The study design was a matched case control study of adults aged 18 years and above at the University Teaching Hospital (UTH) in Lusaka, Zambia. The target population was adult patients seen at UTH either as in-patients or out-patients via Adult Medical Emergency Unit (AMEU), Clinic five, Adult Infectious Diseases Centre (AIDC), and Physiotherapy departments.
A total of 52 HIV + ve patients with ischaemic stroke were prospectively compared with control groups for occurrence of PS, PC deficiencies and hyperhomocysteinaemia. The definitions of PS, PC deficiencies and hyperhomocysteinaemia were as recommended by the International Federation of Clinical Chemistry (IFCC) . The control groups comprised an equal number of consecutive matched for race, sex and age at +/− 5 years interval HIV negative (HIV-ve) and HIV + ve patients with and without ischaemic stroke respectively. Ischaemic stroke was confirmed by clinical assessment and brain imaging [computer tomography (CT) scan or magnetic resonance (MRI)]. Patient recruitment was done after the acute phase of stroke with stroke duration more than 48 h, but up to 1 month and the recruitment period was July 2014 to February 2015.
Hypertension was defined in this study as current use of antihypertensive medication, history of being diagnosed as hypertensive by a doctor prior to stroke, documented blood pressure of greater than or equal to 140 mmHg systolic or 90 mmHg diastolic before the stroke or persisting more than a week after the acute event (World Health Organization) or evidence of left ventricular hypertrophy on ECG or Echocardiography.
Diabetes mellitus was diagnosed if a patient was taking anti-diabetic drugs prior to stroke; if a doctor had diagnosed type I or type II diabetes before stroke; if a patient had a documented non fasting blood glucose of greater than 11.1 mmol/L, or a fasting blood glucose of greater than or equal to 7.0 mmol/L after the acute phase of stroke.
Cigarette smoking was classified as smoker (current or former smoker for more than 1 year) or non smoker, and alcohol consumption as non drinker or drinker (ex-drinker for more than 1 year or current alcohol use).
Laboratory investigations such as Full Blood Count (FBC) measured using FBC SYSMEX 2000 and 4000, CD4 count measured using FACS Calibur, Liver Function Tests (LFTs), cholesterol, urea and creatinine measured using Beckman Coulter AU480 were done at UTH as part of all patient routine work up. The HIV antibody tests were done on all recruited patients using a combination of Determine and Bioline test kits, with the Unigold kit as a tie-breaker. For PS, PC deficiencies and hyperhomocysteinaemia, venous blood sample was collected and immediately transported on ice at a temperature of 2 – 8 Degrees Celsius to a private lab (Lancet-Nkanza Laboratory) for separation of plasma and cellular components before the samples were flown to Lancet Laboratory in Johannesburg, South Africa, for analysis using Hemosil™ immunoassays.
The examination included a detailed neurological assessment with measurement of blood pressure, and assessment for any evidence of vascular disease including hypertensive end-organ damage affecting the heart or fundal vessels and any potential sources of emboli. Assessment for signs of focal neurological deficit (hemiparesis, hemi-sensory loss, cranial nerves palsy, aphasia, level of consciousness, alexia, agraphia, and apraxia) was done together with the patient’s National Institutes of Health Stroke Scale (NIHSS) at the time of examination. Patients were reviewed with all data collection sheets, scans and other investigations, and assigned a final diagnosis plus stroke type using the Trial of Org 10,172 in acute stroke treatment (TOAST) classification. Instances where no cause of ischaemic stroke was found because some investigations had not been done before the patient died or the patient had no funds to pay for some investigations which were not covered by the study were classified as incomplete evaluation under TOAST classification [16, 17].
Patients with haemorrhagic stroke, Sickle Cell Disease (SCD), no neuro-imaging or focal neurological deficit of non-vascular origin were excluded from the study. In addition, taking anticoagulant drugs, contraceptive pills, hormone replacement therapy (HRT), pregnancy, liver disease and refusal to do HIV test were part of the exclusion criteria.
The sample size was calculated by first making the assumption that 19% of all HIV + ve ischaemic strokes were due to coagulopathies as reported by Tipping et al.  Then, the occurrence of coagulopathies in the general population was estimated at 4% taking into account Remkova’s postulation that hereditary deficiencies of Antithrombin III, PC or PS can be found in fewer than 5% of unselected patients . The confidence interval was set at 95% and the power to detect a difference at 80%. There were three arms with the case being HIV + ve ischaemic stroke and two controls – one control being HIV-ve ischaemic stroke and the other being HIV + ve no stroke. A total of 51 subjects were required in each of the three arms.
All the data was entered and analysed on Statistical Package for the Social Sciences (SPSS) statistics 2012 version 21. Data were analysed in contingency tables using Paired t test for means and McNemar’s Chi square test for proportions. Conditional logistic regression step-down model was used to measure for association. The variables for logistic regression were selected using biological plausibility on the basis of association with hypercoagulability state and also variables with significant p values after comparison between cases and controls. For the conditional logistic regression analysis of HIV + ve compared to HIV-ve ischaemic stroke, the selected variables included age, hypertension, obesity, hyperhomocysteinaemia, PS deficiency, smoking, previous ischaemic stroke/TIA and major risk factor for stroke. For ischaemic stroke compared to no stroke HIV + ve patients, PC deficiency, age, alcohol, ART use, hypertension and Diabetes Mellitus were the variables used for step-down analysis. A p-value of less than 0.05 was taken as the level of statistical significance.
Ethical approval was obtained from ERES Converge International Research Board (IRB) (ref. No. 2013 – Dec – 009) and permission was obtained from UTH management to conduct the study. Written informed consent to participate in the study as well as for publication was obtained from each of the study participants. In the event that the participant had altered mental status, informed consent was obtained from the surrogate who was a care-giver and close relative of the participant.