Khalil Ismail A.Mohamed1, Mohammed Sami Khadhum2,
Huda Q. Mohammed Abu-Al-ess1, Saad Hasan Mohammed Ali1,
Suha A.AL. Fukhar1, Wifaq M. Ali AL-Wattar1 and Jinan M. Mousa1

1Clinical Communicable Diseases Research Unit, College of Medicine,
University of Baghdad, Baghdad-Iraq.
2Department of Basic Sciences, College of Dentisty, University of Baghdad, Baghdad-Iraq.

Abstract

The study was carried out during a period of February 2016-September2016 to detection of Entamoebahistolyticain(66) patients with age range 21 -60 years who attended Al –Fayed clinical laboratory in Baghdad. The diagnosis done by microscopic examination and Triage (Micro parasite panel test) methods.a blood samples was taken from each patients as well as other(30)healthy control matching in age and gender. The study included measurement the concentration of LeukotreinD4,Interleukin-6 , Acidphoshatase activity ,Zinc and Copper in sera of patients and control .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 significantly. The concentration of copper statistically non significant in both interval ages in patients sera in comparison with healthy control

Keyword: Entamoebahistolytica, LeukotreinsD4, Acidphosphatase, Interleukin-6, Copper, Zinc.

Introduction

Entamoebahistolytica 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).

Most cases are intestinal and 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.histolyticaoccurs invade colonic crypts lamina propria and cause flask shaped ulcer, this ulcer may activate apoptosis in the target cells (3).Entamoebahistolyticatrophozoites 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. histolyticatrophozoites 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-1β, IL-6, IL-8, IL-12, IFN-γ, and TNF-α (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.

Material and Methods

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 wereobtained from a total of 66 patients clinically suspectedwith amoebic dysentery that had been examined anddefined 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 Entamoebahistolyticain stool samples.

Assay Procedure

  1. 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 (00mg) 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 -20c°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).

Results

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).

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

Interleukin-6
The level of IL-6 Increased 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  statisticallynon 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) .the result 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 play important  role in regulation of cellular and humoral immune response included the suppression of macrophages derived TNF-α  and gene expression of interleukin-1 production and the MHC-II and other signal peptide necessary for cell-to cell communication  would alter macrophage –lymphocyte driven reaction and down regulate the local immune response (17,18).The increasing level of IL-6 in patients with Entamoebiasis  in comparison with healthy control(Table-4) may be due to ability of E.histolytica to up regulate of Th2 and down regulate of Th1 to inhibit INF-ɣ (19)INF- ɣ involved  in clearance of infection and correlated with the protection from E.histolytica infection(20,21) The result of the study demonstrate that serum level of zinc  decreased in patients with acute E.histolytica  were a significan difference was not observed for serum copper level(Table-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 ).

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

Methods
No. of samples
No. of positive
%
Microscopic examination
66
66
100%
Triage
66
66
100%

 

Table 2. Concentration of Leukotreins-D4 in patients with E.histolytica  and healthy control

Parameters
Age categories
Leukotreins D4(ng/ml)
20-40 Patients 48.61±3.06
Control 31.65±6.78
40-60 Patients 37.71±1.82
Control 23.25±1.75

 

Table 3. Concentration of Interleukins-6in patients with E.histolytica  and healthy control

Parameters
Age categories
 IL -6(pg/ml)
20-40 Patients 21489±2.76
Control  1470±5.30
40-60 Patients  21449±4.50
Control  1430±2.90

 

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

Parameters
Age categories
Zinc Copper
Patients Control Patients Control
20-40

 

9.8

±

0.6

12.4

+

2.3

 18.3

±

4.1

11.5

±

1.6

40-60 10.6

±

0.4

13.8

+

3.1

17.6

±

2.8

11.9

±

2.3

 

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

Patients
Age categories
 Acid phosphatase(IU/ml)
20-40 Patients 0.9±0.3
Control 0.4+0.2
40-60 Patients 0.1±0.12
Control 0.5±0.1

 

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 .

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