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The immuassay handbook parte88

  1. 1. 901© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. The term hepatitis means inflammation of the liver and is characterized by the presence of inflammatory cells in liver tissues. The onset of disease is generally insidious, leading to jaundice, fatigue, myalgia, anorexia, and mal- aise. Both viral and non-viral etiologies cause hepatitis. The two main causes of non-viral hepatitis are alcohol consumption and ingestion of toxins and/or drugs, while viral hepatitis is caused by a group of five known viruses designated hepatitis A–E. These viruses are responsible for most cases of acute viral hepatitis that are transmitted to human hosts enterically (hepatitis A and E) or percuta- neously via the blood-borne route (hepatitis B, C, and D). Table 1 lists the mode of transmission and characteristics of each virus type. It is well understood that chronic hepatitis caused by blood-borne viruses is associated with prolonged vire- mia, advanced liver disease, end-stage carcinoma, and excessive mortality. The growing viral hepatitis epi- demic presents a variety of clinical and laboratory chal- lenges, yet relatively recent developments in diagnostic testing and targeted therapies offer improved outcomes especially for hepatitis B and hepatitis C viral infections. Vaccines for hepatitis A (HAV) and hepatitis B (HBV) are available; however, a significant portion of the popu- lation remains unvaccinated and susceptible to infection, including the development of cirrhosis and hepatocellu- lar carcinoma. HBV vaccine is the first against a major form of cancer. There are no commercially available vaccines for hepatitis C (HCV), hepatitis D (HDV), and hepatitis E (HEV). Hepatitis A Virus Etiologic and Clinical Manifestations The hepatitis A virus (HAV) is a positive-sense, single- stranded 27nm non-enveloped RNA virus belonging to the family Picornaviridae. Despite genetic heterogeneity there is only one known human genotype. Evidence of HAV has been found in a variety of non-human primates including chimpanzees, macaques, langurs, gibbons, and orangutans. HAV causes a wide spectrum of infections in humans ranging from asymptomatic subacute illness with non-specific symptoms like vomiting, anorexia, dark urine, light stools, and jaundice to fulminant fatal hepati- tis. Most infections occur in early childhood. An appre- ciable proportion of adult infections are asymptomatic. The incubation period ranges from 14 to 60 days. The sero-prevalence of HAV is lowest in developed countries, especially in Scandinavia (13–17%) and highest in devel- oping countries. The virus is transmitted from person to person via fecal-oral route by ingestion of contaminated food and water. For this reason, HAV infection is often called infec- tious hepatitis. Following ingestion, the virus traverses through intestines into the blood stream, which transports it to the liver where it resides and multiplies. The virus is mainly excreted into feces. Excretion of virus in stools is accompanied by liver function abnormalities. High levels of excreted virus are detected before the onset of acute clinical symptoms and the appearance of serum IgM and IgG antibodies. Virus excretion may persist at detectable levels for weeks after the onset of symptoms. The presence of IgM anti-HAV antibodies indicates recent infection. These antibodies usually disappear within 6–12 months. The presence of IgG antibody in the blood means that the acute stage of the illness is past and the person is immune to further infection. IgG antibodies to HAV are also found in the blood following vaccination. Testing for immunity to the HAV virus relies on the detection of these antibod- ies in serum. Diagnosis and Assay Technology There are various viral and biochemical markers available for the diagnosis of suspected HAV hepatitis. Elevated levels of alanine aminotransferase (ALT, SGPT) and Hepatitis Ravi Kaul ( C H A P T E R 9.17 TABLE 1 Characteristics of Different Hepatitis Viruses Virus Type Classification Size (nm) Genome Genome size (kb) Enveloped Route of transmission Chronic Infection HAV Picornavirus 27–32 RNA 7.5 No Fecal-oral No HBV Hepadnavirus 42–50 DNA 3.2 Yes Blood borne (Percutaneous, Permucosal) Yes HCV Flaviviridae (hepacivirus) 55–60 RNA 9.4–9.6 Yes Blood borne (Percutaneous, Permucosal) Yes HDV Viroid like 35–37 RNA 1.7 Yes Blood borne (Percutaneous, Permucosal) Yes HEV Hepaviridae 27–34 RNA 7.5 No Fecal–oral No
  2. 2. 902 The Immunoassay Handbook aspartate aminotransferase (AST, SGOT) are present during the early stages of infection. These enzymes are released by the liver in response to viral damage. The three main markers of HAV infection are viral RNA, anti-HAV IgM, and anti-HAV IgG. They are measured by a variety of direct and indirect (competitive) solid- phase immunoassays and by reverse transcriptase poly- merase chain reaction (RT-PCR). HAV in fecal extracts was initially identified by electron microscopy, however, it is not easily demonstrated because many patients stop shedding virus by the time symptoms appear with the result the detection of HAV in stool or serum samples is unreliable. Amplification of viral RNA by RT-PCR is currently the most sensitive method for the detection of HAV RNA. The mean duration of HAV viremia is 30+/– 19 days. A virus-specific antibody of IgM class has proven to be very useful for the diagnosis of HAV infection dur- ing acute illness. It is mainly comprised of antibodies against capsid proteins. Anti-HAV IgM appears concur- rently with the onset of clinical symptoms in over 99% of individuals infected with HAV and persists during early convalescence as illustrated in Fig. 1. After the appear- ance of anti-HAV IgM, anti-HAV IgG begins to reach significant levels, associated with the recovery and per- sisting immunity. Resolved HAV infection is diagnosed by detection of IgG anti-HAV, however, commercially available assays detect total anti-HAV (both IgG and IgM antibodies). The presence of total anti-HAV in the absence of IgM anti-HAV can be used to differentiate past from the current infection. Detection of anti-HAV antibodies may also be used to differentiate HAV from other hepatitis viruses. Commercially available tests for total anti-HAV are based on the competitive assay for- mat. Samples containing a mixture of IgG and IgM anti- bodies are incubated with a complex of monoclonal HAV antibody and the antigen, immobilized on the solid sur- face using microtiter plates or microsphere beads. The available antibodies from patient sample bind to antigen immobilized on the solid surface (Fig. 2). After a wash step, the complex is incubated with chromophore or fluorophore-conjugated anti-HAV immunoglobulin probe. Anti-HAV antibodies from sample bound to the solid surface in the first step competitively inhibit the binding of conjugated HAV immunoglobulin probe. The labeled immunoglobulin is subsequently measured by an appropriate detection system. The signal intensity is inversely proportional to anti-HAV in the sample. This competitive procedure does not distinguish between the IgM and IgG class of immunoglobulins. Paired serum specimens are needed for diagnosis of HAV infection in order to demonstrate an increase in titer of anti-HAV by immunoassay. A number of methods have been used for the detec- tion of anti-HAV IgM, including radioimmunoassay (RIA), immunochemical staining, ELISA, immunoblot, dot blot immunogold filtration, and more recently che- miluminescence-based microparticle immunoassays. The immunocapture assay format used in the detection of anti-HAV IgM is shown in Fig. 3. The solid-phase anti-IgM is a polystyrene surface or microparticle coated with mu-chain-specific anti-human antibody. In step 1, if anti-HAV IgM is present in a patient’s serum, it is bound by the mu-chain-specific solid-phase antibody. In step 2, HAV antigen is added and becomes attached to it to form complex solid phase Ab-IgM-HAV. This complex is then detected in step 3 by incubating with probe anti-HAV antibody conjugate (Ab-HAV), a chro- mophore or fluorophore-labeled human anti-HAV IgG. Alternatively, Ab-IgM complex from step 1 can react directly to chromophore or fluorophore-labeled HAV antigen to form Ab-IgM-HAV. Irrespective, the result- ing signal is roughly proportional to the amount of IgM concentration in patient serum. The immunoassays for anti-HAV IgG and IgM are not only useful in the dif- ferential diagnosis of hepatitis, but are also useful for the measurement of anti-HAV IgG titer in blood, blood products and sera of vaccinated individuals. Detection of anti-HAV specific IgG and IgM antibodies is also useful for i) understanding the epidemiology of disease, ii) determining the immune status of individuals and iii) controlling HAV infection in institutions and groups with high risks of HAV transmission. FIGURE 1 Host immune response to HAV. (Source: CDC). FIGURE 2 Diagrammatic representation showing the detection of total anti-HAV antibodies (IgG and IgM) by competitive immunoassay. (The color version of this figure may be viewed at
  3. 3. 903CHAPTER 9.17 Hepatitis Further Reading Chernesky, M.A., Gretch, D., Mushahwar, I.K., Swenson, B.D. and Yarbough, P.O. Laboratory Diagnosis of Hepatitis Viruses, Cumitech Series, (ed Young, S.) (American Society for Microbiology, Washington, DC, 1998) Connor, B.A. Hepatitis A vaccine in the last minute traveler. Am. J. Med. 118 (Suppl 10A), 58S–62S (2005). Kwon, O.S., Byun, K.S., Yeon, J.E., Park, S.H., Kim, J.S., Kim, J.H., Bak, Y.T., Kim, J.H. and Lee, C.H. Detection of hepatitis A viral RNA in sera of patients with acute hepatitis A. J. Gastroenterol. Hepatol. 15, 1043–1047 (2000). of_hepatitis_A_viral_RNA_in_sera_of_patients_with_acute_hepatitis_A. Mackiewicz, V., Dussaix, E., Le Petitcorps, M.F. and Roque-Afonso, A.M. Detection of hepatitis A virus RNA in saliva. J. Clin. Microbiol. 42, 4329–4331 (2004). Nainan, O.V., Xia, G., Vaughan, G. and Margolis, H.S. Diagnosis of hepatitis A virus infection: A molecular approach. Clin. Microbiol. Rev. 19, 63–79 (2006). Stapleton, J.D. Host immune response to hepatitis A virus. J. Infect. Dis. 171 (Suppl 1), S9–S14 (1995). Hepatitis B Virus HBsAg, HBeAg, ANTI-HBs, ANTI-HBc, ANTI-HBc IgM, ANTI-HBe Etiologic and Clinical Manifestation Hepatitis B virus (HBV), originally referred to as Austra- lian Antigen, is a large 42–50nm enveloped spherical par- ticle known as the Dane particle. It contains circular, partially double-stranded DNA and DNA polymerase. The HBV DNA is approximately 1.6×105 Da in molecular weight and the circular molecule is single-stranded for 15–45% of its length. The HBV DNA polymerase converts the virion DNA into a fully double-stranded form with a size of approximately 2.1×106 Da, or 3200 base pairs. Dis- ruption of HBV by nonionic detergent in the presence of a reducing agent results in the removal of the outer glycopro- tein coat of the virion. The outer coat of HBV is the hepa- titis B surface antigen, HBsAg, a defective 22nm particle without nucleic acid that contains approximately 100 copies of HBsAg. The detergent treatment of HBV exposes the nucleocapsid, a 28nm spherical particle containing 180–240 copies of the hepatitis B core antigen. HBcAg is not found free in serum, but free core particles are observed in nuclei of infected hepatocytes. Another associated antigen called HBeAg that is expressed from pre-core genes occurs in soluble form in serum during HBsAg antigenemia. The outer coat of HBV consists of three envelope pro- teins designated large (L), medium (M), and small (S), which form homo- and hetero-dimers via disulfide bond- ing. They are expressed from three open reading frames (ORF) in the coding sequence and share the common C-terminal S region. The M protein contains an additional pre-S2 sequence and the L protein a further pre-S1 sequence. HBsAg is a complex lipid and glycoprotein par- ticle exhibiting a group specific determinant, “a”. Two major determinants, “d/y” and “w/r” are specified by amino acid positions 122 and 160. Other subtypes of envelope antigen containing the same antigenic determinant “a” differ in some amino acid positions in their sequences (Table 2). Table 3 lists the geographical distributions of eight dif- ferent HBV genotypes designated A–H that have been characterized based on the nucleotide sequence along with nine different serotypes. The genotypes have a distinct geographical distribution and are used in tracing the evo- lution and transmission of the virus. Antibodies to all three antigenic components of HBV are induced during the course of clinical and subclinical disease and recovery; antibody to HBsAg (anti-HBs), antibody to HBcAg (anti- HBc), and antibody to HBeAg (anti-HBe). Hepatitis B virus is a widespread cause of liver disease. It is estimated that there are more than 2000 million people alive today that have been infected with HBV at some time in their lives. Of these nearly 350 million remain infected chronically and become carriers of the virus. Only about FIGURE 3 Diagrammatic representation showing the detection of anti-HAV IgM antibodies by immunocapture assay. (The color version of this figure may be viewed at TABLE 2 Amino Acid Residues Specifying Determinants of HBsAg Position Amino Acid Specificity 122 Lys d Arg y 160 Lys w Arg r 127 Pro w1*/w2 Thr w3 Leu/Ile w4 *w1 reactivity also requires Arg122, Phe134 and/or Ala159
  4. 4. 904 The Immunoassay Handbook half of HBV-infected patients have clinical symptoms, which are similar to those of hepatitis A infection (see HEP- ATITIS A), however, the onset of symptoms is slower and it may take several months before patients with acute hepa- titis feel well again. About 20% of HBV-infected individu- als have jaundice. A significant proportion of infected patients (10–15%) go on to develop chronic hepatitis. These individuals may be asymptomatic although they can still transmit the disease to others. The chronic state may last for several years and can result in damage to the liver. HBV DNA persists in liver cells where it can subsequently induce hepatocarcinoma. Approximately one million people die from chronic hepatitis, cirrhosis, or primary liver cancer every year. HBV is found in the serum of patients during acute and chronic infection. It is transmit- ted by direct percutaneous inoculation of blood or blood products and also by physical contact with carriers of the virus, presumably by the passage of bodily fluids through cutaneous breaks or through oral and genital membranes. Table 4 shows the concentration of hepatitis B virus in various body fluids. Hemodialysis patients, residents of mental institutions, users of illegal drugs, homosexually active men, and prostitutes have an increased risk of con- tracting the disease. The medical staff are also at risk. All blood products derived from humans must be tested for HBsAg before use, including the serum, plasma, and blood proteins used in immunoassay calibrators and reagents. In countries with a high incidence of hepatitis B, and a correspondingly high incidence of liver cancer, national vaccination programs have been initiated. Hepatitis B was the first sexually-transmitted infection for which a protec- tive vaccine was proposed. Today, many vaccination cam- paigns are being implemented in emerging countries as well. Clinical Applications of HBV Marker Analytes Acute HBV infection There is a characteristic sequence in the appearance of HBV markers following infection. Viral DNA, HBsAg, and HBeAg become detectable first, with a progressive increase in concentration. In acute HBV infection, HBeAg declines before HBsAg, and then is replaced by anti-HBe. The appearance of HBeAg is indicative of high levels of viral replication and high infectivity while the appearance of anti-HBe is indicative of decreasing infectivity. As anti- HBe increases, HBsAg starts to decline. Anti-HBs appears months after the disappearance of HBsAg and remains detectable indefinitely. The appearance of anti-HBs with the absence of HBsAg indicates the resolution of infection and protective immunity. Anti-HBc also appears early on within one to two weeks after the appearance of HBsAg, and before detectable levels of anti-HBs. During the “win- dow period” when there is a gap of 16–32 weeks between the appearance of HBsAg and the appearance of anti-HBs, presence of anti-HBc may provide the only serological evi- dence of current or recent HBV infection (Fig. 4). All three antibodies remain in the blood for many years. Chronic HBV infection In chronic HBV infection, HBsAg and total anti-HBc anti- body remains in the blood for years without the presence of anti-HBc IgM antibody. Patients may also exhibit low levels of HBV DNA and anti-HBs antibody. This phase is also referred to as the replicative phase with periods of maximum infectivity and liver injury. In chronic HBV infection without seroconversion, anti-HBeAg is not detected and levels of HBeAg remain high, while in patients with late seroconversion anti-HBe is eventually detected preceded by a decline in HBeAg. In both types of chronic illness anti-HBc IgM is produced early on followed by anti- HBc IgG (Fig. 5). TABLE 3 Geographical Distribution of HBV Genotypes and HBsAg Serotypes Genotype Subtype Serotype Geographical Distribution A A1 adw2, aywl Africa, Asia A2 adw2, ayw2 Northern Europe, North America B B1 adw2 Japan B2 adw2, adw3 Rest of Asia B3 adw2, ayw1 Indonesia, China B4 adw1, adw2 Vietnam, Cambodia C C1 adrq+, ayr, adw2, ayw1 Far East C2 adrq+, ayr Far East C3 adrq−, adrq+ Pacific Islands D D1 ayw2, adw1, ayw1 Europe, Middle East, Egypt, India, Asia D2 ayw3, ayw1 Europe, Japan D3 ayw3, ayw2, ayw4 Europe, Asia, South Africa, USA D4 ayw2, ayw3 Australia, Japan, Papua New Guinea E ayw4, ayw2 Sub-Saharan Africa, UK, France F FIa adw4, ayw4 Central America FIb ayw4 Argentina, Japan, Venezuela, USA FII ayw4 Brazil, Venezuela, Nicaragua FIII ayw4 Venezuela, Panama, Columbia FIV ayw4 Argentina, Bolivia, France G adw2 USA, Germany, Japan, France H adw4 USA, Japan, Nicaragua (Source: CDC). TABLE 4 Concentration of Hepatitis B Virus in Various Body Fluids High Moderate Low/Not Detectable Blood Semen Urine Serum Vaginal fluid Feces Wound exudates Saliva Sweat Tears Breast milk
  5. 5. 905CHAPTER 9.17 Hepatitis HBsAg HBsAg is the most common hepatitis B test. The presence of HBsAg in serum indicates that the patient has con- tracted HBV infection. The test is used to identify those at risk of spreading the disease, e.g., blood donors, pregnant women, intravenous drug abusers, healthcare workers, institutionalized people, transplant donors and recipients, and donors of semen for artificial insemination. A quanti- tative HBsAg test can be used to monitor the treatment of chronically infected patients. HBsAg screening assays are normally supported by confirmatory tests, which are used to confirm repeatable reactive (positive) results. Typically the confirmatory test involves neutralization of the HBsAg in the sample by >50% using a human anti-HBs antibody. Anti-HBc and anti-HBc IgM Anti-HBc IgM is the first antibody to be detected following infection. There is a period of time immediately after acute infection when HBsAg and anti-HBs are both undetect- able, and anti-HBc or anti-HBc IgM kits may be used to monitor the course of the disease. Anti-HBc (total) kits measure IgM and IgG antibodies to the core protein. Missed occult HBsAg patients are frequently HBc positive. Anti-HBs and vaccination Anti-HBs testing is used to monitor the immune response of vaccinated individuals, usually one month after the final dose and then every few years, to check whether a booster dose is required. On assays standardized by the WHO ref- erence preparation, a level of 10mIU/mL is typically indicative of protective immunity. HBeAg and anti-HBe HBeAg is found during the early phase of HBV infection, soon after HBsAg is first detected. The presence of HBeAg correlates with an increased number of HBV particles and indicates that patients are at increased risk of transmitting the virus to their contacts. Persistence of HBeAg in HBV carriers is often associated with chronic active hepatitis. The presence of anti-HBe, following seroconversion, cor- responds to reduced levels of infectivity and replication. Diagnosis and Assay Technology HBsAg assay Direct solid-phase immunoassay for the detection of HBsAg is carried out by attaching anti-HBs antibodies to the surface of a solid support such as microparticles, polystyrene beads, test tubes, or microtiter wells. Immobilized antibody is then utilized in a one or two-step reaction for HBsAg detection in serum or other body fluids. The initial step involves incuba- tion of a specimen with the anti-HBs–coated solid phase. If sample contains HBsAg, then it is captured by anti-HBs anti- body on the solid phase. In the second step of the procedure a highly purified labeled probe, usually non-radioactive, using colorimetric, fluorometric, or luminescent signal generation systems based on enzyme labels, is used in an ‘inquiry’ reac- tion whereby labeled antibody will bind to the available free antigenic sites on captured antigen. Upon removal of the excess labeled antibody by thorough washing, the amount of bound labeled antibody is measured and it is roughly propor- tional to the amount of antigen in the original serum speci- men (Fig. 6). This direct solid-phase sandwich technique is generally applicable to all macromolecules and complex structures possessing two or more antibody binding sites. Similar assays have been constructed for HBeAg determina- tions. Numerous HBsAg variants exist across the world and more are being discovered. It is important that antibody(ies) bound to the solid surface and/or used as conjugate are able to detect all HBsAg variants. Most commercial kits utilize a mix- ture of antibodies to capture and identify all HBsAg variants. Confirmatory HBsAg assays The HBsAg confirmatory test uses the principle of antibody neutralization to confirm the presence of HBsAg in patient specimens. Although false-positive HBsAg results are infre- quent, confirmation utilizing human anti-HBs is manda- tory. This is accomplished by incubating the sample with human anti-HBs that binds to the antibody binding site on HBsAg. This prevents subsequent interaction of HBsAg with anti-HBs immobilized on the solid surface leading to signal loss when compared to non-treated sample. A sample is confirmed positive if the signal of the treated sample is 50% of the non-neutralized sample (Fig. 7). FIGURE 4 Typical clinical and laboratory features of acute infection progressing to chronic infection (Source: CDC). FIGURE 5 Typical laboratory features of chronic HBV infection. (Source: CDC).
  6. 6. 906 The Immunoassay Handbook Anti-HBs assay A direct solid-phase sandwich immunoassay for the detec- tion and quantification of anti-HBs antibodies is illustrated in Fig. 8. In this assay, the serum is incubated with native or highly purified recombinant HBsAg coated on the solid phase. If anti-HBs is present in the serum, it complexes with HBsAg immobilized on the solid surface. In the sec- ond step, labeled HBsAg probe is added to the reaction that reacts with antibodies. The amount of anti-HBs anti- bodies present in the serum is correlated to the recorded signal. Indirect (competitive) immunoassays for the detection of HBV antibodies The generic indirect competitive solid-phase immunoas- say for antibody detection is illustrated in Fig. 9. In this assay, labeled antibodies are utilized to assay for serum antibodies. The solid phase is coated with an appropriate antigen. If antibodies are present in the specimen during the first step of the reaction, they are captured by the solid- phase antigen. The labeled antibodies present in the sec- ond inquiry step of the reaction will have fewer antigen-binding sites for reaction, and the final signal of the assay is inversely proportional to the amount of anti- body in the sample. Total anti-HBc and anti-HBc (IgM) assay The solid-phase reagent in this assay uses recombinant HBcAg. The protocol for the detection of anti-HBc antibodies is similar to the detection of anti-HBs anti- bodies except it uses labeled HBc antigen as a detection probe. Some commercial assays use a competitive assay protocol with labeled anti-HBc IgG in a second step that competes with native antibodies present in patient sample. The degree of competition between native anti- bodies and labeled probe is an indication of the amount of anti-HBc. Detection of HBc IgM employs the immu- nocapture assay protocol as described previously for HAV assay. Anti-HBe assay In this assay the unknown sample is mixed with an equal volume of standardized HBeAg-positive serum containing a predetermined quantity of HBeAg, termed the neutral- izing reagent. The mixture is then incubated overnight at room temperature with a solid phase coated with high titered human anti-HBe. The solid phase is then incubated with labeled anti-HBe, and a significant ( 50%) reduction of detection signal indicates the presence of anti-HBe in the test sample. FIGURE 6 Immunoassay for the detection of HBsAg or HBeAg. (The color version of this figure may be viewed at FIGURE 7 HBsAg confirmatory test sequence. (The color version of this figure may be viewed at FIGURE 8 Detection of anti-HBs antibodies. (The color version of this figure may be viewed at
  7. 7. 907CHAPTER 9.17 Hepatitis Interpretation of HBV serologic markers Unlike many other viral diseases, HBV infection is charac- terized by several distinctive serologic and immunologic responses. The temporal profiles of HBV infection can serve as a useful guide for monitoring the course of disease and also provide a serologic correlation with the disease’s progress. Table 5 shows serological test interpretation for hepatitis B virus infection. With the advent of multiplex- ing technology it is possible that a battery of HBV markers will be tested simultaneously with high accuracy and effi- ciency from a single blood draw. The particular combina- tion of markers can be of prognostic value in assessing future clinical courses of the disease and level of infectivity. Recent studies have confirmed the importance of HBV serology for identifying blood bank donors with low levels of HBV DNA. These positive donors would otherwise go undetected by conventional NAT assay. Further Reading Dienstag, J.L. Acute viral hepatitis In: Harrison’s Principles of Internal Medicine, (eds Longo, D.L., Fauci, A.S., Kasper, D.L., Hauser, S.L., Jamerson, J.L., Loscalzo, J.) 18th edn, (New York, McGraw-Hill, Chapter 304 2012). Ganem, D. and Varmus, H.E. The molecular biology of hepatitis B virus. Ann. Rev. Biochem. 56, 651–693 (1987). Kuhns, M.C., Kleinman, S.H., McNamara, A.L., Rawal, B., Glynn, S. and Busch, M.P. Lack of correlation between HBsAg and HBV DNA levels in blood donors who test positive for HBsAg and anti-HBc: Implications for future HBV screening policy. Transfusion 44, 1332–1339 (2004). Short, J.M., Chen, S., Roseman, A.M., Butler, P.J. and Crowther, R.A. Structure of hepatitis B surface antigen from subviral tubes determined by electron cryomi- croscopy. J. Mol. Biol. 390, 135–141 (2009). Stramer, S.L., Zou, S., Notari, E.P., Foster, G.A., Krysztof, D.E., Musavi, F. and Dodd, R.Y. Blood donation screening for hepatitis B virus markers in the era of nucleic acid testing: Are all tests of value?. Transfusion 52, 440–446 (2012). Hepatitis C Virus Etiologic and Clinical Manifestations Hepatitis C virus (HCV) is a small, positive-sense, 55–65nm single-stranded enveloped RNA virus belong- ing to family Flaviviridae. The virus particle consists of RNA core surrounded by core protein encased in a lipid envelope comprising glycoproteins E1 and E2. The genome, 9600 nucleotides long, encodes a large polypep- tide of 3000 amino acids that is processed to produce smaller active proteins. The virus produces structural pro- teins including core protein, E1 and E2 along with non- structural (NS) proteins NS2a, NS2b, NS3, NS4a, NS4b, NS5a, and NS5b. The structural proteins are located at the 5′ end of the viral genome with NS proteins occupying the remainder of the genome in the order described above. Both core and NS proteins are used in the serological diagnosis of HCV. Based on genetic differences, HCV is divided into seven genotypes with several subtypes that FIGURE 9 Detection of anti-HBs antibodies by competitive immunoassay. (The color version of this figure may be viewed at TABLE 5 Typical Interpretation of Serological Test Results for HBV Infection Markers Anti-HBc IgM Anti-HBc Anti-HBs Status Depending on HBs Ag+ HBs Ag− Anti HBc Pos total and IgM + + − Acute Early Recovery Anti HBc Pos − + − Chronic Distantly immune and/or low level chronic infection. Anti HBs not detected, passive transfer to infants born to HBsAg positive mother Anti HBc Pos, anti HBc Pos − + + Chronic Immune due to vaccination Anti HBs Pos − − + Chronic Immune due to vaccination All Pos + + + Late acute, Recovering Recovering acute All Neg − − − Early acute Never infected (Source: CDC).
  8. 8. 908 The Immunoassay Handbook exhibit inter-group divergence of nearly 30%. Subtypes are further broken down into quasispecies or swarms of closely related but different viruses. Infection with one genotype does not confer immunity against others and concurrent infection with two strains is possible. Approxi- mately 60% of infected people worldwide belong to sub- types 1a and 1b. Pathogenesis HCV infection is a major public health problem and a leading cause of chronic liver disease. The worldwide prevalence of HCV infection is estimated to be approxi- mately 3%, corresponding to 170 million people. It is expected that the mortality associated with HCV infec- tion will increase in the near future. The incubation period for HCV varies widely from 2–26 weeks. Only few HCV patients can resolve their infection. Approxi- mately 75–85% of afflicted individuals with acute HCV infection will progress to chronic hepatitis, with 20–30% chronic carriers progressing to cirrhosis of the liver. There is no vaccine currently available for HCV infec- tion, however, bi-therapy with pegylated interferon and ribavirin, and tri-therapy with pegylated interferon, rib- avirin, and viral protease inhibitors are believed to stop the progression of disease during the early stages of liver fibrosis. Diagnosis and Assay Technology HCV is found in the serum of patients during acute and chronic infection. It is transmitted by direct percutaneous inoculation of blood or blood products and also by close physical contact with carriers of the virus, presumably by the passage of bodily fluids through cutaneous breaks or through oral and genital membranes. Table 6 shows risk factors for HCV infection. Patients with acute HCV infection are generally asymptomatic and only 20–30% exhibit jaundice. Serological tests like enzyme immunoas- say (EIA), ELISA, and chemiluminescence immunoassays that detect specific antibody to HCV (anti-HCV) are used for the detection of HCV infection. In addition, supple- mental tests such as the recombinant immunoblot assay (RIBA) have been developed to resolve false-positive test results associated with EIA. It is important to note that third generation anti-HCV tests are highly sensitive and specific, eliminating the need for confirmatory RIBA. The third generation serological assays use antigens from the HCV core, NS3, NS4, and NS5 gene products. While EIA assays use multiple HCV recombinant antigens to capture anti-HCV antibodies, the immunoblot assay uti- lizes recombinant HCV-encoded antigens as well as syn- thetic HCV-encoded peptides immobilized as individual bands onto test strips. Commercially available serology assays are not designed to distinguish between acute and chronic HCV infection. Coupled to these observations is the fact that anti-HCV antibodies are detected 4–10 weeks after initial HCV infection. However, with the availability of HCV antigen–antibody combination testing it is pos- sible to detect infection earlier than with the antibody stand alone test. It is recommended to follow up all posi- tive anti-HCV results with an HCV RNA test. This helps to rule out false-positive anti-HCV results in patients with persistently normal serum ALT and no risk factors as well as patients with anti-nuclear antibodies (Fig. 10). Molecular testing for HCV RNA includes both qualita- tive and quantitative analysis. Qualitative tests for HCV RNA can be used to confirm viremia and to screen blood donations while quantitative HCV RNA tests are used to guide therapy in the treatment of patients with chronic HCV infection (Table 7). With the availability of highly sensitive polymerase chain reaction (PCR) and transcrip- tion-mediated amplification (TMA) assays, there is no longer a need for qualitative assays. TABLE 6 Risk Factors for HCV Infection Risk Factors Injection drug use Transfusion and transplant from infected donor Receipt of blood from HCV positive donor Long term hemodialysis Accidental injuries with needles and sharps Sexual/household exposure to anti-HCV positive contact Multiple sex-partners or history of sexually-transmitted infections Child of an HCV positive woman Source: Albeldawi et al., 2010. FIGURE 10 Serological pattern of acute HCV infection with progression to chronic infection. (The color version of this figure may be viewed at (Source: CDC). TABLE 7 Interpretation of HCV Test Results Anti-HCV HCV RNA Interpretation Negative Negative No infection Positive Positive Acute or chronic infection Negative Positive Acute infection, chronic infection (in immunosuppressed patients) Positive Negative Resolved and/or treated infection Source: Albeldawi et al., 2010.
  9. 9. 909CHAPTER 9.17 Hepatitis Further Reading Albeldawi, M., Ruiz-Rodriguez, E. and Carey, W.D. Hepatitis C virus: Prevention, screening and interpretation of assays. Cleve. Clinic. J. Med. 77, 616–626 (2010). Alter, M.J., Mast, E.E., Moyer, L.A. and Margolis, H.S. Hepatitis C. Infect. Dis. Clin. North Am. 12, 13–26 (1998). Alter, M.J., Seeff, L.B., Bacon, B.R., Thomas, D.L., Rigsby, M.O. and DiBisceglie, A.M. Testing for hepatitis C virus infection should be routine for persons at increased risk for infection. Ann. Intern. Med. 141, 715–717 (2004). CDC Viral Hepatitis Surveillance United States, (2009). Statistics/2009Surveillance/PDFs/2009HepSurveillanceRpt.pdf. Accessed Feb 2012. Clarke, B. Molecular virology of hepatitis C virus. J. Gen. Virol. 78, 2397–2410 (1997). Couroucé, A-M. Development of screening and confirmation tests for antibodies to hepatitis C virus. In: Hepatitis C Virus (ed. Reesink, H.W.) pp. 64–75 (Karger, New York, NY, 1998). EASL clinical practice guidelines: Management of chronic hepatitis B virus infec- tion. J. Hepatol. 57, 167–185 (2012). Ghany, M.G., Strader, D.B., Thomas, D.L. and Seeff, L.B. Diagnosis, management and treatment of Hepatitis C: An update. AASLD Practice guidelines. Hepatology 49, 1335–1374 (2009). Lambert, N., Value of HCV antigen-antibody combined HCV assays in hepatitis C diagnosis. In: Advances in Transfusion Safety Vol IV, (eds Dax, E.M., Famigia, A., Vyas, G.) 113–121 (Development Biology (Basel), Kerger 127, 2007). Lee, J.A., Payette, M. and Osiecki, J. Viral hepatitis: targeted tests and therapies contribute to improved outcomes. Med. Lab. Obs. 44, 18–20 (2012). Nakano, T., Lau, G.M., Lau, G.M., Sugiyama, M. and Mizokami, M. An updated analysis of hepatitis C virus genotypes and subtypes based on the complete coding region. Liver Int. 32, 339–345 (2012). Strader, D.B., Wright, T., Thomas, D.L. and Seeff, L.B. American Association for the study of liver diseases: Diagnosis, management, and treatment of hepatitis C. Hepatology 39, 1147–1171 (2004). Hepatitis D Virus Etiologic and Clinical Manifestation Hepatitis D virus (HDV) is a small 36 nm single-stranded negative sense RNA virus that requires the presence of hepatitis B virus (HBV) for its assembly and replication. Co-infection and super-infection represent two types of HDV known infections. During co-infection, the patient acquires HDV and HBV at the same time while super-infection occurs when a patient with chronic HBV infection becomes infected with HDV. Hepatitis D virion is composed of an outer lipoprotein envelope made of the surface antigen of the HBV (HBsAg) and an inner ribonucleoprotein structure in which the HDV genome resides. HDV produces one protein with two forms; a 27 kDa large-HDAg (delta-Ag-L), and a small- HDAg of 24 kDa (delta-Ag-S). The N-terminals of the two forms are identical; they differ by an additional 19 amino acids in the C-terminal of the large HDAg. These two proteins play diverging roles during the course of an infection. HDAg-S is produced in the early stages of an infection and enters the nucleus supporting viral replication. HDAg-L, in contrast, is produced dur- ing the later stages of an infection, acts as an inhibitor of viral replication, and is required for the assembly of viral particles. Eight different genotypes of HDV have been identified, each with different geographic distribution and distinct clinical course. Genotype I shows wide geo- graphic distribution including Europe and the United States. Pathogenesis The symptoms of a co-infected HBV–HDV patient are similar to those of HBV alone but can be more severe than most single viral hepatitis infections. The course of HDV infection depends primarily on the presence of acute or chronic HBV infection. Simultaneous co-infection with HBV and HDV generally leads to an acute self-limited episode with transient HBs- and HD-antigenemia and eventual recovery. However, the liver damage of each agent is likely to be additive. It has been reported that ful- minant hepatitis occurs more frequently in patients with either acute delta co-infection with HBV, or acute delta super-infection with HBV, than in patients with acute HBV infection alone. The mortality rate for acute delta hepatitis ranges from 2 to 20% compared to less than 1% for acute HBV infection. Infection of chronic HBV carri- ers with HDV super-infection often leads to severe chronic disease with HDV cirrhosis in about 75% of patients com- pared to 15–30% of patients with chronic HBV infection alone. Diagnosis and Assay Technology The modes of HDV transmission are similar to those for HBV. HDV infection is most common among injecting drug abusers, hemophiliacs, and recipients of multiple blood transfusions and is relatively uncommon in men who have sex with men. Perinatal HDV acquisition is rare. The molecular and serologic markers of HDV infec- tion are serum HDV RNA, serum and liver HDAg, serum anti-HD IgM, and serum anti-HD IgG. Immunofluores- cence, immunoperoxidase, and ELISA methods have been used for the localization of HDAg in serum and liver tissue. While serum anti-HD IgG tests are commer- cially available in the United States, HDV RNA and anti- HD IgM tests are available in research and reference laboratories only. Anti-HDV IgG antibodies are detected by RIA or EIA kits. EIA kits utilize a competitive assay format with non-labeled anti-delta antibodies from patient serum competing with a constant amount of human anti-delta labeled IgG for a limited number of HDAg-binding sites. Thus, the proportion of labeled anti-HD bound to the solid phase is inversely propor- tional to the concentration of anti-delta in the test speci- men. Anti-HD IgM is carried out in a similar fashion to the anti-HAV IgM test described under the HAV section. In acute co-infection with HBV and HDV, the following sequential markers appear in the serum: HBsAg, HDV RNA, HDAg, anti-HD IgM, and anti-HD IgG. Both antibodies are generally of low concentration and of short duration. In super-infected patients, there is a persistence of HBs- and HD-antigenemia and HDV RNA in the serum and liver of infected individuals. Generally, anti-HD IgM is short-lived, while anti-HD IgG is present at higher titer. Figures 11 and 12 show typical serological courses during HBV–HDV co-infection and super-infection. It is believed that anti-HD IgM is elevated in response to HDV-induced damage and thus represents a valid surro- gate marker of liver damage which is immunopathologi- cally related to HDV infection. Thus, besides providing diagnostic information, serum anti-HD IgM is the best predictor of impending resolution of chronic HDV
  10. 10. 910 The Immunoassay Handbook disease, whether spontaneous or interferon-induced. While there is no HDV-specific vaccine the most useful tool for preventing HBV–HDV co-infection is the immu- nization with Hepatitis B vaccine since HDV is depen- dent on HBV for replication. Further Reading Bichko, V., Netter, H.J., Wu, T.T. and Taylor, J. Pathogenesis associated with replication of hepatitis delta virus. Infect. Agents. Dis. 3, 94–97 (1994). Birkenmeyer, L.G. and Mushahwar, I.K. Detection of hepatitis A, B, and D virus by the polymerase chain reaction. J. Virol. Methods 49, 101–112 (1994). Borghesio, E., Rosina, F., Smedile, A., Lagget, M., Niro, M.G., Marinucci, G. and Rizzetto, M. Serum immunoglobulin M antibody to hepatitis D as a surrogate marker of hepatitis D in interferon-treated patients and in patients who under- went liver transplantation. Hepatology 27, 873–876 (1998). Hadziyannis, S.J. Review: Hepatitis delta. J. Gastroenterol. Hepatol. 12, 289–298 (1997). Ji, J., Sundquist, K. and Sundquist, J. A population based study of hepatitis D virus as potential risk factor for hepatocellular carcinoma. J. Natl. Cancer Inst. 104, 790–792 (2012). Negro, F. and Rizzetto, M. Diagnosis of hepatitis delta virus infection. J. Hepatol. 22, 136–139 (1995). Niro, G.A. and Smedile, A. Current concept in the pathophysiology of hepatitis delta infection. Curr. Infect Dis. Rep. 14, 9–14 (2012). Polish, L.B., Gallagher, M., Fields, H.A. and Hadler, S.C. Delta hepatitis: Molecular biology and clinical and epidemiological features. Clin. Microbiol. Rev. 6, 211–229 (1993). Hepatitis E Virus Etiologic and Clinical Manifestation Hepatitis E virus (HEV) is the second major cause of epi- demic hepatitis and acute sporadic hepatitis in adults in many developing countries. The virus was isolated, cloned, and fully sequenced in the early 1990s. HEV is a 32nm non- enveloped single-stranded RNA virus. It belongs to the fam- ily Calciviridae and genus Calcivirus. It is largely a waterborne epidemic disease. Animals, especially swine, may represent an important reservoir for HEV. A number of HEV-like sequences have been isolated from swine worldwide. The viral genome consists of a positive-sense, single-stranded, poly-adenylated RNA molecule of approximately 7.6kb. It possesses 3 discontinuous open reading frames (ORF) that encode polypeptides of 1693 amino acids (ORF 1), 660 amino acids (ORF 2), and 123 amino acids (ORF 3) respec- tively. The genome is organized with the structural genes located at the 3′ end and the non-structural genes at the 5′ end. Analysis of the nucleotide sequence reveals that ORF 1 contains several conserved motifs such as an RNA helicase, a methyl transferase, a papain-like proteinase, and an RNA- dependent RNA polymerase. ORF 2 encodes for the capsid protein, while the function of ORF 3 is unknown. Both ORF 2 and ORF 3 proteins, however, are highly antigenic. Five different genotypes of HEV have been described with geno- types 1 and 2 identified in humans, types 3 and 4 from humans and swine and type 5 from chicken. Genotypes 1 and 2 occur in a younger population, while genotypes 3 and 4 occur in older age groups that may be HIV positive. Pathogenesis HEV infection causes symptoms of a self-limiting, acute, icteric disease. Chronic liver disease or persistent viremia has not been observed. Among HEV-infected individuals, the most common complaints are fatigue accompanied by gastrointestinal disorders such as anorexia, nausea, vomit- ing, and abdominal discomfort. Diarrhea, fever, and epi- staxis (bleeding from the nose) are less common symptoms. Most patients become jaundiced. The incubation period ranges between 2 and 9 weeks. Some patients progress to fulminant hepatitis with a 1–3% mortality rate, especially among pregnant women (15–20%) who acquire HEV infection during their third trimester. The clinical mani- festations of HAV and HEV are practically indistinguish- able. The main points of difference for HEV are: the incubation period is longer, cholestasis is more predomi- nant, and the mortality rate during pregnancy is high, especially if HEV is acquired during the late pregnancy period. The highest attack rate is observed in young to middle-aged adults. The occurrence of subclinical cases in younger individuals is suspected, but not yet documented. There is currently no vaccine or immune globulin available to prevent the transmission of HEV. Epidemiology Hepatitis E virus has a worldwide distribution. An esti- mated 20 million cases of HEV are reported each year resulting in 70,000 deaths and 3000 still births. Disease Jaundice Symptoms ALT Total anti-HDV IgM anti-HDV HDV RNA HBsAg Post exposure time Titer FIGURE 12 . Typical serological course during HBV-HDV super-infection. (Source: CDC). FIGURE 11 Typical serological course during HBV-HDV co-infection. (Source: CDC).
  11. 11. 911CHAPTER 9.17 Hepatitis incidence is highest during and after the rainy season in Southeast Asia and during late autumn in Central Asia. Surveys have shown that the sporadic form of HEV is also spread globally and that outbreaks and clusters of cases can occur in any situation where drinking water may be fecally contaminated. In industrialized countries, HEV infection occurs occasionally as imported sporadic cases, although a novel closely related virus, designated swine hepatitis E virus (swine HEV), was identified in pig herds from the Midwestern United States as well as from Asian countries. Most infections occur in early childhood. A variable pro- portion of adult infections are asymptomatic. Hepatitis A and E may coexist in some patients. Diagnosis and Assay Technology There are no FDA-approved serological tests available for commercial use in the United States; however, several reliable tests are available for research and clinical use in specialty and reference laboratories. Generally, the intact virion (HEV) is excreted in feces during the incubation period prior to the onset of symptoms. Analysis of fecal antigen by RT-PCR is more practical than by immune electron microscopy (IEM). However, collection of fecal material prior to knowledge of the onset of symptoms or exposure is not commonly applicable to clinical practice. The availability of highly purified recombinant HEV antigens and synthetic peptides enables scientists to develop sensitive and specific ELISAs for the detection of both IgM and IgG-class antibodies to HEV. An IgM test is marketed in Asia. This test uses recombinant HEV anti- gens derived from the carboxyl terminus of the capsid protein, ORF 2, and ORF 3. In addition, several research laboratories have developed IgM tests based on alterna- tive recombinant HEV (rHEV) antigens expressed in bac- teria or the baculovirus expression system. The presence of anti-HEV IgM antibody in the serum denotes acute infection although IgM can remain positive for more than 1year. IgG antibody peaks soon after IgM antibody and remains detectable for as long as 20 months. Analysis of serum specimens collected from HEV-infected patients during the incubation period, acute phase, convalescence, and recovery phase of the disease have shown that the order of appearance in serum is HEV RNA, anti-HEV IgM, and anti-HEV IgG. While serum HEV RNA and anti-HEV IgM appeared at the peak of ALT elevation, the two markers disappeared after 10 and 20 days respec- tively. Generally, the IgG response is much stronger in individuals with a recent HEV infection. Analysis of sequential samples taken from experimentally-infected rhesus monkeys up to 86 weeks has confirmed this sero- logical pattern. Further Reading Balayan, M.S. Epidemiology of hepatitis E virus infection. J. Viral. Hepat. 4, 155–165 (1997). Dalton, H.R., Bendall, R., Ijaz, S. and Banks, M. Hepatitis E: An emerging infec- tion in developed countries. Lancet Infect. Dis. 8, 698–709 (2008). Meng, X.J., Purcell, R.H., Halbur, P.G., Lehman, J.R., Webb, D.M., Tsareva, T.S., Haynes, J.S., Thacker, B.J. and Emerson, S.U. A novel virus in swine is closely related to the human hepatitis E virus. Proc. Natl. Acad. Sci. (USA) 94, 9860–9865 (1997). Mushahwar, I.K., Dawson, G.J. and Reyes, G.R. Hepatitis E virus: Molecular biol- ogy and diagnosis. Europ. J. Gastroenterol. Hepatol. 8, 312–318 (1996). Schlauder, G.G., Dawson, G.J., Erker, J.C., Kwo, P.Y., Knigge, M.F., Smalley, D.L., Rosenblatt, J.E., Desai, S.M. and Mushahwar, I.K. The sequence and phyloge- netic analysis of a novel hepatitis E virus isolated from a patient with aute hepatitis reported in the United States. J. Gen. Virol 79, 447–456 (1998). Wedemeyer, H., Pischke, S. and Manns, M.P. Pathogenesis and treatment of hepatitis E virus infection. Gastroenterology 142, 1388–1397 (2012). Tam, A.W., Smith, M.M., Guerra, M.E., Huang, C.C., Bradley, D.W., Fry, K.E. and Reyes, G.R. Hepatitis E virus (HEV): Molecular cloning and sequencing of the full-length viral genome. Virology 185, 120–131 (1991). Wierzba, TF, Panzner, U: Report on the international symposium on hepatitis E, Seoul, South Korea, 2010, Emerg. Infect. Dis. [serial on the internet], May 2012. Zhang, J., Ge, S.X., Huang, G.Y., Li, S.W., He, Z.Q., Wang, Y.B., Zheng, Y.j., Gu, Y., Ng, M.H. and Xia, N.S. Evaluation of antibody-based and nucleic acid-based assays for diagnosis of hepatitis E virus infection in a rhesus monkey model. J. Med. Virol. 71, 518–526 (2003).