We performed a secondary analysis of stored samples collected from HIV-1 infected patients enrolled in two large multi-centre, open-label clinical trials prospectively evaluating the efficacy and toxicities associated with approved ART regimens available through the China National Free AIDS Treatment Program respectively from 2008 to 2010 (cohort 1, NCT00872417) and 2012-2014 (cohort 4, NCT01844297). The studies were carried out by the China AIDS Clinical Trial Network in 11 different municipalities/provinces in China, including Beijing, Fujian, Guangdong, Guangxi, Henan, Hunan, Liaoning, Shanghai, Shanxi, Sichuan, and Yunnan provinces. The inclusion and exclusion criteria and study-related procedures of these clinical trials have been previously described in detail [18-20].
The additional criteria for patient inclusion in the present study were as follows: completed at least 96 weeks of follow up with HIV-1 RNA levels maintained ≤ 50 copies/mL after 48 weeks of ART, 12 patients (10 in CRF01_AE group, 2 in non-CRP01_AE group) experienced viral blips (HIV-1 RNA ≤ 200 copies/mL) were allowed in this study.
As part of the parent studies, sociodemographic and clinical data including sex, age, ethnicity, route of transmission, marital status, and smoking history were collected at the time of study enrolment. Baseline CD4+ and CD8+ T cell counts, HIV-1 RNA load, and HIV-1 DNA level were also measured for all participants. Follow-up evaluations were performed at weeks 0/12/24/48/96. Peripheral blood samples (plasma, whole blood) were collected at each time point and stored at -80℃.
HIV-1 nucleic acid quantification
HIV-1 total DNA was extracted from the peripheral blood by using MagNA Pure LC DNA Isolation Kit and MagNA Pure LC instrument (Roche Molecular Biochemicals, Mannheim, Germany), amplified and quantified using LTR gene primers with a real-time fluorescence-based HIV detection kit (SUPBIO, Guangzhou, China) ,the reaction system contains: reaction mixture (44.2μL), enzyme (0.8μL), HIV-1 DNA (5μL). Finally, HIV-1 DNA per 106 PBMCs was calculated by dividing the proportion of lymphocytes and monocytes in the routine complete blood count. The quantification range for HIV-1 DNA using this assay was 20 to 5,000,000 copies/106 WBCs.
HIV-1 RNA was extracted from plasma using the QIAamp RNA mini kit (QIAGEN, Hilden, Germany) and then amplified using the COBAS Ampliprep/TaqMan48 real-time RT-PCR Test (Roche Diagnostics, Indianapolis, Indiana, USA). The detection range of HIV-1 RNA was 40 to 10, 000, 000 copies/mL.
HIV-1 subtype analysis
The HIV Pol gene was amplified with PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China) and then sequenced. PCR primers used for sequencing have been reported previously . Subtypes of HIV-1 were then determined through the Recombinant Identification Program (http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html) and confirmed by neighbour joining phylogenetic analysis via sequence alignment of the Pol gene with reference sequences from the Los Alamos National Laboratory (http://www.hiv.lanl.gov/content/index). In order to further confirm the HIV-1 subtype, sequencing of the V3 loop was also carried out in patients (n = 76) preliminarily determined by Pol gene sequencing.
The patients’ baseline clinical and demographic characteristics were compared between HIV-1 subtype groups using the Student’s t-test for parametric continuous variables, and Wilcoxon Rank Sum test for non-parametric continuous variables.
Patients were classified into 3 categories according to their HIV-1 DNA levels at 96 weeks (high: ≥ 3 log10, moderate: 2-3 log10, and low: ≤ 2 log10 copies/106 PBMCs). Differences in the proportion of patients in each group by subtype CRF01_AE and non-CRF01_AE were analysed by Chi squared analysis. Univariate and multivariable logistic regression using the forward entry method to assess the correlation between HIV-1 total DNA level at 96 weeks (≤ 2 log10 copies/106 PBMCs). Variables for which P < 0.2 in the univariate analyses were included in the subsequent multivariable regression analysis.
All analyses were performed using SPSS version 19.0 (IBM Corporation, Armonk, New York, USA) and GraphPad Prism 6.0 (GraphPad Software, Inc. La Jolla, CA, USA). P values < 0.05 were considered statistically significant.