Marc Wirden

Factors associated with proviral DNA HIV-1 tropism in antiretroviral therapy-treated patients with fully suppressed plasma HIV viral load: implications for the clinical use of CCR5 antagonists

OBJECTIVESnMost treated HIV-1 patients have undetectable viral loads and the strategies for managing long-term side effects may involve a new class of antiretroviral-like CCR5 antagonists. Tropism determination based on proviral DNA sequence is necessary for patients with a fully suppressed plasma viral load, as assays analysing DNA phenotypes have yet to be developed. We aimed to analyse HIV-1 tropism using proviral DNA sequencing and the associated factors, in a group of patients on antiretroviral (ARV) treatment with an undetectable viral load in plasma.nnnMETHODSnBlood samples of 140 HIV-1-infected ARV-treated patients with a plasma viral load of <40 copies/mL were studied. All patients had never been treated with CCR5 antagonists. Co-receptor usage was determined using proviral DNA from the V3 env region sequence by Geno2Pheno (false positive rate 10%) and PSSM algorithms.nnnRESULTSnAmong 140 patients treated using ARV therapy with a fully suppressed plasma viral load, at least 70% of patients had proviral R5-tropic virus. Among all the studied factors (time since HIV infection diagnosis, treatment duration, time since viral load undetectability, HIV subtype, current treatment, age, number of treatment types, sex, genotypic susceptibility score, and nadir and current CD4 cell counts), nadir CD4 T cell count alone was associated with tropism during a multivariate analysis. R5X4/X4 tropism was present in all nadir CD4 categories.nnnCONCLUSIONSnProviral DNA tropism determination should be required, even for patients with a CD4 nadir cell count >350 cells/mm(3), before CCR5 antagonists are used in patients with a fully suppressed plasma viral load.

Impact of Discrepancies between the Abbott RealTime and Cobas TaqMan Assays for Quantification of Human Immunodeficiency Virus Type 1 Group M Non-B Subtypes

ABSTRACT Viral loads in 249 clinical samples from individual patients infected with human immunodeficiency virus type 1 non-B subtypes were determined with both the Abbott RealTime and Cobas TaqMan assays. The differences exceeded 0.5 log for about 20% of samples and 1 log for 3%, with higher values always from the Abbott assay in the latter cases.

Comparative Performances of HIV-1 RNA Load Assays at Low Viral Load Levels: Results of an International Collaboration

ABSTRACT Low-level viremia during antiretroviral therapy and its accurate measurement are increasingly relevant. Here, we present an international collaboration of 4,221 paired blood plasma viral load (pVL) results from four commercial assays, emphasizing the data with low pVL. The assays compared were the Abbott RealTime assay, the Roche Amplicor assay, and the Roche TaqMan version 1 and version 2 assays. The correlation between the assays was 0.90 to 0.97. However, at a low pVL, the correlation fell to 0.45 to 0.85. The observed interassay concordance was higher when detectability was defined as 200 copies/ml than when it was defined as 50 copies/ml. A pVL of ∼100 to 125 copies/ml by the TaqMan version 1 and version 2 assays corresponded best to a 50-copies/ml threshold with the Amplicor assay. Correlation and concordance between the viral load assays were lower at a low pVL. Clear guidelines are needed on the clinical significance of low-level viremia.

Similar Evolution of Cellular HIV-1 DNA Level in Darunavir/Ritonavir Monotherapy versus Triple Therapy in MONOI –ANRS136 Trial over 96 Weeks

Background A higher proportion of intermittent viremia (to have a HIV-1 RNA viral load>50 copies/mL not confirmed) was reported in the boosted protease inhibitor monotherapy arm in some studies including MONOI trial, and that could have an impact on the replenishment of the HIV-1 DNA reservoirs. The HIV-1 DNA level is an interesting marker which reflects the size of cellular HIV reservoir. Our objectives were to study the impact of 96 weeks of Darunavir/ritonavir monotherapy versus a triple standard combination on the HIV-1 blood reservoir and factors associated with HIV-1 plasma DNA at baseline in MONOI trial sub-study. Methodology/Principal Findings This sub-study is focused on 160 patients (79 patients in monotherapy arm and 81 in tritherapy arm) for whom blood cells were available both at baseline and at week 96 (W96). Baseline HIV-1 plasma DNA was associated with CD4 nadir, pre therapeutic HIV-1 RNA viral load and baseline HIV-1 RNA measured by ultrasensitive assay. A similar median delta HIV-DNA was observed between D0 and W96 in both arms; 0.35 log copies/106 leucocytes in monotherapy arm versus 0.51 log copies/106 leucocytes in tritherapy arm. Conclusion/Significance Despite a higher proportion of intermittent viremia in monotherapy arm, a similar evolution of cellular HIV-1 DNA level was observed between mono and triple therapy arm. Trial Registration ClinicalTrials. gov NCT00421551

Resistance profiles of emtricitabine and lamivudine in tenofovir-containing regimens

OBJECTIVESnTo compare the frequency of the selection of the M184V/I resistance mutation in HIV-infected patients who experienced virological failure while receiving emtricitabine (FTC) or lamivudine (3TC), administered with tenofovir disoproxil fumarate (TDF) and either efavirenz (EFV) or a ritonavir-boosted protease inhibitor (PI; lopinavir or atazanavir).nnnMETHODSnPatient data held at two clinical centres in France were analysed retrospectively. Eligible patients had experienced virological suppression (plasma HIV RNA <200 copies/mL) for ≥ 6 months before experiencing their first virological failure (at least two measurements of plasma HIV RNA ≥ 200 copies/mL).nnnRESULTSnOf the 880 patients eligible for the study, 278 patients had experienced virological failure while receiving FTC + TDF + ritonavir-boosted PI, 257 while receiving FTC + TDF + EFV, 178 while receiving 3TC + TDF + EFV and 167 while receiving 3TC + TDF + ritonavir-boosted PI. Proportions of patients harbouring the M184V/I mutation were 24% (n = 62) for those who received FTC + TDF + EFV versus 51% (n = 91) for 3TC + TDF + EFV (P < 0.0001; Fishers exact test); proportions were 11% (n = 30) for FTC + TDF + ritonavir-boosted PI versus 22% (n = 37) for 3TC + TDF + ritonavir-boosted PI (P = 0.002; Fishers exact test). The use of lamivudine versus emtricitabine (P = 0.001), non-nucleoside reverse transcriptase inhibitors versus ritonavir-boosted PIs (P = 0.01) and the level of viral load at the time of virological failure (P = 0.01) were associated with selection of the M184V/I mutation (logistic regression analysis).nnnCONCLUSIONSnEmtricitabine and lamivudine showed differing resistance profiles when administered in combination with tenofovir disproxil fumarate and either efavirenz or a ritonavir-boosted PI. The prevalence of the M184V/I resistance mutation was significantly lower in patients who received emtricitabine and tenofovir disoproxil fumarate than in those who received lamivudine and tenofovir disoproxil fumarate.

Upgraded Cobas Ampliprep-Cobas TaqMan Version 2.0 HIV-1 RNA Quantification Assay Versus First Version : Correction of Underestimations.

ABSTRACT The large underestimations of HIV RNA quantification observed in 17 patients with the first version of Cobas TaqMan assay have been successfully corrected in the upgraded version 2.0. In comparison with the Abbott RealTime assay, the mean difference that was 1.18 log10 copies/ml is now zero. The discrepancies have disappeared.

Combination of two pathways involved in raltegravir resistance confers dolutegravir resistance

OBJECTIVESnHIV-1 integration can be efficiently inhibited by strand-transfer inhibitors such as raltegravir, elvitegravir or dolutegravir. Three pathways conferring raltegravir/elvitegravir cross-resistance (involving integrase residues Q148, N155 and Y143) were identified. Dolutegravir, belonging to the second generation of strand-transfer compounds, inhibits the Y143 and N155 pathways, but is less efficient at inhibiting the Q148 pathway. The aim of this study was to characterize the combination of two pathways involved in raltegravir resistance described in one patient failing a dolutegravir regimen for their propensity to confer dolutegravir resistance.nnnMETHODSnIn this study, a patient first failing a regimen including raltegravir was treated with dolutegravir and showed an increase in viruses carrying a combination of two pathways (N155 and Q148). Impacts of these mutations on integrase activity and resistance to strand-transfer inhibitors were characterized using both in vitro and virological assays.nnnRESULTSnOur data showed that the combination of N155H, G140S and Q148H mutations led to strong resistance to dolutegravir.nnnCONCLUSIONSnCombination of N155H, G140S and Q148H mutations originating from two distinct resistance pathways to raltegravir or elvitegravir led to a high level of dolutegravir resistance. Due to its high genetic barrier of resistance, it would be reasonable to use dolutegravir in first-line therapy before emergence of raltegravir or elvitegravir resistance.

Risk factors for raltegravir resistance development in clinical practice

OBJECTIVESnTo investigate the best conditions of raltegravir use to avoid the selection of resistance mutations in the three main genetic pathways: 148, 155 and 143.nnnMETHODSnA total of 161 patients failing on raltegravir with two consecutive HIV-1 viral loads >20 copies/mL were studied. Ten parameters [HIV-1 RNA and CD4 at baseline and failure, genotypic sensitivity score (GSS) of treatment associated with raltegravir, protease inhibitors used, time spent on raltegravir, subtype, sex and age] were tested in univariate and multivariate logistic regression analyses and compared with the emergence of resistance mutations to raltegravir at failure. Phenotypic susceptibility to raltegravir was studied in 16 patients without the main resistance mutations to raltegravir at failure.nnnRESULTSnAt raltegravir failure, 46/161 patients (28.6%) had integrase resistance mutations, whereas 115/161 (71.4%) had no resistance mutations. High HIV-1 viral load level at failure (ORu200a=u200a2.81, 95% CI 1.8-4.6, Pu200a<u200a0.001) and low GSS of treatment associated with raltegravir (ORu200a=u200a11.6, 95% CI 4.5-36.4, Pu200a<u200a0.001) were independently associated with the selection of raltegravir mutations. The percentages of patients with integrase resistance mutations were 7.7% (6/78) versus 48.1% (40/83) when viral load is ≤200 or >200 copies/mL and 47.5% (39/82) versus 8.9% (7/79) when GSS is <2 or ≥2. Among patients without main resistance mutations, two patients showed raltegravir phenotypic resistance, one naturally with F121Y at baseline and the other acquiring G118R at failure.nnnCONCLUSIONSnOur results show that to avoid the selection of raltegravir resistance mutations, patients have to be treated with at least two active drugs in combination with raltegravir and to maintain a viral load ≤200 copies/mL.

Virological failure of patients on maraviroc-based antiretroviral therapy

OBJECTIVESnVirological failure (VF) in patients on maraviroc-based treatment has been associated with altered HIV tropism and resistance to maraviroc. This multicentre study aimed to characterize VF in patients treated with maraviroc.nnnMETHODSnWe analysed 27 patients whose treatment failed between 2008 and 2011. They had been screened for HIV tropism before maraviroc initiation using population-based V3 genotyping. HIV-1 tropism and resistance of R5 viruses to maraviroc at VF and at baseline were determined retrospectively using an ultrasensitive recombinant virus assay (RVA).nnnRESULTSnViruses from 27 patients given maraviroc on the basis of the R5 genotype were characterized at the time of treatment failure. The RVA indicated that 12 patients harboured CXCR4-using viruses and 15 (56%) had pure R5 viruses at failure. One-third of those harbouring CXCR4-using viruses (4/12) were infected with R5X4/X4 viruses according to the RVA before maraviroc initiation. We analysed the phenotypic resistance to maraviroc of four patients harbouring R5 viruses at failure; two harboured viruses whose maximum percentage inhibition was reduced by 65%-90%, while the other two were infected with susceptible viruses. All patients had effective concentrations of drugs.nnnCONCLUSIONSnHalf of the maraviroc-treated patients who experienced VF harboured CXCR4-using viruses at failure, one-third of them were detected by a phenotypic method before maraviroc initiation. Phenotypic assessment of R5 virus resistance to CCR5 antagonists at failure could help optimize antiretroviral therapy.

E17A mutation in HIV-1 Vpr confers resistance to didanosine in association with thymidine analog mutations.

BACKGROUNDnHIV-1 accessory Vpr protein is involved in the reverse transcription process and has been shown to modulate the virus mutation rate. This process may play a role in the kinetics of appearance of drug resistance mutations under antiretroviral treatment.nnnMETHODSnVpr sequences were analyzed from plasma viruses derived from 97 HIV-1-infected individuals failing antiretroviral treatment and 63 antiretroviral-naïve patients. Vpr genetic variability was analyzed for association with specific drug treatment and drug resistance mutations. Biological and virological experiments were employed to characterize a mutation in Vpr found to be associated with virological failure.nnnRESULTSnE17A mutation located in the first α-helix of Vpr was more prevalent in HAART-treated individuals compared to untreated individuals. E17A was associated with thymidine analog mutations (TAMs) in reverse transcriptase M41L, L210W and T215Y and with the use of didanosine in the patients treatment histories. E17A had no impact on the biochemical and functional properties of Vpr, and did not affect kinetics of replication of wild-type or TAMs-containing viruses. However, its association with TAMs and the use of didanosine was consistent with phenotypic susceptibility assays showing a significant 3-fold decrease in didanosine susceptibility of viruses harboring Vpr E17A combined with TAMs compared to viruses harboring TAMs alone.nnnCONCLUSIONnThese findings highlight a novel role of Vpr in HIV-1 drug resistance. Vpr E17A confers resistance to didanosine when associated with TAMs. Whether Vpr E17A facilitates excision of didanosine is still to be determined.