Cumitech 18A: Laboratory Diagnosis of Hepatitis Viruses

NOVEMBER

1998

Laboratory Diagnosis Hepatitis Viruses

of

MAX A. CHERNESKY, DAVID GRETCH, ISA K. MUSHAHWAR, PAUL D. SWENSON, AND PATRICE 0. YARBOUGH COORDINATING

STEPHEN

EDITOR

YOUNG

Cumitech CUMULATIVE

TECHNIQUES

AND PROCEDURES

IN CLINICAL

MICROBIOLOGY

Cumitech 1B Cumitech 2B

Blood Cultures III l AprzlZ997 Laboratory Diagnosis of Urinary

Cumitech 3A Cumitech 4A Cumitech 5A Cumitech 6A Cumitech 7A

Quality Control and Quality Assurance Practices in Clinical Microbiology l May 1990 Laboratory Diagnosis of Gonorrhea l Aprrl 2 993 Practical Anaerobic Bacteriology l December 1991 New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide l February

Cumitech Cumitech Cumitech Cumitech

8 9 10 11

Cumitech Cumitech Cumitech Cumitech

12A 13A 14A 15A

Cumitech Cumitech Cumitech

16A 17A 18A

Cumitech Cumitech Cumitech

19 20 21

Cumitech Cumitech Cumitech

22 23 24

Cumitech Cumitech Cumitech

25 26 27

Cumitech

28

Cumitech Cumitech

29 30

Cumitech

31

Cumitechs Cumitech Editorial Jamison,

Tract Infections

l

November

1998

Laboratory Diagnosis of Lower Respiratory Tract Infections l September 1987 Detection of Microbial Antigens by Counterimmunoelectrophoresis l December

2978

Collection and Processing of Bacteriological Specimens l August 2 979 Laboratory Diagnosis of Upper Respiratory Tract Infections l December 2979 Practical Methods for Culture and Identification of Fungi in the Clinical Microbiology August 1980 Laboratory Diagnosis of Bacterial Diarrhea l April 1992 Laboratory Diagnosis of Ocular Infections l September 1994 Laboratory Diagnosis of Central Nervous System Infections l March 2 993 Laboratory Laboratory Laboratory

Diagnosis Diagnosis Diagnosis

Laboratory Laboratory Therapeutic

Diagnosis of Hepatitis Viruses l November 1998 Diagnosis of Chlamydial and Mycoplasmal Infections l August 1984 Drug Monitoring: Antimicrobial Agents l October 2 984

1992

Laboratory

l

of Viral Infections l August 1994 of the Mycobacterioses l October 2994 of Female Genital Tract Infections l Jtlne 1993

Laboratory Diagnosis of Viral Respiratory Disease l March 2 986 Immunoserology of Staphylococcal Disease l Atrgust 2 987 Infections of the Skin and Subcutaneous Tissues l June 2 988 Rapid Detection of Viruses by Immunofluorescence l August 2 988 Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing . December 1988 Laboratory Diagnosis of Viral Infections Producing Enteritis l September 2989 Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and Laboratory Animals l February 1996 Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals l February 1996 Laboratory Safety in Clinical Microbiology l July 2 996 Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract l September 1996 Verification

and Validation

of Procedures in the Clinical

should be cited as follows, e.g.: Chernesky, 18A. Laboratory diagnosis of hepatitis viruses.

M. A., D. Gretch, I. K. Mushahwar, Coordinating ed.. S. Young. American

Board for ASM Cumitechs: Frederick S. Nolte, Chairman; Vickie Baselski, Karen Krisher, Brenda McCurdy, Allan Truant, Alice S. Weissfeld. Stephen

The purpose of the Cumitech series is to provide consensus recommendations procedures for clinical microbiology laboratories which may lack the facilities given are not proposed as “standard” methods. Copyright 0 1998 American Society 1325 Massachusetts Avenue NW Washington, DC 20005-4171

for Microbiology

Microbiology

l

February 1997

P. D. Swenson, and P. 0. Yarbough. Society for Microbiology, Washington,

Lorraine Clarke, A. Young.

by the authors for fully evaluating

Laboratory

Curt A. Gleaves.

Janet

Hindler,

1998. D.C. Richard

as to appropriate state-of-the-art operating routine or new methods. The procedures

Laboratory Diagnosis of Hepatitis Viruses Max A. Chernesky McMaster

University,

Hamilton,

Ontario,

Canada

L8N

1 Y4

David Gretch University

of Washington

Viral Discovery

Group,

School

of Medicine,

Seattle,

Washington

98195

Isa K. Mushahwar Abbott

Laboratories,

North

Chicago,

Illinois

60064

Paul D. Swenson Seattle-King

County

Department

of Public

Health,

Seattle,

Washington

982 04

Patrice 0. Yarbough Genelabs

Technologies,

Inc., Redwood

City,

California

94063

COORDINATING EDITOR: Stephen Young University

Introduction Properties

of New

Mexico,

Albuquerque,

New

Mexico

87131-5274

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*................................................. of the Hepatitis Viruses . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . ..‘.....................

2 2

HAV . ..~...~.............~......~....~~.............................................~.~......~....................~......~... HBV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..~...............................................~.................................... HDV . . ..*.......*................................................. . . . . . . . . . . . . . . . . . . . . . ..*...........................*...*... HCV ~...........................~....~~.~...........................~~.~...........~.........~. ... ... ... ... ... .. ... ... ... ... . HEV .......................................................................................................................... G BV-C ......................................................................................................................

Clinical

Findings

...................................................................................

Markers

and lmmunodiagnosis

.............................................

HAV ......................................................................................................................... HBV ......................................................................................................................... HDV ....................................................................................................................... HCV ........................................................................................................................ HEV ........................................................................................................................ G BV-C .....................................................................................................................

Nucleic

Acid Diagnosis

.....................

. .....................

and Treatment

. ........... . ................

...................................................................

......... . .......................................................

16 16 16 16 16 17 17

18 18 18 19 20 20 21

HAV ........................................................................................................................ HBV ........................................................................................................................ HDV ....................................................................................................................... HCV ........................................................................................................................ HEV ........................................................................................................................ G BV-C .....................................................................................................................

References

9 9 9 12 12 14 15

HAV ........................................................................................................................ t HBV . . . . . . . . . . . . . . . . . . . . . . . . . ..*.....*.................................................................*...............*.... HDV .,.....,....,......,..............................*............,.........................*.............*..,.,.......... HCV ........................................................................................................................ HEV ........................................................................................................................ G BV-C .....................................................................................................................

Prevention

5 5 6 6 6 7 8

HAV . . . . . . . . . ..*.........*.........................................................*........*............................*... HBV . . . . . . . . . . ..*..........................................................................................................*. HDV . . . . . . . . . ..*......................................*....................................................................*. HCV ......................................................................................................................... HEV .......................................................................................................................... G BV-C ......................................................................................................................

Serological

2 2 3 3 4 5

..““~ ...................

21

2

Chemesky

CUMITECH

et al.

INTRODUCTION

c

ince Cumitech 18 was published in 1984, significant advances have occurred in the understan d’mg of the viruses that cause hepatitis. u These advances include laboratory diagnosis, prevention, and the treatment of viral hepatitis. Hepatitis (liver inflammation) has many etiologies, one of which is viral infection. A number of viral infections can cause hepatitis (Table l), but currently five viruses, and possibly a sixth virus, are associated primarily with liver disease. Collectively, these viruses are known as hepatitis viruses, and individually they have been designated hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and GB virus C (GBV-C). Infections with HCV and possibly GBV-C were formerly designated as either parenterally or transfusion associated non-A, non-B (NANB) hepatitis. Hepatitis E virus infection was formerly designated either epidemic or enterically transmitted NANB hepatitis. Although the differences in the clinical course of infection with each of the hepatitis viruses give some indication as to the viral etiology, diagnosis is usually laboratory based. Laboratory diagnosis of these infections is based on serological or nucleic acid detection techniques, because members of this group of viruses grow either poorly or not at all in cell culture. Except for HBV and HDV, each of these viruses has distinct genetic and biophysical properties. The lack of similarity between five of the viruses means that there is not partial protection from infection by one hepatitis virus with any of the others. Therefore, protection against the hepatitis viruses will require developing an effective vaccine for each individual agent. PROPERTIES

OF THE HEPATITIS

18A

acid sequence of the virus and from similarities between the genomic organization of HAV and other well-known picornaviruses ( 12 1) . HAV differs from all known picornaviruses in several respects, including its strong tropism for the liver, a low G+ C content of only 38 %, a truncated VP4 with possibly a short leader peptide, and a very low overall nucleotide sequence relatedness to viruses in each of the other genera (139). HAV survives heating to 60°C for a prolonged period, indefinite cold storage (SOC), acidic conditions (pH 3), and nonionic detergents. There is remarkably little genetic variation, with homogeneity of HAV antigens isolated from different strains (122). HAV multiplies in several types of primate cell cultures, but unlike poliovirus it induces neither shutdown of host cell macromolecular synthesis nor a cytopathic effect (24, 41, 106). The virus remains largely cell associated in most infected cell cultures. Immunofluorescent staining of infected cells reveals a characteristic granular accumulation of HAV antigen within the cytoplasm. Wild-type virus grows poorly in all cell types. Primary isolation is difficult and may require weeks before the first appearance of intracellular antigen. With subsequent passage, the virus gradually adapts to cell culture. Analysis of the genetic relatedness of wild-type viruses and cell culture-adapted strains by oligonucleotide mapping, nucleic acid hybridization, and nucleic acid sequencing indicates that nucleotide sequence diversity among strains usually amounts to not more than 1 to 4%, occasionally exceeding 10% (18, 77, 143). HBV HBV is a 42-nm spherical particle consisting of a 7-nm outer shell and a 27-nm inner core that encloses the viral DNA and DNA polymerase (57). The outer

VIRUSES

HAV

Table

1.

Viral

causes

HAV is a small, nonenveloped, RNA-containing virus belonging to the family Picornauiridae (enterovirus 72). The virions sediment at 156s and band around 1.33 g/ml in CsCl. The genome is a linear, singlestranded RNA of messenger-sense polarity with a covalently linked 5’-terminal protein and a 3’-terminal poly(A) tract that appears to follow the classical L434 organizational and translational strategy of picornaviruses (95). A total of 11 structural and nonstructural proteins are encoded by a single open reading frame resembling that described for members of the four distinct picornavirus genera-the enterovirus, rhinovirus, cardiovirus, and aphthovirus genera. Three structural proteins, VPl, VP2, and VP3, ranging between 33 and 24 kDa, have been positively identified. A fourth capsid protein, VP4, only about 2.5 kDa in size, has been predicted from the nucleic

Hepatrtls viruses HAV HBV HCV HDV HEV G BV-C Other viruses Coxsackievtrus Cytomegalovlrus Dengue virus Epstein-Barr virus Herpes simplex virus Lassa virus Measles virus Parainfluenza viruses Reovlrus Rubella virus Varicella-zoster virus Yellow fever virus

of human

hepatitis

CUMITECH

Hepatitis

18A

surface component of the complete virion, sometimes referred to as the Dane particle, contains hepatitis B surface antigen (HBsAg), and the inner core component contains hepatitis B core antigen (HBcAg). In addition to complete virus particles, numerous HBsAg-containing, 22-nm spherical particles and tubular structures of the same diameter and variable length circulate in the blood of HBV-infected individuals. HBV strains have been classified into nine different subtypes, aywl, ayw2, ayw3, ayw4, ayr, adw2, adw4, adrq+, and adrq-, according to the antigenic determinants and subdeterminants of their HBsAg (21, 23). HBV has also been classified into six genotypes, A to F, based on nucleotide sequence differences (96, 98). HBcAg, unlike HBsAg, is not directly detectable in the serum, but removal of the outer HBsAg component of the complete HBV particle with nonionic detergents results in the release of serologically reactive HBcAg (57). Solubilization with sodium dodecyl sulfate and 2-mercaptoethanol of either the inner HBcAg component of HBV particles or core components from infected liver results in the appearance of hepatitis B e antigen (HBeAg) and the disappearance of HBcAg reactivity, suggesting that HBeAg is a structural component of the inner core of the HBV particle (136, 155). HBeAg is also present as a readily detectable soluble protein in the serum of HBV-infected individuals who have high levels of viremia. During HBV infection, HBsAg, HBcAg, and HBeAg can induce an antibody response in the host with the development of anti-HBs, anti-HBc, and anti-HBe, respectively. The inner core component of the HBV virion contains an endogenous DNA polymerase with reverse transcriptase activity and a unique viral genome that consists of partially single-stranded, noncovalently closed circular DNA and is only 3,200 nucleotides (nt) in length. The complete nucleotide sequence of the HBV genome and the coding sequences for all of the HBV proteins have been identified (57). The unique characteristics of HBV classify it as the prototype member of the hepadnavirus family of hepatitis B-like viruses (87), which includes hepatitis viruses infecting woodchucks ( 134), Beechey ground squirrels (82), and Pekin ducks (84). These viruses have several characteristics in common, including ultrastructure, antigenic composition (except for the duck virus), DNA size and structure, DNA polymerase, a tropism for liver, an association with persistent infection, and, for the woodchuck virus and HBV, carcinoma. Transmission of primary hepatocellular HBV in humans is primarily through intimate physical contact or, less frequently, through exchange of blood products, such as transfusion or hemodialysis.

Viruses

3

HDV HDV is a defective RNA virus that requires a helper function provided by HBV to establish infection in humans (55,103). HDV was discovered in a study of liver biopsy specimens from patients with chronic HBV infection (116). The HDV virion is a spherical particle with a diameter of 35 to 37 nm. The external coat of the HDV virion consists of HBsAg, which is provided by the helper HBV. Enclosed within the HBsAg coat is hepatitis delta antigen (HDAg) and the RNA genome. HDAg is the only known protein encoded by HDV. The single-stranded circular RNA genome of HDV is only 1,700 nt in length and has the ability to fold on itself to form an essentially doublestranded, rod-like structure. The structure of the HDV genome is unique among animal viruses, but it closely resembles that of plant viroids, satellite RNAs, and satellite viruses. Three major genotypes of HDV (I, II, and III) have been identified (15). HDV has been transmitted to chimpanzees infected with HBV and also to woodchucks infected with another hepadnavirus, woodchuck hepatitis vir us. In human .s, transmission of HDV occurs via percutaneous or permucosal routes. HDV is highly endemic in southern Italy and parts of Africa and South America. In the United States, HDV infection most commonly affects injection drug users and hemophiliacs. HCV Since its discovery in 1989, HCV has become recognized as the major cause of both transfusion-associated and sporadic chronic NANB hepatitis. After nearly a decade of effort, HCV was discovered through an elegant series of experiments in which the virus was partially purified from a pool of high-titer chimpanzee plasma. A portion of the viral RNA genome was isolated using recombinant DNA techniques. The HCV-specific clone was identified by immunoscreening of chimpanzee cDNA libraries using chronic-phase serum and plasma as a source of HCV antibodies. This discovery led to the introduction of screening assays for HCV prior to blood donation, which has consequently resulted in a dramatic reduction in the worldwide incidence of posttransfusion chronic hepatitis. Sequencing of the HCV genome also provided impetus for the development of powerful new molecular assays for detecting HCV in humans. These molecular assays have greatly improved the diagnosis and management of chronic hepatitis C and have resulted in a significant expansion of our understanding of this medically important chronic viral infection. HCV is an enveloped, positive-strand RNA virus with a genome size of approximately 9,400 nt. This RNA encodes a single viral polyprotein of approximately 3,011 amino acids, which is apparently

4

Chernesky

et al.

cleaved into a series of structural and nonstructural peptides during infection. Based on structural organization and sequence homology, HCV has been classified within the family Flaviviridae, which also includes the flaviviruses and pestiviruses. Efficient propagation of HCV in tissue culture has been difficult, and the only suitable nonhuman model for studying HCV infection has been the endangered chimpanzee. Despite these limitations, HCV virions have been described by electron microscopy as ranging from 40 to 60 nm in size, with the buoyant density of viral particles ranging from 1.09 to 1 .ll g/cm3 by density gradient centrifugation ( 10). Sequence analysis of HCV genomes isolated from infected patients has demonstrated a high degree of genetic variability. Based on phylogenetic analysis of various HCV genes, it has been possible to provisionally classify current HCV isolates into at least six major genotypes and numerous subgenotypes, and several nomenclature systems have been proposed, as discussed in a recent review (123). The spectrum of genetic diversity of HCV, the worldwide distribution pattern of HCV genotypes, and the clinical and virological relevance of HCV genetic classification schemes remain importa .nt questions to resolve and are all areas of active research. The most prevalent HCV genotype in the United States is genotype 1, accounting for well over 50% of infections. However, HCV genotypes 2 and 3 each account for a fairly significant proportion of HCV infections in the United States. In contrast, infections with HCV genotype 4, 5, or 6 are uncommon in the United States. Recent technological advances have made HCV genotype determination readily available in the research setting. Based on early data, it seems possible that HCV genotype determination might prove useful for selecting HCV-infected patients for therapy. However, in a recent Consensus Development Conference on hepatitis C held at the National Institutes of Health (NIH), it was recommended that all patients with significant liver disease associated with chronic hepatitis C should be considered for alpha interferon (IFN-ar) therapy regardless of their HCV genotype. Based on that recommendation, there does not appear to be a strong clinical indication for HCV genotype determination at this time, other than for patient education and counseling purposes. After optimal therapeutic regimens are defined for the different HCV genotypes, molecular determination of HCV genotype may become part of the routine laboratory evaluation of a patient with chronic hepatitis C. The genome of HCV also contains a region of extremely high genetic diversity within the second envelope gene termed a hypervariable region, which undergoes extensive genetic change during the course of infection in humans and chimpanzees (14). The

CUMITECH

18A

HCV hypervariable region is similar to that described for human immunodeficiency virus (HIV), and these viruses have been referred to as quasispecies because of their ability to exist as multiple genetic forms within an infected individual. The hypervariable regions are thought to encode important targets of the immune system, and mutation within this region is thought to result in “immune escape” and viral persistence. The significance of HCV quasispecies to viral persistence and pathogenesis in humans remains an important question to resolve, particularly as it relates to the design and testing of experimental vaccines. Furthermore, tracking HCV quasispecies may ultimately prove useful for acquiring a better understanding of HCV transmission in humans. HEV The virion morphology of spherical nonenveloped particles of 27 to 34 nm (average diameter of 30 to 32 nm) led to the early characterization of HEV as a calicivirus-like virus (11). The intact virus has a sedimentation coefficient of 183s in sucrose gradients and a buoyant density of 1.29 g/cm3 in potassium tartrateglycerol gradients. The physiochemical properties of HEV distinguished it from picornaviruses. Although there is currently no finalized classification for HEV, it has been provisionally classified as a member of the calicivirus family. The Burmese strain of HEV was molecularly cloned from the bile of an experimentally infected cynomolgus monkey (114,137). Subsequent sequencing of the two most divergent strains, HEV Burma (137) and HEV Mexico (58), showed that the viral genomes are closely related. The genomic organization and protein expression strategies used are identical for the two viral isolates. Molecular analysis demonstrates that HEV is different from the other RNA viruses known to cause hepatitis. The virus is a single-stranded RNA virus of approximately 7.6 kb. HEV is a polyadenylated, positive-strand RNA virus with three major open reading frames (ORFs) ( 114,13 7). The genomic organization of HEV localizes the nonstructural genes on the 5’ end and the structural genes at the 3’ end of the genome (137). ORFl is believed to encode the viral nonstructural proteins including the RNA-dependent RNA polymerase and helicase. ORF2 (660 amino acids) is believed to encode the putative capsid protein, which is incorporated into the virus particle. The small ORF3 (123 amino acids) was identified by an immunoscreening protocol (151). Although it is known to encode an immunodominant epitope, the function of ORF3 is currently undetermined. ORF3, which overlaps both ORFl and ORF2, demonstrated that the virus utilizes all three reading frames for encoding viral proteins. Although the use of three overlapping reading

CUMITECH 18A

frames has not been previously described in other single-stranded, positive-sense, nonenveloped RNA viruses, it has been observed for certain enveloped viruses. Particular features of HEV indicate its relatednessto the enveloped alphaviruses. Computer-assisted assignment of the putative functional domains in the nonstructural region of HEV shows the best alignment with rubella virus and furovirus within the alphavirus-like virus superfamily (65,111). The seven motifs of HEV, including the methyltransferase, papain-like protease, polyproline region, RNA helicase, and RNA-dependent RNA polymerase, have the greatest sequencesimilarity to alphavirus-like viruses. Although the overall genomic organization of HEV is similar to that of the feline calicivirus (l37), a conserved motif in the cysteine protease region is missing in HEV. Although electron micrographs and physicochemical characterization of HEV tentatively grouped the virus with calicivirus, sequence homology places HEV in the alphavirus-like superfamily of viruses. The final classification of HEV remains undetermined. GBV-C GBV-C is a flavivirus-like, enveloped, capsidlessparticle (127) that measures 50 to 100 nm in diameter and bands in sucrose gradients at a peak density of 1.08 to 1.13 g/ml (88). The particle is highly associated with lipoproteins with moderate carbohydrate moieties on its surface. The genome is a singlestranded, positive-sense RNA virus of approximately 8,600 nt (127). It possesses a long ORF that encodes a polyprotein of about 2,900 amino acids (73). The genome is organized with structural genes located at the 5’ end and nonstructural genes at the 3’ end. Analysis of the nucleotide sequencereveals a pattern of serine protease and conserved helicase sequences and sequencescoding for an RNA-dependent RNA polymerase. Genotypes of GBV-C have already been identified by analysis of the entire 5’ nontranslated region (5’-NTR) sequence. Phylogenetic analysis of these sequencesdemonstrated the presence of three major types of GBV-C isolates (i.e., 1,2, and 3) as well as subtypes (i.e., la, lb, 2a, and 2b) that correlated with geographic origin (92). The hepatitis G virus (HGV) newly reported by Linnen et al. (80) is actually a member of one of these genotypes and is indeed a different isolate of GBV-C (92, 157). This conclusion is based on the alignment of the 5’-NTR nucleotide sequence of HGV with that of GBV-C. GBV-C differs from all known flaviviruses except GB virus A (GBV-A) in several respects. These include absence of a nucleocapsid, relatively lengthy 5 ‘- and 3’-NTRs, fewer N-linked glycosyl sites in both envelope 1 and 2 structural proteins, and an extraordinarily weak internal ribosome entry site (126).

Hepatitis

CLINICAL

Viruses

5

FINDINGS

HAV HAV usually causes a minor or unnoticed illness in children and young adults, and it is probable that on a worldwide scale fewer than 5% of cases are recognized clinically. In the individual case, the signs and symptoms of hepatitis A are indistinguishable from other types of viral hepatitis (76,121). HAV hepatitis is usually a self-limiting, benign inflammation of the liver. Its clinical course is characterized by a short phase of prodromal illness comprised of mostly general manifestations such as fatigue, malaise, fever, chills, and headache, followed by more specific symptoms of nausea, vomiting, and abdominal discomfort (40). In about two-thirds of clinically overt cases, these symptoms are accompanied by the appearance of jaundice. The laboratory findings in HAV infection of elevated serum transpeptidases are often accompanied by nonspecific elevations of immunoglobulins (IgM and IgG), anti-smooth muscle antibody, and the acute-phase proteins. The IgM is mainly polyclonal, with only a small proportion directed against HAV. The extent of elevation of serum transpeptidases does not appear to be of prognostic significance. The blood lymphocyte count, initially high, usually falls with diseaseevolution, and the red cell volume tends to rise (partially because of mild hemolysis); in recovery there may be a mild pancytopenia. Minor coagulation defects and depressedfibrinogen levels are common, but are clinically and prognostically significant only in those running a fulminant or subfulminant course. The disease usually lasts 2 to 3 weeks but may be followed by a prolonged period of convalescence. It was thought that only occasional patients experience a more protracted course with bimodal or even polyphasic elevation of liver enzymes, but recent evidence suggests that some affected individuals may relapse and that relapses might be associated with reactivation of virus shedding. Persistent infections and chronic hepatitis A have not been documented. Severe, fulminant hepatitis A with a fatal outcome is rare. The overall mortality following infection is as low as 0.1 to 0.5% (49). HAV is shed in the feces early in the incubation period (usually 3 to 4 weeks in duration), reaching a maximum just as symptoms begin to develop. The virus is efficiently transmitted under conditions of low hygienic standards. Efficient vehicles for the virus include contaminated drinking water and food, and shellfish harvested from estuaries or coastal regions polluted with human feces and unprocessedwastewater. Person-to-person spread among household contacts, preschool children in day care, residents of institutions for the mentally handicapped, male homosexuals, and, with apparently increasing fre-

6

Chernesky

CUMITECH

et al.

quency, intravenous drug addicts contribute significantly to transmission of the disease. During early infection in chimpanzees, HAV antigen has been identified by immunofluorescence in the cytoplasm of 5 to 10% of hepatocytes (86). Electron microscopy revealed the presence of viral particles within cytoplasmic vesicles (120). Antigen may be found in the liver before its appearance in the feces and may persist throughout the period of enzyme elevations. Fecal shedding of virus is temporally correlated with the appearance of antigen in the liver. Later in the infection, antigen becomes localized to only a few hepatocytes and Kupffer cells. Antigen has also been detected by immunofluorescence in abdominal lymph nodes, the spleen, and the kidney, where it followed a distribution suggestive of the deposition of immune complexes along the glomerular basement membrane. The finding of viral particles in bile during acute infection has led to the current hypothesis that HAV found in feces is derived from the hepatocyte and reaches the intestines by way of the bile ducts. The virus is hyperendemic in undeveloped countries where poor sanitary standards facilitate the spread of the virus and most children acquire infection in the first few years of life, when the infection is usually anicteric and frequently even asymptomatic. Because infection leads to a long-lasting immunity, clinically overt hepatitis A is usually not a prominent problem among the populations of such countries. Seroepidemiologic studies carried out within North America indicate that the likelihood of prior HAV infection is related to age and socioeconomic status but not to race or sex. Approximately 10 to 20% of North Americans have antibody by the age of 20, and 50% have antibody by the age of 50. The age-related nature of the seroprevalence curve appears to be due to a cohort effect in the presence of a declining incidence of hepatitis A. Thus, older persons are more likely to have antibody, because the incidence of HAV infection was higher during the early years of their lives (29).

WA

brown urine followed several days later by pale stools and jaundice. A chronic HBsAg carrier state develops in 5 to 10% of adults after either symptomatic or subclinical acute hepatitis B (54). Up to 90% of infants who acquire HBV infection from HBsAg-positive mothers become chronically infected. Chronic HBV infection may progress to chronic persistent hepatitis, chronic active hepatitis with or without cirrhosis, or even primary hepatocellular carcinoma. HDV Acute HDV infection occurs either as a simultaneous coinfection with HBV or as a superinfection in a chronic HBV carrier (55). Patients with acute HDV coinfection often have a biphasic illness characterized by separate elevations in serum aminotransferase levels. Acute delta coinfection with HBV is usually selflimited. Less than 5% of patients with acute HDV coinfection go on to develop chronic HDV infection. In contrast, acute HDV superinfection of chronic HBV carriers usually results in chronic HDV infection. More than 80% of patients with acute HDV superinfection develop chronic delta hepatitis. Acute delta hepatitis resembles other types of acute viral hepatitis, but it tends to be more severe. Fulminant hepatitis occurs more frequently in acute HDV coinfection with HBV or acute delta superinfection of HBV carriers than with acute HBV infection alone. The mortality rate for acute delta hepatitis ranges from 2 to 20%, compared to less than 1% for acute hepatitis B. Chronic delta hepatitis is also more severe than other types of chronic viral hepatitis. As many as 70 to 80% of patients with chronic delta hepatitis develop cirrhosis and complications of portal hypertension, compared to only 15 to 30% of patients with chronic hepatitis B. Rapid progression to cirrhosis occurs in 15% of patients with chronic delta hepatitis within 2 years after the onset of symptoms. Such a rapidly progressive course is unusual in chronic hepatitis B or C. HCV

HBV The incubation period for HBV is usually about 3 months, but varies from 4.5 to 180 days. Approximately 30 to 40% of adults infected with HBV develop symptomatic acute hepatitis, and fulminant hepatitis resulting in death occurs in 1 to 3% of these individuals. At least 50% of persons infected with HBV exhibit a transient subclinical infection. In symptomatic HBV infection, the onset of acute hepatitis is preceded by a short prodromal phase characterized by fever, fatigue, malaise, anorexia, myalgia, nausea, and vomiting. The icteric phase usually begins within 10 days of the initial prodromal symptoms and is characterized by the appearance of dark, golden-

HCV is now recognized as the major cause of chronic NANB hepatitis in the world, and recent epidemiologic studies have estimated the prevalence of HCV infection in the United States to be approximately 1.8 % of the general population. Although the major route of HCV transmission appears to be parenteral, both sexual and vertical transmission may also occur, albeit at lower frequencies than that described for other parenteral agents such as HBV and HIV. Several risk factors for acquiring HCV infection have been described, including prior blood transfusion or blood product use, a history of intravenous drug use, renal disease requiring chronic renal dialysis, and a history of close personal contact with an HCV-infected indi-

CUMITECH

18A

vidual. However, in many sporadic cases of hepatitis C, no risk factor for infection can be identified, and nonparenteral transmission of hepatitis C remains an important and unresolved question. The virological course of HCV infection differs substantially from other forms of viral hepatitis. In adults, the vast majority of infected individuals develop chronic persistent viremia, despite an absence of clinical symptoms in most cases. Thus, when highly sensitive molecular methods such as reverse transcription-PCR (RT-PCR) are used, between 80 and 95 % of seropositive individuals will test positive for HCV RNA in serum (i.e., the individuals are viremic), depending on the patient population. Although most adults with chronic HCV infection remain asymptomatic for long periods of time, approximately 60 % develop persistent abnormalities in serum aminotransfera se levels, which indicate ongoing liver injury. The biochemical abnormalities associated with chronic hepatitis C are characteristically mild even when liver disease 1s severe, and thus serum aminotransferase levels provide unreliable indicators of either HCV infection status or activity of liver disease. In a significant percentage of cases, chronic HCV infection is associated with progressive liver disease; histologic studies of HCV-infected patients have demonstrated that advanced liver disease (cirrhosis) develops in 20 to 30% of cases, with the mean time to cirrhosis estimated to be 21 years (2). Based on a 1994 survey of liver transplant programs in the LJnited States, HCV infection is now the leading indication for liver transplantation in this country (147). Finally, patients with chronic HCV infection are at increased ri sk for the de velopment of hepatocellular carcinoma, although the mechanisms of carcinogenesis associated with HCV infection are uncertain. For these reasons, all patients who test positive for HCV antibodies should have additional virological testing, regardless of the results of biochem .ical tests. Chronic HCV infection has also been strongly associated with at least two extrahepatic disease syndromes, essential mixed cryoglobulinemia (EMC) types II and III, and membranoproliferative glomerulonephritis (MPGN). Patients with EMC have the following laboratory findings: cryoglobulinemia (serum precipitates during refrigeration), positive rheumatoid factors, and hypocomplementemia. Variable clinical findings include vasculitis (blood vessel damage and skin lesions), polyneuritis (nerve damage) and nephritis (kidney damage), which are thought to result from precipitation of antibody within the infected individual (59). Because cryoprecipitates contain HCV RNA and anti-HCV antibody, and the clinical syndrome is at least partially alleviated by anti-HCV therapy, the current evidence suggests HCV is causative in the majority of cases of EMC types II and III.

Hepatitis

Viruses

7

HCV is also associated with MPGN, a severe form of chronic renal disease. As in patients with HCV-associated EMC, many patients with HCV-associated MPGN show a clinical benefit from specific antiviral therapy (i.e., renal disease improves), which supports the hypothesis that HCV replication plays a critical role in the disease process. Because approximately 60% of patients with HCV-associated MPGN have cryoglobulinemia and hypocomplementemia, some of these cases probably reflect a variation of HCV-associated EMC with isolated renal disease. HEV Hepatitis E is transmitted by the fecal-oral route and can be rapidly spread by contaminated water (Table 2). The causative agent, HEV, is responsible for major outbreaks of acute hepatitis in developing countries. Upon infection, HEV enters the blood by the gastrointestinal tract, replicates in the liver, and is released from the hepatocytes to the bile and blood (13). Subsequently, the virus is excreted in the feces. Successful fecal-oral transmission (6) of HEV to a human volunteer yielded 27- to 34-nm particles in acutephase stool specimens. Virus-like particles (VLPs) reacted with human sera taken from geographically distinct hepatitis E epidemics. This observation established one major serotype of HEV. The serial passage of HEV from infected human stool suspensions to monkeys and chimpanzees demonstrated that the virus replicated in the livers of infected animals (12). In this primate model, the disease typically results in elevated liver enzymes and the detection of VLPs in both stool and bile. Consumption of fecally contaminated water is the most common route for hepatitis E infection. The incubation period is 2 to 9 weeks (146). Hepatitis E manifests itself in subclinical cases, acute hepatitis, and fulminant hepatitis. Hepatitis E is more likely to be subclinical, anicteric, and symptom free in infants and young chi .ldren (142). Symptoms include fever, chills, nausea, vomiting, joint pain, headache, and abdominal distention. Histologic changes in the liver include focal necrosis and cellular and lobular cholestasis (109). Biopsies show portal inflammation and hepatocyte ballooning. The preicteric phase lasts 1 to Table

2.

Epidemiologic

features

of hepatitis

E

Predominantly an entencally transmitted disease Subclinical forms of anicteric disease Self-limited rctenc disease Mortality (up to 20%) by fulmrnant hepatrtrc failure in infected pregnant women Epidemic cases preferentially among adults In developrng countries Sporadic cases among children and adults In regions of endemicity Sporadrc cases among travelers from Western countries

8

Chernesky

et al.

10 days with symptoms of nausea, vomiting, and epigastric pain. The icteric phase lasts for 15 to 40 days accompanied by jaundice, dark urine, and claycolored stools. The overall course of disease entails virus excretion in the feces, viremia, liver b enzyme eleva tion (alanine transaminase [ALT]), and seroconversion, followed by the clearing of the virus and resolution of disease, Upon recovery, biopsy returns to normal and liver enzyme tests return to baseline. Clinically, hepatitis E is similar to hepatitis A, typically a self-limiting disease without chronic illness. The clinical attack rate during epidemics has been reported at 5%, with the most severe disease observed in young adults (13). Previously thought to be a disease only of adults, severe overt disease in young children has been established (46). During epidemics, limited outbreaks of jaundice last about 7 weeks with few reported secondary cases. Unlike hepatitis A, person-to-person transmission does not appear to occur routinely (62,63). A unique characteristic of hepatitis E is its high case fatality rate, 1 to 2% of hospitalized patients from the general population. There is a high rate of fulminant hepatitis in pregnant women in the third trimester, with 10 to 20% mortality (109). The mechanism for fulminant hepatic failure during pregnancy is unknown; endotoxin-mediated cytotoxicity is implicated but not proven. HEV is also implicated in causing more than 50% in of sporadic acute viral hepatitis cases worldwide regions where the virus is endemic. HEV infection in developed countries is infrequent (149). Most confirmed sporadic cases in the U.S. and Western Europe have been in persons traveling to areas where the disease is endemic. GBV-C The predominant route of transmission of GBV-C appears to be parenteral through contaminated blood and blood products. However, because recent reports have shown that both human saliva (17) and semen (102) of GBV-C infected individuals are positive for GBV-C RNA, the possibility exists that both horizontal and vertical transmissions may play a key role in the spread of GBV-C infections. In fact, cases of vertical transmission of GBV-C from infected mothers to newborns have already been reported (35, 37). Current epidemiologic data (125) show the presence of GBV-C RNA in sera obtained from a variety of sources. The sources include hemophiliacs; thalassedrug abusers; multiply mic patients; intravenous transfused individuals; persons with transfusion-associated hepatitis; volunteer blood donors with both normal and elevated serum transaminase levels; chronic HBV and HCV carriers; acute and chronic non-A-E hepatitis patients; kidney, liver, and bone marrow transplant recipients and donors; patients

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with fulminant hepatitis; and patients on maintenance hemodialysis. Analyses of serum specimens collected from healthy volunteer blood donors from different global areas confirmed the presence of GBV-C RNA in a remarkable 1 to 4% of the specimens (25,36,85, 89, 131, 148). Most GBV-C infections are mild, transient, and self-limiting, with slight or no elevation of liver ALT levels. Most of these subclinical cases resolve after loss of serum GBV-C RNA with a concomitant appearance of antibody to envelope E2 of GBV-C (antiGBV-C E2, or GBV-C E2 antibody [32]). These types of GBV-C infections are hardly noticeable and very difficult to evaluate when studied in multitransfused patients or patients with a more serious and superimposed HBV or HCV infection. GBV-C is capable of inducing persistent infection in about 15 to 25% of GBV-C-infected individuals. Masuko et al. (85) monitored 8 hemodialysis patients with GBV infection retrospectively for 7 to 16 years. In two patients, the virus was present at the start of hemodialysis. One had a history of transfusion, and GBV-C RNA persisted over a period of 16 years; the other cleared GBV-C RNA after 10 years. In five patients, GBV-C RNA was first detected 3 to 20 weeks after blood transfusion and persisted for up to 13 years. Elucidating the viral mechanisms that lead to the establishment and maintenance of the persistent state is crucial for our understanding of the pathogenesis of GBV-C (71). The role of GBV-C in the etiology of fulminant hepatitis is not fully established. Further convincing studies are needed to confirm a definite link between fulminant hepatitis and GBV-C infection. One Japanese study (154) documented the presence of GBV-C RNA in 3 of 6 (50%) of fulminant hepatitis patients without evidence of infection with known hepatitis viruses. Since that report, questions were raised concerning the association of GBV-C with acute liver failure (3). Specifically, questions were raised as to whether GBV-C was an “innocent bystander” transmitted through transfusions given to the three patients prior to the onset of fulminant hepatitis. Additional studies (l53), however, showed that only a few of the 63 fulminant hepatitis patients studied had received therapeutic transfusion before the onset of fulminant hepatitis. In a similar study also carried out in Japan (138), GBV-C RNA was detected in 3 (20%) of 15 patients with HBV infection and 3 (12%) of 25 patients without markers of hepatitis A-E infection. Overall, GBV-C RNA was detected in 6 (14%) of 44 patients with fulminant hepatitis at a frequency significantly higher (P < 0.001) than that in 3 (0.9%) of 326 blood donors matched for age with the patients. Of the 6 patients with GBV-C RNA, only 3 (50%) had a history of transfusion and all of these were coin-

CUMITECH 18A

fected with HBV. These results, according to the authors, indicate a role of GBV-C in inducing fulminant hepatitis either by itself or in concert with other hepatitis viruses. A unique study (36) showing histological features in liver samples from patients infected with GBV-C alone has been reported. GBV-C was implicated in a significant number of acute and chronic casesof nonA-E hepatitis. Among the six chronic hepatitis patients positive for GBV-C RNA, the histology of the liver samplesrevealed chronic active hepatitis in one patient and chronic persistent hepatitis in the five others. All chronic patients had elevated ALT levels between 89 and 478 U/liter. In contrast, among the 11 acute hepatitis cases positive for GBV-C RNA, the ALT levels varied between 615 and 2,477 U/liter. Colombatto et al. (20) studied GBV-C in 67 patients with liver diseasewithout any markers for hepatitis A-E. They report that the spectrum of liver diseaseassociated with GBV-C infection in these patients is wide, with a large variety of serious histologic liver lesions (steatosis, fibrosis, and cirrhosis). Of interest, nonspecific inflammatory bile duct lesions were found in 50% of patients with only GBV-C infection. They also suggest that GBV-C infection is present significantly more often with elevated cholestatic enzymes, namely, gamma glutamyl transpeptidase and alkaline phosphatase. Again, the above-mentioned studies point out the importance of studying individuals positive for GBV-C RNA without concomitant HBV or HCV coinfection. Several reports have shown unequivocally the lack of association or involvement of GBV-C infection with many other known diseases. These included autoimmune liver disease, rheumatoid arthritis, aplastic anemia, hepatocellular carcinoma, non-Hodgkin’s lymphoma, common variable immunodeficiency, X-linked agammaglobulinemia, porphyria cutanea tarda, Kawasaki disease,oral cancer, and oral lichen planus.

SEROLOGICAL MARKERS AND IMMUNODIAGNOSIS HAV Elevated total levels of serum immunoglobulins, especially IgM, are common during acute hepatitis A. Specific antibody is almost always present by the time of onset of symptoms. The IgM anti-HAV response is typically short-lived, but very sensitive antibody capture immunoassays have demonstrated its persistence for 6 to 12 months after hepatitis A (75). Serumneutralizing activity against the virus appears in parallel with antibody detected by immunoassay and may be present 3 to 5 days before the onset of symptoms. Viral antigen continues to be shed in feces after the

Hepatitis

Viruses

9

development of serum neutralizing antibody, and in some patients persists well into the second week of illness. There are no commercially available antigen detection assays for HAV. Although both IgG and IgM antibodies possessneutralizing activity against the virus, some acute-phase serum samplescontaining IgM anti-HAV have also been found to contain infectious virus. Serum IgG anti-HAV persists for long periods after infection, perhaps for life. Earlier assaysfor HAV involved complement fixation, immune electron microscopy, and immune adherence hemagglutination, which were subsequently replaced by solid-phase immunoassays using either radioisotopes (radioimmunoassay [RIA] ) or enzymes (enzyme immunoassay [EIA]) for the measurement of IgG responses (usually in paired sera). Because immune adherence hemagglutination antibodies rise later than those measured by solid-phase immunoassays, the absence of the former and presence of the latter during symptoms may suggest infection with HAV if HBV has been ruled out. Because anti-HAV can plateau rapidly after acute infection, it is more useful to use one of the solid-phase immunoassays for the detection of anti-HAV IgM. There has been a renewed activity in recent years to detect IgM in serum using erythrocytes as the indicator system in either a hemagglutination test (33) or a solid-phase hemadsorption or hemadherence assay (133). These types of assays, as well as the more traditional EIAs, have also been shown to perform well on saliva and urine specimens (99, 101). Tests measuring total anti-HAV immunoglobulins may be used to determine immunity by testing a single serum, and a diagnostic rise may be demonstrated in some casesin paired sera. Solid-phase &M-specific immunoassays are most appropriate to use for diagnosis. They are accurate in measuring anti-HAV IgM responsesup to 4 to 6 months after an acute infection. Commercially prepared testing kits are available and the results are easily interpreted. Serum IgM analysis allows laboratory testing on the same specimen used for hepatitis B testing. HBV BecauseHBV is not readily propagated in cell cultures or in animals other than high-order primates, the diagnosis of HBV infection is accomplished with sensitive immunoassays for the identification of HBVassociated antigens and antibodies in the blood. Third-generation immunoassays including RIAs, EIAs, microparticle enzyme immunoassays (MEIAs), and a reversed passive hemagglutination (RPHA) test are commercially available for the detection of HBsAg, anti-HBs, anti-HBc, IgM anti-HBc, HBeAg, and anti-HBe (Table 3). Additional serum markers for HBV infection include the following: DNA polymer-

10

Chernesky

CUMITECH

et al.

Table 3. Commercial sources kits for detection of hepatitis Serologrc marker

of currently licensed test B virus serologic markers

Method(s)

HBsAg

EIA, MEIA,

Source

RIA, RPHA

EIA, RIA EIA EIA

EIAb

Anti-H Bc

EIA, RIA EIA, RIA EIA EIAC

IgM anti-HBc Anti-H Bs HBeAg/Anti-HBe ‘All DlaSonn ‘Manufactured “Manufactured

EIA, EIA, EIA, EIA, EIA, EIA,

MEIA, RIA RIA RIA RIA RIA

assays are manufactured by Genetic Systems, by DlaSonn

RIA

Abbott Laboratones, North Chicago, III. DiaSonn, Strllwater, Mann.” Organon Teknrka, Durham, N C. Ortho Dragnostrc Systems, Rantan, N.J Sanofr Diagnostrcs Pasteur, Chaska, Mann. Abbott Laboratones DraSonn Ortho Diagnostic Systems Sanofr Dragnostrcs Pasteur Abbott Laboratones DraSorrn Abbott Laboratones DraSorin Abbott Laboratones DraSonn

by DlaSonn, Saluggla, Redmond, Wash

Italy.

ase (61), HBcAg (43), and HBV DNA (see “Nucleic Acid Diagnosis”). Tests for the presence of these markers do not add substantially to the information provided by the commercially available assays for HBeAg and are thus not included in most routine diagnostic evaluations. Measuring HBV DNA levels may be useful in chronic hepatitis B patients who are receiving antiviral therapy. This complicated system of HBV-associated serological markers may define the phase of infection, degree of infectivity, prognosis, and immune status of the patient. Patterns of HBVassociated serological markers commonly associated with the various forms and phases of HBV infection are illustrated in Table 4. HBsAg is the first serological marker to appear after HBV infection. HBsAg becomes detectable during the late incubation period, 2 to 5 weeks before the onset of symptoms, and persists for 1 to 5 months in symptomatic acute hepatitis B (54). A typical serological profile of HBV-associated markers after onset of acute hepatitis B is illustrated in Fig. 1. Although HBsAg is detectable in the sera of most patients with symptomatic acute hepatitis B, it may be undetectable by current third-generation assays in more than 10% of these cases (78). HBsAg is frequently absent, or present for only days, in transient subclinical HBV infection. In chronic HBV infection, HBsAg usually persists at high titer for a minimum of 6 months to as

18A

long as decades. The presence of HBsAg should always be considered indicative of ongoing HBV infection Some individuals with chronic HBV infection have undetectable levels of circulating HBsAg. Such persons are referred to as “low-level carriers” (54). HBeAg appears at the same time as or shortly after HBsAg, and its presence correlates with early and still active disease as well as high infectivity in acute HBV infection. HBeAg persists for 1 to 9 weeks after the onset of symptoms in symptomatic acute hepatitis, and although it may occasionally remain detectable for several weeks after the clearance of HBsAg, it usually disappears before HBsAg. Persistence of HBeAg for more than 10 weeks after the onset of illness predicts the development of chronic HBV infection (1). HBeAg remains detectable for several months to years in HBsAg-positive chronic HBV infection and will eventually disappear in many patients with persistent HBsAg (56). HBeAg-positive chronic HBsAg carriers tend to be young, to have developed the carrier state within the previous 10 years, to have some evidence of chronic liver disease, and to have high titers of hepatitis B virions (54, 56). Mother-toinfant transmission of HBV infection is highly associated with the presence of HBeAg (8, 140). Anti-HBc appears shortly after the appearance of HBsAg and HBeAg and shortly before or at the onset of clinical symptoms (54). In acute hepatitis, anti-HBc is frequently the only detectable serological marker of HBV infection other than anti-HBe. This serological pattern may occur during the acute phase in persons who do not develop detectable HBsAg and HBeAg, or during the convalescent phase after the disappearance of HBsAg but before the appearance of anti-HBs. Anti-HBc, alone or together with anti-HBe, is sometimes observed in individuals who have recovered from past HBV infection but did not develop or subsequently lost detectable anti-HBs. Anti-HBc initially consists of both IgG and IgM antibodies, but IgM anti-HBc decreases in titer and disappears within several months (19,135) to one or more years after acute Table virus

4. Serological infections

Serological marker HBsAg HBeAg HBV DNA Anti-H Bc IgM Total Anti-HBe Anti-HBs ‘+, detectable, ‘Patients with or anti-HBe.

Incubation period

diagnosis Acute HBV Infectron

+a + +

+ + +

-

+ + -

of hepatitis Past HBV Infectron

+ -I-

B

Chronrc HBV infection

+ ++

Hepatitis B vaccination

-

+ kb

-, not detectable, +, may be detectable chronic HBV Infection usually have detectable

HBeAg

CUMITECH

Hepatitis Viruses

18A

Period

Acute

Disease

Early

11

Late

Important Diagnostic Tests I

I 7

I

I

I

1

I

I

I

2

3

4

5

6

7

8

Relative Concentration Of Reactants

Level Of Detection Months After Exposure SGPT (ALT) Symptoms

1

I

I

I

I

1

1

I

1

2

3

4

5

6

7

8

’ c’

FIGURE 1 Typical serologlcal reference 53 with permlsslon.

and cllnlcal

course

of acute

HBV InfectIon.

infection (44,70,78,117), depending on the sensitivity of the assay. Since IgG anti-HBc can persist for years or even a lifetime, the detection of IgM anti-HBc distinguishes HBsAg-negative, anti-HBc-positive patients with current or recent acute HBV infection from those with past infection. IgM anti-HBc has been demonstrated in more than 50% of acute hepatitis patients who are negative for both HBsAg and antiHAV IgM but are positive for total anti-HBc, regardless of the presence or absence of anti-HBs (69). In chronic infections the initial anti-HBc response also consists of both IgG and IgM antibodies, but unlike the situation in acute infection, IgM anti-HBc titers decreasevery slowly and low titers persist for years while total anti-HBc titers remain very high. Anti-HBc may be the only detectable serological marker in chronically infected HBsAg-negative individuals. The demonstration of IgM anti-HBc and high titers of total anti-HBc in these low-level carriers is indicative of chronic HBV infection (117). Because IgM antiHBc can be detected in chronic HBV infection, its presence does not always distinguish between acute and chronic hepatitis B. However, the sensitivity of commercial RIAs, EIAs, and MEIAs for IgM anti-HBc has been adjusted to a level where patients with current or recent acute HBV infection are IgM anti-HBc

SGPT, serum

glutamlc

pyruvlc

transamlnase

Reprinted

from

positive, but those with chronic infection are negative or only weakly positive. Anti-HBe usually becomes detectable 2 to 6 weeks after the disappearance of HBeAg and 2 to 4 weeks before the clearance of HBsAg, and it may persist for months or years after acute infection. Anti-HBe positivity after acute infection has a duration generally less than that of anti-HBc and anti-HBs and may persist for only 6 months or lessin about 5% of acute HBV infections (93). Early appearance of anti-HBe often predicts an uncomplicated course of acute hepatitis, whereas a delay of more than 6 weeks after the clearance of HBeAg may indicate a prolonged course or development of chronic hepatitis ( 1). Seroconversion from HBeAg to anti-HBe frequently occurs several years after the development of HBsAg-positive chronic hepatitis and is usually accompanied by the spontaneous resolution of chronic liver disease(56). Anti-HBe-positive chronic HBsAg carriers tend to be older than HBeAg-positive carriers, tend to have been chronically infected for many years, tend to have little or no evidence of chronic liver disease,and tend to be minimally infectious (54). Anti-HBs usually appears several months after the disappearance of HBsAg in acute hepatitis, although it may be present together with HBsAg as early as the

12

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et al.

onset of symptoms in 6% of cases (135). Seroconversion from HBsAg to anti-HBs usually reflects termination of HBV infection, clinical recovery, and immunity against reinfection by any HBsAg subtype by virtue of the common antigenic determinant (83). Anti-HBs persists for years after recovery from acute infection and is usually detected together with antiHBc and anti-HBe, although it may be found alone in some individuals who have recovered from a past HBV infection with the subsequent loss of detectable anti-HBe and anti-HBc. Anti-HBs is also the only detectable marker in individuals who have been immunized with the hepatitis B vaccine rather than infected with the virus. Anti-HBs is not usually detectable during chronic HBsAg-positive hepatitis, but it may occasionally be present due to previous or subsequent infection with a different HBsAg subtype (21) or as a result of antigen-antibody complexes (4,3O, 97). Tests for the detection of HBsAg, anti-HBc, and anti-HBs should be performed routinely for the laboratory diagnosis of current or past HBV infection. IgM anti-HBc, HBeAg, and anti-HBe determinations should only be performed when indicated by the results of HBsAg, anti-HBc, and anti-HBs assays. IgM anti-HBc testing can be used to distinguish between acute and chronic HBV infection in HBsAg-positive individuals. HBeAg and anti-HBe detection should be attempted on HBsAg-positive sera when the relative infectivity of blood is of clinical significance or when early seroconversion from HBeAg to anti-HBe after acute HBV infection is used as a prognostic indicator of transient rather than chronic infection. The detection of IgM anti-HBc or anti-HBe may be diagnostic of recent or current acute HBV infection when it is found together with total anti-HBc as the only detectable HBV-associated serological markers during the “window” phase of the infection between the disappearance of HBsAg and the appearance of anti-HBs. HBeAg and anti-HBe determinations may have therapeutic importance in chronic HBsAg-positive hepatitis because it has been suggested that antiviral therapy be reserved for HBeAg-positive patients with chronic liver disease (56).

HDV The laboratory diagnosis of HDV infection can be accomplished by a variety of methods including detection of HDAg in serum or liver tissue, total antibodies to HDAg (anti-HDV), IgM anti-HDV, and HDV RNA in serum (55, 103). HDAg can be detected in hepatocyte nuclei of patients with acute or chronic delta hepatitis by immunofluorescence or immunoperoxidase staining of liver tissue sections. HDAg can be detected in the serum of patients with acute HDV infection with an RIA, EIA, or immunoblot assay. Unlike the RIA and EIA for detection of HDAg, the

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immunoblot assay is also able to detect HDAg in the serum of patients with chronic HDV infection in spite of the presence of high titers of total anti-HDV. Total anti-HDV and IgM anti-HDV can be detected in the serum of patients with recent or current HDV infection by EIA and RIA. HDV RNA can be detected in the serum of patients with HDV infection by nucleic acid hybridization and PCR assays (see Nucleic Acid Diagnosis). The only serologic tests for diagnosis of HDV infection that are commercially available in the United States are an EIA and an RIA for detection of total anti-HDV (Abbott Laboratories, North Chicago, Ill.). Tests for IgM anti-HDV and HDAg are available in Europe and can be imported to the United States for research use only. Tests for the detection of HDV RNA in the serum, HDAg in the liver, and HDAg in the serum by immunoblot are only available in research laboratories. In acute coinfection with HBV and HDV, HBsAg is first detectable in the serum during the incubation period, followed shortly by HDV RNA and HDAg. After the onset of clinical symptoms, these serologic markers decline in titer, with eventual clearance as the acute liver disease resolves. IgM anti-HDV and total anti-HDV become detectable after the onset of clinical symptoms, but the IgM and total anti-HDV response is often low in titer and short-lived in patients with self-limited, acute HDV coinfection. In acute superinfection of an HBV carrier with HDV, chronic delta hepatitis frequently develops with persistence of HBsAg and HDV RNA in the serum, HDAg in the liver and serum, low levels of IgM anti-HDV, and high titers of total anti-HDV (Fig. 2). Testing for markers of HDV infection should be considered in HBsAg-positive patients with symptoms of acute or chronic viral hepatitis, particularly those with fulminant hepatitis or at high risk for HDV infection. For most clinical situations, testing for HBsAg, IgM anti-HBc, and total anti-HDV is sufficient for the laboratory diagnosis of HDV infection (Table 5). Patients with acute HDV coinfection should be positive for HBsAg, IgM anti-HBc, and total anti-HDV. Patients with acute HDV superinfection are also positive for HBsAg and total anti-HDV, but they are usually negative for IgM anti-HBc. Therefore, in acute delta hepatitis, coinfection can be distinguished from superinfection by testing for IgM anti-HBc. Although tests for HDV RNA in serum and HDAg in the serum or liver are onlv available in research la.boratories, they may be useful for monitoring the response to antiviral therapy in patients with chronic delta hepatitis.

HCV The diagnosis of chronic viral hepatitis should be considered in any patient with elevated serum amino-

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Hepatitis

Viruses

13

Time After Exposure

FIGURE

2.

Typical

serologlcal

course

of acute

HDV supennfectlon

transferase levels (alanine aminotransferase or aspartate aminotransferase) that persist for greater than 6 months. In addition, the diagnosis should be considered in all at-risk patients, including those patients with a significant history of exposure to HCV. The second-generation screening assay for HCV infection (EIA2), which was approved by the Food and Drug Administration (FDA) in 1992, uses a combination of recombinant HCV core antigen and two nonstructural antigens, NS3 and NS4, in a microtiter format to screen for anti-HCV. Since 1992, the risk of posttransfusion HCV infection has been greatly reduced by EIA2 screening of blood products. Although the sensitivity of HCV immunodiagnosis has been greatly improved, a small percentage of infected individuals may remain negative for anti-HCV by EIA2. Table 5. Serological virus infections

diagnosis

of hepatitis Acute HDV superinfection

delta

Diagnosttc marker

Acute HDV coinfectlon

HBsAg IgM anti-HBc HDAg Liver Serum Anti-HDV

+a +

+ -

+ +

++ -b

km Total HDV RNA

+ + +

+ ++ +

a+, detectable, -, not detectable. bHDAg IS not detectable by RIA or EIA but munoblot

IS

usually

Chronic delta hepatltls

detectable

by Im-

In an HBsAg

carrier.

Reprinted

from

reference

55 with

permlsslon.

These individuals would include patients in the “window” period preceding seroconversion and patients with concomitant HCV and HIV infections who show an unusually high rate of HCV “seroreversion” (i.e., loss of detectable HCV antibody). The specificity of EIA2 is also greatly improved relative to the firstgeneration HCV assay, although false-positive results remain a problem in a population with low prevalence, for example, the blood donor population (156). There is currently little evidence that newer-generation HCV antibody screening assays will improve the serodiagnosis of hepatitis C, and given the high sensitivity of EIA2 as a screening assay, the specificity of any new tests for HCV should be carefully assessed relative to EIA2. The FDA recently licensed a supplemental serological test for HCV, the recombinant immunoblot assay (RIBA-II). RIBA-II uses the same HCV antigens as EIA2 in an immunoblot format, and thus is technically not a confirmatory assay. The RIBA-II format allows the identification of nonspecific reactivity against the recombinant fusion proteins of HCV and thus reduces false positives. The recombinant HCV antigens are designated C22 (core), C33 (NS3), C-l 00 (NS4), and 5-l-l (NS4). A positive result in the RIBA-II assay, defined as reactivity against two or more recombinant HCV antigens without reactivity to recombinant superoxide dismutase (SOD), supports the diagnosis of HCV infection, while a negative result in RIBA-II makes infection unlikely. Reactivity to a single recombinant protein is interpreted as inde-

14

Chernesky

et al.

terminate, as is a multiband result in the context of positive SOD reactivity. Analysis of large numbers of specimens with indeterminate RIBA-II results has shown that approximately 15% of such specimens contain HCV RNA. However, specimens with C-100 or 5-l-l indeterminate patterns had a very low chance of being positive for HCV RNA (less than l%), while C22 or C33 indeterminate patterns predicted the presence of HCV RNA in 25 to 50% of specimens. Thus, RIBA-II testing is most appropriate for EIA-positive specimens obtained from low-risk individuals (i.e., potential blood donors). In high-risk populations and liver disease clinics, less than 1% of EIA-positive specimens will be false positives. Therefore, RIBA-II testing in high-risk populations is not necessary for the diagnosis of hepatitis C (47). It is important to emphasize that many HCV-seropositive individuals may have normal or mildly abnormal serum transaminase levels. However, it is impossible to predict either the presence of HCV viremia or the extent of liver disease based on the detection of HCV antibody or on serum aminotransferase levels. Considering the clinical relevance of HCV infection as previously reviewed, confirmation of HCV viremia in all seropositive individuals has become an important concern for the clinical virology laboratory. HEV The “gold standard” diagnosis of HEV infection has been based on detection by immune electron microscopy (IEM) of VLPs in fecal specimens from persons diagnosed with acute viral hepatitis in the absence of serologic response to HAV, HBV, HCV, HDV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) (12, 13). IEM was essential for identifying HEV in clinical specimens; antibody-coated particles were collected and detected by electron microscope. HEV VLPs recovered from stools obtained from hepatitis patients in Mexico and the former Soviet Union were shown to aggregate with acute-phase antibody from other hepatitis E cases in Burma, Nepal, Pakistan, the former Soviet Union, Sudan, Somalia, and Mexico (13). It was concluded by IEM that these cases of enterically transmitted hepatitis were all caused by one serotype of virus. Although highly specific, IEM is difficult to perform and most clinical specimens do not contain sufficient VLPs to be detected. Krawczynski and Bradley (68) developed a direct immunofluorescence assay to detect HEV antigen in infected hepatocytes. An immunofluorescence blocking assay to detect antibody to HEV in sera (67) was developed, with cryostat liver sections from infected nonhuman primates as the source of virus-specific antigen. Both methods are cumbersome and neither assay can discriminate between recent and past infections. Sequencing of the HEV genome and antibody

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screening of HEV clones paved the road to identifying regions of the virus useful for serodiagnostics. The isolation of two immunodominant epitopes from the HEV Mexico and Burma strains ( 15 1) and their crossreactivity with human, chimpanzee, and cynomolgus monkey sera indicated that these epitopes would have utility in the diagnosis of HEV infection. The identification of type-common viral epitopes within the structural regions of HEV reinforced the concept of a single serotype of HEV shared by the most divergent isolates from Asia and North America. Serodiagnostic assays were developed using these broadly reactive epitope regions. The development of Western blot assays for antibody to HEV has been useful in examining the dynamics of anti-HEV IgM and IgG antibodies following infection (Table 6). Antibodies to HEV have been detected with recombinant antigens from truncated ORF2 proteins containing 42 amino acids (151), 327 amino acids (150), and 436 amino acids (112) and from a full-length ORF2 of 660 amino acids (51). While the IgM antibody to HEV is transient, IgG HEV antibody to ORF2 has been shown to persist for several years. In experimentally infected monkeys, antibodies to ORF2 coincide with partial immunity (112). ORF3 has also been shown to be useful in the detection of acute-phase and convalescent-phase antibodies to HEV (51, 150). Although antibodies to both ORF2 and ORF3 were recognized in human sera of acute hepatitis patients, recombinant ORF2 appears to be a more sensitive diagnostic antigen than the ORF3 antigen. In rhesus monkeys experimentally infected with HEV (79), the immune response to ORF3 has been shown to be more transient, waning within a few months after ALT levels return to normal. These data suggest that the antibodies directed to ORF2 and ORF3 have different roles in the immune response to HEV. Because Western blotting is not suitable for largescale serosurveys, and HEV peptide EIAs are limited in IgM antibody detection, an HEV enzyme-linked immunosorbent assay (ELISA) was developed using ORF2 and ORF3 virus-specific proteins from HEV Mexico and HEV Burma isolates (150). The test is convenient, sensitive, and specific for anti-HEV. The ELISA has been tested for possibl e cross-reactions Table

6.

Laboratory

Procedure RT-PCR lmmunofluorescence blocking assay Western blot Peptide

EIA

tests

for diagnosis

of HEV

infection

Detecting molecule

Specimen

HEV RNA HEV RNA Anti-H EV IgG

Feces Serum Serum

Anti-H EV IgG Antt-HEV IgM Antl-H EV IgG

Serum Serum Serum

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with antibodies to hepatitis A, hepatitis B, hepatitis C, hepatitis D, rubella, and Norwalk viruses and shown to be specific for anti-HEV (26). The HEV antigens will bind acute-phase and convalescent-phase antibodies. By using appropriate secondary antibodies as detector molecules, the ELISA methodology allows the detection of IgG- and IgM-specific anti-HEV and is therefore useful in diagnosing acute and past HEV infection. In testing acute-phase patient sera from outbreaks of hepatitis E, 95% were reactive when tested for anti-HEV IgG. The seroreactivity among healthy persons in low-risk populations is 1 to 2%. The specificity of the HEV IgG ELISA is 99%. The seropositivity among individuals within regions of endemicity can be as high as 20%, often because of long-lived antibodies. IgG antibody to HEV has been shown to persist for up to 14 years in some infected persons (64). The recent development of the HEV IgM ELISA to measure acute-phase antibody improves the diagnosis of recent infection. The sensitivity of the IgM ELISA to detect IgM anti-HEV was determined using patient sera from persons infected with HEV and still shedding virus in feces. The sensitivity of the assay is 93.5% for sera collected within 14 days of the onset of illness. The specificity of the IgM ELISA is 97% as determined by testing blood donors from regions in Singapore and the United States where the virus is nonendemic. The HEV IgG ELISA and HEV IgM ELISA (Genelabs Diagnostics) are commercially available for worldwide distribution. The HEV EIA for IgG anti-HEV (Abbott Laboratories) is currently available in Europe. GBV-C Five different prokaryotically expressed recombinant proteins were employed for serologic studies utilizing ELISA and Western blotting (25, 125). Although some regions of GBV-C have been identified as immunogenic, efforts to develop a screening immunoassay for the detection of GBV-C have been unsuccessful because only about 25% of GBV-C-infected individuals develop antibodies to these recombinant proteins. Most seropositive individuals produce antibodies against only a single antigen, and no single antigen has been identified that is consistently recognized by individuals exposed to GBV-C (25). Because of this, structural GBV-C E2 protein was expressed in Chinese hamster ovary cells (32). The proteins were purified and used in a solid-phase ELISA as described by Dille et al. (32). A population of intravenous drug abusers, acute and chronic non-A-E hepatitis patients, volunteer and commercial blood donors, West Africans, and several chronic HCV patients were examined for GBV-C E2 antibodies (Table 7). None of the specimens that were positive for antibodies to GBV-C E2 were positive for GBV-C RNA. Of 100 volunteer

Hepatitis Table 7. Prevalence various populations

studies

Category

No.

Volunteer donors Commercial donors West Af rlca Acute non-A-E Chronic non-A-E Acute HAV Acute HBV Acute HCV I DAs”

100 711 30 57 157 34 47 41 65

‘All were negattve for GBV-C RNA ‘Only 487 of the 711 samples have c IDAs, rntravenous drug abusers.

of GBV-C

exposure

No. G BV-C RNA posltwe

No. antiG BV-C E2” posltive

1 85b IO 3 12

3 195 4 14 18 15 20 20 43

8 IO been

tested

15

Viruses in

Total Pd 4 280 14 17 30 I5 20 28 53

(4) (39) (47) (30) (19) (44) (42) (68) (82)

by PCR.

blood donors, 1 was RT-PCR positive and 3 were ELISA positive for a total incidence of 4%. In contrast, the incidence in commercial blood donors was 39%. Total prevalences of 14,15,17,20,28,30, and 60% were shown for West Africans, acute HAV patients, acute non-A-E hepatitis patients, acute HBV patients, acute HCV patients, chronic non-A-E hepatitis patients and intravenous drug users, respectively. These data suggest that the exposure to GBV-C is much higher than would have been determined by RT-PCR studies alone and that testing for GBV-C E2 antibodies should greatly extend the ability of RTPCR to define the epidemiology and clinical significance of GBV-C. Other studies (50) have shown that antibodies to GBV-C E2 appear to be long-lived (greater than 3 years) with a fairly constant titer (ranging in reciprocal endpoint dilution from 336 to 21,504) and that anti-GBV-C E2 is associated with recovery (clearance) of GBV-C infection. Two profiles (one of an acute hepatitis followed by recovery with the appearance of GBV-C E2 antibody and the other of an acute hepatitis progressing to chronicity with the persistence of GBV-C RNA) have been reported. These reports resulted from analysis of the serial serum specimens from the six recipients of GBV-C RNA-positive blood donors together with other serial serum specimens from GBV-C RNA-positive individuals collected during the incubation period, recovery or chronic acute phase, convalescence, disease state. Of importance is the recent evidence for the protective immunity of anti-GBV-C E2 (89) among kidney recipients. The data show that anti-GBV-C E2-positive kidney recipients did not acquire GBV-C infection after transplantation from GBV-C RNA-positive donors, while anti-GBV-C E2-negative kidney recipients did acquire GBV-C infection after transplantation from GBV-C RNA-positive donors.

16

NUCLEIC

Chernesky

et al.

ACID DIAGNOSIS

HAV In most cases it is unproductive to examine feces for HAV particles or antigens because most patients have few, if any, in this specimen during overt illness, and what is present may be heavily masked by antibody. Conversely, in an epidemic outbreak of HAV, examination by IEM or solid-phase immunoassay of feces from contacts of individuals with acute disease may alert medical officers to the spread of virus by those in prodromal stages of disease. To date, there have been no published efforts to use nucleic acid amplification technology on clinical specimens such as feces to diagnose HAV infections.

HBV Nucleic acid hybridization assays have been developed for the detection of HBV DNA in the serum of patients with HBV infection (9, 119, 144). Two (research use only) hybridization assays for the quantitation of HBV DNA in human serum are commercially available: a liquid-phase molecular hybridization assay (Genostics HBV DNA Assay; Abbott Laboratories) and a “sandwich” nucleic acid hybridization assay (Quantiplex HBV-DNA Assay; Chiron Corporation, Emeryville, Calif.). Slot or dot blot hybridization assays have a detection limit of about 5 pg of HBV DNA per ml, corresponding to approximately 1.5 X lo6 viral genomes per ml (110). The branchedchain DNA (bDNA) hybridization assay from Chiron Corporation can detect as few as 700,000 HBV DNA equivalents per ml (approximately 2.5 pg/ml) (52) and the liquid-phase hybridization assay from Abbott has a detection limit of 400,000 HBV DNA equivalents per ml (approximately 1.5 pg/ml) (72). The loss of detectable HBV DNA by a solution hybridization assay is an earlier indicator of response to treatment than loss of HBeAg in patients receiving antiviral therapy (72). More recently, PCR assays have been developed for the detection of HBV DNA in the serum (5,43,60,132). PCR has been shown to detect as little as low3 pg of HBV DNA per ml, or approximately 100 to 1000 HBV DNA equivalents per ml, and is currently the most sensitive method available for the detection of HBV DNA in the serum. Most HBsAgpositive individuals have detectable HBV DNA in the serum, especially those who are also HBeAg positive. HBeAg-positive individuals usually have high levels of HBV DNA in the serum that are easily detectable by hybridization assays and PCR. HBsAg-positive individuals who are negative for HBeAg usually have lower levels of HBV DNA in the serum that may be undetectable by hybridization assays, but most of these individuals have detectable HBV DNA by PCR. HBeAg-negative patients with detectable serum HBV

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DNA have higher serum alanine aminotransferase levels than those who are PCR negative. HBV DNA may persist for several months after the clearance of HBsAg from patients with acute HBV infection. HBV DNA is also detectable in some patients with chronic HBV infection who are anti-HBc positive but HBsAg negative. Finally, in patients with chronic hepatitis B who become HBsAg negative following IFN-a treatment, HBV DNA may remain detectable by PCR for many months after the clearance of HBsAg. Thus, the PCR assay for HBV DNA appears to be more sensitive than HBsAg assays for detecting low-level viremia. The clinical significance of low levels of HBV DNA in HBsAg-negative patients is uncertain because HBV DNA eventually becomes undetectable in most of these patients.

HDV Nucleic acid hybridization assays have been developed for the detection of HDV RNA in the serum of patients with HDV infection (27, 129, 130). The hybridization assay for HDV RNA is a less invasive method for monitoring patients with chronic HDV infection than detection of HDAg in liver biopsy tissue and is more sensitive than any of the immunoassays for detection of HDAg in the serum, including the immunoblot assay. The hybridization assay for HDV RNA has been considered to be the most sensitive method for the detection of HDV viremia. However, a more sensitive reverse transcription PCR assay has been recently developed that detects HDV RNA in 93% of patients with chronic delta hepatitis, compared to only 76% for the hybridization assay (7, 128) .

HCV The most accurate method for diagnosing active HCV infection is the detection of viral RNA in serum by RT-PCR assay. Detection of HCV RNA by RT-PCR assay using primers derived from the highly conserved 5’ noncoding region is more accurate than either HCV antibody testing or serum aminotransferase testing. This test is reliable for detecting liver disease in humans, and infectivity studies with chimpanzees and retrospective studies with humans receiving infected blood products have established that HCV transmissibility is best predicted by RT-PCR positivity. RTPCR allows the detection of as few as 10 molecules of purified HCV RNA when performed optimally. This level of sensitivity is sufficient to detect HCV viremia in more than 99% of seropositive individuals with active viral hepatitis. In our experience, a negative RT-PCR assay is highly unusual in patients with histologic findings consistent with active hepatitis C. A negative RT-PCR assay in the absence of liver disease is consistent with resolved infection, although peri-

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odic follow-up (i.e., once or twice per year) seems prudent. It is important to recognize several potential technical problems associated with RT-PCR testing for HCV RNA in the clinical laboratory setting. Although exquisitely sensitive, the RT-PCR assay requires numerous precautions to prevent specimen contamination during the PCR process, which would lead to false-positive results. Both the routine use of negative controls and a proficiency-testing program are essential to ensure assay specificity. Because PCR contamination problems are typically sporadic in nature and often difficult to detect, blinded proficiency testing is essential for evaluating possible systematic problems. An equally important yet often overlooked problem with RT-PCR testing is that the genome of HCV is unstable under improper collection or assay conditions, which results in false-negative testing. Common modifications of PCR assays designed to prevent falsepositive testing may result in a net decrease in assay sensitivity. Because of these problems, RT-PCR assays should include both a low-copy and a high-copy HCV RNA-positive control serum in each run. Given these technical limitations of RT-PCR testing, combined with the clinical importance of documenting HCV viremia as previously described, it is currently recommended that RT-PCR testing only be performed in laboratories with experience and adequate documentation of proficiency in RT-PCR technology. Quantitative tests for measuring the levels of HCV RNA in serum, such as the bDNA assay (Chiron) and the Monitor assay (Roche), have recently been developed and are currently available as research assays. The measurement of HCV RNA levels may provide a direct assessment of the quantity of circulating virus in an infected individual and thus appears to be particularly useful for monitoring antiviral therapy. The second-generation bDNA assay (bDNA 2.0) is both precise and reproducible; however, the analytical sensitivity for detecting HCV RNA is approximately 2,500-fold less than that of the best RT-PCR assays. Therefore, the bDNA assay should not be used to “rule out” HCV viremia in the diagnostic setting. One successful approach to therapeutic monitoring for hepatitis C is to use the bDNA 2.0 test in combination with qualitative or semiquantitative RT-PCR on all bDNA-negative specimens. The Roche Monitor assay provides better sensitivity than the bDNA assay (approximately 1,000 HCV RNA copies per ml for the Roche assay versus 250,000 HCV RNA copies per ml for the bDNA assay). However, high assay variability, as well as a potential bias in underreporting HCV RNA levels with certain HCV genotypes, has been reported (124). Several studies have suggested relationships betureen HCV RNA levels and both clinical stage of

Hepatitis

Viruses

17

disease and response to IFN therapy; however, careful prospective longitudinal studies are needed before the exact relationship between HCV replication level, HCV genotype, and stage of liver disease can be defined. HEV HEV can be detected in acute-phase stools and acutephase sera by RT-PCR. After recovery of the HEV viral RNA from stool or sera, cDNA is synthesized from RNA, followed by amplification by PCR using sense and antisense primers. PCR primers for the RNA-dependent RNA polymerase (RDRP) region (114) or the capsid protein in the ORF2 structural gene (1.50) are most often used to amplify target cDNA. HEV PCR products are analyzed by agarose gel electrophoresis and confirmed by Southern blot analys is with sequence-specific ra .diolabeled or bioti .nylated sequence-specific probes. Because viremia 1s usually short and often peaks before clinical signs of disease, some acute-phase sera may not contain sufficient virus to be amplified. Failure to detect the HEV viral genome in some stool specimens has been attributed to the timing of the virus excretion and possible degradation of the virus in the stool. Precautions must be taken to store the samples at - 70°C and to minimize the freeze and thaw cycles of the specimens. Special procedures must be followed to separate preamplification and postamplification steps to minimize cross contamination. Although it is a sensitive procedure, the labor-intensive RT-PCR assay is not well suited for large-scale analysis. GBV-C Because of the failure thus far to identify and express an immunodominant epitope( s) useful for serological diagnosis of an ongoing GBV-C infection, three RTPCR assays were developed to detect GBV-C RNA in human biological specimens. The first assay utilizes oligonucleotide primers to amplify the NS3 helicase domain (74). This assay effectively detects GBV-C RNA from GBV-C-infected individuals. However, because the target of this assay is a coding region of the virus, silent mutations noted in the original GBV-C isolates (127) necessitated the use of degenerate primers. A second assay targeting highly conserved regions within the 5’-NTR of GBV-C was utilized (91). Both the NS3 and 5’-NTR RT-PCR assays rely on Southern hybridization (SH) for the detection of specific PCR product. PCR products are separated on a 2% agarose gel. Southern blots are performed with radiolabeled probes specific for the appropriate region. Typically, for a specimen to be considered positive for GBV-C RNA it is necessary that both primer sets generate a hybridizable product of the expected size. Specimens that are positive with only one of the primer sets are

18

Chernesky

et al.

retested. In most cases, positive results are also confirmed by sequence analysis. Because testing a large number of samples by these two assay ‘s is tedious, labor-intensive, and time-consum w, a thi .rd assay was developed utilizing an automated RT-PCR format with oligomer hybridization (OH) for the detection of sequences within the 5’-NTR. This system, RT-PCR/OH, is highly recommended for use by clinical laboratories and will soon be available for GBV-C RNA detection. It is a single-tube assay that only requires the addition of serum-derived nucleic acids to a prealiquoted reaction vial. Reverse transcription, PCR amplification, and oligomer hybridization occur in the same tube containing recombinant Thermus thermophilus polymerase, adamantane-labeled sense and antisense oligonucleotide primers for the Y-NTR of GBV-C, and carbazole-labeled OH probe. Detection of GBV-C product employs the automated LCx detection system (Abbott Laboratories) utilizing a microparticle enzyme immunoassay. Two studies have been performed comparing the automated GBV-C RT-PCR/OH with 5’-NTR and NS3 RT-PCR/SH assays. The first study evaluated 285 samples, 47 of which contained GBV-C sequences as determined by both 5’-NTR and NS3 assays. When tested in the RT-PCR/OH format, all 47 RT-PCR/SH-positive samples were successfully detected as positive (data not shown). Thus, this highly simplified assay appears to provide results identical to those obtained by the tedious and labor-intensive Southern analysis. In a second study, 371 samples were tested by 5’-NTR and NS3 RT-PCR/SH, as well as by RT-PCR/OH. GBV-C RNA was detected in 56 of these samples by all 3 assays. Five additional samples were found positive by the 5’-NTR assay and one additional sample by the NS3 assay. Interestingly, all six samples were found to contain GBV-C sequences by RT-PCR/OH. In this particular study, the RTPCR/OH assay appears to be superior to the RTPCR/SH assay. More impressively, the RT-PCR/OH assay format provided the data within 4 h, as opposed to the minimmm of 2 days required for the RTPCR/SH format.

PREVENTION

AND TREATMENT

HAV Pooled human immune serum globulin (ISG) has been used for a long time to provide protection against hepatitis A (76). ISG may prevent infection altogether or ameliorate the symptoms of hepatitis A in infected persons in which the net effect is permanent immunity after an infection. Early administration of ISG relative to exposure appears more likely to prevent infection completely and the proportion of asymptomatic infec-

CUMITECH

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tions may be markedly increased after the administration of ISG in epidemic settings. The isolation, growth, and serial passage of HAV in cell culture (106) led to the development of a live attenuated vaccine and a prototype, formalin-inactivated vacci .ne derived from virus propagated in cell culture (108). Further passage and adaptation of strain CR326F of HAV in MRC-5 cell cultures produced seed virus used to manufacture one of the current preparations of inactivated hepatitis A vactine. Both three-dose (reinjection after 1 or 2 months and after 6 months) and two-dose (reinjection after 6 months) regimens of the inactivated vaccine have been evaluated in healthy adults and children. The vaccine has been shown to be well tolerated and immunogenic (38,145). A single dose induces high titers of antibodies within 2 to 4 weeks. Geometric mean antibody levels increase about lo-fold with each additional injection. IgM antibody to hepatitis A can be detected shortly after the first or second injection. Extended clinical studies are under way with several vaccines. In the absence of adjuvant, purified HAV antigen seems to have slightly reduced immunogenicity. Vaccineinduced antibody titers remain detectable at a significant level over a period of at least 3 years, and specific immunity to HAV can be easily stimulated by a single booster injection. Recent efforts have combined HAV and HBV antigens into a killed vaccine, achieving elevated titers of antibody to both immunogens (39). The clinical success of candidate attenuated vaccines has been limited to date. Early trials of the Merck CR326 strain vaccine, during which virus at various cell culture passage levels was administered to human volunteers, suggested that immunogenicity and hepatovirulence were closely linked (108). Virus that had been passaged sufficiently to eliminate elevations in serum liver enzymes following parenteral administration was found to be poorly immunogenic and to induce relatively low levels of antibody. Antibody responses were significantly delayed following infection, raising substantial questions about the fate of the inoculated virus. Other possible approaches to vaccine development include the in vitro expression of viral antigens from recombinant DNA in prokaryotic or eukaryotic vectors, or the chemical synthesis of oligopeptides corresponding to immunoreactive epitopes on the virus, Recombinant or synthetic subunit vaccines should be expected to share many of the potential disadvantages of inactivated whole-virus vaccines, including the probable need for multiple immunizations and the limited duration of protection. HBV IFN-a is the only approved treatment for chronic hepatitis B. Although several other antiviral agents

CUM11

ECH 18A

have been evaluated for treating chronic hepatitis B, none have been proved effective. The results of preliminary studies suggested that approximately 30% of patients treated with IFN-(x become negative for HBeAg and HBV DNA. In a recent controlled study, treatment with IFN-(x at 5 million units per day for 16 weeks resulted in HBeAg seroconversion in 36% of patients and a sustained loss of HBV DNA by hybridization assay in 45% of patients with chronic hepatitis B (100). More than one-third of treated patients lost both HBeAg and HBV DNA and showed biochemical and histologic improvement. Furthermore, HBsAg disappeared from the serum in 10% of treated patients and in nearly one-third of those who responded. The strongest predictor of a response to treatment was the amount of HBV DNA in the serum. About 50% of patients with less than 100 pg of HBV DNA per ml responded to treatment. In contrast, only 7% of patients with more than 200 pg of HBV DNA per ml responded. In another recent study, 36% of patients with chronic hepatitis B responded to IFN-a treatment and 65% of those who remained negative for HBeAg and HBV DNA also became negative for HBsAg (66). The most promising new antiviral therapies for hepatitis B are the new nucleotide analogs lamivudine and famciclovir. In a preliminary trial of lamivudine in patients with chronic hepatitis B, 12 weeks of therapy were well tolerated and daily doses of 100 mg reduced HBV DNA to levels undetectable by hybridization assay, but HBV DNA reappeared in most patients after therapy was completed (3 1). In a preliminary trial of famciclovir in chronic HBV carriers, a lo-day course of oral famciclovir reduced HBV DNA levels by more than 90% (8 1). Lamivudine and famciclovir may become the preferred treatments for chronic hepatitis B if subsequent trials demonstrate that long-term therapy results in a sustained response rate similar to those found with IFN. The commercial HBV DNA hybridization assays previously mentioned are particularly useful for monitoring the effectiveness of antiviral therapy in infected individuals. Finally, liver transplantation may be offered to patients who develop liver failure as a result of severe acute or chronic hepatitis B. Unfortunately, HBV is frequently transmitted to the transplanted liver. The most effective means of preventing HBV infection is immunization of susceptible persons with hepatitis B vaccine. In the United States, it is recommended that all pregnant women be screened for HBsAg during routine visits for prenatal care. Infants born to mothers who are HBsAg positive should receive hepatitis B vaccine and hepatitis B immune globulin (HBIG) within 12 h of birth. Without prophylaxis against HBV infection, such infants are at high risk of becoming infected by perinatal transmission of HBV and those who become infected have a 90% risk of

Hepatitis

Viruses

19

chronic infection. Hepatitis B vaccine should also be given to all infants born to HBsAg-negative mothers during routine visits for well-child care as part of a comprehensive strategy to eliminate the transmission of HBV infection in the United States (16). Previous recommendations limiting hepatitis B vaccination to persons at high risk of HBV infection failed to reduce the incidence of hepatitis B. Adolescents and adults who are at high risk of HBV infection should also receive hepatitis B vaccine because they account for most HBV infections in the United States. Finally, persons who have percutaneous or permucosal exposure to HBsAg-positive blood, sexual exposure to an HBsAg-positive person, or household contact with a person who has acute or chronic HBV infection should receive appropriate postexposure prophylaxis, which usually includes hepatitis B vaccine alone or together with HBIG. HDV There is no completely satisfactory therapy for chronic delta hepatitis. Early studies indicated that a 3- to 4-month course of IFN-(x suppressed HDV replication and reduced liver disease in some patients, but almost all patients relapsed after therapy was discontinued. Subsequent studies with longer courses of IFN-a have shown more promising results (28, 118). Approximately one-half of patients with chronic delta hepatitis who were treated with 9 million units of IFN-oc three times a week for 48 weeks had a complete response to treatment. A positive response includes a return of serum alanine aminotransferase levels to normal, clearance of HDV RNA from the serum by hybridization assay, and histologic improvement on liver biopsy (34). Half of the patients who had a biochemical response to treatment still had normal alanine aminotransferase levels more than 4 years after discontinuation of therapy. However, none of the treated patients had a sustained clearance of HDV RNA from the serum. While IFN therapy may be helpful in many patients with chronic delta hepatitis, liver transplantation may be the only treatment option for patients with fulminant delta hepatitis or with advanced disease and decompensated cirrhosis. Because HDV can establish infection and cause disease only in individuals who are also infected with HBV, prevention of HBV infection is the most effective way to prevent HDV infection. Persons who are immune to HBV as the result of past infection or vaccination with the hepatitis B vaccine are also protected against HDV infection. Individuals at high risk for HDV infection who are susceptible to HBV infection should receive the hepatitis B vaccine to prevent coinfection with HBV and HDV. Postexposure prophylaxis following percutaneous or permucosal exposure to both HBV and HDV is the same as that for

20

Chernesky

et al.

exposure to HBV alone. Prevention of HDV superinfection in persons with chronic HBV infection is more difficult because it must depend on avoiding percutaneous or permucosal exposure to HDV. HCV IFN-oc is the only FDA-approved therapy for the management of chronic hepatitis C. Numerous studies have reported IFN to be effective in reducing the signs of hepatitis and in reducing or eliminating HCV viremia in 40 to 50% of patients, although it is apparent that at least half of the patients who initially respond to standard therapy will relapse after the discontinuation of therapy. Still, it is important to emphasize that up to 20% of patients show long-term responses to standard IFN therapy with some evidence of prolonged viral clearance. Innovative approaches to IFN dosing, and the use of newer antiviral preparations, promise to improve the therapeutic management of chronic hepatitis C. For example, it is now apparent that extending the duration of IFN therapy from 6 months to 12 months dramatically reduces patient relapse rates in those who showed an initial response (103). The FDA has recently approved the 12-month course of IFN as therapy for chronic hepatitis C, based on these data. One main advance in the treatment of HCV infection has been the ability to monitor the antiviral activity of drugs by direct virological testing (i.e., measurement of HCV RNA levels), providing a direct measurement of ongoing viral replication. Drugs that inhibit HCV replication dramatically reduce the absolute levels of HCV RNA in serum. Likewise, patients who fail to respond to therapy show persistence of HCV RNA. Several studies have demonstrated the use of quantitative nucleic acid assays to monitor antiviral activity during therapy for hepatitis C and to monitor sustained response versus relapse after therapy (47). Such assays should ultimately allow IFN treatment regimens to be tailored to individual patients. Other studies have shown that pretreatment HCV RNA levels are an important predictor of sustained response to IFN therapy in standard protocols; responders had significantly lower mean and median HCV RNA levels in serum before treatment than nonresponders (47, 156). Because HCV genotype may also influence response to IFN therapy, further study is needed before HCV RNA levels can be used to predict treatment response. The factors that influence the progression of chronic hepatitis C are poorly understood, and the long-term prognosis for individuals with mild hepatitis C is not known with certainty. Thus, it remains controversial whether patients with mild HCV infection (minimal histologic evidence of liver disease)

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should be treated with recombinant IFN. Earlier treatment with IFN may lead to better response rates, but cost and toxicity issues have been raised. There is presently no vaccine available for the prevention of HCV infection, and prophylactic immunoglobulin therapy has no proven efficacy for preventing primary infection among exposed individuals. HEV The control of hepatitis E in regions where HEV is endemic will result from clean water and improved health and sanitation at the local level, because the principal mechanism of transmission for HEV is by the fecal-oral route. For persons who travel to regions of endemicity, the control of hepatitis E will result from education on risk factors for hepatitis E. Individual protection through passive immunoprophylaxis is questionable. In a single study using an Indian source of immune serum globulin, the dose administered failed to prevent transmission of disease to case contacts (63). A vaccine for HEV could prevent large outbreaks in developing countries, reduce the morbidity and mortality of pregnant women, and provide pro tection to persons that travel to regions where HEV is endemic. Several reports on successful passive and active immunizations of cynomolgus monkeys against hepatitis provide hope for an efficacious human vaccine against hepatitis E. Passive immunization with high-titer, early-convalescent-phase monkey sera protected cynomolgus monkeys against hepatitis disease, but protection against infection was partial as shown by viral genomes detected in the serum and feces after challenge (141). Full protection against hepatitis E disease and complete protection against infection were achieved in monkeys immunized with the capsid protein expressed from ORF2 ( 141). After challenge with 1,000 CID,, (cynomolgus monkey 50% infectious doses) of a homologous wild-type strain of HEV, immunized animals failed to develop hepatitis or viremia and failed to shed virus in the stool. Complete protection was conferred after two doses of an alumadjuvant vaccine. In an independent study, the highly antigenic capsid protein protected animals from hepatitis after challenge with a heterologous HEV strain (42). Postchallenge, the levels of ALT liver enzymes did not rise, nor was there any histopathologic evidence for hepatocellular damage in any of the immunized animals. The -potential to develop an effective vaccine for hepatitis E capable of providing cross-protection for the most divergent HEV strains isolated and characterized to date has been established. The antigenic and immunogenic properties of the putative capsid protein of HEV show promise as a subunit vaccine candidate. A vaccine would be valuable to prevent hepa-

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WA

titis E epidemics, prevent sporadic cases of hepatitis E in developing countries, protect pregnant women at high risk, and provide protection to travelers. GBV-C At present, attempts to grow GBV-C in tissue culture or to infect convenient and available laboratory animals have been unsuccessful. For these reasons, the development of a live attenuated vaccine and ISG has been difficult to achieve. However, becausehigh titers of GBV-C E2 antibodies have been shown to be protective in humans, the possibility of using human or animal ISG for such a purpose is intriguing. Other possibilities, including the use of recombinant antigens produced in eukaryotic vectors, such as GBV-C E2 glycoprotein or synthetic subunit vaccines, should be explored, if in the future GBV-C is proven to be hazardous to the public. Several studies indicate that GBV-C is susceptible to IFN (45, 94, 149). These studies have shown that in most cases, GBV-C is sensitive to IFN while the patient is on treatment. However, most casesrelapse on cessation (withdrawal) of therapy. It is obvious that future studies should concentrate on many key variables for the establishment of an effective IFN therapy of GBV-C-infected individuals. These variables should include such parameters as duration, level of IFN treatment, viral load, and different GBV-C genotypes (92). REFERENCES 1. Aldershvile, J., G. G. Frosner, J. 0. Nielsen, F. Hardt, F. Dienhardt, I?. Skinhoj, and the Copenhagen Hepatitis Acuta Programme. 1980. Hepatitis B e antigen and antibody measured by radioimmunoassay in acute hepatitis B surface antigen-positive hepatitis. J. Infect. Dis. 141:293-298. 2. Alter, M. J. 1993. The detection, transmission, and outcome of hepatitis C virus infection. Infect. Agents Dis. 2:155-166. 3. Alter, H. J. 1996. The cloning tions of HGV and HGBV-C. 334:1536-1537.

and clinical implicaRT. Etigl. J. Med.

4. Anh-Tuan, N., and E. Novak. 198 1. Hepatitis B surface antigen circulating immune complexes (HBsAgCICs) in patients with hepatitis B and asymptomatic HBsAg carriers. Clin. Exp. Immunol. 43r246 -253. 5. Baker, B. L., A. M. DiBisceglie, S. Kaneko, R. Miller, S. M. Feinstone, J. G. Waggoner, and J. H. Hoofnagle. 1991. Determination of hepatitis B virus DNA in serum using the polymerase chain reaction: clinical significance and correlation with serological and biochemical markers. Hepatology 13:632-636. 6. Balayan, M. S., A. G. Andzhaparidze, S. S. Savinskaya, E. S. Ketiladze, D. M. Braginskys, A. I?. Savinov, and V. F. Poleschuk. 1983. Evidence for a virus

in non-A, non-B hepatitis transmitted oral route. Intervirology 20:23-3 1.

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