We conducted a nested case–control study utilizing data from a long term observational study (Clinical trial.gov, NCT 00411983) for HIV-infected individuals who had previously participated in clinical studies and continued into a long term cohort after the completion of the specific study. The study participants were scheduled for follow-up at The HIV Netherlands Australia Thailand Research Collaboration or HIV-NAT clinic in Bangkok, Thailand on a semi-annual basis to monitor their long-term outcomes for AIDS-defining and non-AIDS defining illnesses as well as adverse events from ARV treatment. Medical history, physical examination and history of ARV treatment were recorded. Subjects underwent blood draw for complete blood count (CBC), serum alanine aminotransferase level (ALT) and serum creatinine level. CD4+ T lymphocyte counts and HIV-1 RNA levels were also measured at each study visit.
Our study focused on liver complications in HIV without hepatitis B or C co-infection. The inclusion criteria were HIV-infected individuals, older than 18 years of age, negative hepatitis B surface antigen (HBs Ag) or HBV DNA and negative HCV antibody (anti-HCV) or HCV RNA. Exclusion criteria were patients who did not have a recorded viral hepatitis serology result, had an abnormal ALT level at their baseline visit or prior to ARV treatment (ALT ≥40 IU/L), unavailable baseline ALT level or had less than 12 months of follow up. Written, informed consent was obtained from all enrollees. This specific study was approved by the Ethics Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
For this case–control study, the cases, or chronic hepatitis was defined as those with elevated ALT ≥ 40 IU/L at 2 consecutive visits 6 months apart after the initiation of ARV [7, 8]. The controls was defined as those patients who never had two consecutive ALT ≥ 40 IU/L and still had normal ALT at the last visit. Both groups had normal ALT at pre-ARV initiation (baseline).
The case and control were matched 1:1 for duration from ARV initiation (± 6 months).
All clinical data and laboratory testing for comparison were selected by utilizing data at baseline and at the time chronic hepatitis occurred in the cases and the data at the same period (± 6 months) for the matched controls.
All analytic data for this study were censored on July 1, 2012.
The duration of the study was calculated from the time of initiation of ARV to the last follow-up visit.
The onset of chronic hepatitis was calculated from the time of ARV initiation to the diagnosis of chronic hepatitis.
HIV infection was defined by a positive result for HIV-specific antibodies by enzyme-linked immunosorbent assay (ELISA) and/or HIV-1 RNA by the Roche Amplicor HIV-1 Monitor Test v1.5.
Hepatitis B virus infection was defined by a positive result for HBsAg using the ARCHITECT HBsAg qualitative assay (ABBOTT Max-Planck-Ring 2, Germany) and/or detectable HBV DNA by polymerase chain reaction (PCR).
Hepatitis C virus infection was defined by a positive result for HCV-specific antibodies using the ARCHITECT Anti-HCV assay or detectable HCV RNA by PCR.
Severity grading of chronic hepatitis was classified according to the following ranges of ALT level: grade 1, 1.0 – 2.5 times the upper limit of normal (1.0-2.5 × ULN); grade 2, 2.6–5.0 × ULN; grade 3, 5.1–10 × ULN; grade 4, >10 × ULN. Severe hepatitis was defined by at least grade 3 ALT elevation .
Severity of HIV infection was classified by Centers for Disease Control and Prevention (CDC) 1993 guidelines .
Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. BMI categories specific for Asian individuals were assigned as follows: BMI <18.5 (underweight), BMI = 18.5 – 22.9 (normal), BMI = 23–24.9 (overweight) and BMI ≥25.0 (obese) .
Dyslipidemia was defined as serum triglyceride level ≥ 150 mg/dL, HDL-cholesterol ≤ 40 mg/dL for males, ≤ 50 mg/dL for females, LDL-cholesterol ≥ 130 mg/dL and total cholesterol ≥200 mg/dL [15, 16].
Elevated blood pressure was defined as a systolic blood pressure > 130 mmHg or diastolic blood pressure > 85 mmHg, or known history of hypertension .
Impaired fasting plasma glucose was defined as a fasting plasma glucose ≥ 100 mg/dL or known history of diabetes mellitus .
Metabolic syndrome was defined as having at least 3 of 5 criteria serum triglyceride level ≥150 mg/dL, serum HDL-cholesterol ≤40 mg/dL for male or ≤50 mg/dL for females, elevated blood pressure, impaired fasting plasma glucose and BMI ≥ 23 kg/m2 or waist circumference ≥ 90 cm. for male or ≥ 80 cm. for females .
Clinically-diagnosed lipodystrophy was defined as those with lipoatrophy or lipohypertrophy, truncal obesity or facial lipodystrophy .
The main risk factors that were evaluated included clinical characteristics [age, gender, sexual risk behavior, HIV-related illness and disease severity, baseline CD4+ T lymphocyte counts, plasma HIV-1 RNA level and metabolic diseases] and clinical parameters and laboratory assessments at an event visit [previous history of ARV treatment, concomitant medications, clinical and laboratory findings of HIV-related illnesses and metabolic diseases].
Statistical and data analysis
Clinical characteristics were described as frequency and percentage for categological data. Continuous data were reported as mean (x̄) and standard deviation (SD) if normally distributed or as median and inter-quartile range (IQR) if not normally distributed . For inferential data analysis, the McNemar’s test was used for categorical data. For continuous data, the paired student’s t-test was used if the data had normal distribution, while the Wilcoxon signed-rank test was used if they showed non-normal distribution. Factors with p value less than 0.2 from univariate analysis were selected to the model of multivariate analysis. Conditional stepwise, logistic regression was used to evaluate the risk factors of chronic hepatitis. Odds ratio (OR) and adjusted OR with 95% confidence intervals (CI) were reported to demonstrate an association between significant factors with chronic hepatitis. Statistical significance was defined as a p value less than 0.05. We used STATA/LC version 11.2 for Windows.