Biochemistry & Molecular Biology الكيمياء الحيوية والأحياء الجزيئية
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- ItemMolecular Detection and Genotyping of Hydatid Cysts in Slaughtered Livestock in the West Bank, Palestine(Al-Quds University, 2025-01-08) Walaa Habes Mohammad Yones; ولاء حابس محمد يونسDespite the widespread occurrence of CE, there is a significant gap in understanding the specific Echinococcus genotypes present in various regions, including the West Bank of Palestine. This lack of knowledge hampers effective disease management and control strategies. Identifying and genotyping the parasites affecting the intermediate hosts is crucial for epidemiological surveillance, Public Health Implications by informing health authorities and policymakers to implement effective control measures based on local epidemiological data. This study aimed to identify and genotype the Echinococcus parasite genotypes causing CE in the West Bank of Palestine, specifically targeting the intermediate hosts (IMHs) such as sheep and goats. Materials and Methods: A total of 54 cyst tissue samples were collected from livestock in the West Bank, 52 sheep samples from different slaughterhouses from areas known for high CE incidence in the West Bank consisting of hydatid cyst tissues were isolated from infected organs during examinations of the intermediate host, and two are human cysts tissue collected from hospitals after surgery as accidental intermediate hosts. DNA was extracted and amplified by PCR to ensure high-quality genomic DNA for sequencing using the EgG1 Hae III sequence (266 bp). The extracted DNA was subjected to NGS using two primers pools, allowing for comprehensive genetic analysis. Targeting all SNPs of both mitochondrial genes at once: COX-1 (Cytochrome c oxidase subunit 1) and NAD-1 (NADH dehydrogenase subunit 1) for the first time in the present study, which were selected for their variability and are useful for distinguishing between different Echinococcus species and genotypes. Finally, using the Variant Call Analysis platform for identifying and genotyping of Echinococcus specific sequences among thousands of sequences through online bioinformatics software Galaxy/Europe. Results: An EgG1 Hae III sequence of 266 bp was identified in 98% of samples. NAD1 and COX-1 specific fragments were successfully amplified and sequenced through NGS platform from 53 cyst tissue samples. Genotyping analysis was successfully performed in 84% of sequenced samples, revealing that 58% were the G1 genotype and 42% were for the G3 genotype. Conclusion: This study was to distinguish between s.s genotypes G1 and G3 in our isolates based on seven different informative regions together for the first time using NGS with high-quality outputs confirmed the predominance of the G1 genotype while also indicating a significant presence of the G3 genotype among the intermediate hosts in endemic areas of Palestine. While the sensitivity of NGS is widely acknowledged in the scientific community, our findings emphasize the importance of understanding the local epidemiology of CE. The identification of both G1 and G3 genotypes in this region provides crucial data that can inform future public health strategies and control measures.
- ItemScreening of mutations in Polynucleotide Kinase 3' - Phosphatase gene causing Microcephaly, Seizures, and Developmental Delay in Palestine(Al-Quds University, 2025-01-05) Maysa "Mohammad Amer" Kamal Natsheh; ميساء "محمد عامر" كمال نتشةMicrocephaly, Seizures, and Developmental Delay (MCSZ) is an uncommon genetic disorder with an undetermined prevalence. It is inherited in an autosomal recessive pattern associated with either a homozygous or compound heterozygous mutation in the PNKP gene. This gene plays a crucial role in various DNA repair mechanisms, and its mutation leads to the continuous activation of the DNA damage response. This persistent activation is a consequence of the accumulation of double-strand breaks within the affected cells, which may result in the death of sensitive neuronal cells, potentially contributing to the pathogenesis of MCSZ. The disorder is primarily characterized by microcephaly, or an unusually small head size, along with a range of neurological impairments. Purpose: This research aims toscreen PNKP mutations in several Microcephaly, Seizure and Developmental Delay (MCSZ) disorder patients from Palestine. Methods: Three patients from different families were studied, who fulfilled our inclusion criteria: (1) Patients (males and females of different ages) having the general characteristics of MCSZ. (2) Their parents, especially if they are consanguineous in marriage determine if they are carriers for PNKP mutation, and/or family members. Genomic DNA was extracted after obtaining a blood samples which drawn for PNKP gene detection using PCR and Sanger sequencing. PNKP mutations were screenedand targeted the most common published exons (Exon 11, 14 and 15). Results:In this research, we identified a female patient exhibiting microcephaly, significant developmental delays, and early-onset refractory seizures, attributed to a homozygous mutation in the PNKP gene. This mutation, classified as likely pathogenic, is a missense variant denoted as NM_007254.4: c.968 C > T: p. (Thr323Met),located in exon 11 of thePNKP genes. The screening of first-degree relatives revealed that this genetic variant was inherited from both the father and mother who were identified as heterozygous for the MCSZ variant (GA). A pedigree was constructed following the screening of the affected individuals’ first-degree relatives. In contrast, samples from the other two patients did not exhibit any mutations in the PNKP gene. These samples were subsequently subjected to Whole Exome Sequencing, which confirmed the absence of mutations in the PNKP gene for both patients. Conclusion: Microcephaly, Seizures, and Developmental Delay (MCSZ) is underdiagnosed and undertreated in our population. Even simple knowledge among families toward consanguineous marriages, genetic counseling and cascade screening are essentialfor the diagnosis and prevention of MCSZ early in life. The results in this research will be considered as preliminary results on that disease among our population and more cohorts studiesand advanced genetic analysis are still needed.
- Itemmolecular Epidemiology: Prevalence of Human Cutaneous Leishmaniasis in palestine in the period between 2016 and 2024 using Next Generation Sequencing(Al-Quds University, 2025-01-12) Hanan Abdelmajeed Al-Jawabreh; حنان عبد المجيد عبد العزيز المناصرة /الجوابرةBackground: Leishmaniasis is a vector-borne disease caused by protozoan parasite of the genus Leishmania. The infection is transmitted by bite of infected female sandflies of the genus Phlebotomus in the old world or Lutzomyia in the new world. The diagnosis is made by stained smear microscopy, in-vitro culture, and DNA-based detection methods. Materials and Methods: In this study that spanned from 2016 to 2024 included patients from eleven districts in the West Bank of Palestine. The diagnostic methods used in the study included microscopy of Giemsa-stained touch smears, in-vitro culture using NNN medium, and ITS1-PCR. The amplicon-based next-generation sequencing of ITS1-219 was included in the comparison part of the study. The comparison arm of the study compared two groups, CL-confirmed group in w were positive by any of the tests used in the epidemiologic part of the study and a non-CL group which were negative by all diagnostic methods. The NGS1-219-PCR was compared to ITS1-PCR, which is considered as a gold standard owing to its superiority over microscopy and in-vitro culture. Results: The positivity rate during the study period was 17% (213/1262) with a prevalence of 7.0 per 100,000. The annual incidence rate 0.84 per 100,000 (25 cases per year) with approximately equal distribution between males (52%) and females (48%). The age range between 0 to 14 yrs was the most affected by CL. The CL lesions primarily affected the head (45%), followed by the upper extremities (38%) and the lower extremities (17%). The choropleth mapping showed that Jericho is still the district with highest annual incidence rate (21 per 100,000). The study revealed that Leishmania tropica is the predominating species with L. major restricted mainly to Jericho. The year 2017 was the last year to witness a CL peak in Palestine with Jericho contributing to 40% of peak cases. More than half (52%) of the cases appeared in the months of January to March. As for the comparison arm of the study, ITS1-219-NGS was shown to have a higher sensitivity of 94% compared to the imperfect gold standard (ITS1-PCR) of 86%. The agreement between the two tests was fair (Kappa-0.24) only agreed on 56% of the cases only. Furthermore, the standard ITS1-PCR was unable to genotype 29 CL cases but were genotyped by ITS1-219- NGS and confirmed by BLAST search. Unlike the ITS1-PCR, imperfect gold standard, ITS1-219-NGS genotypes all its positive results correctly as confirmed by BLAST search. Moreover, 13 ITS1-PCR weak positive CL cases were found to be negative by ITS1-219- NGS and upon BLAST search shown to be contamination of human and bacterial origin. Conclusion: Jericho area remains the main focus of CL in Palestine with L. tropica as the main species in the country and L. major restricted to Jericho area. Incidence rate has dropped compared to the two decades preceding this study as result of control measures implemented. Amplicon-based NGS is a feasible, highly sensitive, and high throughput diagnostic method with accurate species identification that can be used in clinical practice and epidemiologic survey.
- ItemMolecular Characterization of Borrelia Species as Causative Agents for Tick-Borne Relapsing Fever from Ixodidae and Argasidae in Palestine(Al-Quds University, 2025-01-09) Abbas Khader Hussin Masalma; عباس خضر حسين مسالمةIntroduction: Tick-Borne Relapsing Fever (TBRF) is primarily caused by several Borrelia species that are transmitted through the bites of ticks. It is mainly characterized by multiple recurrences of nonspecific signs and symptoms, including fever, headache, myalgia, arthralgia, and even some neurologic complications. These Borrelia species are mostly vectored by hard ticks like Rhipicephalus genus and soft ticks like Ornithodoros tholozani which is known as the main vector of B. persica in Palestine. However, Molecular based data on Borrelia species and their hosts in Palestine are very scarce as B. persica is the only Borrelia species that had been studied.Objectives: This study aimed to investigate the molecular epidemiology of Borrelia species in ectoparasite hard and soft ticks, collected from different animal hosts throughout Palestine.Methodology: Ticks samples were collected from different animal hosts residing in different districts through Palestine, all ticks were identified at the genus and species levels based on taxonomic keys. DNA extraction was carried out, then screening for the presence of Borrelial DNA was carried out by PCR targeting flagellin (fla B) gene. All positive samples were confirmed by 16sRNA – PCR. PCR products were loaded onto agarose gel for band detection at 250 bp for fla B and 523 bp for 16sRNA, positive samples were sequenced and analyzed for BLAST identification. Statistical analysis was carried out using IBM SPSS v27.0 software. Results: Among all tick samples: 86% (n= 734) were identified as Ixodidae (hard ticks) and 14% (n= 117) were Argasidae (soft ticks). Overall, 76% of the identified hard ticks belonged to the genus Rhipicephalus (92% R. sanguineous and 5.5% R. Turanicus), 12% of Haemaphysalis ticks (80% H. parva and 20% H. adleri), 11% were Hyalomma ticks (84% H. dromedarri, 6% H. impeltatum, and 5% H. eagyptium). All collected soft ticks (n= 117) were identified as Ornithodoros tholozani. Out of 734 hard ticks, 2% (n= 13) were detected to be positive for Borrelia DNA by PCR. Sequence analysis revealed the presence of B. persica in 77% (n=10) of positive samples, while 15% (n=2) were B. turcicae, and 8% (n=1) was B. Lonestari. On the other hand, 9% of soft ticks (n=17) were found to harbor B.persica.Conclusions: Our results highlight the presence of Borrelia persica among hard ticks despite its’ usual presence among soft ticks, this could indicate an incidental acquisition or ecological overlaps. B. persica genotype was also observed in soft ticks. Notably, in Palestine, R. sanguineus is the major hard ticks which vector Borrelia species among hard ticks, while O. tholozani is the major one among soft ticks.
- ItemMolecular and phylogenetic analysis of Enterobius- vermicularis from appendectomy specimens in the West Bank, Palestine(Al-Quds University, 2024-12-22) Issam Bassam Ahmed Jawabra; عصام بسام احمد جوابرهBackground A parasitic nematode known as Enterobius vermicularis (human pinworm) frequently causes gastrointestinal symptoms and, in some cases, acute appendicitis. Despite its widespread prevalence, Palestinian populations have limited molecular data on E. vermicularis. This study aims to investigate the molecular and phylogenetic characteristics of E. vermicularis in appendectomy specimens across Palestine. The study will concentrate on the COX1 gene to comprehend the genetic diversity and transmission patterns. Methods A number of 55 formalin-fixed, paraffin-embedded (FFPE) appendectomy specimens previously collected from 2018-2023, were confirmed for E. vermicularis infection. Samples originated from three government hospitals: Queen Alia Hospital in Hebron, Rafidia Hospital in Nablus, and Beit Jala Hospital in Bethlehem. DNA extraction using FFPE sections was carried out, followed by polymerase chain reaction (PCR) targeting the COX1 gene. Phylogenetic, sequence, and haplotype analysis were performed... Results The male-to-female ratio was 1.2:1, with 54.5% of patients being male and 45.5% female. The average age of patients was 13.3 years. There are no sequence variations among the sequenced samples, suggesting the conservation of this population's genetic structure. The phylogenetic analysis revealed the existence of Type B E. vermicularis in the Palestinian samples, which is exclusively associated with human hosts. Similarly, the haplotype analysis using median-joining network group all Palestinian samples in Group B. Statistical analysis of demographic and clinical variables; including age, gender, WBC, and neutrophil counts, showed no significant correlations with the infection Conclusion The study revealed a strong genetic uniformity among E. vermicularis samples from appendectomy samples in Palestine, suggesting limited transmission dynamics. All samples were identified as Type B, exclusive to human hosts when compared with global patterns. No significant associations were found between demographic or clinical manifestations and infection, indicating stable infection patterns across the population. The findings of the current research provide baseline molecular information about E. vermicularis infection in Palestine and may direct public health efforts by the Palestinian Ministry of Health in future research on the transmission and epidemiology of Enterobius.