Síntesis de la intervención clínica

Each of the study participants' recent CD4⁺ counts (within a period of six months) was obtained from their folders and recorded in the questionnaires.

(a) Albuminuria assessment (macroalbuminuria and microalbuminuria)

Ten millilitres of urine was collected from each participant into a plain bottle on the first day of contact and tested for proteinuria using combi 10 test strips. A strip was taken from the container and dipped into the urine sample so that the test area made contact with the urine. It was removed after a second and while being removed, the edge of the strip was wiped against the rim of the sample container to remove excess urine. After 60 seconds, the reaction colours of the test areas were compared with the colours of the label and the value for the nearest colour block was assigned to each entity tested for.

Those that were negative for protein at the initial urinalysis were tested for microalbuminuria, using early morning urine samples. Participants were given plain bottles to take home for the collection of early morning urine samples. They were instructed to bring the sample to the hospital within one hour of collection. The microalbuminuria test was done by an optically

51 read immunoassay semi quantitative method, using Micral-Test II Strips. The micral test strip was immersed in the urine, such that the urine level was between the two black bars for five seconds.

The strip was then placed horizontally across the urine container for 60 seconds. The colour change in the test zone was then compared with a colour scale provided by the manufacturers.

Microalbuminuria was graded as mild (20-50mg/L), moderate (50-100mg/L) or severe (100-300mg/L), depending on the colour change on the strip. The presence of any of the grades was termed positive. Those who tested positive to the microalbuminuria test at the initial test had a repeat microalbuminuria test done within 2 to 4 weeks and those who had a positive test on the two occasions were regarded as having microalbuminuria.

(b) Assessment of fasting lipid profile and fasting plasma glucose

Eight millilitres of fasting venous sample was collected from each participant, after an overnight fast of at least 8 hours and not later than 10 hours. Five millilitres of the sample was collected into plain bottles, for fasting lipid profile estimation, while 3mls was collected into fluoride oxalate bottles for fasting blood glucose estimation. The samples were centrifuged and sera/plasma separated, and stored at a temperature of -20^oc in the hospital's deep freezer. The stored samples were analyzed at the Chemical Pathology laboratory of the University of Uyo Teaching Hospital within a week of collection.

Fasting Lipid Profile was performed for each participant with the use of Randox cholesterol enzymatic end point method. This uses enzymatic hydrolysis and oxidation to determine cholesterol concentration in serum or plasma.⁹³ Total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were estimated by the enzymatic method, while LDL was estimated with the Friedewald formula as follows; LDL (mg/dl) = TC-HDL-(TG/5).⁹⁴ Where

52 TG was > 4.5mmol/L (400mg/dl), LDL-C was measured directly. Fasting blood glucose was assessed using the glucose oxidase method (Trinder method).⁹⁵ Total cholesterol (TC)

≥5.2mmol/L, LDL-C > 3.3mmol/L, TG >1.7mmol/L, HDL-C < 1.0mmol/L were regarded as elevated and the presence of at least one of the above was termed dyslipidaemia.⁹⁶ A fasting blood glucose of 6.1 to 6.9mmol/L was termed Impaired Fasting Glucose (IFG).⁸⁶ Detailed laboratory procedure for the estimation of fasting lipid profile and fasting plasma glucose is attached (Appendix VI and Appendix VII respectively)

(c) The oral glucose tolerance test

This was performed by me, the principal investigator, on the participants that had impaired fasting glucose at the initial blood glucose assay. The participants were booked for the test within one week after the initial FPG estimation, so as to prepare them adequately for the procedure.

Those who did not have money for transportation were provided with money to take care of their transportation to and from the hospital for the test. Participants who were resident at distant locations were made to sleep overnight in the medical wards of the hospital.

The WHO recommendation was used for the test.⁸⁶ The test was conducted in the morning, between 7am and 9am after a fast of 8 to 14 hours, preceded by 3 days of unrestricted carbohydrate diet and physical activity. Participants were advised not to smoke or drink caffeine in the morning of the test. An intravenous cannula was inserted into a peripheral vein of the participants and two milliliters (2mls) of venous blood specimen was collected into flouride oxalate bottles for fasting plasma glucose (FPG) estimation. Seventy-five grams of glucose D was dissolved in 200mls of water and given to each participant, to sip over five minutes. Participants remained seated throughout the test and were allowed to drink more water if they wished. Two millilitres of venous

53 blood was drawn from the insitu cannula into fluoride oxalate bottles, two hours after administration of the glucose drink. Analysis of the blood samples for glucose was done using the method of Trinder.⁹⁵ Random plasma glucose within 7.8 to less than 11.1mmol/L (140 to less than 200mg/dl) two hours after the glucose drink was termed Impaired Glucose Tolerance (IGT), while values ≥11.1 mmol/L (200mg/dl), was classified as diabetes mellitus.⁸⁶

(d) Ouality control of Assays for glucose and Lipids:

This was done by determining the intra-assay and inter-assay coefficient of variation (CV)% for the glucose and lipid measurements respectively. The intra-assay coefficients of variation for the glucose assays and lipid measurements were obtained respectively by determining the concentration of each sample of low, medium and high plasma concentration ten times in one assay run, while the inter-assay coefficients of variation were determined from four consecutive assay runs. The intra-assay and inter-assay precision were expressed as coefficient of variation (CV)%, calculated by dividing the standard deviation of the set of measurements by the mean of the set and multiplying the obtained value by 100. An intra-assay coefficient of variation of <10%

is acceptable, while for inter-assay coefficient of variation, a value of <15% is acceptable.⁹⁷