This cross-sectional study was done in 2015-2016 and targeted three adjacent villages, Nassarieh, Beit-Hassan, and Al Aqrabaneieh, in the Palestinian part of the Jordan Valley located 50km north of Jericho and 15km west of Nablus. The populations of An-Nassariya, Beit-Hassan, and Al ‘Aqrabaniya numbered 1,923, 1,360, and 1,215 inhabitants, respectively , most of whom were active farmers and livestock breeders. The villages stand on the foothills east of Nablus, approximately, at an latitude of 32.2437782287598 north and longitude of 35.391918182373 east (Epi Info version 18.104.22.168) and an elevation ranging from -40m below sea level to 20m above sea level.
The 104 individuals surveyed and examined parasitologically were selected randomly by knocking on the doors of houses without prior arrangement. After obtaining informed consent, members of households were interviewed by filling in a pre-tested questionnaire that took 15 minutes to complete. Four previously trained personnel carried out the interviews. The questionnaire solicited: demographic data such as age, sex, and place of residence; socioeconomic status (SES) data that included level of education, occupation and income. The questionnaire also questioned personal hygienic behavior such as hand washing as well as eating and drinking habits.
The head of each household was given a clean, labeled, wide-mouth screw cap container for each family member and thereby 102 early-morning fecal specimens were collected and transported to the diagnostic laboratory within one hour. Each fecal specimen was divided into three parts. Approximately 2g of each specimen were transferred into 2ml microcentrifuge tube and stored at -20°C for DNA analysis. The second part was used in making the wet preparation for microscopical examination. The third part was mixed with 10% formalin preservative at a ratio of 1:3 of specimen to preservative and stored for examination after concentration by either ethyl acetate sedimentation or by zinc sulfate floatation.
Microscopy of wet mount fecal preparations
For each fecal specimen, two wet mount preparations were made on a clean glass slide and under 22x22mm cover slips. For one preparation, the specimen was emulsified with normal saline (0.85% (w/v) NaCl) to enable detection of motile forms of parasites such as trophozoites. For the other preparation, the specimen was emulsified with D’Antoni’s Iodine (1% (w/v) KI and 1.5% (w/v) I2, in distilled water) to enable detection of immotile forms of parasites like cysts. The entire area under the cover slip was examined systematically, using a 10x objective, confirming the presence of parasites, using a 40x objective . For quality control and to prevent observer bias, all samples were examined microscopically by two experienced laboratory technicians. One hundred and one samples were examined by microscopy (Fig 1).
Fecal concentration methods
Two concentration procedures were used to detect parasites present in small numbers that might have been missed when scanning the wet preparations: ethyl acetate sedimentation and zinc sulphate floatation, as described elsewhere, using the fecal samples preserved in 10% formalin . Ninty- two samples were examined by sedimentation and 93 were examined by floatation (Fig 1).
Genomic DNA was extracted from 0.25 to 0.5g of fecal specimen, using a Nucleospin® Soil (Machery Nagel GmbH, Düren-Germany) commercial kit with slight modifications that included two pre-treatment steps: the tube containing fecal suspension together with the ceramic beads was incubated at 95°C for 10 min and was then beaten with a disruptor Genie (Scientific Industries, USA) for 5 min at 2800 rpm before proceeding with manufacturer’s instructions.
PCR primers were selected for the 18S rRNA gene sequences of the species E. histolytica, E. coli, E. dispar, and E. muris; GenBank accession numbers AB426549.1 and AB282660.1 for E. histolytica; AB445018.1 for E. muris, AB282661.1 for E. dispar; AF149915.1, AF149914.1, and AB444953.1 for E. coli. More versions of the sequence from the same species but of different accession numbers were included to check the stability of the polymorphic regions among the species. The sequences of all the targeted species of Entamoeba were aligned, using the Multialin website (http://multalin.toulouse.imra.fr/multalin/multalin.html) . Primers were designed, using primer 3 on-line software (http://frodo.wi.mit.edu/primer3), in which the conditions were to exclude the polymorphic regions and produce products for the entire sequence (Table 1).
The amplification reactions for Entamoeba spp considered here were performed in a volume of 25µl with PCR-Ready™-High Specificity (Syntezza Bioscience Ltd., Jerusalem), 1μl of 10µM of each primer, 2μl of the DNA template and 21μl nuclease-free water. Positive and negative controls were included. PCRs for the parasites of the species Giardia lamblia, Hymenolpis nana and Cryptosporidium spp. were performed, using the sets of primers and profiles described in Table 1 for the detection and identification of intestinal parasites. Conventional PCRs for the parasites of Giardia lamblia and those of the species of Cryptosporidium spp were conducted for quantitative real-time PCR (qPCR) to confirm results by subsequent nucleotide Sanger sequencing from both directions followed by a nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Amplification was done, using a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc. Hercules, California 94547, USA). The PCR mix was subjected to the thermal cycling profile given in Table 1. PCR products were separated by electrophoresis at 100V for 30 minutes, using a 1.8% agarose gel containing ethidium bromide at a concentration of 0.6µg/ml (LE Seakam Agarose, Lonza Group Ltd, Muenchen, Steinerstrasse 38 CH-4002, Basel, Switzerland) and Tris-Acetate-EDTA (TAE, pH 8.0) as the running buffer The gel was visualized under a UV viewer with a GeneRuler 100 bp DNA Ladder (Thermo Fisher Scientific, USA) as size marker.
Copro-PCR, qPCR and conventional PCR
Specific primers and probes were used for amplification as described by Verweij et al. . Standard curves were generated, using pure DNA from parasites of the species G. lamblia and those of Cryptosporidium spp, by plotting cycle threshold (Ct) values against the log of the DNA concentration of pure samples. To generate the standard curves, the standard DNA sample was adjusted to known concentrations of 5 to 6 points in duplicates in tenfold-serial dilutions at different concentrations of DNA from parasites of the species G. lamblia and those of Cryptosporidium. Low Ct values corresponded to high amounts of parasite-specific DNA in the samples tested. Samples with Ct above the standard curve were considered positive. qPCR reactions and conditions are given in Table 1.
For inclusion in applying the corpo-PCR, the DNA from the parasites of randomly selected infected cases done by the real-time PCR was amplified by a conventional PCR and the PCR product was DNA sequenced (Hylab, Rehovot) for species identification (Table1). One hundred and three samples were tested by copro-PCR (Fig 1).
Data management and statistical analysis
Data were analyzed, using mainly the EpiInfo statistical package and Prism online calculators. Analysis included distribution, 2x2 contingency tables, and frequency tables. A heat map was constructed based on the bivariate Pearson correlation between types versus number of parasites. Risk predictor variables for parasitic infection were analyzed by the Mid-P exact test as it is less conservative and more powerful than Fisher’s exact test, however, the latter was used whenever a cell was <5. Chi square and odds ratio with a 95% confidence interval were also calculated and confirmed by multiivariate analysis, employing the logistic regression model to calculate the adjusted odds ratio (AOR). Cochrane’s Q, McNemar post hoc test and Dunn’s post hoc test were used to assess performance of the diagnostic methods. The level of statistical significance was P<0.05.