Genetic characterization of Mycobacterium tuberculosis in the West Bank, Palestinian Territories

BACKGROUND: The World Health Organization (WHO) declared human tuberculosis (TB) a global health emergency and launched the “Global Plan to Stop Tuberculosis” which aims to save a million lives by 2015. Global control of TB is increasingly dependent on rapid and accurate genetic typing of species of the Mycobacterium tuberculosis (MTB) complex including M. tuberculosis. The aim of this study was to identify and genetically characterize the MTB isolates circulating in the West Bank, Palestinian Territories. Genotyping of the MTB isolates from patients with pulmonary TB was carried out using two molecular genetic techniques, spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) supported by analysis of the MTB specific deletion 1 (TbD1). FINDINGS: A total of 17 MTB patterns were obtained from the 31 clinical isolates analyzed by spoligotyping; corresponding to 2 orphans and 15 shared-types (SITs). Fourteen SITs matched a preexisting shared-type in the SITVIT2 database, whereas a single shared-type SIT3348 was newly created. The most common spoligotyping profile was SIT53 (T1 variant), identified in 35.5 % of the TB cases studied. Genetic characterization of 22 clinical isolates via the 15 loci MIRU-VNTR typing distinguished 19 patterns. The 15-loci MIT144 and MIT145 were newly created within this study. Both methods determined the present of M. bovis strains among the isolates. CONCLUSIONS: Significant diversity among the MTB isolates circulating in the West Bank was identified with SIT53-T1 genotype being the most frequent strain. Our results are used as reference database of the strains circulating in our region and may facilitate the implementation of an efficient TB control program

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

MDR tuberculosis and non-compliance with therapy

The emergence of multidrug-resistant (MDR) tuberculosis is a worldwide public health problem. We reported a case of pulmonary MDR tuberculosis in a 41-year-old man with diabetes from the West Bank, Palestine. 1 The bacteria belonged to the Beijing family, which is highly virulent and easily disseminated. 2 The patient withdrew from treatment after 2 years while still sputum-positive. Despite persistent efforts of the tuberculosis directly observed treatment programme, he disappeared and cannot be located. The patient might have fled to a neighbouring country, and there is a risk that he will pass on MDR tuberculosis to people with whom he has contact

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 PCRbased 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.Acknowledgments We thank L. F. Schnur for reviewing the manuscript. Authors’ contributions AA, conception of the research, study design, data analysis and drafting of the manuscript. SE and AN, molecular biological testing and analysis. KD and HA collection of samples and conventional examination. ZA, data analysis and interpretation. All the authors have read and approved the final manuscript. Funding This research is a self-funded work by the researchers

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.Acknowledgments Not applicable. Authors’ contributions AN, SE, AA-J and ZA conceived and designed the experiments. AN, SE, HA-J, NA-L and AA-J performed the experiments. SE, AN and AA-J analyzed the data. AN, AA-J and SE wrote the first draft of the manuscript. AN, SE, NA-L, HA, AA-J and ZA competed the final revision of the manuscript to be published. All authors read and approved the final manuscript. Funding This research received no financial support

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.Acknowledgments The authors gratefully acknowledge the UNRWA outpatient clinic members (Hebron and Ramallah, Palestine) who contributed to the patients’ recruitment, and all subjects who participated to this study. Funding The deanship of scientific research-Al-Quds University provided the financial support

Characterization of Leishmania Ulcers Microbiota Using Next Generation Sequencing

The human skin microbiome is a major source of bacteria in cutaneous leishmaniasis (CL) ulcers following the fall of the crust and the subsequent formation of a shallow depression in the epidermis and dermis of the skin. As a result, secondary bacterial infections are frequently observed which impair the healing process. Our study aimed at studying the bacterial communities in CL lesions using next-generation sequencing. A total of 298 patients (178 males and 120 females; median age of 17) presenting ulcerated skin lesions suspected with CL were included in this study. CL was confirmed in 153 (51%) cases by ITS1-PCR and/ or microscopy. Based on bacterial 16S rRNA-PCR, 92 samples were positive for the presence of bacteria, while 206 samples were negative and excluded from the microbiome study. A total of 925 Operational Taxonomic Units (OTUs) were identified and assigned to 215 genera. Despite insignificant difference in the microbiome composition between CL and non-CL lesions, the phylum level analysis revealed that Actinobacteria was significantly higher in CL ulcers while Protoeobacteria was significantly higher in non-CL ulcers (X2, P=0.039). The relative abundance of the most commonly encountered skin pathogens i. e E coli, Pseudomonas aeruginosa, Enterobacter, Enterococcus and Acinetobacter species were significantly higher in non-CL ulcers (X2, P<0.05) compared to Staphylococcus aureus and Proteus mirabilis which were higher in CL ulcers (P<0.05). Our data showed that bacterial communities did not cluster according to the Leishmania infection. Nonetheless, bacterial diversity was lower in CL compared to non-CL lesions. Presence of pathogenic bacteria in CL lesions such as S. aureus might exacerbate lesions, hinder diagnosis, and delay healing

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.The authors thank each of Baraah Farun, Peter Bael, aya siaj, aseel Nassar, alhareth amro, Yahya abu Jwaid, Bayan awad, adan Rabayah, Basel Zaben, Natalie Khamashta, Mo’men alashwas, anas Barabrah, anas toqan, Mohammad shehadeh and Raya amer for their assistance in the data collection for this project

New Clinicoepidemiologic Profile of Cutaneous Leishmaniasis, Morocco

During the past 20 years, cutaneous leishmaniasis has emerged as a major public health threat in Morocco. We describe distribution of Leishmania major and L. tropica in Morocco and a new focus of cutaneous leishmaniasis due to L. infantum. We recommend using molecular techniques to diagnose suspected leishmaniasis cases

First-Time Detection of Mycobacterium bovis in Livestock Tissues and Milk in the West Bank, Palestinian Territories

Background: Bovine tuberculosis, bTB, is classified by the WHO as one of the seven neglected zoonontic diseases that cause animal health problems and has high potential to infect humans. In the West Bank, bTB was not studied among animals and the prevalence of human tuberculosis caused by M. bovis is unknown. Therefore, the aim of this study was to estimate the prevalence of bTB among cattle and goats and identify the molecular characteristics of bTB in our area. Methodology/principal findings: A total of 208 tissue samples, representing 104 animals, and 150 raw milk samples, obtained from cows and goats were examined for the presence of mycobacteria. The tissue samples were collected during routine meat inspection from the Jericho abattoir. DNA was extracted from all samples, milk and tissue biopsies (n = 358), and screened for presence of TB DNA by amplifying a 123-bp segment of the insertion sequence IS6110. Eight out of 254 animals (3.1%) were found to be TB positive based on the IS6110-PCR. Identification of M. bovis among the positive TB samples was carried out via real time PCR followed by high resolution melt curve analysis, targeting the A/G transition along the oxyR gene. Spoligotyping analysis revealed a new genotype of M. bovis that was revealed from one tissue sample. Significance: Detection of M. bovis in tissue and milk of livestock suggests that apparently healthy cattle and goats are a potential source of infection of bTB and may pose a risk to public health. Hence, appropriate measures including meat inspection at abattoirs in the region are required together with promotion of a health campaign emphasizing the importance of drinking pasteurized milk. In addition, further studies are essential at the farm level to determine the exact prevalence of bTB in goats and cattle herds in the West Bank and Israel.Financial support was provided by the Dutch government; project M27-072NVHU 2009 02 ‘Vector-Borne Pathogens in Israel and the Palestinian Authority.’ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript