Papers by Pascale Trimoulet
Clinical Microbiology and Infection
OBJECTIVE We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -r... more OBJECTIVE We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.
Journal of Hepatology, 2001
Gastroentérologie Clinique et Biologique, 2005
Journal of Clinical Virology, 2015
Background. The NS5A protein of the hepatitis C virus has been shown to be involved in the develo... more 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 A c c e p t e d M a n u s c r i p t 4 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.
Background: Mathematical models are widely used for studying the dynamic of infectious agents suc... more Background: Mathematical models are widely used for studying the dynamic of infectious agents such as hepatitis C virus (HCV). Most often, model parameters are estimated using standard least-square procedures for each individual. Hierarchical models have been proposed in such applications. However, another issue is the leftcensoring (undetectable values) of plasma viral load due to the lack of sensitivity of assays used for quantification. A method is proposed to take into account left-censored values for estimating parameters of non linear mixed models and its impact is demonstrated through a simulation study and an actual clinical trial of anti-HCV drugs. Methods: The method consists in a full likelihood approach distinguishing the contribution of observed and leftcensored measurements assuming a lognormal distribution of the outcome. Parameters of analytical solution of system of differential equations taking into account left-censoring are estimated using standard software. Results: A simulation study with only 14% of measurements being left-censored showed that model parameters were largely biased (from-55% to +133% according to the parameter) with the exception of the estimate of initial outcome value when left-censored viral load values are replaced by the value of the threshold. When leftcensoring was taken into account, the relative bias on fixed effects was equal or less than 2%. Then, parameters were estimated using the 100 measurements of HCV RNA available (with 12% of left-censored values) during the first 4 weeks following treatment initiation in the 17 patients included in the trial. Differences between estimates according to the method used were clinically significant, particularly on the death rate of infected cells. With the crude approach the estimate was 0.13 day-1 (95% confidence interval [CI]: 0.11; 0.17) compared to 0.19 day-1 (CI: 0.14; 0.26) when taking into account left-censoring. The relative differences between estimates of individual treatment efficacy according to the method used varied from 0.001% to 37%. Conclusion: We proposed a method that gives unbiased estimates if the assumed distribution is correct (e.g. lognormal) and that is easy to use with standard software.
Journal of Viral Hepatitis
We investigated the seroprevalence and incidence of hepatitis E virus (HEV) infection in men who ... more We investigated the seroprevalence and incidence of hepatitis E virus (HEV) infection in men who have sex with men (MSM) who have been exposed to pre‐exposure prophylaxis (PrEP) against HIV as sexual transmission of HEV has been suggested. A total of 147 PrEP‐using MSM and 147 blood donors matched for sex, age and geographical area were tested for anti‐HEV IgG and IgM. Among them, 135 have been followed for 1 year, at the end of which serological tests for HEV were performed retrospectively on stored samples. Laboratory data on sexual transmitted infections (STIs) and viral hepatitis, including hepatitis A virus (HAV), were collected. Baseline seroprevalence rates in PrEP users were 42.2% (anti‐HEV IgG) and 3.4% (anti‐HEV IgM). Those of the control blood donors were similar (anti‐HEV IgG 43.5% and anti‐HEV IgM 4.1%). There was no incident of HEV infection despite the rates of bacterial STIs (incidence rate (IR) = 46.6%) and HAV infection (IR = 15.8%). Age was the only risk factor associated with anti‐HEV IgG seropositivity at baseline and at the end of follow‐up. Sexual transmission does not seem to be a major route of HEV infection in MSM, unlike HAV.
Viruses, Jul 9, 2018
Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic de... more Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-d sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection ( = 0.001). Results were confirmed after stratification for HBeAg-status and patients’ age. A Sn = 0 at specific positions in the C...
BMC infectious diseases, 2018
HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote ... more HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from litera...
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, Jan 19, 2017
Hepatitis B viral load monitoring is an essential part of managing patients with chronic Hepatits... more Hepatitis B viral load monitoring is an essential part of managing patients with chronic Hepatits B infection. Beckman Coulter has developed the VERIS HBV Assay for use on the fully automated Beckman Coulter DxN VERIS Molecular Diagnostics System.1 OBJECTIVES: To evaluate the analytical performance of the VERIS HBV Assay at multiple European virology laboratories. Precision, analytical sensitivity, negative sample performance, linearity and performance with major HBV genotypes/subtypes for the VERIS HBV Assay was evaluated. Precision showed an SD of 0.15 log10 IU/mL or less for each level tested. Analytical sensitivity determined by probit analysis was between 6.8-8.0 IU/mL. Clinical specificity on 90 unique patient samples was 100.0%. Performance with 754 negative samples demonstrated 100.0% not detected results, and a carryover study showed no cross contamination. Linearity using clinical samples was shown from 1.23-8.23 log10 IU/mL and the assay detected and showed linearity with...
Antiviral therapy, Jan 21, 2017
Recent data have suggested that failure to achieve sustained virological response with direct-act... more Recent data have suggested that failure to achieve sustained virological response with direct-acting antiviral therapy is usually due to relapse and is primarily associated with the emergence of resistance-associated substitutions. The aim of this study was to investigate the prevalence and characterization of non-structural-5A resistance-associated substitutions in patients infected with hepatitis C virus genotypes 1, 3, and 4 treated by direct-acting antiviral therapy, including anti- non-structural-5A, and to characterize the preexisting resistance-associated substitutions in subjects treated with anti- non-structural-5A inhibitors. From January 2014 to March 2016, 2995 patients infected with hepatitis C virus genotypes 1, 3, and 4 were exposed to non-structural-5A inhibitors. Sequencing results at the time of virologic failure were available for 61 patients; sequencing at baseline was available for 35 of these patients. Among the 35 patients with sequencing results available at ...
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, Jul 1, 2017
Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has deve... more Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System.(¥) OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test a...
Journal of clinical microbiology, Jul 19, 2017
The analytical performance of the VERIS HIV-1 Assay for use on the new, fully automated Beckman C... more The analytical performance of the VERIS HIV-1 Assay for use on the new, fully automated Beckman Coulter DxN VERIS Molecular Diagnostics System(¥) was evaluated at 10 European Virology laboratories. Precision, analytical sensitivity, performance with negative samples, linearity, and performance with HIV-1 Groups/Subtypes were evaluated. Precision for the 1 mL assay showed an SD of 0.14 log10 cp/mL or less and CV ≤6.1% for each level tested. The 0.175 mL assay showed an SD of 0.17 log10 cp/mL or less and CV ≤5.2% for each level tested. Analytical sensitivity determined by probit analysis, was determined to be 19.3 cp/mL for the 1 mL assay and 126 cp/mL for the 0.175 mL assay. Performance with 1,357 negative samples demonstrated 99.2% not detected. Linearity using patient samples was shown from 1.54 - 6.93 log10 cp/mL. The assay performed well, detecting and showing linearity with all HIV-1 genotypes tested. The VERIS HIV-1 Assay demonstrated comparable analytical performance to curren...
PloS one, 2017
The emergence of new strains in RNA viruses is mainly due to mutations or intra and inter-genotyp... more The emergence of new strains in RNA viruses is mainly due to mutations or intra and inter-genotype homologous recombination. Non-homologous recombinations may be deleterious and are rarely detected. In previous studies, we identified HCV-1b strains bearing two tandemly repeated V3 regions in the NS5A gene without ORF disruption. This polymorphism may be associated with an unfavorable course of liver disease and possibly involved in liver carcinogenesis. Here we aimed at characterizing the origin of these mutant strains and identifying the evolutionary mechanism on which the V3 duplication relies. Direct sequencing of the entire NS5A and E1 genes was performed on 27 mutant strains. Quasispecies analyses in consecutive samples were also performed by cloning and sequencing the NS5A gene for all mutant and wild strains. We analyzed the mutant and wild-type sequence polymorphisms using Bayesian methods to infer the evolutionary history of and the molecular mechanism leading to the duplic...
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology, May 1, 2017
Beckman Coulter has developed the VERIS HCV Assay for use on the new fully automated DxN VERIS Mo... more Beckman Coulter has developed the VERIS HCV Assay for use on the new fully automated DxN VERIS Molecular Diagnostic System(¥) for HCV viral load monitoring. Evaluate the clinical performance of the new quantitative VERIS HCV Assay. Comparison was performed on 279 plasma specimens from HCV infected patients tested with the VERIS HCV Assay and COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test and 369 specimens tested with the VERIS HCV Assay and RealTime HCV Assay. Patient monitoring sample results from four time points were also compared. The average bias between the VERIS HCV Assay and the COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test was 0.04 log10IU/mL, while between the VERIS HCV Assay and the RealTime HCV Assay average bias was 0.21 log10IU/mL. Bias, however, was not consistent across the measuring range. Analysis at the lower end of quantification levels 50, 100, and 1000IU/mL showed a predicted bias for VERIS HCV Assay versus COBAS(®) Ampliprep/COBAS(®) Taqman(®) HCV Test betwee...
Oncotarget, 2017
Background: An impaired HBsAg-secretion can increase HBV oncogenicproperties. Here, we investigat... more Background: An impaired HBsAg-secretion can increase HBV oncogenicproperties. Here, we investigate genetic-determinants in HBsAg correlated with HBVinduced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation. Methods: This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry.
Journal of clinical microbiology, Apr 1, 2017
The analytical performance of the VERIS HCV Assay for use on the new, fully automated Beckman Cou... more The analytical performance of the VERIS HCV Assay for use on the new, fully automated Beckman Coulter DxN VERIS Molecular Diagnostics System (DxN VERIS System)(¥) was evaluated at 10 European virology laboratories. Precision, analytical sensitivity, specificity and performance with negative samples, linearity, and performance with HCV genotypes were evaluated. Precision for all sites showed an SD of 0.22 log10 IU/mL or lower for each level tested. Analytical sensitivity determined by probit analysis was between 6.2 - 9.0 IU/mL. Specificity on 94 unique patient samples was 100% and performance with 1089 negative samples demonstrated 100% not detected results. Linearity using patient samples was shown from 1.34 - 6.94 log10 IU/mL. The assay demonstrated linearity upon dilution with all HCV genotypes. The VERIS HCV Assay demonstrated comparable analytical performance to currently marketed HCV assays when tested across multiple European sites.
Journal of Clinical Virology, 2015
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Papers by Pascale Trimoulet