Phylogenetic analysis of HIV-1 archived DNA in blood and gut-associated lymphoid tissue in two patients under antiretroviral therapy.

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.

ART-Treated Patients Exhibit an Adaptive Immune Response against the HFVAC Peptides, a Potential HIV-1 Therapeutic Vaccine (Provir/Latitude45 Study).

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.

Estimating HIV-1 Genetic Diversity in Brazil Through Next-Generation Sequencing.

Approximately 36.7 million people were living with the human immunodeficiency virus (HIV) at the end of 2016 according to UNAIDS, representing a global prevalence rate of 0.8%. In Brazil, an HIV prevalence of 0.24% has been estimated, which represents approximately 830,000 individuals living with the virus. As a touristic and commercial hub in Latin America, Brazil harbors an elevated HIV genetic variability, further contributed by the selective pressure exerted by the host immune system and by antiretroviral treatment. Through the progress of the next-generation sequencing (NGS) techniques, it has been possible to expand the study of HIV genetic diversity, evolutionary, and epidemic processes, allowing the generation of HIV complete or near full-length genomes (NFLG) and improving the characterization of intra- and interhost diversity of viral populations. Greater sensitivity in the detection of viral recombinant forms represents one of the major improvements associated with this development. It is possible to identify unique or circulating recombinant forms using the near full-length viral genomes with increasing accuracy. It also permits the characterization of multiple viral infections within individual hosts. Previous Brazilian studies using NGS to analyze HIV diversity were able to identify several distinct unique and circulating recombinant forms and evidenced dual infections. These data unveiled unprecedented high rates of viral recombination and highlighted that novel recombinants are continually arising in the Brazilian epidemic. In the pooled analysis depicted in this report, HIV subtypes have been determined from HIV-positive patients in five states of Brazil with some of the highest HIV prevalence, three in the Southeast (Rio de Janeiro, São Paulo, and Minas Gerais), one in the Northeast (Pernambuco) and one in the South (Rio Grande do Sul). Combined data analysis showed a significant prevalence of recombinant forms (29%; 101/350), and a similar 26% when only NFLGs were considered. Moreover, the analysis was able to evidence the occurrence of multiple infections in some individuals. Our data highlight the great HIV genetic diversity found in Brazil and unveils a more accurate scenario of the HIV evolutionary dynamics in the region.

Provir/Latitude 45 study: A step towards a multi-epitopic CTL vaccine designed on archived HIV-1 DNA and according to dominant HLA I alleles.

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).

HDV Can Constrain HBV Genetic Evolution in HBsAg: Implications for the Identification of Innovative Pharmacological Targets.

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' 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 < 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.

A New HIV-1 Circulating Recombinant Form (CRF98_cpx) Between CRF06_cpx and Subtype B Identified in Southwestern France.

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.

Diversity of HIV-1 in Aquitaine, Southwestern France, 2012-2016.

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.

A European multicenter study on the analytical performance of the VERIS HBV assay.

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.

Therapeutic Vaccine Against HIV, Viral Variability, Cytotoxic T Lymphocyte Epitopes, and Genetics of Patients.

The scientific and medical community is seeking to cure HIV. Several pathways have been or are being explored including therapeutic vaccination. Viroimmunological studies on primary infection as well as on elite controllers have demonstrated the importance of the cytotoxic CD8 response and have mainly oriented research on vaccine constructs toward this type of response. The results of these trials are clearly not commensurate with the hope placed in them. Might there be one or more uncontrolled variables? The genetics of patients need to be taken into consideration, especially their human lymphocyte antigen (HLA) alleles. There is a need to find a balance between the conservation of cytotoxic T lymphocyte (CTL) epitopes and presentation by HLA alleles. The pathway is a narrow one between adaptation of the virus to HLA I restriction and the definition of conserved proviral CTL epitopes presentable by HLA I alleles. It is likely that the genetics of patients will need to be considered for HIV-1 vaccine studies and that multidisciplinary collaboration will be essential in this field of infectious diseases.

Sanger and Next-Generation Sequencing data for characterization of CTL epitopes in archived HIV-1 proviral DNA.

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.

A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS® TAQMAN® HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit.

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 log10IU/mL between Veris and RealTime, -0.36 log10IU/mL between Veris and Versant, and -0.12 log10IU/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.

A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

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 1000cp/mL showed predicted bias to be <0.3 log10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log10cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log10cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. 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.

European Multicenter Study on Analytical Performance of Veris HIV-1 Assay.

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 log10 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 log10 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 log10 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.).

A European multicentre study on the comparison of HCV viral loads between VERIS HCV assay and COBAS® TaqMan® HCV Test and RealTime HCV 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 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 between -0.42 and -0.22 log10IU/mL and for VERIS HCV Assay versus RealTime HCV Assay between 0.00 and 0.13 log10IU/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.

European Multicenter Study on Analytical Performance of DxN Veris System HCV Assay.

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 log10 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 log10 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.

Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro.

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 <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001). CONCLUSIONS: Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

Detection of prions in the plasma of presymptomatic and symptomatic patients with variant Creutzfeldt-Jakob disease.

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.

Contribution of next generation sequencing to early detection of cytomegalovirus UL97 emerging mutants and viral subpopulations analysis in kidney transplant recipients.

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.