Impact of Intestinal Entamoeba histolytica on Sera of Leukotreins D4, Interleukin -6, Acid phosphatase and other Some Trace Elements

The study was carried out during the period of February 2016 September 2016 for the detection of Entamoeba histolytica in (66) patients with age group ranges from 21 -60 years who attended Al –Fayed clinical laboratory in Baghdad. The diagnosis done by microscopic examination and Triage (Micro parasite panel test) methods. Blood samples were taken from patients as well from other (30) healthy control matching in age and gender. The study included measurement of concentration of Leukotrein D4, Interleukin-6, Acidphoshatase activity, Zinc and Copper in sera of patients and control. The results indicated presence of parasites in all patients in both methods. The concentration of LTD4, IL-6, ACP increased significantly. The concentration of Zinc decreased significantly. The concentration of copper statistically non significant in both interval ages in patients sera in comparison with healthy control

Entamoeba histolytica is the causative organism of amoebic dysentery, a disease which affects a large number of people every year in the tropical regions of the world.This organism invades the human gut by first adhering to the intestinal mucosa and then secreting enzymes for cytolysis 1 .
M o s t c a s e s a r e i n t e s t i n a l a n d asymptomatic.Symptoms, when occur, are multiple and varied, ranging from mild abdominal discomfort and diarrhea (often with blood and mucus) alternating with periods of remission or constipation, to severe illness with fever, chills, and significant bloody or a mucoid diarrhea .Amoebic colitis may be confused with inflammatory bowel disease such as ulcerative colitis 2 .The Infection of E.histolytica occurs invade colonic crypts lamina propria and cause flask shaped ulcer, this ulcer may activate apoptosis in the target cells 3 .Entamoeba histolytica trophozoites reaching the liver create their unique abscesses, which are well circumscribed region of cytolysed cells, liquefied cells, cellular debris, the lesions are surrounded by connective tissue enclosing few inflammatory cells paranchymal cells adjacent to the lesion are often unaffected, however lysis of neutrophil by E. histolytica trophozoites might release mediators that lead to death of liver cells, and extended damage to hepatocyte 4 . of liver, ruptured into the lung; the other way is by lymph channels from amoebic hepatitis, and the last way is through the systemic circulation 5 .Cell mediated response have been characterized by lymphocytes proliferation and lymphokines secretion especially in patients with amoebic liver abscess.Suggestion a role of body immune system cells.the production of inflammatory cytokines, including IL-1b, IL-6, IL-8, IL-12, IFN-g, and TNF-a 6,7,8 .IECs are the second line of barriers against pathogens after the mucosal layer and the first line of host cells to encounter microbial/parasite antigens, they express an array of pathogen recognition receptors (PRRs), including TLRs 9 .
Recently, experimental studies show that zinc alter functionality of E.histolytica and reflect decrease in replication and adhesion manifested by inhibition of amoebic pathogenicity 10 .E.histolytica acid phosphatase activity is significantly inhibited by copper suggesting a possible roles in amoebic dysentery 11 .In this study the level of Leukotreins D4,Interleukin-6, Acidphosphatase and Copper with Zinc determined in patients with acute Amoebiasis as inflammatory mediators in patients with healthy control group matched in age and gender.

Studied groups
The study carried out during the period from (February2016-November 2016), the age of patients extended from (21 -60) years, two studied groups were involved Suspected patients: Blood and stool samples were obtained from a total of 66 patients clinically suspected with amoebic dysentery that had been examined and defined as suspected cases by specialized physician and healthy control

Samples collection
Stool sample from each patient was collected in a clean, dry tight cover container and examined with a half an hour.The samples were examined for the presence of E. histolytica.

Stool sample examination Macroscopic examination
It was performed by observing the consistency of stool, presence of blood, mucous and other substances.

Microscopic examination
For each stool sample, wet mount preparation slide was examined by clean, dry slides by obtaining one drop of normal saline and small amount of stool from different places of stool by using clean wooden stick, especially when blood or mucous were noticed, then mixed gently with normal saline and covered with cover slip, the slide was examined under the low (10x) and high power (40x) of microscope 12 .

Specific test for E.histolytica (Triage) Cassette
This test is based on quantitative Immunochromatographic assay for determination of Entamoeba histolytica in stool samples.Assay Procedure The cap of the stool collection tube was taken out and used the stick to pick up sufficient sample quantity.

2.
Introduced the stick once into 4 different parts of the stool sample (≈ 100mg) and added it to the stool collection tube.

3.
For liquid samples(≈ 100mg)was added in the stool collection tube by using a micropipette, closed the tubes of the diluents and stool samples.4.
Proceeded to shake the stool collection tube in order to assure good sample dispersion.

5.
The Entamoeba card test was removed from its sealed bag just before use.

6.
The stool collection tube was taken, cut the end of the cap, and dispensed 4 drops in the circular window marked with letter S. Avoid adding solid particles with the liquid.7.
Read the results at 10 minutes.

Blood samples
Five mL of Venus blood was obtained from each patient and collected in sterilized screw cap plastic tube, blood samples were left for 30 min.at room temperature, then centrifuge at 3000 rpm for five minute, then the serum for each sample was collected in eppendorf tubes and stored in deep freeze at -20°C until the time for using.The current study included Immunological & Clinical biochemical aspects.the level of interleukin -6(IL-6) estimated by ELISA according to manual procedure of cusabio Biotech(Germany) and Leukotreins D4 were estimated by ELISA according to the manual procedure of Creative -Diagnostic Company.
Copper, Zinc and acidphosphatase Concentration determined according to manufactures instructions of Biosystem(Spain).

Statistical Analysis
The results were analyzed using statistical system SPSS version -18 (T-testing).

Diagnosis of E.histolytica
The result of E.histolytica show prevalence using direct microscopic Examination and Triage (Micro parasite panel test) that 66 patient with a percent of 100% infected with Entamoebiasis (Table 1).

LeukotreinsD4
The level of Leukotreins D4 increased significantly(p ≤ 0.05) in patients with E.histolytica in comparison with healthy control in both interval ages till reach to 48.61,37.71for patients and 31.75,23.25 for healthy control respectively (Table-2) .

Interleukin-6
T h e l e v e l o f I L -6 I n c r e a s e d siginifigantly(p≤0.05) in patients with E.histolytica in comparison with healthy control in both interval ages the value 21489,21449 pg ml for patients and 11470,11430 pgmlfor healthy control respectively (Table 3) .

Zinc and Copper
The concentration of zinc decreased significantly (p≤0.05) in both interval ages of patients with Entamoebiasis in comparison with healthy control (Table 4).While the result of copper statistically non significant in both interval ages of patients and healthy control.

Acidphosphatase activity
The activity of acid phosphatase increased significantly p≤0.05 in both interval ages of patients in comparison with healthy control (Table-5).

DISCUSSION
The presence of E.histolytica by using direct microscopic examination and Triage (Table 1) .theresult show no difference between the two methods.In spite of the microscopic examination of stool samples considered to be the gold standard for diagnosis of Entamoebiasis and other parasites 13 .However, microscopy has several important dis advantages among these (I) Correct identification depend greatly on experience and skills of microscopist (II) Sensitivity is low and therefore ,examination of multiple samples is required(III).E.histolytica cannot be differentiated from the other nonpathogenic E.disapr simply on the basis of the morphology of the cyst and small trophozoites 14 .The Triage is immunoassay to diagnosis the stool for antigens for the parasites 15 .The increasing level of LeukotreinsD4(LTD4) in patients with E.histolytica in comparison with healthy control may be associated with the impairment of the immune system especially during the acute phase of disease by the appearance of suppressor CD8 lymphocyte ,than, defect in cell mediated immune response which occur in amoebic infection.However, the mechanism of immunosuppression in Entamoebiasis occur by induce macrophages eicosanoides in both Cycloxygenase and 5-Lipoxygenase pathway to produce prostaglandins and Leukotreins 16 which  -4).However, acute phase of infection with Entamoebiasis causes increased metallothionein mediated hepatic uptake of serum zinc ,leading to hepatic accumulation of zinc than decreased serum zinc level via interleukin -1 mediated mechanism 22 than increase the severity of disease ,altered immune status and impaired antioxidant system 22 on the other hand the immune response up regulate of Ceruloplasmin gene and synthesis Ceruloplasmin -CU complex in the blood.A Ceruloplasmin contain 95% of total serum copper and this may at least partly explain lack of a significant increase or decrease in serum Copper concentration.Acid phoshatase increased significantly in patients with Entamoebiasis (Table 5) in a general ,ACP considered as a virulence factor in some pathogenic microorganism or may be important for management of disease severity 24 .

CONCLUSION
The result indicated presence the parasites in all patients in both methods .The concentration of LTD4, IL-6, ACP increased significantly.The concentration of Zinc decreased significant.The concentration of copper statistically non-significant in both interval ages in patients sera in comparison with healthy control .

Table 1 .
Distribution of Entamoeba histolytica infection according to microscopic examination and Triage

Table 4 .
Zinc and Copper concentration(mmol/L) in patients with E.histolytica and healthy control

Table 5 .
Acid phoshatase activity in patients with E.histolytica and healthy control