Hepatitis B virus

This chapter defines the essential components of a laboratory safety program. The key to a safe laboratory environment is the development, implementation, and enforcement of a high-quality safety management program that considers worker safety as a responsibility of the facility. The risk from biological, chemical, physical, and radiological hazards is related to exposure levels, duration of exposure, toxicity or pathogenicity of the hazardous material, safety controls present, and other factors such as the general health or age of the laboratory worker. The Occupational Safety and Health Administration (OSHA) identifies a number of practices that should be implemented to protect the worker from exposure to blood-borne pathogens, including an exposure control and risk assessment plan. The chapter describes various modes of hazard prevention such as handwashing, barrier protection and immunization. While disinfectants destroy all microorganisms, but not necessarily their spores on inanimate surfaces, sterilants are agents that kill all microbial life, including spores, on inanimate surfaces. The chapter provides discusses spill management, both for biological and chemical spills. A training program should include recognition and evaluation of fire hazards, planning to reduce the risk of fire, and all actions to take when a fire occurs. For effective and efficient management of infectious wastes, a comprehensive management plan is essential to ensure the safety of the employees handling the waste, and implementation of cost-effective strategies for waste disposal.

This chapter discusses the major features of the current healthcare environment. It describes the current regulations affecting laboratory practice. The chapter explains future regulations and political and economic events impacting pathology and laboratory practice. A high level of volatility and uncertainty, reflecting the political, economic, and social instability of our current environment, characterizes healthcare in the United States in the second decade of the 21st century. Funding and support for healthcare are diminishing, and the economic future of healthcare is hostage to some identifiable trends and concerns that include: a growing federal deficit and concerns about the future sustainability of Medicare and Medicaid; and a healthcare insurance and delivery system plagued by problems of access, quality, and safety. The “four horsemen” of federal laws and regulations that are particularly pertinent to pathology and laboratory medicine services are CLIA ’88, HIPAA, the Occupational Safety and Health Administration (OSHA) standards for occupational exposure to blood-borne pathogens, and the so-called Stark regulations that prohibit self-referral by healthcare providers. The increasing research and clinical demands for validated biospecimens can be expected to lead to significant growth in the number and size of biorepositories. Two other areas of concern to research and clinical laboratory practice are restrictive patents and excessive restraints on the use of human tissue and data for research and clinical purposes.

Hepatitis B virus (HBV) was identified and characterized after the discovery of the Australia antigen by Blumberg and colleagues in 1965. The Australia antigen, now designated hepatitis B surface antigen (HBsAg), is detected in the sera of patients with both acute and chronic HBV. HBV infects hepatocytes, leading to an acute infection that resolves or a chronic infection lasting years. Safe and effective vaccines against HBV have been available since 1982. HBV infection is diagnosed by serological and molecular markers using serum or plasma. The laboratory diagnosis of HBV uses a combination of tests that detect virus-specific protein and nucleic acid as well as the host immune response to infection. The methods for identification of HBV infection use a combination of molecular, antigenic, and serological methods. Serologic tests for HBV-specific antibodies are used to determine the stage of disease and to establish immunity due to vaccination. Patients with chronic hepatitis that are hepatitis B e antigen (HBeAg) negative are more likely to have more advanced liver disease in spite of lower serum HBV DNA levels. The majority of individuals vaccinated for HBV have detectable levels of anti-HBs, but some test negative due to waning levels of anti-HBs. Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of HBV for its replication. The laboratory diagnosis of HDV depends on the detection of specific antibodies, HDAg, and HDV RNA.

Molecular methods are now firmly established as new diagnostic gold standards for most of the clinically important viruses. Molecular methods have changed the face of clinical virology and created new opportunities for laboratories to impact diagnosis and management of patients with viral infections. This chapter discusses the thoughts and impressions on the current practice and future trends in diagnostic molecular virology. Real-time PCR methods have largely replaced conventional, end-point PCR methods in clinical laboratories, with the attendant benefits of speed, broad dynamic range and increased precision for target quantitation, and reduced contamination. Multiplex, real-time amplification methods are limited to three to four different targets by the small number of available fluorescent reporter dyes. These new uses for hepatitis C virus (HCV) viral load measurements provide motivation for patients to complete therapy and permit the individualization of therapy duration. There has also been significant progress in the development of specifically targeted antiviral therapy for hepatitis C, such as NS5 polymerase and NS3/4 protease inhibitors. Phylogenetic genotyping has a more limited role in the clinical management of patients with chronic hepatitis B than it does in patients with chronic hepatitis C .

Test validation is the ongoing process of ensuring that the expected performance of an assay is consistently met in testing clinical specimens. Test (or assay) validation is an integral part of quality assessment (QA), which includes quality control, quality improvement, and method validation. QA encompasses routine quality control, proficiency testing, technical staff competency, instrument calibration, and clinical correlation. Quality management of molecular testing begins with the test request and continues through specimen collection, transport, processing, analytical testing, result generation, result review, test interpretation, and reporting. This chapter focuses mainly on the analytical phase of testing. The types of molecular testing included in the chapter are qualitative, quantitative, multiplex, and microarray methods. Quantitative molecular testing consists of numeric values with defined units in the test result. Quality assurance includes quality control, quality improvement, and method validation. Validation of the analytical phase not only includes ensuring adequate and acceptable training on the test but also relies upon evaluation by actual observation of the technologist performing the test on a recurrent basis (operator competency assessment). This should include validation of staff adherence to the standard operating procedure (SOP) exactly as stated, biosafety, patient confidentiality, result interpretation, reporting, and quality control documentation. The use of quality controls, proficiency testing, and monitoring of technical staff competency and equipment and instrument performance are all essential parts of this process.

During viral infection, cells of the innate immune system such as monocytes, dendritic cells (DCs), natural killer (NK) cells, and polymorphonuclear leukocytes serve several functions: alerting the host to invading pathogens, providing early containment of pathogens, mediating antimicrobial effects against pathogens, directing the nature of the immune response, and participating in wound healing and tissue repair. During viral infection, antigen-presenting cells (APCs) sense viral nucleic acids via members of the Toll-like receptor family (TLR3, 7, 8, and 9) and intracellular sensors such as the RNA helicases RIG-I, mda5, and LGP2. Ablating the pleiotropic activity of type I interferons (IFNs) during viral infection in mice containing a genetic deletion of the type I interferon receptor resulted in higher virus replication and severe pathology or death. The potent antiviral responses of NK cells that directly clear certain viral infections to promote host survival unmistakably demonstrate how crucial these cytotoxic cells are; at the same time, the importance of NK cell activity is equally demonstrated by the discovery of the many strategies different viruses employ to specifically evade detection by NK cells. Although viruses evolve because of the pressures exerted by cells of the immune system, there is evidence that the mammalian immune system also adapts in response to viral evasion mechanisms. As more viral ligands of activating NK cell receptors are uncovered, we will have a greater appreciation of the evolution of mammalian receptors on NK cells at the population level driven by viruses.

While the term immunopathology was originally used to describe an aberrant immune response to autoantigen resulting in pathology, more recent developments in our understanding of virus-induced immunopathology have revealed that this can be linked to both an aberrant immune response to the virus itself as well as unfavorable virus-induced changes to the immune response as a whole; for example, lymphopenia following certain types of infections as well as the specific loss of CD4 T cells in HIV infection. Perhaps the most common virus-induced pathology is T-cell-induced destruction of host tissues. T-cell mediated pathologies occur through both direct and indirect mechanisms including the targeted cytolysis of the infected cell by effector T cells, the induction of apoptosis in infected cells and generalized tissue damage due to bystander effects of T-cell activation. Difficulties in establishing an animal model of hantavirus pulmonary syndrome (HPS) had greatly hampered progress in understanding the factors involved in pathogenesis. Perhaps the most widely examined example of antibody dependent enhancement (ADE) can be found in dengue virus infections. The process by which viruses may induce or enhance autoimmunity is hypothesized to occur by a number of mechanisms. Recent clinical trials for several different vaccines, where vaccinated groups had elevated risk for infection and or disease than their naive cohorts, have highlighted the necessity for continued basic science research into the precise mechanisms of virus-induced immunopathology. One example of this can be found in the vastly different outcomes achieved with different strategies in respiratory syncytial virus (RSV) vaccination.

An appreciation of the role of the fifth human hepatitis virus, hepatitis E virus (HEV), as an important cause of acute, and occasionally chronic, hepatitis in humans has increased in recent years. Hepatitis viruses have long been implicated as the cause of substantial morbidity and mortality in the developing world, through both sporadic and epidemic disease. The predictable seasonal outbreaks, coincident with the monsoon rains, have provided numerous opportunities to study risk factors for HEV infection, the clinical course of hepatitis E disease, and the characteristics of the virus. In 1980, by excluding hepatitis A virus (HAV) and hepatitis B virus (HBV) as causes of a large 1955 to 1956 hepatitis epidemic in India, two groups independently reported the hypothesis of a novel, enterically transmitted non-A, non-B (ET-NANB) virus. As HEV shares certain morphologic and biophysical properties with members of the Caliciviridae family, it was initially classified as a calicivirus. Electron microscopy has been used to detect virus-like particles in the livers of patients with HEV-attributable fulminant hepatitis. The age- and gender-matched control group in a case control study in Sudan found a high (18%) anti-HEV IgG seroprevalence among children in the general population. Immunologic changes of pregnancy, such as the down-regulation of TH1 cytokines, and hormonal changes may also influence pathogenesis. A recent case report of fulminant hepatitis due to infection with an HEV genotype 3 strain in a patient in Spain has suggested that an important cofactor was the usage of hormonal contraceptives.