Iran J Parasitol: Vol. 12, No. 3, Jul-Sep 2017, pp. 364-373 Iran J Parasitol Tehran University of Medical Open access Journal at Iranian Society of Parasitology Sciences Public a tion http:// ijpa.tums.ac.ir http:// isp.tums.ac.ir http:// tums.ac.ir Original Article Prevalence and Risk Factors of Human Intestinal Parasites in Roudehen, Tehran Province, Iran Nasrin HEMMATI 1, *Elham RAZMJOU 1, Saeideh HASHEMI-HAFSHEJANI 1, Abbas MOTEVALIAN 2, Lameh AKHLAGHI 1, *Ahmad Reza MEAMAR 1 1. Dept. of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 2. Dept. of Epidemiology, School of Public Health, Iran University of Medical Sciences, Tehran, Iran Received 10 Nov 2016 Abstract Accepted 15 Jun 2017 Background: Intestinal parasitic infections are among the most common infec- tions and health problems worldwide. Due to the lack of epidemiologic informa- tion of such infections, the prevalence of, and the risk factors for, enteric parasites Keywords: were investigated in residents of Roudehen, Tehran Province, Iran. Blastocystis, Methods: In this cross-sectional study, 561 triple fecal samples were collected Entamoeba, through a two-stage cluster-sampling protocol from Jun to Dec 2014. The samples Intestinal parasite, Pre- were examined by formalin-ether concentration, culture, and with molecular me- valence, thods. Protozoa Results: The prevalence of enteric parasites was 32.7% (95% CI 27.3–38). Blasto- cystis sp. was the most common intestinal protozoan (28.4%; 95% CI 23.7–33.0). The formalin-ether concentration and culture methods detected Blastocystis sp., En- *Correspondence tamoeba coli, Giardia intestinalis, Dientamoeba fragilis, Iodamoeba butschlii, Entamoeba com- Emails: plex cysts or trophozoite, Chilomastix mesnilii, and Enterobius vermicularis. Single-round [email protected] PCR assay for Entamoeba complex were identified Entamoeba dispar and E. [email protected] moshkovskii. E. histolytica was not observed in any specimen. Multivariate analysis showed a significant association of parasites with water source and close animal contact. There was no correlation between infections and gender, age, occupation, education, or travel history. Protozoan infections were more common than hel- minth infections. Conclusion: This study revealed a high prevalence of enteric protozoan parasite infection among citizens of Rodehen. As most of the species detected are transmit- ted through a water-resistant cyst, public and individual education on personal hy- giene should be considered to reduce transmission of intestinal parasites in the population. 364 Available at: http://ijpa.tums.ac.ir Hemmati et al.: Prevalence and Risk Factors of Human Intestinal Parasites … Introduction ntestinal parasite infection remains a It is 1850 m above sea level and has an area of health problem, especially in developing 50 km2 with a population of 21477 in 7393 I countries, in spite of efforts by the households (12). WHO and governments to eliminate parasites and prevent and treat parasitic disease. Study design and sampling Worldwide, over 3.5 billion people are in- The cross-sectional study was conducted fected with an intestinal parasite annually, and from Jun to Dec 2014. The triple faecal sam- 4.5 million exhibit clinical symptoms (1). Para- ples from 561 residents were collected in a sitic infections may be asymptomatic or ex- two-stage cluster-sampling scheme. In the first tend to morbidity and mortality, depending on stage, the city was divided into 82 blocks, and the nutrition and health status of affected in- 23 blocks were randomly selected. In the dividuals (2, 3). second stage, 10 households within each block The prevalence of intestinal parasitic infec- were randomly selected. All members of se- tions has been related to low levels of educa- lected households were invited to participate tion, health practices, malnourishment, con- in the study. If in the selected household, no taminated food and/or water, climate, popula- one answered to investigators on two separate tion growth, and socioeconomic and health dates, or if all individuals in the household conditions as well as close contact with ani- refused to participate in the study, the next mals (2-5). Intestinal parasitic diseases are household was invited. among the most important infectious diseases The aim and design of the study were that have a direct relationship to personal and explained to household members. Agreeable public hygiene (4, 5). individuals and the parents of children under A dramatic decrease in the prevalence of pa- age 15 gave written consent and filled a rasitic infections has been reported in Iran (6), questionnaire form including sex, age, although the occurrence of enteric protozoans occupation, education, drinking water source, is still common and remain a health problem and history of travel and animal contact. in some parts of the country (4, 7-9). Prior Three samples were collected from each studies reported Giardia intestinalis and Enta- participant with one-day interval. moeba coli to be the most common intestinal parasites in Iran (8, 9), but recently Blastocystis Laboratory procedures sp. has been suggested to be the most preva- Stool samples were transferred immediately lent (10, 11). after collection to the Department of Parasi- The goal of this study was to determine the tology and Mycology, Iran University of Med- prevalence of, and the risk factors for, intes- ical Sciences, for examination and identifica- tinal parasites in Roudehen, Tehran Province, tion of parasites. Iran. Parasite examination Materials and Methods Pea-sized pieces of fecal samples were sub- mitted to formalin-ether concentration tech- Study Area nique to identify ova, cysts or oocysts of para- Roudehen is a city in Damavand County, sites. The remaining sample was emulsified in Tehran Province; northern Iran located 35 km PBS and passed through a two-layer gauze east of Tehran, in the Alborz Mountains. filter to remove larger particulates, and the Roudehan has a mountain temperate climate. suspension was centrifuged at 1000 × g for 5 Available at: http://ijpa.tums.ac.ir 365 Iran J Parasitol: Vol. 12, No. 3, Jul-Sep 2017, pp. 364-373 min. The sediments were separated into sepa- distilled water was included as a negative rate portions for culture and molecular study. control and the mix of genomic DNA of E. One hundred mg of the sediment was histolytica reference strain HM-1, IMSS Clone 6 resuspended in 200 µl 2% polyvinylpolypyrro- (ATCC® Number, 50527 ™), E. dispar refer- lidone in PBS and stored at -80 °C until sub- ence strain SAW 760 (ATCC® Number, mitted to DNA extraction and 500 µl of PRA260™), and E. moshkovskii reference which was cultured in a biphasic medium, strain Laredo (ATCC® Number, 30042 ™) horse serum slant overlaid with 5 ml of Rin- were used, as positive control, to validate the ger’s solution, 200 µl of rice starch (5 mg/ml) results of multiplex PCR. The PCR products and penicillin-streptomycin incubated at were detected on ethidium bromide stained 35.5 °C and examined three times at 48 h in- 1.5% agarose gels. tervals (13). Samples were considered positive if helminth eggs, larvae, or cysts, and/or tro- Data management and analysis phozoites of protozoans were detected by at The sampling weights were calculated by least one of the two techniques. multiplication of three weighting components: 1) inverse sampling weight, 2) inverse of Molecular examination nonresponse for each block, and 3) post- All samples containing Entamoeba tropho- stratification weights calculated by dividing zoites or cysts were submitted to molecular census-derived population weights by sample study. A single-round PCR assay (14) was per- weights for each of 18 age/sex groups (Table formed for discrimination of Entamoeba spe- 1). cies (Entamoeba dispar, Entamoeba moshkovskii, For descriptive analysis, frequency and per- Entamoeba histolytica). DNA was extracted from centage rates were used to describe characte- 200 µl of frozen sediment of stool samples ristics of the studied population, including the using QIAamp DNA minikit (Qiagen, Hilden, prevalence of intestinal parasites. All variables Germany) according to manufacturer’s in- were included in multiple logistic regression struction. PCR of Entamoeba complex was per- analyses to identify the adjusted odds ratios formed using the forward primer EntaF and (aOR) of risk factors for intestinal parasite reverse primers EhR, EdR, and EmR, specific infection. Stata v.10.0 (StataCorp LP, Texas, for E. histolytica, E. dispar, and E. moshkovskii, USA) was used to conduct statistical proce- respectively (14). The PCR reaction was de- dures. signed to amplify a 166 bp PCR product with E. histolytica DNA, a 752-bp PCR product with Ethical considerations and treatment E. dispar DNA, and a 580 bp product with E. The study protocol was reviewed and ap- moshkovskii DNA of a small-subunit rRNA proved by the Ethics Committee of Iran Uni- gene (14). In the PCR reaction, a 25 µl PCR versity of Medical Sciences (IUMS) with the reaction mixture consisted of 2 µl of template code number: IR.IUMS.REC 93-04-30-25381, DNA, 0.25 µM of each the forward primer following the revised Helsinki Declaration of EntaF and reverse primers EhR, EdR, and 2008. Each participant was asked to sign an EmR primers, 12.5 µl Taq DNA polymerase informed consent. For children under 15 yr, mastermix (Ampliqon, Denmark), and 6.5 µl one parent was required to agree and sign the H2O. PCR was carried out with the following consent form. At the end of the study, a la- amplification conditions: 1 cycle at 94 °C for 3 boratory report was provided to each partici- min followed by 30 cycles at 94 °C for 60 sec, pant, and, if indicated, they were referred for 58 °C for 60 sec, and 72 °C for 60 sec, and a medical care. final extension of 72 °C for 7 minutes.