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

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

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

Incidence of Echinococcus granulosus in Domestic Dogs in Palestine as Revealed by Copro-PCR

Hydatidosis or echinococcosisis considered a neglected zoonotic disease despite its high burden in the livestock industry and the high risk of infection by humans in endemic areas. In a cross-sectional study we estimated the copro-Incidence and also genotyped Echinococcus granulosus isolates from domestic dogs using polymerase chain reaction (PCR). Medical archives in nine major hospitals in Palestine were reviewed to determine incidence of E. granulosus infection detected in humans during surgery. Faecal samples were collected from 93 domestic dogs in three districts with the highest number of human cases: Al- Khalil (Hebron), Tubas and Jenin. Genomic DNA was extracted from dog faecal samples and amplified by PCR targeting the repeat DNA sequence (EgG1 Hae III) followed by sequencing of five positive samples. Genotyping was determined by sequencing and BLAST searching of mitochondrial cytochrome c oxidase subunit (CO1). The incidence of E. granulosus infection detected in humans at surgery was 1.2 per 100,000 in the West Bank and 1.0 per 100,000 in Gaza Strip. Seventeen of 93 domestic dogs (18%) were positive, based upon comparison with the Echinococcus DNA control. The five sequenced samples were confirmed to be E. granulosus. Successfully genotyped sample belonged to E. granulosus sensu stricto (formerly G1-G3 complex, sheep strain). For domestic dogs, age group (13-24 months) and sex were identified as two risk factors for contracting E. granulosus. The study identified the high incidence of E. granulosus sensu stricto in dogs in Palestine. AuthorWe thank the Arab American University in Jenin-Palestine for the fund received under grant number 2013-104, cycle 2. Also, the study received support from the Netherlands Ministry of Foreign Affairs, The Hague, The Netherlands and NVHU under grant reference number 2014.52146. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Molecular epidemiology of human cutaneous leishmaniasis in Jericho and its vicinity in Palestine from 1994 to 2015

Cutaneous leishmaniases (CL) are vector-borne parasitic diseases endemic inmany countries of the Middle East including Palestine. Between 1994 and 2015, 2160 clinically suspected human cases of CL from the Jericho District were examined. Stained skin tissue smears and aspirates were checked by microscopy and cultured for promastigotes, respectively. For leishmanial species identification, amplification products from a PCR-ITS1 followed by RFLP analysis using Hae III. Data were analyzed using Epi Info free-software. The overall infection rate was 41.4% (895/2160), 56.3% (504/895) of the cases were male, 43.7% (391/895) female, 60.5% (514/849) children under age 14, 41.3% (259/627) of the cases were caused by Leishmania major and 57.3% (359/627) by Leishmania tropica. The case numbers peaked in 1995, 2001, 2004, and 2012. Statistically-significant clusters of cases caused by L. major were restricted to the Jericho District; those caused by L. tropica were from the districts of Jericho, Bethlehem, Nablus and Tubas. CL is seasonal and trails the sand fly season. Distribution of cases was parabolicwith fewest in July. Themonthly total number of cases of CL and just those caused by L.major correlated significantly with temperature, rainfall, relative humidity, evaporation, wind speed and sunshine (P b 0.05, r2= 0.7–0.9 and P b 0.05, r2=0.5–0.8, respectively). Cases caused by L. tropica, significantly, had a single lesion compared to cases caused by L. major (P=0.0001), which, significantly, had multiple lesions (P=0.0001). This and previous studies showed that CL is present in all Palestinian districts. The surveillance of CL has increased public awareness and molecular biologicalmethodology for leishmanial species identification is an essential addition to classical diagnosis. The overall results are discussed, correlated to climatic and environmental changes and largescale human activities.This work received financial support from grants of the Deutsche Forschungsgemeinschaft (DFG), Scho 448/6-1-3, Scho 448/8-1, Scho 448/8-2 that extended from 1998 until 2015. It also received support fromEurNegVeg COST Action TD1303 (Cost 037/13). At one time during the study WHO Eastern Mediterranean Region (EMRO), Division of Communicable Diseases (DCD) and the WHO Special Programme for Research and Training in Tropical Diseases (TDR): the EMRO/DCD/TDR Small Grants Scheme for Operational Research in Tropical and Communicable Diseases financially supported this work. We thank Dr. L. F. Schnur for going over our manuscript

The clinical burden of human cystic echinococcosis in Palestine, 2010-2015

Background Cystic echinococcosis (CE) is classified by the WHO as a neglected disease inflicting economic losses on the health systems of many countries worldwide. The aim of this caseseries study was to investigate the burden of human CE in Palestine during the period between 2010 and 2015. Methodology/Principal findings Records of surgically confirmed CE patients from 13 public and private hospitals in the West Bank and Gaza Strip were reviewed. Patients' cysts were collected from surgical wards and formalin-fixed paraffin-embedded (FFPE) blocks were collected from histopathology departments. Molecular identification of CE species /genotypes was conducted by targeting a repeat DNA sequence (EgG1 Hae III) within Echinococcus nuclear genome and a fragment within the mitochondrial cytochrome c oxidase subunit 1, (CO1). Confirmation of CE species/genotypes was carried out using sequencing followed by BLAST analysis and the construction of maximum likelihood consensus dendrogram. CE cases were map-spotted and statistically significant foci identified by spatial analysis. A total of 353 CE patients were identified in 108 localities from the West Bank and Gaza Strip. The average surgical incidence in the West Bank was 2.1 per 100,000. Spot-mapping and purely spatial analysis showed 13 out of 16 Palestinian districts had cases of CE, of which 9 were in the West Bank and 4 in Gaza Strip. Al-Khalil and Bethlehem were statistically significant foci of CE in Palestine with a six-year average incidence of 4.2 and 3.7 per 100,000, respectively. Conclusions/Significance To the best of our knowledge, this is the first confirmation of human CE causative agent in Palestine. This study revealed that E. granulosus sensu stricto (s.s.) was the predominating species responsible for CE in humans with 11 samples identified as G1 genotype and 2 as G3 genotype. This study emphasizes the need for a stringent surveillance system and risk assessment studies in the rural areas of high incidence as a prerequisite for control measures.The research that has led to these results has been technically supported by the European Community's Seventh Framework Programme under the grant agreement 602051 (Project HERACLES: Human cystic Echinococcosis ReseArch in CentraL and Eastern Societies; http://www. Heracles-fp7.eu/)

Laboratory study of enteroviral central neurous system infections

Human enteroviruses (HEV) belong to the family Picornaviridae. They are associated with a broad spectrum of clinical features including acute respiratory illness, myocarditis, “hand, foot and mouth disease”, conjunctivitis, neonatal multi-organ failure, acute flaccid paralysis and CNS infection with aseptic meningitis cases accounting for the vast majority of the hospitalizations worldwide. The aim of present study was: a) the isolation and the detection of enteroviruses that circulated in Greece and caused CNS infections, such as aseptic meningitis, encephalitis and other CNS infections in respect to clinical manifestations and CSF laboratory findings. b) Optimization molecular methods for the detection and typing the enteroviruses directly from the CSF, in comparison to the conventional methods. c) to study the molecular characterization and epidemiology of the enteroviruses that have been circulated during the study period in comparison with the enteroviruses that isolated in Greece in the previous years as well as in Europe and other countries using sequence and phylogenetic analysis During the period of March 2003-September 2006, 167 Greek patients with CNS infections were tested for enteroviruses (86 with symptoms of aseptic meningitis, 60 with encephalitis and 21 with other CNS infections). In addition, 34 CSF samples from Palestinian patients with aseptic meningitis during an outbreak that occurred in summer 2005 in Palestine were tested. Tissue culture with neutralization test, serology as well as molecular methods that companied with the phylogenetic analysis, were optimized in the present study. Overall, enterovirus was detected in 45 (26.9%) from Greek patients and in 3 (8.8%) from Palestinian patients. Analytically, 33 came from patients with aseptic meningitis, 10 from patients with encephalitis and 2 from patients with other CNS infections. Regarding the cases from Palestine, the three patients had aseptic meningitis. Enteroviruses were isolated only from some cases with aseptic meningitis and no enterovirus was isolated from CSF samples with encephalitis. The molecular techniques in combination with the phylogenetic analysis were found to be more sensitive and specific for direct diagnosis and typing the enteroviruses directly from CSF samples. Most (72,7%) of the EV aseptic meningitis cases were children less than 15 years old, while 70% of the EV encephalitis occurred in adults. The male to female ratio was 2.4:1. More specifically, the male to female ratio for the EV aseptic meningitis was 2:1 and 9:1 for the cases with EV encephalitis. Most of the EV infections were reported during summer and autumn months. During the study period, an outbreak of aseptic meningitis in Patra district in autumn 2005 was reported. In general, the outcome of EV infections in our study was mild to moderate and the final outcome was favorable with no reported death or sequelae. However, the cases with EV encephalitis were found to be more complicated where two of them died. The most predominant enterovirus that circulated and caused CNS infections during the study period was Echo 15 (50%), followed by Echo 11 and Coxsackie B5 with 13.3% of each and then by Echo 6 with 10%, followed with very small frequency (3.3%), of Echo 5, Echo 9, Echo 14 and Coxsackie A9. More specifically for Echo 15, a strong evidence of recombination was observed. Regarding the EVs which were isolated and/or detected from the Palestinian cases, they were belonging to serotypes Echo 6, Echo 14 and Echo 30.Οι Ανθρώπειοι Εντεροϊοί (HEV) ανήκουν στην οικογένεια Picornaviridae. Αυτοί είναι το αίτιο για ένα ευρύ φάσμα νοσημάτων που περιλαμβάνει οξεία αναπνευστική λοίμωξη, μυοκαρδίτιδα, νόσο χειρός- ποδός και στόματος, επιπεφυκίτιδα, νεογνική πολυοργανική ανεπάρκεια, οξεία χαλαρή παράλυση και λοιμώξεις του ΚΝΣ, με τις περιπτώσεις της άσηπτης μηνιγγίτιδας να προσμετρούν τη πλειονότητα των ασθενών που χρειάζεται να νοσηλευθούν σε νοσοκομείο παγκοσμίως. Σκοπός της παρούσας μελέτης ήταν: α) η απομόνωση και η ανίχνευση των εντεροϊών που κυκλοφορούν στην Ελλάδα και προκαλούν λοιμώξεις του ΚΝΣ, όπως άσηπτη μηνιγγίτιδα, εγκεφαλίτιδα και άλλες, και η συσχέτισή τους με τις κλινικές εκδηλώσεις και τα εργαστηριακά ευρήματα. β) Η εφαρμογή μοριακών μεθόδων για τη διάγνωση και την τυποποίηση των εντεροϊών άμεσα στο ΕΝΥ, καθώς και η σύγκριση αυτών με τις συμβατικές μεθόδους, και γ) Ο μοριακός χαρακτηρισμός και η επιδημιολογία των εντεροϊών που κυκλοφορούσαν κατά τη διάρκεια της μελέτης στην Ελλάδα, μετά από σύγκριση με τα επιδημιολογικά δεδομένα τυποποίησης εντεροϊών προηγουμένων ετών στην Ελλάδα, την Ευρώπη και άλλες χώρες, με μελέτη της αλληλουχίας των βάσεων και τη φυλογενετική ανάλυση. Κατά το χρονικό διάστημα Μαρτίου 2003 - Σεπτεμβρίου 2006, ελέγχθηκαν για λοίμωξη από εντεροϊό 167 Έλληνες ασθενείς με λοίμωξη του ΚΝΣ (86 με συμπτώματα άσηπτης μηνιγγίτιδας, 60 με συμπτώματα εγκεφαλίτιδας και 21 με άλλη λοίμωξη του ΚΝΣ). Επιπλέον, μελετήθηκαν 34 δείγματα ΕΝΥ από ασθενείς με συμπτώματα άσηπτης μηνιγγίτιδας που πάρθηκαν κατά τη διάρκεια μίας επιδημίας που παρατηρήθηκε στην Παλαιστίνη το 2005. Εφαρμόστηκαν οι κλασσικές μέθοδοι καλλιέργειας σε συνδυασμό με εξουδετερωτικές δοκιμασίες, ορολογικές μέθοδοι, καθώς και μοριακές μέθοδοι συνοδευόμενες από φυλογενετική ανάλυση. Συνολικά, εντεροϊός ανιχνεύτηκε σε 45 (26,9%) Έλληνες ασθενείς και σε 3 (8,8%) από την Παλαιστίνη. Αναλυτικά, 33 θετικά δείγματα προερχόταν από ασθενείς με άσηπτη μηνιγγίτιδα, 10 από ασθενείς με εγκεφαλίτιδα και 2 ασθενείς με άλλη λοίμωξη του ΚΝΣ. Όσον αφορά την ομάδα των ασθενών από την Παλαιστίνη, και οι τρεις ασθενείς είχαν άσηπτη μηνιγγίτιδα. Εντεροϊοί απομονώθηκαν σε καλλιέργειες κυττάρων μόνο από περιπτώσεις άσηπτης μηνιγγίτιδας και σε καμία από τις περιπτώσεις εγκεφαλίτιδας. Οι μοριακές τεχνικές σε συνδυασμό με τη φυλογενετική ανάλυση βρέθηκε να είναι οι πλέον ευαίσθητες και ειδικές για την άμεση διάγνωση και τυποποίηση των εντεροϊών κατευθείαν από δείγματα ΕΝΥ. Τα περισσότερα (72,7%) περιστατικά άσηπτης μηνιγγίτιδας από εντεροϊό παρατηρήθηκαν σε παιδιά ηλικίας μικρότερης των 15 ετών, ενώ εγκεφαλίτιδα παρατηρήθηκε κυρίως στους ενήλικες (70%). Η αναλογία ανδρών/γυναικών με λοιμώξεις του ΚΝΣ οφειλόμενων σε εντεροϊό ήταν 2,4:1. Ειδικότερα, για τα περιστατικά άσηπτης μηνιγγίτιδας η αντίστοιχη αναλογία ήταν 2:1 και για τα περιστατικά εγκεφαλίτιδας ήταν 9:1. Οι περισσότερες περιπτώσεις λοίμωξης με εντεροϊό παρατηρήθηκαν τους καλοκαιρινούς και φθινοπωρινούς μήνες. Κατά τη διάρκεια της μελέτης παρατηρήθηκε επίσης μία επιδημία άσηπτης μηνιγγίτιδας στην περιοχή της Πάτρας το φθινόπωρο του 2005. Γενικά, η πρόγνωση των λοιμώξεων από εντεροϊό ήταν καλή. Ωστόσο βαρύτερη εικόνα παρατηρήθηκε στους ασθενείς με εγκεφαλίτιδα, και δύο από τους δέκα απεβίωσαν. Ο συχνότερος εντεροϊός που κυκλοφορούσε στην Ελλάδα και προκάλεσε λοιμώξεις ΚΝΣ την περίοδο της μελέτης ήταν ο ιός Echo 15 (50%), και ακολουθούσαν οι τύποι Echo11 (13,3%), Coxsackie B5 (13,3%) και Echo 6 10%. Σε πολύ μικρότερη συχνότητα (3,3%) βρέθηκαν οι ιοί Echo 5, Echo 9, Echo 14 και Coxsackie A9. Ειδικότερα για τον ιό Echo 15 υπήρξε ισχυρή ένδειξη ότι επρόκειτο για ανασυνδυασμένο στέλεχος. Όσον αφορά τα στελέχη των εντεροϊών που ανιχνεύτηκαν στην Παλαιστίνη, αυτά ανήκαν στους τύπους Echo 6, Echo 14, και Echo 30

Molecular detection and genotyping of enteroviruses from CSF samples of patients with suspected sepsis-like illness and/or aseptic meningitis from 2012 to 2015 in West Bank, Palestine.

BACKGROUND:Human enteroviruses (HEVs) are the most frequently reported cause of aseptic meningitis with or without CSF pleocytosis in childhood. Rapid detection and genotype of HEVs is essential to determine the causative agent and variant causing sepsis-like illness and/or aseptic meningitis. AIM:To investigate the molecular epidemiology of enteroviruses (EVs) among patients with sepsis-like illness and/or aseptic meningitis admitted to three major hospitals in West Bank, Palestine from 2012 to 2015. METHODS:During the study period, 356 CSF samples were collected from patients with sepsis-like illness and/or aseptic meningitis. Two RT-nested PCR assays targeting a partial part of 5'UTR for direct diagnosis and the VP1 region for genotyping by sequence analysis of the viral genome were used. RESULTS:HEV RNA was detected in 66 of 356 (18.5%) of CSF samples. Age distribution showed that 64% (42/66) were infants (<1 year), 18% were children between 1 and 5 years old, 12% were children between 5 and 10 years old, and 6% were more than 10 years old. Of the 66 EV cases, 12 were successfully genotyped. Five different EV genotypes were identified. All of them belonged to HEV-B species. The study showed that echovirus 6 genotype accounted for 42% of the sequenced cases. The HEV infections in the present study tended to show slight seasonal pattern with more cases occurring during spring and summer, yet still significant numbers were also reported in fall and winter seasons. CONCLUSION:HEV was isolated from a significant number of children with sepsis-like illness and/or aseptic meningitis. In addition, the molecular method utilized for direct diagnosis and genotyping of HEV from CSF revealed that more than one HEV type circulated in the West Bank, Palestine during the study period

Distribution of HEV genotypes.

<p>Distribution of HEV genotypes.</p

HEV cases included in the study (2012–2015) with Ministry of Health-reported sepsis like illness and/or viral meningitis cases and incidence rates (2000–2015).

<p>HEV cases included in the study (2012–2015) with Ministry of Health-reported sepsis like illness and/or viral meningitis cases and incidence rates (2000–2015).</p

Demographic data and clinical manifestations of the study population.

<p>Demographic data and clinical manifestations of the study population.</p