SUMMARY
SUMMARY Hepatitis B virus (HBV) is an enveloped virus with a small (3.2-kb) partially double-stranded DNA genome that causes acute and chronic infections. The impact of these infections on public health worldwide is enormous, with an estimated prevalence of 2 billion acute infections and 360 million chronic infections globally. This review focuses on chronic hepatitis B and the molecular assays used in its diagnosis and management. Background information, including that about features of the hepatitis B virion, viral replication, and epidemiology of infection, that is important for understanding chronic hepatitis B and molecular diagnostic tests for HBV is provided. To facilitate an understanding of the utility of molecular testing for chronic hepatitis B, the four stages of chronic hepatitis B infection that are currently recognized, as well as an additional entity, occult hepatitis B, that can be diagnosed only by sensitive nucleic acid amplification methods, are reviewed in detail, including available therapeutic agents. The molecular diagnostic content focuses on tests for HBV DNA quantification, genotyping, and mutation detection (including precore/core promoter and antiviral resistance mutations). The discussion of these tests encompasses their current utility and performance characteristics, drawing from current clinical guidelines and other studies from the literature. In recognition of the continual evolution of this field, the final section describes emerging molecular markers with future diagnostic potential.
INTRODUCTION
Hepatitis B virus (HBV) causes a highly complex chronic infection that impacts a significant proportion of the world's population. Diagnostics for chronic hepatitis B have evolved from the simple detection of HBsAg through the complex antibody response against individual viral proteins and to the detection and quantification of viral DNA. Implementation of increasingly sensitive methods of HBV DNA quantification has greatly aided the diagnosis and management of disease. Assays are also available to determine HBV genotypes and to detect the presence of viral mutants, including those that confer drug resistance and others that downregulate HBV e antigen. In this review, an overview of the virus and chronic hepatitis B infection is provided. The current utility of the different types of molecular diagnostic tests is discussed, and the performance characteristics of the available assays are described.
HBV PROTEINS AND REPLICATION
HBV is an enveloped virus containing a 3.2-kb, partially double-stranded, relaxed circular genome. The genomic coding scheme is extraordinarily efficient; every nucleotide is a part of at least one open reading frame. The major viral proteins (polymerase, core, envelope, X, and e antigen) and their activities are shown in Table 1. HBsAg and HBeAg are particularly important in the management of chronic hepatitis B. Detectable HBsAg in serum is a marker of chronic infection. HBeAg in serum is a marker of high viral replication levels in the liver. Loss of HBeAg in serum and emergence of anti-HBeAg antibody (termed HBeAg seroconversion) is associated with clinical improvement of hepatitis (reduced HBV DNA, normalized serum aminotransferase levels, and quiescence of inflammation in the liver) (9).
HBV proteins
HBV replicates primarily in human hepatocytes, although viral DNA can be found in peripheral blood mononuclear cells. Entry is mediated by envelope binding to an unknown receptor. After entry and virion uncoating, nucleocapsids are translocated into the nucleus, where cellular DNA repair enzymes complete virion DNA synthesis. The resultant covalently closed circular DNA (cccDNA) is the template for viral mRNA transcription, which is mediated by host polymerase. Replication-competent nucleocapsids comprised of core protein, encapsidated full-length pregenomic RNA, and viral polymerase are assembled in the cytoplasm. Genomic DNA is synthesized by reverse transcription of pregenomic RNA by viral polymerase. Encapsidated, relaxed, open circular DNA can be transported to the nucleus to become cccDNA and additional mRNA template, or it can be released from the host cell via a process that requires cytosolic packaging (along with polymerase) by envelope glycoproteins, budding into endoplasmic reticulum, and release after Golgi transit.
HBV infection is noncytolytic. Clearance of infected cells is believed to be mediated in part by the noncytolytic intracellular activity of cytokines secreted by T cells (23). Cytotoxic T lymphocytes also lyse infected hepatocytes and induce liver injury (23).
EPIDEMIOLOGY
The worldwide burden of HBV is enormous. The World Health Organization (WHO) currently estimates that 2 billion people have been infected with HBV and that 360 million are chronically infected (153). HBV is a significant contributor to morbidity worldwide. Current estimates suggest that it causes 30% of cirrhosis and approximately 50% of hepatocellular carcinoma (HCC) globally (121).
HBV can be transmitted perinatally, percutaneously, and sexually. Routes of transmission are dependent on the regional prevalence of infection (131). In areas of high endemicity such as Asia and the South Pacific, where seroprevalence is ≥8%, most infections are acquired perinatally or horizontally during childhood. In regions of intermediate seroprevalence (2 to 7%), such as sub-Saharan Africa, Alaska, the Mediterranean, and India, infection is transmitted during childhood (perinatally or horizontally) and later in life (sexually, by intravenous drug use, or by unsafe health care-related injection practices). In low-prevalence regions (most of the developed world and parts of Central and South America; seroprevalence, <2%), HBV is usually transmitted sexually and by intravenous drug