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Objective: Drug-resistance mutations are routinely detected using standard Sanger sequencing, which does not detect minor variants with a frequency below 20%. The impact of detecting minor variants generated by ultra-deep sequencing (UDS) on HIV drug-resistance interpretations has not yet been studied., Design: Fifty HIV-1 patients who experienced virological failure were included in this retrospective study., Methods: The HIV-1 UDS protocol allowed the detection and quantification of HIV-1 protease and reverse transcriptase variants related to genotypes A, B, C, F and G. DeepChek-HIV simplified drug-resistance interpretation software was used to compare Sanger sequencing and UDS., Results: The total time required for the UDS protocol was found to be approximately three times longer than Sanger sequencing with equivalent reagent costs. UDS detected all of the mutations found by population sequencing and identified additional resistance variants in all patients. An analysis of drug resistance revealed a total of 643 and 224 clinically relevant mutations by UDS and Sanger sequencing, respectively. Three resistance mutations with more than 20% prevalence were detected solely by UDS: A98S (23%), E138A (21%) and V179I (25%). A significant difference in the drug-resistance interpretations for 19 antiretroviral drugs was observed between the UDS and Sanger sequencing methods. Y181C and T215Y were the most frequent mutations associated with interpretation differences., Conclusion: A combination of UDS and DeepChek software for the interpretation of drug resistance results would help clinicians provide suitable treatments. A cut-off of 1% allowed a better characterization of the viral population by identifying additional resistance mutations and improving the drug-resistance interpretation.

The objective of this study was to assess the analytical and clinical relevance of minority variants using a new pyrosequencing (PSQ) assay and to detect minor variants with frequencies below the current 20% clinical setting limit. A PSQ approach for detecting and quantifying mutations was developed for the analysis of 14 codons of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene below the limit of conventional sequencing. Ten patients who experienced virological failure (VF) after a first-line regimen of lamivudine, tenofovir, and either efavirenz, nevirapine, or etravirine, as well as 10 controls patients without VF, were included in this retrospective study. Baseline plasma and plasma from the time of VF were assessed using Sanger sequencing and PSQ methods. The analytical sensitivity for the detection of minor sequence variants is 5%. At baseline, no minority variant was detected in 10/10 patient controls using both the Sanger sequencing and PSQ assays, whereas, two patients who failed therapy had baseline non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations that were not detected by the standard genotyping. At the time of VF, standard genotyping detected mutations in four out of the 10 VF patients, whereas, PSQ detected mutations in five out of the 10 VF patients. Clinically, minority mutations at a 10% level of detection can be assessed efficiently by pyrosequencing and used as a suitable predictor of the evolution of viral populations. These traits allow for a better interpretation of data analysis, which can help clinicians in providing a suitable treatment for HIV., (© 2013 Wiley Periodicals, Inc.)

Background: The risk of male-to-female intravaginal HIV-1 transmission is estimated at about 1 event per 200-2000 coital acts. The aim of this study was to assess the residual risk of HIV presence in semen in patients under HAART therapy., Methods and Findings: The study took place in France from October 2001 to March 2009. 394 paired blood and semen samples were provided from 332 HIV-1 infected men. The Roche Cobas AMPLICOR Monitor HIV assay was used to quantify HIV-1 RNA in blood and in seminal plasma. Three percent of 394 HIV-1 infected men enrolled in an assisted reproductive technology program harbored detectable HIV-1 RNA in semen, although they had no other sexually transmitted disease and their blood viral load was undetectable for at least 6 months under antiretroviral treatment., Conclusion: These data suggest that undetectable plasma HIV RNA means a lower risk of viral transmission through seminal fluid on a population level, but not necessarily at the level of the individual.

Objective: To monitor the disappearance of resistance-associated mutations and reappearance of wild-type (WT) virus during structured treatment interruptions (STI) using DNA sequencing and line probe assay., Methods: Eleven HIV-1-infected patients participating in the MUTAVIR study undergoing a 3-month STI after multi-HAART failure were monitored biweekly. Genotypes were assessed by sequencing and VERSANT HIV-1 Resistance Assays (LiPA)., Results: At treatment interruption, 54 mutations in total were identified with both methods among the patients. LiPA provided a result for 93.3% of the codons at baseline. For 37 mutations, a complete reversion of mutant to WT was observed with one of the two methods. Among these, LiPA detected mutations in 23 codons for 7 to 52 days longer, in 10 codons for the same period, and in four codons for a shorter time than sequencing. Similarly, LiPA detected 35 WT codons 8 to 86 days earlier, and 15 at the same time point as sequencing. A sharp reduction in the number of mutations was observed at the time of viral load increase in five of the 11 patients. Taking only the codons detected by LiPA into consideration, two patients showed a complete reversion to WT according to both genotyping assays at the end of the STI period., Conclusions: The determination of the timepoint at which a virus population of an HIV-1 patient undergoing STI reverts to WT is dependent on the assay used. The viral load increase in most patients is compatible with the outgrowth of virus with fewer or no mutations.

Objective: To analyse the presence of genotypic and phenotypic resistance in lymph node mononuclear cells from patients with sustained undetectable plasma HIV-1 RNA with highly active antiretroviral therapy., Design: Cross-sectional study on 27 HIV-infected patients receiving triple therapy for a mean period of 232.8 +/- 22.1 weeks., Methods: HIV-1 RNA was measured in plasma and lymph node cells using PCR. Reverse transcriptase and protease genes were sequenced from HIV-1 RNA obtained from lymph node cells and from peripheral blood mononuclear cell proviral DNA using a commercially available kit (TruGene). Phenotypic resistance was assessed by using a recombinant virus assay (AntiVirogram)., Results: Mutations were not found in lymph node mononuclear cell RNA in six out of nine patients on first-line regimens although they were detected in 15 out of 18 who received prior suboptimal combinations. Phenotypic resistance was confirmed in most of these cases. These patterns of resistance were closely related to patients' history of antiretroviral therapy and genotypic analysis of plasma HIV-1 RNA taken just before initiation of the current regimen. In half the patients analysed, resistance mutations found in lymph nodes were not always detected in archival proviral DNA from blood cells. Mean levels of HIV-1 RNA in lymph node cells were not different in patients exhibiting resistance compared with those harbouring wild-type viruses., Conclusion: These data demonstrate that resistant HIV-1 is produced in lymphoid tissues for prolonged periods despite effective therapy. The mechanism could represent a release from previously infected cells rather than new cycles of cellular infection.

Objective: To measure the evolution of proviral HIV-1 DNA levels in patients receiving highly active antiretroviral therapy (HAART) compared to those treated with HAART plus interleukin-2 (IL-2) and hydroxyurea., Design: Prospective randomised trial., Methods: Twenty-two HIV-1 infected patients were randomly assigned to a five-drug antiretroviral regimen for 72 weeks, with or without IL-2, followed by a three-drug regimen up to week 120 with additional hydroxyurea in patients having received IL-2. HIV-1 DNA levels in peripheral blood mononuclear cells (PBMC) were measured regularly using the Amplicor Monitor kit from Roche Diagnostics (Meylan, France). Potentially infectious HIV-1 was cultured in enhanced conditions from circulating CD4 T cells at week 120., Results: During the study period of 120 weeks, HIV-1 DNA levels in PBMC decreased by -1.1 log in patients treated with HAART only compared with -1.8 log in patients with additional IL-2 and hydroxyurea. A two-phase decay rate was observed, with an inflexion point at 12 weeks. The second decay was slow, with mean half-lives of 130.1 +/- 21.3 weeks and 95.1 +/- 26.3 weeks for patients on HAART and those receiving additional IL-2 and hydroxyurea, respectively. At week 120, one out of 11 patients with HAART alone compared to six out of 11 in the group with IL-2 and hydroxyurea had undetectable proviral DNA levels and three of them had unsuccessful recovery of replication-competent HIV-1 from blood CD4 T cells., Conclusion: Therapeutic strategies combining HAART and immune interventions have higher potency to decrease the number of infected cells than HAART alone.