Viral and clinical factors associated with surface gene variants among hepatitis B virus carriers.

BACKGROUND: Understanding the prevalence of potential antigenic variation of the hepatitis B virus (HBV) surface antigen (HBsAg) is fundamental for assay design and to future changes in vaccine formulation. In this study, the nature and frequency of HBsAg polymorphisms occurring in France in chronic carriers and in newly diagnosed patients were determined. We focused on variations in the major hydrophilic region (MHR), the central core of HBsAg known to be exposed on the surface and involved in antibody binding. METHODS: Two patient groups were identified: 51 chronic HBV carriers followed at our institution for > 1 year; and 129 newly diagnosed patients (63 of whom had a first HBsAg-positive result at our hospital laboratory and 66 a first positive result in a private laboratory). DNA sequences of HBsAg were obtained from these 180 patients and compared with consensus sequences built with 168 full-length HBV sequences imported from GenBank. Polymorphisms of the MHR of HBsAg were analysed with the Mutation Master Software. Literature review and BLOSUM scores were used to define potentially altered antigenicity. RESULTS: The global frequency of MHR variants was 27.8%. Occurrence of MHR variants was independent of viral load, HBeAg status and sex, but was associated with the chronic carriers' group, advancing age, the presence of antibodies to HBsAg, immunoprophylaxis administration, antiviral treatment and genotypic resistance to antivirals. In multivariate analysis, the independent variables associated with MHR variants were advancing age and the presence of genotypic resistance to nucleoside or nucleotide analogues. CONCLUSION: Most MHR variants emerge with longer disease duration and upon indirect selective pressure. Variation of the MHR may serve to restore virus replication of resistant strains. Combined envelope and polymerase variants could impair diagnostic assays and limit treatment alternatives.

Could the new HIV combined p24 antigen and antibody assays replace p24 antigen specific assays?

The performance of twelve HIV combined p24 antigen and antibody assays available in Europe were compared. The assays were examined with a total of 1983 samples that included 1005 unselected HIV negative samples, 7 HIV-1 p24 Ag reference samples with HIV-1 Ag, 10 samples of a HIV antigen sensitivity commercial panel, 124 samples of 31 p24 antigen panels of different HIV-1 subtypes, 168 members of 24 HIV-1 seroconversion panels, 559 HIV-1 (groups M and O) antibody positive samples and 110 HIV-2 antibody positive samples. The specificity ranged from 99.4 to 100%. Ten of the 12 assays detected all anti-HIV positive samples irrespective of genotype while two assays missed one sample each (one subtype F and one subtype C). The combined assays could be classified into three groups. The first includes two assays (Enzygnost HIV Integral and Vironostika Ag/Ab) that have a clinical sensitivity similar to the two antibody only assays. The second includes the seven assays that detected infection after the p24 antigen only assay and show a delay from 3.3 to 5.17 days after HIV-1 RNA. The third group detected the infection before the p24 antigen assay and less than 3 days after nucleic acid testing (NAT). The improved ability to detect p24 Ag, at levels similar to specific HIV Ag assays, suggests that these new HIV combined Ag/Ab assays could replace p24 antigen only assays in situations for blood or organ screening when NAT is not feasible or not affordable.

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.

Evaluation of the sensitivity and specificity of six HIV combined p24 antigen and antibody assays.

In this study, we evaluated the performance of six HIV combined p24 antigen and antibody (Ag/Ab) assays versus two third-generation anti-HIV antibody assays. The assays were evaluated using p24 antigen panel of 31 HIV-1 subtypes (n = 124), 25 HIV-1 seroconversion panels (n = 176), HIV-1 antibody positive samples including group M subtypes and group O (n = 559), HIV-2 positive samples (n = 110), and unselected HIV negative samples from four French private laboratories (n = 1005). The results showed that overall HIV combined Ag/Ab assays present better performance, when compared to antibody-only assays. However, some differences were observed in the sensitivity of the six HIV combined Ag/Ab assays evaluated. The AxSYM and Murex Combo assays had the best sensitivity score in this study and reduced the window period by 2.0-2.35 days relative to antibody only assays and 1-2.17 days relative to the other combined Ag/Ab assays. Among combined HIV Ag/Ab assays, Genscreen Plus and AxSYM Combo presented the highest specificity, with 99.9% and 99.8%, respectively.

The variable sensitivity of HIV Ag/Ab combination assays in the detection of p24Ag according to genotype could compromise the diagnosis of early HIV infection.

BACKGROUND: In France, HIV infection diagnosis was modified by a decree, published in 2010, that requires the use of HIV Ag/Ab assays able to detect at least 2 IU/ml of p24Ag. This measure raises the concern of the capacity of these assays to equally detect all HIV variants. OBJECTIVES: To assess the performance of HIV Ag/Ab assays for the detection of p24Ag from diverse HIV isolates. STUDY DESIGN: Ten HIV Ag/Ab assays were compared using two p24Ag reference standards, 297 samples from 99 HIV-1 and HIV-2 cell-culture derived isolates including various subtypes and groups, and 9 native specimens from subjects with primary HIV infection. RESULTS: The p24Ag limit of detection (LOD) ranged from 0.505 IU/ml to 1.90 1 IU/ml and, from 11.9 pg/ml to 33.5 pg/ml when using WHO and French national standards, respectively. The overall percentage of positive samples ranged from 26.8% to 74.5%. Five assays failed to detect all dilutions of at least one group M subtype, three missed all group O and six all the group P samples. Three assays were able to detect 2-10 of the 30 HIV-2 samples. The distribution of LODs for each group M isolate showed a wide dispersion between the assays. Percentage of isolates detected at a p24Ag level less than 2 IU/ml varied from 22% to 98.7%. CONCLUSION: This study demonstrated that, even though their analytical sensitivity fulfills the requirements, many of HIV Ag/Ab assays could fail to detect HIV primary infection due to HIV-1 non-B, non-M and HIV-2 strains.

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.

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.