Patients were followed for at least 1 year after initiation of therapy (Table 1). CD4 cells were isolated from 10 mL of whole blood [collected from patients in ethylenediaminetetraacetic acid (EDTA)] by an immunomagnetic method using anti-CD4 coated magnetic beads (Dynabeads M450 CD4; Dynal AS, Oslo, Norway) according to the manufacturer’s protocol and were stored at −80 °C until

use. The purity of the CD4 cell preparation was approximately 99% as estimated using Becton Dickinson FACScan Flow Cytometer technology (Franklin Lakes, New Jersey, USA) (data not shown). The 8E5 cell line was cultured in RPMI-1640 medium and the GDC-0973 chemical structure cells were counted using a Coulter automated haematology analyser, diluted to 106 cells per aliquot and stored at −80 °C. The 8E5 cell-free virus present in the culture supernatant was diluted and stored in aliquots of 30 000 HIV-1 RNA copies/mL. The HIV-1 RNA plasma viral load was assessed using the Versant HIV-1 RNA 3.0 assay (Siemens Medical

Diagnostics Solutions, Tarrytown, NY, USA) according to the manufacturer’s instructions. This assay is based on branched DNA (bDNA) technology. It requires 1 mL of sample and has a dynamic range of 50–500 000 copies/mL. Plasma samples and the 8E5 cell culture supernatant were frozen at −80 °C until tested for HIV-1 RNA viral load. A viral load result of <50 copies/mL was regarded as 50 copies/mL when calculating the mean viral load of a given patient group. DNA was extracted from purified patient CD4 cells diluted in 200 μL of phosphate-buffered Selleckchem CYC202 saline (PBS), using the High Pure® PCR Template Preparation

Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s recommendations. Erastin To concentrate the HIV-1 target gene in patient samples, DNA was eluted in a volume of 50 μL. DNA from 106 T-lymphoblastoid 8E5 cells, used as positive control, was eluted in 200 μL. A negative control (in which the template was replaced with nuclease-free water) was included in each polymerase chain reaction (PCR) run. Particular attention was paid to using DNAse- and RNAse-free materials. Depending on the number of samples, the whole DNA purification process required approximately 1 h. HIV RNA was extracted from plasma and from the 8E5 cell culture supernatant using the QIAmp Viral Mini Kit™ (Qiagen, Leiden, the Netherlands) according to the manufacturer’s directions. Viral RNA and proviral DNA genotypic antiretroviral drug resistance mutations were identified using an in-house reverse transcriptase–polymerase chain reaction (RT-PCR) method applied to the RT and PR genes (adapted from Schmit et al. [37]). Direct cycle sequencing with BigDye terminator chemistry was carried out on the ABI Prism 310 sequencer (Applied Biosystems). In cases of PCR failure, samples were analysed using a TRUGENE HIV-1 Genotyping Kit (Siemens Medical Diagnostics Solutions).