Introduction
Viral hepatitis continues to account for significant global disease and high mortality from liver cancer and cirrhosis. In 2019, the World Health Organization estimated that 296 million people were living with chronic hepatitis B virus (HBV) infection worldwide and that there are about 1.5 million new hepatitis B infections each year, despite the availability of a highly effective vaccine [
1].
The course of HBV serum markers during a typical acute self-limited HBV infection is usually depicted as a composite of traditional HBV marker data from studies of blood donors [
2‐
4]. Results are displayed as relative concentrations along with the mean lengths of the various phases of acute infection. The order of appearance of serum HBV markers follows a consistent pattern with an eclipse phase preceding the release of virions into the blood, followed by the first detectable levels of HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and antibodies to HBV core and HBeAg antigens (anti-HBc IgM, total anti-HBc, and anti-HBe). Resolution of infection and recovery is marked by loss of serum HBsAg and HBV DNA and the appearance of antibody to HBsAg (anti-HBs).
The development of persistent infection is indicated by the continued detection of HBsAg for more than six months. Models of the natural history of HBV infection are critical to our understanding of the risk of transmission and reactivation and the sensitivity requirements for assays used in diagnostics and blood screening. In addition, knowledge of the complexities of HBV markers during acute self-limited infection or development of persistent infection is relevant to understanding resolution of chronic hepatitis B in response to anti-viral therapy and in studies of occult HBV infection.
The current study updates and expands our knowledge of acute hepatitis B with data on longitudinal samples from five plasmapheresis donors with acute HBV infection, four with self-limited infection and one with development of persistent infection. We report detailed serological and molecular data on the course of hepatitis B seroconversion using state of the art assays for quantitation of HBsAg and HBeAg, a new highly sensitive HBsAg assay, and novel biomarkers for quantitation of HBV pregenomic RNA (pgRNA) and HBV core related antigen (HBcrAg).
Discussion
The current study provides detailed serological and molecular profiles for longitudinal samples from five plasmapheresis donors with acute HBV infection. Included were four seroconversion series with acute resolving infection and one acute series leading to persistent infection. The panels in the current study are uniquely informative in that they encompass the entire seroconversion profile from early acute infection, through the peak levels of HBV DNA and HBsAg, to the decline and loss of detectable DNA and HBsAg and development of anti-HBe and anti-HBs. Although the current report is limited to five panels, it is important to note that HBV seroconversion panels that span the entirety of acute HBV infection are extremely rare. Informative panels require samples obtained during the ascending, peak, and descending portions of the HBV biomarker seroconversion curves through resolution of acute infection and detection of anti-HBs. In addition, sampling intervals should be less than two weeks, preferably one week, with sufficient volume to allow testing of many different biomarkers. A survey of 85 HBV seroconversion panels commercially available over the past decade revealed that only four panels fulfilled the above criteria and none demonstrated progression to chronic infection. The seroconversion profiles of two of the four commercial panels (SCP-HBV-001 from DiaMex, Heidelberg, Germany and PHM935 from SeraCare, Milford MA) were comparable to panel 43527-3453 in the current study, i.e., prolonged declining surface antigenemia. The profiles for the other two commercial panels (6281 from Zeptometrix, Franklin, MA and SCP-HBV-002 from DiaMex) were similar to seroconverter 13867-3482 from the current study.
The current study is distinctive in using quantitative assays for HBV DNA, HBsAg, and HBeAg; a new highly sensitive HBsAg assay; and quantitative assays for two new HBV biomarkers, HBV pgRNA and HBcrAg. Quantitative assays for HBsAg and HBeAg are now widely available. These assays are standardized against WHO International Reference Standards; results are expressed as IU/ml facilitating comparison of data across studies. Previously, depictions of HBV seroconversion relied on results from qualitative assays, which limited the accuracy of determining the timing and concentrations of peak antigen levels. In the current study, the quantitative assays showed that the peak surface antigenemia occurred from 4 days before to 9 days after the HBV DNA peak (Figs.
1,
2). HBeAg levels consistently peaked after HBV DNA ranging from 2 to 20 days later. A wide range of maximum HBV DNA and HBsAg levels were observed in this study. Panels 26022-14518 and 13867-3482 with relatively short durations of infection had the lowest peak levels of HBV DNA and HBsAg (2.2 × 10
5–3.4 × 10
5 HBV DNA IU/ml and 7.9–134.3 HBsAg IU/ml) (Table
1). HBV DNA and HBsAg levels in panels 1807–3463, 43527-3453, and 0994-3457 were orders of magnitude higher (1.7 × 10
9–2.7 × 10
9 HBV DNA IU/ml and 9.7 × 10
4–1.1 × 10
5 HBsAg IU/ml). Maximum HBeAg levels also demonstrated orders of magnitude differences among the panels (Table
1). For comparison, mean HBV DNA levels of 10
7–10
8 IU/ml and mean HBsAg levels of 2.3 × 10
4–9 × 10
4 IU/ml have been reported for patients with HBeAg positive chronic infection [
14].
Previous studies have estimated the mean duration of HBsAg positivity using an assay with 0.02 IU/ml sensitivity. One study provided an estimate of 63 days based on the numbers of HBV nucleic acid test (NAT) positive blood donor samples with detectable HBsAg [
4]. Another study reported a mean of 92.7 days based on evaluation of 10 seroconversion panels [
15]. The duration of HBsAg positivity is dependent on the sensitivity of the HBsAg assay used. The HBsAgNx assay with 0.005 IU/ml sensitivity has been shown to reduce the early acute HBsAg negative window by 4.1–4.6 days and lengthen duration of HBsAg detection in the late acute phase in comparison with an assay with 0.02 IU/ml sensitivity [
5,
6,
10]. In the current study, series 26022-14518 and 13867-3482 had relatively short infections based on the duration of HBsAgNx positivity (37 and 43 days, respectively) while the duration was longer in panel 1807-3463 (159 days) (Table
2).
Interestingly, only two samples were HBeAg positive in the 26022-14518 panel: at days 0 and 2 relative to the DNA peak. The possibility of such a short period of HBeAg positivity should be considered when interpreting patient test results when evaluation of HBV infection depends on only one sample or limited follow-up. HBcrAg results were positive for both of the HBeAg positive samples and for samples immediately preceding and following.
In contrast to the panels with short or intermediate durations of infection, a prolonged course was observed in panel 43527-3453 where HBsAgNx was positive for 314 days before becoming negative 266 days after the HBV DNA peak; anti-HBs was detected at day 317 (Fig.
2, Table
3). By the criterion for classification of chronic infection (HBsAg positivity exceeding six months), this panel could represent a chronic infection which spontaneously resolved. Alternatively, the observation that HBV DNA was still intermittently detectable at very low levels after the loss of HBsAg suggests that this could represent an early chronic infection that became occult (anti-HBc positive, low level HBV DNA positive, HBsAg negative, with low level anti-HBs). The continued high levels of HBV DNA and HBsAg with no detectable anti-HBs in panel 0994-3457 clearly indicate the development of persistent infection.
Although it is common for HBV DNA to be detectable for about 10 days after HBsAg clearance (the so-called post-HBsAg or second window period), intermittent detection of very low levels of HBV DNA in serum years after clearance of serum HBsAg and recovery from self-limited infection has been reported [
2]. The existence of HBV DNA PCR positive, HBsAg negative sera from cases of resolving HBV infection and the continued presence of HBV cccDNA and HBV RNA in liver years after HBsAg clearance has been documented [
16,
17]. Using the strategy of replicate testing to enhance detection, we found that three of the panels in this study (43527-3453, 26022-14518, and 13867-3482) had intermittent low-level HBV DNA (estimated at ≤ 3 IU/ml) in late follow-up samples. In panels 26022-14518 and 13867-3482, samples with very low DNA levels were observed in the presence of relatively low anti-HBs (25–312 mIU/ml in 13867–3482) and in samples with high anti-HBs (> 1000 mIU/ml in 26022-14518).
Serum HBV pgRNA has emerged as a new biomarker reflecting the levels and activity of intrahepatic HBV covalently closed circular DNA (cccDNA) [
13]. HBV pgRNA can be released into the serum within enveloped virions. A dual-target RT-PCR assay has been developed to quantitate HBV pgRNA [
8]. HBV pgRNA results from the current study augment and support our previous report showing similar kinetics for HBV DNA and HBV pgRNA during seroconversion, including panel 0994-3457 which progressed to persistent infection [
8]. Concentrations of pgRNA were an average of 2.2 logs lower than HBV DNA in the present study, as was found previously. To the authors’ knowledge, these are the only studies reporting the kinetics of HBV pgRNA for longitudinal samples from acute HBV infections. Interestingly, panel 26022-14518 in which we observed intermittently detectable low-level HBV DNA after HBsAg clearance, also had low levels of HBV pgRNA during the same time period, suggesting continued transcriptional activity of HBV cccDNA in the liver.
HBcrAg is comprised of three antigens expressed from the pre-core/core gene: HBcAg, HBeAg, and a 22 kD pre-core precursor protein [
11]. The three proteins overlap, sharing an identical 149 amino acid sequence. HBcrAg can be detected in virions containing HBV DNA, DNA-negative particles, and possibly in pgRNA-containing particles. HBeAg is the predominant component detected by the HBcrAg assay in HBeAg positive sera. HBcrAg has been shown to correlate with HBV cccDNA and has been extensively studied relative to the phases of chronic hepatitis B infection and in monitoring antiviral therapies [
13,
18]. In the current study of acute hepatitis B, HBcrAg kinetics were similar to HBeAg and HBV DNA and HBcrAg correlated with both markers. Thirty HBV DNA-positive HBeAg-negative samples were positive by the HBcrAg assay, likely due to the multiple proteins detected by the HBcrAg assay. The lack of an international standard and the inability to distinguish among the different HBcrAg components limits further analysis.
An interesting observation from this study is the heterogeneity in the duration of HBsAg and HBV DNA positivity. Although few similar panels are offered by commercial vendors, the seroconversion series in the current study nevertheless are representative of acute HBV infection as supported by comparison with four other commercial panels (discussed above) and by the kinetics of HBV DNA and HBsAg during the ramp-up phase. Biswas et al. reported a mean HBV viral load doubling time of 2.56 days with HBsAg increasing in parallel with HBV DNA during the ramp-up phase for 23 seroconversion panels [
9]. For the five panels in the current study, viral load doubling times ranged from 2.0 to 2.7 days with HBsAg increasing in parallel with HBV DNA (Figs.
1,
2) [
19]. Thus, the observed differences in duration of surface antigenemia may reflect the natural variability among individuals with acute HBV and may be instructive in appreciating the spectrum of acute profiles relative to the typical composite seroconversion profile and in the interpretation of patient results.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.