F. B. Hollinger

Occult hepatitis B virus infection: a covert operation.

Summary.  Detection of occult hepatitis B requires assays of the highest sensitivity and specificity with a lower limit of detection of less than 10 IU/mL for hepatitis B virus (HBV) DNA and <0.1 ng/mL for hepatitis B surface antigen (HBsAg). This covert condition is relatively common in patients with chronic hepatitis C virus (HCV) that seems to exert some influence on the replicative capacity and latency of HBV. Detection of virus‐specific nucleic acid does not always translate into infectivity, and the occurrence of primer‐generated HBV DNA that is of partial genomic length in immunocompetent individuals who have significant levels of hepatitis B surface antibody (anti‐HBs) may not be biologically relevant. Acute flares of alanine aminotransferase (ALT) that occur during the early phase of therapy for HCV or ALT levels that remain elevated at the end of therapy in biochemical nonresponders should prompt an assessment for occult hepatitis B. Similarly, the plasma from patients with chronic hepatitis C that is hepatitis B core antibody (anti‐HBc) positive (±anti‐HBs at levels of <100 mIU/mL) should be examined for HBV DNA with the most sensitive assay available. If a liver biopsy is available, immunostaining for hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) should be contemplated and a portion of the sample tested for HBV DNA. This is another reason for optimal collection of a specimen (e.g. two passes with a 16‐guage needle under ultrasound guidance). Transmission of HBV to immunosuppressed orthotopic liver transplant recipients by donors with occult hepatitis B (OHB) will continue to occupy the interests of the transplant hepatologist. As patients with OHB may have detectable HBV DNA in serum, peripheral blood mononuclear cells (PBMC) and/or liver that can be reactivated following immunosuppression or intensive cytotoxic chemotherapy, the patient needs to be either monitored or treated depending on the pretreatment serological results such as an isolated anti‐HBc reaction or a detectable HBV DNA.

Hepatitis B virus genetic diversity and its impact on diagnostic assays

Summary.  Hepatitis B virus (HBV) circulates in blood as closely related, but genetically diverse molecules called quasispecies. During replication, HBV production may approach 1011 molecules/day, although during peak activity this rate may increase 100–1000 times. Generally, DNA polymerases have excellent fidelity in reading DNA templates because they are associated with an exonuclease which removes incorrectly added nucleotides. However, the HBV‐DNA polymerase lacks fidelity and proofreading function partly because exonuclease activity is either absent or deficient. Thus, the HBV genome and especially the envelope gene, is mutated with unusually high frequency. These mutations can affect more than one open reading frame because of overlapping genes. The S gene contains an exposed major hydrophilic region (residues 110–155), which encompasses the ‘a’ determinant that is important for inducing immunity. Nucleotide substitutions in this region are common and result in reduced binding or failure to detect hepatitis B surface antigen (HBsAg) in diagnostic assays. Adaptive immunity also depends on the recognition of HBsAg by specific antibody and variants pose a threat if they interfere with binding to antibody. Finally, genomic hypervariability allows HBV to escape selection pressures imposed by antiviral therapies, vaccines and the host immune system, and is responsible for creating genotypes, subgenotypes and subtypes.

Occult hepatitis B virus infection and hepatocellular carcinoma: a systematic review

Occult hepatitis B (OHB) infection has been reported to play an important role in the development of hepatocellular carcinoma (HCC). In this systematic review, a significantly higher prevalence of OHB was observed in patients with HCC in the presence or absence of HCV infection when compared with control populations without HCC. Correspondingly, among adequately designed prospective studies, the cumulative probability of developing HCC was significantly greater among patients with OHB than among HBV DNA‐negative patients in the presence or absence of HCV infection. Study design, inclusion criteria, treatment options, methodology and potential confounding variables were evaluated, and immunopathogenic mechanisms that could be involved in OHB as a risk factor in HCC were reviewed. From this analysis, we conclude that although OHB is an independent risk factor in HCC development in anti‐HCV‐negative patients, a synergistic or additive role in the occurrence of HCC in HCV‐coinfected patients is more problematic due to the HCC risk attributable to HCV alone, especially in patients with advanced fibrosis and cirrhosis.

A comparison of reverse transcription–polymerase chain reaction and branched-chain DNA assays for hepatitis C virus RNA in patients receiving interferon treatment

Measurement of hepatitis C virus (HCV) RNA may be beneficial in managing the treatment of patients with chronic HCV infection. In a phase 3 study comparing consensus interferon (IFN) and IFN‐α2b treatment in patients with chronic HCV infection, serum samples were assayed for HCV RNA using two different assays: a quantitative multicycle reverse transcription–polymerase chain reaction (RT–PCR) method and the Quantiplex branched‐chain DNA (bDNA) method. Lower and upper detection limits were 100 copiesml–1 and 5×106 copiesml–1, respectively, for the RT–PCR method, and 3.5×105 and 4×107 genome equivalentsml–1, respectively, for the bDNA method. The two assays were generally concordant over the common range of detectability. The major discrepancy was where PCR still indicated detectable virus in the sample but the bDNA result was negative. Assessment of serum samples during IFN treatment demonstrated that 37% of samples were negative for HCV RNA by bDNA but positive by RT–PCR. Differences were also noted in the quantification of baseline HCV RNA by genotype. These data suggest that HCV patients could be categorized as treatment responders by the bDNA assay when the more sensitive RT–PCR assay indicates lack of complete viral response.

Detection of antibodies to hepatitis C virus in seronegative patients using an immune complex dissociation assay

Summary. Sera from a small percentage of hepatitis C virus (HCV)‐infected blood donors do not react in the currently available assays for detection of antibody to HCV (anti‐HCV) and, as a consequence, hepatitis C may develop in recipients of this blood. One possible explanation for this phenomenon is that antibody is present but cannot be detected because it is sequestered in circulating immune complexes. To test this hypothesis, an immune complex dissociation (ICD) assay was developed to disrupt any immune complexes that might be present in these anti‐HCV‐negative, HCV RNA‐positive sera. A positive result in this test would indicate that antibody is present in these patients but is not detectable under routine anti‐HCV testing conditions. Nine chronic and two acute HCV patients, all negative for antibody but positive for HCV RNA by reverse transcriptase‐polymerase chain reaction (RT‐PCR) were tested, together with appropriate controls. Three of the nine study patients with chronic HCV had evidence of anti‐HCV after immune complex dissociation compared with none of the two patients with acute HCV. Although the number of patients tested was small, the negative results in the patients with acute HCV presumably indicates that anti‐HCV seroconversion had not yet occurred. Incorporation of an ICD step into existing anti‐HCV assays may enable blood banks to detect those rare instances of patients with chronic HCV who are antibody negative; this would minimize potential cases of post‐transfusion hepatitis in recipients.

Current tests for antibody to hepatitis B core antigen used to screen donors for non‐A, non‐B hepatitis are comparable to the original radioimmunoassay for hepatitis B core antigen

ABSTRACT: Prospective studies have shown a relationship between the transfusion of donor blood which is positive for antibodies to hepatitis B core antigen (anti‐HBc) and an increased incidence of non‐A, non‐B hepatitis. The anti‐HBc test was selected on the assumption that epidemiologic circumstances predisposing donors to hepatitis B infection also might favor exposure to non‐A, non‐B hepatitis. Current radioimmunoassays (RIA) and enzyme‐linked immunoassays (EIA) for anti‐ HBc utilize hepatitis B core antigen (HBcAg) prepared by recombinant DNA technology, whereas the original RIA anti‐HBc assay used HBcAg derived from hepatitis B virions. In the current study, 1329 sera were evaluated of which 23.3 percent were anti‐HBc positive. The results indicate that sensitivity, specificity, and positive and negative predictive values of the current EIA and RIA tests for anti‐HBc (Abbott Diagnostic Laboratories) are virtually identical to the original RIA test kit. In addition, all donor samples (128 specimens) administered to 57 cases of non‐A, non‐B hepatitis that were prospectively followed at Baylor College of Medicine for the Transfusion‐Transmitted Viruses (TTV) Study group were retested with the EIA‐recombinant anti‐HBc assay. All 21 samples which were reactive in the original RIA anti‐HBc test also were positive by the current EIA procedure. One sample was EIA positive/RIA negative, and 106 other samples were negative by both assays. Thus, commercial anti‐HBc kits based on HBcAg derived by recombinant DNA technology, should retain their predictive value for reducing the incidence of non‐A, non‐B hepatitis as described in the prospective studies.