Roche Diagnostics) could potentially improve genotypic resistance assessments because it detects the same range of mutants than Sanger viral population sequencing, but with higher sensitivity [2]. Studies have shown that pre-existing minority drug-resistant mutants increase the risk of virological failure to first-line antiretroviral therapy with non-nucleoside reverse transcriptase inhibitors (NNRTIs) [3,4,5,6,7]. Conversely, low-frequency drug- resistant variants do not affect virological outcomes of first-line therapy including drugs with high genetic barrier, like ritonavir- boosted protease inhibitors (PIr) [8]. Whereas most studies addressing the role of minority variants have been performed in antiretroviral-na¨ıve subjects [2,3,4,5,6,7,9,10,11,12,13], less infor- mation exists on the clinical significance of minority mutants in antiretroviral-experienced individuals. [14,15,16] It is particularly uncertain if ultrasensitive genotypic tests could provide clinically relevant information in heavily pre-treated HIV- 1-infected subjects beyond that provided by standard population sequencing genotypic tests. On one hand, detection of additional minority drug-resistant mutants could improve the assessment of viral susceptibility to drugs with intermediate or high drug genetic barrier. On the other hand, the presence of extensive drug resistance could compromise the ability of deep sequencing to add relevant genotypic information to that already obtained with population sequencing, particularly when alternative treatment options are severely limited. Moreover, mutant fixation during virological failure in the presence of ART pressure could potentially complicate the detection of additional low-frequency variants [16]. We therefore sought to explore the potential of deep sequencing to provide additional, clinically relevant genotypic information that could be used to improve treatment decisions in heavily pre- treated HIV-1-infected subjects, relative to population sequencing. Methods Subjects This proof-of-concept observational study included HIV-1- infected adults with previous virological failure (VF) to protease inhibitors (PIs), nucleos(t)ide (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), who developed virological failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Virological failure was defined as the presence of HIV-1 RNA levels .200 copies/mL 24 weeks after salvage therapy initiation or beyond. Adherence was self- reported by the patient and collected from medical charts. Adequate adherence was defined as intake of all medication doses. Partial adherence was defined as the presence of missed doses during treatment. Treatment interruption meant the complete interruption of therapy during follow-up. The Institu- tional Review Board of the Hospital Universitari Germans Trias i Pujol, Badalona, Spain, approved the study; participants provided written informed consent for retrospective sample testing. HIV RNA extraction and reverse transcription HIV-1 RNA was extracted from 1 mL of plasma within 4 weeks before initiation of deep salvage therapy (baseline) and at virological failure (QIAamp UltraSens Virus KitTM, QIAGEN, Valencia, CA). Three One-Step RT-PCRs (SuperScriptH III One-Step RT-PCR System with PlatinumH Taq High Fidelity, Invitrogen, Carlsbad, CA) were performed in parallel per each sample. Primers used were 1571-L23 (HXB2: 1417R1440) 59-ATT TCT CCT ACT GGG ATA GGT GG-39 and 5464-L27 (HXB2: 5464R5438) 59-CCT TGT TAT GTC CTG CTT GAT ATT CAC-39. Cycling conditions were: 30 min. at 52uC, 2 min. at 94uC; 30 sec. at 94uC, 30 sec. at 56uC, 4 min. at 68uC, for 20 cycles; followed by 5 454 in Heavily Pre-Treated HIV+ Subjects min. at 68uC. Triplicate RT-PCR products were pooled, column- purified (QIAquick PCR Purification Kit, QIAGEN, Valencia, CA) and used for both viral population and deep HIV-1 sequencing. Viral population genotyping Triplicate nested PCRs were performed in parallel (PlatinumH Taq DNA Polymerase High Fidelity, Invitrogen, Carlsbad, CA) with primers 2084-U26 (HXB2: 2084R2109) 59-ATT TTT TAG GGA AGA TCT GGC CTT CC-39 and 5456-L26 (HXB2: 5456R5431) 59-TGT CCT GCT TGA TAT TCA CAC CTA GG-39. Cycling conditions were: 2 min. at 94uC; 30 sec. at 94uC, 30 sec. at 56uC, 4 min. at 68uC, for 20 cycles; followed by 5 min. at 68uC. Nested PCR products were pooled and column-purified. Protease (PR), reverse transcriptase (RT) and integrase (IN) were sequenced in-house (BigDye v3.1, Applied Biosystems, Foster City, CA, USA) and resolved by capillary electrophoresis (ABI 7000, Foster City, CA, USA). Deep HIV-1 sequencing Pooled purified RT-PCR products were used as template to generate eight overlapping amplicons covering PR and RT and 3 amplicons covering codons 51 to 215 in IN. Each codon was interrogated by at least 2 independent amplicons, which were generated in triplicate during 20 cycles of PCR amplification (PlatinumH Taq DNA Polymerase High Fidelity, Invitrogen, Carlsbad, CA) followed by pooling and purification of triplicate PCR products (QIAquick PCR Purification Kit, QIAGEN, Valencia, CA). PCR reactions were pooled and purified using the Agencourt AMPure Kit (Beckman Coulter, Benried, Germany) to eliminate primer-dimers. The number of molecules was quantified by fluorometry (Quant-iT PicoGreen dsDNA assay kit, Invitrogen, Carlsbad, CA). When amplicon concentrations were below 5 ng/ mL, amplicon size and primer-dimer content were analysed by spectrometry using BioAnalyzer (Agilent Technologies Inc., Santa Clara, CA). Emulsion PCR was carried out as in [17]. Amplicon Sequencing was performed at 454 Life Sciences, Branford, Connecticut, USA, using a Genome Sequencer FLX. Sequences were extracted and aligned against a consensus sequence from all the reads for each sequences region. Clonal sequences resulting with ,90% similarity with the consensus sequence were discarded. Sequencing errors in homopolymeric regions were manually inspected and corrected. Sequences that included a stop codon were removed. Unique sequences (haplotypes) were identified and quantified. The frequency of mutants with a Stanford HIV Database [18] score . 5 was determined. Using a conservative approach, only mutations present in $ 1% of the virus population where considered in this study. An in-house analysis of 992 pNL43 clonal sequences obtained by deep sequencing under the same PCR conditions used to generate patient samples showed a mean (SD) nucleotide mismatch rate of 0.07% (0.13%), which is almost identical to previous reports [14] and corresponds to a variability rate of 1.69610-5, within the range of the expected PCR error. The mean (SD) error rate for any aminoacid mismatch was 0.14% (0.19%), but the mean (SD) error rate for actual drug resistance mutations was 0.03% (0.07%). The 99th percentile of mismatches would establish the threshold for nucleotide errors in 0.61% and the limit for identifying true drug resistance mutations in 0.20%. Assessment of discrepancies between population and deep sequencing Genotypes were interpreted with the HIVdb v6.0.8 [18], Rega v8.0.2 [19] and ANRS v18 [20] algorithms. Only mutations with a PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19461