A semi–structured questionnaire containing the biodata of the subjects, demographic and socioeconomic characteristics of the study subjects (such as occupation, ethnicity, educational level, income), medical history, clinical examination and laboratory evaluation was administered to all subjects (participants and controls) who had given an informed consent (appendix B). This was conducted by research assistants who were trained and supervised by the researcher over a span of 10 months. A physical examination was conducted on each subject including their weight (kg) which was taken using a clinical weighing scale with minimum clothing. Height (m) was
measured using standiometer. The BMI was then calculated as weight/height2: kg/m2.10mls of venous blood was taken once from an ante–cubital vein for the assessment of blood glucose, creatinine, albumin, lipids, retroviral screen, haemoglobin concentration (Hb) and CD4 count, while urine and blood samples were taken twice, 3 months apart for subjects who had at least +1 proteinuria or abnormal ACR > 30mg/g, or eGFR of <60ml/min/1.73m2 respectively.
Early morning sample of urine was collected for spot urine determination of AlbuminCreatinine-Ratio (ACR) and also for urinalysis which was done using dipstick for spot urine detection of protein, glucose and blood using multistix urine testing strips-Medi Test Combi 10.
Procedure and Use: One reagent strip was removed from the container and the container immediately capped, minimizing the exposure of the remaining test strips to light and air. The strip was completely immersed with its reagent pads in the urine sample for two seconds and then removed immediately to avoid dissolving out the reagent pads. While removing the reagent strip the edge of the strip was wiped against the rim of the specimen container to remove excess urine.
The strip was held in a horizontal position to prevent possible cross contamination of chemicals located in adjacent reagent pads. The color change of reagent pads was compared to the corresponding color chart on the bottle label and the results read exactly after 60 seconds (Leukocytes after 90-120 seconds).
Outcome measures were:
1. Albumin-Creatinine-Ratio (ACR): ACR in the urine was assayed using the Bayer Micro albustix™ reagent strip [Batch number: 04960872]. A sterile urine bottle was given to each consenting subject to provide freshly voided spot mid-stream urine. This was tested with Micro albustix™ reagent strips according to the specified procedure. A
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stopwatch was used to time the readings of the albumin and creatinine tests. The albumin test was read at 50 seconds after dipping and the creatinine test at 60 seconds, after which the ACR was calculated. Micro albuminuria is a reliable marker of cardiovascular and chronic kidney disease, and is detected much earlier than an increase in serum creatinine.21 An albumin level of less than 30 mg albumin/g creatinine was regarded as representing a normal ACR. Micro albuminuria is indicated at a ratio of 30 to 300 mg/g (abnormal ACR), and clinically overt albuminuria/proteinuria at a ratio of > 300 mg/g was regarded as macroalbuminuria.21
2. Proteinuria: Defined as presence of ≥ +1 Protein (excretion of greater than 30mg/day) 3. Glycosuria: Defined as presence of glucose in the urine. The presence of more than 2+
(greater than 150mg/dl) is indicative of glycosuria.
4. Haematuria: Detected on dipstick testing of urine (microscopic haematuria). Red blood cells more than 2+ (greater than 50 erythrocytes per microlitre) is indicative of haematuria.
5. Hyperglycemia: Criteria for diagnosis was fasting blood glucose greater than 7 mmol/l or random blood glucose greater than 11.1 mmol/l.
6. Blood pressure: Blood pressures were taken in both the sitting and erect positions on the dominant arm of the patient using Accuson’s mercury sphygmomanometer, manufactured by A.C. Cossor and Sons Ltd, Accuson works, London, with an appropriate standard cuff of 12-13cm by 55cm size. Hypertension was diagnosed in those with systolic and or diastolic blood pressure of >140mmhg and >90 mmhg respectively.
7. Dyslipidemia: Quantitative determination of HDL- Cholesterol and triglycerides from plasma using the Roche Cobas C311 autoanalyzer was done. LDL and VLDL was then precipitated when the serum was combined with polythene glycol reagent. (PEG 6000).
Total cholesterol was considered to be high if it was > 5.17 mmol/l. LDLc was considered to be raised if it was >3.34 mmol/l, triglycerides was considered to be high if it was >1.7 mmol/l and HDLc was considered to be low if it was <1.03 mmol/l and <1.3 mmol/l in men and women, respectively.105
Serum Creatinine: the normal range of serum creatinine concentration is 0.8 to 1.3mg/dl ( 70 to 114 mmol/l ) in men and 0.6 to 1.0 mg/dl ( 53 to 88.5 mmol/l ) in women.103 Serum creatinine measurement was done using Kinetic method : Jaffe reaction
Serum creatinine measurement using Jaffe’s reaction method Reagent used: Picric acid and sodium hydroxide.
Procedure: 0.5ml blank (distilled water), a standard and 1ml of serum were pipetted into separate test tubes. 0.5ml of trichloroacetic acid was then added to the distilled water and the standard which was supplied in the kit. 1ml of the reagent was then added to each of the test tubes. The contents of the test tubes were mixed and allowed to stand at room temperature for 20 minutes. The absorbance of the standard (Ast) and sample (Asp) were measured against the distilled water using a colorimeter.
Serum creatinine (umol/l) = Asp 2 Ast
8. Serum Albumin: measurement was done with Roche Cobas C311 automated analyzer.
Hypoalbuminaemia was determined if the serum albumin was less than 35mg/dl.
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9. HIV seropositivity was determined by Double Enzyme Linked Immunosorbent Assay (ELISA) test. Determine HIV-1 and 2 kits, Abbot Laboratories, USA; and Immunocomb HIV-1 and 2 kits, Organics, Israel were used.
10. CD4+ T-lymphocyte quantification using cyflow: Principle: Using CD4+ monoclonal antibody to tag the CD4+ lymphocytes. The CD4+ cells are counted based on their
optical densities.
Procedure: 20µl CD4+ PE antibody, and 20µl well mixed whole blood (EDTA) were added into a partec test tube. They are mixed well and incubated for 15 minutes at room temperature. The sample was mixed every 5 minutes during the incubation. Then 0.8ml of CD4+ buffer was added into the partec test tube and the partec test tube was plugged to the cyflow machine for counting using the observed peaks generated on the screen and the CD4+ cells count was read.
11. Estimated glomerular filtration rate (eGFR) less than 60ml/min/1.73m2 was the criteria for diagnosis of CKD.The serum creatinine concentration of all the subjects was analyzed by using the Roche Cobas C311 analyzer. The value of serum creatinine obtained from the laboratory was used to calculate the glomerular filtration rate of each subject. The eGFR was calculated using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration)103:
The CKD-EPI equation, expressed as a single equation is:
eGFR =141 x min (SCr/k,1)a x max (SCr/k,1)-1.209 x 0.993 Age x (1.018 if female) x( 1.159 if Black) where SCr is serum creatinine (mg/dL), k is 0.7 for females and 0.9 for males, a is -0.329 for females and -0.411 for males, min indicates the minimum of SCr/k or 1 and max indicates the maximum of SCr/k or 1.103
Evidence of Chronic Kidney disease was defined if a subject had any of the following over a 3 month period 21:
1. At least +1 proteinuria or Subjects with abnormal ACR > 30mg/g 2. eGFR of <60ml/min/1.73m2
All patients with abnormal ACR > 30mg/g, proteinuria (≥1+) on dipstick and those with estimated GFR< 60 mls/min had their urinalysis and eGFR repeated respectively after 3 months to establish chronicity and determine the prevalence of CKD.
3.7 Data analysis
Statistical analysis was performed using a computer software SPSS version 20.The data from each participant was expressed using descriptive statistics and percentages. Quantitative data were presented using means and standard deviations while qualitative variables were presented as percentages. Student t and Chi square tests were used to compare means and proportions respectively. The above analysis was followed by a multivariate logistic regression analysis, where the potential predictor variables (risk factors) identified from the initial univariate analysis (t-tests, chi-square) was included in a logistic regression model to determine the variables that are associated independently with CKD in HIV positive patients. p-values of <0.05 were taken as statistically significant.
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CHAPTER FOUR
RESULTS