Genotypic Sensitivity Score Research Articles

DORAVIR: a French national survey of people with HIV-1 treated with an antiretroviral regimen including doravirine.

Doravirine is the latest NNRTI to be approved for the treatment of HIV-1 and has a different resistance profile from first-generation NNRTIs. Our aim was to investigate the virological efficacy of antiretroviral treatment including doravirine in people living with HIV-1 (PLWHIV), the factors associated with virological failure (VF) and those associated with the emergence of reverse transcriptase (RT) mutations in the case of VF. A retrospective national survey of PLWHIV who were either naive or experienced on antiretroviral treatment including doravirine was conducted. VF was defined as two consecutive plasma viral loads (VLs) of ≥50 copies/mL or one VL of ≥200 copies/mL. Genotypic resistance tests were interpreted using the Stanford (v9.4.1) and ANRS (v33) algorithms. Of the 589 PLWHIV treated with a doravirine-containing regimen, 8.5% were naive and 91.5% had prior antiretroviral experience; 56.9% were infected with HIV-1 B subtype. Overall, 88.3% and 85.1% of participants were virologically controlled at Month (M)3 and M6 of doravirine treatment, respectively. In multivariable analysis, CRF02_AG subtype, higher zenith plasma HIV-1 RNA VL, doravirine initiation in the context of failure and baseline V179D mutation presence were associated with VF. Among 88 PLWHIV who experienced virological failure at M6, 15.9% had a median of 2 (IQR 1-3) HIV RT mutations. In multivariable analysis, the only factor associated with the occurrence of mutations was a genotypic sensitivity score that was not fully sensitive. This study is one of the largest to characterize the virological efficacy of doravirine-containing regimens in clinical practice and to identify factors associated with VF or emergence of resistance mutations that should be considered in clinical management.

Minimal Cross-resistance to Tenofovir in Children and Adolescents Failing ART Makes Them Eligible for Tenofovir-Lamivudine-Dolutegravir Treatment.

Background:Fixed-dose combination of dolutegravir (DTG) with tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) likely improves adherence and has a favorable resistance profile. We evaluated predicted efficacy of TLD (TDF-3TC-DTG) in children and adolescents failing abacavir (ABC), zidovudine (AZT), or TDF containing regimens.Methods:Drug resistance mutations were analyzed in a retrospective dataset of individuals <19 years of age, failing ABC (n = 293) AZT (n = 288) or TDF (n = 69) based treatment. Pol sequences were submitted to Stanford HIVdb v8.9. Genotypic susceptibility scores were calculated for various DTG-containing regimens.Results:Genotypes were assessed for 650 individuals with a median age of 14 years (IQR 10-17 years). More individuals failed a protease inhibitor (PI)-based (78.3%) than a non-nucleoside reverse transcriptase inhibitors (NNRTI)-based (21.7%) regimen. Most individuals in the AZT group (n = 288; 94.4%) failed a PI-based regimen, compared with 71.0% and 64.2% in the TDF (n = 69) and ABC group (n = 293). Genotypic sensitivity scores <2 to TLD were observed in 8.5% and 9.4% of ABC- and AZT-exposed individuals, compared with 23.2% in the TDF group. The M184V mutation was most often detected in the ABC group (70.6%) versus 60.0% and 52.4% in TDF and AZT groups. The presence of K65R was rare (n = 13, 2.0%) and reduced TLD susceptibility was commonly caused by accumulation of nucleoside reverse transcriptase inhibitor (NRTI) mutations.Conclusions:Cross-resistance to TDF was limited, further reducing concerns about use of transition to TLD in children and adolescents. The NADIA trial has subsequently shown that patients failing a TDF/3TC/EFV regimen can safely be transitioned to a TLD regimen but we do not have data for patients failing an ABC/3TC/NNRTI or PI regimens. Frequent virological monitoring is recommended after switch to DTG, especially in children continuing ABC in the backbone. Clinical studies correlating predicted resistance with clinical outcomes, especially in settings without access to genotyping, are required.

Factors associated with the emergence of integrase resistance mutations in patients failing dual or triple integrase inhibitor-based regimens in a French national survey.

Successful 2-drug regimens (2DRs) for HIV were made possible by the availability of drugs combining potency and tolerability with a high genetic barrier to resistance. How these deal with resistance development/re-emergence, compared with 3DRs, is thus of paramount importance. A national survey including patients who were either naive or experienced with any 2DR or 3DR but failing integrase strand transfer inhibitor (INSTI)-containing regimens [two consecutive plasma viral load (VL) values >50 copies/mL] was conducted between 2014 and 2019. Genotypic resistance tests were interpreted with the v28 ANRS algorithm. Overall, 1104 patients failing any INSTI-containing regimen (2DRs, n = 207; 3DRs, n = 897) were analysed. Five hundred and seventy-seven (52.3%) patients were infected with a B subtype and 527 (47.3%) with non-B subtypes. Overall, 644 (58%) patients showed no known integrase resistance mutations at failure. In multivariate analysis, factors associated with the emergence of at least one integrase mutation were: high VL at failure (OR = 1.24 per 1 log10 copies/mL increase); non-B versus B subtype (OR = 1.75); low genotypic sensitivity score (GSS) (OR = 0.10 for GSS = 2 versus GSS = 0-0.5); and dolutegravir versus raltegravir (OR = 0.46). Although 3DRs versus 2DRs reached statistical significance in univariate analysis (OR = 0.59, P = 0.007), the variable is not retained in the final model. This study is one of the largest studies characterizing integrase resistance in patients failing any INSTI-containing 2DR or 3DR in routine clinical care and reveals factors associated with emergence of integrase resistance that should be taken into consideration in clinical management. No difference was evidenced between patients receiving a 2DR or a 3DR.

Drug resistance mutations in HIV provirus are associated with defective proviral genomes with hypermutation.

HIV proviral sequencing overcomes the limit of plasma viral load requirement by detecting all the 'archived mutations', but the clinical relevance remains to be evaluated. We included 25 participants with available proviral sequences (both intact and defective sequences available) and utilized the genotypic sensitivity score (GSS) to evaluate the level of resistance in their provirus and plasma virus. Defective sequences were further categorized as sequences with and without hypermutations. Personalized GSS score and total GSS score were calculated to evaluate the level of resistance to a whole panel of antiretroviral therapies and to certain antiretroviral therapy that a participant was using. The rate of sequences with drug resistance mutations (DRMs) within each sequence compartment (intact, defective and plasma viral sequences) was calculated for each participant. Defective proviral sequences harbored more DRMs than other sequence compartments, with a median DRM rate of 0.25 compared with intact sequences (0.0, P = 0.014) and plasma sequences (0.095, P = 0.30). Defective sequences with hypermutations were the major source of DRMs, with a median DRM rate of 1.0 compared with defective sequences without hypermutations (0.042, P < 0.001). Certain Apolipoprotein B Editing Complex 3-related DRMs including reverse transcriptase gene mutations M184I, E138K, M230I, G190E and protease gene mutations M46I, D30N were enriched in hypermutated sequences but not in intact sequences or plasma sequences. All the hypermutated sequences had premature stop codons due to Apolipoprotein B Editing Complex 3. Proviral sequencing may overestimate DRMs as a result of hypermutations. Removing hypermutated sequences is essential in the interpretation of proviral drug resistance testing.

Persistent low-level viraemia in antiretroviral treatment-experienced patients is not linked to viral resistance or inadequate drug concentrations.

To assess genotypic sensitivity scores (GSSs), plasma antiretroviral concentrations (PACs) and immunovirological outcomes at Week 96 (W96) in patients with persistent low-level viraemia (LLV). On 1 January 2017, we analysed data from patients on three-drug regimens with persistent LLV defined as at least two consecutive plasma viral loads (pVLs) between 21 and 200 copies/mL (including one pVL of ≥50 copies/mL), at the Pitié-Salpêtrière Hospital. Outcomes were: GSS, PACs and HIV-DNA load at study entry; and virological status and proportion of patients with resistance-associated mutations (RAMs) at W96. Fifty-seven patients were included, with median age of 52.6 years (IQR 45.2-57.9), last CD4 count of 658 cells/mm3 (IQR 462-909) and total ART duration of 10.2 years (IQR 5.7-15.2). LLV duration was 14.0 months (IQR 5.5-22.3). GSS was 3 in 46/57 (81%) patients and PACs were adequate in 53/57 (93%) patients. Median total HIV-DNA was 2.65 log10 copies/106 cells (IQR 2.44-2.86). During follow-up, 26/57 (46%) had experienced ART modifications. At W96, 38/57 (67%) patients remained with LLV, 15/60 (26%) had achieved confirmed pVL of <20 copies/mL and 4/57 (7%) had virological failure. The four virological failures were due to three ART interruptions and one incomplete adherence (selection of Y181C RAM). No factors (patient characteristics at study entry, GSS, PACs, total HIV-DNA load and ART modification) were associated with W96 viral outcome, except for time from HIV diagnosis and the LLV duration at study entry. A substantial number of patients harbouring LLV had no resistance to ART and adequate PACs. Two-thirds of these patients remained with this LLV status.

Resistance to integrase inhibitors: a national study in HIV-1-infected treatment-naive and -experienced patients.

To describe integrase strand transfer inhibitor (INSTI) resistance profiles and factors associated with resistance in antiretroviral-naive and -experienced patients failing an INSTI-based regimen in clinical practice. Data were collected from patients failing an INSTI-containing regimen in a multicentre French study between 2014 and 2017. Failure was defined as two consecutive plasma viral loads (VL) >50 copies/mL. Reverse transcriptase, protease and integrase coding regions were sequenced at baseline and failure. INSTI resistance-associated mutations (RAMs) included in the Agence Nationale de Recherches sur le SIDA genotypic algorithm were investigated. Among the 674 patients, 359 were failing on raltegravir, 154 on elvitegravir and 161 on dolutegravir therapy. Overall, 90% were experienced patients and 389 (58%) patients showed no INSTI RAMs at failure. The strongest factors associated with emergence of at least one INSTI mutation were high VL at failure (OR = 1.2 per 1 log10 copies/mL increase) and low genotypic sensitivity score (GSS) (OR = 0.08 for GSS ≥3 versus GSS = 0-0.5). Patients failing dolutegravir also had significantly fewer INSTI RAMs at failure than patients failing raltegravir (OR = 0.57, P = 0.02) or elvitegravir (OR = 0.45, P = 0.005). Among the 68 patients failing a first-line regimen, 11/41 (27%) patients on raltegravir, 7/18 (39%) on elvitegravir and 0/9 on dolutegravir had viruses with emergent INSTI RAMs at failure. These results confirmed the robustness of dolutegravir regarding resistance selection in integrase in the case of virological failure in routine clinical care.

Prevalence and determinants of resistance mutations in HIV-1-infected patients exposed to integrase inhibitors in a large Italian cohort.

The aim of the study was to analyse the prevalence of integrase resistance mutations in integrase strand transfer inhibitor (INSTI)-experienced HIV-1-infected patients and its predictors. We selected HIV-1 integrase sequences from the Antiviral Response Cohort Analysis (ARCA) database, derived from INSTI-experienced patients between 2008 and 2017. Differences in the prevalence of resistance to raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG) were assessed by χ2 test and predictors of resistance were analysed by logistic regression. We included 462 genotypes from INSTI-exposed individuals: 356 'INSTI-failing' patients and 106 'previously INSTI-exposed' patients (obtained a median of 42 weeks after INSTI discontinuation [interquartile range (IQR) 17-110 weeks]). Overall, at least low-level resistance (LLR) to any INSTI (Stanford 8.5 algorithm) was detected in 198 (42.9%) cases. The most frequent INSTI resistance mutation was N155H, followed by Q148H/K/R, G140A/C/S, E138A/K/T and Y143C/H/R. Y143R and E138A were more prevalent in viral subtype B versus non-B [5.2 versus 1.5%, respectively (P = 0.04), and 3.1 versus 0%, respectively (P = 0.02)]. Overall, the Q148H/K/R plus G140A/C/S and/or E138A/K/T pattern, defining an intermediate level of resistance to DTG, was detected in 70 (15%) cases. Independent predictors of at least LLR to any INSTI were current use versus past use of INSTIs, a lower genotypic sensitivity score (GSS) for contemporary antiretroviral drugs used, and having an integrase sequence obtained in calendar year 2016 as compared to 2008-2009. The results support integrase resistance testing in INSTI-experienced patients. Emergence of INSTI resistance is facilitated by the reduced genetic barrier of the regimen as a consequence of resistance to companion drugs. However, INSTI resistance may become undetectable by standard population sequencing upon INSTI discontinuation.

Assessment of Transmitted HIV-1 Drug Resistance Mutations Using Ultra- Deep Pyrosequencing in a Turkish Cohort.

Antiretroviral treatment (ART) reduces morbidity and mortality caused by human immunodeficiency virus (HIV) infection; however, the emergence of drug-resistant strains poses an important obstacle to treatment success. Using conventional sequencing methods to determine antiretroviral resistance, mutations present in ≥20% of quasispecies can be identified, but drug-resistant minority variants can lead to virologic failure. We aimed to assess transmitted drug resistance mutations (TDRMs) within minority variants using ultra-deep pyrosequencing (UDPS). Treatment-naive adult patients were included in this observational study. Surveillance TDRMs were classified as ≥20% or at minority variant level (≥2% - <20%). Genotypic sensitivity score calculated by using all pre-treatment drug resistance mutations (PDRMs) was also evaluated. Thirty-six patients were analyzed. Any TDRM at ≥20% level was detected in 8.3% of the patients (n=3). This prevalence increased to 30.6% (n=11) with the inclusion of minority variants. All non-nucleoside reverse transcriptase inhibitor and protease inhibitor-related TDRMs were within minority variants. The genotypic sensitivity score of rilpivirine-based regimens was considerably diminished when minority variants were included in the PDRM analysis. UDPS was used for the first time to assess TDRM in a Turkish HIV cohort and uncovered several mutations hidden within minority variants. UDPS may be preferred to detect PDRMs for avoiding virologic failure with rilpivirine-based ART regimens.

HIV-1 drug resistance testing at second-line regimen failure in Arua, Uganda: avoiding unnecessary switch to an empiric third-line.

The number of patients on second-line antiretroviral therapy is growing, but data on HIV drug resistance patterns at failure in resource-constrained settings are scarce. We aimed to describe drug resistance and investigate the factors associated with extensive resistance to nucleoside/nucleotide reverse transcriptase inhibitors (NRTI), in patients failing second-line therapy in the HIV outpatient clinic at Arua Regional Referral Hospital, Uganda. We included patients who failed on second-line therapy (two consecutive viral loads ≥1000 copies/mm3 by SAMBA-1 point-of-care test) and who had a drug resistance test performed between September 2014 and March 2017. Logistic regression was used to investigate factors associated with NRTI genotypic sensitivity score (GSS) ≤1. Seventy-eight patients were included: 42% female, median age 31years and median time of 29months on second-line therapy. Among 70 cases with drug resistance test results, predominant subtypes were A (47%) and D (40%); 18.5% had ≥1 major protease inhibitor mutation; 82.8% had ≥1 NRTI mutation and 38.5% had extensive NRTI resistance (NRTI GSS ≤1). A nadir CD4 count ≤100/ml was associated with NRTI GSS ≤1 (OR 4.2, 95% CI [1.3-15.1]). Thirty (42.8%) patients were switched to third-line therapy, composed of integrase inhibitor and protease inhibitor (60% darunavir/r)+/-NRTI. A follow-up viral load was available for 19 third-line patients at 12months: 84.2% were undetectable. Our study highlights the need for access to drug resistance tests to avoid unnecessary switches to third-line therapy, but also for access to third-line drugs, in particular integrase inhibitors. Low nadir CD4 count might be an indicator of third-line drug requirement for patients failing second-line therapy.

Clinically relevant thresholds for ultrasensitive HIV drug resistance testing: a multi-country nested case-control study

Implementation of ultrasensitive HIV drug resistance tests for routine clinical use is hampered by uncertainty about the clinical relevance of drug-resistant minority variants. We assessed different detection thresholds for pretreatment drug resistance to predict an increased risk of virological failure. We did a case-control study nested within a prospective multicountry cohort. Our study included patients from 12 clinical sites in Kenya, Nigeria, South Africa, Uganda, and Zambia. We defined cases as patients with virological failure (ie, those who had either viral load ≥400 copies per mL at 12 months or had switched to second-line antiretroviral therapy [ART] as a result of virological failure before 12 months) and controls as those with viral suppression (viral load <400 copies per mL at 12 months) on first-line non-nucleoside reverse transcriptase inhibitor-based antiretroviral therapy. We assessed pretreatment drug resistance with Illumina MiSeq next-generation sequencing, using the International Antiviral Society (IAS)-USA mutation list or the Stanford HIV Drug Resistance Database (HIVDB) genotypic sensitivity score. We calculated diagnostic accuracy measures and assessed the odds of virological failure using conditional logistic regression for 1%, 5%, and 10% pretreatment drug resistance detection thresholds, compared with the conventional 20% or more used in Sanger-based sequencing. Paired viral load results before ART and at month 12 of follow-up were available from 1896 participants. We identified 178 patients with virological failure and selected 338 matched controls. We excluded 117 patients from pretreatment drug resistance analysis; therefore, 152 cases of virological failure and 247 controls were included in the final analysis. With the IAS-USA mutation list, at a detection threshold of 20% or more in patients with pretreatment drug resistance, the adjusted odds ratio (OR) for virological failure was 9·2 (95% CI 4·2-20·1) compared with those without pretreatment drug resistance. Lowering the threshold resulted in adjusted ORs of virological failure of 6·8 (95% CI 3·3-13·9) at the 10% threshold, 7·6 (3·4-17·1) at the 5% threshold, and 4·5 (2·0-10·2) at the 1% threshold. Lowering the detection threshold from 20% improved the sensitivity (ie, ability to identify cases) from 12% (n=18) to 13% (n=19) at detection threshold 10%, to 15% (n=23) at detection threshold 5%, and to 17% (n=26) at detection threshold 1%, but caused a slight reduction in specificity (ie, ability to identify controls) from 98% (n=241) to 96% (n=238) at the 10% threshold, 96% (n=236) at the 5% threshold, and a larger reduction to 92% (n=227) at the 1% threshold. Diagnostic ORs were 5·4 (95% CI 2·1-13·9) at the 20% threshold, 3·8 (1·7-8·6) at the 10% threshold, 3·8 (1·8-8·1) at the 5% threshold, and 2·3 (1·2-4·2) at the 1% threshold. Use of the Stanford HIVDB genotypic sensitivity scores yielded similar ORs for virological failure, sensitivities, specificities, and diagnostic ORs. Ultrasensitive resistance testing for pretreatment drug resistance improved identification of people at risk of virological failure; however, this came with a reduction in our ability to identify people with viral suppression, especially at very low thresholds. Further modelling is needed to estimate the optimal trade-off for the 5% and 20% thresholds, balancing improved case finding against unnecessary regimen switching. The Netherlands Ministry of Foreign Affairs, IrsiCaixa, and European Union.

Drug resistance patterns following pharmacy stock shortage in Nigerian Antiretroviral Treatment Program

BackgroundFor patients on antiretroviral therapy (ART), treatment interruptions can impact patient outcomes and result in the accumulation of drug resistance mutations leading to virologic failure. There are minimal published data on the impact of an ART stock shortage on development of drug resistance mutations (DRMs). In this report, we evaluate data from patients enrolled in the Government of Nigeria National ART Program that were receiving treatment at the time of a national drug shortage in late 2003.MethodsWe conducted a cross-sectional evaluation of samples collected between December 2004 and August 2005 from ART patients in virologic failure that either had a treatment interruption or did not during the late 2003 drug shortage period at the Jos University Teaching Hospital (JUTH). Plasma virus was genotyped, sequence data were edited and analyzed, and mutation profiles were categorized to evaluate predicted drug susceptibility. Data were analyzed to examine factors associated with development of resistance mutations. A genotypic sensitivity score to the alternate recommended regimen was computed to assess drug susceptibility if regimens were changed.ResultsA total of 56 patients were included in this evaluation (28 interrupted, 28 uninterrupted). Patients in the interrupted group had more DRMs than those in the uninterrupted group (p < 0.001); interrupted patients were more likely than uninterrupted patients to have one or more TAM-2 mutations (57.1% interrupted vs. 21.3% uninterrupted; p = 0.04). There was a statistically significant difference in resistance to both d4T (53.7% interrupted vs. 17.9 uninterrupted; p = 0.011) and AZT (64.3% interrupted vs. 25.0% uninterrupted; p = 0.003) by drug interruption status. Examining genotypic sensitivity scores, we found that 67.9% of the interrupted patients, as compared to 25.0% of the uninterrupted patients, did not have full susceptibility to one drug in the regimen to which guidelines recommended they be switched (p = 0.001).DiscussionIn this small observational study, we found evidence of a difference in resistance profiles and ART susceptibility between those that were stocked-out of drug versus those that were not. We believe that these data are relevant for many other low- and middle-income countries (LMIC) that also experienced similar ART shortages as they rapidly scaled up their national programs.

Drug resistance testing through remote genotyping and predicted treatment options in human immunodeficiency virus type 1 infected Tanzanian subjects failing first or second line antiretroviral therapy.

IntroductionAntiretroviral therapy (ART) has been successfully introduced in low-middle income countries. However an increasing rate of ART failure with resistant virus is reported. We therefore described the pattern of drug resistance mutations at antiretroviral treatment (ART) failure in a real-life Tanzanian setting using the remote genotyping procedure and thereafter predicted future treatment options using rule-based algorithm and the EuResist bioinformatics predictive engine. According to national guidelines, the default first-line regimen is tenofovir + lamivudine + efavirenz, but variations including nevirapine, stavudine or emtricitabine can be considered. If failure on first-line ART occurs, a combination of two nucleoside reverse transcriptase inhibitors (NRTIs) and boosted lopinavir or atazanavir is recommended.Materials and methodsPlasma was obtained from subjects with first (n = 174) or second-line (n = 99) treatment failure, as defined by clinical or immunological criteria, as well as from a control group of ART naïve subjects (n = 17) in Dar es Salaam, Tanzania. Amplification of the pol region was performed locally and the amplified DNA fragment was sent to Sweden for sequencing (split genotyping procedure). The therapeutic options after failure were assessed by the genotypic sensitivity score and the EuResist predictive engine. Viral load was quantified in a subset of subjects with second-line failure (n = 52).ResultsThe HIV-1 pol region was successfully amplified from 55/174 (32%) and 28/99 (28%) subjects with first- or second-line failure, respectively, and 14/17 (82%) ART-naïve individuals. HIV-1 pol sequence was obtained in 82 of these 97 cases (84.5%). Undetectable or very low (<2.6 log10 copies/10−3 L) viral load explained 19 out of 25 (76%) amplification failures in subjects at second-line ART failure. At first and second line failure, extensive accumulation of NRTI (88% and 73%, respectively) and NNRTI (93% and 73%, respectively) DRMs but a limited number of PI DRMs (11% at second line failure) was observed. First line failure subjects displayed a high degree of cross-resistance to second-generation NNRTIs etravirine (ETR; 51% intermediate and 9% resistant) and rilpivirine (RPV; 12% intermediate and 58% resistant), and to abacavir (ABC; 49% resistant) which is reserved for second line therapy in Tanzania. The predicted probability of success with the best salvage regimen at second-line failure decreased from 93.9% to 78.7% when restricting access to the NRTIs, NNRTIs and PIs currently available in Tanzania compared to when including all approved drugs.DiscussionThe split genotyping procedure is potential tool to analyse drug resistance in Tanzania but the sensitivity should be evaluated further. The lack of viral load monitoring likely results in a high false positive rate of treatment failures, unnecessary therapy switches and massive accumulation of NRTI and NNRTI mutations. The introduction of regular virological monitoring should be prioritized and integrated with drug resistance studies in resource limited settings.

Dolutegravir in HIV-2-Infected Patients With Resistant Virus to First-line Integrase Inhibitors From the French Named Patient Program.

Dolutegravir has shown in vitro activity against human immunodeficiency virus type 2 (HIV-2). We report safety and efficacy data of regimens containing dolutegravir (50 mg twice daily) in antiretroviral-experienced, HIV-2-infected patients. HIV-2-infected patients experiencing virological failure to raltegravir received dolutegravir with optimized background antiretroviral combinations within the French Named Patient Program (NPP). Plasma HIV-2 RNA (pVL) was assessed at time of dolutegravir initiation (baseline), month 3, and month 6. Antiretroviral trough plasma concentrations (C12h) were determined using liquid chromatography coupled with tandem mass spectrometry. Thirteen HIV-2-infected-patients, with a median duration of 15 years' infection and given 16 previous antiretroviral regimens, were included in NPP. Median follow-up was 9 months (min-max, 3-15 months). Median baseline pVL and CD4 cell count were 9544 copies/mL (inter quartile range [IQR], 3096-23 120 copies/mL) and 100 cells/µL (IQR, 77-171 cells/µL), respectively. Available integrase genotypic resistance patterns were Y143C/G/H/R (n = 5), Q148R/K (n = 2), and N155H (n = 4). Optimized background antiretroviral regimens conferring a genotypic sensitivity score ≤2 in 10 patients included nucleoside reverse transcriptase inhibitors associated with darunavir/ritonavir (n = 12), saquinavir/ritonavir (n = 2), and maraviroc (n = 3). At months 3 and 6, pVL was undetectable in 6 of 13 and 4 of 12 patients, respectively, and median CD4 count was 161 (101-188) cells/µL and 167 (135-1353) cells/µL, respectively. Median dolutegravir C12h was 4086 (1756-5717 ng/mL) ng/mL in 9 patients. No serious events were notified except 1 death from progressive multifocal leukoencephalopathy at month 4. Optimized dolutegravir-containing antiretroviral regimens supported by good plasma exposure provide a substantial initial efficacy rate for salvage therapy in heavily antiretroviral-experienced HIV-2-infected patients with virus harboring resistance to first-generation integrase inhibitors. Larger numbers of patients and longer follow-up are needed to confirm these findings.

Improved detection of resistance at failure to a tenofovir, emtricitabine and efavirenz regimen by ultradeep sequencing.

Resistant minority variants present before ART can be a source of virological failure. This has been shown for NRTIs, NNRTIs and CCR5 inhibitors. However, very few data are available for the detection of such minority resistant variants that could be selected at virological failure and not detected using classical Sanger sequencing. We studied 26 patients treated with tenofovir, emtricitabine and efavirenz with their first virological failure (defined as two consecutive viral loads >50 copies/mL). We performed standard Sanger sequencing and ultradeep sequencing (UDS; Roche 454(®) Life Sciences) in plasma at failure. For UDS, mutations >1% were considered. We compared the presence of reverse transcriptase mutations between the two techniques, using the latest ANRS algorithm. UDS detected more resistance mutations in 38.5% of cases (10/26 patients) and the genotypic sensitivity score (GSS) was reduced for 6 of them (23.1%). The GSS was impacted more often for NRTIs than for NNRTIs, for which most mutations were already detected by Sanger sequencing. Resistant minority variants were detected even in patients with low viral load at failure. These results strongly argue for the use of next-generation sequencing in patients failing on an NRTI+NNRTI regimen, as UDS has the potential to modify the choice of the subsequent regimen.

Baseline HIV-1 resistance, virological outcomes, and emergent resistance in the SECOND-LINE trial: an exploratory analysis

WHO-recommended second-line antiretroviral therapy (ART) of a pharmacologically enhanced (boosted) protease inhibitor plus nucleoside or nucleotide reverse transcriptase inhibitors (NtRTIs) might be compromised by resistance. Results of the 96 week SECOND-LINE randomised trial showed that NtRTI-sparing ART with ritonavir-boosted lopinavir and raltegravir (raltegravir-group) provided non-inferior efficacy to ritonavir-boosted lopinavir and two or three NtRTIs (NtRTI-group) in participants with virological failure composed of a first-line regimen of a non-nucleoside reverse transcriptase inhibitor plus two NtRTIs. We report the relation of baseline virological resistance with virological failure and emergent resistance on study. As part of the randomised open-label SECOND-LINE trial, second-line ART NtRTI selection was made by either genotype (local laboratory) or algorithm. Genotypic resistance for the entire cohort at baseline was assessed on stored samples at a central laboratory. Virological failure was defined as plasma viral load greater than 200 copies per mL. Baseline viral isolates were assigned genotypic sensitivity scores (GSSs) by use of the Stanford HIV Database version 6.3.1: a global GSS (gGSS), defined as the combined GSS for lamivudine or emtricitabine, abacavir, zidovudine, stavudine, didanosine, and tenofovir and a specific GSS (sGSS) defined as the GSS for the ART regimen initiated by a specific participant. Emergent resistance was reported on samples with a viral load greater than 500 copies per mL. We used multivariate logistic regression with backward elimination to assess predictors of virological failure and emergent resistance. From April 19, 2010, to July 22, 2013, 271 patients were included in the NtRTI group and and 270 in the raltegravir group. In the NtRTI group 215 had available baseline sequence data, and 240 had viral load measurements at 96 weeks; in the raltegravir group 236 had baseline sequence data and 255 had viral load measurements at 96 weeks. Median (IQR) gGSS was 3.0 (1.3-4.3) in the NtRTI group and 3.0 (1.0-4.3) in the raltegravir group. The median sGSS in the NtRTI group was 1.0 (0.5-1.8). Multivariate analysis showed significant associations between virological failure and less than complete adherence at week 4 (odds ratio [OR] 2.18, 95%CI 1.07-4.47; p=0·03) and week 48 (2.49, 1.09-5.69; p=0.03), baseline plasma viral load greater than 100,000 copies per mL (3.43, 1.70-6.94; p=0.0006), baseline gGSS >4.25 (4.73, 1.94-11.6; p=0.0007), and being Hispanic (3.13, 1.21-8.13; p=0.02) or African (3.49, 1.68-7.28; p=0.0008) rather than Asian. We observed emergent major mutations in one (1%) of 129 participants for protease (both groups), eight (13%) of 64 for reverse transcriptase (NtRTI group) and 16 (20%) of 79 for integrase. Emergent resistance was associated with the raltegravir group (OR 2.47, 95% CI 1.02-5.99; p=0.05), baseline log10 viral load (1.83, 1.12-2.97; p=0.02), and absence of the Lys65Arg (K65R) or Lys70Glu (K70E) mutation at baseline (3.18, 1.12-9.02; p=0.03). Poor adherence was a major determinant of virological failure in people on second-line ART. In settings with limited resources, investment in optimisation of adherence rather than implementation of drug resistance testing might be advisable. University of New South Wales Australia, Merck, AbbVie, and the Foundation for AIDS Research.

Cross-resistance to elvitegravir and dolutegravir in 502 patients failing on raltegravir: a French national study of raltegravir-experienced HIV-1-infected patients.

The objectives of this study were to determine the prevalence and patterns of resistance to integrase strand transfer inhibitors (INSTIs) in patients experiencing virological failure on raltegravir-based ART and the impact on susceptibility to INSTIs (raltegravir, elvitegravir and dolutegravir). Data were collected from 502 treatment-experienced patients failing a raltegravir-containing regimen in a multicentre study. Reverse transcriptase, protease and integrase were sequenced at failure for each patient. INSTI resistance-associated mutations investigated were those included in the last ANRS genotypic algorithm (v23). Among the 502 patients, at failure, median baseline HIV-1 RNA (viral load) was 2.9 log10 copies/mL. Patients had been previously exposed to a median of five NRTIs, one NNRTI and three PIs. Seventy-one percent harboured HIV-1 subtype B and the most frequent non-B subtype was CRF02_AG (13.3%). The most frequent mutations observed were N155H/S (19.1%), Q148G/H/K/R (15.4%) and Y143C/G/H/R/S (6.7%). At failure, viruses were considered as fully susceptible to all INSTIs in 61.0% of cases, whilst 38.6% were considered as resistant to raltegravir, 34.9% to elvitegravir and 13.9% to dolutegravir. In the case of resistance to raltegravir, viruses were considered as susceptible to elvitegravir in 11% and to dolutegravir in 64% of cases. High HIV-1 viral load at failure (P < 0.001) and low genotypic sensitivity score of the associated treatment with raltegravir (P < 0.001) were associated with the presence of raltegravir-associated mutations at failure. Q148 mutations were selected more frequently in B subtypes versus non-B subtypes (P = 0.004). This study shows that a high proportion of viruses remain susceptible to dolutegravir in the case of failure on a raltegravir-containing regimen.

Efficacy, tolerability and virological consequences of long-term use of unboosted atazanavir plus 2 NRTIs in HIV-infected patients.

Switch to unboosted atazanavir (ATV) is an attractive option due to convenience and tolerability in HIV-positive patients. With limited available data we investigated the determinants of long-term efficacy and the consequences of virological failure of unboosted atazanavir-based regimens. Retrospective analysis in two Italian large outpatient clinics including demographic, immunovirological, resistance and pharmacokinetic data. 249 patients receiving atazanavir (400 mg once-daily) plus 2 NRTIs were included; 163 were males (65.5%) and median age was 47 years (42-51.5). Median CD4+ T-cell count was 396/uL (261-583); 146 (58.6%) presented a viral load < 50 copies/mL. Over a median follow up of 157 weeks (106-203) 193 patients (77.5%) were still on treatment with 10 (4%) and 2 (0.8%) stopping for virological failure or toxicity, respectively. Ten patients with virological failure presented newly selected resistance associated mutations (RAMs) for NRTIs (2/10) or ATV (4/10, one I50L). Total cholesterol and triglycerides showed significant decreases at 48 [-4 mg/dL and -41 mg/dL] and 96 weeks [-14 mg/dL and -54 mg/dL] as compared to baseline. At multivariate analysis a genotypic sensitivity score ≤ 1, atazanavir RAMs > 1 and suboptimal adherence were independently associated with virological failure; in lamivudine/emtricitabine-treated patients the presence of M184V (without other NRTI RAMs) was not associated with virological failure. Unboosted-atazanavir containing regimens were efficacious (with uncommon virological failures) and well-tolerated (with improvements in lipid profile over time) treatments in HIV-positive patients. Isolated M184V in lamivudine/emtricitabine recipients was not associated with higher failure rates supporting the use of functional ATV-based dual therapies as maintenance strategies.

Improved virological and immunological efficacy of resistance-guided switch in antiretroviral therapy: a Frankfurt HIV cohort analysis.

To evaluate the treatment outcome of antiretroviral therapy, depending on the use and utility of a concept of resistance-guided switch, patients from the Frankfurt HIV cohort have been followed for 24weeks. If available, prior resistance data have been evaluated and patients were grouped into their expected viral response. The data of 354 patients were thus analysed, taking into account the genotypic sensitivity score of the administered medication (> or ≤2). When looking at the proportion of patients who achieved a viral load of <50/ml, the response rates differed significantly better for patients with a favourable resistance scoring as compared to an unfavourable one (71.9% as compared to 56.0%, p=0.008). Interestingly, patients with a favourable resistance score also showed a better immunological response, as measured by median CD4 cell count of 391/µl [interquartal range (IQR) 250-530/µl] against 287/µl (IQR 174-449/µl) and a larger total increase of 141/µl against 38/µl. A significant virological and immunological benefit could be demonstrated for patients of a cohort with resistance-guided antiretroviral therapy adjustments.

Improved Prediction of Salvage Antiretroviral Therapy Outcomes Using Ultrasensitive HIV-1 Drug Resistance Testing

The clinical relevance of ultrasensitive human immunodeficiency virus type 1 (HIV-1) genotypic resistance testing in antiretroviral treatment (ART)-experienced individuals remains unknown. This was a retrospective, multicentre, cohort study in ART-experienced, HIV-1-infected adults who initiated salvage ART including, at least 1 ritonavir-boosted protease inhibitor, raltegravir or etravirine. Presalvage ART Sanger and 454 sequencing of plasma HIV-1 were used to generate separate genotypic sensitivity scores (GSS) using the HIVdb, ANRS, and REGA algorithms. Virological failure (VF) was defined as 2 consecutive HIV-1 RNA levels ≥200 copies/mL at least 12 weeks after salvage ART initiation, whereas subjects remained on the same ART. The ability of Sanger and 454-GSS to predict VF was assessed by receiver operating characteristic (ROC) curves and survival analyses. The study included 132 evaluable subjects; 28 (21%) developed VF. Using HIVdb, 454 predicted VF better than Sanger sequencing in the ROC curve analysis (area under the curve: 0.69 vs 0.60, Delong test P = .029). Time to VF was shorter for subjects with 454-GSS < 3 vs 454-GSS ≥ 3 (Log-rank P = .003) but not significantly different between Sanger-GSS < 3 and ≥3. Factors independently associated with increased risk of VF in multivariate Cox regression were a 454-GSS < 3 (HR = 4.6, 95 CI, [1.5, 14.0], P = .007), and the number of previous antiretrovirals received (HR = 1.2 per additional drug, 95 CI, [1.1, 1.3], P = .001). Equivalent findings were obtained with the ANRS and REGA algorithms. Ultrasensitive HIV-1 genotyping improves GSS-based predictions of virological outcomes of salvage ART relative to Sanger sequencing. This may improve the clinical management of ART-experienced subjects living with HIV-1. NCT01346878.

A comparison of computational models with and without genotyping for prediction of response to second‐line HIV therapy

We compared the use of computational models developed with and without HIV genotype vs. genotyping itself to predict effective regimens for patients experiencing first-line virological failure. Two sets of models predicted virological response for 99 three-drug regimens for patients on a failing regimen of two nucleoside/nucleotide reverse transcriptase inhibitors and one nonnucleoside reverse transcriptase inhibitor in the Second-Line study. One set used viral load, CD4 count, genotype, plus treatment history and time to follow-up to make its predictions; the second set did not include genotype. Genotypic sensitivity scores were derived and the ranking of the alternative regimens compared with those of the models. The accuracy of the models and that of genotyping as predictors of the virological responses to second-line regimens were compared. The rankings of alternative regimens by the two sets of models were significantly correlated in 60-69% of cases, and the rankings by the models that use a genotype and genotyping itself were significantly correlated in 60% of cases. The two sets of models identified alternative regimens that were predicted to be effective in 97% and 100% of cases, respectively. The area under the receiver-operating curve was 0.72 and 0.74 for the two sets of models, respectively, and significantly lower at 0.55 for genotyping. The two sets of models performed comparably well and significantly outperformed genotyping as predictors of response. The models identified alternative regimens predicted to be effective in almost all cases. It is encouraging that models that do not require a genotype were able to predict responses to common second-line therapies in settings where genotyping is unavailable.