Tikrit Journal of Pure Science

B ackground: Entamoeba histolytica are very common protozoan parasites in Iraq and are causative agents of gastrointestinal diseases especially among children. Objective: To show the distribution of Entamoeba histolytica and Giardia lamblia among children aging 6-12 years old in Tikrit City. Materials and Methods: A cross sectional study was carried on (1100) school children attended Tikrit Primary Heath Care Centers for period from February 2010 to June 2012. For each child the clinical examination was carried on by physicians in addition to anthropometric measurements, laboratory investigations including haematological, biochemical and general stool examination. Results: Stool samples were collected from 1100 children from both sexes, the rate of infections were: E. histolytica 9.3%, G. lamblia 8.9%.The overall rate of E. histolytica and G. lamblia infections was highest among 6-7 years (20.7%) followed by >10-12 (18.7%) and >7-10 years (17.2%) years. The distribution of


Introduction
Amoebiasis is a common problem of human civilization, it is caused by parasitic protozoan infection, most common in developing countries especially among immunocompromised individual and also distributed among monosexual.It is acquired by ingestion of food or water containing the cyst form of the parasite Entamoeba histolytica [1].Entamoeba histolytica is known as pathogenic intestinal ameba, its range of symptoms varies and depends on location of parasite in the host and the extent of tissue infection.Among symptomatic intestinal amoebiasis, patients exhibit nondescript abdominal symptoms or may complain of more specific symptoms including diarrhea, abdominal pain and cramping, chronic weight loss, anorexia, chronic fatigue and flatulence.Secondary bacterial infection may develop after the formation of flaskshaped amebic ulcers.Some cases develop into amebic dysentery [2].Extra-intestinal amebiasis may take place in the liver, lung, pericardium, spleen, skin and brain.Venereal amebiasis may occur [3&4].In Iraq, the distribution of amoebiasis were fluctuated depending on sample size, seasonal assessments, and laboratory techniques and other factors (5, 6, 7, 8, 9 & 10).Giardiasis is a disease caused by Giardia lamblia occur worldwide and may infect up a third of the population in developing countries.Approximately about 200 million of people in the world have clinically manifested giardiasis, with 500.000New cases per year [11].It is transmitted by swallowing cysts in contaminated food and water or fecal-oral contact.While the parasite can be spread in different ways, water (drinking water and recreational water) is the most common mode of transmission [12].Giardiasis varies in severity, the disease can be acute or chronic and asymptomatic carriers are common.Children are more frequently affected than adults, although all ages may acquire the infection [13].Following incubation period of 2 to 3 weeks, water and smelly diarrhea, abdominal cramps, flatulence and anorexia, sometimes accompanied by nausea may occur.Fat soluble vitamin and folic acid deficiencies , weight loss, malabsorption syndrome as well as steatorrhea may occur [14].In Iraq, there are different reports about distribution of giardiasis in various parts of country with different rate of infection (15, 16, 17, 18, 19, &20).The present study was carried on to show the distribution of E. histolytica and G. lamblia Materials and Methods: Study population: The present study was done in Tikrit city on 1100 children aged from 6-12 years old (445 males and 655 females).Those were children attended to the clinical centers during the time of the study suffering from diarrhea.All the children were exposed to complete clinical examination in the three Medical Centers and private medical laboratories, including laboratory investigations.The study started from beginning of February 2010 until end of June 2012.A special questionnaire form was arranged to collect information, using direct interviewing to the eligible adults for the individuals, and with children parents.Anthropometric measurements: The body weight of children were measured by electronic balance distributed by (WHO/UNICEF to primary health care's) with minimal clothing.The height of the children measured to the nearest 0.5cm with a vertical measuring scale fixed to the metal bar connected to the beam balance.The skin fold thickness (SFT) was measured using Harpenden skin fold caliper of a constant pressure of 10gm/sq.mm was used to serve as an index of subcutaneous fat.The mid upper arm circumference (MUAC) was measured to the nearest 0.1 centimeter with a non-elastic fiber glass measuring tape, which was placed gently but firmly round the middle of the left upper arm to avoid compression of the soft tissue [21].Collection of the specimen: One-three stool samples were collected from each patient in a tightly covered wide mouth plastic disposable container.These were labeled with sample numbers, date and name of the patient.The sample was examined within 1 hour from collection to detect the parasitic infection.The samples were examined by direct wet mount method and formal-ether concentration techniques [13].Blood samples were obtained, about 10 ml from each patient, 9 ml of blood without EDTA and 1 ml of blood added with EDTA-was drawn intravenously from each subject.The former was used for T-and Blymphocyte counts, biochemical tests, and zinc concentration.The blood with EDTA used for hemoglobin estimation, total and differential WBC count.Hematological parameters: The hemoglobin estimation was done by Sahli method, the test is done by dilution of 0.02 ml. of blood with 2 ml of 0.1 mol/hydrochloric acid converting the hemoglobin to acid hematin.The white blood cell numbers were counted under light microscope (10X and 40X) using Neubauer counting chamber.It was estimated by diluting of whole blood in Turk's solution (glacial acetic acid, 1% with 1-2 drop methyl blue to give purple colour).The differential leucocytes count was made by counting 200 leukocytes in blood smears, stained with Leishman' s stain.These were differentiated into neutrophils, eosinophils, basophils, lymphocytes and monocytes and the percentage of each was calculated using oil lens microscope (100X).Biochemical tests: The total serum protein and albumin were measured by using kits from Randox Laboratories Ltd., U.K. The value of serum globulin was estimated by subtracting the value of serum albumin from the total protein.The alkaline phosphatase (ALP) was estimated by colorimetric method using kit of bioMerieux sa Marcyl Etoile-France.The serum zinc level was estimated using atomic absorption spectrophotometer (Varian, Australia) after dilution of serum samples with deionized distilled water 1:4 to bring the metal concentration within the working range of atomic absorption spectrophotometer by direct aspiration of the samples.Statistical analysis: Both Chi-square and student's ttest were used according to the requirement of the test [22].

Results
The overall rate of E. histolytica and G. lamblia infections among school children was 18.2 %.The frequency of E. histolytica 9.3%, the highest rate was among 6-7 years (11.3%)followed by >10-12 (10.9%) and >7-10 (7.8%).In G. lamblia was 8.9 %, the highest rate was among 6-7 (9.4%), and >7-10 years (9.3%), the lowest was among >10-12 years (7.8%).The overall rate of both infections, was (20.7%) among 6-7 year , followed by >10-12 years (18.7%) and >7-10 years (17.2%).statistically there was no significant difference between infected and non-infected children; the haemoglobin value was within normal range.The total white blood cell counts in infected groups was significantly higher than non-infected ones (<0.05) and also its value was within normal range.and G. lamblia infected children and non-infected ones.In infected children, the T-rosette and B lymphocyte were lower while the Null cells was higher than non-infected children.

Discussion
The overall rate of E. histolytica and G. lamblia among 1100 children aged from 6-12 years old were 18.2%.This reflects that protozoan infections are common in Tikrit.Salman et al. [20] examined 266 found the overall rate of parasitic was 64.28% in 171 stool samples, this rate was contributed five protozoan parasites: E. histolytica 94(34.21%),G. lamblia 47(17.66%).These infections are endemic worldwide and are a major public health problem specially in tropical and subtropical countries [23].
Comparing the rate of infections with those from different parts of the country and neighboring countries, showed a considerable difference could be found in prevalence of infections.The differences can be explained by the influence of level of sanitation, environmental conditions, human behavior, laboratory diagnosis used for detection of infections and health education among school children [24].Transmission of these protozoan occur via fecal-oral route either directly or indirectly via eating or drinking focally transmitted food and water, in addition, asymptomatic carriers of these infections are a constant risk for transmission in a community [18].High infection rates were found in children aged from 6-7 and >7-10 years old and low infection rates were found among children aged from >10-12 years.There were no significant difference between infected and non-infected ones in relation to age group.This finding is almost identical to that reported by [18] in Baghdad City, who reported that the rate of intestinal parasites among children was 57.9%., the highest prevalence (71.4%) was recorded among 6-11 years age group followed by age group 12-18 years (55.1%), while the group below than 2 were the less affected group (30.6%).In Dohuk [15] examined 1261 stool specimens of children, found the age group 10-12 years had the highest rate (81.2%) and 7-9 years the lowest (22.9%).In Kadhmiyah Hospital in Baghdad [8] the rate of E. histolytica was 9.80% and G. lamblia 1.51%.In Kirkuk, [19] examined 417 stool samples from internal displaced persons living in schools, houses under construction and rented accommodation and houses in Kirkuk; recorded the rate of G. lamblia was 10.31%, E. histolytica 1.67%.They showed high rate among those aging from 1-10 years and no significant difference between males and females.Regarding the distribution of infections according to gender, although the rate of both infection in males were greater than females, but statistically there was no significant difference between sexes.In Baghdad City, it has been reported that there was no significant difference between gender and infectivity rate of E. histolytica and G. lamblia in children aging from 1 month to 2 years (8).In Kirkuk [9] examined 943 stool samples of children attended Kirkuk Pediatric hospital, she found the rate of E. histolytica/E.diapar was 30.22%, the highest rate was among 5-6 years (43.96%).Infection rate among illiterate children was higher than educated ones.G. lamblia is recognized as the most common intestinal protozoan parasite of human beings in Iraq [25] especially in Kirkuk [26].In Duhok city-Iraq, [15], examined 451 stool samples from primary school children, they found the rate of Giardia lamblia infection was 41.2%, which was higher among boys (41.6%) than girls (35.6%).In Baghdad, [18], found the prevalence of G. lamblia was (31.)%), the rate of intestinal parasites was slightly higher in males (59.9%) than females (56.2%), but statistically there was no significant difference between sexes.The higher rate of infection in males is due to males tend to spend their time in street, and playing in sand but females tend to spend most of time in homes.This finding is contrast to [27] who found the rate of intestinal parasites was higher in females than in males.The lower anthropometric measurements in infected children indicate that protozoan infections lead to growth retardation, this finding is in agreement with that reported by [28] in Hawija district, Kirkuk governorate and also with [29] who studied the prevalence of B. hominis in Turkey and in Tikrit City [10].
In the present study, although the hemoglobin concentration was lower in all infected children groups than non-infected ones, but statistically there was no significant difference between infected and non-infected groups.The lower hemoglobin concentration among infected children reflect that protozoal infections affect the general health of those children.The increase in white blood cells during protozoal infections is also reported in B. hominis infection [10].The white blood cells main function is to fight infection defend the body by phagocytosis against invasion by foreign organisms, and to produce or at least transport and distribute antibodies in the immune response.In this study the white blood cells count was higher among infected children than noninfected ones.This finding is also reported in other study in Baghdad [8] in G. lamblia and E. histolytica [30].Blood serum contains large amounts of protein.Two major groups of proteins in blood serum are albumin and globulins.A total serum protein test measures the total amount of protein in blood serum as well as the amount of albumin and globulin.Because the total protein represents the sum of albumin and globulin.It is important to know which protein fraction is high or low than the total protein.In this study the values of serum total protein and immunoglobulin did not differ between infected and non-infected children.Similar finding is reported by [10] in B. hominis infection.Decrease albumin may also be explained by malnutrition or a low protein diet.

Tikrit Journal of Pure Science 23 (6) 2018 ISSN: 1813 -1662 (Print) E-ISSN: 2415 -1726 (On Line)
Giardia lamblia and Entamoeba histolytica were related to delirious consequence may result from protein energy malnutrition.In the present study the total protein and albumin were significantly lower in both infections [31].In Tikrit [32] recorded significantly lower level of serum total protein and albumin in G. lamblia infected patients comparison to control.In Erbil, [5] recorded no significant difference in levels of total serum protein and albumin in infected and non-infected groups (6.82+0.59,3.8+o.3)(7.23+0.4,3.82+0.27gm/dl) respectively.In Kalar town [31], showed that the concentration of serum globulin did not vary significantly between E. histolytica infected and noninfected children.The value of alkaline phosphatase, total protein, albumin did not vary significantly between infected and non-infected children.Although the the globulin value did not vary significantly between children infected with E. histolytica but in G. lamblia infected children was lower than non infected ones.It has been reported that alkaline phosphatase and leucocyte counts elevated in amoebic liver abscess [33] Higher concentration of serum globulin among G. lamblia infected patients than non-infected ones is identical to study reported by [34] who recorded higher concentration of total globulin in serum in G. lamblia infected 2.57gm/dl compared to control 1.83gm/dl.They found that the concentration of serum globulin did not vary significantly between children infected with E. histolytica and non-infected ones.Zinc is essential trace element for a wide range of biochemical, immunological and clinical infections.Zinc deficiency is due to lack of intake of animal protein, high dietary phytate that limit zinc absorption and intake, in addition to increased fecal losses during diarrhea [10].The lower zinc concentration in infected children than non infected ones is in agreement with [35] who reported decrease in zinc level in serum of children aged 6 years infected with diarrhea.This is also identical to that reported in G. lamblia infection in Turkey [36] and in B. hominis infection among primary school children in Baghdad [37].

Table 3
, shows the anthropometric measurements values (mean weight, height, skin fold thickness and mid upper arm circumference) among children infected with E. histolytica and G. lamblia infections were lower than non-infected controls of different age groups.The difference between infected and non infected groups in all parameters in both infections was statistically significance (P<0.05).Table (3 ) The anthropometric measurements values among E. histolytica and G. lamblia infected childrenMean ±S.D.

Table 6 ,
shows some biochemical parameters among infected and non-infected children.The alkaline phosphatase, total protein and albumin values did not differ significantly between infected and non-infected children.Although the globulin value did not vary significantly between children infected with Entamoeba histolytica, and non-infected children but in G. lamblia infected children was lower than non-infected children (P<0.01).Table (6) Biochemical parameters among E.histolytica and G. lamblia infected children Mean± S.D.