Frequency of Intestinal Parasites in Patients With Gastrointestinal Disorders , in Different Parts of Iran During 2012-2013

1Department of Medical Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran 2Department of Parasitology, Iran University of Medical Sciences, Tehran, IR Iran 3Department of Community and Health, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran 4Department of Immunology, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran 5Department of Medical Parasitology, School of Medicine, Jundishapur University of Medical Sciences, Ahvaz, IR Iran


Background
Intestinal parasitic infections are being considered among the most common infections in the world.It was estimated that 60% of people worldwide were infected with intestinal parasites (1,2).Recent estimates indicate that at least more than one-quarter of the world's population are infected with intestinal parasites and most of them are people who live in developing countries (2,3).These parasites can cause severe illness and the mortality in endemic areas (4).Parasitic diseases are important hygiene problems and the impediment to economic and social development in many countries, particularly in developing world.Poverty, illiteracy, low levels of hygiene, lack of approach to safe drinking water and humid tropical climate are the most common factors of intestinal infec-tions.In Turkey, people who had unsanitary disposal of feces the prevalence of pathogenic parasites were high (5).
Parasitic infections of the gastrointestinal tract are a major cause of morbidity in developing countries and are increasingly important in certain populations from the developed countries (6).Approximately 39 disability adjusted life years (DALY) has been ascribed to intestinal parasitic infections (7,8).Therefore represents a considerable economic burden and death particularly among invasive amoebiasis patients (8).Epidemiology of intestinal parasitic infections suggests that these parasites are found in every age group and both sexes.But in some areas, the incidence is higher in children (9).Intestinal parasitic infections caused by worms and protozoan par-asites are the most common human infections in developing countries (10).
The pathogenic protozoa Entamoeba histolytica, Giardia intestinalis, and ryptosporidium spp.have a worldwide distribution and are a common cause of diarrhea in human.In Pakdasht, Tehran Province, protozoan parasites infections were reported the highest rate (41.5%) (11).In Ardabil, northwestern Iran, a total of 10 (14%) species were identified with Giardia intestnalis, Blastocystis hominis (10%) and Entamoeba coli (4.1%) being the most common parasites (12).According to the World Health Organization estimates, 50 million of people worldwide suffer from invasive amoebiasis per annum (13).It is prevalent in West and South-east Africa, China, and Mexico (14).E. histolytica is more abundant in the tropical and subtropical, however, the range of infection in temperate climates and cooler, with poverty of sanitary is the same as what is found in tropical climate.As in the United States of America, G. intestinalis is the most common intestinal parasite of humans; also it is a very common infection in developing countries (10).
In recent years, the isolation of parasitic DNA from fecal samples and PCR techniques, have been improved and simplified.Moreover, the introduction of real-time PCR has made it possible to multiplex different targets into one reaction (14).Recently, in different parts of Iran, several studies have been conducted to reveal the intestinal parasites prevalence.According to literature review, there is a sharp decline in the prevalence of human helminthes infections (15).

Objectives
The aim of the study was to determine the frequency of intestinal parasites in patients with gastrointestinal disorders, in different parts of Iran.

Population Study
During April 2012 to May 2013, by non-randomize simple sampling selection, 1520 stool samples were collected from patients with gastrointestinal disorders who had been referred to clinical laboratories of: Fardis central laboratory, Fardis, Shahid Taleghani hospital, Tehran, Health care centers of Amol and Varamin and Tamin Ejtemaee hospital, Ahvaz, Iran (Table 1).

Stool Examination 3.2.1. Direct Microscopy (Wet Mount)
The stool samples were macroscopically examined for consistency, color, the presence of blood and mucus, adult intestinal helminthes.All specimens examined by using normal saline (0.85% NaCl) for the presence of trophozoites and lugol's iodine staining was used for detection of cysts of intestinal protozoa under light microscope (× 400).

Formalin-Ether Concentration
About, 5 g of preserved stool sample by using an applicator was put in a clean beaker containing 7 mL formalin.The sample was dissolved and blended thoroughly with applicator stick.The resulting suspension was filtered through cotton gauze into a 15 mL conical centrifuge tube.The debris captured on the sieve was discarded.After adding 3 mL of ether to the mixture and hand shaken, the content was centrifuged at 2000 rpm for 3 minutes.The supernatant was discharged.The pellets put on slide and covered with cover slip.The complete area under the cover slip was examined using 100 objective magnifications.Also the sediments were examined for the ova of helminthes, as well as for the cysts and the trophozoites of protozoa under light microscope (× 400).

Ziehl-Neelsen Staining
Second smear from sedimentation was made and stained using the modified Ziehl-Neelsen staining method for the detection of Cryptosporidium.Finally, the stained smears were microscopically examined using 1000x magnification (16).

Culture Method
Positive stools specimens of amoeba were cultured in Horse Serum ringer-starch medium (HSr-s) (17) to detect the presence of Entamoeba histolytica/dispar/moshkovskii.Cultures were incubated at 35-37°C for four days, and a drop of culture medium was examined under a 400 × objective magnification.

Polymerase Chain Reaction (PCR)
DNA-based method used to detect the three species of amoeba.Genomic DNA was extracted from samples using the QIAamp DNA stool mini kit (QIAgen, Germany).100 mg of the positive stool samples of infected patient mixed with 1 mL distilled water (DW) in a 1.5 mL micro tube, washed with DW twice by centrifugation at 4000 rpm and 14000 rpm for Entamoeba and Cryptosporidium, respectively.Then extraction of DNA from pellets was done according to the manufacture's recommendation.The multiplex PCR assay was used to detect and differentiate the Entamoeba spp.A conserved forward primer Int J Enteric Pathog.2015;3(1):e22682 was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species.The sequence of a forward primer used was conserved in all three Entamoeba spp., whereas the specific reverse primers were specific for E. histolytica, E. dispar, E. moshkovskii, respectively (Table 2) (18).Also we used the single-plex PCR for detection of Cryptosporidium.Forward and reverse primers were derived from the small-subunit rRNA gene (Table 2) (19).PCR was performed using amplicon (Bie and Berntsen A/s, Denmark) as a ready-made solution.The reaction mixture contained 5 μL distilled water, 7.5 μL amplicon, 20 pmol forward and reverse primers, and about 5-10 ng of extracted DNA template to achieve a final volume of 15 µL.Amplification of each species-specific DNA fragment started with an initial denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, with a final extension at 72°C for 7 minutes.Amplified products were visualized after electrophoresis on 1.5% agarose gels.

Statistical Analysis
All data from stool study was collected and analyzed with Statistical Package for the Social Sciences (SPSS) version 16.0.Statistical significance was determined with the chi-square (x 2 ) test.Data analyzed allowed us to compare prevalence of intestinal parasites compared according to age and sex.

Discussion
Intestinal parasites comprise some of the most common and important parasites of human and they are regarded as an important public health problem of most communities, especially those situated in tropical and subtropical areas.However, patterns of intestinal parasites infections in the population may be altered on account of changes in man behavior and life styles during a time and prevalence of these varies with different geographical regions (7,20,21).
Several studies have carried out on prevalence of intestinal parasites in Iran.The current study was performed to determine the frequency of the parasitic infections during 2012-2013 in patients with gastrointestinal disorders.The prevalence of intestinal parasites was 10.06%.Our results showed that the prevalence of protozoan infection (9.7%) was higher than the (0.3%) of helminthes infection.The higher incidence rate of than helminthes may be attributed to the fact that being infected with the protozoan cyst through contaminated water and food will be readily, thus make easy their acquisition of the infections.Some studies from Iran and some parts of the world found that intestinal protozoan infection had higher prevalence than intestinal helminthes infection (12,(22)(23)(24)(25).
The relatively high incidence of intestinal protozoan infections in the study highlights the methods of preventing the transmission and their expansion must be considered.Results of this survey in the population in order of protozoa frequencies indicated that Blastocystis spp.(4.73%) and G. intestinalis (2.3%) were the most commonly detected organisms.Several studies in other areas of Iran have previously revealed rates of intestinal protozoan parasitic infection of 0.5 to 30% (21,(26)(27)(28)(29)(30)(31).In period of 2004-2005, the distributions of intestinal protozoan parasites were characterized, in rural population of Mazandaran, northern Iran and results proved that the G. intestinalis (10.2%) and Blastocystis spp.(9.8%) were the most detected protozoan (22).
Based on our findings the frequency of G. intestinalis was highest in children with peak values among the ≤ 1-10 years age group (42.86%).Also in the sex-related incidence and intensity of the infection, male gender 22/35 (62.86%) recorded a higher incidence and intensity of the infection than female gender 13/35 (37.14%).However age and sex did not show any significant correlation with the prevalence of G. intestinalis (P > 0.05).
In this study for one microscopy-positive E. histolytica/E.dispar/moshkovskii sample, multiplex PCR was performed for the accurate differential diagnosis of Entamoeba spp.It was identified as E. dispar.Studies show that E. dispar is maybe 10 times more prevalent than E. histolytica worldwide (32).It seems that E. dispar is main species particularly in the central and northern areas of Iran and amoebiasis as a result of E. histolytica is a rare infection in the country.Haghighi et al. (27) showed that of the eight microscopy-positive E. histolytica/E.dispar samples, six were identified as E. dispar by PCR/gel electrophoresis, whereas E. histolytica was not detected at all.Nazemalhosseini Mojarad et al. in a study among patient with gastrointestinal complaint indicated that of 22 microscopy-positive samples for histolytica/E.dispar complex, 21 (95.45%)samples were diagnosed as E. dispar by PCR (33).
In our study, Cryptosporidium as a coccidian parasite was detected by microscopy (modified Ziehl Neelsen staining method) in one patient (0.06%).We also used DNAbased methods to detect Cryptosporidium spp., when this case was analyzed with single round PCR; the presence of Cryptosporidium was confirmed.Ghobadi and et al. from Sanandaj, Iran reported 8% frequency for Cryptosporidium in their study (34).Kia et al. and Berenji et al. in their studies did not find any Cryptosporidium in patients (21,24).Currently, there are various methods for the detection of Cryptosporidium, including special staining, IFAT, and molecular techniques.In the present study, among helminthic intestinal infection we found Hymenolepis nana 3 (0.19%), Dicrocoelium dendriticum 2 (0.13%).Heterodera ova along with its larva were also seen which is consistent with Ashrafi study (35).
With regard to the egg of these parasites in fecal samples can not necessarily confirm the real infection, therefore, the foregoing report is provided only for laboratory observations.Meanwhile, due to the lack of adequate access to information about their supply of Dicrocoelium eggs found in the feces of patients cannot be accurately commented on the true occurrence of the infection caused by this parasite.
Blastocystis and G. intestinalis were the predominant intestinal parasites detected in patient with gastrointestinal disorders.The results indicated that the intestinal parasites, particularly helminth infections have been significantly declined in recent years.

Figure 1 .
Figure 1.Single-Round PCR Amplification Products With the (A) 752 bp and (B) 749 bp Sizes for E. dispar and Cryptosporidium spp.Respectively

Table 1 .
Clinical Laboratories Which Stool Samples Were Collected From Patients With Gastrointestinal Disorders

Table 2 .
Details of Oligonucleotide Primers Used in This Study

Table 3 .
Prevalence of Intestinal Parasites in Patients With Gastrointestinal Disorders, in Different Parts of Iran