Intestinal Parasites Among Unity Primary School Pupils, In Oraifite, Ekwusigo L.g.a., Anambra State, Southeastern Nigeria

CHAPTER THREE

MATERIALS AND METHODS

Study area

The study was undertaken in Oraifite Community, Ekwusigo Local Government Area (LGA) Anambra State, Southeastern Nigeria. It has a tropical continental climate with distinct wet and dry seasons. The average relative humidity is about 80% reaching 90% during rains. There are wide divergences in the composition of the soil from rich loamy soil to sandy soil with immense agricultural potentialities. The inhabitants are predominantly farmers and traders. Rain water stored in tanks and boreholes are their sources of drinking water. Method of faecal disposal in the primary school includes pit latrine and defecation in buhes surrounding the school premises. Only one public health centre offer health services to the host community.

Subjects

The subjects were pupils of Unity Primary School in Oraifite Community. The consents of their parents, guardians and school authority were sought and obtained after they were briefed on the importance and significance of the study by the researcher.

Collection of stool sample

Each pupil was given a clean, caped, dry, well labeled specimen container. Pupils were instructed a day before to collect stool specimens with the container at home next morning with the help of their parents and return it immediately. All the stool samples were examined in the Parasitology Laboratory unit of Nnamdi Azikiwe University Teaching Hospital (NAUTH), Nnewi, Anambra State. Of the 622 pupils issued a specimen container, 462 returned their stool specimen for examination between January and March 2009. A questionnaire was administered to each pupil from whom personal information on age, sex, and occupation of parents/guardian was derived.

In the laboratory, each sample was first examined for its consistency, colour, and presence of blood, mucous, adult worms, proglottids of tapeworms, with an applicator stick.

Saline and iodine wet mount, concentrated saturated sodium chloride floatation and formol-ether concentration techniques were used according to Chessbrough (1999) and WHO (1991) were used for the microscopic examination of each stool sample. Antigen testing was not performed; therefore the differentiation of Entamoeba histolytica from Entamoeba dispar was not possible.

PARASITOLOGICAL METHODS

Materials and reagents

Coverslips

Dropping bottles containing: saline solution, and isotonotic lugol’s iodine (1% solution)

Microscope slides

Markers for labeling

Applicator sticks

Procedure

The patient’s stool sample was given a reference number, which was also indicated on the slide. A drop of saline was placed at the center of the right half of the same slide. An applicator stick was used to pick-up a small portion of the specimen and mix with the drop of saline. Similarly, a small portion of the stool was also picked and mixed with the drop of iodine. The drop of iodine and saline were covered with a coverslip by holding at an angle and touching the edge of the drop and gently lowering on to the slide. This was to reduce the chance of including air bubbles in the mount. Examination of the slide was first done using X10 objective and the X40 objective.

3.5.2 . Egg Floatation Method Materials and methods

-coverslip

-dropping bottles containing saturated saline solution.

-microscope slides -pens for labeling.

-applicator sticks

-test-tubes

Procedure

7ml of saturated sodium chloride solution was added to approximately 1g of faeces in a test-tube and stirred using an applicator stick, until a slightly cloudy suspention was seen. The test-tube was filled to the brim with saturated sodium chloride solution before covering with slide. After 10 minutes, the slide was removed for examination under a coverslip.

3.5.3. Formol-ether Concentration Technique Materials and reagents

Applicator stick

Centrifuge with head and cups to hold 15 ml conical tubes

Test tube rack

Coverslips and microscope slides

Funnel, and surgical gauze for seiving

Pipettes, Pasteur with rubber bulb

Ether

Lugol’s iodine (1%) and normal saline

To approximately 1g of faeces, 10ml of 10% formalin was added and stirred until a cloudy suspension is formed. Gauze was fitted into a funnel and the funnel placed on top of the centrifuge tube. The faecal suspension was placed through the filter into the centrifuge tube until the 7ml mark was discarded with the lumpy residue. Then, 3ml of ether was added and well mixed for one minute before centrifuging for 3 minutes. After centrifugation, there were four layers in the tube. The first was that of ether, followed by the debris, formalin solution layer and the layer containing the eggs and cysts of parasites (the sediment). The fatty debris at the interface was then loosed with applicator stick and the supernatant was quickly poured off by inverting the tube. The small deposit at the bottom of the centrifuge was shaken before poured on to a slide and examined.