INTRODUCTION:

Amebic dysentery is an human infection caused by parasitic protozoal as Entamoeba histolytica, or other Entamoeba group as Entamoeba dispar; Entamoeba coli; Entamoeba bangladeshi; Entamoeba moshkovskii; Entamoeba polecki; Entamoeba hartmanni; Iodamoeba butschlii; Endolimax nana and Dientamoeba fragilis that causes Dientamoebias is (Visvesvara et al., 2007, Al-abodi et al., 2018), which causing amebic colitis, brain abscess; liver abscess (Mandell, and Bennett's, 2015,

Shaker et al., 2018), also it can be causing appendicitis (inflammation of the appendix) and granulomatous inflammation, that cause pseudo tumour which lead to bowel obstruction (Choudhuri and Rangan 2012), E. histolytica is a single-celled protozoan that can enters the patient's body when ingesting the cysts through drink contaminated water or eating food or due directly contact with the fecal matter. The symptoms of amebiasis including stomach pain, loose stool and abdominal cramping. moreover, most patients with amebiasis won’t Suffers significant symptoms (Al-Mayaliand Al-abodi, 2017).

Entamoeba histolytica remains endemic, therefore in developed countries the diagnosis of amebiasis was uncommon (Ohnishi et al., 2004, Al-abodi, 2015), also makes diagnosis of it by antibody detection was difficult, because of difficulty to corroborate the present from the past infection (Parija et al., 2014), therefore can be used many methods in diagnosis E histolytica as based on the patient’s history, stool examination; serological test; real-time PCR and fecal antigen testing (Marn et al., 2012 and Wuerz et al., 2012), but stool microscopic assessment was low sensitivity (10-50)% also cannot differentiate the E histolytica from noninvasive; nonpathogenic E. moshkovskii and E. dispar (Hamzah and Jameel, 2017).

MATERIAL AND METHODS:

During (February–May) 2018, Collected the sample from teaching Hospital, Baghdad city.

1. Questioner :

Collected the patient information as age, sex, educational level, Residency and Income level.

2. Stool specimens:

Collected two hundred thirty-three (233) sample from patients suffering from diarrhea, then examined all specimens within thirty min. for investigating presence trophozoite of E. histolytica.so used two type of direct wet film as Normal saline 0.85% for detecting motility of trophozoite as well as Lugol's iodine 5% for structures of E. histolytica.

General stool examination:

Macroscopic examination:

The stool sample were examined by the eye to investigate consistency, mucus, blood, smell and colour (David & William, 2006).

Microscopic examination:

A-Direct physiological normal saline smear:

The stool sample were examined by mixed small amount of stool (0.5gm) different area of stool (especially mucus and blood) with one drop of normal saline by wooden stick, then put cover slide and examined under microscopic in 20x,40x (Plorde,1983).

B-Direct iodine-lugol solution smear:

The stool sample were examined by mixed small amount of stool (0.5 gm) different area of stool (especially mucus and blood) by wooden stick with one drop of iodine lugol solution, then put cover slide and examined under microscopic (David& William, 2006).

3. Blood specimens:

In this study collected ( 5 ml) from patients infected by Entamoeba histolytica and centrifuged for 5 minute at 3000 rpm for separate the serum and kept at -20 C° (deep freeze ) until used.

1) Immunological tests:

Determined the concentration of immunoglobulin and complement (C3 & C4 ) by using Single Radial Immuno-diffusion (SRID) (Tektook et al., 2015 ).

RESULTS:

Table (1): Distribution of study groups according to age and gender.

Study groups	Age Group (years) (No., %)			Gender (No., %)		Total
Study groups	Group1(1-15)	Group2(16-20)	Group3(21-25)	Male	Female	Total
Patients No. (%)	59(45)	30 (23)	42 (32)	70(53.4)	61(46.6)	131(100)
Healthy Control group No. (%)	15 (30)	15(30)	20(40)	25 (50)	25(50)	50(100)

Results in table (1) were found higher percentage of patients in the age groups1(No=59 as 45%), whilst decreased in age groups 3 (No.= 42 as 32%, also results showed highly percentage in male (53.4) % than female (46.6) %.

Table (2): Demographic characteristic of Amoebiasis in study groups

Demographic characteristic	*Study group (Amoebiasis) No. (%)*	Control group (Healthy) No. (%)
Education level
Non – study (illiterate people)	45 (34.4)	7 (14)
Primary	34(26)	13 (26)
Secondary	48(36.6)	16 (32)
University	4 (3)	14 (28)
P- value	0.05
Residency
Urban	98 (74.8)	30(60)
Rural	33 (25.2)	20(40)
P- value	0.05
Income level
Low	70 (53.5)	18 (36)
Good	35 (24)	22 (44)
High	26 (19.5)	10 (20)
P- value	0.05

Table-2 showed the percentage of infection with Entamoeba histolytica was highest in Secondary as 48 (36.6)% followed by Non–study (illiterate people) as 45 (34.4)% and primary as 34(26)%, while the lowest percentage of infection was in patients whose had university education as 4(3), as well as results according to residential are as showed urban 98(74.8) % more than rural 33(25.2) and low Income level 70(53.5)% more than both good and high income level as 35, (24%) and 26 (19.5%) respectively, when compared between patients groups and control group there was significant differences (p≤0.05).

Our results are agreed with (Al-khafaji, 2011), who found a high amoebiasis infection in rural area 77% than urban area 23%, also this study agreed with results of (Al-abodi, 2015and Al-Nassari et al., 2007) who explained many important factors as contamination drink, lower both educational and health levels in rural population may be don’t diagnosis the causes of diarrhea in medical laboratories (Al-Nassari, and Awad,. 2007; Al-abodi et al.,2018), so high prevalence of amoebiasis patients with a lower level of education may be lack in health care and in housing conditions (Tomas et al,.2011).

Table (3): Concentration (Mean± S.D) of Immunoglobulin, Complement in sera Amoebas patient's and healthy (control group).

Parameters	Study group *(Amoebiasis)* (mean ± S.D)	Control group (Healthy) (mean ± S.D)	P. Value
IgG	1010.21±.460.61	775.48±310.47	0.001
IgM	601.01±6.10	105.21±1.41	0.001
IgA	184.55±1.24	12.28±0.01	0.001
Complement			P. Value
C3	140.21±40.91	85.71±34.01	0.001
C4	32.91±11.10	25.10±9.21	0.001

The mean± S.D concentrations of immunoglobulin and Complement were found high significant (p<0.001) in patients group (Amoebiasis) than control group (healthy person), IgG for study group (1010.21±.460.61mg/dl) compare with control group (775.48±310.47mg/dl), so IgM concentration in study group was (601.01±6.10 mg/dl) compare to control group (105.21±1.41mg/dl), also IgA was (184.55±1.24 mg/dl) in patients compare to control group (12.28±0.01 mg/dl), so both complement C 3&C4 were (140.21±40.91and 32.91±11.10) respectively in patients compare to control group as shown in table (3).

Al Quraishi et al., (2017) showed in his study highly concentration of immunoglobulin IgM, IgG and IgA as (294.9, 707.4 and 387.9) mg/dl respectively, also he explained high specificity and sensitivity in relationship with E. histolytica infection via high concentration of IgM, therefore it can be used in early diagnosis of infections as amoebiasis (Al Quraishi et al., 2017 and Tektook, N.K., 2017). Quantitative determination of serum immunoglobulins; complement and C –reactive protein in Diabetic Retinopathy patients with and without UTIs. first international scientific conference of the college of biotechnology al-qasim green university). So the highly concentration of both complement (C3 and C4) may be because the activate of complement system by E. histolytica (Reed et al., 1995), as well as Al–Kubassi, 2002 results showed high concentration of IgA in group patients than control group, because the immunoglobulin A (IgA) has important activity in humeral immunity against the parasite, that participated in reduced infection and preventing the repeated it (Haque et al., 2003).

DISCUSSION:

Results of current were found higher percentage of patients in the age groups (1-15), also results showed highly percentage in male than female (46.6) %. and infected with Entamoeba histolytica was highest in Secondary study, followed by Non–study (illiterate people), also in urban more than rural and low Income level more than both good and high income level. as well as when compared between patients groups and control group there was significant differences (p≤0.05). Our results are agreed with (Al-khafaji, 2011), who found a high amoebiasis infection in rural area 77% than urban area 23%, also this study agreed with results of (Al-abodi, 2015and Al-Nassari et al., 2007) who explained many important factors as contamination drink, lower both educational and health levels in rural population may be don’t diagnosis the causes of diarrhea in medical laboratories (Al-Nassari, and Awad,. 2007, Al-abodi et al., 2018), so high prevalence of amoebiasis patients with a lower level of education may be lack in health care and in housing conditions (Tomas et al,.2011).

The mean±S.D concentrations of immunoglobulin and Complement were found high significant (p<0.001) in patients group (Amoebiasis) than control group (healthy person), Al Quraishi et al., (2017) showed in his study highly concentration of immunoglobulin IgM, IgG and IgA as (294.9, 707.4 and 387.9) mg/dl respectively, also he explained high specificity and sensitivity in relationship with E. histolytica infection via high concentration of IgM, therefore it can be used in early diagnosis of infections as amoebiasis (Al Quraishi et al., 2017 and Tektook, N.K., 2017). So the highly concentration of both complement (C3 and C4) may be because the activate of complement system by E. histolytica (Reed et al., 1995), as well as Al–Kubassi, 2002 results showed high concentration of IgA in group patients than control group, because the immunoglobulin A (IgA ) has important activity in humeral immunity against the parasite, that participated in reduced infection and preventing the repeated it (Haque et al., 2003.)

CONCLUSIONS:

Present results study, conclusions the following:

1. The infection with E. histolytica were highest in young old with age range from (1-15) years who live in rural areas.

2. The high rate concentration of IgG; IgM and IgE in sera of amoebiasis patients than control group as well as the complement levels (C3&C4).

RECOMMENDATIONS:

Based on the findings of Current study, can be recommended as the study the level of cytokines in amoeboasis patients.

DECLARATIONS SECTION :

Ethical Approval and Consent to participate:

Ethical clearance was taken from patients admitted to the Hospital as well as all patients were informed about the study and provided consent to participation. Ethical clearance also was taken from College of Medical and Health Technology committee, the informed consent was obtained from the authors’ institution; the patient/ participant, and all the authors in this search.

SOURCE OF FUNDING:

Self.

CONFLICT OF INTEREST:

No Conflict of interest.

AVAILABILITY OF SUPPORTING DATA:

From Research and Manufacturing of Alternatives Unit, College of Medical & Health Technology.

COMPETING INTERESTS:

No Competing interests.

FUNDING /SUPPORT:

This work was supported by our self only.

AUTHORS' CONTRIBUTIONS:

First author (Lecturer Eptissam Younan Pirko) had full access to all data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis.

ACKNOWLEDGMENTS:

We would like to thank Research and Manufacturing of Alternatives Unit, College of Medical and Health Technology, for assistance in our study.

REFERENCES:

1. Al - Kubassi , A.B.H. ( 2002 ) . Immunological , epidemiological study of patients infected with Entamoeba histolytica Ph.D . thesis. Coll. Sci. Al- Mustansiriya Univ.

2. Al-Quraishi, M. A. and Al- Sultany, S. H.(2017). Immunoglobulin profile of E. histolytica and E. dispar in human sera. J. Bio. Innov 6(2):203-213.

3. Al-abodi, H. R., Tharef, M.F. and Tektook, N.K. and Jaffar, Z. A.(2018). Molecular Investigation of Entamoeba dispar in Patients with diarrhea refers to Al-Kadhimiya hospital in Baghdad. Conference College of Medicine.

4. Al-abodi, H. R. J. (2015). Phylogenetic sequeincing for some species of Entamoeba sp. in Al-Qadisiya Province. Ph.D. thesis, College of Sciences, University of Al-qadisiyah.

5. AL-khafaji, M. (2011).Thesis of parasite immunology: Pp52-58.

6. Al-Mayali, H. M. H. and Al-abodi, H. R. J. (2017). Molecular characterization of Entamoeba spp. In Al-Qadisiya province, Iraq. Al-Kufa University Journal for Biology, VOL.9 / NO.1 : 374-386

7. Al-Nassari, Mubderabd Awad (2007). Epidemilogical study on pathogensis of Entamoeba histolytica for children reviews and fallen sleep from the post age of birth until 10 years in general BEIJI Hospital and the effect of extracts alcoholic and water to some plants on them. Masterthesis, Faculty of Education university of Tikrit pp:4773.

8. Choudhuri G. and Rangan, M. (2012). Amebic infection in humans. Indian J Gastroenterol. ;31:153–62.

9. David,T.J. and William, A.P. (2006). Markell and Voges Medical parasitology text book. (lumened welling protozoa).9th ed. Chap3.:27-37.

10. Hamzah, H. M. and Jameel, H. R. (2017). Epidemiology study of Entamoebaspesies in AL-Qadisiya governorate. Al-qadisiyah Journal of Pure Science, Vol.22, No. 3.

11. Hussien , A. A.and Shani ,W.S. Shani.2006.Serum levels of IL – 4 Immunoglobulines and complements during human amoebiasis, Basrah Journal of Science (B) Vol.24(1),112-119.

12. Löe H. The Gingival Index, the Plaque Index and the Retention Index Systems. J Periodontol. 1967 Nov-Dec, 38(6): Suppl:610-6.

13. Mandell, D. and Bennett's .(2015).Principles and Practice of Infectious Diseases (Eighth Edition).

14. Marn H, Ignatius R, Tannich E, Harms G, Schurmann M, Dieckmann S. Amoebic liver abscess with negative serologic markers for Entamoeba histolytica: Mind the gap! Infection. 2012;40:87–91.

15. Ohnishi, K.; Kato, Y.; Imamura, A.; Fukayama, M., Tsunoda, T., Sakaue,Y., et al. (2004) Present characteristics of symptomatic Entamoeba histolytica infection in the big cities of Japan. Epidemiol Infect. ;132:57–60.

16. Parija, S.C.; Mandal, J., and Ponnambath, D. K.(2014).Laboratory methods of identification of Entamoeba histolytica and its differentiation from look-alike Entamoeba spp. Trop Parasitol.; 4(2): 90–95.

17. Plorde, J.J. (1983). Harrisons principle of internal medicine. Text book. (Amoebiasis and Giardiasis)10th ed.Chap.217.1182-1186.

18. Reed, S.L. ; Ember, J.A. ; Herdman , D.S. ; Di Scipio , R.G. ; Hugli , T.E. and Gigli , I. ( 1995 ) . The extracellular neutral cysteine proteinase of Entamoeba histolytica degrades anaphylatoxins C3a and C5a . J. Immunol. 155 ( 1 ) : 266 – 274 .

19. Samie, A., Barrett, L. J., Bessong, P. O. , et al. (2010). Seroprevalence of Entamoeba histolytica in the context of HIV and AIDS: The case of Vhembe district, in South Africa’s Limpopo province. Ann Trop Med Parasitol. ;104:55–63.

20. Shaker, E. M., Al-Shaibani, K. T. M. and Al-abodi, H. R. J. (2018). Effect of Alcohol extract of green tea plant Camellia sienensis as a therapeutic of parasite Entamoeba histolytica. Plant Archives Vol. 18 No. 1, pp. 953-959.

21. Tektook , N.K.(2015). Investigation of some genes responsible for Biofilm Formationin Staphylococcus epidermidis Isolated From clinical samples. Ph. D. dissertation ( immunology). College of Science. Al-Mustansiriyah University Iraq.

22. Tektook , N.K.(2017).Quantitative determination of serum immunoglobulins; complement and C –reactive protein in Diabetic Retinopathy patients with and without UTIs. first international scientific conference of the college of biotechnology al-qasim green university.

23. Tomas M.L. Eagan, Amund Gulsvika, , Geir E. Eide,, Per S. Bakke.(2011) .The effect of educational level on the incidence of asthma and respiratory symptoms Department of Thoracic Medicine, Haukeland University Hospital, N-5021 Bergen, Norway for macrophage function : Effect of zinc deficiency on uptake and Killing of protozoan parasite .Immunology , 68Pp : 114 – 119 1.

24. Visvesvara GS, Moura H, Schuster FL (June 2007). "Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthiamandrillaris, Naegleriafowleri, and Sappiniadiploidea". FEMS Immunol. Med. Microbiol. 50 (1): 1–26.

25. Wolfs TG, Buurman WA, van Schadewijk A, de Vries B, Daemen MA, Hiemstra PS et al. (2002). Role NF_K ß Immunol 168Pp: 1286–1293.

26. Wuerz, T., Kane J. B. and Boggild, A. K. .(2003). A review of amoebic liver abscess for clinicians in a nonendemic setting. Can J Gastroenterol. 2012;26:729–33.

Received on 04.03.2019 Modified on 18.04.2019

Research J. Pharm. and Tech. 2019; 12(7):3555-3558.

DOI: 10.5958/0974-360X.2019.00606.1