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BACKGROUND: Rapid diagnostic tests (RDT) have been developed for the detection of hepatitis B surface antigen (HBsAg). They represent a promising alternative to enzyme immunoassays and a powerful tool for large-scale screening and diagnosis of HBV infection, especially in regions without easy access to serological and molecular testing. OBJECTIVES: The aims of the present study were to evaluate the characteristics and clinical performance of a new CE-marked HBsAg RDT, DRW-HBsAg v2.0 assay (Diagnostics for the Real World™, Ltd., USA), in various patient populations, including those chronically infected with HBV, patients with severe acute hepatitis of unknown origin and pregnant women with unknown HBV serological status at delivery. RESULTS: The lower limit of detection of the assay, evaluated in 21 clinical samples, ranged from 0.30±0.07 to 0.97±0.26 international units/mL (using Abbott Architect as a reference), depending on the HBV genotype. The assay tested positive in 100% of patients with chronic hepatitis B, 96.3% of HBsAg-positive acute hepatitis patients, and 95.2% of HBsAg-positive pregnant women. Its specificity was 98.8% in HBsAg-negative patients, 98.7% in HBsAg-negative patients with acute hepatitis of unknown origin and 97.8% in HBsAg-negative pregnant women. Amino acid substitutions in the HBsAg major hydrophilic region did not affect HBsAg detection by DRW-HBsAg v2.0. CONCLUSIONS: The new DRW-HBsAg v2.0 assay is a simple, rapid, easy-to-run and highly sensitive assay that can be used in both high- and low-risk populations for the diagnosis of HBsAg carriage. It appears to be a promising new tool for large-scale screening and diagnosis of HBV infection.

Background In France, there are no consistent data estimating hepatitis delta virus (HDV) prevalence in the general population. Objectives To better characterize HDV/HBV infection and its trends over a 15-years period from 1997 to 2011, we used data retrieved from the National Epidemiological Donors database including viral and demographic characteristics of all French HBV infected blood donors. Study design Of the 39,911,011 donations collected over the 15 year-study-period, 6214 (1.56 in 10 4 donations) were confirmed positive for HBV from which 72.3% were tested for HDV antibodies (Ab). HDV viral load was performed using a real-time PCR assay on positive HDV Ab samples and HDV genotype determined for each positive viremic sample. Results Among the 4492 HBV donations, 89 (1.98%) were HDV Ab positive. After being stable around 1.1% from 1997 to 2005, this rate has continuously increased to reach 6.5% in 2010, before declining to 0.85% in 2011. Of the 61 investigated HDV Ab positive individuals, 22.9% were viremic with a viral load ranging from 10 4 to 9.8 × 10 7 copies mL −1 . Genotyping revealed 12 HDV-1, 1 HDV-6 and 1 HDV-7 in accordance with the geographical origin of individuals. Conclusion Such a study gives unexpected features of HBV–HDV infection in the population of blood donors which is a priori, a healthy population. The increase of HDV prevalence mainly linked to migration of population from endemic countries, demonstrates that there is still no complete control of HBV infection and must encourage HBV vaccination campaigns and systematic screening for HDV in HBV-infected.

Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents.

The risk of hepatitis B virus (HBV) transmission by transfusion is higher than that of other blood-borne viruses. In France, before the introduction of HBV nucleic acid testing (NAT) in 2010, blood donations were tested for hepatitis B surface antigen (HBsAg) and antibodies against hepatitis B core antigen, and the residual risk of HBV transfusion related to preseroconversion acute phase was estimated at 0.54 per million donations. The additional value of the implementation of a highly sensitive HBV NAT to prevent such transmissions is discussed.Two lookback investigations based on HBV seroconversion of repeat donors were performed. Donors and recipients were followed up in multiple samples that were tested for HBV serologic and molecular markers.The recipients have shown posttransfusion HBsAg seroconversion. The archived samples from the implicated donations were positive for HBV DNA at extremely low viral load in both cases. HBV isolates from donors and recipients of each case were organized in the same cluster with 100% identities into Genotypes A2 and B4, respectively. One recipient spontaneously recovered from infection while the second was successfully treated.The present cases highlight the importance of introducing highly sensitive HBV NAT to prevent transmission. Moreover, the lookback studies based on appropriate molecular and serologic investigations of patients transfused with previous donations from newly identified HBV-infected repeat donors offer the opportunity to treat a recently infected recipient.

Most industrialized countries maintain surveillance programs for monitoring transmissible infection in blood donations, revising approaches to methodology and risk assessment as new threats emerge. A comparison of programs in the United States, Canada, France, the UK, and Australia indicates that they have similar function, although the structure of blood programs vary as does the extent and nature of formal ties with public health. The emergence of HIV in the late 1970s and early 1980s was key in recognizing that surveillance systems specific to blood transfusion were essential. Hence, most industrialized countries monitor transfusion-transmissible infections in donors and evaluate the impact of new testing and of predonation screening strategies. Emerging infections since HIV have had different transmission pathways and challenged blood programs to draw upon resources for a rapid and effective response, with recognition that the original focus on sexual/drug-related risk of HIV and hepatitis was inadequate. The focus of surveillance programs on new and emerging pathogens fulfills a key role in risk assessment and policy formulation. The precise nature of such activities varies by country because of the structure of the blood programs and surveillance systems, the strategic focus of the blood programs, and the epidemiology of disease in each country.

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.

The epidemic history of HCV genotype 5a is poorly documented in France, where its prevalence is very low, except in a small central area, where it accounts for 14.2% of chronic hepatitis C cases. A Bayesian coalescent phylogenetic investigation based on the E1 envelope gene and a non-structural genomic segment (NS3/4) was carried out to trace the origin of this epidemic using a large sample of genotype 5a isolates collected throughout France. The dates of documented transmissions by blood transfusion were used to calibrate five nodes in the phylogeny. The results of the E1 gene analysis showed that the best-fitting population dynamic model was the expansion growth model under a relaxed molecular clock. The rate of nucleotide substitutions and time to the most recent common ancestors (tMRCA) of genotype 5a isolates were estimated. The divergence of all the French HCV genotype 5a strains included in this study was dated to 1939 [95% HPD: 1921–1956], and the tMRCA of isolates from central France was dated to 1954 [1942–1967], which is in agreement with epidemiological data. NS3/4 analysis provided similar estimates with strongly overlapping HPD values. Phylodynamic analyses give a plausible reconstruction of the evolutionary history of HCV genotype 5a in France, suggesting the concomitant roles of transfusion, iatrogenic route and intra-familial transmission in viral diffusion.

International audience; AIM: To screen hepatitis B virus (HBV) genotypes and associated basal core promoter (BCP; T1762A/A1764) and precore (PC; A1896) mutations among the 100 HBV surface antigen (HBsAg) positive voluntary blood donors in France.METHODS: HBV genotypes were determined by using direct sequence analysis. Three methods were used to detect G1896A mutation: non-commercial real-time PCR (PCRTR°, line probe assay (InnoLiPA HBV PreCore, INNOGENETICS(®)) and direct sequencing of precore gene. HBV viral load was quantified with two commercial real-time PCR (COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV Test/Roche and Real Time HBV/M2000/Abbott).RESULTS: The mean age of donors was 30 (18-64). Patients were from Africa (42%), Europa (50%), and Asia (8%). HBV/D was the most predominant (37%) genotype followed by HBV/A (31%) and HBV/E (22%). PC and BCP mutants were found in 57% with Inno-LIPA HBV test and 59% with both PCRTR and sequencing methods. A significant difference in the viral load of blood donors with wild and PC mutants was observed with the Taqman Cobas real time PCR (3,19 Log(10) UI/ml versus 4,93 Log(10) UI/ml, pCONCLUSIONS: Non-Caucasian genotype E was present in the French blood donors. PC mutation was more common than BCP mutations in this study. As HBV infected blood donors were more often asymptomatic carriers, we could speculate that the G1896A mutation may favour the asymptomatic state, supporting previous observations.; Buts du travailPlusieurs études internationales ont décrit une prévalence élevée de la mutation précore G1896A (PC) chez les donneurs de sang. Nous avons mené un projet en collaboration avec l’Institut national de transfusion sanguine sur la prévalence des génotypes et des mutants précore chez les donneurs de sang en France.Patients et méthodesL’effectif comprenait 100donneurs de sang nouvellement dépistés AgHBs positifs. La mutation PC a été recherchée par trois méthodes différentes: une technique de PCR temps réel (PCR TR) développée dans notre laboratoire et ciblée sur la mutation PC, le séquençage direct et l’hybridation moléculaire inverse (Kit Inno-LIPA HBV Precore, INNOGENETICS®).RésultatsLa moyenne d’âge dans la population étudiée était de 30ans (extrêmes 18–64). L’origine géographique était africaine dans 42% des cas. La prévalence de la mutation G1896A isolée ou associée à du virus précore sauvage était de 57% en technique Inno-LIPA et de 59% par séquençage ou PCR TR. Les donneurs infectés par un virus précore muté avaient des charges virales VHB significativement plus basses que les patients porteurs d’un virus sauvage (3,19Log10UI/ml versus 4,93Log10UI/ml, pConclusionsNous confirmons les données de la littérature à savoir une prévalence relativement élevée de mutation PC chez les donneurs de sang français. Sachant que ces sujets sont la plupart du temps asymptomatiques et avec des charges virales VHB inférieures au seuil défini par l’EASL pour le portage inactif, la question du rôle de la mutation G1896A dans la sévérité des lésions hépatiques reste à préciser.

Recently antibodies to hepatitis C virus were detected in sera of chronic active hepatitis patients, with anti-smooth muscle autoantibodies or with anti-liver/kidney microsomal type 1 autoantibodies. As the latter were used to differentiate autoimmune chronic active hepatitis from chronic non-A, non-B virus hepatitis, it was mainly important to discover if autoantibodies were associated with chronic hepatitis C virus infection. The sera of 272 chronic hepatitis C patients were screened by indirect immunofluorescence for non-organ specific autoantibodies. Antinuclear anti-bodies and anti-smooth muscle autoantibodies were more frequent in chronic hepatitis C patients than in blood donors ( n = 100). Anti-liver/kidney microsomal type 1 autoantibodies were not detected in the sera of the blood donors, in the 74 hepatitis B patients or in the 30 alcoholic hepatitis or cirrhotic patients' sera tested as controls. They were detected in 14 chronic hepatitis C patients. These antibodies were compared in immunodiffusion to anti-liver/kidney microsomal type 1 autoantibodies sera obtained from type-2 autoimmune chronic active hepatitis patients and an identity reaction was observed. Chronic hepatitis C patients without or with anti-liver/kidney microsomal type 1 autoantibodies, did not differ in age, sex ratio, transaminases and gammaglobulin level, risk factors for hepatitis C virus infection, association with other autoimmune diseases. These patients differed significantly from type-2 autoimmune chronic active hepatitis patients. We conclude that: (i) in some chronic hepatitis C patients the pattern and the titer of autoantibodies may create confusion with an autoimmune chronic active hepatitis; (ii) There is no serological evidence for a hepatitis C virus infection in true type-2 autoimmune chronic active hepatitis.

Droplet digital PCR (ddPCR), which has been recently developed to provide an absolute quantitation of target molecules without relying on the use of standard curves ([1][1]), might be an interesting alternative to conventional real-time PCR assays used for viral load (VL) determination ([2][2], [3][

The new version of the INNO-LiPA HBV genotyping assay (Innogenetics) developed to identify all hepatitis B virus (HBV) genotypes, A to H, has been evaluated in comparison with sequencing of PCR-amplified HBV DNA from 200 samples before or after cloning. The genotyping data obtained with INNO-LiPA were in agreement with those from direct sequencing in the 179 samples characterized by the two methods. INNO-LiPA revealed 28 mixed infections. However, sequencing after molecular cloning confirmed only 15 of them and did not identify any that were of genotype H ( n = 9). Our study demonstrates that INNO-LiPA overestimates mixed infections as a result of erroneous genotype H detection.

Sub-Saharan Africa remains the epicenter of the human immunodeficiency virus (HIV) pandemic. However, there is a lack of multicenter data on the risk of transfusion-transmitted HIV from blood centers in sub-Saharan Africa.The incidence of HIV infections in the blood donations collected in the main blood banks of five countries (Burkina Faso, Congo, Ivory Coast, Mali, and Senegal) was determined to estimate the current transfusion risk of HIV infection using the incidence rate/window period model.The risk of transfusion-transmitted HIV infections associated with the window period varied from 1 in 90,200 donations (Senegal) to 1 in 25,600 (Congo). Considering the five participating blood centers as a whole, the incidence rate of HIV-positive donors per 100,000 person-years was 56.6 (95% confidence interval [CI], 47.1-67.9); the residual risk (RR) was 34.1 (95% CI, 7.8-70.7) per 1 million donations, which represents 1 in 29,000 donations (95% CI, 1/128,000-1/14,000).RR estimates varied according to the country. This is potentially due to a lower incidence of HIV infection in the general population or to a more efficient selection of blood donors in the countries with the lowest risk. The estimates of the transfusion risk of HIV infection in each country are important, both to assess the impact of current preventative strategies and to contribute data to policy decisions to reinforce transfusion safety.

Human parvovirus 4 infections are primarily associated with parenteral exposure in western countries. By ELISA, we demonstrate frequent seropositivity for antibody to parvovirus 4 viral protein 2 among adult populations throughout sub-Saharan Africa (Burkina Faso, 37%; Cameroon, 25%; Democratic Republic of the Congo, 35%; South Africa, 20%), which implies existence of alternative transmission routes.

Improving blood safety without introducing nucleic acid testing in blood screening may be possible using antigen/antibody (Ag/Ab) combination assays, especially in resource-poor countries.To evaluate the potential reduction of human immunodeficiency virus (HIV)-transmitted infection using such an assay, a study was carried out aimed to compare the routine strategy for blood screening (S1), combining a rapid test (Determine HIV-1/-2, Inverness) and an enzyme immunoassay (human HIV-1/-2, Human Diagnostic) with an HIV Ag/Ab assay (S2, Genscreen ULTRA HIV Ag-Ab, Bio-Rad) in 2000 blood donations tested in Cameroon. Western blot and HIV RNA polymerase chain reaction were used to confirm the infection in reactive donors, and genotype was determined in HIV RNA-positive samples.Of the 2000 donors, 3.1% were positive with S1 and 4.3% with S2. Of the 97 samples positive with S1 and/or S2 tested for confirmation, 54.7% were positive, 38.1% indeterminate, and 7.2% negative. There were significant differences between S1 and S2 in terms of sensitivity (S1, 79.2%; S2, 100%), irrespective of the genotype, and in specificity (S1, 99.0; S2, 98.3%). The most frequent genotype (49%) was CRF02_AG.A testing strategy using Genscreen ULTRA HIV Ag/Ab could prevent 55 HIV transmissions per 10,000 donations. However, this would be at a cost of discarding 170 per 10,000 negative donations that would test false positive, showing that the implementation of new techniques in blood screening need an optimization before routine use. Using confirmatory assays, HIV Ab prevalence in blood donors in Cameroon was estimated at 2.65%.

Hepatitis B virus (HBV) diversity is characterized by eight genotypes correlated to eight hepatitis B surface antigen (HBsAg) subtypes, which differ in their geographical distribution.To establish virologic characteristics and the evolution of HBV diversity, we carried out a study over a 9-year period in HBV-infected French blood donors. HBsAg subtyping based on specific antibody method concerned 2901 donors, from whom 940 have been analyzed by an S-gene sequencing to determine genotypes and S-gene mutations.HBsAg subtypes were distributed as follows: ayw2, 34.4%; adw2, 25.7%; ayw1, 10.2%; ayw4, 14.9%; adr, 7.8%; ayw3, 6.4%; and adw4, 0.7%. Ayw4 (Genotype E) proportion increased over time in correlation with an increased proportion of subjects originated from sub-Saharan Africa. The genotype observed with the highest proportion was D (43.0%), then A (26.2%), E (17.5%), B (6.5%), C (6.4%), and F (0.4%). Genotype B had the highest proportion of hepatitis B e antigen (39.2%) and the highest viral loads (VLs). Forty-three (5.5%) isolates presented one (n=35) or multiple (n=8) amino acid envelope substitutions. Donors infected with mutated isolates had lowest VLs. rtA181T/sW172 stop mutation associated with resistance to nucleos(t)ide analogs was detected in two donors suggesting a transmission of these isolates.This extensive study shows that HBV genotype evolution is closely linked to the geographical origin of subjects and that the occurrence of viral envelope mutants is not an exceptional event in healthy HBV chronic carriers. Blood donors rarely recruited in HBV studies provide further relevant information on the characteristics of HBV diversity.

Author(s): Laperche, Syria; Boukatou, Genevieve; Kouegnigan, Leonard; Nebie, Yacouba; Boulahi, Mohamed Ould; Tagny, Claude Tayou; Yahaya, Rakia; Tapko, Jean-Baptiste; Murphy, Edward; Lefrere, Jean Jacques | Abstract: BackgroundFollowing World Health Organization recommendations that a quality control (QC) system be implemented in African blood centers, a pilot study of the performance of human immunodeficiency virus antibody (anti-HIV), hepatitis B surface antigen (HBsAg), and hepatitis C virus antibody (anti-HCV) testing by several Sub-Saharan African blood centers was initiated.Study design and methodsA reference laboratory sent a panel of 25 samples to six African blood center laboratories. The panel included eight negative samples; four anti-HIV-1–, one anti-HIV-2–, four anti-HCV–, and five HBsAg-positive samples; and three samples consisting of mixtures of two sera to mimic coinfections. Sensitivity, specificity, and overall quality (correct positive or negative status) scores were calculated.ResultsFrom the 21 sets of results obtained (seven for each virus), eight were from rapid tests (two for HIV, three for HBV, and three for HCV) and 13 were from enzyme immunoassays (EIAs; all HIV EIAs were antigen/antibody combination assays). Overall assay sensitivity was 98% for HIV, 75% for HBV, and 88% for HCV; agreement between blood centers using the same assay was good. Sensitivity of rapid tests was notably poorer than EIAs, with overall sensitivity quality scores of 64.5% for rapid tests (20% for HBsAg rapid tests) compared to 100% for EIAs. The overall specificity quality scores were 98.3 and 94.5% for EIAs and rapid tests, respectively.ConclusionsThis pilot QC study organized for blood centers of Sub-Saharan Africa showed the feasibility of the approach despite some logistic constraints. Although interlaboratory variability was small, the poor performance of rapid tests, especially for HBsAg, raises policy questions about their use as the only screening assay.

Resume L’existence d’un passage sanguin asymptomatique de certains agents infectieux entraine un risque de transmission de ces agents lors d’une transfusion sanguine. Si ce risque est actuellement bien maitrise pour certains agents infectieux (VIH, HTLV, VHC, VHB), il n’est en revanche pas documente et quantifie pour d’autres agents pathogenes, responsables d’infections non depistees ou encore indetectables par serologie au moment du don. Ce risque est generalement faible en situation endemique, mais il augmente dans le temps et l’espace lors de la survenue de cas groupes ou d’epidemies. Des mesures de prevention peuvent alors etre mises en place (suspension de la collecte, mise en quarantaine des dons…). Ces mesures pouvant avoir d’importantes repercussions, notamment en limitant l’approvisionnement des etablissements de soins en produits sanguins labiles, il est important qu’elles soient adaptees au risque de transmission par transfusion. Des estimations quantitatives du risque de contamination d’un don de sang peuvent ainsi permettre de les orienter. C’est dans ce contexte que l’Institut de veille sanitaire (InVS) a initie, debut 2005, un projet visant a disposer a priori d’estimations quantitatives du risque de contamination d’un don de sang par des agents infectieux pour differents scenarii en termes d’incidence et de distribution spatiotemporelle. L’objectif de cet article est d’actualiser les dernieres estimations du risque residuel de contamination d’un don de sang par les virus depistes de facon systematique (VIH, HTLV VHC, VHB) et de presenter le travail realise par le groupe de travail « Estimation quantitative du risque de contamination d’un don de sang par des agents infectieux ».

International audience; The existence of hepatitis C virus (HCV) proteins encoded by alternate reading frames overlapping the core-encoding region has been suggested. Several mechanisms of production have been postulated, and the functions of these proteins in the HCV life cycle remain unknown. We analyzed cases of seroconversion to an alternate reading frame protein in a group of 17 patients infected by one of the two HCV genotype 1b strains during an outbreak in a hemodialysis unit. Three patients seroconverted, and antibodies were transiently detected in another patient. Three of these patients were infected by one of the two HCV strains, whereas the strain infecting the remaining patient could not be identified. Quasispecies sequence analysis of the core-coding region showed no differences in the core or +1 reading frame sequences that could explain alternate reading frame protein seroconversion in some but not all of the patients infected by one of the HCV strains, and no such difference was found between the two strains. Because differences in the structure of RNA elements could play a role in frameshift events, we conducted a predictive analysis of RNA folding. No difference was found between the patients who did and did not seroconvert to alternate reading frame protein. Conclusion: Our findings prove that alternate reading frame proteins can be produced during acute HCV infection. However, seroconversion does not occur in all patients for unknown reasons. Alternate reading frame protein could be generated by minority quasispecies variants or variants that occur transiently.

Quantitative PCR assays used to monitor hepatitis B virus (HBV) load differ in their ability to detect different HBV variants. This study evaluated the performance of the Abbott RT PCR assay for quantitating DNA from different HBV genotypes and from HBV variants bearing HBsAg gene mutations. The study was performed on a randomly-selected sample with a viral load >6logIU/mL for each genotype and on 25 HBsAg variants. Each sample was assayed using the Abbott RT assay and with the Roche Cobas AmpliPrep-Cobas TaqMan as a reference method. All HBV genotypes were detected with the Abbott RT assay with an equivalent dynamic range (1-8logIU/mL). For each genotype, the data suggest that the assay was linear over the entire dilution range (r(2): 0.985-0.995). For the 25 HBsAg variants, viral titres determined with the two assays correlated well (r(2): 0.929). The mean difference between the two methods was -0.295 (95% CI: -0.520 to -0.071). The difference was lower than 1log unit in all but two cases. In conclusion, the Abbott RT assay can detect and quantify DNA from different HBV variants with equivalent performance and is thus suitable for routine monitoring of patients with chronic HBV infections.

The subtype distribution of 142 genotype 2 and 97 genotype 4 hepatitis C virus (HCV) isolates from the sera of 1,319 volunteer blood donors in France was determined by gene sequencing and by phylogenetic analysis of the NS5B region and E1 envelope. Findings underlined a wide range of subtypes in both genotypes, that is, 20 in HCV-2 and 11 in HCV-4. Eighteen of these 31 subtypes had not been defined previously. Some subtypes, that is, 2a, 2b, 2c, 2i, 2k, 4a, and 4d, showed numerous strains while subtypes in donors from West Africa or Central Africa showed an endemic profile with only a few strains. A Bayesian coalescence approach was used to estimate the demographic history of each HCV subtype. The estimated mean dates of the most recent common ancestors (MRCA) were 1,889 (confidence interval (CI), 1,842-1,930) for HCV-2a, 1,886 (CI, 1,843-1,921) for HCV-2b, 1,791 (CI, 1,699-1,848) for HCV-2c, 1,846 (CI, 1,803-1,878) for HCV-2i, 1,911 (CI, 1,879-1,937) for HCV-4a, and 1,957 (CI, 1,943-1,967) for HCV-4d. The period of spread for subtype 2b, 2c, and 2i was between 1900 and 1960 whereas rapid exponential spread for subtype 2a, 4a, and 4d occurred in the 1960s. The inferred histories of population growth indicated that transmission rates differed according to HCV subtype. These results may help to predict the future burden of HCV in France.

Being an orphan virus despite a large number of investigations, hepatitis G virus is a blood-borne agent for which screening is not required in blood donations. The in vivo efficacy of pathogen inactivation methods could be assessed by the absence of hepatitis G virus markers after transfusion of pathogen-inactivated blood products, in recipients susceptible to infection before the transfusion.

Hepatitis B virus (HBV) DNA quantification is used to establish the prognosis of chronic HBV-related liver disease, to identify those patients who need to be treated, and to monitor the virologic response and resistance to antiviral therapies. Real-time PCR-based assays are gradually replacing other technologies for routine quantification of HBV DNA in clinical practice. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of the real-time PCR Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform for HBV DNA quantification. Specificity was satisfactory (95% confidence interval, 98.1 to 100%). Intra-assay coefficients of variation ranged from 0.22% to 2.68%, and interassay coefficients of variation ranged from 1.31% to 4.13%. Quantification was linear over the full dynamic range of quantification of the assay (1.7 to 8.0 log 10 IU/ml) and was not affected by dilution. The assay was accurate regardless of the HBV genotype. Samples containing HBV DNA levels above 4.5 log 10 IU/ml were slightly underestimated relative to another accurate assay based on branched-DNA technology, but this is unlikely to have noteworthy clinical implications. Thus, the CAP/CTM HBV DNA assay is sensitive, specific, and reproducible, and it accurately quantifies HBV DNA levels in patients chronically infected by HBV genotypes A to F. Samples with HBV DNA concentrations above the upper limit of quantification need to be diluted and then retested. Broad use of fully automated real-time PCR assays should improve the management of patients with chronic HBV infection.

Hepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5′-untranslated region (5′UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5′UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5′UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5′UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.

A national evaluation study was performed in 14 specialized laboratories with the objective of assessing their capacities to provide (i) hepatitis B virus (HBV) viral loads (VL), (ii) HBV genotypes, and(iii) identification of precore/core mutants. The panel consisted of 12 HBV DNA-positive samples with VLs from 2.8 to 9.1 log10copies/ml, different HBV genotypes (A to F), and 3 mutant and 9 wild-type samples at nucleotide 1896. The coefficients of variation of the mean VLs ranged from 2.4% to 10.4% with the Cobas HBV Monitor assay, from 1.8% to 5.5% with the CobasTaqMan 48, from 1.5 to 26.2% with RealArt HBV PCR, and from 0 to 7% with branched DNA (bDNA). The Cobas Monitor assay underestimated the VLs of genotype F samples, with differences ranging from 1.4 to 2.4 log10copies/ml. The accuracies of genotype determinations ranged from 33% to 100%, and those of precore mutant determinations ranged from 25 to 100%. This study showed some drawbacks of two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping and the determination of precore/core mutants need to be clearly stated to justify the standardization of these methods.

Mutations in hepatitis B virus surface antigen (HBsAg) involving amino acid substitution within the immunodominant “a” determinant may affect the performance of commercial HBsAg assays. The performances of four HBsAg assays that recently received Conformité Européene marking, Advia Centaur HBsAg (Bayer), Monolisa HBsAg Ultra (Bio-Rad), Liaison HBsAg (Dia Sorin), and Vidas HBsAg Ultra (bioMérieux), were compared with that of the routinely used HBsAg assay AxSYM HBsAg V2 (Abbott). Assays were evaluated for (i) analytical sensitivity performance with a national reference HBsAg panel (including 10 samples with calibrated HBsAg concentrations from 0.04 to 2.24 ng/ml) and (ii) the detection of HBsAg mutants by studying a panel of 35 HBsAg mutants (23 collected from patients and 12 recombinant mutants). The limits of detection of these assays were

A multicenter study of NS5b hepatitis C virus (HCV) genotype determination involving 12 laboratories demonstrates that any laboratory with expertise in sequencing techniques would be able to provide a reliable HCV genotype for clinical and epidemiological purposes as long as they are provided a consensus reference sequence database.

BACKGROUND: A new enzyme immunoassay based on the simultaneous detection of nucleocapsid proteins of hepatitis C virus (HCV) and anti-HCV (Monolisa HCV antigen-antibody Ultra, Bio-Rad) was evaluated as an alternative to nucleic acid testing (NAT) for the diagnosis of HCV infection during the window period in blood donations. STUDY DESIGN AND METHODS: The study included 107 sequential samples from 10 HCV seroconversion commercial panels; 81 samples were in the preseroconversion phase, and 26 were collected after seroconversion. All samples were tested with HCV antigen-antibody assay and the two minipool (MP) NAT procedures that are routinely used in France (transcription-mediated amplification in pools of 8 and COBAS AmpliScreen HCV test [Roche Diagnostic] in pools of 24 donations). RESULTS: From the 44 samples collected during window period that were MP-NAT–positive, 31 (70.5%) were also positive with the Monolisa HCV antigen-antibody assay. The mean delay in detecting HCV infection between these two methods was 5.1 days (range, 0-24 days). The Monolisa HCV antigen-antibody assay led to a reduction in the window period of 26.8 days (range, 0-72 days). All samples collected after seroconversion were detected with the HCV antigen-antibody assay. The specificity analyzed in 2503 consecutive blood donations was estimated at 99.88 percent. CONCLUSION: This new developed assay presents an improvement for the detection of HCV infection, especially in the early phase of infection when antibodies are undetectable. Although less sensitive than NAT, this assay could be a suitable solution for blood screening in developing countries where NAT (or HCV core antigen–specific assay) is not affordable or its implementation is not feasible.

To evaluate whether a new enzyme immunoassay developed for the simultaneous detection of hepatitis C virus (HCV) core antigen (Ag) and anti-HCV antibodies (anti-HCV Ab) (Monolisa HCV Ag/Ab ULTRA; Bio-Rad) could improve the early detection of HCV infection, we compared its sensitivity to that of anti-HCV, HCV core Ag, and HCV RNA assays. The populations studied included 12 blood donor samples positive for HCV RNA and HCV core Ag but negative for anti-HCV antibodies and 23 hemodialysis patients who developed anti-HCV Ab (seroconversion) during the follow-up. From these 23 individuals, 83 samples sequentially collected prior to seroconversion and 108 samples collected after seroconversion were tested. Six of 12 blood donations were positive by the HCV Ag/Ab assay. In the hemodialysis cohort, the 24 HCV RNA-negative samples were negative by the HCV Ag/Ab assay and 23 of the 59 HCV RNA-positive samples (39%) were positive. The HCV Ag/Ab assay detected HCV infection on average 21.6 days before the most sensitive antibody assay. The HCV Ag/Ab assay did not detect HCV infection as early as the HCV RNA assay (mean delay, 30.3 days) or HCV Ag assay (mean delays, 27.9, and 16.3 days by the HCV core Ag quantification assay and the HCV Ag blood screening assay, respectively). This new assay provides a notable improvement for the early detection of HCV infection during the so-called window period compared with anti-HCV Ab assays and could be a useful alternative to HCV RNA detection or HCV core Ag assays for diagnosis or blood screening when nucleic acid technologies or HCV core Ag detection are not implemented.

A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5′ noncoding (5′ NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5′ NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5′ NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.

The main objective of the implementation of NAT for the screening of blood-borne viruses was to compensate for the failure of serologic assays during the window period. Because this new screening procedure theoretically covers the entire period of infectivity, the necessity for maintaining serologic assays in blood screening strategy could become questionable.To investigate this issue, a panel of 35 samples has been studied by NAT. These samples had been collected from HIV-1 antibody-positive individuals presenting a persistently low viral RNA load (400 copies/mL) in the absence of antiviral therapy. All samples were analyzed with the minipool (x8) NAT routinely used in blood bank setting (HIV-1 and HCV assay based on transcription-mediated amplification) and with single-donation testing.The minipool NAT failed to detect the presence of HIV RNA in 15 of the 35 samples (11 remained negative when retested). Single-donation testing gave negative results in 4 samples (3 remained negative when retested). Fourteen of the 18 samples with a viral load greater than 50 copies per mL were positive by minipool NAT versus 6 of the 17 samples with fewer than 50 copies per mL (p = 0.02).The results clearly demonstrate that anti-HIV screening should not be withdrawn from biologic qualification procedures of blood donations, even when single NAT is performed.

An EIA based on immune complex disassociation of nucleocapsid proteins of HCV has been developed to detect and quantify HCV core antigen.To evaluate whether this new assay (trak-C, Ortho Clinical Diagnostics) could be an alternative to NAT during the window period, its sensitivity in this context was assessed, and its performance was compared with that of a first-generation HCV core antigen assay dedicated to the blood screening (HCV core antigen ELISA). Studied populations included nine HCV RNA-positive, HCV antibody-negative blood donors and 23 hemodialysis patients who underwent an HCV seroconversion. From these individuals, 81 samples (23 HCV RNA-negative and 58 HCV RNA-positive) sequentially collected during the phase before seroconversion were tested.The nine blood donor samples were positive for the presence of HCV core antigen by the trak-C, and 6 of 8 tested were positive for the presence of HCV core antigen by blood screening ELISA. In the hemodialysis cohort, the 23 HCV RNA-negative samples were negative with the two HCV core antigen assays. Among the 58 HCV RNA-positive samples, 46 of 57 (80.7%) tested were positive for the presence of HCV core antigen with the blood screening assay, and 57 of 58 (98.2%) were positive for the presence of HCV core antigen with the trak-C. The mean delays in detecting HCV infection between trak-C and the appearance of HCV antibodies, between HCV RNA testing and trak-C, and between trak-C and HCV core antigen ELISA were 58.2, 0.24, and 3.33 days, respectively.Trak-C was more sensitive than the blood screening assay and had similar performance to HCV RNA assay in the window period. Trak-C could constitute an alternative to NAT for the diagnosis of HCV infection during the window period, especially when molecular biology procedures cannot be implemented.

International audience; The aim of this study was to determine whether multicentre quality controls for the detectability of viral genomes could contribute to the improvement of diagnostic performance in the participating laboratories. The study was carried out during two successive rounds, during which 18 laboratories specialized in nucleic acid testing analyzed, through a polymerase chain reaction (PCR) assay, a common panel of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA-positive and -negative samples. During the first round, the laboratories used either an 'in-house' PCR procedure or a partly standardized commercial test. After decoding the results of the first round, the procedures of the participating laboratories were compared in order to establish a consensus procedure deduced from those of the laboratories which provided the best results. During the second round, each participating laboratory could use the resulting consensus procedure, or its own procedure, or both. The results of this quality control study indicated that, whatever method used, even specialized and trained laboratories may give false-negative or false-positive results. The commercial assay did not guarantee a systematic high quality level of results. The striking heterogeneity of results observed among laboratories using the same commercial assay confirm that molecular biology methods need skilled technicians. The results of this quality control study suggest that full standardization of viral genome detection, including all steps of the procedure, is necessary and that the laboratories performing PCR should participate in repeated quality control studies, whatever technique is being used.

Background & Aims Strains responsible for acute hepatitis B infections (AHB) in France have not been characterized. This study was first designed to analyze the molecular epidemiology of AHB and second to describe the differences between AHB and chronic hepatitis B (CHB) exacerbations. Methods This prospective study was based on the French mandatory notification system for AHB. 147 samples corresponding to declared cases were shipped to a central laboratory for classification as AHB or CHB according to the level of anti-HBc IgM and anti-HBc avidity. Results Based on biological marker values and file examination, 75 cases (59%) were classified as AHB. Independently of the acute or chronic status, genotype A (57%), D (22%) and E (14%) were the most prevalent and no phylogenetic clustering was observed among HBV sequences (n=68). Precore or basal core-promoter variants were not particularly associated with disease severity but were more prevalent in CHB. No antiviral resistant strains or immune-escape HBsAg was observed. HBV viral loads in AHB or CHB were comparable but with opposite distributions. ALT levels reached 10 times the upper normal value in 94% of AHB but only in 24% of CHB. Conclusions After rigorous classification, no major difference at the genetic level was found between HBV strains isolated from AHB and CHB. Absence of potentially deleterious variant detection is reassuring. When based upon HBsAg and anti-HBc IgM determination, AHB notification may falsely include more than 40% CHB, leading to an important risk of bias in national surveillance programs of AHB.