Validation of the INNO-LiPA HBV DR assay (version 2) in monitoring hepatitis B virus-infected patients receiving nucleoside analog treatment.

Hepatitis B virus (HBV) antiviral drug resistance mutations prevent successful outcome of treatment and lead to worsening of liver disease. Detection of its emergence permits opportune treatment with alternative drugs. Unfortunately, the use of newly approved antivirals, including adefovir dipivoxil, emtricitabine, and telbivudine, is also associated with the development of drug resistance, albeit to a lesser extent than the use of lamivudine. The objectives of this work were to assess the performance characteristics (sensitivity and accuracy) of an updated drug resistance test, the INNO-LiPA HBV DR v2, which includes detection of mutations associated with lamivudine, adefovir, emtricitabine, and telbivudine resistance, and to compare the results with consensus sequencing of serum samples from patients treated with HBV antivirals. Diagnostic sensitivity, defined as detection of a positive amplification line on the line probe assay (LiPA) strip, was 94.8% (95% confidence interval [CI], 89.7 to 97.9) after initial testing, increasing to 96.3% (95% CI, 91.6 to 98.8) after repeat test 1 and to 100% (95% CI, 97.3 to 100.0) after repeat test 2. In diagnostic accuracy determinations, full concordance was observed between sequencing and LiPA for 77.0% of the codons tested (620/805 codons [95% CI, 74.0 to 79.9]), whereas LiPA and sequencing were partially concordant 22% of the time (177/805 codons). In 167 out of 177 cases, LiPA detected a wild-type/mutant mixture whereas sequencing detected only one of the two results. Performance testing of the new LiPA test, the INNO-LiPA HBV DR v2, showed convincing diagnostic sensitivity and accuracy. The ability of the test to detect mixed infections and minority viral populations associated with resistance to the current generation of antivirals, including adefovir, emtricitabine, and telbivudine, makes it a useful tool for HBV therapy monitoring.

Interferon therapy for hepatitis C.

Initial trials indicated that around 50% of patients respond to recombinant alpha interferon by normalizing alanine aminotransferase (ALT) at the end of therapy and that half of these relapsed within 6 months following cessation of treatment. Both dose and duration of treatment are critical in the response to therapy. Higher doses and longer duration have been suggested to be more effective than the current recommendations of 3 MUI thrice weekly for 6 months based on results of these initial studies which used ALT and histological scores to evaluate the efficacy of interferon therapy. Following studies using virological markers have shown that improvements in clinical features of disease are associated with decrease or loss of hepatitis C virus (HCV) from serum and liver. The heterogeneity of the response rates between clinical centers using identical protocol emphasizes that the selection of the patients treated was as important for the outcome that the therapy regimen itself with better responses in cases without cirrhosis and with low levels of HCV RNA. Furthermore, the genotype of HCV seems to be also critical for the response rate. Virological evaluations appears therefore crucial to assess not only HCV infection but also for the indication and monitoring of therapy.

Lack of pre-C region mutation in woodchuck hepatitis virus from seroconverted woodchucks.

Woodchuck hepatitis virus, which shares a large degree of homology with human HBV, was examined for indications of mutational variants. No alteration in the pre-C region was found, but as in HBV, viral DNA could still be detected by PCR after seroconversion to anti-WHe.

Hepatitis B, C, and D virus infections in patients with chronic hepatitis, cirrhosis, and hepatocellular carcinoma: a comparative study in Niger.

Eighty-nine Sahelian African patients with chronic active hepatitis (CAH) (14), cirrhosis (49), hepatocellular carcinoma (HCC) (26), and 47 controls were tested for hepatitis B virus (HBV, hepatitis B surface antigen [HBsAg]) and hepatitis D virus (HDV, anti-HDV antibody). Seventy-three percent of the patients were positive for HBsAg versus 29.8% of the controls (P < 0.0001). With anti-HDV test, 55.0% of the patients were positive versus 17.0% of the controls (P < 0.0001). To assess the prevalence of antibody to hepatitis C virus (HCV), we used an enzyme-linked immunosorbent assay for screening (anti-HCV2): 19.1% of the patients were positive versus 6.4% of the controls (P < 0.05). An association between HBsAg and anti-HDV-positive test results was found in 46.1% of the patients versus 6.4% of the controls (P < 0.0001). A combination of HBsAg and anti-HCV2-positive test results was found in 13.5% of the patients versus 2.2% of the controls (P < 0.05). Anti-HDV and anti-HCV2 test results were positive in 13.5% of the patients versus 2.2% of the controls (P < 0.05). Triple-positive test results (HBsAg, anti-HDV, and anti-HCV2) were found in 11.2% of the patients but in none of the controls (P < 0.025). Triple-negative test results were found in 14.6% of the patients versus 57.4% of the controls (P < 0.0001). The predominant association of the chronic HBV infection with CAH, cirrhosis, and HCC is confirmed in Sahelian Africa. The HDV superinfection (chronic HBV plus HDV infections) may be a major etiology.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of the cross-reactivity with hepatitis B virus antigens and antibodies for the demonstration of a woodchuck hepatitis virus 'e' antigen-antibody system.

Woodchucks hepatitis virus (WHV)-associated antigens and antibodies were studied using current sensitive radio- or enzyme immunoassays (RIA, EIA). A significant cross-reactivity was observed between hepatitis B surface antigen (HBsAg) and woodchuck hepatitis surface antigen (WHsAg) using RIA or EIA (Abbott Laboratories, North Chicago, Ill., U.S.A.) although not with two other commercial EIA tested (Organon Technika, Oss, The Netherlands; Behringwerke AG, Marburg, F.R.G.). A weak but significant reactivity was also found when woodchuck sera positive for WHsAg or anti-WHs by immunodiffusion were tested for HBeAg and anti-HBe by RIA, suggesting the existence of a WHeAg-anti-WHe system in infected woodchucks. The specificity of this e-anti-e reactivity in the woodchuck was further confirmed by successful absorption experiments. WHsAg and WHeAg could be distinguished serologically by immunodiffusion and separated from each other by ultracentrifugation and ammonium sulphate precipitation. A WHeAg preparation was used to boost the presumed natural antibody activity of an immune woodchuck. The specific anti-HBe response detected by RIA during the immunization experiments demonstrated the existence of a soluble WHeAg cross-reacting with the human HBe-anti-HBe system. This was confirmed in immunodiffusion by a partial identity between the precipitin lines formed by the WHeAg-anti-Whe and HBeAg-anti-HBe reaction. Whether the WHe-Ag-anti-WHe system wil mimick HBeAg and anti-HBe in all their clinico-pathological correlations, deserves further study.

A novel vector for the study of hepatitis delta virus replication.

In order to study HDV replication without the difficulties caused by the use of a multimeric construction and to obtain a selectable expression vector, a minimal amount of antigenomic HDV cDNA, sufficient to initiate RNA dependent replication was cloned into the plasmid pUTSV1. The first plasmid, pUTdelta1.7, contained 1.7 genomes of HDV cDNA. After transfection of pUTdelta1.7 into HuH7 cells, antigenomic HDV RNA was produced, processed and could enter into the replicative cycle of HDV. However, after transfection of an antigenomic ribozyme mutant (pUTdelta1.7(AGR)) constructed on the same model, plasmid DNA dependent production of genomic HDV RNA was observed, especially in COS7 cells. It seems that a promoter within vector sequences downstream from the HDV insert may initiate counter-clockwise transcription of the plasmid. The presence of two genomic ribozymes in the insert permits the excision of a genome length genomic HDV RNA from this counter-clockwise transcript. In order to allow quantitative analysis of HDV replication, this problem was eliminated by removing the second genomic ribozyme from the insert to give the vector pUTdelta1.5. This vector can be used conveniently for transfection experiments to explore HDV biology.

High affinity human monoclonal antibodies directed against hepatitis B surface antigen.

Peripheral blood mononuclear cells from donors immunized with hepatitis B vaccine (Pasteur Hevac B) were transformed with Epstein-Barr virus. Two polyclonal cell lines, producing antibodies to hepatitis B surface antigen were established and cloned. Seven clones were isolated; they secreted between 10 and 20 micrograms/ml of HBs specific IgG1 kappa or lambda antibody with anti-HBs titer of 300-800 IU/ml. These human antibodies expressed the anti 'a' specificities and had high affinity and avidity; their potential use as reagents for hepatitis B virus detection and for passive immunotherapy is under study.

Direct cloning and expression of PCR amplified DNA and RNA sequences: application to the hepadnaviruses nucleocapsid proteins.

Gene amplification may benefit from the construction of primers that augments the speed at which cloning and protein expression proceeds. Such primers include EcoRI or HindIII linkers as well as an in phase initiation or termination codon. PCR was carried out directly from viral particles of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) without DNA purification and from RNA extracted from WHV infected liver. Amplified products were directly cloned in the pKK223-3 expression vector under the control of the tac promoter. The characterization of the recombinant clones expressing the nucleocapsid protein (C protein) was done by direct incubation of the filter with 125I-labelled anti-HBc and confirmed by radioimmunoassay and Western-blot analysis. This procedure allows easy selection of recombinant clones expressing a given protein and could be applied to many other genes.

Identification and detection of long incubation non-A, non-B hepatitis virus and associated antigens or antibodies.

Three distinct antigen/antibody systems supposedly associated with an HBV-like virus of non-A, non-B hepatitis have been identified. Because of previously demonstrated cross-reactivity with HBe/3 and HBc antigens and other analogies the following terminology is tentatively used. 1. The previously reported serum antigen has been redesignated non-A, non-B e antigen, since it is equivalent to HBe/3 Ag and cross-reacts with it. Non-A, non-BeAg or Ab were detected in 51/62 post-transfusion and 11/56 sporadic acute non-A, non-B hepatitis cases, and in 12/14 cases affecting staff members. In non-A, non-B chronic persistent or active hepatitis and cryptogenic cirrhosis, the prevalence was similarly high: 14/18, 22/48 and 12/18 respectively. Ten out of 26 implicated blood donors were found positive for non-A, non-BeAg accounting for 7 out of 8 post-transfusion cases. A high prevalence of non-A, non-BeAg was also found in haemophiliacs (11/48) and haemodialysed patients (6/42), whereas anti-non-A, non-Be was respectively detected in 4/48 and 6/42 of these cases. 2. Using immunofluorescence, a second antigen termed non-A, non-BcAg has been identified in liver biopsies from 55/84 non-A, non-B chronic hepatitis or cryptogenic cirrhosis cases. All 8 positive biopsies examined by electron microscopy revealed clusters of 22--25 nm intranuclear particles identical to those described in chimpanzees. Anti-non-A, non-Bc detectable by counter-electrophoresis and indirect immunofluorescence was found in the serum of all patients of which biopsy was positive for non-A, non-BcAg. Anti-non-A, non-Bc was also detected in 5/5 non-A, non-BeAg positive cases of post-transfusion hepatitis, 2--6 weeks after onset end remained positive for the 6 month follow-up period. 3. A third antigen, tentatively designated non-A, non-BsAg, has been found less frequently than non-A, non-BeAg in serum. However, it was detectable in 3/18 and 2/12 washed ultracentrifugation pellets of sera positive for non-A, non-BeAg or anti-non-A, non-Be, respectively.

[Spontaneous and experimental infection of alpine marmots (Marmota marmota) by the North American woodchuck hepatitis virus (Marmota monax). Initial results]. / Infection spontanée et expérimentale de la marmotte alpine (Marmota marmota) par le virus de l'hépatite de la marmotte nord-américaine (Marmota monax). Premiers résultats.

Summer's discovery in 1978 of a DNA virus, very close to human Hepatitis B virus in a woodchuck population in the U.S.A. (Pennsylvania) was a confirmation of the first description made by Snyder at Penrose Research Laboratory (Philadelphia). It was the first animal model of human B hepatitis infection. The comparative study of morphological, ecological and ethological characteristics of the marmot (Marmota marmota) and the woodchuck (Marmota monax) enables an easy distinction between these two species. The natural infection of M. monax by the WHV shows that the woodchuck is a good model for human B hepatitis and should be extended to M. marmota. A sample of 24 marmots caught in the Alpes of Haute-Provence has not revealed any spontaneous infection in these animals by the woodchuck virus. The failure of experimental inoculation of the marmot (24 animals) with the WHV confirms the refractory status of this species (no viremia and very low and short serological response with or without an immunosuppressive treatment). These preliminary results require a confirmation in other animals of different age and geographical region and also by using more specific tests such as molecular hybridization, research on DNA polymerase and direct transfection trials.

[Significance of the expression of pre-S proteins in mononuclear blood cells in chronic hepatitis caused by the hepatitis B virus]. / Signification de l'expression des protéines pré-S dans les cellules mononucléées du sang au cours des hépatites chroniques dues au virus de l'hépatite B.

In chronic viral type B hepatitis, the presence in the serum of pre-S proteins of hepatitis B virus (HBV) envelope reflects viral replication. As peripheral blood mononuclear cells (PBMC) are known to be target cells for HBV replication, the aim of our study was to investigate the clinical relevance of pre-S protein expression in PBMC. Fifty-seven patients with chronic type B hepatitis and HBs antigenemia were studied. Following separation using the Ficoll gradient, the PBMC were lysed and studied for pre-S proteins by Western blot. HBs Ag and HBc/e Ag were assayed in parallel by radioimmunoassay. HBs Ag was detected in PBMC in 86 percent of cases, HBc/e Ag in 28 percent of cases and pre-S proteins in 34 percent of cases. A statistically significant association was found between the presence of HBc/e Ag in PBMC and both the serum HBe Ag (chi 2 test, p less than 0.01) and the serum viral DNA/DNA polymerase (t test, p = 2.10(-4)). The pre-S protein expression in PBMC was significantly associated with higher levels of DNA/DNA polymerase activity (chi 2 test, p less than 0.05). The expression of pre-S proteins in PBMC appears therefore to correlate with the HBV viral replication phase. The HBc/e Ag and pre-S protein detection in PBMC therefore offers a reliable non invasive approach to tissular viral replication. The clinical relevance of pre-S testing in PBMC was illustrated by the study of 12 cases of chronic active hepatitis positive for anti-HBe but with no or low level of serum DNA polymerase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hepatitis B virus reactivation after allogeneic bone marrow transplantation in a patient previously cured of hepatitis B]. / Réactivation virale B après greffe de moelle allogénique chez un malade précédemment guéri d'une hépatite virale B.

The presence of antibodies to HBs and HBc antigens indicates previous infection with hepatitis B virus but does not necessarily reflect viral clearance. Immunosuppression such as that observed in patients with bone marrow transplantation may be responsible for viral reactivation followed by acute exacerbation after withdrawal of immunosuppressive therapy. We report a case in a patient with natural immunity to hepatitis B who had undergone allogenic bone marrow transplantation with an identical sibling donor one year before for the chronic myelogenous leukemia in the first chronic phase. Ganciclovir treatment resulted in control of hepatitis virus B replication and in biochemical remission. We suggest that prevention relies on serological evaluation and therapy with active or passive immunisation or antiviral drugs in case of a rapid decline of anti-HBs Ab titers to undetectable levels.

[Prevalence of viral hepatitis B markers in the region of Tataouine (southern Tunisia)]. / Prévalence des marqueurs du virus de l'hépatite B dans la région de Tataouine (Sud Tunisien).

Tataouine (South Tunisia) has been subject to several viral hepatitis epidemics during these last years. Prospective study has been conducted to define the prevalence of HVB infection. It has examined 511 cases, the age of whom was between 1 month and 70 years, reported in groups through the OMS recommendations, taking into account the number of inhabitants in each area. The global prevalence was 6.2% for HBs Ag and 18.5% for HBs antibody. These results were not different from the frequencies observed within the Tunisian population in general. The analysis of this study according to these groups, shows that in three areas (Ras El Oued, Bir 30, Dhibet) 60 to 80% of cases have at least one of HVB marquers, whereas the prevalence in other areas (Rogba) was very weak. The geographic repartition of HVB infection corresponds approximatively to areas that have been the most infected by the last hepatitis epidemics. A second study has completed and confirmed the results of the first one, taking into consideration a more important number of cases from areas of strong and weak endemicity. 596 sera have been examined in Rogba which counts 2000 inhabitants and is a weak endemicity area. 528 sera have been examined in the three areas of strong endemicity: 199 in Bir 30, 201 in Dhibet and 128 in Ras El Oued, which count around 2000 inhabitants, too. The percentage of cases presenting at least one of HVB serological marquers reaches 84 in Dhibet, 70 in Ras El Oued, 50 in Bir 30 and 24 in Rogba. This HBs Ag frequency is 3.8% in Rogba, 15% in Bir 30, 24% in Ras El Oued and 26.3% in Dhibet. It seems that the HVB is the virus of hepatitis epidemics observed in Tataouine at least in the three strong endemicity areas.

Risk of hepatitis B surface antigen seroreversion after allogeneic hematopoietic SCT.

Allogeneic hematopoietic SCT (HSCT) increases the risk of hepatitis B virus (HBV) reactivation in hepatitis B surface antigen (HBsAg) carriers but the incidence, risk factors and course of HBV reactivation after HSCT in HBsAg-negative/anti-hepatitis B core antigen (anti-HBc)-positive recipients are not well known. A total of 50 HBsAg-negative/anti-HBc-positive HSCT recipients with onco-hematological diseases, underwent sequential clinical and laboratory examinations, including serum HBsAg, during follow-up. Serum HBV DNA collected at HSCT was retrospectively amplified by a sensitive PCR assay. During 17 months of follow-up, six (12%) patients had seroreverted to HBsAg, 7-32 months after HSCT, with 1- and 5-year cumulative rates of 13 and 22%. HBsAg seroreversion was associated with serum HBeAg higher than 8 log10 copies per ml HBV DNA and a 1.5 to 36 fold increase of serum alanine aminotransferase leading to HBeAg-positive chronic hepatitis B in all patients. Patients with chronic onco-hematological disease and long-lasting immunosuppression following HSCT had a higher risk of HBsAg seroreversion independently of serum HBV DNA levels at HSCT. HBsAg-negative/anti-HBc-positive HSCT recipients with chronic onco-hematological disease carry a significant risk of HBsAg seroreversion and HBeAg-positive chronic hepatitis B, independently of serum levels of HBV DNA at transplantation.

In vitro characterization of the anti-hepatitis B virus activity and cross-resistance profile of 2',3'-dideoxy-3'-fluoroguanosine.

The fluorinated guanosine analog 2',3'-dideoxy-3'-fluoroguanosine (FLG) was shown to inhibit wild-type (wt) hepatitis B virus (HBV) replication in a human hepatoma cell line permanently expressing HBV. Experiments performed in the duck model of HBV infection also showed its in vivo antiviral activity. In this study, we investigated the mechanism of inhibition of FLG on HBV replication and its profile of antiviral activity against different HBV or duck hepatitis B virus (DHBV) drug-resistant mutants. We found that FLG-triphosphate inhibits weakly the priming of the reverse transcription compared to adefovir-diphosphate in a cell-free system assay allowing the expression of an enzymatically active DHBV reverse transcriptase. It inhibits more potently wt DHBV minus-strand DNA synthesis compared to lamivudine-triphosphate and shows a similar activity compared to adefovir-diphosphate. FLG-triphosphate was most likely a competitive inhibitor of dGTP incorporation and a DNA chain terminator. In Huh7 cells transiently transfected with different HBV constructs, FLG inhibited similarly the replication of wt, lamivudine-resistant, adefovir-resistant, and lamivudine-plus-adefovir-resistant HBV mutants. These results were consistent with those obtained in the DHBV polymerase assay using the same drug-resistant polymerase mutants. In conclusion, our data provide new insights in the mechanism of action of FLG-triphosphate on HBV replication and demonstrate its inhibitory activity on drug-resistant mutant reverse transcriptases in vitro. Furthermore, our results provide the rationale for further clinical evaluation of FLG in the treatment of drug-resistant virus infection and in the setting of combination therapy to prevent or delay drug resistance.

European multicenter evaluation of high-density DNA probe arrays for detection of hepatitis B virus resistance mutations and identification of genotypes.

Polymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 10(3) genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B.

[Identification of a virus similar to hepatitis B virus in non-A non-B hepatitis]. / Identification d'un virus semblable au virus de l'hépatite B dans les hépatites NON-A NON-B.

Hepatitis B virus-like particles (including DANE particles) with DNA polymerase activity but negative for HBs Ag have been identified in NON-A, NON-B hepatitis sera positive for HC Ag. Although specifically associated with the particles, HC Ag is not a surface antigen of the hepatitis C virus identified here for the first time. The relationship of this agent with HBV seems obvious, and deserves further study.

Heterogeneity of hepatitis B virus (HBV) core gene in a patient with HBV-associated cirrhosis and serum negativity for anti-HBc.

AIMS: We describe here the case of a patient suffering from severe chronic hepatitis B associated with an unusual hepatitis B virus serology: HBsAg and HBeAg were both positive while anti-HBc was negative by radioimmunoassay. METHODS: A very sensitive anti-HBc ELISA (IMx CORE) was performed and was able to detect anti-HBc sporadically throughout the clinical course. Molecular characterization of hepatitis B virus strains in this patient enabled us to explain this particular serological and clinical pattern of hepatitis B virus infection. RESULTS: Hepatitis B virus genotype determined by size polymorphism of the core gene and the pre-S region was found to be D/E and consistent with the results of serological subtyping (HBV ayw2-4). DNA sequence analysis of the pre-C/C region showed the presence of significant nucleotide changes. In association with a wild type hepatitis B virus strain, we could detect at least four hepatitis B virus variants with nucleotide deletions leading to a frameshift in the core gene. According to the position of the mutations, these hepatitis B virus core variants are expected to be defective for B-cell epitopes and TH-cell epitopes. CONCLUSIONS: These mutations explain the low level production of anti-HBc antibody. It is noteworthy that the absence of detectable anti-HBc in serum was associated with severe liver damage, suggesting that the deficient humoral response to HBcAg was not accompanied by a cellular immune tolerance to HBc/eAg, the supposed target for cytotoxic T-cell lysis.

[Demonstration of a serum and liver antigen in acute or chronic non A-non B viral hepatitis]. / Mise en évidence d'un antigène sérique et hépatique au cours des hépatites virales non A-non B aiguës ou chroniques.

A new precipitating antigen-antibody system possibly was demonstrated by immuno-diffusion in the serum of patients suffering from non A-non B hepatitis. The antigen appears during the first four weeks of transaminases elevation. In acute cases was transient antigenemia (average 3 weeks). Antibodies appeared rapidly after the disappearance of antigen. The same antigen was also detected, by immunodiffusion and by immunofluorescence, in the liver nuclei of infected hepatocytes. This antigen specific appears for non A-non B hepatitis since it is neither found in the serum of normal subjects nor in that of patients with cirrhosis toxic or viral that hepatitis A or B. The hypothesis of a virus associated antigen is the most likely explanation.

Diagnostic markers of viral hepatitis B and C.

Hepatitis B virus (HBV) serology has become extremely refined. As well as the recognised hepatitis B surface (HBs), hepatitis B core (HBc), and hepatitis B e (HBe) antigen-antibody systems, new markers have been introduced including pre-S1, pre-S2 for the envelope and the functional X protein. New automates have been introduced allowing flexibility in the different tests according to precise needs. The monitoring of pre-S1 antigen provides a relevant correlate of viral replication. The quantitative determination of HBV-DNA, pre-S1 Ag, and IgM anti-HBc seem most useful for the decision to use, and the monitoring of, antiviral treatment. Second generation ELISAs detect antibodies to three sets of hepatitis C virus (HCV) protein including the c22 core, and c33, and c100, which correspond to the non-structural regions (NS3 and NS4, respectively). Second generation ELISAs require confirmation by supplement assays, but their biggest limitation is the delayed appearance of anti-HCV after primary infection. In addition 10% of chronic infections with liver disease still remain seronegative despite circulating HCV RNA in serum or liver, or both. Much progress still has to be made before HCV serology can reach the level of sophistication of HBV.