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One of the approaches to cure human immunodeficiency virus (HIV) is the use of therapeutic vaccination. We have launched the Provir/Latitude 45 study to identify conserved CTL epitopes in archived HIV-1 DNA according to the HLA class I alleles in aviremic patients under antiretroviral therapy (ART). A HIV-1 polypeptidic therapeutic vaccine based on viral sequence data obtained from circulating blood was proposed; here, our aim was to compare the proviral DNA in blood and gut-associated lymphoid tissue (GALT). Peripheral blood mononuclear cells and gut biopsies were obtained from two HIV-1 infected patients under successful antiretroviral therapy. Total DNA was extracted including the proviral DNA. The HIV-1 reverse transcriptase was sequenced in both compartments using next generation sequencing followed by single genome sequencing; phylogenetic trees were established and compared. The proviral sequences of both compartments intra-patient exhibited a very low genetic divergence while it was possible to differentiate the sequences inter-patients; single genome sequencing analysis of two couples of samples confirmed that there was no compartmentalization of the sequences intra-patient. We conclude that, considering these two cases, the proviral DNA sequences in blood and GALT are similar and that the epitope analysis of HIV-1 provirus in blood should be considered as relevant to that observed in the GALT, a hard-to-reach major compartment, and can therefore be used for therapeutic vaccine approaches.

International audience; Background: Global HIV-1 genetic diversity and evolution form a major challenge to treatment and prevention efforts. An increasing number of distinct HIV-1 recombinants have been identified worldwide, but their contribution to the global epidemic is unknown. We aimed to estimate the global and regional distribution of HIV-1 recombinant forms during 1990-2015.Methods: We assembled a global HIV-1 molecular epidemiology database through a systematic literature review and a global survey. We searched the PubMed, Embase (Ovid), CINAHL (Ebscohost), and Global Health (Ovid) databases for HIV-1 subtyping studies published from Jan 1, 1990, to Dec 31, 2015. Unpublished original HIV-1 subtyping data were collected through a survey among experts in the field who were members of the WHO-UNAIDS Network for HIV Isolation and Characterisation. We included prevalence studies with HIV-1 subtyping data collected during 1990-2015. Countries were grouped into 14 regions and analyses were done for four time periods (1990-99, 2000-04, 2005-09, and 2010-15). The distribution of circulating recombinant forms (CRFs) and unique recombinant forms (URFs) in individual countries was weighted according to the UNAIDS estimates of the number of people living with HIV in each country to generate regional and global estimates of numbers and proportions of HIV-1 recombinants in each time period. The systematic review is registered with PROSPERO, CRD42017067164.Findings: Our global data collection yielded an HIV-1 molecular epidemiology database of 383 519 samples from 116 countries in 1990-2015. We found that the proportion of recombinants increased over time, both globally and in most regions, reaching 22·8% (7 978 517 of 34 921 639) of global HIV-1 infections in 2010-15. Both the proportion and the number of distinct CRFs detected increased over time to 16·7% and 57 CRFs in 2010-15. The global and regional distribution of HIV-1 recombinants was diverse and evolved over time, and we found large regional variation in the numbers (0-44 CRFs), types (58 distinct CRFs), and proportions (0-80·5%) of HIV-1 recombinants. Globally, CRF02_AG was the most prevalent recombinant, accounting for 33·9% (2 701 364 of 7 978 517) of all recombinant infections in 2010-15. URFs accounted for 26·7% (2 131 450 of 7 978 517), CRF01_AE for 23·0% (1 838 433), and other CRFs for 16·4% (1 307 270) of all recombinant infections in 2010-15. Although other CRFs accounted for small proportions of infections globally (

International audience; We proposed a new HIV-1 therapeutic vaccine based on conserved cytotoxic T lymphocyte (CTL) epitopes of archived HIV-1 DNA according to their affinity to the dominant HLA-A and -B alleles of the population investigated. Our proposal (Hla Fitted VAC, HFVAC) was composed of 15 peptides originating from the RT, gag and nef parts of proviral DNA. Our aim was to investigate baseline immune reactivity to the vaccine in HIV-1 chronically infected patients at success of antiretroviral therapy (ART) who would be eligible for a therapeutic vaccine. Forty-one patients were tested. Most of them had been infected with HIV-1 subtype B and all had been receiving successful ART for 2 to 20 years. The predominant HLA-A and -B alleles were those of a Caucasian population. ELISPOT was carried out using the HFVAC peptides. In 22 patients, the PD-1 marker was investigated on CD4+ and CD8+ T cells by flow cytometry in order to evaluate global T cell exhaustion. ELISPOT positivity was 65% overall and 69% in patients exhibiting at least one HLA allele fitting with HFVAC. The percentages of CD4+ and CD8+ T cells expressing PD-1 were high (median values 23.70 and 32.60, respectively), but did not seem to be associated with an impairment of the immune response investigated in vitro. In conclusion, reactivity to HFVAC was high in this ART-treated population with dominant HLA alleles, despite potential cellular exhaustion associated with the PD-1 marker.

One of the approaches to cure HIV is the use of therapeutic vaccination. We have launched the Provir/Latitude 45 study to identify conserved CTL epitopes in archived HIV-1 DNA according to the HLA class I alleles in aviremic patients under antiretroviral therapy (ART). A HIV-1 polypeptidic therapeutic vaccine based on viral sequence data obtained from circulating blood was proposed; here, our aim was to compare the proviral DNA in blood and gut-associated lymphoid tissue (GALT) at two different levels : nucleotide sequences and potential CTL epitopes. The reverse transcriptase was sequenced in both compartments using next generation sequencing (NGS) in samples from nine individuals, two of which had also single genome sequencing (SGS) performed; phylogenetic trees were established and compared; CTL epitopes were also identified according to their potential affinity for the HLA alleles. The proviral sequences of both compartments intra-patient exhibited a very low genetic divergence while it was possible to differentiate the sequences inter-patients; SGS analysis of two couples of samples confirmed that there was not a compartmentalization of the sequences intra-patient.When we simulated the CTL epitopes which can be presented by the corresponding HLA alleles in both compartments, no significant difference was observed. We conclude that the proviral DNA sequences in blood and GALT are similar and that the epitope analysis in blood can be considered as relevant to that observed in the GALT, a hard-to-reach major compartment, and can therefore be used for therapeutic vaccine approaches.

Dans le cadre de l'enseignement de l'Automatique continue et échantillonnée à l’école d’ingénieurs ESIX Normandie département MeSN (Mécatronique et Systèmes Nomades) et à l'UFR des Sciences à Caen en cycles Licence (L3) et Master EEEA (Electronique, Energie Electrique, Automatique), nous présentons une solution à faible coût permettant de faire de l’acquisition de données et de la commande de procédés en temps réel en utilisant l’environnement logiciel Matlab/Simulink. Cette solution est basée sur l’utilisation d’une carte de type Raspberry Pi associée à une carte de conversion AN/NA. Le spectre des fonctionnalités de la carte Raspberry Pi est assez large. Nous proposons ici de l’utiliser pour répondre à différents aspects d’un problème d’Automatique en temps réel (identification et commande).

Background Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System. 1 Objectives To evaluate the clinical performance of the Veris HBV Assay at multiple EU laboratories Study design Method comparison was performed with a total of 344 plasma specimens from HBV infected patients tested with Veris and COBAS® TaqMan® HBV Test (Cobas), 207 specimens tested with Veris and RealTime HBV Assay (RealTime), 86 specimens tested with Veris and VERSANT® HBV Assay (Versant), and 74 specimens tested with Veris and artus® HBV RG PCR kit (artus). Results Bland-Altman analysis showed average bias of −0.46 log10 IU/mL between Veris and Cobas, −0.46 log10 IU/mL between Veris and RealTime, −0.36 log10 IU/mL between Veris and Versant, and −0.12 log10 IU/mL between Veris and artus. Bias was consistent across the assay range. Patient monitoring results using Veris demonstrated similar viral load trends over time to Cobas, RealTime, and artus. Conclusions The VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris.

Background 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. Study design 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. Results 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 1000 cp/mL showed predicted bias to be Conclusions The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay.

Background 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. Objectives Evaluate the clinical performance of the new quantitative VERIS HCV Assay. Study design 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. Results The average bias between the VERIS HCV Assay and the COBAS® Ampliprep/COBAS® Taqman® HCV Test was 0.04 log10 IU/mL, while between the VERIS HCV Assay and the RealTime HCV Assay average bias was 0.21 log10 IU/mL. Bias, however, was not consistent across the measuring range. Analysis at the lower end of quantification levels 50, 100, and 1000 IU/mL showed a predicted bias for VERIS HCV Assay versus COBAS® Ampliprep/COBAS® Taqman® HCV Test between −0.42 and −0.22 log10 IU/mL and for VERIS HCV Assay versus RealTime HCV Assay between 0.00 and 0.13 log10 IU/mL. Patient monitoring of HCV viral load over time demonstrated similar levels between VERIS HCV Assay results and COBAS® Ampliprep/COBAS® Taqman® HCV Test (52 samples from 13 patients) and RealTime HCV Assay (112 samples from 28 patients). Conclusions VERIS HCV Assay for use on the DxN VERIS Molecular Diagnostic System represents a reliable new tool for easy sample to result HCV RNA viral load monitoring.

The analytical performance of the Veris HCV Assay for use on the new and 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 hepatitis C virus (HCV) genotypes were evaluated. Precision for all sites showed a standard deviation (SD) of 0.22 log 10 IU/ml or lower for each level tested. Analytical sensitivity determined by probit analysis was between 6.2 and 9.0 IU/ml. Specificity on 94 unique patient samples was 100%, and performance with 1,089 negative samples demonstrated 100% not-detected results. Linearity using patient samples was shown from 1.34 to 6.94 log 10 IU/ml. The assay demonstrated linearity upon dilution with all HCV genotypes. The Veris HCV Assay demonstrated an analytical performance comparable to that of currently marketed HCV assays when tested across multiple European sites.

Challenging Diagnosis of Zika Virus Infection Zika virus infection is associated with birth defects. This letter describes the challenging diagnosis of Zika virus infection in a pregnant woman.

One of the approaches by which the scientific community is seeking to cure HIV is the use of therapeutic vaccination. Previous studies have highlighted the importance of the virus-specific CD8+ T cell cytotoxic responses for the immune control of HIV and have oriented research on vaccine constructs based on CTL epitopes from circulating HIV-1 strains. The clinical trials with therapeutic vaccines to date have had limited success likely due to (i) a discrepancy between archived CTL epitopes in the viral reservoir and those in circulating viruses before antiretroviral therapy (ART) initiation and (ii) the lack of strong affinity between the selected CTL epitopes and the HLA grooves for presentation to CD8+ cells. To overcome these limitations, we launched the Provir/Latitude 45 study to identify conserved CTL epitopes in archived HIV-1 DNA according to the HLA class I alleles of aviremic patients, most of whom are under ART. The near full-length genomes or Gag, Pol and Nef regions of proviral DNA were sequenced by Sanger and/or Next Generation Sequencing (NGS). The HLA-A and B alleles were defined by NGS or molecular analysis. The TuTuGenetics software, which moves a sliding window of 8 to 10 amino acids through the amino acid alignment, was combined with the Immune Epitope Data Base (IEDB) to automatically calculate the theoretical binding affinity of identified epitopes to the HLA alleles for each individual. We identified 15 conserved epitopes in Pol (11), Gag (3), and Nef (1) according to their potential presentation by the dominant HLA-A and B alleles and now propose to use the corresponding conserved peptides in a multi-epitopic vaccine (HLA-fitted VAC, HFVAC).

Background Cytomegalovirus (CMV) is the major opportunistic virus encountered after transplantation, and resistant variants challenge antiviral treatment. We studied the emergence and evolution of the canonical UL97 L595S mutation in four kidney recipients by comparing Sanger sequencing with a specific next-generation sequencing (NGS) assay, and assessed the global evolution of UL97 gene variability. Study design Plasmids harbouring wild-type and/or L595S mutated UL97 genes were used to assess the analytical performances of NGS assay. UL97 gene was retrospectively analysed in patients’ samples drawn during CMV infection follow-up, Shannon entropy (Sn) was calculated and phylogenetic analyses were performed. Results Wild-type and L595S plasmids PCR products were mixed to obtain L595S concentrations of 0, 1, 2, 5, 10, 20 and 100%. Mean triplicate NGS results were 0, 0.71, 1.79, 5.30, 13.17, 17 and 100%, respectively, while Sanger sequencing only detected L595S when above 20%. The NGS mean error rate was 0.196 ± 0.07%. In the four patients, emergence of L595S mutation under ganciclovir treatment was followed-up. After foscarnet rescue therapy, leading to undetectable CMV viral load, in two patients, L595S mutant re-emerged, but was only detected by NGS technology (14% and 9.6%). Using NGS data, phylogenetic trees and Sn showed a complex evolution of concomitant viral subpopulations. Conclusions NGS technology allowed a deeper discrimination of the emergence and persistence of a drug resistance mutation, which could be pertinent to investigate when routine Sanger sequencing detects only wild-type strains. Moreover, NGS improved sensitivity helps in studying viral abundance, dynamics and diversity, less approachable with Sanger sequencing.

A major obstacle to using next generation sequencing (NGS) technology in clinical routine practice is reliable data analysis. Thousands of sequences need to be aligned and validated, to exclude sequencing artifacts and generate accurate results. We compared two analysis pipelines for Roche 454 ultradeep pyrosequencing (UDPS) raw data generated from HIV-1 clinical samples: a commercial and fully automated Web-based software NGS HIV-1 Module (SmartGene, Zug, Switzerland) vs. the Amplicon Variant Analyzer software (AVA, 454 Life Sciences; Roche). Results were also compared to those obtained with Sanger sequencing. HIV-1 reverse transcriptase and protease genes from 34 plasma samples were submitted to Sanger sequencing and GS Junior UDPS. Raw UDPS data (sff files) from all samples were analyzed with AVA 2.7 software plus manual review of the alignments and the fully automated SmartGene NGS HIV-1 Module prototype (SMG). Results obtained with both analysis pipelines showed good correlation (85.0%). Divergent results were mainly observed at homopolymer positions, such as K101, where the frame-aware alignment and error corrections of the automated approach were more efficient and more accurate, both in terms of detecting and quantifying drug resistance mutations. Our study shows that NGS data can easily be analyzed via a fully automated analysis pipeline, here the SmartGene NGS HIV-1 Module, thus minimizing the need for manual review of alignments by the user, otherwise essential to ensure accurate results. Such automated analysis pipelines may facilitate the adoption of NGS platforms in the routine clinical laboratory.

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.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.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 (p0.05), 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%, p0.01, 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).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.

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 (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients&rsquo, age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p &lt, 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection.

We have estimated the prevalence of the different viral subtypes between January 2012 and December 2016 in HIV-1-infected patients of the Aquitaine region (southwest part of France) who had a routine HIV-1 genotype resistance testing (GRT) centralized at the Bordeaux University Hospital. GRT was performed on viral RNA (1,784 samples) before treatment initiation or at failure, whereas proviral DNA was used as template (1,420 samples) in the event of a treatment switch in patients with viral load below 50 copies/mL. Pol and integrase sequences were obtained; subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs) were assigned by combining the results of SCUEAL, REGA, COMET, and HIV BLAST. Globally, subtype B was predominant with 71.7%, whereas non-B subtypes accounted for 28.3%. Within the non-B viruses, CRF02_AG was the most prominent (11.6%) followed by non-B non-URF (13.5%), A, CRF01_AE, G, CRF06_cpx, F, C, D, H, J, and finally URF (3.2%). The analysis of the two compartments separately showed that RNA exhibits higher percentages of non-B viruses than DNA. This study reveals a high degree of diversity of HIV-1 non-B subtype strains in Aquitaine, with an increasing prevalence of CRF02_AG and URF in the population investigated for viral RNA, that is, including more recently detected HIV-1-infected patients. Future studies should attempt to identify the transmission clusters while paying special attention to URF, since they seem to be increasing in the population and could potentially host CRF.

During a recent study on the sequencing data of our database between 2012 and 2016 in Southwestern France, we observed that eight patients harbored what seemed to be the same virus. Indeed, routine genotyping at the time of HIV diagnosis showed that protease and reverse transcriptase were related to CRF06_cpx and subtype B, respectively. The integrase sequences (available for three patients) were clustering with CRF06_cpx and envelope (Env) gp120 sequences (available for two patients) with subtype B. Since such a recombinant has not been recorded in the Los Alamos database, we decided to characterize the full-length genome of this virus. The data suggest the identification of a new circulating recombinant form (CRF) between CRF06_cpx and subtype B, the structure of which is very complex with multiple breakpoints. We will refer this CRF as CRF98_cpx.

Background 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. Study design Precision, analytical sensitivity, negative sample performance, linearity and performance with major HBV genotypes/subtypes for the VERIS HBV Assay was evaluated. Results 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 major HBV genotypes/subtypes. Conclusions The VERIS HBV Assay demonstrated comparable analytical performance to other currently marketed assays for HBV DNA monitoring.

Hepatitis B viral load testing is essential to treatment and monitoring decisions in patients with chronic Hepatitis B. Beckman Coulter has developed the VERIS HBV Assay (Veris) for use on the fully automated DxN VERIS Molecular Diagnostics System.Bland-Altman analysis showed average bias of -0.46 logThe VERIS HBV Assay demonstrated comparable clinical performance, with varying degrees of negative bias, compared to other currently marketed assays for HBV DNA monitoring. This negative bias should be taken into consideration if switching monitoring methods to Veris.

One of the strategies for curing viral HIV-1 is a therapeutic vaccine involving the stimulation of cytotoxic CD8-positive T cells (CTL) that are Human Leucocyte Antigen (HLA)-restricted. The lack of efficiency of previous vaccination strategies may have been due to the immunogenic peptides used, which could be different from a patient's virus epitopes and lead to a poor CTL response. To counteract this lack of specificity, conserved epitopes must be targeted. One alternative is to gather as many data as possible from a large number of patients on their HIV-1 proviral archived epitope variants, taking into account their genetic background to select the best presented CTL epitopes. In order to process big data generated by Next-Generation Sequencing (NGS) of the DNA of HIV-infected patients, we have developed a software package called TutuGenetics. This tool combines an alignment derived either from Sanger or NGS files, HLA typing, target gene and a CTL epitope list as input files. It allows automatic translation after correction of the alignment obtained between the HxB2 reference and the reads, followed by automatic calculation of the MHC IC50 value for each epitope variant and the HLA allele of the patient by using NetMHCpan 3.0, resulting in a csv file as output result. We validated this new tool by comparing Sanger and NGS (454, Roche) sequences obtained from the proviral DNA of patients at success of ART included in the Provir Latitude 45 study and showed a 90% correlation between the quantitative results of NGS and Sanger. This automated analysis combined with complementary samples should yield more data regarding the archived CTL epitopes according to the patients' HLA alleles and will be useful for screening epitopes that in theory are presented efficiently to the HLA groove, thus constituting promising immunogenic peptides for a therapeutic vaccine.

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. The precision, analytical sensitivity, performance with negative samples, linearity, and performance with HIV-1 groups/subtypes were evaluated. The precision for the 1-ml assay showed a standard deviation (SD) of 0.14 log 10 copies/ml or less and a coefficient of variation (CV) of ≤6.1% for each level tested. The 0.175-ml assay showed an SD of 0.17 log 10 copies/ml or less and a CV of ≤5.2% for each level tested. The analytical sensitivities determined by probit analysis were 19.3 copies/ml for the 1-ml assay and 126 copies/ml for the 0.175-ml assay. The performance with 1,357 negative samples demonstrated 99.2% with not detected results. Linearity using patient samples was shown from 1.54 to 6.93 log 10 copies/ml. The assay performed well, detecting and showing linearity with all HIV-1 genotypes tested. The Veris HIV-1 assay demonstrated analytical performance comparable to that of currently marketed HIV-1 assays. (DxN Veris products are Conformité Européenne [CE]-marked in vitro diagnostic products. The DxN Veris product line has not been submitted to the U.S. FDA and is not available in the U.S. market. The DxN Veris molecular diagnostics system is also known as the Veris MDx molecular diagnostics system and the Veris MDx system.)

// Romina Salpini 1, * , Matteo Surdo 1, * , Nadia Warner 2 , Maria Francesca Cortese 1 , Danny Colledge 2 , Sally Soppe 2 , Maria Concetta Bellocchi 1 , Daniele Armenia 1 , Luca Carioti 1 , Fabio Continenza 3 , Domenico Di Carlo 1 , Patrizia Saccomandi 1 , Carmen Mirabelli 1, 4 , Michela Pollicita 1 , Roberta Longo 5 , Sara Romano 5 , Giuseppina Cappiello 5 , Alberto Spano 5 , Pascale Trimoulet 6 , Herve Fleury 6 , Jacopo Vecchiet 7 , Nerio Iapadre 8 , Angelo Barlattani 9 , Ada Bertoli 1 , Terenzio Mari 10 , Caterina Pasquazzi 11 , Gabriele Missale 12 , Cesare Sarrecchia 13 , Elisa Orecchini 14 , Alessandro Michienzi 14 , Massimo Andreoni 13 , Simona Francioso 15 , Mario Angelico 15 , Jens Verheyen 16 , Francesca Ceccherini-Silberstein 1 , Stephen Locarnini 2 , Carlo Federico Perno 1 , Valentina Svicher 1 1 Department of Experimental Medicine and Surgery, University of Rome “Tor Vergata” Rome, Italy 2 Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia 3 Laboratory of Monitoring Antiviral Drugs, National Institute for Infectious Diseases (INMI) “Lazzaro Spallanzani” Rome, Italy 4 Institut Pasteur, Unite de Biologie des Virus Enteriques, Paris, France 5 Unit of Microbiology, “S. Pertini Hospital”, Rome, Italy 6 Laboratoire de Microbiologie Fondamentale et Pathogenicite, Hopital Pellegrin Tripode, Bordeaux, France 7 Department of Medicine and Aging Sciences, “SS Annunziata” Hospital, Chieti, Italy 8 Infectious Diseases Unit, “S Salvatore” Hospital, L'Aquila, Italy 9 Hepatology Unit, “S Giacomo” Hospital, Rome, Italy 10 Hepatology Unit, “Regina Margherita” Hospital, Rome, Italy 11 Hepato-Infectivology Unit, “S Andrea” Hospital, Rome, Italy 12 Hospital of Parma, Parma, Italy 13 Tor Vergata University Hospital, Infectious Diseases Unit, Rome, Italy 14 Department of Biomedicine and Prevention, University of Rome “Tor Vergata” Rome, Italy 15 Tor Vergata University Hospital, Hepatology Unit, Rome, Italy 16 Institute of Virology, University Hospital, University of Duisburg-Essen, Essen, Germany * These authors have contributed equally to this work Correspondence to: Carlo Federico Perno, email: cf.perno@uniroma2.it Valentina Svicher, email: valentina.svicher@uniroma2.it Keywords: hepatitis B, hepatocellular carcinoma, hepatitis B surface antigen, HBsAg mutations, cell proliferation Received: November 25, 2016 Accepted: December 27, 2016 Published: February 01, 2017 ABSTRACT Background: An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced 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. Results: Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P

Hepatitis C virus (HCV) protease inhibitors (PIs) and polymerase inhibitors: nucleos(t)ide inhibitors (NS5B-NIs) and non-nucleos(t)ide inhibitors (NS5B-NNIs) have been recently developed to inhibit protease (NS3) or polymerase (NS5B) activities. The drawback of antiviral treatment is the emergence of resistance mutations to the drugs. The prevalence of such mutations conferring resistance to PIs, NS5B-NIs, and NS5B-NNIs before treatment has not been investigated so far in the Tunisian population. The aim of this study was to investigate the prevalence of known substitutions conferring resistance to HCV-PIs, NS5B-NIs, and NS5B-NNIs in 149 untreated patients naive of any novel or investigational anti-HCV drugs and infected with HCV genotype 1 (genotype 1a = 7; genotype 1b = 142). Twelve sequences (9.2%) of the 131/149 HCV NS3 sequences analyzed showed amino-acid substitutions associated with HCV PIs resistance mutations (T54S, n = 4 (3%); V55A, n = 2 (1.5%); Q80K, n = 4 (3%); R155K, n = 1 (0.7%); A156V, n = 1 (0.7%)). One (1%) of the 95/149 HCV NS5B sequences analyzed showed the substitution V321I conferring resistance to NS5B-NIs, while 34 of 95 (35.8%) showed substitutions conferring resistance to NS5B-NNIs (C316N, n = 2 (2%); M414L, n = 1 (1%); A421V, n = 8 (8.5%); M423A, n = 1 (1%); M423T, n = 2 (2%); I424V, n = 5 (5.2%); C445F, n = 1 (1%); I482T, n = 2 (2%); V494A, n = 1 (1%); P496A, n = 1 (1%); V499A, n = 15 (16%); S556G, n = 5 (5.2%)). Naturally occurring substitutions conferring resistance to NS3 or NS5B inhibitors exist in a substantial proportion of Tunisian treatment-naive patients infected with HCV genotype 1. Their influence on treatment outcome should be assessed. J. Med. Virol. 86:1350–1359, 2014. © 2014 Wiley Periodicals, Inc.

Beckman Coulter has developed the VERIS HCV Assay for use on the new fully automated DxN VERIS Molecular Diagnostic SystemEvaluate 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 COBASThe average bias between the VERIS HCV Assay and the COBASVERIS HCV Assay for use on the DxN VERIS Molecular Diagnostic System represents a reliable new tool for easy sample to result HCV RNA viral load monitoring.

Financial and operational constraints limit low-resource countries in the screening of high-risk genital human papillomaviruses (HR-HPV), the etiological agents of cervical cancer. With its simple storage, conservation and shipping, dried cervical sample (DCS) could represent an efficient tool. The aim of the study was to evaluate the reliability of HPV genotyping from DCS. Cervical samples were obtained from 50 women infected with HIV-1 in Cote d'Ivoire. After DNA extraction from both DCS and matched liquid cervical samples (LCS), HPV genotyping was performed and the concordance of genotyping results was evaluated. HPV prevalence was 88% in LCS and 78% in DCS. Kappa statistic was 0.51 for the presence of any genotype (95% confidence interval, 0.25-0.77) and 0.73 for HR-HPV (0.45-0.99). Out of 50 samples, 45 were HPV-positive for DCS and/or LCS, and HR-HPV were detected in 37 samples (74%) with 36 HR-HPV multiple infections. Any genotype and HR genotype identification was concordant/compatible in 86% (43/50) and 88% (44/50) of samples, respectively. In most instances, kappa statistics for detection of type-specific HPV was over 0.6 (including HPV-16, -18, -31, -33). An excellent agreement (kappa statistic ≥ 0.81) was found for eight genotypes (HPV-6, -31, -35, -40, -56, -58, -66, and -82). In spite of interfering factors (multiple infections, different HPV loads, amplification competition, different inputs), DCS and LCS led to concordant/compatible results in most cases. DCS could represent an efficient tool for epidemiological field studies in resource-limited settings, and more importantly for improving the screening coverage and care management in women infected with HPV.

Introduction: Community acquired respiratory virus (CARV) commonly infects patients following hematopoietic stem cell transplantation (HSCT), causing acute infection but also long-term decline in lung function, as a trigger to alloimmune lung syndrome. We conducted a 3-year case-control study to determine the effect of CARV detected by multiplex PCR on airflow decline. Methods: The PFT (Pulmonary functional test) of symptomatic adult HSCT recipients who had a positive PCR for CARV between September 2012 and May 2015 were compared to PFT of those with negative PCR. The viruses detected were Influenza, Respiratory Syncytial Virus, Metapneumovirus, Coronavirus, Rhinovirus, Enterovirus, Parainfluenza, Adenovirus and Bocavirus . Results: During the study period, 112 cases and 39 controls were considered. Cases underwent 179 infectious episodes confirmed by PCR, 40,7% were complicated by lower respiratory tract infection and 47,5% developed GVHD (Graft versus Host disease) post infection. Of 78 cases with positive PCR and complete PFTs, 42% developed persistent FEV-1 decline >5% more than 6 weeks after the episode, versus 18% in the control group (OR 3,35; CI 95% [0,063-0,42]; p = 0,009) and 23% developed persistent FEV-1 decline >10%, versus 7,7% in the control group (OR 3,60; CI 95% [0,062-0,030]; p = 0,041). Conclusion: CARV infection were associated with long term airflow decline in adult HSCT recipients. The role of associated factors like type of virus, stem cell source, smoking status, lymphopenia, hypogammaglobulinemia, treatment by corticosteroids or other immunosuppressive drug, former GVHD or multiple infections will be evaluated in a multivariate analysis.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from the consumption of meat products contaminated by the agent causing bovine spongiform encephalopathy. Evidence supporting the presence of a population of silent carriers that can potentially transmit the disease through blood transfusion is increasing. The development of a blood-screening assay for both symptomatic vCJD patients and asymptomatic carriers is urgently required. We show that a diagnostic assay combining plasminogen-bead capture and protein misfolding cyclic amplification (PMCA) technologies consistently detected minute amounts of abnormal prion protein from French and British vCJD cases in the required femtomolar range. This assay allowed the blinded identification of 18 patients with clinical vCJD among 256 plasma samples from the two most affected countries, with 100% sensitivity [95% confidence interval (CI), 81.5 to 100%], 99.2% analytical specificity (95% CI, 95.9 to 100%), and 100% diagnostic specificity (95% CI, 96.5 to 100%). This assay also allowed the detection of silent carriage of prions 1.3 and 2.6 years before the clinical onset in two blood donors who later developed vCJD. These data provide a key step toward the validation of this PMCA technology as a blood-based diagnostic test for vCJD and support its potential for detecting presymptomatic patients, a prerequisite for limiting the risk of vCJD transmission through blood transfusion.

The emergence of viral-resistant strains is a major problem for the medical management of HIV-infected individuals. The aim of this study was to characterize viral subtypes and drug-resistance mutations (DRMs) in HIV-1 isolates from patients failing antiretroviral therapy (ART). A total of 45 HIV-1-infected patients failing ART were enrolled. The viral RT and Prot genes were amplified and sequenced to determine subtypes and potential DRMs. The subtype distribution was 74% subtype B, 11% subtype A, 9% CRF02-AG, 4% subtype G, and 2% subtype C. Virus samples from 34% of the patients had no DRM while 53%, 27%, and 2% of samples carried at least one DRM conferring resistance to drugs of one, two, or three classes, respectively. DRMs were observed in 50% of the patients infected with non-B strains. The prevalence of nucleoside transcriptase inhibitor (NRTI) mutations was 48%, M184V being largely predominant. The prevalence of nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations was 13%, with K103N present in 57% of samples from NNRTIs-exposed patients. The prevalence of protease inhibitor (PI) mutations was 22%, with major mutations V82A and M46I seen in 16% and 11% of viruses from PI-exposed individuals, respectively. Our study shows the emergence of DRMs in HIV-1 isolates from Moroccan patients failing ART. Although not surprising, the data plead for longitudinal surveys of the dynamics of emergence of DRMs (with a focus on multidrug resistance) in treated patients and circulation of resistant HIV-1 strains in this country.