Resistance profile and treatment outcomes in HIV-infected children at virological failure in Benin, West Africa.

Background: In Africa a high percentage of HIV-infected children continue to experience HIV treatment failure despite enormous progress. In Benin (West Africa), there are currently no data on HIV drug resistance at failure in paediatric populations. Objectives: To assess the frequency and patterns of HIV drug resistance among children with virological ART failures. Methods: Dried blood spots from 62 HIV-infected children with virological failure were collected at the paediatric clinic of the National Hospital Center in Cotonou for genotyping and plasma drug concentration determination. Results: Characteristics of the population show a median age of 10 years (IQR 6-13) and a median duration on ART of 5 years (IQR 3-7). Viruses from 53 children were successfully amplified. Of these, 76% of patients were on an NNRTI-based regimen and 24% on a boosted PI-based regimen. NRTI, NNRTI and dual-class resistance was present in 71%, 84% and 65% of cases, respectively. Only 4% of the children had major resistance mutations to PIs and none had major resistance mutations to integrase inhibitors. Among the participants, 25% had undetectable antiretroviral concentrations. Conclusions: Our results showed that the development of drug resistance could be one of the main consequences of high and continuous viral replication in HIV-infected children in Benin. Thus, inadequate attention to monitoring lifelong ART in children may prevent achievement of the goal of the United Nations Program on HIV and AIDS (UNAIDS) of 90% viral suppression among patients receiving ART.

Prevalence and clinical impact of minority resistant variants in patients failing an integrase inhibitor-based regimen by ultra-deep sequencing.

Background: Integrase strand transfer inhibitors (INSTIs) are recommended by international guidelines as first-line therapy in antiretroviral-naive and -experienced HIV-1-infected patients. Objectives: This study aimed at evaluating the prevalence at failure of INSTI-resistant variants and the impact of baseline minority resistant variants (MiRVs) on the virological response to an INSTI-based regimen. Methods: Samples at failure of 134 patients failing a raltegravir-containing (n = 65), an elvitegravir-containing (n = 20) or a dolutegravir-containing (n = 49) regimen were sequenced by Sanger sequencing and ultra-deep sequencing (UDS). Baseline samples of patients with virological failure (VF) (n = 34) and of those with virological success (VS) (n = 31) under INSTI treatment were sequenced by UDS. Data were analysed using the SmartGene platform, and resistance was interpreted according to the ANRS algorithm version 27. Results: At failure, the prevalence of at least one INSTI-resistant variant was 39.6% by Sanger sequencing and 57.5% by UDS, changing the interpretation of resistance in 17/134 (13%) patients. Among 53 patients harbouring at least one resistance mutation detected by both techniques, the most dominant INSTI resistance mutations were N155H (45%), Q148H/K/R (23%), T97A (19%) and Y143C (11%). There was no difference in prevalence of baseline MiRVs between patients with VF and those with VS. MiRVs found at baseline in patients with VF were not detected at failure either in majority or minority mutations. Conclusions: UDS is more sensitive than Sanger sequencing at detecting INSTI MiRVs at treatment failure. The presence of MiRVs at failure could be important to the decision to switch to other INSTIs. However, there was no association between the presence of baseline MiRVs and the response to INSTI-based therapies in our study.

Emerging resistance mutations in PI-naive patients failing an atazanavir-based regimen (ANRS multicentre observational study).

Background: Atazanavir is a PI widely used as a third agent in combination ART. We aimed to determine the prevalence and the patterns of resistance in PI-naive patients failing on an atazanavir-based regimen. Methods: We analysed patients failing on an atazanavir-containing regimen used as a first line of PI therapy. We compared the sequences of reverse transcriptase and protease before the introduction of atazanavir and at failure [two consecutive viral loads (VLs) >50 copies/mL]. Resistance was defined according to the 2014 Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) algorithm. Results: Among the 113 patients, atazanavir was used in the first regimen in 71 (62.8%) patients and in the first line of a PI-based regimen in 42 (37.2%). Atazanavir was boosted with ritonavir in 95 (84.1%) patients and combined with tenofovir/emtricitabine or lamivudine (n = 81) and abacavir/lamivudine or emtricitabine (n = 22). At failure, median VL was 3.05 log10 copies/mL and the median CD4+ T cell count was 436 cells/mm3. The median time on atazanavir was 21.2 months. At failure, viruses were considered resistant to atazanavir in four patients (3.5%) with the selection of the following major atazanavir-associated mutations: I50L (n = 1), I84V (n = 2) and N88S (n = 1). Other emergent PI mutations were L10V, G16E, K20I/R, L33F, M36I/L, M46I/L, G48V, F53L, I54L, D60E, I62V, A71T/V, V82I/T, L90M and I93L/M. Emergent NRTI substitutions were detected in 21 patients: M41L (n = 2), D67N (n = 3), K70R (n = 1), L74I/V (n = 3), M184V/I (n = 16), L210W (n = 1), T215Y/F (n = 3) and K219Q/E (n = 2). Conclusions: Resistance to atazanavir is rare in patients failing the first line of an atazanavir-based regimen according to the ANRS. Emergent NRTI resistance-associated mutations were reported in 18% of patients.

Usefulness of an HIV DNA resistance genotypic test in patients who are candidates for a switch to the rilpivirine/emtricitabine/tenofovir disoproxil fumarate combination.

OBJECTIVES: In the context of a rilpivirine/emtricitabine/tenofovir disoproxil fumarate switch in HIV-1-infected patients with at least 1 year of virological success, we determined whether proviral DNA is an alternative to plasma HIV RNA for resistance genotyping. METHODS: Resistance-associated mutations (RAMs) in DNA after at least 1 year of virological success [viral load (VL) <50 copies/mL] were compared with those identified in the last plasma RNA genotype available. Rilpivirine/emtricitabine/tenofovir disoproxil fumarate RAMs studied were K65R, L100I, K101E/P, E138A/G/K/R/Q, V179L, Y181C/I/V, M184V/I, Y188L, H221Y, F227C and M230I/L in the RT. We studied patients without virological failure (VF) and with at least 1 VF (two consecutive VLs >50 copies/mL). Kappa's coefficient was used to measure agreement between the DNA and RNA genotypes. RESULTS: In patients without VF (n = 130) and with VF (n = 114), RNA and DNA showed resistance to at least one drug of the rilpivirine/emtricitabine/tenofovir disoproxil fumarate combination in 8% and 9% and in 60% and 45%, respectively. For rilpivirine RAMs, correlation between RNA and DNA was higher in patients without VF than in patients with VF (kappa = 0.60 versus 0.19, P = 0.026). Overall, the prevalence of RAMs was lower in DNA than in RNA. CONCLUSIONS: Incomplete information provided by the DNA genotypic test is more notable in patients with VF, suggesting that all resistance mutations associated with prior VF have not been archived in the proviral DNA or decreased to a level below the threshold of detection. In the case where no historical plasma genotypic test is available, DNA testing might be useful to rule out switching to rilpivirine/emtricitabine/tenofovir disoproxil fumarate.

Performance of genotypic algorithms for predicting tropism of HIV-1CRF02_AG subtype.

BACKGROUND: Several genotypic rules for predicting HIV-1 non-B subtypes tropism are commonly used, but there is no consensus about their performances. OBJECTIVES: Three genotypic methods were compared for CRF02_AG HIV-1 tropism determination. STUDY DESIGN: V3 env region of 178HIV-1 CRF02_AG from Pitié-Salpêtrière and Saint-Antoine Hospitals was sequenced from plasma HIV-1 RNA. HIV-1 tropism was determined by Geno2Pheno algorithm, false positive rate (FPR) 5% or 10%, the 11/25 rule or the combined criteria of the 11/25 and net charge rule. RESULTS: A concordance of 91.6% was observed between Geno2pheno 5% and the combined criteria. The results were nearly similar for the comparison between Geno2pheno 5% and the 11/25 rule. More mismatches were observed when Geno2pheno was used with the FPR 10%. A lower nadir CD4 cell count was associated with a discordance of tropism prediction between Geno2pheno 5% and the combined criteria or the 11/25 rule (p=0.02 and p=0.03, respectively). A lower HIV-1 viral load was associated with some discordance for the comparison of Geno2pheno 10% and the combined rule (p=0.02). CONCLUSION: Geno2pheno FPR 5% or 10% predicted more X4-tropic viruses for this set of CRF02_AG sequences than the combined criteria or the 11/25 rule alone. Furthermore, Geno2pheno FPR 5% was more concordant with the 11/25 rule and the combined rule than Geno2pheno 10% to predict HIV-1 tropism. Overall, Geno2pheno 5% could be used to predict CRF02_AG tropism as well as other genotypic rules.

Antiretroviral resistance at virological failure in the NEAT 001/ANRS 143 trial: raltegravir plus darunavir/ritonavir or tenofovir/emtricitabine plus darunavir/ritonavir as first-line ART.

OBJECTIVES: To describe the pattern of drug resistance at virological failure in the NEAT001/ANRS143 trial (first-line treatment with ritonavir-boosted darunavir plus either tenofovir/emtricitabine or raltegravir). METHODS: Genotypic testing was performed at baseline for reverse transcriptase (RT) and protease genes and for RT, protease and integrase (IN) genes for patients with a confirmed viral load (VL) >50 copies/mL or any single VL >500 copies/mL during or after week 32. RESULTS: A resistance test was obtained for 110/805 (13.7%) randomized participants qualifying for resistance analysis (61/401 of participants in the raltegravir arm and 49/404 of participants in the tenofovir/emtricitabine arm). No resistance-associated mutation (RAM) was observed in the tenofovir/emtricitabine plus darunavir/ritonavir arm, and all further analyses were limited to the raltegravir plus darunavir arm. In this group, 15/55 (27.3%) participants had viruses with IN RAMs (12 N155H alone, 1 N155H + Q148R, 1 F121Y and 1 Y143C), 2/53 (3.8%) with nucleotide analogue RT inhibitor RAMs (K65R, M41L) and 1/57 (1.8%) with primary protease RAM (L76V). The frequency of IN mutations at failure was significantly associated with baseline VL: 7.1% for a VL of <100,000 copies/mL, 25.0% for a VL of ≥100,000 copies/mL and <500,000 copies/mL and 53.8% for a VL of ≥500,000 copies/mL (PTREND = 0.007). Of note, 4/15 participants with IN RAM had a VL < 200 copies/mL at time of testing. CONCLUSIONS: In the NEAT001/ANRS143 trial, there was no RAM at virological failure in the standard tenofovir/emtricitabine plus darunavir/ritonavir regimen, contrasting with a rate of 29.5% (mostly IN mutations) in the raltegravir plus darunavir/ritonavir NRTI-sparing regimen. The cumulative risk of IN RAM after 96 weeks of follow-up in participants initiating ART with raltegravir plus darunavir/ritonavir was 3.9%.

Rapid plasma viral suppression in naive HIV-infected patients with high CD4 cells and low viraemia initiating a dual nucleoside reverse transcriptase inhibitor strategy: a proof-of-concept study.

OBJECTIVES: To evaluate whether a dual nucleoside reverse transcriptase inhibitor (NRTI) strategy can control HIV replication in antiviral therapy (ART)-naive HIV-infected patients with a high CD4 cell count and a low viral load (VL). METHODS: This observational study included all HIV-infected treatment-naive patients with a CD4 cell count >300 cells/mm(3), a plasma HIV RNA between 1000 copies/mL and 30,000 copies/mL and wild-type virus who initiated dual NRTI ART between January 2008 and December 2012. HIV RNA and CD4 cell count were assessed at Day 0, Week (W) 4, W12, W24 and W48. The primary endpoint was the proportion of patients with a plasma VL (pVL) <50 copies/mL at W24. RESULTS: Twenty patients were included. The median (IQR) baseline characteristics were: time since HIV diagnosis, 25 months (8-66 months); CD4 cell count, 592 cells/mm(3) (405-798 cells/mm(3)); HIV RNA, 10,395 copies/mL (4106-16,566 copies/mL); and HIV DNA, 464 copies/10(6) peripheral blood mononuclear cells (195-1168 copies/10(6) PBMC). Nineteen patients received tenofovir/emtricitabine and one patient received abacavir/lamivudine. At W12, 88% of the patients with available data (n = 16/18, 95% CI 0.65-0.99) had a pVL <50 copies/mL. Overall, the proportion of patients with a pVL <50 copies/mL was 100% (n = 20/20, 95% CI 0.83-1.0) at W24 and 95% (n = 18/19, 95% CI 0.74-0.99) at W48 (with one patient lost to follow-up and one patient with poor treatment compliance). The median increase in CD4 cells was 83 cells/mm(3) (40-310 cells/mm(3)). There was no discontinuation of antiretroviral therapy for any reason such as lack of efficacy or toxicity. CONCLUSIONS: This pilot study suggests that, in patients with a high CD4 cell count and a low VL, a dual NRTI strategy may represent a potentially effective treatment strategy to control HIV replication. This needs to be confirmed in larger controlled clinical studies.

High level of HIV-1 resistance in patients failing long-term first-line antiretroviral therapy in Mali.

OBJECTIVES: In resource-limited settings, few data are available on virological failure after long-term first-line antiretroviral therapy. This study characterized the genotypic resistance patterns at the time of failure after at least 36 months of a first-line regimen in Mali, West Africa. METHODS: Plasma samples from 84 patients who were receiving first-line antiretroviral treatment and with an HIV-1 RNA viral load (VL) >1000 copies/mL were analysed. Genotypic resistance testing was performed and HIV-1 drug resistance was interpreted according to the latest version of the National Agency for HIV and Hepatitis Research algorithm. RESULTS: At the time of resistance testing, patients had been treated for a median of 60 months (IQR 36-132 months) and had a median CD4 cell count of 292 cells/mm(3) (IQR 6-1319 cells/mm(3)), a median HIV-1 RNA level of 28266 copies/mL (IQR 1000-2 93 495 copies/mL) and a median genotypic susceptibility score of 1 (IQR 1-4). The prevalence of nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations was 78% and 82%, respectively. Viruses were resistant to at least one drug in 92% of cases. Although etravirine and rilpivirine were not used in the first-line regimens, viruses were resistant to etravirine in 34% of cases and to rilpivirine in 49% of cases. The treatment duration, median number of NRTI and NNRTI mutations and some reverse transcriptase mutations (T215Y/F/N, L210W, L74I, M41L and H221Y) were associated with the VL at virological failure. CONCLUSIONS: This study demonstrated a high level of resistance to NRTIs and NNRTIs, compromising second-generation NNRTIs, for patients who stayed on long-term first-line regimens. It is crucial to expand the accessibility of virological testing in resource-limited settings to limit the expansion of resistance and preserve second-line treatment efficacy.

Rilpivirine, emtricitabine and tenofovir resistance in HIV-1-infected rilpivirine-naive patients failing antiretroviral therapy.

OBJECTIVES: In the context of simplification strategies, it is essential to know the feasibility of a switch to a rilpivirine-based therapy. The aim of this study was to describe rilpivirine, tenofovir and emtricitabine resistance in HIV-1-infected patients who experienced virological failure during their previous antiretroviral treatment. PATIENTS AND METHODS: The studied population included two groups of patients, all rilpivirine naive, tested for resistance by bulk sequencing from 2008 to 2011: the first group (n = 998) failing a nucleoside reverse transcriptase inhibitor (NRTI) plus boosted protease inhibitor (PI)-based regimen and the second group (n = 3733) failing an NRTI plus non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen. RESULTS: In the first group, the frequency of rilpivirine mutations and resistance to rilpivirine (5.1%) was similar to that in antiretroviral-naive HIV-1-infected patients. Among the 1605 patients from the second group with at least one NNRTI mutation in their HIV, the prevalence of viruses 'resistant' or 'possibly resistant' to efavirenz, nevirapine and etravirine was 78%, 79% and 74%, respectively, while 59% were resistant to rilpivirine. Resistance to rilpivirine was significantly more frequent in non-B subtype versus B subtype viruses. Among pretreated patients with viruses with at least one NNRTI mutation (other than for rilpivirine), 22% of sequences were susceptible to the combination rilpivirine/emtricitabine/tenofovir disoproxil fumarate. CONCLUSIONS: In patients failing an NRTI plus NNRTI-based regimen, to know the feasibility of a switch to rilpivirine/emtricitabine/tenofovir disoproxil fumarate, reliable resistance information should be available at the time of use of concurrent NNRTI therapy.

Genetic barrier to the development of resistance to rilpivirine and etravirine between HIV-1 subtypes CRF02_AG and B.

OBJECTIVES: It has been demonstrated for some drugs that the genetic barrier, defined as the number of genetic transitions and/or transversions needed to produce a resistance mutation, can differ between HIV-1 subtypes. We aimed to assess differences in the genetic barrier for the evolution of resistance to the second-generation non-nucleoside reverse transcriptase inhibitors etravirine and rilpivirine in subtypes B and CRF02_AG in antiretroviral-naive patients. METHODS: An analysis was undertaken of 25 substitutions associated with etravirine and rilpivirine resistance at 12 amino acid positions in 267 nucleotide sequences (136 HIV-1 B and 131 HIV-1 CRF02_AG subtypes) of the reverse transcriptase gene. RESULTS: The majority (7/12) of amino acid positions studied were conserved between the two HIV-1 subtypes, leading to a similar genetic barrier. Different predominant codons between the subtypes were observed in 5/12 positions (90, 98, 179, 181 and 227), with an effect on the calculated genetic barrier only at the V179D and V179F codons (2.5 versus 3.5 for V179D, and 2.5 versus 5 for V179F, respectively, for subtype B versus subtype CRF02_AG). CONCLUSIONS: The majority of amino acids involved in etravirine and rilpivirine resistance showed a high degree of conservation of the predominant codon between the B and CRF02_AG subtypes. For rilpivirine, the genetic barrier was the same between the two subtypes. Nevertheless, subtype CRF02_AG showed a higher genetic barrier to acquiring mutations V179D and V179F (mutations associated with resistance to etravirine) compared with subtype B, suggesting that it would be more difficult to produce resistance to etravirine in the CRF02_AG subtype than the B subtype.

Prevalence of pre-existing resistance-associated mutations to rilpivirine, emtricitabine and tenofovir in antiretroviral-naive patients infected with B and non-B subtype HIV-1 viruses.

OBJECTIVES: The prevalence of rilpivirine, emtricitabine and tenofovir resistance-associated mutations (RAMs), described in vitro and in vivo, was determined in antiretroviral-naive patients. PATIENTS AND METHODS: From 2008 to 2011, 1729 treatment-naive patients were tested for resistance by bulk sequencing. We studied the primary rilpivirine RAMs (K101E/P, E138A/G/K/Q/R, V179L, Y181C/I/V, H221Y, F227C and M230I/L) and other potential rilpivirine-associated mutations (V90I, L100I, K101T, E138S, V179D/I, Y188L, V189I, G190A/E/S and M230V). We also studied the M184V/I and K65R mutations for emtricitabine and tenofovir, respectively. RESULTS: Among 1729 sequences, half of patients had B-subtype viruses and the other half non-B (with 26.7% CRF02, n=461). Primary rilpivirine RAMs were infrequent (4.6%, n=79) and the most prevalent were E138A (3%, n=52), E138K, (0.3%, n=5), H221Y (0.3%, n=5), E138G (0.2%, n=4) and Y181C (0.2%, n=4). The frequency of the primary rilpivirine RAMs was similar between B and non-B subtypes. The other potential rilpivirine-associated mutations that were most prevalent were V179I (8.4%, n=145), V90I (3.8%, n=65) and V189I (2.3%, n=40). The common V179I, V189I and V90I polymorphisms have not been associated with virological failure in Phase 3 clinical studies. By the ANRS algorithm, 4.9% (n=84) of samples were resistant to rilpivirine, 3.7% (n=32) of B-subtype viruses versus 6% (n=52) of non-B-subtype viruses (P=0.02, χ(2) test). The prevalence of K65R and M184I/V was 0.06% (1/1729) and 1% (18/1729), respectively. The prevalence of K103N was 2% (35/1729). CONCLUSIONS: The prevalence of rilpivirine, emtricitabine and tenofovir resistance mutations was very low in antiretroviral-naive patients. The prevalence of resistance to rilpivirine (4.9%, n=84) was not statistically different from the prevalence of efavirenz and nevirapine resistance in our population.

Long-term efficacy of darunavir/ritonavir monotherapy in patients with HIV-1 viral suppression: week 96 results from the MONOI ANRS 136 study.

OBJECTIVES: Long-term results at week 96 are needed to evaluate the capacity of the darunavir/ritonavir monotherapy strategy to maintain a sustained control of the HIV-1 viral load. METHODS: MONOI is a prospective, open-label, non-inferiority, randomized, 96 week trial comparing darunavir/ritonavir monotherapy versus a darunavir/ritonavir triple-therapy strategy to maintain HIV-1 viral load suppression in HIV-1-infected patients. CLINICAL TRIAL REGISTRATION: NCT00412551. RESULTS: From 225 randomized patients, 219 patients reached the 48 week follow-up and 211 reached the 96 week follow-up (106 patients in the darunavir monotherapy arm and 105 in the darunavir triple-therapy arm). Baseline characteristics were well balanced between the two treatment groups. At week 96, in intent-to-treat analysis, 91/103 patients (88%, 95% CI 81-94) allocated to the darunavir/ritonavir monotherapy arm and 87/104 patients (84%, 95% CI 75-90) allocated to the darunavir triple-therapy arm achieved an HIV-1 viral load <50 copies/mL, with no statistical difference between the two groups. Throughout the 96 week follow-up, 66/112 patients (59%, 95% CI 49-68) and 79/113 patients (70%, 95% CI 61-78) consistently had HIV-1 RNA <50 copies/mL with darunavir/ritonavir monotherapy and darunavir/ritonavir triple therapy, respectively. CONCLUSIONS: The MONOI study establishes darunavir/ritonavir monotherapy as durable and efficacious for maintaining virological suppression in HIV-1 patients. Darunavir/ritonavir monotherapy should be considered as a (tailored) treatment option for standard triple-therapy patients who have had a substantial period of viral suppression.

Pharmacokinetics, protein-binding-adjusted inhibitory quotients for atazanavir/ritonavir 300/100 mg in treatment-naïve HIV-infected patients.

OBJECTIVES: Studies have shown the importance of having a high protein-binding-adjusted inhibitory quotient (IQ) for protease inhibitors (PIs) boosted with ritonavir. The objective of this study was to explore the virological response when combination atazanavir/ritonavir was administered to treatment-nai¨ve patients. METHODS: Protein-binding-adjusted IQs were calculated in 100 treatment-nai¨ve patients initiating therapy with atazanavir 300 mg/ritonavir 100 mg plus two nucleoside reverse transcriptase inhibitors. RESULTS: The median atazanavir trough level was 635 ng/mL [interquartile range (IQR) 342-1000] and the median atazanavir protein-binding-adjusted IQ was 45 (IQR 24-71). Eighty-four per cent of patients had a successful virological response, and those who failed did not develop resistance. The IQ for boosted atazanavir is high, resulting in rare treatment failure without resistance mutations. CONCLUSIONS: This study showed that the protein-binding-adjusted IQ of atazanavir is close to those measured for lopinavir and darunavir used once daily in first-line treatment. Finally the selection of resistance in the case of virological failure (plasma viral load 4400 HIV-1 RNA copies/mL) to atazanavir/ritonavir used in first-line therapy seems uncommon, as it is for all boosted PIs.