Kinetics of acute hepatitis B virus infection in humans.

Using patient data from a unique single source outbreak of hepatitis B virus (HBV) infection, we have characterized the kinetics of acute HBV infection by monitoring viral turnover in the serum during the late incubation and clinical phases of the disease in humans. HBV replicates rapidly with minimally estimated doubling times ranging between 2.2 and 5.8 d (mean 3.7 +/- 1.5 d). After a peak viral load in serum of nearly 10(10) HBV DNA copies/ml is attained, clearance of HBV DNA follows a two or three phase decay pattern with an initial rapid decline characterized by mean half-life (t(1/2)) of 3.7 +/- 1.2 d, similar to the t(1/2) observed in the noncytolytic clearance of covalently closed circular DNA for other hepadnaviruses. The final phase of virion clearance occurs at a variable rate (t(1/2) of 4.8 to 284 d) and may relate to the rate of loss of infected hepatocytes. Free virus has a mean t(1/2) of at most 1.2 +/- 0.6 d. We estimate a peak HBV production rate of at least 10(13) virions/day and a maximum production rate of an infected hepatocyte of 200-1,000 virions/day, on average. At this peak rate of virion production we estimate that every possible single and most double mutations would be created each day.

A rapid real-time quantitative polymerase chain reaction for hepatitis B virus.

Quantification of hepatitis B virus (HBV) DNA in serum is important for monitoring treatment. A rapid and cost effective alternative to the methods available currently was developed based on a real-time quantitative polymerase chain reaction (PCR) done in the LightCycler apparatus. Primers and a probe for sequences of the surface gene of HBV were designed and quantification achieved by reference to standards containing known concentrations of the target sequence. A single copy of the HBV genome could be detected if present in the reaction mixture. The quantitative range of the assay was from 4 x 10(2) to 1.3 x 10(10) surface gene copies/ml serum. Nested PCR was required for quantification in the lower part of this range (<10(5) copies). The real-time PCR and Amplicor Monitor (Roche) tests performed comparably at virus concentrations below 10(6) copies/ml. The commercial test underestimated higher concentrations of virus.

Monitoring the emergence of hepatitis B virus polymerase gene variants during lamivudine therapy using the LightCycler.

Treatment of chronic hepatitis B virus (HBV) infection with lamivudine is associated with the appearance in the circulation of HBV variants with mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the polymerase gene. Fluorometric real-time PCR with the LightCycler assay was used for the detection of resistant variants. Differences in the hybridization melting curve kinetics of probes bound to the sequences encoding the wild-type or the mutant YMDD motifs (YIDD or YVDD in which the methionine residue is altered to an isoleucine or a valine, respectively) distinguished the single-base changes responsible for the resistance phenotype. The LightCycler probe hybridization assay was applied to 40 serum specimens from 19 patients, and the results were correlated with the nucleotide sequences determined for the corresponding PCR products. All three variants could be identified in the specimens. PCR clones obtained from four patients early in the course and prior to lamivudine therapy were investigated for the appearance of YIDD and YVDD variants with the LightCycler assay. In one patient, a transient appearance of the YIDD variant was observed 6 weeks into therapy. Subsequently, after 11 months of lamivudine therapy, the YVDD variant emerged in that patient.

Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms.

After hepatitis B virus (HBV) infection, liver injury and viral control have been thought to result from lysis of infected hepatocytes by virus-specific cytotoxic T cells. Patients are usually studied only after developing significant liver injury, and so the viral and immune events during the incubation phase of disease have not been defined. During a single-source outbreak of HBV infection, we identified patients before the onset of symptomatic hepatitis. The dynamics of HBV replication, liver injury, and HBV-specific CD8+ and CD4+ cell responses were investigated from incubation to recovery. Although a rise in alanine transaminase (ALT) levels was present at the time of the initial fall in HBV-DNA levels, maximal reduction in virus level occurred before significant liver injury. Direct ex vivo quantification of HBV-specific CD4+ and CD8+ cells, by using human leukocyte antigen (HLA) class I tetramers and intracellular cytokine staining, showed that adaptive immune mechanisms are present during the incubation phase, at least 4 weeks before symptoms. The results suggest that the pattern of reduction in HBV replication is not directly proportional to tissue injury during acute hepatitis B in humans. Furthermore, because virus-specific immune responses and significant reductions in viral replication are seen during the incubation phase, it is likely that the immune events central to viral control occur before symptomatic disease.

Molecular epidemiology of a large outbreak of hepatitis B linked to autohaemotherapy.

BACKGROUND: Unregulated skin-piercing procedures potentially facilitate the transmission of bloodborne pathogens. In February, 1998, a patient who had recently received autohaemotherapy at an alternative medicine clinic in the UK was diagnosed with acute hepatitis B. The autohaemotherapy procedure involved the drawing of 1 mL of the patient's blood, mixing with saline, and reinjection of the autologous blood mixture. We investigated the extent of hepatitis B virus (HBV) infection in patients and staff of the clinic. METHODS: Patients who had attended the clinic between January, 1997, and February, 1998, were tested for serological markers of HBV, and for HBV DNA by PCR. HBV DNA was sequenced to assess the relatedness of the virus identified in the cases. We analysed the number and dates of visits with regard to HBV status. FINDINGS: Serum samples were received from 352 patients and four staff members. Serological evidence of exposure to HBV was found in 57 (16%). Of the 33 patients and staff who were positive for hepatitis B surface antigen, 30 (91%) showed complete nucleotide identity in the DNA segments derived from the surface and core genes. Five patients with linked infection had markers of chronic hepatitis B, and one of these was regarded as the likely source of the outbreak. The attack rate was associated with the number of visits (p<0.0001) and the week of visit (p=0.011). Contaminated saline in a repeatedly used bottle was the probable vehicle of transmission. INTERPRETATION: We have described a large community-based outbreak of hepatitis B due to transmission by a single HBV variant. Our findings emphasise the continuing risk of transmission of bloodborne viruses in all health-care settings where skin-piercing procedures are used.