HIV co-receptor tropism prediction remains stable over time in treatment-naïve patients.

HIV co-receptor tropism determination is essential before prescribing the CCR5 antagonist maraviroc. British HIV Association guidelines suggest tropism testing may remain valid for only 90 days in antiretroviral-naïve patients. We aimed to determine the accuracy of this figure. Tropism was assessed in 26 antiretroviral-naïve patients with ongoing viral replication, sampled yearly from first clinic visit. The V3 region of HIV-1 was sequenced in triplicate, then tropism predicted using the Geno2Pheno system. Baseline tropism prediction remained valid for a median of 52 months (range 7-81). For 19/26 individuals baseline tropism remained unchanged throughout a median of 54 months follow-up; 18 R5 tropic and 1 X4 tropic. In seven patients (27%) baseline tropism switched at least once (range 1-4 switches) during follow-up; however, their baseline tropism prediction remained valid for a median of 45 months. Co-receptor tropism in treatment-naïve patients with ongoing viral replication appears highly stable over time, suggesting that baseline genotypic tropism prediction may be valid for a longer duration in patients delaying ART initiation. In this study, baseline tropism prediction remained valid for a median of 52 months, suggesting current guidelines recommending repeat testing after 90 days may be excessively conservative in their assessment of tropism stability.

Prevalence of baseline polymorphisms for potential resistance to NS5A inhibitors in drug-naive individuals infected with hepatitis C genotypes 1-4.

BACKGROUND: The non-structural 5A (NS5A) protein of HCV is a multifunctional phosphoprotein involved in regulation of viral replication and virion assembly. NS5A inhibitors targeting domain I of NS5A protein have demonstrated high potency and pan-genotypic antiviral activity, however they possess a low genetic barrier to resistance. At present, only genotype 1, the most prevalent HCV genotype has been studied in detail for resistant variants. METHODS: Utilizing a panel of genotypic-specific resistance assays, population sequencing was performed on plasma-derived viral RNA isolated from 138 patients infected with HCV genotypes 1-4 and not treated with direct-acting antiviral agents. Amino acid changes in HCV NS5A domain I at codon positions 28, 30, 31, 32 and 93, reported to confer reduced susceptibility to certain NS5A inhibitors were examined. Additionally, genotypic outcome based on NS5A sequences were compared with VERSANT HCV Genotype Assay (LiPA) 1.0 (Siemens Healthcare Diagnostics, Surrey, UK) and Abbott m2000 RealTime HCV genotype II assay (Abbott Molecular, Maidenhead, Berkshire, UK). RESULTS: Amino acid substitutions associated with moderate to high level resistance to NS5A inhibitors were detected in 2/42 (4.76%) HCV-1a, 3/23 (13.04%) HCV-1b, 4/26 (15.38%) HCV-2, 1/24 (4.17%) HCV-3 and 1/23 (4.35%) HCV-4 infected patients who had not been treated with NS5A inhibitors. Genotype prediction based on NS5A sequences were concordant with LiPA and/or Abbott RealTime for 97.10% of cases. CONCLUSIONS: Primary resistance mutations associated with resistance to first-generation NS5A inhibitors such as daclatasvir were observed in all genotypes, albeit at low frequencies. An excellent correlation based on NS5A genotyping and LiPA or Abbott RealTime was achieved.

IL28B genotype predicts response to chronic hepatitis C triple therapy with telaprevir or boceprevir in treatment naïve and treatment-experienced patients other than prior partial- and null-responders.

Single nucleotide polymorphisms (SNPs) in the IL28B gene were shown to have limited utility in predicting response to telaprevir and boceprevir in treatment of chronic HCV infection in clinical trials. Data outside of the clinical trial setting are lacking. We assessed the value of single and combined IL28B SNPs rs12979860 and rs8099917 genotypes in predicting sustained virological response 12 weeks after cessation of triple therapy (SVR12) with telaprevir or boceprevir in a single-centre cohort of treatment-naïve and treatment-experienced patients with genotype 1 HCV mono-infection (n = 105). The overall SVR12 rate was 65.7%. By unadjusted bivariate logistic regression analysis, rs12979860-CC and rs8099917-TT were significantly associated with SVR12 in the subgroup of patients including all naïve patients and all treatment-experienced patients with the exception of partial- and null-responders to previous HCV therapy. The predictive value of rs12979860-CC was stronger than rs8099917-TT and only rs12979860-CC remained significantly predictive of treatment success when the two variants were assessed by adjusted logistic regression analysis in the whole study cohort. In patients presenting the rs12979860-CC variant, the additional determination of rs8099917 genotype had no value. IL28B rs12979860-CC remained significantly associated with SVR12 also in the multivariate analysis including the other baseline characteristics associated to SVR12 in the bivariate analysis (i.e., female gender, HCV genotype 1b, baseline viral load <800,000 IU/mL, advanced liver fibrosis and prior partial- or null-response to HCV therapy). Our study suggests that testing for the IL28B rs12979860 genotype may still be useful in predicting response to triple therapy with boceprevir and telaprevir in naïve patients and treatment-experienced patients other than partial and null-responders.

Evaluation of sequencing of HCV core/E1, NS5A and NS5B as a genotype predictive tool in comparison with commercial assays targeting 5'UTR.

BACKGROUND: Hepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options. OBJECTIVE: To establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B. STUDY DESIGN: Genotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA. RESULTS: Of 100 samples from 50 patients, ≥ 2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively. LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available. Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be subtyped by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold. CONCLUSION: Genotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA.

The utility of different bioinformatics algorithms for genotypic HIV-1 tropism testing in a large clinical cohort with multiple subtypes.

OBJECTIVES: HIV-1 tropism needs to be determined before the use of CCR5 antagonist drugs such as maraviroc (MVC), which are ineffective against CXCR4-using HIV-1. This study assessed how different computational methods for predicting tropism from HIV sequence data performed in a large clinical cohort. The value of adding clinical data to these algorithms was also investigated. DESIGN AND METHODS: PCR amplification and sequence analysis of the HIV-1 gp120 V3 loop region was performed on triple replicates of plasma viral RNA or proviral DNA extracted from peripheral blood monocytes (PBMCs) in 242 patients. Coreceptor usage was predicted from V3 sequences using seven bioinformatics interpretation algorithms, combined with clinical data where appropriate. An intention-to-treat approach was employed for exploring outcomes and performance for different viral subtypes was examined. RESULTS: The frequency of R5 predictions varied by 22.6%, with all seven algorithms agreeing for only 75.3% of tests. The identification of individuals likely to fail was poor for all algorithms. The addition of clinical data improved this, but at the expense of their ability to predict success. The clinical algorithms varied across subtypes, whereas other algorithms were more consistent. Furthermore, individuals with discordant clonal and clinical predictions were more likely to fail MVC treatment. CONCLUSION: Eligibility for MVC varied depending on the algorithm method used. The addition of clinical parameters alongside sequence data may help predict X4 emergence during treatment. It could be that V3 loop analysis in isolation may not be the best method for selecting individuals for MVC.

Telaprevir or boceprevir based therapy for chronic hepatitis C infection: development of resistance-associated variants in treatment failure.

The use of triple-therapy, pegylated-interferon, ribavirin and either of the first generation hepatitis C virus (HCV) protease inhibitors telaprevir or boceprevir, is the new standard of care for treating genotype 1 chronic HCV. Clinical trials have shown response rates of around 70-80%, but there is limited data from the use of this combination outside this setting. Through an expanded access programme, we treated 59 patients, treatment naïve and experienced, with triple therapy. Baseline factors predicting treatment response or failure during triple therapy phase were identified in 58 patients. Thirty seven (63.8%) of 58 patients had undetectable HCV RNA 12weeks after the end of treatment. Genotype 1a (p=0.053), null-response to previous treatment (p=0.034), the rate of viral load decline after 12weeks of previous interferon-based treatment (p=0.033) were all associated with triple-therapy failure. The most common cause of on-treatment failure for telaprevir-based regimens was the development of resistance-associated variants (RAVs) at amino acids 36 and/or 155 of HCV protease (p=0.027) whereas in boceprevir-based regimens mutations at amino acid 54 were significant (p=0.015). SVR12 rates approaching 64% were achieved using triple therapy outside the clinical trial setting, in a patient cohort that included cirrhotics.