Study design and patient enrollment
We conducted a prospective single centre study in the Department of Cardiology at Chris Hani Baragwanth Hospital, Soweto, South Africa. The protocol was approved by the ethics committee of the University of the Witwatersrand and adheres to the Declaration of Helsinki. All patients gave informed consent before study entry. Between March 2004 and February 2008, 30 consecutive black HIV patients presenting with ACS (ACS+: HIV+ group) were enrolled. For each HIV patient with ACS we selected the first presenting non-HIV black patient with ACS as a case-control comparator (ACS+: HIV- group). In addition a second control group consisting of 30 asymptomatic HIV patients matched for age, sex and ethnicity (ACS-: HIV+ group) were recruited from the HIV clinic. ACS was defined as either ST-elevation myocardial infarction, non ST-elevation myocardial infarction or unstable angina. Patients were categorized as having diabetes, hypertension or dyslipidemia when being treated chronically for these conditions or when diagnosed with the condition on admission. Patients were classified as having "other" coronary risk factors if any of the following conditions were present: i) Family history of premature CAD (men < 55 yrs, women < 65 yrs), ii) chronic kidney disease, iii) post menopausal state and iv) abdominal obesity (abdominal circumference > 102 cm in men and 88 cm in women). Demographic data was recorded for each patient and anthropometric measurements including weight, height, body mass index (BMI), waist to hip ratio and abdominal circumference (AC) were measured on admission according to guidelines set out in the Interheart study [8]. Infection with HIV was diagnosed with a standard enzyme linked immunosorbent assay and Western blot techniques after obtaining consent and offering pretest counseling. In the HIV group, Plasma HIV RNA level was determined by quantitative polymerase chain reaction. CD4 count was determined by flow cytometry and patients were staged according to the CDC staging system [9]. Patients with ACS were managed according to accepted guidelines set out by the European Heart Association [10, 11] and followed up at the Chris Hani Baragwanath cardiac clinic.
Laboratory methods
Blood was obtained by clean venipuncture with seated subjects and non-fasting venous blood was drawn into plastic tubes and allowed to clot at 37 degrees and then cetrifuged at 2500 × g for 8 minutes for sera preparation. The serum was immediately stored at -80 degrees until use. All sera were thawed only once in a water bath at 37 degrees for 15 minutes before testing. The analysis of aPL was performed at Lancet Laboratories, Johannesburg. Antiphospholipid antibodies including aCL, anti- β2-GP-1, and aPT (IgG, IgM, IgA) were measured at baseline in all patients and at least 12 weeks later in the ACS groups using commercial enzyme linked immunosorbent assay (ELISA) kits (Orgentec Diagnostika, Mainz, Germany). The results were expressed in units, according to the manufacturer's instructions. IgG, IgM and IgA aCL were expressed as U/mL. To establish normal values of aPL in our population we used the blood samples of 100 asymptomatic HIV negative black South African blood donors matched for age and sex to the study population. Results for each of the aPL measured were considered positive when the optical density obtained for each patient exceeded that of the mean value plus 2 standard deviations (SD) of the 100 sera from black South African normal healthy subjects. The normal values obtained were as follows: aCL IgG and IgA < 10 U/mL, IgM < 7 U/mL; anti- β2-GP-1 IgG, IgA, IgM < 8 U/mL; aPT IgG, IgA, IgM < 10 U/mL. The antiphospholipid syndrome (APS) was diagnosed in patients with ACS who had the presence of aPL (aCL IgG/IgM and/or anti- β2-GP-1 IgG/IgM) in titres greater than the mean plus 2SD from normal healthy subjects on two occasions at least 12 weeks apart [12].
Stastistical analysis
Statistical analysis was performed using SAS 9.1 software (SAS, Cary, NC, USA). Normally distributed continuous data are presented as the mean (± standard deviation), and variables with non-Gaussian distribution as the median (min-max range). Categorical data are presented as frequencies and percentages. The initial analysis compared variables between the 3 groups using the one way anova test for continuous variables with normal distribution and the Kruskal Wallis test in case of non-normal distribution. For categorical variables the Chi square test was performed with a Fisher exact test when necessary. Significant differences between variables in the 3 groups was assumed at p < 0.05. Subgroup analysis with multiple pairwise comparisons was then performed applying the Bonferroni correction with a p value < 0.0166 considered significant. Univariate logistic regression was performed to determine predictors of the variables: aCL IgG, aPT IgG and APS: data presented as odds ratios (OR) with 95% confidence intervals (CI).