A Prevalence study of Entamoeba spp. in Basrah Province using Different Detection Methods

This study aims to determine the prevalence of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii by three methods of diagnosis (microscopic examination, cultivation and PCR) that were compared to obtain an accurate diagnosis of Entamoeba spp. during amoebiasis. Total (n=150) stool samples related to patients were (n = 100) and healthy controls (n= 50). Clinically diagnosed stool samples (n=100) were collected from patients attending the consultant clinics of different hospitals in Basrah during the period from January 2018 to January 2019. The results showed that 60% of collected samples were positive in a direct microscopic examination. All samples were cultivated on different media; the Brain heart infusion agar showed high efficiency and was the most suitable in cultivating the parasite. Data and results of molecular study were indicated by DNA extraction from stool samples and used in PCR technique with specific primers. This study identifies different infection percentage for the three species. The highest infection in Basrah patients was Entamoeba moshkovskii 15% followed by Entamoeba dispar 10% and Entamoeba histolytica, which was 5%.


Introduction:
In many countries, amoebiasis has been considered one of the major public health problems. It is caused by the genus Entamoeba, which includes several species, such as E. histolytica, E. dispar and E. moshkovskii. One of the main reasons that this disease is widely spread in developing countries is the lack of hygienic measurements and the supply of clean drinking water. In addition, the disregarding of parasitic infections in such countries increases the number of these infections (1). Many studies worldwide investigate asymptomatic amoebiasis. There is an idea of some researchers that the pathogenesis of the parasite differs according to the species and strains, while others believe that the severity of the parasite pathogenicity and the condition of host may increase the clinical symptoms. The prevalence of E. histolytica is normally over estimated, this is because of the overlap of the epidemiology of E. dispar and E. moshkovskii which are similar in morphology to E. histolytica (2).
Generally, the prevalence of Entamoeba spp. is not well described in different parts of the world. The only well-known side is in human that E. histolytica can cause invasive diseases, while the other two species (E. dispar and E. moshkovskii) are considered as non-pathogenic and free-living amoeba until now. Even though, there are many studies that have suggested the pathogenicity of these two species in human. The diagnosis of amoebiasis requires more sensitive method than the ones used now as trophozoites that have low viability in feces sample and the cysts, which are irregularly excreted in the sample in asymptomatic (3) Although E.histolytica, E. dispar, and E. moshkovskii have identical appearance, the pathogenicity of the E. dispar and E. moshkovskii have not been well understood. The main laboratory diagnosis of Entamoeba spp. in human stool is microscopic examination but it is not possible to distinguish between these types depending on it (4).
The morphological similarity between Entamoeba spp. is a major concern, which may lead to mistreating the patients. Therefore, the need of accurate diagnostic method is important, this method is represented by the molecular diagnosis using PCR technique in different regions around the world (5).
Molecular diagnosis has showed that the distribution of E. dispar was 10 times higher than that of E. histolytica worldwide; many studies reported the relationship between E. dispar and the clinical symptoms (6). Most molecular studies used polymerase chain reaction (PCR) techniques in differentiating E. histolytica and E. dispar. Whereas the diagnosis of E. moshkovskii was ignored, although recent studies have shown that E. moshkovskii cause gastrointestinal disorders (GIDs) in human, which is an appropriate host for this species (7). In Basrah province many studies were conducted regarding Entamoeba spp. i.e., Al-Yaquob (8) who used the PCR technique for the detection and differentiation of Entamoeba spp. and compared it with microscopically examination. Moreover, Al-Abodi (9) studied phylogenetic sequencing for species E. histolytica, E. dispar, and E. moshkovskii in Al-Qadisiya province. Therefore, this study aims to clarify an accurate method for evaluating the prevalence of E. histolytica, E. dispar and E. moshkovskii in Basrah, to the south of Iraq, using three methods of diagnosis (microscopic examination, cultivation and PCR).

Samples collection
The study was subjected a 150 stool samples related to a 100 patients and a 50 for healthy controls. Clinically diagnosed stool samples were collected from outpatients suffering from gastrointestinal disorders, abdominal pain and diarrhea, those patients were attending the consultant clinics of different hospitals in Basrah. The collection took place during the period from January 2018 to January 2019. All patients ranged from 1 -70 years old with both genders. The samples were collected in a labeled sterile plastic container and brought directly to the laboratory of parasitology in the Department of Biology/ College of Science/ University of Basrah. Each sample was divided into two parts, the first one for direct microscopic observation and culturing, while the second part kept at -20C • for DNA extraction.

Microscopic examination of Entamoeba spp.
The microscopic examination of 150 stool samples (100 patients and a 50 for healthy controls) were performed by direct wet smear method (which included a diagnostic examination for trophozoite by normal saline solution and cyst stages by using Lugols iodine solution of Entamoeba spp.) and concentration methods using simple sedimentation and formalin-ether technique. In order to get a precise identification for Entamoeba spp., the parasites were stained by trichrome stain including Basic and Modified by using a test tube instead of slide (10). Cultivation of Entamoeba spp.
In order to obtain a clear growth of Entamoeba spp. a 150 stool samples (100 patients and a 50 for healthy controls were cultivated in different media, which were xenic and diphasic (11).
A. Basic culture media: Locke ' s egg medium, Robinson ' s medium, Trypticase yeast extract iron serum (TYI-S-33) medium (11). B. New culture media for Entamoeba spp. including 1. Brain heart infusion agar (Hi media, India) 2. Sheep testes agar Sheep test extract: five gm. of sheep testes was taken, added to 150 ml of distilled water and then put in a mixture for 15 min to be filtrated. This medium was prepared from mixing 1.5g agar with 100 ml sheep testes extract.

Buffalo milk agar
This medium was prepared from mixing 1.5g agar with 100ml buffalo milk.

Sheep liver agar
Sheep liver extract: five gm. of sheep liver was taken, added to 150 ml of distilled water and then put in a mixture for 15 minute to be filtrated. This medium was prepared from mixing 1.5g agar with 100 ml sheep liver extract.

Chicken liver agar
Chicken liver extract: five gm. of chicken liver was taken, added to 150ml of distilled water and then put in a mixture for 15 min to be filtrated it. This medium was prepared from mixing 1.5g agar, protein 1 g with 100 ml chicken liver extract.
Then 15 ml from each media was added to tubes. Two type of antibiotics were added to all the media above, Erythromycin 0.5g /100 ml and Nystatin 0.5g/100ml, both antibiotics were sterilized by Millipore filtration and stored at 4 • C.

Inoculation of Entamoeba spp. in culture media
Samples (n=150) were cultivated by using the different culture media. Each media was supplied with the inoculation of Nystatin 0.25mg/ml, Erythromycin 0.25mg/ml, phosphate buffer saline (pH 7.4) 2 ml, Human serum 7 ml and suspension of parasite 1 ml (which contains 750  (12,13).

Molecular diagnosis of Entamoeba spp.
At first, molecular methods were used to diagnose all samples (n=150, a 100 for patients and a 50 for healthy as controls). After that, the molecular analysis was repeated for samples that were succeeded by cultivated methods (n=10). DNA extraction DNA was extracted from stool samples according to the procedure of Presto TM Stool DNA Extraction Kit (Geneaid, Taiwan).

Diagnosis of Entamoeba spp. by PCR
The PCR amplification reaction was performed to the final volume of 25 µl. The reaction was optimized to combine the specific primers, for E. histolytica (SPEH): forward primer 5 ΄ -ATGCACGAGAGCGAAAGCAT-3΄ and reverse primer 5 ΄ -GATCTAGAAACAATGCTT CTCT-3΄, for E. dispar (SPED): forward primer 5 ΄ -ATGCACGAGAGCGAAAGCAT-3΄and reverse primer 5 ΄ -CACCACTTACTATCCCTACC-3΄, for E. Moshkovskii (SPEM): forward primer 5 ΄ -ATGCACGAGAGCGAAAGCAT-3΄and reverse primer 5΄-TGACCGGAGCCAGAGACAT-3΄. The volumes for a single reaction were 1.5 µl of each forward and reverse primer , Go Tag Green master mix 12 µl , Nuclease free water 7 µl and 3 µl of extracted DNA samples. The thermal cycler (Thermo cycler, Applied Bio system, USA) program used to amplify each species-specific DNA fragment was started with an initial denaturation at 95 • C for 4 min., followed by 35 cycles of 95 • C for 35 sec., 55 • C for E. histolytica , 53 • C for E.dispar and 52.7 • C for E. moshkovskii for 35 sec. and 72 • C for 35 sec. with a final extension at 72 • C for 7 min. PCR products were detected by 2 % agarose gel electrophoresis ,stained with (0.2 µl) ethidium bromide and visualized by UV transilluminator (14).

Calculate sensitivity to diagnostic methods for clinical samples (n=100)
For the three diagnosis methods (direct microscopic examination, cultivation and molecular diagnosis), the sensitivity of detecting Entamoeba spp. were calculated according to the equation below (15).

Results: Prevalence of Entamoeba spp. infection according to microscopic examination
In general, the present study showed that 60% was positive samples in direct microscopic examination, regarding sex. The infection was found at 56.6% for male patients and 43.4% for female patients.
The results showed that percent infection of Entamoeba spp. was different according to age groups. The highest percentage infection of Entamoeba spp. was in group1 (1-10 years) and group 7 (61-70 years) with 31.6% and 23.3%. While, the lowest percentage was associated with group 3 (21-30 years) and group 4 (31-40 years) with 1.6 % for each.
The results also showed a difference in the percentage of infections between months of the year, the highest infection was in July 18.3%, while the lowest infection was in December 1.6%. Moreover, there were differences in the stool appearance, which differ from bloody, mucous, fatty, and semisolid. The mucus samples were the most frequent with 31.6% followed by semi-solid samples 23.3%.
Direct microscopic examination showed positive results, the trophozoite was clearly shown as a static colorless or moving using normal saline method and the cysts were diagnosed by Lugol's Iodine stain ( Fig. 1 A and B), the diagnostic method by both basic and modified trichrome stain showed the Entamoeba spp. trophozoite as in (Fig. 2 A   Cultivation of Entamoeba spp. for clinical samples Generally, clinical samples (n=100) were cultivated, however, only ten samples (10%) were positive in culture. These 10 samples were also positive in direct microscopic examination.

Cultivation of Entamoeba spp. for control samples
Control isolates (50) were free from 3 species infection

Parasite cultivation in basic media
Three culture media were used in order to obtain the best medium for Entamoeba spp. The best growth period for each culture medium after incubation for 48h. was determined by confirming that the parasite stags alive for the longest period. Locke ' s egg medium showed that this period reaches a maximum to 4 days with parasite alive, while Robinson ' s and TYI-S-33 media recorded a maximum to 2 days that the parasite is still alive ( Table 1). From these results it is clear that Robinson ' s and TYI-S-33 media are less efficient than Locke's egg medium in maintaining the parasites growth.

Open
Chicken liver agar

Molecular diagnosis of Entamoeba spp. DNA extraction
The genomic DNA showed in Fig. 3.

Polymerase Chain Reaction of Entamoeba spp. from clinical samples by specific primers A. Diagnosis of E. histolytica
Five isolates out of 100 samples (5%) were diagnosed as E. histolytica using the specific primer. The gene band size was 166bp as shown in Fig. 4.

B. Diagnosis of E. dispar
Ten isolates out of the100 samples (10%) were diagnosed as E. dispar using the specific primer. The gene bands size was 752bp as shown in

C. Diagnosis of E. moshkovskii
Fifteen isolates out of the 100 samples (15%) were diagnosed as E. moshkovskii using the specific primer. The gene bands size was 580 bp as shown in Fig. 6.

Polymerase Chain Reaction of Entamoeba spp. from samples that were succeeded by cultivated methods
Out of 10 isolates cultivated, 3 isolates were identified as E. moshkovaskii, while 7 isolates were negative in PCR.

PCR of Entamoeba spp. from controlled samples
Control isolates (50) were free from 3 species infection

Sensitivity of diagnostic methods for clinical samples
In terms of the sensitivity, the highest sensitivity was present in the microscopic examination (60%), while the lowest sensitivity value was occurred in cultivation (10%). The molecular diagnosis showed a sensitivity of (30%).

Discussion:
Generally, the most common intestinal parasites in Iraq are Entamoeba spp. these species are similar to each other in morphology but they are genetically different (14). The present study showed prevalence infection of Entamoeba spp. in Basrah patients (60%) by testing the direct microscopic examination, similar findings were reported by other researchers in Al-Qadisiyah province as they found that the prevalence of Entamoeba spp. was 61.26% (9). A variation of amoebiasis prevalence was found among Iraqi provinces, the highest percentage (68%) was reported in a study conducted in Al-Qadisiyah (16), while Kadir et al. (17) reported that the lowest percentage in Salah Al-Din 9.3%. This variability may be due to differences in the levels of sanitation, personal hygiene, population density, lifestyle and the climatic conditions suitable for the survival of cysts for a long time, which increase the possibility of causing such infection.
Results regarding sex showed a higher percentage for males than females, this result was confirmed by Al-Kaeebi and Al-Difaie (18) who stated that the infection found in males 58.3% and 41.6% for females in Al-Qadisiyah province. The variations in infection percentages between males and females might be caused by the different social behavior between the two sexes, as males normally are the working sex in the society which made them in contact with the environment (19).
The increased percentages of infection in age group 1 (1-10) yrs. and group 7 (61-70) yrs., could be explained by the influence of different factors as for group1 the behavior of children might be involved less cautious regarding their personal hygiene. In addition, they are more exposed to food from different sources. The high infection percentage in group 7 (61-70) might be resulting from the fact that elderly people exposed to many infectious agents during their life as their immune system is weaken (20).
Our results also reported that summer season associated with increased infection. These results may be attributed to the availability of proper environmental factors for parasites growth, as intestinal parasites were dominant at tropical area. Moreover, the need of fast consumption of drinking water that contains the infective stage of these parasites (cysts) can be considered as another cause (21). Another reason is represented by our living habit in Basrah city, because of the increased percentages of salt in the tap water and the contamination of drinking water tanks with the infective stages (22). Direct examination gave a confirmation of Entamoeba spp. existence in both trophozoite and cysts stages. However, the use of modified trichrome stain in the test tube was efficient in getting good quality stained samples free of contaminants (Bacteria and debris). The repeated centrifugation in this methods helped the sedimentation of parasites to be precipitated in the bottom of the tube and led to a clear isolation with high purity.
Regarding the parasite cultivation method, in our study stool samples were used, the results showed that it was less sensitivie than the microscopic examination. In addition, there is a high risk of contamination with fungi, bacteria and other protozoa, therefore it was not recommended as a routine examination (9). The Locke's egg medium (basic media) was the best among the other media. This is because it contained nutrients that helped parasites to secrete proteases in order to digest proteins, which was essential for rapid growth (23). These nutrients include the eggs that contained the high protein contents necessary for parasite growth and reproduction compared to the presence of peptone found in Robinson medium, which is a simple structured protein. The high protein content allows Entamoeba spp. to get nutrients before their consumption by bacteria. In addition to this, it also contained vitamins that stimulated Entamoeba spp. for metabolic reactions (24). Regarding Robinson medium which has benzoic acid as one of its components, this compound acts as antibacterial and used to preserve food (25). It has an inhibitory effect on the growth for some microorganisms. For the first time we used 5 new culture media, which were highly efficient in cultivating Entamoeba spp. and easy to prepare from different animal sources. The results showed that Brain heart infusion agar was the most efficient medium to give dense growth and increase the number of parasites with a high purity for ten to eleven days period. This efficiency may be due to the high chemical contents of the nutrients necessary for parasite's growth and reproduction because Brain heart infusion agar contains amino acids, glucose, mineral salts, highly lipid complex and cholesterol (23). The Sheep testes agar came in the second place for the efficient medium in parasite isolation and growth, this might be due to its contents of proteins and carbonate, in addition to the presence of high lipid (26). Moreover, the Buffalo milk agar, which showed a lower growth rate compared with Brain heart infusion agar and Sheep testes agar media may be related to their components which were less suitable for parasite growth (27). The same reason applied for Sheep liver agar and Chicken liver agar media, which have less percentages of fat and proteins, respectively (28,29). The human serum was added to all media during the culture process, because this serum is normally obtained from media rich with lipoproteins, which had an important role to grow the parasite in pure culture (30).
The DNA was extracted in order to diagnose the parasites; the technique of PCR was used as a highly sensitive way to differentiate between Entamoeba spp. The results showed that the high efficiency of differentiation among the three species was based on the specific primer of each parasite .The highest infection was E. moshkovskii 15% followed by E. dispar 10% and E. histolytica, which was 5 %. Al-Abodi (9) also recorded different infection percentages for the Entamoeba spp. in the province of Al-Qadisiya, where E. histolytica had the highest prevalence (74%) followed by E. dispar with (26%), while E. moshkovskii was with only 7%. On other part of Asia i.e., in Iran 0.58% was the highest prevalence of E. dispar, while the least prevalence was for E. moshkovskii 0.07% (5). The specific primers used in this study were derived from the middle of the small-subunit rRNA gene since it was conserved, found in all organism, used to identify and classify organism due to unique characters for each species (15). The differences found in PCR results may be attributed to different reasons, firstly due to the variations in DNA extraction methods from stool samples. Secondly differences in the number of parasites in stool samples. Finally, the fixative and preservative agents used in the study may interfere with cell components resulting in effecting DNA (4).
Regarding the sensitivity of the diagnosis methods, the microscopic examination had the highest sensitivity for detection of parasites trophozoite and cysts in stool samples followed by the molecular diagnosis then cultivation method. The negative results by using PCR or cultivation methods of some positive samples in microscopic examination may be explained by several reasons. Firstly, in microscopic examination there was a chance in misdiagnosing the parasite by way of some times the parasite is misidentified with WBCs as well as with other stages of Entamoeba spp. such as E.moshkovskii, E. histolytica and E. dispar (31). Secondly, the presence of some inhibitory substances in the stool such as urea, bile salt and polysaccharides, which may be bond to DNA polymerase and inhibited its work in replicating the DNA (32).

Conclusion:
In conclusion although microscopic examination is a traditional method for the diagnosis of Entamoeba spp. the probability of misdiagnosis of these species is high, the molecular diagnosis method is considered a precise diagnostic method despite the low sensitivity which is due to the presence of both alike forms in the sample