Study Area
Four hospitals covering the three senatorial zones of Ebonyi state were used for the study. They include Ebonyi State University Teaching Hospital (EBSUTH) Abakaliki, Federal Medical Centre (FMC) Abakaliki, Mile-4 Hospital Isieke and Mater Miserecordiae Hospital (MMH) Afikpo. Ebonyi State is located in the South-Eastern part of Nigeria. It is defined by longitude 8o61611 E and latitude 6o2212811 N, elevated at 115.824m above sea level and covers an area approximately 51km2. The vegetation characteristic is that of the tropical rain forest with an average annual rainfall of about 1600mm and an atmospheric temperature of 30oC. There are two distinct seasons, the wet and the dry seasons, the former taking place between April and October, while the other occurs from November to March each year. The people of Ebonyi State are predominantly Rice, Yam and Cassava farmers.
Study Population
The study population comprised of 1000 apparently healthy blood donors aged between 18-47 years who visited the above mentioned hospitals for blood donation from June 2008 to May 2010.The donors were made up of 914 males and 86 females. They were volunteers who satisfied the criteria for blood donation, commercial blood donors and those who have patients with conditions that require blood transfusion. Following informed consent, a well structured questionnaire (Appendix II) was used to obtain socio-demographic information from the subjects involved in the study.
Sample Collection
A large drop of blood specimen was obtained from each donor by finger prick using a sterile lancet. With the aid of a pipette, the collected specimen was then transferred to a grease-free microscopic slide before screening for malaria parasite. The procedure was done according to Cheesbrough, (2005).
Tests for Malaria Parasite (MP)
The screening of participants for parasitemia was done using Giemsa staining technique as described by Cheesbrough (2005).
Procedure for thick Blood Film Staining Technique
A thick film was made by placing a large drop of blood (about 15mm in size) on the centre of grease-free microscopic slide. Without delay, the blood was spread with a glass spreader held at a steep angle to achieve a thick smear covering an area of 15 x 15mm. This was allowed to air dry in a horizontal position. Then a Giemsa stain (3% solution diluted with buffer for 30 minutes staining) was applied on the thick film and allowed for 30 minutes. After this time, the stain was washed off using distilled water and again allowed to air-dry. The dried stained thick smear was finally viewed under a light microscope using 100x oil immersion objective lens. The chromatin of the parasites appeared as dark red dots while the cytoplasm appeared as blue rings.
Procedure for thin Blood Film Staining Technique
A thin film was made by placing a small drop of blood on the centre of a grease-free microscopic slide. The drop of blood was then spread with a glass spreader held at an angle of 30o to obtain a thin film with a smooth tail end. This was allowed to air dry in a horizontal position and then fixed with absolute methanol for two minutes. After this, a Giemsa stain diluted with buffer for 30 minutes staining was applied on the thin film and allowed to air dry for 30 minutes. The stain was then washed off using distilled water and also air dried. The stained thin film was viewed under a light microscope using 100x oil immersion objective lens. Identification of different Plasmodium species was based on referencing to the distinctive characteristics of the various stained morphologic forms of each species (Appendix IV-VII).
Index for Reporting Level of Parasitemia
The index for reporting the level of parasitemia among malaria parasite positive subjects were based on the number of parasites seen in 100 high power fields as described by Cheesbrough (2005), (Appendix I).
Statistical Analysis
The relationship between parasitemia and socio-demographic factors were determined using Chi-square table of contingency. Statistical significance was achieved if P<0.05