Study setting and sampling

This cross-sectional study was conducted from June 2015 to November 2016 on 1383 individuals, attended to 14 medical laboratories in Kurdistan Province, Iran. After collecting faecal samples from the medical laboratories and completing the questionnaires, the samples were directly transferred to the Research Laboratory of the Department of Parasitology and Mycology (School of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran) for daily microscopic examinations.

Questionnaire

A structured questionnaire was used to collect information on the following causes of referral: (i) routine evaluation (i.e. check-up and receiving a health certificate); (ii) diagnosing agents of GIDs (i.e. diarrhoea, abdominal pain, weight loss, loss of appetite, vomiting, nausea, etc.); and (iii) detecting diseases other than GIDs (e.g. cancer, diabetes, autoimmune diseases, immunodeficiency disorders, etc.).

Microscopic examination

All faecal samples were examined to detect Entamoeba cysts or trophozoites, using direct wet mount examination and formalin–ether sedimentation technique under microscopic observation (Zeiss, Germany, 40× magnification). Following that, trichrome staining was performed for determining and confirming Entamoeba samples under high-power microscopic observation (Zeiss, 100× magnification).

All microscopy-positive isolates and those resembling the Entamoeba complex, in addition to 55 microscopy-positive isolates for Entamoeba coli, Endolimax nana and/or negative stool samples, were kept in 70% alcohol at 4 °C for DNA extraction and molecular analysis.

Molecular investigations were conducted in the Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences (Tehran, Iran). However, due to funding limitations, we were unable to perform PCR assays for all the samples.

Genomic DNA extraction

Almost 300 µl of faecal specimens were washed three times with triple-distilled water through centrifugation to remove any traces of alcohol. Then, genomic DNA was extracted directly from the samples, using FavorPrep® Stool DNA Isolation Mini Kit (YTA, FavorGen, Cat. No YT9032, Taiwan) with slight modifications. After adding a glass milk matrix and 1 ml of lysis buffer, the samples were frozen in liquid nitrogen and thawed at 90 °C in a water bath. The genomic DNA was then eluted in 50 µl of elution buffer and stored at −20 °C until PCR amplification.

DNA amplification by PCR

A nested multiplex PCR assay using species-specific primers was performed to amplify the region of 18S rRNA gene for the Entamoeba complex. The sensitivity and specificity of this method for the detection of the Entamoeba complex have been examined in the literature [Reference Khairnar and Parija13]. The first pair of primers, E-1 (5′-TAAGATGCACGAGAGCGAAA-3′) and E-2 (5′-GTACAAAGGGCAGGGACGTA-3′), was used to amplify about 900 bp of 18S rRNA gene. For the second round of nested multiplex PCR, the reaction conditions were optimised for amplifying species-specific product sizes (439, 553 and 174 bp for E. histolytica, E. moshkovskii and E. dispar, respectively).

The PCR assay was performed in a multiplex reaction mixture under similar conditions by combining three pairs of primers: EH-1 (5′-AAGCATTGTTTCTAGATCTGAG-3′) and EH-2 (5′-AAGAGGTCTAACCGAAATTAG-3′); Mos-1 (5′-GAAACCAAGAGTTTCACAAC-3′) and Mos-2 (5′CAATATAAGGCTTGGATGAT-3′); and ED-1 (5′-TCTAATTTCGATTAGAACTCT-3′) and ED-2 (5′-TCCCTACCTATTAGACATAGC-3′). The primer sequences were examined for specificity by conducting Basic Local Alignment Search Tool (BLAST) searches in the National Center for Biotechnology Information (NCBI). The primers were synthesised using the Macrogen® system (South Korea). For confirmation of the multiplex PCR, single-round PCR was also carried out using the described primers. The PCR assay was repeated four times in the samples (twice by multiplex-nested PCR and twice by single-nested PCR) under similar conditions.

The first PCR reaction was performed in a final volume of 25 µl, containing 12.5 µl of 2X PCR kit master mix (Ampliqon ApS, Literbuen 11, DK-2740 Skovlunde, Denmark), 15 ρM of each primer and 10 ng of extracted DNA. The second PCR reaction was performed in a final volume of 30 µl, containing 15 µl of 2X PCR master mix, 15 ρM of each primer and 10 ng of the first PCR product. The reaction conditions for the second PCR were optimised to combine the primers of E. histolytica (EH-1 and EH-2) with E. dispar (ED-1 and ED-2) and E. moshkovskii (Mos-1 and Mos-2) primers in a single reaction mixture under the same conditions.

For the first PCR assay, amplification was carried out in a thermocycler (Techne Ltd., Cambridge, UK) at 95 °C for 5 min; followed by 30 cycles at 94 °C for 30 s, at 58 °C for 30 s and at 72 °C for 30 s; and a final extension at 72 °C for 5 min. In addition, nested amplification included 35 cycles at 94 °C for 30 s, at 55 °C for 30 s and at 72 °C for 30 s under identical conditions for the initial denaturation and final extension.

Both positive and negative controls were included in each round of PCR to validate the results. Then, 3 µl of PCR products was electrophoresed on agarose gel 1.5%, stained with ethidium bromide and visualised under UV light. The positive control DNA was collected from axenically grown E. histolytica HM-1:IMSS, E. dispar SAW760 and E. moshkovskii Laredo strains. All positive control DNAs were provided by Dr Seiki Kobayashi (Department of Tropical Medicine and Parasitology, School of Medicine, Keio University, Tokyo, Japan) for A. Haghighi.

Sequencing of PCR products

The PCR-amplified products were subjected to direct sequencing, using a BigDye Terminator Cycle Sequencing Kit (PE Biosystems, Foster City, CA, USA) and a genetic analyser (3130 × 1; ABI Prism). The sequence chromatograms were observed using Chromas Version 1.0 (Technelysium Pty Ltd, Unit 406, 8 Cordelia St, South Brisbane QLD 4101, Australia). The nucleotide sequences were manually edited, and the sequence representatives for each identified species were submitted to the GenBank/EMBL/DDBJ database under accession numbers KY884295 and KY823418 to KY823428.

Statistical analysis

Data were entered in Microsoft Excel and analysed in STATA version 12.0 (StataCorp LP). The proportion percentage was measured to describe the characteristics of the participants, including the frequency of Entamoeba complex infection according to variables including age, sex, etc. The χ ² test or Fisher's exact test was used to analyse the association between the Entamoeba complex and different subgroups. The odds ratios (OR) and 95% confidence intervals (CI) were also determined, based on the binary logistic regression analysis to identify the potential contribution of each variable to the acquisition of Entamoeba complex infection. P-value <0.05 was considered statistically significant.