Eve Todesco

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

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

No impact of HIV-1 protease minority resistant variants on the virological response to a first-line PI-based regimen containing darunavir or atazanavir

Objectives To evaluate, in a clinical cohort of HIV-1-infected patients, the prevalence of PI minority resistant variants (MRV) at ART baseline and their impact on the virological response to a first-line PI-based regimen. Patients and methods In an observational single-centre cohort, we assessed all ART-naive patients initiating a first-line regimen including two NRTI and one boosted PI, darunavir/ritonavir or atazanavir/ritonavir, between January 2012 and March 2015. Ultra-deep sequencing of the pol gene was performed using Illumina® technology. Protease mutations were identified using the WHO transmitted drug resistance list and major PI resistance mutations (IAS-USA drug resistance mutations list). Results Ninety-four and 16 patients initiating a darunavir/ritonavir-based regimen and an atazanavir/ritonavir-based regimen, respectively, were assessed. Twenty-eight percent of the patients were HIV-1 subtype B, 39% CRF02_AG and 33% other non-B subtypes. Thirteen patients (13.8%) in the darunavir group and three patients (18.8%) in the atazanavir group experienced a virological failure (VF). Overall, 13 (11.8%) subjects had PI MRV at baseline in the median proportion of 1.3% (IQR = 1.1-1.7). The most prevalent PI MRV were G73C (n = 5) and M46I (n = 3). The proportion of patients harbouring baseline PI MRV was similar between those with virological success (10.6%) and those experiencing VF (18.8%) (P = 0.40). No difference was observed in the rate of PI MRV by viral subtype (P = 0.51) or by PI drug (P = 0.40). Conclusions This study showed a prevalence of 11.8% of PI MRV among 110 ART-naive subjects, without significant impact on the virological response to a first-line PI-based regimen containing darunavir or atazanavir.

Ultradeep sequencing detection of the R263K integrase inhibitor drug resistance mutation

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Disparities in HIV-1 transmitted drug resistance detected by ultradeep sequencing between men who have sex with men and heterosexual populations

Transmitted drug resistance (TDR) can impair the response to first‐line antiretroviral therapy. In treatment‐naïve patients chronically infected with HIV type 1 (HIV‐1), it was previously shown through Sanger sequencing that TDR was more common in men who have sex with men (MSM) than in other transmission risk groups. We aimed to compare two HIV‐1 transmission groups in terms of the presence of TDR mutations.

Low-level HIV-1 viraemia in patients on HAART: risk factors and management in clinical practice

OBJECTIVES Characterization of the conditions favouring HIV-1 low-level viraemia (LLV) during treatment is required to guide strategies for prevention and cure. METHODS The characteristics and treatments of 171 patients experiencing a confirmed LLV of 50-1000 copies/mL (PLLVs) were compared with those of 146 patients with persistently controlled viraemia. We analysed the risk factors for LLV, the parameters affecting the level of viraemia and the presence of resistance-associated mutations (RAMs). We compared outcomes for PLLVs on fully effective HAART as a function of treatment modifications. RESULTS LLV was <500 copies/mL in at least 90% of cases. A higher zenith viral load (VL) (5.27 versus 4.91 log10 copies/mL, OR 2.23; P = 0.0003), a shorter time on continuous HAART (4.3 versus 6.8 years, OR 0.88; P = 0.0003) and previously detected RAMs (43% versus 23%, OR 2.42; P = 0.0033) were independent predictors of LLV. NNRTIs were less frequently used in PLLVs and were associated with more stable treatment. The presence of any RAM during LLV was associated with a lower zenith VL and a higher LLV. In the absence of resistance, virological success was achieved in similar proportions of patients with and without treatment modification. CONCLUSIONS Viraemia >500 copies/mL should no longer be considered to be LLV. In patients with a high zenith VL, several years on continuous HAART may be required to decrease the HIV reservoir and prevent LLV. Resistance testing is useful to detect RAMs, leading if necessary to treatment modifications. In the absence of resistance, treatment changes seemed dispensable.

Multicenter comparison of the new Cobas 6800 system with Cobas Ampliprep/Cobas TaqMan and Abbott RealTime for the quantification of HIV, HBV and HCV viral load

BACKGROUND The new Roche Cobas 6800 platform (C6800) has been recently introduced for viral load (VL) measurement. OBJECTIVES Comparing C6800 to Cobas Ampliprep/Cobas TaqMan v2.0 (CAP/CTM) for the quantification of HIV, HBV and HCV viremia, and to the Abbott RealTime assay (ABB) for HCV quantification. STUDY DESIGN We analysed 121 samples for HBV, and 139 for HIV-1 including 2 groupO and 137 group M viruses (36.5% subtype B, 27.0% CRF02_AG, 22.6% from other clades, and 14% subtype not available). For the 100 HCV samples compared with CAP/CTM, 42% were genotype 1 and 17% were genotype 4. For the 68 HCV samples compared with ABB, 52.9% were genotype 1 and 22.1% were genotype 4. RESULTS C6800 results correlated well with those of CAP/CTM for all three viruses (R2: 0.97-0.99). However, C6800 can yield different viraemia results: higher for HIV (mean difference: +0.11 log10copies/mL, p<0.0001), and lower for HBV (mean difference:-0.11 log10 IU/mL, p<0.0001). Differences exceeded 0.5 log10 for 6.5% of HIV-1 samples and 7.4% of HBV samples. For HCV quantification, C6800 gave mostly lower values than the other assays towards the bottom of the range, and higher values in the upper part of the range, especially in comparisons with ABB, for which 28% of differences exceeded 0.5 log10 IU/mL. No particular HCV genotype was identified as responsible for these differences. CONCLUSION Overall, the comparison tests between C6800 and CAP/CTM systems are satisfactory for the three viruses. Frequent discrepancies were observed between C6800 and ABB for HCV.

Qualitative and quantitative HIV antibodies and viral reservoir size characterization in vertically infected children with virological suppression

Background Absence of detectable viraemia after treatment cessation in some vertically HIV-infected (VHIV) children suggests that early initiation of HAART could lead to functional cure. Objectives We described the factors associated with HIV antibody levels and the viral reservoir size in HAART-treated VHIV children. Methods Study included 97 VHIV children with virological suppression, in Bamako, Mali. The anti-gp41 antibody activities and HIV serostatus were assessed. The viral reservoir size was measured by quantifying total cell-associated HIV DNA. Results Among the children studied, the median total HIV DNA level was 445 copies/10 6 cells (IQR = 187-914) and the median anti-gp41 antibody activity was 0.29 OD (IQR = 0.18-0.75). Low activity of anti-gp41 antibodies was associated with a younger age of HAART initiation ( P  =   0.01). Overall, eight HIV-1 seroreversions were identified. Conclusions Study identified potential candidates with low viral reservoir and low antibody levels or activities for future trials aiming to reduce HIV-1 reservoir to limit HAART duration.

Evaluation of different analysis pipelines for the detection of HIV-1 minority resistant variants

Objective Reliable detection of HIV minority resistant variants (MRVs) requires bioinformatics analysis with specific algorithms to obtain good quality alignments. The aim of this study was to analyze ultra-deep sequencing (UDS) data using different analysis pipelines. Methods HIV-1 protease, reverse transcriptase (RT) and integrase sequences from antiretroviral-naïve patients were obtained using GS-Junior® (Roche) and MiSeq® (Illumina) platforms. MRVs were defined as variants harbouring resistance-mutation present at a frequency of 1%–20%. Reads were analyzed using different alignment algorithms: Amplicon Variant Analyzer®, Geneious® compared to SmartGene® NGS HIV-1 module. Results 101 protease and 51 RT MRVs identified in 139 protease and 124 RT sequences generated with a GS-Junior® platform were analyzed using AVA® and SmartGene® software. The correlation coefficients for the MRVs were R2 = 0.974 for protease and R2 = 0.972 for RT. Discordances (n = 13 in protease and n = 15 in RT) mainly concerned low-level MRVs (i.e., with frequencies of 1%–2%, n = 18/28) and they were located in homopolymeric regions (n = 10/15). Geneious® and SmartGene® software were used to analyze 143 protease, 45 RT and 26 integrase MRVs identified in 172 protease, 69 RT, and 72 integrase sequences generated with a MiSeq® platform. The correlation coefficients for the MRVs were R2 = 0.987 for protease, R2 = 0.995 for RT and R2 = 0.993 for integrase. Discordances (n = 9 in protease, n = 3 in RT, and n = 3 in integrase) mainly concerned low-level MRVs (n = 13/15). Conclusion We found an excellent correlation between the various UDS analysis pipelines that we tested. However, our results indicate that specific attention should be paid to low-level MRVs, for which the use of two different analysis pipelines and visual inspection of sequences alignments might be beneficial. Thus, our results argue for use of a 2% threshold for MRV detection, rather than the 1% threshold, to minimize misalignments and time-consuming sight reading steps essential to ensure accurate results for MRV frequencies below 2%.

Week 96 efficacy of lopinavir/ritonavir monotherapy in virologically suppressed patients with HIV: a randomized non-inferiority trial (ANRS 140 DREAM)

Background Sparing of antiretroviral drug classes could reduce the toxicity and cost of maintenance treatment for HIV infection. Objectives To evaluate the non-inferiority of efficacy and the safety of lopinavir/ritonavir (r) monotherapy versus a single-tablet regimen of efavirenz, emtricitabine and tenofovir (EFV/FTC/TDF) over 2 years. Methods Adults on stable ART with plasma HIV-1 RNA viral load <50 copies/mL for the past 12 months and no documented treatment failure were randomized to receive either lopinavir/r or EFV/FTC/TDF for 2 years. The primary endpoint was the proportion of patients without treatment failure at week 96 (viral load <50 copies/mL at week 96, confirmed at week 98), without study treatment discontinuation, a new AIDS-defining illness, or death. Results In the ITT analysis, the primary endpoint was reached by, respectively, 64% and 71% of patients in the lopinavir/r (n = 98) and EFV/FTC/TDF arms (n = 97), yielding a difference of -6.8% (lower limit of the 95% two-sided CI: -19.9%). Sanger and UltraDeep sequencing showed the occurrence of PI mutations in the lopinavir/r arm (n = 4) and of NNRTI and/or NRTI mutations in the EFV/FTC/TDF arm (n = 2). No unexpected serious clinical events occurred. Conclusions Lopinavir/r monotherapy cannot be considered non-inferior to EFV/FTC/TDF. PI resistance rarely emerged in the lopinavir/r arm and did not undermine future PI options. Two years of lopinavir/r monotherapy had no deleterious clinical impact when compared with EFV/FTC/TDF.

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.