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.

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

BACKGROUND: Dyslipidemia 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. METHODS: A 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. RESULTS: There 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). CONCLUSIONS: The 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.

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.

Common FTO rs9939609 variant and risk of type 2 diabetes in Palestine.

BACKGROUND: Genetic and environmental factors play a crucial role in the development of type 2 diabetes mellitus (T2DM) and obesity. This study aimed to investigate the association of the fat-mass and obesity-associated gene (FTO) rs9939609 variant with T2DM and body mass index (BMI) among Palestinian population. METHODS: A total of 399 subjects were recruited, of whom 281 were type 2 diabetic patients and 118 normoglycemic subjects. All of them were unrelated, aged > 40 years and recruited within the period 2016-2017. The A allele of FTO rs9939609 was identified by PCR-RFLP. RESULTS: Significant association of the minor allele A of FTO rs9939609 and T2DM risk was observed with an allelic odd ratio of 1.92 (95% CI [1.09-3.29], p = 0.02) adjusted for age and gender, this association partly attenuated when adjusted for BMI with OR of 1.84, (95%CI [1.04-3.05], p = 0.03). Stratified data by glycemic status across FTO genotypes showed that A allele was marginally associated with increased BMI among diabetic group (p = 0.057) but not in control group (p = 0.7). Moreover, no significant association was observed between FTO genotypes and covariates of age, gender, T2DM complications or any tested metabolic trait in both diabetic and nondiabetic individuals (p > 0.05). CONCLUSIONS: The variant rs9939609 of the FTO gene was associated with T2DM in Palestine. This is the first study conducted on this gene in the Palestinian population and provides valuable information for comparison with other ethnic groups. Further analysis with larger sample size is required to elucidate the role of this variant on the predisposition to increased BMI in Palestinians.

Isolation and characterization of phenol degrading bacterium strain Bacillus thuringiensis J20 from olive waste in Palestine.

This study aimed at isolation of phenol degrading bacteria from olive mill wastes in Palestine. The efficiency of phenol removal and factors affecting phenol degradation were investigated. A bacterial strain (J20) was isolated from solid olive mill waste and identified as Bacillus thuringiensis based on standard morphological, biochemical characteristics and 16SrRNA sequence analysis. The strain was able to grow in a phenol concentration of 700 mg/L as the sole carbon and energy source. The culture conditions showed a significant impact on the ability of these cells to remove phenol. This strain exhibited optimum phenol degradation performance at pH 6.57 and 30 °C . Under the optimized conditions, this strain could degrade 88.6% of phenol (700 mg/L) within 96 h when the initial cell density was OD600 0.2. However, the degradation efficiency could be improved from about 88% to nearly 99% by increasing the cell density. Immobilization of J20 was carried out using 4% sodium alginate. Phenol degradation efficiency of the immobilized cells of J20 was higher than that of the free cells, 100% versus 88.6% of 700 mg/L of phenol in 120 h, indicating the improved tolerance of the immobilized cells toward phenol toxicity. The J20 was used in detoxifying crude OMWW, phenolic compounds levels were reduced by 61% compared to untreated OMWW after five days of treatment. Hence, B. thuringiensis-J20 can be effectively used for bioremediation of phenol-contaminated sites in Palestine. These findings may lead to new biotechnological applications for the degradation of phenol, related to olive oil production.

Detection and molecular identification of Hepatozoon canis and Babesia vogeli from domestic dogs in Palestine.

Dogs serve as hosts for a great number of parasites, which may affect their health and wellbeing. This study aimed to observe tick borne pathogens in dogs from Palestine including Hepatozoon canis and Babesia species. The prevalence of both H. canis and Babesia species infections in apparently healthy dogs, from ten districts of the West Bank was surveyed. DNA was extracted from blood samples obtained from dogs (n = 362) and ticks (n = 213) collected from dogs (n = 77). A primer set that amplifies a partial sequence of the Babesia and Hepatozoon 18S rRNA gene was used for PCR and the DNA sequences of the PCR products of all samples were determined. Twenty-nine (8·0%) of the dogs were found infected including 20 with H. canis (5·5%), seven with Babesia vogeli (1·9%) and two with undefined Babesia spp. (0·6%). Twelve Rhipicephalus sanguineus s.l ticks were pathogen-positive, including ten with H. canis (4·7%), one with B. vogeli (0·5%), and one with Hepatozoon felis (0·5%). The results indicated that a wide range of tick borne pathogens is circulating in the canine population in the surveyed region. This study is the first report on the prevalence of H. canis, B. vogeli and Babesia spp. in dogs in Palestine and its results will assist in the management of diseases associated with these blood parasites.

Identification of Risk Factors for the Development of Diabetic Retinopathy Among Palestinian Adults With Type 2 Diabetes Mellitus: A Cross-Sectional Study.

INTRODUCTION: Although risk factors linked to diabetic retinopathy (DR) among patients with Type 2 diabetes mellitus (T2DM) have been extensively studied globally, the specific determinants of these factors in relation to DR in Palestine are presently not well understood. METHODS: This retrospective cross-sectional study included patients who underwent DR screening with a fundus camera (VersaCam a). The study included patients aged ≥18 with T2DM, excluding those with other types of diabetes or a history of malignancies. Univariable and multivariable logistic regressions were used to identify factors associated with DR. RESULTS: A total of 1163 patients with T2DM were included in this study. Of these, 211 (18.1%) patients were classified in the DR group, 761 (65.4%) in the no DR group and 191 (16.4%) were ungradable. Among the included patients, 434 (37.3%) were male. A secondary level of education or higher and a BMI ≥30 kg/m2, compared with <25 kg/m2, were independently and inversely associated with DR, with odds ratios (ORs) of 0.46 (p < 0.001) and 0.58 (p = 0.046), respectively. A 5-year increase in the duration of T2DM correlated with 45% higher odds of having DR (p < 0.001). Patients with DR were more likely to have HbA1c >7%, be physically inactive and use insulin, with ORs of 1.63 (p = 0.02), 2.05 (p < 0.001) and 1.53 (p = 0.03), respectively. Age, gender, occupational status, hypertension and hyperlipidaemia were not independent predictors of DR (p < 0.05). CONCLUSION: Longer duration of T2DM, HbA1c >7%, physical inactivity and insulin use were all independently associated with the presence of DR. Furthermore, a secondary or higher educational level and obesity demonstrated independent and inverse associations with the development of DR.

Epidemiological Characterization and Genetic Variation of the SARS-CoV-2 Delta Variant in Palestine.

The emergence of new SARS-CoV-2 variants in Palestine highlights the need for continuous genetic surveillance and accurate screening strategies. This case series study aimed to investigate the geographic distribution and genetic variation of the SARS-CoV-2 Delta Variant in Palestine in August 2021. Samples were collected at random in August 2021 (n = 571) from eight districts in the West Bank, Palestine. All samples were confirmed as positive for COVID-19 by RT-PCR. The samples passed the quality control test and were successfully sequenced using the ARTIC protocol. The Delta Variant was revealed to have four dominant lineages: B.1.617 (19%), AY.122 (18%), AY.106 (17%), and AY.121 (13%). The study revealed eight significant purely spatial clusters (p < 0.005) distributed in the northern and southern parts of Palestine. Phylogenetic analysis of SARS-CoV-2 genomes (n = 552) showed no geographically specific clades. The haplotype network revealed three haplogroups without any geographic distribution. Chronologically, the Delta Variant peak in Palestine was shortly preceded by the one in the neighboring Israeli community and shortly followed by the peak in Jordan. In addition, the study revealed an extremely intense transmission network of the Delta Variant circulating between the Palestinian districts as hubs (SHR ≈ 0.5), with Al-Khalil, the district with the highest prevalence of COVID-19, witnessing the highest frequency of transitions. Genetic diversity analysis indicated closely related haplogroups, as haplotype diversity (Hd) is high but has low nucleotide diversity (π). However, nucleotide diversity (π) in Palestine is still higher than the global figures. Neutrality tests were significantly (p < 0.05) low, including Tajima's D, Fu-Li's F, and Fu-Li's D, suggesting one or more of the following: population expansion, selective sweep, and natural negative selection. Wright's F-statistic (Fst) showed genetic differentiation (Fst > 0.25) with low to medium gene flow (Nm). Recombination events were minimal between clusters (Rm) and between adjacent sites (Rs). The study confirms the utility of the whole genome sequence as a surveillance system to track the emergence of new SARS-CoV-2 variants for any possible geographical association and the use of genetic variation analysis and haplotype networking to delineate any minimal change or slight deviation in the viral genome from a reference strain.

Systolic inter-arm blood pressure difference and estimated glomerular filtration rate in type 2 diabetic patients in Palestine: a cross-sectional study.

OBJECTIVES: This study aimed to investigate the association between systolic inter-arm blood pressure difference (IABPD) and the estimated glomerular filtration rate (eGFR), as well as chronic kidney disease (CKD), in patients with type 2 diabetes mellitus (T2DM). PATIENTS AND METHODS: This cross-sectional study included 189 Palestinians diagnosed with T2DM. Data were collected through personal interviews, medical records and three separate blood pressure measurements from both arms. Patients were stratified in two ways: based on systolic IABPD ≥15 mmHg and the presence of CKD, indicated by an eGFR of <60 mL/min/1.73 m2 over a three months period. We used simple and multiple linear regression analyses to clarify the association between systolic IABPD (mmHg) and eGFR and to identify independent predictors for eGFR. RESULTS: The mean age was 61.3 years, with a female percentage of 57.7%. The prevalence of systolic IABPD ≥15 mmHg and CKD was 27.5% and 30.2%, respectively. Among patients with eGFR <60 mL/min/1.73 m2, the median systolic IABPD was 12.5 mmHg (interquartile range (IQR), 13.5 mmHg), whereas in patients with eGFR ≥60 mL/min/1.73 m2, it was 7.5 mmHg (IQR, 9.8 mmHg) with a significant difference (p = .021). The results of the multiple linear regression model did not reveal an independent association between systolic IABPD and eGFR, with an unstandardized coefficient (B) of -0.257 (95% confidence interval (CI), -0.623 to 0.109; p = .167). However, older age (B, -0.886; 95% CI, -1.281 to -0.49; p < .001), hypertension (B, -12.715; 95% CI, -22.553 to -2.878; p = .012) and a longer duration of DM (B, -0.642; 95% CI, -1.10 to -0.174; p = .007) were significantly and negatively associated with eGFR. CONCLUSIONS: Systolic IABPD did not exhibit an independent association with eGFR in T2DM patients. However, older age, a previous history of hypertension, and a longer duration of DM were all significantly associated with lower eGFR.

Aldose reductase (-106) C/T gene polymorphism and associated risk factors with proliferative diabetic retinopathy in Palestine: A cross sectional study.

Background and Aims: Genetic variants play a crucial role in the development of diabetic retinopathy (DR). Therefore, our study aimed to investigate the relationship between aldose reductase (ALR2) (C106T) polymorphism with proliferative DR and associated risk factors in Palestinian type 2 diabetic patients. Methods: A cross sectional study was conducted at St John Eye Hospital-East Jerusalem in 2020-2021 on patients with DR. All subjects had fundus examination by ophthalmologists and classified according to the severity of retinopathy. Genomic DNA was extracted from whole blood samples and genotyped by amplicon based next generation sequencing. Results: A total of 155 patients were included, of them, 103 (66.5%) were diagnosed with non-proliferative DR (NPDR) and 52 (33.5%) with proliferative DR (PDR). The PDR group had a significantly lower median age (59.5 [IQR: 13.3]) compared to the NPDR group (62 [IQR: 11.5]) (p = 0.04). Additionally, the duration of diabetes was higher in the PDR group (20 [IQR: 9]) compared to the NPDR group (15 [IQR: 10]) (p < 0.001). Conversely, the mean value of diastolic blood pressure was significantly lower in the PDR group (79.2 ± 11.1) compared to the NPDR group (83.4 ± 10.3) (p = 0.02). Logistic regression analysis, revealed that the odds for patients with dyslipidemia to develop PDR were 2.74 times higher than those with NPDR (95% CI: 1.08-6.98) (p = 0.034). Furthermore, the probability of a patient with ≥20 years of diabetes to develop PDR was seven times higher than other patients (95% CI: 1.98-27.91) (p = 0.003). The genotypes distribution of ALR2 gene and its allele frequency showed no statistical differences between the two groups (p > 0.05). Conclusions: The present study showed that duration of diabetes and dyslipidemia were strong indicators for PDR progression, while ALR2 (C106T) polymorphism was not associated with severity of DR.

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.

Association of DNA methylation and genetic variations of the APOE gene with the risk of diabetic dyslipidemia.

Apolipoprotein E (APOE) is a key regulator of lipoprotein metabolism, and consequently, affects the plasma and tissue lipid contents. The aim of the present study was to investigate the parallel effects of APOE genetic variants and promoter methylation levels of six CpGs on the risk of diabetic dyslipidemia. A total of 204 Palestinian type 2 diabetes (T2D) patients (mean age ± SD: 62.7±10.2) were enrolled in the present study (n=96 with dyslipidemia and n=108 without dyslipidemia). Next generation sequencing was performed to analyze five single nucleotide polymorphisms: Two variants rs7412 and rs429358 that determine APOE ε alleles, and three variants in the promoter region (rs769446, rs449647, and rs405509). For all subjects, the most common genotype was ε3/ε3 (79.4%). No statistical differences were observed in the APOE ε polymorphisms and the three promoter variants among T2D patients with and without dyslipidemia (P>0.05). A comparison of lipid parameters between ε3/ε3 subjects and ε4 carriers in both groups revealed no significant differences in the mean values of LDL-C, HDL-C, TG, and TC levels (P>0.05). Six CpG sites in the APOE promoter on chromosome 19:44905755-44906078 were identified, and differential DNA methylation in these CpGs were observed between the study groups. Logistic regression analysis revealed a significant association of DNA methylation level at the six CpGs with an increased risk of diabetic dyslipidemia (odds ratio, 1.038; 95% confidence interval, 1.012-1.064; P=0.004). In conclusion, the present study revealed that DNA methylation levels in six CpGs in the APOE promoter region was associated with the risk of diabetic dyslipidemia independently of the APOE ε4 variant which could be a potential therapeutic target to reverse the methylation of the APOE promoter.

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.

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.

Pulmonary tuberculosis in the West Bank, Palestinian Authority: molecular diagnostic approach.

OBJECTIVE: To compare the effectiveness and feasibility of an insertion sequence (IS6110)-based polymerase chain reaction (PCR) assay with conventional methods of detecting Mycobacterium tuberculosis and to analyse mutations present in the hot spot region of the RNA polymerase B subunit (rpoB) gene associated with rifampin resistance by DNA sequencing. METHODS: Ninety-five sputum samples from 84 clinically suspected cases of tuberculosis were tested for mycobacterial infections by Ziehl Neelsen smear examination, Lowenstein-Jensen culture and IS6110-based PCR assay. RESULTS: Sensitivity and specificity of the PCR were 94%; the sensitivity of culture was 65%, and of smear tests, 59%. Both smear microscopy and culture had 100% specificity. DNA sequencing data of the 305-bp fragment of the rpoB gene for nine clinical isolates revealed one point mutation at position I572F and double mutations at position S531F in two isolates obtained from two patients who did not respond to the anti-tuberculosis therapy. CONCLUSION: IS6110-based PCR can be used routinely in clinical laboratories for rapid detection of Mycobacterium tuberculosis and thus allow early diagnosis and treatment of any contacts by the cheapest method currently available in the Palestinian Authority region. Rapid detection of rifampin resistance isolates will enable efficient treatment of patients and assist in eradication of the disease in the Palestinian territories.

Clinical Profile of COVID-19 Patients Presenting with Uveitis - A Short Case Series.

PURPOSE: To present the clinical features of anterior, intermediate and posterior uveitis in patients with COVID-19 and to increase the awareness of the treating physicians to refer patients with COVID-19 who have eye symptoms for ophthalmic exam, in order to diagnose as early as possible and prevent vision-threatening complications. METHODS: Retrospective observational case reports. RESULTS: We report three cases of COVID-19 patients who developed uveitis during or after the course of their sickness with COVID-19. All patients underwent a detailed eye examination, relevant history and investigations did not prove any other cause of uveitis. CONCLUSION: This report presents novel data on the course of subjects with uveitis during the COVID-19 pandemic. Intermediate and posterior uveitis warrant further evaluation with differential diagnosis supported by laboratory tests due to the association with systemic diseases and risk of permanent vision loss. Iridocyclitis, intermediate, and posterior uveitis treatment should be guided by ophthalmologists, particularly uveitis specialists, when possible.

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.

Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis.

Identification and characterization of the Mycobacterium tuberculosis strains are important for clinical and therapeutic management of tuberculosis. Real-time PCR with a high-resolution melt assay was found to improve the diagnostic process. The assay includes differentiation between M. tuberculosis and Mycobacterium bovis based on one single-nucleotide polymorphism (SNP) in the narGHJI and oxyR genes and determination of M. bovis based on the region of differences 1 (RD1). This assay correctly identified the 7 tested Mycobacterium reference strains and 52 clinical samples with a sensitivity of 2 pg DNA. This assay will help in prescribing adequate treatment and monitoring disease dynamics.