The assay was highly specific, and all DNA references were successfully identified to the species level. RESULTS: Among the wild-caught sand flies (n = 187), Phlebotomus spp. represented 95% of the collected samples (177/187), including Ph. sergenti (147/187, 79%), Ph. papatasi (19/187, 10.2%), Ph. perfiliewi (3/187, 1.6%), Ph. tobbi (2/187, 1.2%) and Ph. syriacus (6/187, 3.2%). Sergentomyia spp. represented only 5% (10/187) of the collected samples and included S. dentata (n = 6), S. fallax (n = 2), S. schwetzi (n = 1) and S. ghesquiere (n = 1). The study observed strong positive correlation between sand fly identification results of the Amp-NGS and morphological identification method (r = 0.84, df = 185, P < 0.001). Some discrepancies between the two methods in the identification of closely related species (i.e. Ph. perfiliewi, Ph. tobbi and Ph. syriacus) were observed. Leishmania DNA was detected and identified as L. tropica in 14 samples (14/187, 7.5%). CONCLUSIONS: Our assay was sensitive to detect (limit of detection was 0.0016 ng/reaction) and identify Leishmania DNA in sand flies, thus representing a new tool for studying sand flies and their associated Leishmania parasites in endemic areas.

Tracking of SARS-CoV-2 Alpha variant (B.1.1.7) in Palestine.

As surges of the COVID-19 pandemic continue globally, including in Palestine, several new SARS-CoV-2 variants have been introduced. This expansion has impacted transmission, disease severity, virulence, diagnosis, therapy, and natural and vaccine-induced immunity. Here, 183 whole genome sequences (WGS) were analyzed, of which 129 were from Palestinian cases, 62 of which were collected in 11 Palestinian districts between October 2020 and April 2021 and sequenced completely. A dramatic shift from the wild type to the Alpha variant (B 1.1.7) was observed within a short period of time. Cluster mapping revealed statistically significant clades in two main Palestinian cities, Al-Khalil (Monte Carlo hypothesis test-Poisson model, P = 0.00000000012) and Nablus (Monte Carlo hypothesis test-Poisson model, P = 0.014 and 0.015). The phylogenetic tree showed three main clusters of SARS-CoV-2 with high bootstrap values (>90). However, population genetics analysis showed a genetically homogenous population supported by low Wright's F-statistic values (Fst <0.25), high gene flow (Nm > 3), and statistically insignificant Tajima's D values (Tajima's test, neutrality model prediction, P = 0.02). The Alpha variant, rapidly replaced the wild type, causing a major surge that peaked in April 2021, with an increased COVID-19 mortality rate, especially, in the Al-Khalil and Nablus districts. The source of introduction remains uncertain, despite the minimal genetic variation. The study substantiates the use of WGS for SARS-CoV-2 surveillance as an early warning system to track down new variants requiring effective control.

First Report of the HEV Seroprevalence and the Risk Factor Assessment in the West Bank, Palestine, during the Period of 2015-2017.

Hepatitis E virus is emerging viral hepatitis with hyperendemicity in many countries. Data on the burden of disease is not available in Palestine. This study aims to determine the seroprevalence and the risk factors of the HEV among the general population of the West Bank, Palestine. In this cross-sectional study, a total of 432 sera samples from 40 localities in the eleven districts of the West Bank and Jerusalem, Palestine, during the period of March 2015 to March 2017, were tested for HEV-IgG. A structured questionnaire was used to collect data of the participants' demographics and disease risk factors. The overall seroprevalence was 3.7%. Level of education was significantly inversely associated with HEV seropositivity (P=0.04). Purely spatial analysis did not detect any significant cluster related to the distribution of HEV-IgG cases; however, living in the southern West Bank is shown to be significantly associated with HEV. Age was also associated with HEV seropositivity. The young (<19 years) and adults (>40 years) had the highest prevalence, compared to those between 20 to 39 years old (P=0.12). Furthermore, males and those in contact with animals were associated with HEV seropositivity (P=0.1 and 0.3, respectively). In conclusion, the seroprevalence of HEV IgG in the West Bank, Palestine is low. Several well-investigated risk factors cannot be supported by our results due to the small number of the positive HEV-IgG samples. Finally, this study is useful for providing a first look into the seroepidemiology of HEV in Palestine.

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.

Genetic diversity, haplotype analysis, and risk factor assessment of hepatitis A virus isolates from the West Bank, Palestine during the period between 2014 and 2016.

BACKGROUND: Hepatitis A virus (HAV) infection is one of the major causes of acute viral hepatitis. HAV genotypes and its genetic diversity is rarely investigated in our region as well as worldwide. AIMS: The aims of the present study were to determine the HAV genotypes and its risk factors and to investigate the genetic diversity of the HAV isolates in the West Bank, Palestine. STUDY DESIGN: A cohort of 161 clinically and laboratory-confirmed HAV (IgM-positive) cases and 170 apparently healthy controls from all the districts of the West Bank, Palestine during the period of 2014 to 2016 were tested for HAV infection using IgM antibodies, RT-PCR and sequence analysis of the VP3/VP1 junction region of the HAV genome. Phylogenetic analysis, genetic diversity and haplotypes analysis were used to characterize the VP3/VP1 sequences. RESULTS: All the 34 sequences of the HAV were found to be of HAV-IB sub-genotype. The phylogenetic analysis showed four main clusters with cluster III exclusively consisting of 18 Palestinian isolates (18/23-78%), but with weak bootstrap values. A high haplotype diversity (Hd) and low nucleotide diversity (π) were observed. Cluster III showed high number of haplotypes (h = 8), but low haplotype (gene) diversity (Hd = 0.69). A total of 28 active haplotypes with some consisting of more than one sequence were observed using haplotype network analysis. The Palestinian haplotypes are characterized by closely related viral haplotypes with one SNV away from each other which ran parallel to cluster III in the phylogenetic tree. A smaller Palestinian haplotype (4 isolates) was three SNVs away from the major haplotype cluster (n = 10) and closer to others haplotypes from Iran, Spain, and South Africa. Young age, low level of parent's education, infrequent hand washing before meals, and drinking of un-treated water were considered the major HAV risk factors in the present study. CONCLUSION: Haplotype network analysis revealed haplotype variation among the HAV Palestinian sequences despite low genetic variation and nucleotide diversity. In addition, this study reconfirmed that age and parent's level of education as HAV risk factors, while hand washing and treating drinking water as protective factors.

Case Report: Autochthonous Case of Human Visceral Leishmaniasis in the West Bank, Palestine.

Human visceral leishmaniasis (HVL) is a parasitic disease infecting children in the Mediterranean region. Here, we portray a case of a 2-year-old child with an epidemiological description of the situation surrounding the case. The patient was suffering from recurrent fever, weakness, and abdominal discomfort associated with loss of appetite. Routine blood investigations showed pancytopenia, whereas examination revealed hepatomegaly. A diagnosis of HVL was made by demonstrating amastigotes in a Giemsa-stained smear from a bone marrow aspirate followed by genotyping by PCR and sequencing. In conclusion, early detection of VL infection followed by appropriate treatment protocols is essential to saving the patient.

Author Correction: Molecular Detection of Theileria ovis and Theleiria equi in Livestock from Palestine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Prevalence of Trypanosoma evansi in livestock in Palestine.

BACKGROUND: Trypanosoma evansi is the causative agent of surra, a disease that occurs in many animal species. The disease is responsible for substantial losses in global production and can be fatal if not diagnosed early. This study aims to determine the prevalence of T. evansi in livestock, equids and dromedary camels in Palestine. METHODS: Blood samples were collected during 2015-2017 from domesticated animals (n = 259 animals; 77% females and 23% males) including camels (n = 87), horses (n = 46), donkeys (n = 28), mules (n = 2), sheep (n = 49) and goats (n = 48) from eight districts: Ariha (Jericho), Nablus, Bethlehem, Deir Al Balah, Jenin, Rafah, Tubas, and Khan Yunis. Parasite prevalence was determined using PCR and blood smear microscopy. PCR-positive samples were further phylogenetically analyzed using DNA sequences of the 18S ribosomal RNA gene. RESULTS: The overall infection prevalence was 18% (46/259). The positivity rates according to PCR and microscopy examination were 17% (45/259) and 2.7% (7/259), respectively. The infection rates were as follows: camels, 26/61 (30%); horses, 8/46 (17%); donkeys, 3/28 (11%); mules, 1/2 (50%); sheep, 2/42 (4%); and goats, 6/42 (13%). Phylogenetic analyses of the 18S rRNA gene showed that 24 positive T. evansi samples from Palestine formed a monophyletic cluster with seven T. evansi sequences from Africa, Asia and South America, and three T. brucei sequences from Africa retrieved from GenBank. The spatial analysis showed three statistically significant foci of T. evansi infection in Jenin, Tubas (P = 0.02) and Ariha (Jericho) (P = 0.04). No statistically significant foci were detected in the Gaza Strip. CONCLUSIONS: To the best of our knowledge, this is the first confirmation of high levels of infection with T. evansi as a causative agent of surra in Palestine. Our study emphasizes the need for a stringent surveillance system and risk assessment studies as prerequisites for control measures. Further investigations focusing on vectors and evaluation of risk factors are needed.

Prevalence of selected intestinal protozoan infections in marginalized rural communities in Palestine.

BACKGROUND: Intestinal parasitic infections are common in rural areas with poor infrastructure and low socioeconomic status. The aim of this study was to estimate the prevalence of selected parasitic infections in marginalized rural areas in the northern part of the Palestinian West Bank Region, using conventional and PCR-based methods, and also to assess risk predictors of infection. METHODS: A cross-sectional study was conducted on 104 individuals from three rural villages in the Jordan Valley. Stool samples were collected and examined by a battery of tests that included microscopy of wet fecal samples in normal saline with iodine, concentration by ethyl acetate sedimentation and also by zinc sulfate floatation, a conventional PCR and a real-time PCR (qPCR). Risk factors were assessed that included demographic, socioeconomic, and behavioral characteristics. Data on method performance was analyzed by kappa-statistic, Cochrane's Q, and McNemar post hoc test. Mid-P exact test and odds ratio were used to discern association between outcome and risk predictors. RESULTS: The overall prevalence of intestinal parasitic infections was 48% (49/102). The predominant parasites were Giardia lamblia at 37% (37/102) and Hymenolepis nana at 9% (9/102). To concentrate cysts and eggs, sedimentation can be used as an alternative to floatation with a loss of 1% of positive cases. The methods employing PCRs proved crucial as it increased the detected infection rate of G. lamblia approximately three-fold from 13% by the conventional methods to 37% by the qPCR. Multiple infections were present in 13% (13/102) of the study group, which included double (10%) and triple (3%) infections. Regarding the genus Entamoeba, E. dispar and E. coli were detected at rates of 2 and 8%, respectively. While none of the individuals were infected with the pathogenic E. histolytica, E. nana (4%) was detected for the first time in the area. Age was a risk predictor for infection (OR = 2.61, CI 95% 1.05-6.45, P = 0.038). CONCLUSIONS: The increased prevalence of intestinal parasitic infections in children in marginalized rural areas in Palestine is worrying. The addition of PCR-based methods is important for the diagnosis of such infections as, with cautious interpretation, it increases proficiency and overcomes underestimation and misdiagnosis of cases. Control measures including education on personal hygiene and environmental sanitation, should be introduced to reduce the prevalence of the intestinal parasites and, thus, the infections they cause in this and other areas.

Molecular Detection of Theileria ovis and Theleiria equi in Livestock from Palestine.

Theileria and Babesia are intracellular protozoan parasites infecting a wide range of animals. In Palestine, there is limited information on the prevalence of Theileria and Babesia spp. in livestock. We used PCR of the 18S ribosomal RNA gene followed by DNA sequencing to detect and identify parasite DNA in blood samples from sheep (n = 49), goats (n = 48), horses (n = 40), camels (n = 34), donkeys (n = 28) and mules (n = 2) from four districts of Palestine. DNA of T. ovis and T. equi was detected in 19 and 2 ovine blood samples, respectively. None of the camels, donkeys, and goats were positive for T. ovis. Sheep had a significantly higher rate of infection than other animals (P < 0.05). Theileria ovis is highly prevalent in sheep, while T. equi DNA was detected in a small proportion of the equids in Palestine.

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 region.

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

BACKGROUND: 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.

Genetic diversity of the enteroviruses detected from cerebrospinal fluid (CSF) samples of patients with suspected aseptic meningitis in northern West Bank, Palestine in 2017.

BACKGROUND: Human enterovirus genus showed a wide range of genetic diversity. OBJECTIVES: To investigate the genetic diversity of the enteroviruses isolated in 2017 in northern West Bank, Palestine. STUDY DESIGN: 249 CSF samples from aseptic meningitis cases were investigated for HEV using two RT-PCR protocols targeting the 5' NCR and the VP1 region of the HEV genome. The phylogenetic characterization of the sequenced VP1 region of Echovirus18 (E18) and Coxsackievirus B5 (CVB5) isolated in Palestine along with 27 E18 and 27 CVB5 sequences available from the Genbank were described. RESULTS: E18 and CVB5 account for 50% and 35% of the successfully HEV types, respectively. Phylogenetic tree of E18 and CVB5 showed three main clusters, with all Palestinian isolates uniquely clustering together with those from China and from different countries, respectively. Cluster I of E18, with 13 Palestinian and 6 Chinese isolates, showed the lowest haplotype-to-sequence ratio (0.6:1), haplotype diversity (Hd), nucleotide diversity (π), and number of segregating sites (S) compared to clusters II and III. Furthermore, cluster I showed negative Tajima's D and Fu-Li'sF tests with statistically significant departure from neutrality (P<0.01). In both E18 and CVB5 populations, high haplotype diversity, but low genetic diversity was evident. Inter-population pairwise genetic distance (Fst) and gene flow (Nm) showed that the Palestinian E18 and CVB5 clusters were highly differentiated from the other clusters. CONCLUSIONS: The study divulged close genetic relationship between Palestinian HEV strains as confirmed by population genetics and phylogenetic analyses.

Prevalence of Sero-Molecular Markers of Hepatitis C and B Viruses among Patients with ß-Thalassemia Major in Northern West Bank, Palestine.

BACKGROUND: HCV and HBV present a great challenge in the management of ß-thalassemia patients. OBJECTIVE: The present study aimed to determine the prevalence of both HBV and HCV in multitransfused-dependent ß-thalassemia patients in northern West Bank, Palestine, using sero-molecular markers. METHODS: Serum sample from 139 multitransfused ß-thalassemia patients were tested for HBV and HCV markers including HBsAg, anti-HBc, anti-HBs, HBV-DNA, and anti-HCV and HCV-RNA. Demographic data and selected clinical parameters were collected by means of a questionnaire and from the patients' medical files. RESULTS AND CONCLUSION: The mean (±SD) age of patients was 18.1 years (±10.6). The overall prevalence of the HCV was 10% (14/139), which is 50 times higher than the normal Palestinian population (0.2%). Of which, 3 were positive for anti-HCV alone, 7 positives for HCV-RNA alone, and 4 positives for both anti-HCV and PCR-RNA. On the other hand, low prevalence of HBV was detected at a level of 0.7% (1/139). Only one patient had HCV-HBV coinfection. Twenty-five patients (19%) were positive for anti-HBc, while 99 (71%) were immune with the anti-HBs level above 10 IU/mL. Anti-HBc was insignificantly high (P=0.07) in HCV-positive cases. In conclusion, the prevalence of HCV among ß-thalassemia patients is considered high compared to normal population. Determination of HCV prevalence should be based on the detection of both HCV-RNA and anti-HCV. On the contrary, HBV showed a low prevalence. A follow-up schedule and administration of booster dose of HBV vaccine is strongly recommended for ß-thalassemia patients whose anti-HBs level <10 IU/ml.

The Prevalence of PCR-Confirmed Pertussis Cases in Palestine From Archived Nasopharyngeal Samples.

BACKGROUND: Pertussis caused by Bordetella pertussis is a vaccine-preventable disease causing whooping cough in humans of all ages. This study reports infection rate of pertussis in Palestine between the years 2004-2008 from archived nasopharyngeal samples collected from clinically- suspected cases. METHODS: A convenience archived DNA samples collected from 267 clinically-suspected pertussis cases were investigated for B. pertussis. Laboratory diagnosis was done by examining all DNA samples using polymerase chain reaction (PCR). RESULTS: Approximately 49% (130/267) were confirmed by PCR. A pertussis peak was shown to occur in 2008 with 77% (100/130) of PCR-confirmed cases isolated in that year. PCR-confirmed cases existed in all Palestinian districts with highest rate in Ramallah, Bethlehem, Jenin and Al-Khalil. Half of the PCR-confirmed cases (68/130) were less than 2 months old. The positivity rate among who had three doses of vaccine (at 2, 4 and 6 months) was 38%, and became 50% with the fourth dose at 12 months. CONCLUSION: The prevalence of pertussis was found to be significantly high among infants less than 2 months old. Active pertussis surveillance using rapid PCR assays is essential, as it is helpful in prompt diagnosis and treatment of patients with pertussis.

A comparison of the efficiency of three sampling methods for use in the molecular and conventional diagnosis of cutaneous leishmaniasis.

In human cutaneous leishmaniasis (CL), the success of positive diagnoses and species identifications depends, primarily, on how biopsies are taken and then processed and examined. The efficiency of three methods of taking skin biopsies from suspect cases of CL was compared using the classical methods of microscopy of stained smears, in vitro culture of tissue aspirate, and internal transcribed spacer region 1 (ITS1)-polymerase chain reaction in diagnosing positive cases and identifying the species of Leishmania causing them. From 1994-2014, biopsy samples from the skin lesions of 2232 CL-suspected patients were collected as unstained smears, as smears stained with Giemsa's stain and on filter paper, and compared in the diagnostic tests employed. Matched comparison based on testing biopsy samples from 100 patients, microscopy, in vitro culture and ITS1-PCR were also conducted to assess the most suitable combination of methods for diagnosing leishmaniases. In the 100-case-matched comparison, the three different types of sample proved to be equally good with no significant difference (P > 0.05). However, skin tissue imprints on filter paper revealed most cases of CL. The kappa statistic for measuring the degree of agreement among the three samples was 89%, which is considered good. Agreement was highest between imprints on filter paper and unstained smears, and lowest was for stained smears. In the overall comparison between the ITS1-PCR and conventional methods, the ITS1-PCR using samples from filter papers was the most sensitive method but the difference was insignificant (P = 0.32). The combination of microscopy together with ITS1-PCR on samples from filter papers increased the sensitivity significantly to 46%, compared to using the methods individually (P = 0.003-0.0008). On comparing the results of the tests done on the samples from the 2232 patients after applying ITS1-PCRs to their samples from filter papers, unstained smears, in vitro culture, microscopy, and stained smears showed, respectively, test sensitivities of 81, 69, 64, 57 and 48%. Of the tests and samples adjudicated, ITS1-PCRs run on skin tissue samples from filter papers proved best for the routine laboratory diagnosis of CL. Adding microscopy of stained smears to it, improved its diagnostic value significantly.