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.

Genome wide comparison of Ethiopian Leishmania donovani strains reveals differences potentially related to parasite survival.

Leishmania donovani is the main cause of visceral leishmaniasis (VL) in East Africa. Differences between northern Ethiopia/Sudan (NE) and southern Ethiopia (SE) in ecology, vectors, and patient sensitivity to drug treatment have been described, however the relationship between differences in parasite genotype between these two foci and phenotype is unknown. Whole genomic sequencing (WGS) was carried out for 41 L. donovani strains and clones from VL and VL/HIV co-infected patients in NE (n = 28) and SE (n = 13). Chromosome aneuploidy was observed in all parasites examined with each isolate exhibiting a unique karyotype. Differences in chromosome ploidy or karyotype were not correlated with the geographic origin of the parasites. However, correlation between single nucleotide polymorphism (SNP) and geographic origin was seen for 38/41 isolates, separating the NE and SE parasites into two large groups. SNP restricted to NE and SE groups were associated with genes involved in viability and parasite resistance to drugs. Unique copy number variation (CNV) were also associated with NE and SE parasites, respectively. One striking example is the folate transporter (FT) family genes (LdBPK_100390, LdBPK_100400 and LdBPK_100410) on chromosome 10 that are single copy in all 13 SE isolates, but either double copy or higher in 39/41 NE isolates (copy number 2-4). High copy number (= 4) was also found for one Sudanese strain examined. This was confirmed by quantitative polymerase chain reaction for LdBPK_100400, the L. donovani FT1 transporter homolog. Good correlation (p = 0.005) between FT copy number and resistance to methotrexate (0.5 mg/ml MTX) was also observed with the haploid SE strains examined showing higher viability than the NE strains at this concentration. Our results emphasize the advantages of whole genome analysis to shed light on vital parasite processes in Leishmania.

Mucosal Leishmaniasis in Travelers with Leishmania braziliensis Complex Returning to Israel.

Mucosal leishmaniasis (ML) is a complication of New World cutaneous leishmaniasis (CL) caused mainly by Leishmania (Viannia) braziliensis. This retrospective study investigated all cases of ML caused by L. (V.) braziliensis in a tertiary medical center in Israel, evaluating the risk factors, clinical presentations, diagnosis, treatment, and outcome of mucosal involvement in ML caused by L. (V.) braziliensis in travelers returning to Israel. During 1993-2015, a total of 145 New World CL cases were seen in travelers returning from Bolivia; among them, 17 (11.7%) developed ML. Nasopharyngeal symptoms developed 0-3 years (median 8 months) after exposure. The only significant risk factor for developing ML was the absence of previous systemic treatment. Among untreated patients, 41% developed ML, compared with only 3% of treated patients (p = 0.005). Systemic treatment for CL seems to be a protective factor against developing ML.

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.

Genetic characterization of spotted fever group rickettsiae in questing ixodid ticks collected in Israel and environmental risk factors for their infection.

This study aimed to genetically characterize spotted fever group rickettsiae (SFGR) in questing ixodid ticks from Israel and to identify risk factors associated with SFGR-positive ticks using molecular techniques and geographic information systems (GIS) analysis. 1039 ticks from the genus Rhipicephalus were collected during 2014. 109/1039 (10·49%) carried SFGR-DNA of either Rickettsia massiliae (95), 'Candidatus Rickettsia barbariae' (8) or Rickettsia conorii (6). Higher prevalence of SFGR was found in Rhipicephalus turanicus (18·00%) compared with Rhipicephalus sanguineus sensu lato (3·22%). Rickettsia massiliae was the most commonly detected species and the most widely disseminated throughout Israel (87·15% of all Rickettsia-positive ticks). GIS analysis revealed that Central and Northern coastal regions are at high risk for SFGR. The presence of ticks was significantly associated with normalized difference vegetation index and temperature variation over the course of the year. The presence of rickettsiae was significantly associated with brown type soils, higher land surface temperature and higher precipitation. The latter parameters may contribute to infection of the tick with SFGR. Health care professionals should be aware of the possible exposure of local communities and travellers to R. massillae. Molecular and geographical information can help professionals to identify areas that are susceptible to SFGR-infected ticks.

Structural basis for selective targeting of leishmanial ribosomes: aminoglycoside derivatives as promising therapeutics.

Leishmaniasis comprises an array of diseases caused by pathogenic species of Leishmania, resulting in a spectrum of mild to life-threatening pathologies. Currently available therapies for leishmaniasis include a limited selection of drugs. This coupled with the rather fast emergence of parasite resistance, presents a dire public health concern. Paromomycin (PAR), a broad-spectrum aminoglycoside antibiotic, has been shown in recent years to be highly efficient in treating visceral leishmaniasis (VL)-the life-threatening form of the disease. While much focus has been given to exploration of PAR activities in bacteria, its mechanism of action in Leishmania has received relatively little scrutiny and has yet to be fully deciphered. In the present study we present an X-ray structure of PAR bound to rRNA model mimicking its leishmanial binding target, the ribosomal A-site. We also evaluate PAR inhibitory actions on leishmanial growth and ribosome function, as well as effects on auditory sensory cells, by comparing several structurally related natural and synthetic aminoglycoside derivatives. The results provide insights into the structural elements important for aminoglycoside inhibitory activities and selectivity for leishmanial cytosolic ribosomes, highlighting a novel synthetic derivative, compound 3: , as a prospective therapeutic candidate for the treatment of VL.

Abietane-Type Diterpenoid Amides with Highly Potent and Selective Activity against Leishmania donovani and Trypanosoma cruzi.

Dehydroabietylamine (1) was used as a starting material to synthesize a small library of dehydroabietyl amides by simple and facile methods, and their activities against two disease-causing trypanosomatids, namely, Leishmania donovani and Trypanosoma cruzi, were assayed. The most potent compound, 10, an amide of dehydroabietylamine and acrylic acid, was found to be highly potent against these parasites, displaying an IC50 value of 0.37 µM against L. donovani axenic amastigotes and an outstanding selectivity index of 63. Moreover, compound 10 fully inhibited the growth of intracellular amastigotes in Leishmania donovani-infected human macrophages with a low IC50 value of 0.06 µM. This compound was also highly effective against T. cruzi amastigotes residing in L6 cells with an IC50 value of 0.6 µM and high selectivity index of 58, being 3.5 times more potent than the reference compound benznidazole. The potent activity of this compound and its relatively low cytotoxicity make it attractive for further development in pursuit of better drugs for patients suffering from leishmaniasis and Chagas disease.

Synthesis and biological evaluation of 2-arylbenzimidazoles targeting Leishmania donovani.

A set of 56 2-arylbenzimidazoles was designed, synthesized and tested against Leishmania donovani amastigotes. The left- and right-hand side rings of the molecule, as well as the amide linker were modified. Structurally different derivatives were screened on L. donovani axenic amastigotes at concentrations of 5, 15 and 50 µM, and the ten most active derivatives were selected for further testing. 2-Arylbenzimidazole derivative 24 was active against L. donovani-infected THP-1 cells showing 46% parasite inhibition at 5 µM.

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.

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.

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.

Anti-leishmanial activity of disubstituted purines and related pyrazolo[4,3-d]pyrimidines.

We report here results of screening directed to finding new anti-leishmanial drugs among 2,6-disubstituted purines and corresponding 3,7-disubstituted pyrazolo[4,3-d]pyrimidines. These compounds have previously been shown to moderately inhibit human cyclin-dependent kinases. Since some compounds reduced viability of axenic amastigotes of Leishmania donovani, we screened them for interaction with recombinant leishmanial cdc-2 related protein kinase (CRK3/CYC6), an important cell cycle regulator of the parasitic protozoan. Eighteen pairs of corresponding isomers were tested for viability of amastigotes and for inhibition of CRK3/CYC6 kinase activity. Some compounds (9A, 12A and 13A) show activity against amastigotes with EC(50) in a range 1.5-12.4µM. Structure-activity relationships for the tested compounds are discussed and related to the lipophilicity of the compounds.

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 diagnosis of Old World Leishmaniasis by high-resolution melting analysis of the 7SL RNA gene.

High-resolution melt analysis PCR (HRM PCR) for diagnosis of Old World Leishmania was developed using the 7SL RNA gene. Cutaneous leishmaniasis samples were analyzed. Sensitivity and specificity of HRM PCR were significantly better (P < 0.001) than those of internal transcribed spacer 1 PCR and similar to those of kinetoplast DNA PCR.

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.