In order to assess for sex differences in immunologic, virologic, anthropometric, and other responses to antiretroviral regimens, we performed a secondary analysis of data collected as part of Neverest 2, a clinical trial (ClinicalTrials.gov: NCT00117728) assessing the reuse of nevirapine in nevirapine-exposed HIV-infected children conducted from 2005 to 2010 [24, 25]. Children previously exposed to single-dose nevirapine prophylaxis at birth, and <24 months of age at the time of initiation of LPV/r-based ART, were identified at Rahima Moosa Mother and Child Hospital in Johannesburg, South Africa. Those who achieved and sustained plasma HIV-1 RNA <400 copies/mL for ≥3 months during the first 12 months of treatment were eligible for randomization to either continue on LPV/r or switch to nevirapine-based ART in combination with stavudine and lamivudine. Specific treatment regimens have been previously reported [24, 25]. Children were followed for 76 weeks post-randomization and then re-enrolled in extended follow up. In this analysis, we evaluated sex differences pretreatment, in the pre-randomization phase, at the time of randomization, at post-randomization study visits, and at participants’ final visit. This study was approved by the Institutional Review Boards of Columbia University (New York, New York) and the University of the Witwatersrand (Johannesburg, South Africa). Informed consent was provided by each child’s parent or guardian.
Socio-demographic information, medical history, weight (kg), height (cm), and blood samples (for CD4 T-cell determination and HIV-1 RNA quantity) were collected at a pretreatment visit. Subsequent visits were scheduled at 2, 4, 8, 12, and every 12 weeks thereafter up to 52 weeks until randomization and at 2, 4, 8, 16, 24, 36, 52, 64, 76 and every 12 weeks thereafter. Blood samples for plasma viral load tests by quantitative HIV-1 RNA PCR (Roche, Cobas Ampliprep Taqman V2, Branchburg, NJ) were collected at 4, 16, 24, 36, 52, 64, and 76 weeks post-randomization and for absolute CD4 count and percentages (Beckman Coulter Flow Analyzer) at 16, 24, 36, 52, 64, and 76 weeks post-randomization, and for both tests at every 12 weeks thereafter until the final study visit. Lopinavir, ritonavir, and nevirapine plasma concentrations were determined using validated LC-MS/MS assays developed in the Division of Clinical Pharmacology, Cape Town, South Africa. An AB Sciex 4000 mass spectrometer was operated at unit resolution in the multiple reaction monitoring mode. The assays were validated over the concentration range of 0.16-20 μg/ml for lopinavir, 0.10-20 μg/ml for nevirapine, and 0.04-5 μg/ml for ritonavir.
Non-fasting total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides in mg/dL were measured at pretreatment, time of randomization, and 36, 88, and 136 weeks post randomization (Roche, Cobas Integra 400, Branchburg, NJ). At the final study visit following an overnight fast, total cholesterol, LDL, HDL, triglycerides, C-reactive protein in mg/L, and insulin in mg/dL were measured. Venous blood glucose in mg/dL was measured with a handheld glucometer and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated . Classification of biochemical results has been described previously . Assessments for drug-related rashes and hepatotoxicity by alanine aminotransferase (ALT) were performed at post-randomization visits [24, 25].
Weight (kg) and height (cm) were measured using a digital scale and stadiometer, respectively. Weight-for-age (WAZ) and height-for-age z-scores (HAZ) were calculated using World Health Organization growth standards . Underweight was defined as WAZ < −2 and stunting was defined as HAZ < −2. Circumferences at the mid-upper arm, mid-upper thigh, mid-waist, and maximum hip were measured using a flexible tape measure with a spring tension attachment. Bicep, tricep, subscapular, suprailiac, umbilical, and mid-thigh skinfolds were measured with a Harpendon caliper (Baty International, England). As previously reported, total body fat percent (%BF) was estimated by single-frequency bioimpedance analysis (BIA) (Quantum II, RJL Systems, Clinton Township, MI)  and children were classified as having lipodystrophy, possible lipodystrophy, or no signs of lipodystrophy by two physicians (RS, LM) .
All children with HIV-1 RNA >1000 copies/mL were recalled and retested within 4 weeks. Additional counseling was provided if adherence difficulties were detected. Children in the switch group were returned to the LPV/r-based regimen if HIV-1 RNA remained >1000 copies/mL despite enhanced adherence counseling. To evaluate adherence, caregivers were asked to return drug at all scheduled visits. Percentage of medication return was calculated and poor adherence was defined as 20% greater than expected medication return for each drug.