A Highly Prevalent Polymorphism in the Core Region Impairs Quantification of Hepatitis B Virus (HBV) by the cobas TaqMan HBV Assay.

The high genetic variability of hepatitis B virus (HBV) can impair DNA quantification. Here, we investigate a major underquantification of HBV by the cobas TaqMan HBV assay (CTM; Roche). In France, between 2005 and 2017, HBV DNA was detected in 3,102 blood donations by use of the CTM (95% limit of detection [LOD95], 4.8 IU/ml). HBV strains were sequenced in the S region (LOD95, ∼30 IU/ml). Concordant (n = 120) and discordant (n = 45) samples were identified according to the agreement between the plasma viral load (pVL) determined by the CTM and sequencing; all samples were also quantified using the RealTime HBV assay (RTH; Abbott). The viral signature, cloning, and mutagenesis were used to characterize the polymorphism responsible for CTM misquantification. A CTM-RTH discordance (>1 log IU/ml) was found in 14/45 samples that had low pVLs and were successfully genotyped (pVLlow genoS+). PreC/C clones of concordant (C1, C2) and discordant (D1, D2) strains were used to challenge the CTM. Strains D1 and D2 were highly underquantified (42- and 368-fold). In clones, mutating the region corresponding to the CTM reverse primer from a discordant sequence to a concordant sequence restored the levels of quantification to 24% (D1→C1) and 59% (D2→C1) of theoretical levels, while mutating the sequence of a concordant strain to that of a discordant strain led to 78-fold (C1→D1) and 146-fold (C1→D2) decreases in quantification. Moreover, mutating positions 1961 and 1962 was enough to induce a 5-fold underquantification. We conclude that the CTM underestimates pVLs for HBV strains with mutations in the reverse primer target. Specifically, the polymorphism at nucleotides 1961 and 1962 is naturally present in 4.79 and 4.22% of genotype A and D strains, which are highly frequent in Europe, leading to a 5-fold decrease in quantification. Quantification using the new-generation Roche C4800 assay is not affected by this polymorphism.

Two cases of transfusion-transmitted hepatitis B virus (HBV) infection in a low-endemic country before implementation of HBV nucleic acid testing.

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Overestimation of incidence of hepatitis B virus mixed-genotype infections by use of the new line probe INNO-LiPA genotyping assay.

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.

Advances in human B19 erythrovirus biology.

Since its discovery, human parvovirus B19 (B19V), now termed erythrovirus, has been associated with many clinical situations (neurological and myocardium infections, persistent B19V DNAemia) in addition to the prototype clinical manifestations, i.e., erythema infectiosum and erythroblastopenia crisis. In 2002, the use of new molecular tools led to the characterization of three different genotypes of human B19 erythrovirus. Although the genomic organization is conserved, the geographic distribution of the different genotypes varies worldwide, and the nucleotidic divergences can impact the molecular diagnosis of B19 virus infection. The cell cycle of the virus remains partially unresolved; however, recent studies have shed light on the mechanism of cell entry and the interactions of B19V proteins with apoptosis pathways.

Changing epidemiology of human parvovirus 4 infection in sub-Saharan Africa.

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.

National survey of hepatitis B virus (HBV) polymorphism in asymptomatic HBV blood donors from 1999 to 2007 in France.

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples.

Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization.

Performance of HBsAg quantification assays for detection of Hepatitis B virus genotypes and diagnostic escape-variants in clinical samples.

BACKGROUND: The impact of hepatitis B virus (HBV) genomic variability on the measurement of HBsAg level has been poorly evaluated. OBJECTIVE: This study was designed to compare the performance of all the available assays measuring HBsAg level in this setting. STUDY DESIGN: A large selection of wild type HBV genotypes (n=184) and HBsAg strains harboring mutations in the S gene (n=81) from clinical samples was studied with three HBsAg quantification assays: Architect HBsAg (Abbott), LiaisonXL Murex HBsAg Quant (DiaSorin) and the Elecsys HBsAgII (Roche). RESULTS: The overall percentage of positive results was 99.2% for Abbott, 98.9% for DiaSorin and 98.1% for Roche. Abbott and Roche assays provided an excellent concordance in HBsAg quantification (global mean bias of -0.006 logIU/mL). By contrast, DiaSorin underestimated HBsAg level with values 0.112 logIU/ml and 0.103 logIU/ml lower than Abbott and Roche, respectively. By contrast, DiaSorin slightly over quantified gtC (2.5% over the expected value) while Abbott provided values 6.2% lower than expected and 16.2% lower than what observed for the other genotypes. HBsAg quantitative assays were influenced by HBs protein substitutions irrespective to the genotype but no specific protein pattern that would particularly impair the quantification by one technique has been identified. However, Roche seemed to be particularly impacted by substitutions at 145 residue: 75% of under quantified samples carried a substituted 145 residue. CONCLUSION: This head-to-head comparison indicates a good correlation between all current systems used to quantify HBsAg but clearly shows an influence of both the genotype and the presence of "a" determinant variants in the absolute quantification of HBsAg. While these discrepancies may not translate into major clinical consequence, they may explain an absence of detection of weak concentration of HBsAg on some systems.

Increasing prevalence of HDV/HBV infection over 15 years in France.

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.

Variable capacity of 13 hepatitis B virus surface antigen assays for the detection of HBsAg mutants in blood samples.

BACKGROUND: Natural variation and mutations in the envelope protein (S) of hepatitis B virus can translate into HBsAg variants no longer detectable by conventional HBsAg assays. OBJECTIVES: The aim of the study was to assess the performance of 13 commercial assays currently used for screening and clinical analysis of HBsAg variants. STUDY DESIGN: The limit of detection (LOD) for each assay was established using two reference standards (WHO HBsAg 00/588 and the SFTS French reference). Sensitivity was evaluated using different panels. Panel 1 included 25 recombinant HBs variants at three concentrations, panels 2 and 4 included 8 recombinant HBsAg variants and 9 wild-type proteins (genotypes A-F), respectively, panel 3 included 16 natural HBsAg variants. RESULTS: LODs ranged from 0.011 to 0.095 IU/ml with the WHO standard, and from 0.021 to 0.326 ng/ml with the French reference. The overall percentage of positive signals using HBsAg variants ranged from 62.9% to 97.9%. Three substitutions: T123, D144A and G145, were negative at all concentrations with at least one assay. DISCUSSION: Our findings show that, although they fulfil CE requirements for analytical sensitivity (LODs below 0.13 IU/ml), HBsAg assays may vary in their capacity to detect HBsAg variants. This limit in diagnosis performance should encourage the health regulatory agencies to include HBsAg variant panels in the evaluation process.

Impact of hepatitis B virus genotypes and surface antigen variants on the performance of HBV real time PCR quantification.

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.

Sensitivity of hepatitis B virus DNA transcription-mediated amplification testing in hepatitis B surface antigen-positive blood donations.

BACKGROUND: The objective was to evaluate the performance of nucleic acid testing (NAT) in the detection of hepatitis B virus (HBV) infection in hepatitis B surface antigen (HBsAg)-positive blood donations. STUDY DESIGN AND METHODS: A total of 253 HBsAg- and anti-hepatitis B core antigen (HBc)-positive samples (50 hepatitis B e antigen [HBeAg]-positive and 203 anti-HBe-positive) from blood donations collected in France were studied. The samples were investigated with a blood screening assay (Procleix Ultrio, Chiron/Gen-Probe) in minipool (MP; x8) and in individual-donation (ID) testing. All nonreactive samples were retested once, and nonreactive MP samples were assayed for viral load (VL). RESULTS: All 50 HBeAg-positive samples were reactive in MP-NAT and ID-NAT. Of the 203 anti-HBe-positive donations, 80.3 percent were MP- and ID-reactive, 17.2 percent were MP-nonreactive and ID-reactive, and 2.5 percent were nonreactive in ID-NAT. Overall the sensitivity of ID-NAT was 98 percent versus 84 percent for MP-NAT. After retesting, 16 of the 35 MP-nonreactive and/or ID-reactive donations became MP-reactive and 2 of the ID-nonreactive donations became NAT-reactive. The capacity of Procleix Ultrio to detect HBV DNA was not related to HBsAg subtype, but correlated with the VL: the mean VL in the group of MP-nonreactive samples was 1,420 copies per mL vs. 17,000 copies per mL in the group of 40 MP-reactive samples. CONCLUSION: These results demonstrate that HBV-NAT in ID format is far more effective in detecting viremia in chronic HBsAg carriers than in MP-NAT. The sensitivity of the NAT assay needs to be improved to be considered for replacing the current HBsAg assays, especially when anti-HBc testing is not performed.

Sensitivities of four new commercial hepatitis B virus surface antigen (HBsAg) assays in detection of HBsAg mutant forms.

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 <0.15 ng/ml (from 0.089 to 0.121 ng/ml). The sensitivity performances for mutant virus detection varied, ranging from 37.1% to 91.4%. The lack of detection of these mutants by commercial assays was probably due to the epitope recognition of the anti-HBs assay reagents in the capture phase and in the conjugates. The prevalence and clinical impact of HBsAg mutants are under investigation. However, the manufacturers must be vigilant in the design of the assays in order to reduce the risk of missing a broad range of described S gene mutants.

Is an assay for simultaneous detection of hepatitis C virus core antigen and antibody a valuable alternative to nucleic acid testing?

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.

Simultaneous detection of hepatitis C virus (HCV) core antigen and anti-HCV antibodies improves the early detection of HCV infection.

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.