The study was conducted in four geographically distant Schistosoma mansoni endemic areas namely: Wondo Genet about 261Km south of Addis Ababa, located at 07°05′35″N, 038°36′66″E at an altitude of 1755 m above sea level, Ziway about 164Km away from the capital Addis Ababa, located at 07°56′37″N, 038°43′25″E at an altitude of 1642 m above sea level, Sille-Elgo about 525Km away southwest of Addis Ababa located at about 05°28′39″N, 037°26′02″E at an altitude of 1188 m above sea level, and Kemissie about 305 km northeast of Addis Ababa located at 10°43′30″N, 039°04′20″E at an altitude of 1450 m above sea level. Work permission was obtained from local administrative officers, health offices and school principals.
From 2010 to 2011 a cross sectional parasitological study was conducted in the four study areas in order to determine the prevalence and intensity of Schistosoma mansoni infection. Among those found with high intensity of Schistosoma mansoni infection, stool specimens were collected for the second time to harvest egg and hatch miracidia. Each single miracidium was used to determine genetic diversity and population structure of Schistosoma mansoni isolates from the four endemic study areas.
Stool collection, examination, and miracidia hatching
Small plastic sheets were distributed to voluntary study participants and sizable stool specimens were collected and examined using Kato-Katz method (41.7 mg template) . Infection status was determined by the presence or absence of Schistosoma mansoni eggs. Stool specimens were collected from 16 S. mansoni positive subjects in Sille-Elgo, 30 subjects in Wondo Genet, 30 subjects in Kemissie and15 subjects from Ziway, totaling 91. The stool specimens were kept in 0.85 % saline in vial and transported in ice box to the Medical Parasitology Laboratory of Aklilu Lemma Institute of Pathobiology, Addis Ababa University, to harvest miracidia.
In order to stimulate hatching of miracidia, stool samples were homogenized with saline and sieved through tiered sieve of 425, 180 and 140 μm mesh size and kept for about 20 min in dark in order to allow the eggs to settle in the bottom of the flask. The supernatant was poured and the eggs were put in 250 ml flask filled with aged water. The flasks were exposed to artificial light in order to initiate hatching. The flasks were covered with black carbon paper and aluminum foil. This induces the positive phototropic and negative geotropic characteristics of the miracidia which results in their accumulation on the top of the flask . From those specimens that hatched a total of 379 miracidia from 52 patients were collected. These included 81 miracidia from seven individuals in Sille-Elgo, 88 miracidia from 19 individuals in Wondo Genet, 151 from 20 individuals of Kemissie, and 59 from six individuals in Ziway. The miracidia were transferred individually in 2 μl of water using micropipette into Eppendorf tube under a dissecting microscope. Single miracidium was put in Eppendorf tube in 96 % ethanol at −20 °C until processed in the laboratory of Centre de Biologie et d’Ecologie Tropicale et Méditerranéenne, University of Perpignan Via Domitia, France.
DNA extraction from Schistosoma mansoni miracidia was done following Beltran et al.  protocol. In brief, before DNA extraction, miracidia were individually vacuum-dried for 15 min in a Speedvac evaporator. Following, 20 μl of NaOH (250 mM) was added to each tube. After a 15 min incubation period at 25 °C, the tubes were heated in boiling water at 99 °C for 2 min. Then, 10 μl HCl (250 mM), 5 μl of Tris–HCl (500 mM) and 5 μl Triton X-100 (2 %) were added and a second heat shock in boiling water at 99 °C for 2 min was performed. The products were put in room temperature until processed for Polymerase Chain Reaction (PCR).
Polymerase chain reaction
Previously published 11 polymorphic microsatellite markers, namely SMDA28, SMC1, SMDO11 and AF325698 , R95529, SMD57, L46951 and SMD25 , SMBR16 and SMBR10  and SMS7  were used to determine the genetic variation and Schistosoma mansoni population structure of the Ethiopian isolates. To maximize efficiency and minimize cost, these PCRs were performed in three multiplexes . The PCR amplifications loci: R95529, SMC1, SMBR16, SMD57, SMDO11 are Multiplex1; SMDA28, SMS7, SMD28 are Multiplex2, and SMBR10, L46951, SMD25 are Multiplex 3. The PCR reactions were carried out in a total volume of 20 μl containing 4 μl of 5X buffer (10 mM Tris–HCl, pH 9.0 at 25 °C, 50 mM KCl, 0.1 % Triton X-100), 0.2 μM of each oligonucleotide primer, 200 μM of each dNTP (Promega), 1 unit of GoTaq polymerase (Promega, Madison, Wisconsin), 1 μl of extracted DNA and DNase-free water q.s.p. 20 μl. The PCR program consisted an initial denaturation phase at 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s, 57 °C annealing temperature for 20 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min in a thermocycler (Bio-Rad, Hercules, USA). For each marker, the forward PCR primer was 5̍ fluorescein labeled (Proligo, Cambridge, UK) allowing a precise analysis in an automated DNA sequencer. A mix of 40 μl sample loading solution (Beckman Coulter, Villepinte, France) and 0.1875 μl DNA size 400, a red labeled size standard (CEQTM DNA size standard kit, 400 Beckman Coulter), was prepared and 0.75 μl of the microsatellite PCR products were diluted in 39.25 μl sample loading solution. Mineral oil was dropped in each tube and electrophoresed using an automatic sequencer (CEQTM 8000, Beckman Coulter) with CEQTM 8000 sequence analysis software. The sizes of the alleles were calculated using the fragment analyzer package . All loci were tested pairwise based on 4400 permutations and adjusting P value to 0.000227 and there was no linkage disequilibrium detected.
In this study, miracidia from all the patients within a single study site were treated as a population while all miracidia in a single host are treated as infrapopulation.
The allele frequencies were calculated using the program MICROSATELLITE TOOLKIT (software available upon request). Both the expected and observed heterozygosities were also calculated using MICROSATELLITE TOOLKIT and their statistical significance tested using the chi-square test at α̍ =0.05. FSTAT was used to test for deviations from Hardy-Weinberg equilibrium using exact tests, testing the hypothesis that observed diploid genotypes are the product of random union of gametes. An exact test for linkage disequilibrium between pairs of loci was performed using the FSTAT. Mean estimates of F
(inbreeding coefficient) for each population and pairwise F
(between all population pairs) were also calculated following the method of Weir and Cockerham . Deviation of F
values from zero was tested using a permutation test. All F statistics were carried out using FSTAT 22.214.171.124 . Isolation By Distance (IBD) was tested correlating genetic distance (Fst/(1-Fst)) and geographic distance in kilometer. The paired t-test is used to compare two sample means where there is a one-to-one correspondence (or pairing) between the samples while Friedman’s test was used for ordinal data or an interval-scale variable that is not normally distributed . Genetic structuration was assessed using both Principal Component Analysis (PCA) using Genetix software  and bayesian approach using Structure software . Full-sib analyses was assessed by estimating mean number of genetically unique adult worm pairs and its standard deviation for each patient using Colony software .
The study was ethically approved by the Institutional Research and Ethics Committee of the Department of Microbial, Cellular and Molecular biology, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia and by the National Research Ethics Review Committee of Federal Republic of Ethiopia Ministry of Science and Technology, P.O. Box 2490, Addis Ababa, Ethiopia. Informed verbal consent was obtained from all adults. For school age children younger than 18, informed verbal consent was obtained from their parents through health extension workers and school principals. In addition, the children also gave their assent. All study participants found positive for S. mansoni were treated with Praziquantel at a dose of 40 mg/Kg body weight.