Dynamics of drug resistance-associated mutations in HIV-1 DNA reverse transcriptase sequence during effective ART

Dynamics of drug resistance-associated mutations in HIV-1 DNA reverse transcriptase sequence... Abstract Objectives To investigate the dynamics of HIV-1 variants archived in cells harbouring drug resistance-associated mutations (DRAMs) to lamivudine/emtricitabine, etravirine and rilpivirine in patients under effective ART free from selective pressure on these DRAMs, in order to assess the possibility of recycling molecules with resistance history. Patients and methods We studied 25 patients with at least one DRAM to lamivudine/emtricitabine, etravirine and/or rilpivirine identified on an RNA sequence in their history and with virological control for at least 5 years under a regimen excluding all drugs from the resistant class. Longitudinal ultra-deep sequencing (UDS) and Sanger sequencing of the reverse transcriptase region were performed on cell-associated HIV-1 DNA samples taken over the 5 years of follow-up. Results Viral variants harbouring the analysed DRAMs were no longer detected by UDS over the 5 years in 72% of patients, with viruses susceptible to the molecules of interest found after 5 years in 80% of patients with UDS and in 88% of patients with Sanger. Residual viraemia with <50 copies/mL was detected in 52% of patients. The median HIV DNA level remained stable (2.4 at baseline versus 2.1 log10 copies/106 cells 5 years later). Conclusions These results show a clear trend towards clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control, free from therapeutic selective pressure on these DRAMs, reflecting probable residual replication in some reservoirs of the fittest viruses and leading to persistent evolution of the archived HIV-1 DNA resistance profile. Introduction In view of the prolongation of life expectancy for patients living with HIV, the question of optimization of ART, which is still a lifelong treatment, becomes central.1,2 Although most patients achieve virological success, their treatments often need to be optimized to limit adverse events and drug interactions and improve adherence. Circulating HIV-1 resistant variants can be archived in viral reservoirs, where they can persist for an unknown duration and re-emerge in the presence of therapeutic selective pressure.3,4 Residual viraemia (HIV viral load < 50 copies/mL) under effective ART has been found in 60% to 80% of patients in several studies.5 It leads to persistent evolution of the diversity of archived viral quasispecies in PBMCs.6,7 The main objective of this study was to investigate, by ultra-deep sequencing (UDS), the dynamics of decay and the persistence of DNA HIV-1 variants harbouring drug resistance-associated mutations (DRAMs) to key molecules of the antiretroviral strategy, namely lamivudine/emtricitabine, etravirine and rilpivirine, in patients with sustained virological control for at least 5 years under effective ART free from therapeutic selective pressure on these DRAMs. Patients and methods Study population In 2016, we retrospectively selected 25 patients followed up in the Departments of Infectious Diseases and Internal Medicine in Pitié-Salpêtrière Hospital (Paris, France) infected with HIV-1 B subtype, treated in the past with NRTI and/or NNRTI with selection of at least one DRAM to lamivudine/emtricitabine (n = 13 for the NRTI-off group, patients 15 to 25) and/or etravirine and/or rilpivirine (n = 14 for the NNRTI-off group, patients 1 to 14), identified on an RNA sequence in their history (between 2000 and 2010), and with viral suppression (HIV plasma RNA <50 copies/mL) for at least 5 years under a therapeutic regimen excluding all drugs of the resistant class (all NRTI and/or all NNRTI). Two patients met the inclusion criteria of both groups (patients 9 and 11). Analyses were performed retrospectively on frozen samples of whole blood taken during the 5 years of follow-up. Ethics All the patients gave their written informed consent to have their medical chart recorded in the electronic medical record system Nadis® (www.dataids.org; Fedialis Medica, Marly Le Roi, France; CNIL number: 770134, 30 October 2001). Total HIV-DNA quantification Cell-associated HIV-1 DNA was quantified by ultrasensitive real-time PCR as previously described.8 Reverse transcriptase genotypic drug resistance testing by Sanger sequencing and UDS Sanger sequencing and UDS were performed according to the Agence Nationale de recherche sur le SIDA et les hépatites virales (ANRS) consensus and using Illumina technology (Illumina, San Diego, CA, USA), respectively, as previously described.9 The sequence reads were analysed with IDNS® software v3_8_0r4 (©SmartGene), and resistance was interpreted using the latest ANRS resistance algorithm (http://www.hivfrenchresistance.org).9 For UDS, the minimum coverage was set at 50 and the ambiguity filter at 1%. Identification of defective and hypermutated G-to-A viruses G-to-A mutation frequencies were analysed using the Hypermut 2.0 program (www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html) in DNA sequences obtained from Sanger sequencing. Ultrasensitive plasma HIV-1 RNA quantification Plasma HIV-1 RNA viral load was quantified by a real-time PCR assay using the COBAS ® Ampliprep/COBAS ® TaqMan® HIV-1 test, V2.0 (Roche). Below the standard cut-off, the assay indicates the presence/absence of HIV-1 RNA. Results Patients’ characteristics at baseline in 2011 Patients’ characteristics at baseline are described in Table 1. ART is described in Figure 1. Table 1. Patients’ characteristics at baseline in 2011 Characteristic All patients (N = 25) NRTI-off group (N = 13) NNRTI-off group (N = 14) Age (years), median (IQR) 52 (46–59) 56 (49–61) 50 (47–54) Male, n (%) 84 77 93 B subtype, n (%) 100 100 100 Time since HIV diagnosis (years), median (IQR) 21 (16–23) 22 (16–23) 20 (15–21) Nadir CD4+ cell count (cells/mm3), median (IQR) 145 (89–181) 148 (137–192) 91 (18–203) Plasma HIV-1 RNA viral load zenith (log10 copies/mL), median (IQR) 4.8 (4.4–5.4) 4.5 (4.3–5.3) 5 (5–5) Duration of ART (years), median (IQR) 17 (16–21) 18 (16–21) 17 (15–20) ART since initiation (number), median (IQR)  total 14 (11–15) 14 (10–14) 14 (12–15)  NRTI 6 (5–6) 6 (5–6) 5 (4–6)  NNRTI 2 (1–2) 2 (1–2) 2 (1–2)  PI/r 4 (3–6) 4 (2–5) 4.5 (4–6)  INI 1 (1–1) 1 (1–1) 1 (1–2)  other (maraviroc) 0 (0–1) 0 (0–0) 0 (0–1) Duration of NRTI treatment (years), median (IQR) NA 15 (11–18) NA Duration of NNRTI treatment (years), median (IQR) NA NA 4 (2–6) Current ART (number of different drugs), median (IQR)  total 2 (2–3) 2 (2–2) 2 (2–3)  NRTI 0 (0–1) 0 (0–0) 1 (0–2)  NNRTI 0 (0–1) 1 (0–1) 0 (0–0)  PI/r 0 (0–1) 0 (0–1) 0 (0–1)  INI 1 (1–1) 1 (1–1) 1 (1–1)  other (maraviroc) 0 (0–0) 0 (0–0) 0 (0–0) Time since NRTI interruption (years), median (IQR) NA 2 (2–3) NA Time since NNRTI interruption (years), median (IQR) NA NA 4 (2–8) Duration of suppressed HIV viraemia (viral load <50 copies/mL) (years), median (IQR) 2 (2–4) 2 (2–3) 2 (2–4) Cell-associated HIV-1 DNA (log10 copies/106 cells), median (IQR) 2.4 (1.9–2.6) 2.2 (2.0–2.4) 3 (2–3) Time since last resistant RNA genotype (years), median (IQR) 3 (1–6) 2 (1–3) 2 (1–7) Characteristic All patients (N = 25) NRTI-off group (N = 13) NNRTI-off group (N = 14) Age (years), median (IQR) 52 (46–59) 56 (49–61) 50 (47–54) Male, n (%) 84 77 93 B subtype, n (%) 100 100 100 Time since HIV diagnosis (years), median (IQR) 21 (16–23) 22 (16–23) 20 (15–21) Nadir CD4+ cell count (cells/mm3), median (IQR) 145 (89–181) 148 (137–192) 91 (18–203) Plasma HIV-1 RNA viral load zenith (log10 copies/mL), median (IQR) 4.8 (4.4–5.4) 4.5 (4.3–5.3) 5 (5–5) Duration of ART (years), median (IQR) 17 (16–21) 18 (16–21) 17 (15–20) ART since initiation (number), median (IQR)  total 14 (11–15) 14 (10–14) 14 (12–15)  NRTI 6 (5–6) 6 (5–6) 5 (4–6)  NNRTI 2 (1–2) 2 (1–2) 2 (1–2)  PI/r 4 (3–6) 4 (2–5) 4.5 (4–6)  INI 1 (1–1) 1 (1–1) 1 (1–2)  other (maraviroc) 0 (0–1) 0 (0–0) 0 (0–1) Duration of NRTI treatment (years), median (IQR) NA 15 (11–18) NA Duration of NNRTI treatment (years), median (IQR) NA NA 4 (2–6) Current ART (number of different drugs), median (IQR)  total 2 (2–3) 2 (2–2) 2 (2–3)  NRTI 0 (0–1) 0 (0–0) 1 (0–2)  NNRTI 0 (0–1) 1 (0–1) 0 (0–0)  PI/r 0 (0–1) 0 (0–1) 0 (0–1)  INI 1 (1–1) 1 (1–1) 1 (1–1)  other (maraviroc) 0 (0–0) 0 (0–0) 0 (0–0) Time since NRTI interruption (years), median (IQR) NA 2 (2–3) NA Time since NNRTI interruption (years), median (IQR) NA NA 4 (2–8) Duration of suppressed HIV viraemia (viral load <50 copies/mL) (years), median (IQR) 2 (2–4) 2 (2–3) 2 (2–4) Cell-associated HIV-1 DNA (log10 copies/106 cells), median (IQR) 2.4 (1.9–2.6) 2.2 (2.0–2.4) 3 (2–3) Time since last resistant RNA genotype (years), median (IQR) 3 (1–6) 2 (1–3) 2 (1–7) INI, integrase inhibitor; IP/r, protease inhibitor boosted by ritonavir; NA, not applicable. Table 1. Patients’ characteristics at baseline in 2011 Characteristic All patients (N = 25) NRTI-off group (N = 13) NNRTI-off group (N = 14) Age (years), median (IQR) 52 (46–59) 56 (49–61) 50 (47–54) Male, n (%) 84 77 93 B subtype, n (%) 100 100 100 Time since HIV diagnosis (years), median (IQR) 21 (16–23) 22 (16–23) 20 (15–21) Nadir CD4+ cell count (cells/mm3), median (IQR) 145 (89–181) 148 (137–192) 91 (18–203) Plasma HIV-1 RNA viral load zenith (log10 copies/mL), median (IQR) 4.8 (4.4–5.4) 4.5 (4.3–5.3) 5 (5–5) Duration of ART (years), median (IQR) 17 (16–21) 18 (16–21) 17 (15–20) ART since initiation (number), median (IQR)  total 14 (11–15) 14 (10–14) 14 (12–15)  NRTI 6 (5–6) 6 (5–6) 5 (4–6)  NNRTI 2 (1–2) 2 (1–2) 2 (1–2)  PI/r 4 (3–6) 4 (2–5) 4.5 (4–6)  INI 1 (1–1) 1 (1–1) 1 (1–2)  other (maraviroc) 0 (0–1) 0 (0–0) 0 (0–1) Duration of NRTI treatment (years), median (IQR) NA 15 (11–18) NA Duration of NNRTI treatment (years), median (IQR) NA NA 4 (2–6) Current ART (number of different drugs), median (IQR)  total 2 (2–3) 2 (2–2) 2 (2–3)  NRTI 0 (0–1) 0 (0–0) 1 (0–2)  NNRTI 0 (0–1) 1 (0–1) 0 (0–0)  PI/r 0 (0–1) 0 (0–1) 0 (0–1)  INI 1 (1–1) 1 (1–1) 1 (1–1)  other (maraviroc) 0 (0–0) 0 (0–0) 0 (0–0) Time since NRTI interruption (years), median (IQR) NA 2 (2–3) NA Time since NNRTI interruption (years), median (IQR) NA NA 4 (2–8) Duration of suppressed HIV viraemia (viral load <50 copies/mL) (years), median (IQR) 2 (2–4) 2 (2–3) 2 (2–4) Cell-associated HIV-1 DNA (log10 copies/106 cells), median (IQR) 2.4 (1.9–2.6) 2.2 (2.0–2.4) 3 (2–3) Time since last resistant RNA genotype (years), median (IQR) 3 (1–6) 2 (1–3) 2 (1–7) Characteristic All patients (N = 25) NRTI-off group (N = 13) NNRTI-off group (N = 14) Age (years), median (IQR) 52 (46–59) 56 (49–61) 50 (47–54) Male, n (%) 84 77 93 B subtype, n (%) 100 100 100 Time since HIV diagnosis (years), median (IQR) 21 (16–23) 22 (16–23) 20 (15–21) Nadir CD4+ cell count (cells/mm3), median (IQR) 145 (89–181) 148 (137–192) 91 (18–203) Plasma HIV-1 RNA viral load zenith (log10 copies/mL), median (IQR) 4.8 (4.4–5.4) 4.5 (4.3–5.3) 5 (5–5) Duration of ART (years), median (IQR) 17 (16–21) 18 (16–21) 17 (15–20) ART since initiation (number), median (IQR)  total 14 (11–15) 14 (10–14) 14 (12–15)  NRTI 6 (5–6) 6 (5–6) 5 (4–6)  NNRTI 2 (1–2) 2 (1–2) 2 (1–2)  PI/r 4 (3–6) 4 (2–5) 4.5 (4–6)  INI 1 (1–1) 1 (1–1) 1 (1–2)  other (maraviroc) 0 (0–1) 0 (0–0) 0 (0–1) Duration of NRTI treatment (years), median (IQR) NA 15 (11–18) NA Duration of NNRTI treatment (years), median (IQR) NA NA 4 (2–6) Current ART (number of different drugs), median (IQR)  total 2 (2–3) 2 (2–2) 2 (2–3)  NRTI 0 (0–1) 0 (0–0) 1 (0–2)  NNRTI 0 (0–1) 1 (0–1) 0 (0–0)  PI/r 0 (0–1) 0 (0–1) 0 (0–1)  INI 1 (1–1) 1 (1–1) 1 (1–1)  other (maraviroc) 0 (0–0) 0 (0–0) 0 (0–0) Time since NRTI interruption (years), median (IQR) NA 2 (2–3) NA Time since NNRTI interruption (years), median (IQR) NA NA 4 (2–8) Duration of suppressed HIV viraemia (viral load <50 copies/mL) (years), median (IQR) 2 (2–4) 2 (2–3) 2 (2–4) Cell-associated HIV-1 DNA (log10 copies/106 cells), median (IQR) 2.4 (1.9–2.6) 2.2 (2.0–2.4) 3 (2–3) Time since last resistant RNA genotype (years), median (IQR) 3 (1–6) 2 (1–3) 2 (1–7) INI, integrase inhibitor; IP/r, protease inhibitor boosted by ritonavir; NA, not applicable. Figure 1. View largeDownload slide View largeDownload slide Evolution of archived DRAMs in cell-associated HIV-1 DNA under effective ART. (a) NRTI-off group. (b) NNRTI-off group. DNA was sequenced by UDS: results in % (mutation frequency among all reads). RNA was sequenced by Sanger and the genotypes were cumulated: mutations detected (+) or not detected (-). 3TC, lamivudine; ABC, abacavir; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine; ddI, didanosine; ETR, etravirine; RPV, rilpivirine; DRV/r, darunavir/ritonavir; LPV/r, lopinavir/ritonavir; ATV, atazanavir; ATV/r, atazanavir/ritonavir; RAL, raltegravir; DTG, dolutegravir; MVC, maraviroc. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1. View largeDownload slide View largeDownload slide Evolution of archived DRAMs in cell-associated HIV-1 DNA under effective ART. (a) NRTI-off group. (b) NNRTI-off group. DNA was sequenced by UDS: results in % (mutation frequency among all reads). RNA was sequenced by Sanger and the genotypes were cumulated: mutations detected (+) or not detected (-). 3TC, lamivudine; ABC, abacavir; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine; ddI, didanosine; ETR, etravirine; RPV, rilpivirine; DRV/r, darunavir/ritonavir; LPV/r, lopinavir/ritonavir; ATV, atazanavir; ATV/r, atazanavir/ritonavir; RAL, raltegravir; DTG, dolutegravir; MVC, maraviroc. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. DRAM dynamics by UDS in HIV-1 DNA reverse transcriptase sequence archived in cells In the NRTI-off group, 10/13 patients had NRTI DRAMs detectable in their HIV-1 DNA sequence at baseline (patients 9, 19 and 20, had any, not shown), of whom 5 showed progressive clearance of these DRAMs over time, with complete absence of DRAMs in their HIV-1 sequence at 5 years (Figure 1a). The other five patients showed a fluctuating and/or dissociated evolution of the DRAMs, with persistence of some DRAMs (patients 17, 22 and 25), re-emergence of former DRAMs (patient 25) or emergence of new DRAMs (patients 18, 22, 24 and 25) (Figure 1a). In the NNRTI-off group, 11/14 patients had NNRTI DRAMs detectable in their HIV-1 DNA sequence at baseline (patients 1, 9 and 14 had no DRAMs detectable, not shown), of whom 8 showed progressive clearance of these DRAMs (Figure 1b) and 3 showed persistence (patient 4) or emergence (patients 6 and 7) of some DRAMs (Figure 1b). Overall, the analysed DRAMs were no longer detected after 5 years in 18/25 patients (72%), and interpretation with the ANRS resistance algorithm found viruses susceptible to the molecules of interest in 20/25 patients (80%) at 5 years (2/5 patients who still had NRTI DRAMs had viruses susceptible to lamivudine/emtricitabine). Characteristics (age, sex, duration of infection and treatment, viral load zenith, CD4+ cell count nadir, total cell HIV-1 DNA level, time since removal of therapeutic pressure, duration of suppressed viraemia, time since last resistant RNA genotype) of patients with and without detectable DRAMs at 5 years were not significantly different (P > 0.05 for all data with the Mann–Whitney test). Comparison of Sanger sequencing and UDS results In 11/67 (16%) sequencing results, some DRAMs detected by UDS and not by Sanger sequencing, with frequencies between 1.1% and 21.3%, led to a different interpretation of resistance by the ANRS algorithm. Thus, Sanger sequencing found viruses interpreted as susceptible to the molecules of interest in 22/25 patients (88%) at 5 years. Notably, some DRAMs detected by UDS and not by Sanger had frequencies higher than 20%, up to 40.7%. Residual viraemia Residual viraemia was found at least once during the 5 years in 13 patients (52%), of whom 9 showed progressive clearance of archived DRAMs (patients 1, 3, 8, 10, 11, 12, 13, 15 and 23), and 4 showed persistence or (re-)emergence of archived DRAMs (patients 6, 7, 17 and 27). Cell-associated HIV-1 DNA quantification The median total cell-associated HIV-1 DNA load remained stable (2.4 log10 copies/106 cells at baseline versus 2.1 log10 copies/106 cells at 5 years). The total cell-associated HIV-1 DNA load was not detectable (<66 copies/106 cells) in 13 samples. APOBEC3-induced G-to-A mutations and defective viruses Two patients had statistically significant (P < 0.05) G-to-A mutation frequencies in their HIV-1 DNA Sanger sequences: patient 20 with 35 G-to-A mutations in 2011 and patient 23 with 3, 3 and 27 G-to-A mutations in 2013, 2014 and 2016 respectively. Thirteen patients (52%) had stop codons and/or G-to-A DRAMs (184I and 230I) in their UDS sequence reads, with frequencies between 2.3% and 100%. These frequencies increased over time in 69% of them. Sanger sequencing detected fewer stop codons and G-to-A mutations than UDS, with frequencies up to 37.5%. Discussion These results show a clear trend towards progressive clearance over time of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA in these 25 patients with suppressed plasma viral load under a therapeutic regimen free from cross-selective pressure on these DRAMs. This dynamic is probably due mainly to preferential residual replication in some reservoirs of the fittest viruses among the quasispecies and the removal of the therapeutic pressure of an entire drug class. These viruses might be released into the blood from reservoirs, leading to persistent residual viraemia under treatment and replenishment of peripheral reservoirs with these viruses free of DRAMs, with clearance of resistant proviruses.6,7,10 However, some DRAMs to the removed therapeutic class could be linked on the same viral variant to other DRAMs conferring resistance to the current regimen, and could therefore be indirectly selected by the therapeutic pressure,7 thus explaining the emergence of some DRAMs. Indeed, DRAMs can emerge even in the case of minimal viral replication, with a suppressed plasma viral load (<50 copies/mL).3,11 The dynamics of DRAMs archived in HIV-1 DNA did not seem to be correlated to the HIV-1 DNA level in the cell reservoir, as these levels did not vary over the 5 years of follow-up. This 5 year period is indeed probably too short to find a significant decrease in HIV-1 DNA level, especially for patients whose treatment was initiated in the chronic phase of infection.12 This study has several limitations. The number of included patients was low, probably because of strict inclusion criteria and the study’s monocentric nature. The low HIV-1 DNA viral load could induce in UDS many reads of a few genomes, in which case evolution of mutations becomes stochastic rather than biological events. The absence of variations in the HIV-1 DNA viral load and the longitudinal aspect of the study are not in favour of this hypothesis. Furthermore, an analysis of DRAM dynamics in anatomical reservoirs could be interesting, as viral genetic diversity and mutational resistance patterns can vary in some body compartments.3 In conclusion, these results show a clear trend towards progressive clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control free of therapeutic selective pressure on those DRAMs. These results provide a rationale for further clinical evaluation of drug recycling in selected patients with sustained virological control under non-optimal regimens, after genotypic resistance testing by UDS in archived HIV-1 DNA. Acknowledgements We thank the ICM Institute at Pitié-Salpêtrière Hospital for access to the Illumina Platform and Yannick Marie for his technical assistance. Funding This work was supported by ‘Agence Nationale de la Recherche sur le Sida et les hépatites virales’ (ANRS). Transparency declarations None to declare. References 1 Morlat P , Blanc A , Bonnet F et al. Prise en charge médicale des personnes vivant avec le VIH, recommandations du groupe d’experts. (Sous l’égide du CNS et de l’ANRS, 2017 ). https://cns.sante.fr/actualities/prise-en-charge-du-vih-recommendations-du-groupe-dexperts/. 2 Engsig FN , Zangerle R , Katsarou O et al. Long-term mortality in HIV-positive individuals virally suppressed for >3 years with incomplete CD4 recovery . Clin Infect Dis 2014 ; 58 : 1312 – 21 . Google Scholar CrossRef Search ADS PubMed 3 Turriziani O , Andreoni M , Antonelli G. Resistant viral variants in cellular reservoirs of human immunodeficiency virus infection . Clin Microbiol Infect 2010 ; 16 : 1518 – 24 . Google Scholar CrossRef Search ADS PubMed 4 Lambotte O , Chaix ML , Gubler B et al. The lymphocyte HIV reservoir in patients on long-term HAART is a memory of virus evolution . AIDS 2004 ; 18 : 1147 – 58 . Google Scholar CrossRef Search ADS PubMed 5 Palmer S , Maldarelli F , Wiegand A et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy . Proc Natl Acad Sci USA 2008 ; 105 : 3879 – 84 . Google Scholar CrossRef Search ADS PubMed 6 Michelini Z , Galluzzo C , Pirillo MF et al. HIV-1 DNA dynamics and variations in HIV-1 DNA protease and reverse transcriptase sequences in multidrug-resistant patients during successful raltegravir-based therapy . J Med Virol 2016 ; 88 : 2115 – 24 . Google Scholar CrossRef Search ADS PubMed 7 Gantner P , Morand-Joubert L , Sueur C et al. Drug resistance and tropism as markers of the dynamics of HIV-1 DNA quasispecies in blood cells of heavily pretreated patients who achieved sustained virological suppression . J Antimicrob Chemother 2016 ; 71 : 751 – 61 . Google Scholar CrossRef Search ADS PubMed 8 Avettand-Fènoël V , Chaix ML , Blanche S et al. LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01) . J Med Virol 2009 ; 81 : 217 – 23 . Google Scholar CrossRef Search ADS PubMed 9 Lapointe HR , Dong W , Lee GQ et al. HIV drug resistance testing by high-multiplex ‘wide’ sequencing on the MiSeq instrument . Antimicrob Agents Chemother 2015 ; 59 : 6824 – 33 . Google Scholar CrossRef Search ADS PubMed 10 Chun TW , Murray D , Justement JS et al. Relationship between residual plasma viremia and the size of HIV proviral DNA reservoirs in infected individuals receiving effective antiretroviral therapy . J Infect Dis 2011 ; 204 : 135 – 8 . Google Scholar CrossRef Search ADS PubMed 11 Martinez-Picado J , DePasquale MP , Kartsonis N et al. Antiretroviral resistance during successful therapy of HIV type 1 infection . Proc Natl Acad Sci USA 2000 ; 97 : 10948 – 53 . Google Scholar CrossRef Search ADS PubMed 12 Viard JP , Burgard M , Hubert JB et al. Impact of 5 years of maximally successful highly active antiretroviral therapy on CD4 cell count and HIV-1 DNA level . AIDS 2004 ; 18 : 45 – 9 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Dynamics of drug resistance-associated mutations in HIV-1 DNA reverse transcriptase sequence during effective ART

Journal of Antimicrobial Chemotherapy , Volume Advance Article (8) – May 29, 2018

Loading next page...
 
/lp/ou_press/dynamics-of-drug-resistance-associated-mutations-in-hiv-1-dna-reverse-q0JOYhriTZ
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
ISSN
0305-7453
eISSN
1460-2091
D.O.I.
10.1093/jac/dky130
Publisher site
See Article on Publisher Site

Abstract

Abstract Objectives To investigate the dynamics of HIV-1 variants archived in cells harbouring drug resistance-associated mutations (DRAMs) to lamivudine/emtricitabine, etravirine and rilpivirine in patients under effective ART free from selective pressure on these DRAMs, in order to assess the possibility of recycling molecules with resistance history. Patients and methods We studied 25 patients with at least one DRAM to lamivudine/emtricitabine, etravirine and/or rilpivirine identified on an RNA sequence in their history and with virological control for at least 5 years under a regimen excluding all drugs from the resistant class. Longitudinal ultra-deep sequencing (UDS) and Sanger sequencing of the reverse transcriptase region were performed on cell-associated HIV-1 DNA samples taken over the 5 years of follow-up. Results Viral variants harbouring the analysed DRAMs were no longer detected by UDS over the 5 years in 72% of patients, with viruses susceptible to the molecules of interest found after 5 years in 80% of patients with UDS and in 88% of patients with Sanger. Residual viraemia with <50 copies/mL was detected in 52% of patients. The median HIV DNA level remained stable (2.4 at baseline versus 2.1 log10 copies/106 cells 5 years later). Conclusions These results show a clear trend towards clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control, free from therapeutic selective pressure on these DRAMs, reflecting probable residual replication in some reservoirs of the fittest viruses and leading to persistent evolution of the archived HIV-1 DNA resistance profile. Introduction In view of the prolongation of life expectancy for patients living with HIV, the question of optimization of ART, which is still a lifelong treatment, becomes central.1,2 Although most patients achieve virological success, their treatments often need to be optimized to limit adverse events and drug interactions and improve adherence. Circulating HIV-1 resistant variants can be archived in viral reservoirs, where they can persist for an unknown duration and re-emerge in the presence of therapeutic selective pressure.3,4 Residual viraemia (HIV viral load < 50 copies/mL) under effective ART has been found in 60% to 80% of patients in several studies.5 It leads to persistent evolution of the diversity of archived viral quasispecies in PBMCs.6,7 The main objective of this study was to investigate, by ultra-deep sequencing (UDS), the dynamics of decay and the persistence of DNA HIV-1 variants harbouring drug resistance-associated mutations (DRAMs) to key molecules of the antiretroviral strategy, namely lamivudine/emtricitabine, etravirine and rilpivirine, in patients with sustained virological control for at least 5 years under effective ART free from therapeutic selective pressure on these DRAMs. Patients and methods Study population In 2016, we retrospectively selected 25 patients followed up in the Departments of Infectious Diseases and Internal Medicine in Pitié-Salpêtrière Hospital (Paris, France) infected with HIV-1 B subtype, treated in the past with NRTI and/or NNRTI with selection of at least one DRAM to lamivudine/emtricitabine (n = 13 for the NRTI-off group, patients 15 to 25) and/or etravirine and/or rilpivirine (n = 14 for the NNRTI-off group, patients 1 to 14), identified on an RNA sequence in their history (between 2000 and 2010), and with viral suppression (HIV plasma RNA <50 copies/mL) for at least 5 years under a therapeutic regimen excluding all drugs of the resistant class (all NRTI and/or all NNRTI). Two patients met the inclusion criteria of both groups (patients 9 and 11). Analyses were performed retrospectively on frozen samples of whole blood taken during the 5 years of follow-up. Ethics All the patients gave their written informed consent to have their medical chart recorded in the electronic medical record system Nadis® (www.dataids.org; Fedialis Medica, Marly Le Roi, France; CNIL number: 770134, 30 October 2001). Total HIV-DNA quantification Cell-associated HIV-1 DNA was quantified by ultrasensitive real-time PCR as previously described.8 Reverse transcriptase genotypic drug resistance testing by Sanger sequencing and UDS Sanger sequencing and UDS were performed according to the Agence Nationale de recherche sur le SIDA et les hépatites virales (ANRS) consensus and using Illumina technology (Illumina, San Diego, CA, USA), respectively, as previously described.9 The sequence reads were analysed with IDNS® software v3_8_0r4 (©SmartGene), and resistance was interpreted using the latest ANRS resistance algorithm (http://www.hivfrenchresistance.org).9 For UDS, the minimum coverage was set at 50 and the ambiguity filter at 1%. Identification of defective and hypermutated G-to-A viruses G-to-A mutation frequencies were analysed using the Hypermut 2.0 program (www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html) in DNA sequences obtained from Sanger sequencing. Ultrasensitive plasma HIV-1 RNA quantification Plasma HIV-1 RNA viral load was quantified by a real-time PCR assay using the COBAS ® Ampliprep/COBAS ® TaqMan® HIV-1 test, V2.0 (Roche). Below the standard cut-off, the assay indicates the presence/absence of HIV-1 RNA. Results Patients’ characteristics at baseline in 2011 Patients’ characteristics at baseline are described in Table 1. ART is described in Figure 1. Table 1. Patients’ characteristics at baseline in 2011 Characteristic All patients (N = 25) NRTI-off group (N = 13) NNRTI-off group (N = 14) Age (years), median (IQR) 52 (46–59) 56 (49–61) 50 (47–54) Male, n (%) 84 77 93 B subtype, n (%) 100 100 100 Time since HIV diagnosis (years), median (IQR) 21 (16–23) 22 (16–23) 20 (15–21) Nadir CD4+ cell count (cells/mm3), median (IQR) 145 (89–181) 148 (137–192) 91 (18–203) Plasma HIV-1 RNA viral load zenith (log10 copies/mL), median (IQR) 4.8 (4.4–5.4) 4.5 (4.3–5.3) 5 (5–5) Duration of ART (years), median (IQR) 17 (16–21) 18 (16–21) 17 (15–20) ART since initiation (number), median (IQR)  total 14 (11–15) 14 (10–14) 14 (12–15)  NRTI 6 (5–6) 6 (5–6) 5 (4–6)  NNRTI 2 (1–2) 2 (1–2) 2 (1–2)  PI/r 4 (3–6) 4 (2–5) 4.5 (4–6)  INI 1 (1–1) 1 (1–1) 1 (1–2)  other (maraviroc) 0 (0–1) 0 (0–0) 0 (0–1) Duration of NRTI treatment (years), median (IQR) NA 15 (11–18) NA Duration of NNRTI treatment (years), median (IQR) NA NA 4 (2–6) Current ART (number of different drugs), median (IQR)  total 2 (2–3) 2 (2–2) 2 (2–3)  NRTI 0 (0–1) 0 (0–0) 1 (0–2)  NNRTI 0 (0–1) 1 (0–1) 0 (0–0)  PI/r 0 (0–1) 0 (0–1) 0 (0–1)  INI 1 (1–1) 1 (1–1) 1 (1–1)  other (maraviroc) 0 (0–0) 0 (0–0) 0 (0–0) Time since NRTI interruption (years), median (IQR) NA 2 (2–3) NA Time since NNRTI interruption (years), median (IQR) NA NA 4 (2–8) Duration of suppressed HIV viraemia (viral load <50 copies/mL) (years), median (IQR) 2 (2–4) 2 (2–3) 2 (2–4) Cell-associated HIV-1 DNA (log10 copies/106 cells), median (IQR) 2.4 (1.9–2.6) 2.2 (2.0–2.4) 3 (2–3) Time since last resistant RNA genotype (years), median (IQR) 3 (1–6) 2 (1–3) 2 (1–7) Characteristic All patients (N = 25) NRTI-off group (N = 13) NNRTI-off group (N = 14) Age (years), median (IQR) 52 (46–59) 56 (49–61) 50 (47–54) Male, n (%) 84 77 93 B subtype, n (%) 100 100 100 Time since HIV diagnosis (years), median (IQR) 21 (16–23) 22 (16–23) 20 (15–21) Nadir CD4+ cell count (cells/mm3), median (IQR) 145 (89–181) 148 (137–192) 91 (18–203) Plasma HIV-1 RNA viral load zenith (log10 copies/mL), median (IQR) 4.8 (4.4–5.4) 4.5 (4.3–5.3) 5 (5–5) Duration of ART (years), median (IQR) 17 (16–21) 18 (16–21) 17 (15–20) ART since initiation (number), median (IQR)  total 14 (11–15) 14 (10–14) 14 (12–15)  NRTI 6 (5–6) 6 (5–6) 5 (4–6)  NNRTI 2 (1–2) 2 (1–2) 2 (1–2)  PI/r 4 (3–6) 4 (2–5) 4.5 (4–6)  INI 1 (1–1) 1 (1–1) 1 (1–2)  other (maraviroc) 0 (0–1) 0 (0–0) 0 (0–1) Duration of NRTI treatment (years), median (IQR) NA 15 (11–18) NA Duration of NNRTI treatment (years), median (IQR) NA NA 4 (2–6) Current ART (number of different drugs), median (IQR)  total 2 (2–3) 2 (2–2) 2 (2–3)  NRTI 0 (0–1) 0 (0–0) 1 (0–2)  NNRTI 0 (0–1) 1 (0–1) 0 (0–0)  PI/r 0 (0–1) 0 (0–1) 0 (0–1)  INI 1 (1–1) 1 (1–1) 1 (1–1)  other (maraviroc) 0 (0–0) 0 (0–0) 0 (0–0) Time since NRTI interruption (years), median (IQR) NA 2 (2–3) NA Time since NNRTI interruption (years), median (IQR) NA NA 4 (2–8) Duration of suppressed HIV viraemia (viral load <50 copies/mL) (years), median (IQR) 2 (2–4) 2 (2–3) 2 (2–4) Cell-associated HIV-1 DNA (log10 copies/106 cells), median (IQR) 2.4 (1.9–2.6) 2.2 (2.0–2.4) 3 (2–3) Time since last resistant RNA genotype (years), median (IQR) 3 (1–6) 2 (1–3) 2 (1–7) INI, integrase inhibitor; IP/r, protease inhibitor boosted by ritonavir; NA, not applicable. Table 1. Patients’ characteristics at baseline in 2011 Characteristic All patients (N = 25) NRTI-off group (N = 13) NNRTI-off group (N = 14) Age (years), median (IQR) 52 (46–59) 56 (49–61) 50 (47–54) Male, n (%) 84 77 93 B subtype, n (%) 100 100 100 Time since HIV diagnosis (years), median (IQR) 21 (16–23) 22 (16–23) 20 (15–21) Nadir CD4+ cell count (cells/mm3), median (IQR) 145 (89–181) 148 (137–192) 91 (18–203) Plasma HIV-1 RNA viral load zenith (log10 copies/mL), median (IQR) 4.8 (4.4–5.4) 4.5 (4.3–5.3) 5 (5–5) Duration of ART (years), median (IQR) 17 (16–21) 18 (16–21) 17 (15–20) ART since initiation (number), median (IQR)  total 14 (11–15) 14 (10–14) 14 (12–15)  NRTI 6 (5–6) 6 (5–6) 5 (4–6)  NNRTI 2 (1–2) 2 (1–2) 2 (1–2)  PI/r 4 (3–6) 4 (2–5) 4.5 (4–6)  INI 1 (1–1) 1 (1–1) 1 (1–2)  other (maraviroc) 0 (0–1) 0 (0–0) 0 (0–1) Duration of NRTI treatment (years), median (IQR) NA 15 (11–18) NA Duration of NNRTI treatment (years), median (IQR) NA NA 4 (2–6) Current ART (number of different drugs), median (IQR)  total 2 (2–3) 2 (2–2) 2 (2–3)  NRTI 0 (0–1) 0 (0–0) 1 (0–2)  NNRTI 0 (0–1) 1 (0–1) 0 (0–0)  PI/r 0 (0–1) 0 (0–1) 0 (0–1)  INI 1 (1–1) 1 (1–1) 1 (1–1)  other (maraviroc) 0 (0–0) 0 (0–0) 0 (0–0) Time since NRTI interruption (years), median (IQR) NA 2 (2–3) NA Time since NNRTI interruption (years), median (IQR) NA NA 4 (2–8) Duration of suppressed HIV viraemia (viral load <50 copies/mL) (years), median (IQR) 2 (2–4) 2 (2–3) 2 (2–4) Cell-associated HIV-1 DNA (log10 copies/106 cells), median (IQR) 2.4 (1.9–2.6) 2.2 (2.0–2.4) 3 (2–3) Time since last resistant RNA genotype (years), median (IQR) 3 (1–6) 2 (1–3) 2 (1–7) Characteristic All patients (N = 25) NRTI-off group (N = 13) NNRTI-off group (N = 14) Age (years), median (IQR) 52 (46–59) 56 (49–61) 50 (47–54) Male, n (%) 84 77 93 B subtype, n (%) 100 100 100 Time since HIV diagnosis (years), median (IQR) 21 (16–23) 22 (16–23) 20 (15–21) Nadir CD4+ cell count (cells/mm3), median (IQR) 145 (89–181) 148 (137–192) 91 (18–203) Plasma HIV-1 RNA viral load zenith (log10 copies/mL), median (IQR) 4.8 (4.4–5.4) 4.5 (4.3–5.3) 5 (5–5) Duration of ART (years), median (IQR) 17 (16–21) 18 (16–21) 17 (15–20) ART since initiation (number), median (IQR)  total 14 (11–15) 14 (10–14) 14 (12–15)  NRTI 6 (5–6) 6 (5–6) 5 (4–6)  NNRTI 2 (1–2) 2 (1–2) 2 (1–2)  PI/r 4 (3–6) 4 (2–5) 4.5 (4–6)  INI 1 (1–1) 1 (1–1) 1 (1–2)  other (maraviroc) 0 (0–1) 0 (0–0) 0 (0–1) Duration of NRTI treatment (years), median (IQR) NA 15 (11–18) NA Duration of NNRTI treatment (years), median (IQR) NA NA 4 (2–6) Current ART (number of different drugs), median (IQR)  total 2 (2–3) 2 (2–2) 2 (2–3)  NRTI 0 (0–1) 0 (0–0) 1 (0–2)  NNRTI 0 (0–1) 1 (0–1) 0 (0–0)  PI/r 0 (0–1) 0 (0–1) 0 (0–1)  INI 1 (1–1) 1 (1–1) 1 (1–1)  other (maraviroc) 0 (0–0) 0 (0–0) 0 (0–0) Time since NRTI interruption (years), median (IQR) NA 2 (2–3) NA Time since NNRTI interruption (years), median (IQR) NA NA 4 (2–8) Duration of suppressed HIV viraemia (viral load <50 copies/mL) (years), median (IQR) 2 (2–4) 2 (2–3) 2 (2–4) Cell-associated HIV-1 DNA (log10 copies/106 cells), median (IQR) 2.4 (1.9–2.6) 2.2 (2.0–2.4) 3 (2–3) Time since last resistant RNA genotype (years), median (IQR) 3 (1–6) 2 (1–3) 2 (1–7) INI, integrase inhibitor; IP/r, protease inhibitor boosted by ritonavir; NA, not applicable. Figure 1. View largeDownload slide View largeDownload slide Evolution of archived DRAMs in cell-associated HIV-1 DNA under effective ART. (a) NRTI-off group. (b) NNRTI-off group. DNA was sequenced by UDS: results in % (mutation frequency among all reads). RNA was sequenced by Sanger and the genotypes were cumulated: mutations detected (+) or not detected (-). 3TC, lamivudine; ABC, abacavir; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine; ddI, didanosine; ETR, etravirine; RPV, rilpivirine; DRV/r, darunavir/ritonavir; LPV/r, lopinavir/ritonavir; ATV, atazanavir; ATV/r, atazanavir/ritonavir; RAL, raltegravir; DTG, dolutegravir; MVC, maraviroc. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1. View largeDownload slide View largeDownload slide Evolution of archived DRAMs in cell-associated HIV-1 DNA under effective ART. (a) NRTI-off group. (b) NNRTI-off group. DNA was sequenced by UDS: results in % (mutation frequency among all reads). RNA was sequenced by Sanger and the genotypes were cumulated: mutations detected (+) or not detected (-). 3TC, lamivudine; ABC, abacavir; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine; ddI, didanosine; ETR, etravirine; RPV, rilpivirine; DRV/r, darunavir/ritonavir; LPV/r, lopinavir/ritonavir; ATV, atazanavir; ATV/r, atazanavir/ritonavir; RAL, raltegravir; DTG, dolutegravir; MVC, maraviroc. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. DRAM dynamics by UDS in HIV-1 DNA reverse transcriptase sequence archived in cells In the NRTI-off group, 10/13 patients had NRTI DRAMs detectable in their HIV-1 DNA sequence at baseline (patients 9, 19 and 20, had any, not shown), of whom 5 showed progressive clearance of these DRAMs over time, with complete absence of DRAMs in their HIV-1 sequence at 5 years (Figure 1a). The other five patients showed a fluctuating and/or dissociated evolution of the DRAMs, with persistence of some DRAMs (patients 17, 22 and 25), re-emergence of former DRAMs (patient 25) or emergence of new DRAMs (patients 18, 22, 24 and 25) (Figure 1a). In the NNRTI-off group, 11/14 patients had NNRTI DRAMs detectable in their HIV-1 DNA sequence at baseline (patients 1, 9 and 14 had no DRAMs detectable, not shown), of whom 8 showed progressive clearance of these DRAMs (Figure 1b) and 3 showed persistence (patient 4) or emergence (patients 6 and 7) of some DRAMs (Figure 1b). Overall, the analysed DRAMs were no longer detected after 5 years in 18/25 patients (72%), and interpretation with the ANRS resistance algorithm found viruses susceptible to the molecules of interest in 20/25 patients (80%) at 5 years (2/5 patients who still had NRTI DRAMs had viruses susceptible to lamivudine/emtricitabine). Characteristics (age, sex, duration of infection and treatment, viral load zenith, CD4+ cell count nadir, total cell HIV-1 DNA level, time since removal of therapeutic pressure, duration of suppressed viraemia, time since last resistant RNA genotype) of patients with and without detectable DRAMs at 5 years were not significantly different (P > 0.05 for all data with the Mann–Whitney test). Comparison of Sanger sequencing and UDS results In 11/67 (16%) sequencing results, some DRAMs detected by UDS and not by Sanger sequencing, with frequencies between 1.1% and 21.3%, led to a different interpretation of resistance by the ANRS algorithm. Thus, Sanger sequencing found viruses interpreted as susceptible to the molecules of interest in 22/25 patients (88%) at 5 years. Notably, some DRAMs detected by UDS and not by Sanger had frequencies higher than 20%, up to 40.7%. Residual viraemia Residual viraemia was found at least once during the 5 years in 13 patients (52%), of whom 9 showed progressive clearance of archived DRAMs (patients 1, 3, 8, 10, 11, 12, 13, 15 and 23), and 4 showed persistence or (re-)emergence of archived DRAMs (patients 6, 7, 17 and 27). Cell-associated HIV-1 DNA quantification The median total cell-associated HIV-1 DNA load remained stable (2.4 log10 copies/106 cells at baseline versus 2.1 log10 copies/106 cells at 5 years). The total cell-associated HIV-1 DNA load was not detectable (<66 copies/106 cells) in 13 samples. APOBEC3-induced G-to-A mutations and defective viruses Two patients had statistically significant (P < 0.05) G-to-A mutation frequencies in their HIV-1 DNA Sanger sequences: patient 20 with 35 G-to-A mutations in 2011 and patient 23 with 3, 3 and 27 G-to-A mutations in 2013, 2014 and 2016 respectively. Thirteen patients (52%) had stop codons and/or G-to-A DRAMs (184I and 230I) in their UDS sequence reads, with frequencies between 2.3% and 100%. These frequencies increased over time in 69% of them. Sanger sequencing detected fewer stop codons and G-to-A mutations than UDS, with frequencies up to 37.5%. Discussion These results show a clear trend towards progressive clearance over time of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA in these 25 patients with suppressed plasma viral load under a therapeutic regimen free from cross-selective pressure on these DRAMs. This dynamic is probably due mainly to preferential residual replication in some reservoirs of the fittest viruses among the quasispecies and the removal of the therapeutic pressure of an entire drug class. These viruses might be released into the blood from reservoirs, leading to persistent residual viraemia under treatment and replenishment of peripheral reservoirs with these viruses free of DRAMs, with clearance of resistant proviruses.6,7,10 However, some DRAMs to the removed therapeutic class could be linked on the same viral variant to other DRAMs conferring resistance to the current regimen, and could therefore be indirectly selected by the therapeutic pressure,7 thus explaining the emergence of some DRAMs. Indeed, DRAMs can emerge even in the case of minimal viral replication, with a suppressed plasma viral load (<50 copies/mL).3,11 The dynamics of DRAMs archived in HIV-1 DNA did not seem to be correlated to the HIV-1 DNA level in the cell reservoir, as these levels did not vary over the 5 years of follow-up. This 5 year period is indeed probably too short to find a significant decrease in HIV-1 DNA level, especially for patients whose treatment was initiated in the chronic phase of infection.12 This study has several limitations. The number of included patients was low, probably because of strict inclusion criteria and the study’s monocentric nature. The low HIV-1 DNA viral load could induce in UDS many reads of a few genomes, in which case evolution of mutations becomes stochastic rather than biological events. The absence of variations in the HIV-1 DNA viral load and the longitudinal aspect of the study are not in favour of this hypothesis. Furthermore, an analysis of DRAM dynamics in anatomical reservoirs could be interesting, as viral genetic diversity and mutational resistance patterns can vary in some body compartments.3 In conclusion, these results show a clear trend towards progressive clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control free of therapeutic selective pressure on those DRAMs. These results provide a rationale for further clinical evaluation of drug recycling in selected patients with sustained virological control under non-optimal regimens, after genotypic resistance testing by UDS in archived HIV-1 DNA. Acknowledgements We thank the ICM Institute at Pitié-Salpêtrière Hospital for access to the Illumina Platform and Yannick Marie for his technical assistance. Funding This work was supported by ‘Agence Nationale de la Recherche sur le Sida et les hépatites virales’ (ANRS). Transparency declarations None to declare. References 1 Morlat P , Blanc A , Bonnet F et al. Prise en charge médicale des personnes vivant avec le VIH, recommandations du groupe d’experts. (Sous l’égide du CNS et de l’ANRS, 2017 ). https://cns.sante.fr/actualities/prise-en-charge-du-vih-recommendations-du-groupe-dexperts/. 2 Engsig FN , Zangerle R , Katsarou O et al. Long-term mortality in HIV-positive individuals virally suppressed for >3 years with incomplete CD4 recovery . Clin Infect Dis 2014 ; 58 : 1312 – 21 . Google Scholar CrossRef Search ADS PubMed 3 Turriziani O , Andreoni M , Antonelli G. Resistant viral variants in cellular reservoirs of human immunodeficiency virus infection . Clin Microbiol Infect 2010 ; 16 : 1518 – 24 . Google Scholar CrossRef Search ADS PubMed 4 Lambotte O , Chaix ML , Gubler B et al. The lymphocyte HIV reservoir in patients on long-term HAART is a memory of virus evolution . AIDS 2004 ; 18 : 1147 – 58 . Google Scholar CrossRef Search ADS PubMed 5 Palmer S , Maldarelli F , Wiegand A et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy . Proc Natl Acad Sci USA 2008 ; 105 : 3879 – 84 . Google Scholar CrossRef Search ADS PubMed 6 Michelini Z , Galluzzo C , Pirillo MF et al. HIV-1 DNA dynamics and variations in HIV-1 DNA protease and reverse transcriptase sequences in multidrug-resistant patients during successful raltegravir-based therapy . J Med Virol 2016 ; 88 : 2115 – 24 . Google Scholar CrossRef Search ADS PubMed 7 Gantner P , Morand-Joubert L , Sueur C et al. Drug resistance and tropism as markers of the dynamics of HIV-1 DNA quasispecies in blood cells of heavily pretreated patients who achieved sustained virological suppression . J Antimicrob Chemother 2016 ; 71 : 751 – 61 . Google Scholar CrossRef Search ADS PubMed 8 Avettand-Fènoël V , Chaix ML , Blanche S et al. LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01) . J Med Virol 2009 ; 81 : 217 – 23 . Google Scholar CrossRef Search ADS PubMed 9 Lapointe HR , Dong W , Lee GQ et al. HIV drug resistance testing by high-multiplex ‘wide’ sequencing on the MiSeq instrument . Antimicrob Agents Chemother 2015 ; 59 : 6824 – 33 . Google Scholar CrossRef Search ADS PubMed 10 Chun TW , Murray D , Justement JS et al. Relationship between residual plasma viremia and the size of HIV proviral DNA reservoirs in infected individuals receiving effective antiretroviral therapy . J Infect Dis 2011 ; 204 : 135 – 8 . Google Scholar CrossRef Search ADS PubMed 11 Martinez-Picado J , DePasquale MP , Kartsonis N et al. Antiretroviral resistance during successful therapy of HIV type 1 infection . Proc Natl Acad Sci USA 2000 ; 97 : 10948 – 53 . Google Scholar CrossRef Search ADS PubMed 12 Viard JP , Burgard M , Hubert JB et al. Impact of 5 years of maximally successful highly active antiretroviral therapy on CD4 cell count and HIV-1 DNA level . AIDS 2004 ; 18 : 45 – 9 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 29, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off