Elisabeth André-Garnier

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre-emptive therapy.

Two quantitative duplex real‐time PCR assays were developed for co‐amplification of human albumin and cytomegalovirus (CMV) or Epstein Barr virus (EBV) genes after automated extraction on whole blood, and compared two units for expressing viral DNA loads (copies per ml of blood or per 106 peripheral blood leukocytes (PBLs)) on 1,138 positive samples. Both PCRs were characterized by high sensitivity, reproducibility, and linear range. Automated extraction by a MagNA Pure LC Instrument was shown to be more efficient when peripheral blood cell count was inferior to 5 × 109 PBLs/L. Albumin co‐amplification allows the detection of PCR inhibitors and normalization of viral load according to the number of cells calculated in the sample. The two ways of expressing viral load results were highly correlated, but quantitative differences varied in relation to variations of blood cell count. As these two viruses are highly cell associated, viral loads can be underestimated in patients with leucopenia. In the setting of pre‐emptive strategies during CMV infection, the units in which results are expressed can influence clinical management, as illustrated in this article. J. Med. Virol. 81:90–98, 2009.

Nevirapine-raltegravir combination, an NRTI and PI/r sparing regimen, as maintenance antiretroviral therapy in virologically suppressed HIV-1-infected patients.

BACKGROUND Nucleoside reverse transcriptase inhibitor (NRTI) and protease inhibitor (PI)/ritonavir sparing regimens may be useful to some HIV-infected patients. Nevirapine (NVP) and raltegravir (RAL) are both potent antiretrovirals with good long-term safety profiles. METHODS We retrospectively identified from our electronic database all patients with HIV RNA<50 copies/ml for >6 months on an NVP-containing regimen and no prior exposure to integrase strand transfer inhibitors who were switched to RAL plus NVP. Data was collected for 36 months or until discontinuation of RAL plus NVP for any reason. RESULTS A total of 39 patients (30 male) were included in this analysis. Median duration of prior antiretroviral therapy was 14 years (IQR 10-17) and median duration with plasma HIV-1 RNA<50 copies/ml prior to switch was 50 months (IQR 22-96). Switched regimens included mainly a boosted PI (n=24) or tenofovir disoproxil fumarate/emtricitabine (n=12). After switching, the percentages of patients with HIV-1 RNA<50 copies/ml were 87.2% (95% CI 76.7, 97.7) and 82.1% (95% CI 70.0, 94.1) at 6 and 12 months, respectively, in the intent-to-treat-exposed analysis, 97.1% (95% CI 91.6, 100) and 94.1% (95% CI 86.2, 100), respectively, in the per-protocol analysis. All patients with follow-up to month 24 (n=22) or month 36 (n=14) had HIV-1 RNA<50 copies/ml. One virological failure was observed (related to archived non-nucleoside reverse transcriptase inhibitor resistance mutation). During follow-up, no patient experienced Grade 3 or 4 adverse events. Median values of serum creatinine and lipids significantly improved after switch. CONCLUSIONS In patients with prolonged HIV-1 RNA<50 copies/ml, switching to NVP-RAL combination maintained virological suppression throughout 36 months. This combination deserves further evaluation in patients unable to tolerate NRTI or PI/ritonavir agents.

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin

ABSTRACT The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment.

Emergence of HCV resistance-associated variants in patients failing sofosbuvir-based regimens: an observational cohort.

BACKGROUND Real-life effectiveness data of new hepatitis C direct-acting antivirals are now required. The present study aims to assess the rate of sustained viral response (SVR) and virological failure (VF) in patients infected with chronic HCV treated with sofosbuvir (SOF)-based regimens in routine medical practice. METHODS This observational study included a total of 106 patients infected with HCV genotypes (G)1-4, who initiated SOF-based regimens in 2014. Viral load was followed at baseline, week (W)2, W4, W12 (or W24) and W12 post-treatment. For all VFs, resistance-associated variants (RAVs) were determined at baseline and failure by sequencing of NS5A, NS5B and/or NS3 genes, using the Sanger method. RESULTS SVR rate was 85% for the whole cohort, 91% for the patients who underwent the full treatment course. The distribution of HCV genotypes was as follows: G1 n=66 (1a=33, 1b=29; 62%), G2 n=8 (8%), G3a n=20 (19%) and G4 n=12 (11%). The main regimens used were SOF+daclatasvir (37%), SOF+ribavirin (33%), SOF+simeprevir (26%) and SOF+ledipasvir (3%). Twenty-five (23%) patients were HIV-coinfected and 1 was HBV-coinfected. Seventy (65%) patients had a prior treatment experience. All VF were relapses (n=9): 3 G1a, 1 G2, 4 G3a and 1 G4, and mutations conferring resistance to NS5A inhibitors were found but none for NS5B polymerase inhibitors. CONCLUSIONS In a real-life context, the rate of SVR in DAA-treated HCV-infected patients is close to clinical Phase III trial results. RAVs emerged for all patients treated by the anti-NS5A daclatasvir, and persisted several weeks after the end of treatment.

Interpretation of Real-Time PCR Results for Hepatitis C Virus RNA When Viral Load Is Below Quantification Limits

ABSTRACT Hepatitis C virus RNA quantification results obtained in 18 laboratories using real-time PCR methods with 10 negative samples and 22 sample dilutions (viral loads of 0.5 to 500 IU/ml) showed a score of correct results of up to 93.5%. However, 55.6% of the laboratories did not follow the recommendations for the interpretation of their results, leading to ambiguous conclusions.

Phenotypic analysis of HIV-1 E157Q integrase polymorphism and impact on virological outcome in patients initiating an integrase inhibitor-based regimen

Objectives To assess the phenotypic susceptibility of the E157Q polymorphism in HIV-1 integrase (IN) and the virological outcome of patients infected with E157Q-mutated virus initiating an IN inhibitor (INI)-based regimen. Methods This was a multicentre study assessing IN sequences from INI-naive patients among 17 French HIV clinical centres. E157Q site-directed mutants in pNL4.3 and pCRF02_AG contexts were assessed in a recombinant phenotypic assay. Results Prevalence of the E157Q polymorphism was 2.7% among 8528 IN sequences from INI-naive patients and its distribution was 1.7%, 5.6% and 2.2% in B, CRF02_AG and various non-B subtypes, respectively. Thirty-nine INI-naive patients with E157Q-mutated virus initiated an INI-based regimen. Among them, 15 had a viral load (VL) <50 copies/mL at initiation and virological suppression was maintained during the first year of follow-up in all but two exhibiting a viral blip. Twenty-four patients had a VL > 50 copies/mL at the time of INI-based regimen initiation. Among them eight were receiving a first-line regimen and the only two patients who did not reach VL < 50 copies/mL at week 24 were receiving elvitegravir. The 16 remaining patients were ART experienced in virological failure with drug-resistant viruses displaying several virological outcomes independently of the genotypic susceptibility score. Phenotypic analyses showed a fold change in EC50 of 0.6, 0.9 and 1.9 for raltegravir, dolutegravir and elvitegravir, respectively, in a subtype B context, and 1.1, 1.9 and 2.4 for raltegravir, dolutegravir and elvitegravir, respectively, in a CRF02_AG context. Conclusions Assessment of virological response in 39 patients initiating an INI-based regimen with E157Q-mutated virus, in combination with phenotypic analysis, suggests that particular attention should be paid to antiretroviral-naive patients and dolutegravir should be preferentially used in these patients.

Optimizing the virological success of tenofovir DF/FTC/rilpivirine in HIV-infected naive and virologically suppressed patients through strict clinical and virological selection*

Abstract Background: Tenofovir DF/FTC/rilpivirine (TDF/FTC/RPV) is a single tablet regimen considered as safe and efficacious in HIV population as long as food requirements, concomitant PPI administration, and compromised antiviral activity have been carefully reviewed. We evaluated TDF/FTC/RPV in a real-life setting with focus on clinical and virological outcomes. Methods: OCEAN II is a prospective, two-centre observational study. From September 2012 to December 2013, antiretroviral-naive patients with HIV RNA <100,000 copies/mL or wishing to switch for simplification were considered for TDF/FTC/RPV. A systematic review of potential obstacles to TDF/FTC/RPV administration was undertaken during a multidisciplinary meeting, including DNA genotyping to detect archived RPV and/or NRTI-associated resistance mutations if historical RNA resistance testing was lacking. Results: TDF/FTC/RPV was considered for 480 patients, however was not offered to 194 patients (40%), mainly because of risk of insufficient virological efficacy, issues on adherence, patient refusal, meal constraint, or PPI therapy. A total of 286 patients (269 in maintenance; 17 ART-naive) received TDF/FTC/RPV. After a median follow-up of 30 months, virological failure occurred in five patients (1.7%) without the emergence of resistance mutations. Discontinuation of TDF/FTC/RPV occurred in 98 patients, due to adverse events in 43 patients (44%) and non-safety reasons in 55 patients (56%). No grade three-fourth adverse events occurred. Conclusion: In this real-life experience, cohort consisting primarily of virologically suppressed patients, TDF/FTC/RPV usually maintained virologic suppression. Discontinuation of therapy because of intolerability was due to mild adverse events. Strict clinical and virological screening probably explained the low rate of virological failure.

Identification of a duplicated V3 domain in NS5A associated with cirrhosis and hepatocellular carcinoma in HCV-1b patients

BACKGROUND The NS5A protein of the hepatitis C virus has been shown to be involved in the development of hepatocellular carcinoma. OBJECTIVES In a French multicenter study, we investigated the clinical and epidemiological features of a new HCV genotype 1b strain bearing a wide insertion into the V3 domain. STUDY DESIGN We studied NS5A gene sequences in 821 French patients infected with genotype 1b HCV. RESULTS We identified an uncharacterized V3 insertion without ORF disruption in 3.05% of the HCV sequences. The insertion comprised 31 amino-acids for the majority of patients; 3 patients had 27 amino-acids insertions and 1 had a 12 amino-acids insertion. Sequence identity between the 31 amino-acids insertions and the V3 domain ranged from 48 to 96% with E-values above 4e(-5), thus illustrating sequence homology and a partial gene duplication event that to our knowledge has never been reported in HCV. Moreover we showed the presence of the duplication at the time of infection and its persistence at least during 12 years in the entire quasispecies. No association was found with extrahepatic diseases. Conversely, patients with cirrhosis were two times more likely to have HCV with this genetic characteristic (p=0.04). Moreover, its prevalence increased with liver disease severity (from 3.0% in patients without cirrhosis to 9.4% in patients with both cirrhosis and HCC, p for trend=0.045). CONCLUSIONS We identified a duplicated V3 domain in the HCV-1b NS5A protein for the first time. The duplication may be associated with unfavorable evolution of liver disease including a possible involvement in liver carcinogenesis.

The spectrum of HIV mother-to-child transmission risk

With the implementation of combined antiretroviral therapy (cART) and prevention of mother‐to‐child transmission (MTCT) we observed dramatic decreases in rates of perinatal MTCT of HIV, 0.3% in France in women with plasma viral load (pVL) <50 c/mL at delivery. We describe a case of MTCT which occurred despite virologic suppression of the mother at delivery, the first case in our centre since 2002.

Evaluation of residual viremia and quantitation of soluble CD14 in a large cohort of HIV-infected adults on a long-term non-nucleoside reverse transcriptase inhibitor-based regimen.

Beyond virological suppression and immunologic recovery, the objective of long‐term antiretroviral therapy is to suppress maximally viremia and to control for persistent immune activation. Non‐nucleoside reverse transcriptase inhibitor (NNRTI)‐containing regimens are associated with lower residual viremia. The objective of the study was to evaluate the impact of long term NNRTI‐containing treatment on residual viremia and on monocyte activation in a cohort of patients infected with HIV‐1. To identify factors associated with residual viremia, adult patients infected with HIV on nevirapine or efavirenz‐based therapy with viral load <50 copies/ml for >6 months were included. Residual plasma viremia was quantified using an adapted Cobas/Taqman HIV‐1 assay. Viral loads with no detected signal were considered as <1 copy/ml. Monocyte activation was evaluated by quantitation of plasma sCD14 by ELISA assay at the time of residual viremia measurement. Logistic regression was used to determine factors associated with residual viremia <1 copy/ml. In this cohort of 421 patients on long‐term NNRTI regimen, three quarters had a residual viremia <1 copy/ml. In multivariate analysis, duration of plasma viral load below 50 copies/ml was the only factor associated with residual viremia <1 copy/ml. Soluble CD14 was in the normal range although treatment with nevirapine was associated with a significant lower level of sCD14 compared to efavirenz. Residual viremia <1 copy/ml was frequent in this cohort of patients with long term virological control and confirmed the results of previous studies. Apart from its antiviral effect, nevirapine as well as efavirenz could decrease monocyte activation. J Med. Virol. 85:1878–1882, 2013.