Hepatitis B Virus Serum DNA andRNA Levels in Nucleos(t)ide Analog‐Treated or Untreated Patients During Chronic and Acute Infection

Abstract

Treatment of chronic hepatitis B (CHB) patients with nucleos(t)ide analogs (NAs) suppresses hepatitis B virus (HBV) DNA synthesis but does not affect synthesis of HBV pregenomic RNA (pgRNA). Hepatitis B virus pgRNA is detectable in the serum during NA treatment and has been proposed as a marker of HBV covalently closed circular DNA activity within the infected hepatocyte. We developed an automated assay for the quantification of serum HBV pgRNA using a dual-target real-time quantitative PCR approach on the Abbott m2000sp/rt system. We demonstrate accurate detection and quantification of serum HBV RNA. Hepatitis B virus DNA was quantified using the Abbott RealTime HBV viral load assay. We further compared serum nucleic acid levels and kinetics in HBV-positive populations. Samples included on-therapy CHB samples (n = 16), samples (n = 89) from 10 treatment naïve CHB subjects receiving 12 weeks of NA treatment with 8-week follow-up, hepatitis B surface antigen-positive blood donor samples (n = 102), and three seroconversion series from plasmapheresis donors (n = 79 samples). Conclusion: During NA treatment of CHB subjects, we observed low correlation of HBV DNA to pgRNA levels; pgRNA concentration was generally higher than HBV DNA concentrations. In contrast, when NA treatment was absent we observed serum pgRNA at concentrations that correlated to HBV DNA and were approximately 2 log lower than HBV DNA. Importantly, we observe this trend in untreated subject samples from both chronic infections and throughout seroconversion during acute infection. Results demonstrate that the presence of pgRNA in serum is part of the HBV lifecycle; constant relative detection of pgRNA and HBV DNA in the serum is suggestive of a linked mechanism for egress for HBV DNA or pgRNA containing virions.

Hepatitis B Virus Virions Produced Under Nucleos(t)ide Analogue Treatment Are Mainly Not Infectious Because of Irreversible DNA Chain Termination

“…Therefore, the reported “HBV load” in NA‐treated subjects may vary from kit to kit that detects different regions of HBV DNA. Furthermore, as expected, but different from a recent report, the levels of serum HBV RNA are highest in the PreC/C region and declined from the S region to X region. Notably, HBV‐RNA level may also be affected by the RNA splicing‐variants given that some of them lack the S region, particularly after NA treatment .…”

Serum Hepatitis B Virus RNA: A New Potential Biomarker for Chronic Hepatitis B Virus Infection

“…The QuantiGene assay developed by Lam et al uses HBV‐specific probes, designed to hybridize with the X open reading frame (ORF), to enable direct quantification of RNA without cDNA synthesis or PCR amplification . Recently, Butler et al developed an automated high‐throughput assay to quantify serum HBV RNA, which was isolated using RNA selective extraction chemistry (Abbott mSample Preparation System), followed by a multiplex quantitative RT‐PCR procedure for detecting amplicons of hepatitis B X and core region on the m2000 platform (Abbott Molecular) . To further evaluate the automated prototype assay performance, Butler et al conducted a comparative testing of flRNA and found a good correlation between these two methods.…”

Hepatitis B virus pregenomic RNA status can reveal the long‐term prognoses of chronic hepatitis B patients treated with nucleos(t)ide analogues

“…Our previous study showed that CHB patients could achieve HBeAg seroconversion when their HBV RNA levels were below 4.12 log 10 copies/mL before treatment . Another study found a weak correlation between the HBV DNA and pgRNA levels during NA therapy . High serum pgRNA often reflects reverse transcription failure .…”