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KEY TERMS Anti-HBe Anti-HBs Cytomegalovirus (CMV) Epstein-Barr virus (EBV) Hepatitis Hepatitis A virus (HAV) Hepatitis B surface antigen (HBsAg) Hepatitis B virus (HBV) Hepatitis Be antigen (HBeAg) Hepatitis C virus (HCV) Hepatitis D virus (HDV) Hepatitis E virus (HEV) Heterophile antibodies Human T-cell lymphotropic virus type I (HTLV-I) Human T-cell lymphotropic virus type II (HTLV-II) IgM anti-HBc Mumps virus Parenteral Rubella virus Rubeola virus Varicella-zoster virus (VZV) Viruses are submicroscopic pathogens whose size is measured in nanometers. Their basic structure consists of a core of DNA or RNA packaged into a protein coat or capsid. In some viruses, the capsid is surrounded by an outer envelope of glycolipids and proteins derived from the host-cell membrane (Fig. 23–1). It is remarkable that these tiny particles are capable of causing severe, and sometimes lethal, disease in humans, ranging from childhood infections to inflammatory diseases with a predilection for a specific organ, disseminated disease in immunocompromised patients, cancer, and congenital abnormalities. Viruses are obligate intracellular pathogens that rely on the host cell for their replication and survival. They infect their host cells by attaching to specific receptors on the cell surface; penetrating the host cell membrane; and releasing their nucleic acid, which then directs the host cell’s machinery to produce more viral nucleic acid and proteins. These components assemble to form intact viruses that are released by lysis of the cell or by budding off the cell’s surface (Fig. 23–2). Replication can occur quickly in cytolytic viruses that produce acute infections, or slowly in viruses that result in chronic infections. The free virions that are generated can then infect neighboring host cells and begin new replication cycles that promote dissemination of the infection. Thus, viruses can be present in the host as both freely circulating particles and intracellular particles. This chapter briefly addresses the immunologic mechanisms required to attack the virus in its different states. Successful defense against viral infections requires a coordinated effort among innate, humoral, and cell-mediated immune responses (Fig. 23–3). The remainder of the chapter discusses some of the most important viral infections detected by serology and molecular methods. These include the hepatitis viruses, herpes viruses, measles, mumps, rubella, and human T-cell lymphotropic viruses. Laboratory tests for the HIV virus are discussed separately in Chapter 24. FIGURE 23-1 Basic structure of a virus. FIGURE 23-2 Basic steps of a virus life cycle. (1) Attachment of the virus to a receptor on the host cell surface. (2) Penetration, or entry of the virus into the host cell through endocytosis or other mechanisms. (3) Uncoating, or degradation of the viral capsid and subsequent release of viral nucleic acid. With some viruses, the nucleic acid integrates into the host-cell genome. (4) Transcription to produce additional viral nucleic acid. (5) Translation of viral nucleic acid to produce viral proteins. (6) Assembly of the viral components to produce intact virions. (7) Budding off the host-cell membrane or host-cell lysis results in (8) release of viral progeny. Modifications of these steps can occur with different viruses. FIGURE 23-3 Innate defenses provide the initial barrier to viral infection. Infected cells release interferons (IFNs) α and β, which (A) inhibit viral replication in surrounding cells and (B) stimulate natural killer (NK) cells. Both NK and cytotoxic T (Tc) cells destroy virus-infected host cells (C, D), resulting in the release of free virions (E). Virus-specific B cells recognize these free virions (F), as well as virions that have penetrated the epithelium (G), leading to the production of antibodies (H) that bind free virions and mediate virus neutralization, opsonization, complement activation, and antibody-dependent cellular cytotoxicity (ADCC). Immune Defenses Against Viral Infections Innate immunity provides the first line of protection against viral pathogens. Viruses first encounter naturally occurring barriers in the body, such as the skin and mucous membranes. If they are able to invade these barriers, other innate defenses are activated when cells of the innate immune system recognize pathogen-associated molecular patterns (PAMPs) on the surface of, or within, virus-infected host cells. Two important nonspecific defenses against viruses involve type I interferons (IFNs) and natural killer (NK) cells. Virus-infected cells are stimulated to produce IFN-α and IFN-β following recognition of viral RNA by Toll-like receptors (TLRs). IFNs inhibit viral replication by inducing the transcription of several genes that code for proteins with antiviral activity —for example, a ribonuclease enzyme that degrades viral RNA. IFN-α and IFN-β also enhance the activity of NK cells, which bind to virus-infected cells and release cytotoxic proteins such as perforin and granzymes, causing the cells to die and release the viruses (see Fig. 2–7). These cell-free virions are now accessible to antibody molecules. Connections Interferons IFNs “interfere” with the ability of viruses to replicate by stimulating infected host cells to produce proteins that degrade viral nucleic acid and proteins and induce an anti-viral state (see Chapter 6). IFNs exert their effects not only on the original infected cell but also on neighboring uninfected host cells. When innate defenses are insufficient in preventing viral infection, specific humoral and cell-mediated defenses are activated. Virus-specific antibodies are produced by B cells and plasma cells and can attack free virus particles in several ways. Antibodies play a key role in preventing the spread of a viral infection through neutralization. This process involves the production of antibodies that are specific for a component of the virus that binds to a receptor on the host-cell membrane. When these neutralizing antibodies bind to the virus, they prevent it from attaching to and penetrating the host cell. Secretory immunoglobulin A (IgA) antibodies play an especially important role in this process because they neutralize viruses in the mucosal surfaces (e.g., respiratory and digestive tracts), which often serve as entryways for the pathogens. Meanwhile, immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies can bind to viruses in the bloodstream and inhibit dissemination of the infection. In addition, IgG antibodies promote phagocytosis of viruses through their opsonizing activity and promote destruction of viruses through antibody-dependent cellular cytotoxicity (ADCC). IgG and IgM antibodies also activate complement, which can mediate opsonization via C3b or lyse enveloped viruses by inducing formation of the membrane attack complex. IgM antibodies may also inactivate viral particles by agglutinating them. Although antibodies can attack viruses in many different ways, they cannot reach viruses that have already penetrated host cells. Elimination of intracellular viruses requires the action of cell-mediated immunity. Type 1 helper (Th1) cells and cytotoxic T lymphocytes (CTLs) play a key role in this mechanism of defense. Th1 cells produce IFN-g, which induces an antiviral state within the virus-infected cells, and interleukin-2 (IL-2), which assists in the development of effector CTLs. In this process, CD8+ CTLs become programmed to expand in number and attack the virus-infected cells. To recognize the virus-infected host cell, the T-cell receptor (TCR) on the CTL must bind to a viral antigen complexed with class I major histocompatibility complex (MHC) on the surface of the infected cell (see Fig. 4-10 and Fig. 23–3). CD8 is a co-receptor in this interaction. These molecular interactions stimulate the granules in the CTL to release a pore-forming protein called perforin, which produces pores in the membrane of the infected host cell, and proteases called granzymes, which enter the pores. These enzymes activate apoptosis in the host cell, interrupting the viral-replication cycle and resulting in release of assembled infectious virions. The free virions can then be bound by antibodies. The CTL response is powerful and involves a series of cell divisions that can produce up to 50,000 times the original number of cells in a period of 1 to 3 weeks. Viral Escape Mechanisms Viruses can escape the host’s defense mechanisms in several ways. First, viruses are rapidly dividing agents that undergo frequent genetic mutations. These mutations result in the production of new viral antigens, which are not recognized by the initial immune response to the virus. For example, continual antigenic variation in the influenza virus results in the emergence of novel infectious strains that require the development of new vaccines every year to protect the population. Antigenic variation is also seen in other viruses, including rhinoviruses, which cause the common cold, and HIV, which causes AIDS. Second, some viruses can escape the action of components of the innate immune system such as IFNs, complement proteins, or the lysosomal enzymes in phagocytic cells. For example, the hepatitis C virus (HCV) can block IFN-mediated degradation of viral RNA, and herpes simplex viruses (HSV) produce a protein that binds to the complement component C3b, resulting in inhibition of the complement pathways. Third, viruses can evade the host’s defense by suppressing the adaptive immune system. Some viruses, such as the cytomegalovirus (CMV) and HIV, do this by reducing the expression of class I MHC molecules on the surface of virus-infected cells, making them less likely to be recognized by CTLs. Other viruses, such as rubeola, can cause decreased expression of class II MHC molecules, resulting in reduced Th cell activity. Some viruses can alter the function of certain cells of the immune system after directly infecting them. For example, the Epstein- Barr virus (EBV) causes polyclonal activation in B lymphocytes, whereas HIV suppresses the function of CD4 Th cells. EBV can also inhibit immune responses by producing a protein that can suppress Th1 cells because of its similarity to interleukin-10 (IL-10). Finally, some viruses, such as CMV, varicella-zoster virus (VZV), and HIV, can remain in a latent state by integrating their nucleic acid into the genome of the infected host cells. In this situation, the virus is only stimulated to replicate again if the host is exposed to other infectious agents or if the host’s immune defenses decline. Latent viruses can remain silent within host cells for years because they are hidden from the immune system, although reactivation can occur later in life. By using these evasion mechanisms, viruses have established themselves as successful human pathogens that can cause a range of mild to life-threatening diseases. Rapid, reliable laboratory detection of these pathogens is essential for early patient diagnosis and treatment. Laboratory identification also leads to prompt implementation of measures to prevent further spread of the virus to other members of the population. Laboratory Testing for Viral Infections As our knowledge of viruses has increased, so has the development of laboratory assays to detect viral infections. Serological and molecular tests can be easily and rapidly performed by the clinical laboratory. Therefore, they play an essential role in helping physicians establish a presumptive diagnosis so that treatment can be initiated promptly. Serological tests are also important in monitoring the course of infection, detecting past infections, and assessing immune status, whereas molecular tests have enhanced our ability to detect active infection and are essential in guiding antiviral therapy. In general, the presence of virus-specific IgM antibodies in patient serum indicates a current or recent viral infection, whereas IgG antibodies to a virus signify either a current or past infection and, in many cases, immunity. Virus-specific IgM antibody in the newborn’s serum indicates a congenital infection because IgM is actively made during fetal life. In contrast, IgG antibodies in the infant’s serum are mainly maternal antibodies that have crossed the placenta. Current infections in the adult or newborn may also be detected by immunoassays for viral antigens in serum or other clinical samples or by the presence of viral nucleic acids that can be detected by molecular methods. Hepatitis Viruses Hepatitis is a general term that means inflammation of the liver. It can be caused by several viruses and by noninfectious agents, including ionizing radiation, chemicals, and autoimmune processes. The primary hepatitis viruses affect mainly the liver. Other viruses, such as CMV, EBV, and HSV, can also produce liver inflammation, but it is secondary to other disease processes. This section will focus on the primary hepatitis viruses. The hepatitis A virus (HAV) and the hepatitis E virus (HEV) are transmitted primarily by the fecal-oral route, whereas the hepatitis B virus (HBV), the hepatitis D virus (HDV), and the hepatitis C virus (HCV) are transmitted mainly by the parenteral route (i.e., through contact with blood and other body fluids). All of the hepatitis viruses may produce similar clinical manifestations. The early, or acute, stages of hepatitis are characterized by general flu-like symptoms and mild to moderate pain in the right upper quadrant (RUQ) of the abdomen. Progression of the disease leads to liver enlargement (hepatomegaly) and tenderness, jaundice, dark urine, and light feces. Initial laboratory findings typically include elevations in bilirubin and in the liver enzymes, most notably alanine aminotransferase (ALT). These findings are nonspecific indicators of liver inflammation and must be followed by specific serological or molecular tests to identify the cause of hepatitis more definitively. The specific laboratory tests used to detect each type of hepatitis are listed in Table 23–1. Hepatitis A HAV is a nonenveloped, single-stranded ribonucleic acid (RNA) virus that belongs to the Hepatovirus genus of the Picornaviridae family Two major genotypes of the virus are associated with human disease, and both can be detected by the same serological assays (see the text that follows). Hepatitis A is a common infection responsible for an estimated 1.4 million cases of hepatitis worldwide. HAV is transmitted primarily by the fecal-oral route, close person-to-person contact, or ingestion of contaminated food or water. Conditions of poor personal hygiene, poor sanitation, and overcrowding facilitate transmission. Rarely, transmission through transfusion of contaminated blood has been reported and may occur during a short period within the acute stage of infection when a high number of viral particles can be found in the source blood. Following an average incubation period of 28 days, the virus produces symptoms of acute hepatitis in the majority of infected adults; however, most infections in children are asymptomatic. The infection does not progress to a chronic state and is usually self-limiting, with symptoms typically resolving within 2 months. Treatment is mainly supportive, involving bedrest, nutritional support, and medication for fever, nausea, and diarrhea. Massive hepatic necrosis resulting in fulminant hepatitis and death is rare and occurs mainly in those patients with underlying liver disease or advanced age. HAV antigens are shed in the feces of infected individuals during the incubation period and the early acute stage of infection, but they usually decline to low levels shortly after symptoms appear and are not a clinically useful indicator of disease. Therefore, serological tests for antibody are critical in establishing diagnosis of the infection. Hepatitis A antibodies are most commonly detected by automated enzyme immunoassays (EIAs) and chemiluminescent microparticle immunoassays. Acute hepatitis A is routinely diagnosed in symptomatic patients by demonstrating the presence of IgM antibodies to HAV (Fig. 23–4). IgM anti-HAV is detectable at the onset of clinical symptoms and declines to undetectable levels within 6 months in the majority of infected individuals. Because false-positive results can occur, the test should be reserved for symptomatic individuals. Tests for total HAV antibodies also detect IgM but predominantly detect IgG, which persists for life. Thus, a positive total anti- HAV test result in combination with a negative IgM anti-HAV indicates that the patient has developed immunity to the virus, either through natural infection or vaccination. Negative total anti-HAV tests can be used to identify nonimmune individuals who may have been exposed to the virus. Although IgM anti-HAV is the primary marker to detect acute hepatitis A, false-negative results can occur during the early phase of the infection. Molecular methods to detect HAV RNA have been shown to be more sensitive in this situation. The most common format of these methods is the reverse-transcriptase polymerase chain reaction (RT-PCR). Molecular methods can also be used to test samples of food or water suspected of transmitting the virus. Multiplex quantitative polymerase chain reaction (qPCR) methods that can simultaneously detect more than one type of hepatitis virus in clinical samples have also been developed. A vaccine consisting of formalin-killed HAV was licensed in the mid-1990s to prevent hepatitis A. Currently, two inactivated single-antigen vaccines are licensed and available in the United States for hepatitis A prevention. Vaccination has resulted in a significant decrease in the number of HAV infections in the United States and other countries throughout the world. To prevent infection in unimmunized individuals who have been exposed to the virus, prophylactic administration of the hepatitis A vaccine or injections of immune globulin are recommended. The vaccine is the preferred treatment for persons aged 1 to 40 years, but intramuscular injection of immune globulin, a sterile preparation of pooled human plasma that contains antibodies to HAV, can be used to prevent infection in individuals of any age. To be effective, these treatments must be administered within 2 weeks of exposure. Connections Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) In RT-PCR, viral RNA is treated with the enzyme reverse-transcriptase to generate a complementary DNA (cDNA) sequence. The cDNA is then amplified by the PCR to generate millions of copies that can be detected in the laboratory (see Chapter 12). Table 23-1 The Hepatitis Viruses and Their Associated Serological and Molecular Markers SEROLOGICAL PROGRESSION AND HEPATITIS TYPE AND TO CHRONIC MOLECULAR CLI VIRUS FAMILY TRANSMISSION STATE COMPLICATIONS MARKERS SIG Hepatitis A RNA Fecal–oral, direct No Low risk of fulminant IgM anti-HAV Acu (HAV) Picornaviridae contact with liver disease Total anti-HAV Imm infectious individual hep Blood transfusion (rare) HAV RNA Dete clin wat Hepatitis B DNA Parenteral, sexual, Yes 10% to 90% of cases HBsAg Acti (HBV) Hepadnaviridae perinatal may develop chronic infe hepatitis (depending HBeAg Acti on age), with with increased risk for liver infe cirrhosis and hepatocellular IgM anti-HBc Cur carcinoma acu Total anti-HBc Cur hep Anti-HBe Rec hep Anti-HBs Imm hep HBV DNA Acu occ vira use effe ther Hepatitis C RNA Flaviviridae Parenteral, sexual, Yes Eighty-five percent Anti-HCV Cur (HCV) perinatal develop chronic hep infection, with HCV RNA Cur increased risk of infe cirrhosis, may hepatocellular mon carcinoma, or effe autoimmune ther manifestations to d gen Hepatitis D RNA Genus Mostly parenteral, but Yes Increased risk of IgM-anti-HDV Acu (HDV) Deltavirus also sexual, developing fulminant hep perinatal; HBV hepatitis, cirrhosis, or IgG-anti-HDV Rec infection required hepatocellular hep carcinoma chro HDV RNA Acti infe may mon effe ther Hepatitis E RNA Hepeviridae Fecal–oral Blood Yes, in Fulminant liver failure in IgM anti-HEV Cur (HEV) transfusion; vertical immunocompromised pregnant women infe transmission individuals IgG anti-HEV Cur hep HEV RNA Cur infe FIGURE 23-4 Hepatitis A Serology. Typical patterns of IgM and total (IgM plus IgG) anti-HAV. Hepatitis E HEV is a nonenveloped, single-stranded RNA virus that belongs to the genus Hepevirus, in the family Hepeviridae. HEV is a major cause of hepatitis worldwide. The World Health Organization (WHO) estimates that the virus causes 20 million infections, with over 3.3 million cases of acute hepatitis annually. In 2015, approximately 44,000 deaths were reported. Similar to the HAV, HEV is transmitted primarily by the fecal-oral route; however, person-to-person transmission is uncommon. The four genotypes of the virus differ in terms of their epidemiology and source of infection. Genotypes 1 and 2 are associated primarily with the consumption of fecally contaminated drinking water in developing regions of the world with poor sanitation, including parts of Africa, Asia, the Middle East, and Mexico. Outbreaks commonly occur in times of natural disasters such as flooding and earthquakes and can affect thousands of individuals. Genotypes 3 and 4 have been increasingly recognized in developed parts of the world, including Europe, North America, China, Taiwan, and Japan. HEV3 and HEV4 are zoonotic infections in which pigs are the primary host. Deer, wild boars, and other mammals are also known to harbor the virus. These infections are thought to be transmitted mainly by the consumption of infected pork and deer meat and possibly by direct contact with infected animals or fecally contaminated water. HEV has also been detected in the blood supply in several countries and can be transmitted through blood transfusions. Although HEV infections are often silent, all genotypes are capable of causing acute hepatitis with symptoms that are indistinguishable from other types of hepatitis. Following an incubation period of 2 to 10 weeks, HEV infection in most people causes a self-limiting illness, with recovery occurring by 4 to 6 weeks. However, the infection can have severe consequences. Some patients may experience extrahepatic symptoms, including neurological syndromes, renal injury, pancreatitis, and hematologic abnormalities. Pregnant women infected with HEV1 or HEV2 have a mortality rate of 20% to 25% because of obstetric complications or development of fulminant hepatitis, which is associated with rapidly progressing liver disease and failure. The reason for this is unclear, but it may be caused by the hormonal and immunologic changes associated with pregnancy. HEV3 can result in chronic infection in immunocompromised individuals. Chronic infection can progress to liver fibrosis, cirrhosis, and liver failure, which may require liver transplantation. Measures to prevent the infection include the provision of clean drinking water, improvement of sanitation conditions in developing countries, and in the case of HEV3, avoidance of eating undercooked meat, especially pork. A vaccine to prevent HEV1 infection has been licensed for use in the People’s Republic of China. The vaccine consists of virus-like particles that have been genetically modified to express a gene that codes for a key HEV protein. To date, no U.S. Food and Drug Administration (FDA)-cleared vaccine is available in the United States. Because HEV is not easily cultured, diagnosis relies on serology to detect antibodies to the virus and molecular methods to detect HEV nucleic acid. Antibodies to HEV are typically identified by sensitive EIAs that use recombinant and synthetic HEV antigens. Rapid immunochromatographic assays have also been developed. Antibody tests for HEV can detect all four genotypes of the virus because there is only one viral serotype. Acute infection is indicated by the presence of IgM anti-HEV, which is detectable at clinical onset, remains elevated for about 8 weeks, and becomes undetectable in most patients by 32 weeks. HEV-specific IgG antibodies appear soon after IgM, reach peak levels about 4 weeks after symptoms develop, and persist for several years. Immunoassays for IgG anti-HEV may be performed to detect patients in the later stages of infection, determine past exposure, and identify seroprevalence of the infection in a population. Immunocompromised persons often yield negative antibody test results; molecular testing for HEV RNA is recommended in these patients. Quantitation of HEV nucleic acid can be performed by qPCR (the gold standard for diagnosis of acute HEV infections) or a loop-mediated isothermal amplification assay (LAMP), which is suitable for resource-limited settings because it is faster and does not require expensive equipment. These assays can be performed on blood or stool samples. HEV RNA can be detected just before clinical symptoms. It becomes undetectable in the blood about 3 weeks after symptom onset; in the stool, it becomes undetectable at about 5 weeks. Therefore, a negative result for HEV RNA does not exclude the possibility of a recent infection. Hepatitis B Hepatitis B is a major cause of morbidity and mortality throughout the world. The WHO estimates that HBV has infected 2 billion people worldwide, causing approximately 257 million chronic infections. In 2015, the WHO reported 887,000 deaths because of complications from the disease. The virus is highly endemic in the Far East, parts of the Middle East, sub-Saharan Africa, and the Amazon areas. In the United States, which is considered a low-prevalence area, approximately 2.2 million individuals are living with chronic HBV infections. HBV is transmitted through the parenteral route by intimate contact with HBV-contaminated blood or other body fluids, most notably semen, vaginal secretions, and saliva. Transmission has thus been associated with sexual contact, blood transfusions, sharing of needles and syringes by intravenous drug users, tattooing, and occupational needlestick injury. Inapparent transmission of HBV may occur through close personal contact of broken skin or mucous membranes with the virus. Transmission of HBV may also occur via the perinatal route, from infected mother to infant, most likely during delivery. Several measures have been introduced to prevent HBV infection, including screening of blood donors, treating plasma-derived products to inactivate HBV, implementing infection-control measures, and most importantly, immunizing with a hepatitis B vaccine. The current vaccines, consisting of recombinant hepatitis B surface antigen (HBsAg) produced from genetically engineered yeast or mammalian cells, are some of the most widely used vaccines throughout the world. Immunization has been highly successful, resulting in a significant decline in the incidence of acute hepatitis B in the United States since routine immunization was implemented in 1991. Increasingly widespread use of the vaccine will likely continue to reduce the incidence of new HBV infections worldwide. The vaccine can also be administered to individuals thought to be exposed to the virus, along with hepatitis B immune globulin (HBIG), a preparation derived from donor plasma with high concentrations of antibodies to HBV that provides temporary protection. Despite the preventative measures that have been implemented, a substantial number of HBV infections continue to occur, as we previously discussed. Infection with HBV results in an incubation period of 30 to 180 days, followed by a clinical course that varies in different age groups. Over 90% of newborns with perinatal HBV infection remain asymptomatic, whereas typical symptoms of acute hepatitis are observed in about 10% of children aged 1 to 5 years and in approximately one-third of adolescents and adults. Symptoms may last several weeks to several months and are usually managed through bedrest and other supportive treatment. Most HBV- infected adults recover within 6 months and develop immunity to the virus, but about 1% develop fulminant liver disease with hepatic necrosis. This highly fatal condition is treated with intensive life support, antiviral drugs, and in some patients, liver transplantation. Development of chronic HBV infection, in which the virus persists in the body for 6 months or more, occurs in the majority of infected infants, about one-third of young children, and 10% of infected adults. Chronic infection is also more likely to develop in persons who are immunosuppressed and those who have HIV. Chronic infection with the virus results in inflammation and damage to the liver and places the patient at increased risk of developing cirrhosis or hepatocellular carcinoma. Patients with chronic infection can be treated with antiviral drugs to reduce liver inflammation and the risk of developing liver complications. Therapies consist of nucleoside analogues that inhibit the polymerase enzyme needed for viral replication and IFN-α, which enhances the immune response against the virus. The virus responsible for hepatitis B, HBV, is a DNA virus belonging to the Hepadnaviridae family. Eight genotypes, designated A through H, have been identified based on nucleotide-sequence differences in their genomes. The genotypes vary in their geographic distribution, pathogenicity, and response to treatment but can be identified by the same serological assays. The intact HBV virion is a 42-nm sphere consisting of a nucleocapsid core surrounded by an outer envelope of lipoprotein. The core of the virus contains circular, partially double- stranded DNA; a DNA-dependent DNA polymerase enzyme; and two proteins, the hepatitis B core antigen and the hepatitis Be antigen (HBeAg). A protein called the hepatitis B surface antigen (HBsAg) is found in the outer envelope of the virus. HBsAg is produced in excess and is found in noninfectious spherical and tubular particles that lack viral DNA and circulate freely in the blood. These antigens, and antibodies to them, serve as serological markers for hepatitis B and have been used in the differential diagnosis of HBV infection, monitoring the course of infection in patients, assessing immunity to the virus, and screening blood products for infectivity. The levels of these markers vary with the amount of viral replication and the host’s immune response. They are useful in establishing the initial diagnosis of hepatitis B and monitoring the course of infection. Serological markers for hepatitis B are listed in Table 23–1 and are described in the text that follows. Typical patterns of the markers during acute and chronic hepatitis B are shown in Figures 23–5 and 23–6. HBsAg is the first marker to appear, becoming detectable 2 to 10 weeks after exposure to HBV. Its levels peak during the acute stages of infection, then gradually decline as the patient develops antibodies to the antigen and recovers. Serum HBsAg usually becomes undetectable by 4 to 6 months after the onset of symptoms in patients with acute hepatitis B. In patients with chronic HBV infection, HBsAg remains elevated for 6 months or more. Thus, HBsAg is an indicator of active infection and is an important marker in detecting initial infection, monitoring the course of infection and progression to chronic disease, and screening of donor blood. HBeAg appears shortly after HBsAg and disappears shortly before HBsAg in recovering patients. It may be elevated during chronic infection. This marker is present during periods of active replication of the virus and indicates a high degree of infectivity. The hepatitis B core antigen (HBc) is not detectable in serum because the viral envelope masks it. As the host develops an immune response to the virus, antibodies appear. First to appear is IgM antibody to the core antigen, or IgM anti-HBc. This antibody indicates current or recent acute infection. It typically appears 1 to 2 weeks after HBsAg during acute infection and persists in high titers for 4 to 6 months and then gradually declines. IgM anti-HBc is useful in detecting infection in cases in which HBsAg is undetectable—for example, just before the appearance of antibodies to HBsAg (commonly referred to as the “core window” period), in neonatal infections, and in cases of fulminant hepatitis. Therefore, it is used in addition to HBsAg for the screening of donor blood. IgG antibodies to the core antigen are produced before IgM anti-HBc disappears and then persist for the individual’s lifetime. They are the predominant antibodies detected in the test for total anti-HBc and can be used to indicate a past HBV infection. FIGURE 23-5 Typical serological markers in acute hepatitis B. Solid lines represent viral antigen concentrations, whereas dashed lines indicate antibody concentrations. Each antigen shares the same color with its associated antibody. FIGURE 23-6 Typical serological markers in chronic hepatitis B. The appearance of antibodies to the HBe antigen, or anti-HBe, occurs shortly after the disappearance of HBeAg and indicates that the patient is recovering from HBV infection. Antibodies to HBsAg, or anti-HBs, also appear during the recovery period of acute hepatitis B, a few weeks after HBsAg disappears. These antibodies persist for years and provide protective immunity. Anti-HBs are also produced after immunization with the hepatitis B vaccine. Protective titers of the antibody in the serum are considered to be 10 mIU/mL or higher. Anti-HBs are not produced during chronic HBV infection, in which immunity fails to develop. Serological markers for hepatitis B are most commonly detected by commercial immunoassays. These are available in a variety of formats, such as EIA and chemiluminescent immunoassay (CLIA). They are typically automated to ease batch testing in the clinical laboratory and have excellent sensitivity and specificity. An example of an immunoassay for detecting HBsAg is shown in Figure 23–7. Although these methods are highly sensitive and specific, false-positive and false-negative results can occur. Any initial positive results should be verified by repeated testing of the same specimen in duplicate, followed by confirmation with an additional assay, such as an HBsAg neutralization test or a molecular test that detects HBV DNA. Several molecular methods have been developed to detect HBV DNA in serum or plasma and are mostly based on target amplification by traditional or qPCR (method of choice to quantify HBV DNA) or branched DNA (bDNA) signal amplification. HBV DNA can be detected in the serum about 21 days before HBsAg and may be a useful adjunct in detecting early acute HBV infection in certain situations, such as the screening of blood donors, assessing cases of occupational exposure, and evaluating patients with equivocal HBsAg test results. HBV DNA testing is also used to evaluate the effectiveness of antiviral therapy in patients with chronic hepatitis B. Successful treatment is indicated by a 1-log10 reduction in HBV DNA levels by 6 months, whereas persistently elevated HBV DNA levels indicate possible drug resistance and a need to change therapy. Molecular testing is also used to diagnose atypical cases of hepatitis B originating from mutations in the HBV genome that cause HBsAg tests to be negative. Molecular methods to detect HBV genotypes and HBV mutations associated with antiviral drug resistance have also been developed. These tests will likely be used more widely in the future to determine optimal patient therapy. FIGURE 23-7 Detection of the HBs antigen by chemiluminescence microparticle immunoassay. Patient serum or plasma containing HBsAg (A) is mixed with magnetic microparticles coated with anti-HBs (B) and acridinium-labeled anti-HBs conjugate (C). During incubation, complexes form, with the antigen sandwiched in between the antibodies (D). Application of a magnetic field holds the microparticles and bound reagents in the tube while unbound materials are washed away and chemiluminescent reagents are added (E). The magnitude of light produced is measured in a luminometer (F) and is proportional to the concentration of HBsAg in the sample. Hepatitis D Hepatitis D, also known as delta hepatitis, is a parenterally transmitted infection that can occur only in the presence of hepatitis B. HDV is a defective virus that requires the help of HBV for its replication and expression. The only member within the Deltavirus genus, HDV consists of a circular RNA genome and a single structural protein called hepatitis delta antigen within its core, surrounded by a viral envelope that is of HBV origin and contains the HBsAg. Three genotypes have been identified: Genotype I (most common and found worldwide), Genotype II (Japan and Taiwan), and Genotype III (South America). Approximately 15 to 20 million people around the world are believed to be infected with HDV, which is highly prevalent in Mediterranean Europe, the Middle East, the Amazon basin, central Africa, and parts of Asia. The number of new infections appears to be increasing in certain parts of the world. Similar to HBV, HDV is transmitted sexually in semen or vaginal secretions; through blood by intravenous drug use, needlestick injuries, or transfusions; or perinatally from mother to infant. Infection with the virus can occur in one of two ways: HDV can be transmitted simultaneously as a co-infection with HBV or HDV can be contracted as a superinfection of individuals who are already chronic HBV carriers. Clinically, most patients with co- infections experience an acute, self-limited hepatitis in which both viruses are cleared within a few months. Some patients may experience more severe symptoms of acute hepatitis than those infected with HBV alone, but only about 2% of cases progress to a chronic state. In contrast, more than 70% of patients with superinfections develop chronic liver disease with an accelerated progression to cirrhosis and liver failure. Combinations of IFN-α and antiviral drugs can be administered to patients with chronic or severe hepatitis D in an attempt to eradicate the virus. Testing for hepatitis D should be performed in all patients who are HBsAg positive and involves the detection of HDV antibodies and HDV RNA. Antibodies are detected by immunoassays employing the hepatitis D antigen. The presence of IgG anti-HDV antibodies indicates exposure to the virus and can signify an acute, chronic, or past hepatitis D infection. Although IgM anti-HDV is produced during acute hepatitis D infections, its appearance may be delayed, it may persist for only a short period of time, and it may be missed. IgM antibodies to HDV can also persist during chronic infection. Serology testing for hepatitis B can be used to help distinguish HBV and HDV co- infections from HBV and HDV superinfections, which, as previously discussed, have different clinical outcomes. In addition to being positive for HDV antibodies, patients with co-infections are positive for IgM anti-HBc, whereas patients with superinfections are positive for IgG anti-HBc. The detection of hepatitis D has been aided tremendously by the development of molecular methods to detect HDV RNA, a marker of active viral replication that is present in all types of active hepatitis D infections. HDV RNA testing is routinely used to confirm a positive HDV antibody screen. Molecular testing for serum HDV RNA is performed by sensitive, real-time RT-PCR assays. These assays also provide quantitative results that can be used to monitor the response of patients to antiviral therapy. Hepatitis C Hepatitis C is a major public health problem, with an estimated 71 million people infected worldwide. It is the most common bloodborne infection in the United States, with an estimated 2.4 million individuals living with chronic HCV. Hepatitis C is also the most frequent cause of chronic liver infection and the leading indicator for liver transplantation in the United States. HCV, the virus that causes hepatitis C, is responsible for most of the infections previously classified as “nonA– nonB” before the discovery of the virus in 1989. It is an enveloped, single-stranded, positive-sense RNA virus belonging to the family Flaviviridae and the genus Hepacivirus. Scientists have discovered seven different genotypes of the virus, designated 1 through 7, and numerous subtypes for each, indicated by lowercase letters. The genotypes differ in their geographic distribution, pathogenicity, and response to antiviral treatment. Genotype 1, the most common, is responsible for 46% of hepatitis C infections worldwide and approximately 75% of HCV infections in the United States. Genotypes 1, 2, and 3 are predominant in North America, Europe, and Japan; genotypes 3 and 6 are found throughout south and southeast Asia; and genotypes 4, 5, and 7 are most common in parts of Africa. The variability of HCV, along with its ability to undergo rapid mutations within its hosts, has created difficulty in developing an effective vaccine. Hepatitis C is transmitted mainly by exposure to contaminated blood, with intravenous drug use being the main source of infection. Blood transfusion was also a major vehicle of transmission before routine screening of blood donors for HCV antibody was implemented in 1992, but transmission by this means is rare today. Organ transplantation before 1992 was also a route of transmission. Other risk factors for acquiring hepatitis C include occupational exposures to contaminated blood, long-term hemodialysis, and unregulated body piercing or tattooing in environments such as correctional facilities where contaminated needles are likely to be used. Sexual transmission of HCV is thought to be less common but is higher in those who have had multiple sex partners or a history of sexually transmitted diseases. Perinatal transmission has been estimated to occur at a rate of about 6%. HCV has an average incubation period of 7 weeks (range is 2 weeks to 6 months). The majority of infections are asymptomatic, with symptoms of acute hepatitis occurring in only about 20% of cases. Asymptomatic infection is problematic because chronic infection develops in about 70% of infected persons, and up to half of these individuals develop cirrhosis. Cirrhosis occurs slowly over a 25- to 30-year period, causing damage to the liver and posing an increased risk of developing hepatocellular carcinoma. Patients with chronic HCV infection may also develop extrahepatic manifestations, including rheumatological conditions; glomerulonephritis, vasculitis, or other autoimmune manifestations; neuropathy; ophthalmological symptoms; and dermatological symptoms. Early detection would help prevent these complications, but HCV is often missed in its early stages because of the asymptomatic nature of the infection in most individuals. Clearance of the infection may occur spontaneously or may require treatment with antiviral drugs. The standard treatment involves a combination of pegylated IFN-α (PEG IFN-α) and ribavirin. Although this treatment has been successful in 80% of persons infected with genotypes 2 or 3, it is effective in only half of those with genotype 1 and is associated with numerous side effects. Increased understanding of the biology of HCV has led to the development of direct-acting antiviral drugs (DAAs) and host-targeted agents (HTAs) that inhibit specific steps of the viral replication cycle. Combination therapies employing these agents are being evaluated at a rapid pace and are revolutionizing the way hepatitis C is being treated. The laboratory plays an essential role in screening for hepatitis C, monitoring patients known to have HCV infection, and guiding therapy. Between 1998 and 1999, the Centers for Disease Control and Prevention (CDC) issued recommendations that screening for HCV infection be conducted in high-risk individuals, including those who received blood or blood products. In 2012, the CDC extended these recommendations to include a one-time screening of all persons in the United States who were born between 1945 and 1965, regardless of risk factors. In 2013, the U.S. Preventative Task Force endorsed this recommendation. The rationale behind the latest recommendation was that about 75% of individuals living with HCV infection in the United States were born during this time period but are asymptomatic. Identification of these persons could lead to closer monitoring for disease progression and earlier administration of effective antiviral treatment. Screening and diagnosis of hepatitis C begins with serological testing for HCV antibodies. Anti-HCV IgG is most commonly detected by sensitive EIAs or CLIAs that use recombinant and synthetic antigens developed from the conserved domains of the capsid core protein (C) and the nonstructural proteins NS3, NS4, and NS5. Alternatively, a rapid immunoblot assay can be used for point-of-care testing. Antibodies become detectable 8 to 10 weeks after HCV exposure and can remain positive for a lifetime. Thus, a reactive result can indicate the presence of a current HCV infection or a past HCV infection that has resolved. In addition, despite the excellent specificity of these methods, false-positive results may occur because of cross-reactivity in persons with other viral infections or autoimmune disorders. Therefore, any positive results from an anti-HCV screening test should be confirmed to distinguish between the various interpretations of these results. Current CDC guidelines recommend the use of nucleic acid testing (NAT) for HCV RNA for confirmation. If HCV RNA is detected, a current HCV infection is indicated. In contrast, if the NAT is nonreactive, this suggests a past HCV infection or false-positive antibody test result. To distinguish between a true-positive and false-positive result, HCV antibody testing can be repeated using a different assay from the initial test because a biological false-positive result is unlikely to occur in two different methods. Molecular assays for HCV RNA can be classified as qualitative or quantitative. Qualitative tests distinguish between the presence or absence of HCV RNA in a clinical sample. These tests are used to confirm infection in HCV-antibody-positive patients (as previously mentioned), detect infection in antibody-negative patients who are suspected of having HCV, screen blood and organ donors for HCV, and detect perinatal infections in babies born to HCV-positive mothers. Qualitative RT-PCR and transcription-mediated amplification (TMA) methods are commercially available. These tests can detect as low as 5 International Units (IU) of HCV RNA per mL of serum (for TMA) or 50 IU/mL HCV RNA (for RT-PCR) and become positive within 1 to 3 weeks after infection. They are generally positive at the onset of symptoms but, in some patients, can transiently decrease to undetectable levels during the acute phase of the infection. Quantitative tests are performed by RT-PCR, qPCR, or bDNA amplification. Commercial tests can detect a wide range of HCV concentrations, from about 10 IU/mL to 10 million IU/mL. They are used to monitor the amount of HCV RNA, or “viral load,” carried by patients before, during, and after antiviral therapy in chronically infected individuals. The ultimate goal of such therapy is to achieve a sustained virological response (SVR) in which the patient continuously tests negative for HCV RNA 12 or 24 weeks after therapy is completed. The initial viral load level has also been used as a prognostic tool because those with a low initial viral load are most likely to achieve an SVR. Genotyping, to determine the exact genotype and subtype of the virus responsible for the infection, should be performed on all HCV-infected patients before antiviral therapy. It is important to identify the patient’s HCV genotype in order to determine the most effective treatment because HCV genotypes vary in their response to different antiviral drugs. For example, as we previously mentioned, PEG IFN-α/ribavirin treatment is more effective in patients with genotypes 2 or 3 than in patients with genotype 1. Genotyping is also useful in epidemiological studies to determine the source of HCV infection in specific populations. Genotyping can be performed by PCR amplification and sequencing of the target gene, PCR followed by identification of the target gene with genotype-specific probes, or qPCR. PCR/direct sequencing (Sanger sequencing) is the reference method because it provides precise information regarding the genomic variability of the virus in patients during the course of the disease. However, sequencing is primarily performed in research laboratories because of the specialized equipment and analysis software required, whereas clinical laboratories typically use qPCR methods or PCR/probe hybridization. Herpes Virus Infections The herpes viruses are large, complex DNA viruses that are surrounded by a protein capsid, an amorphous tegument, and an outer envelope. These viruses are all capable of establishing a latent infection with lifelong persistence in the host. The Herpesviridae family includes eight viruses that can cause disease in humans: the herpes simplex viruses (HSV-1 and HSV-2); VZV; EBV; CMV; and the human herpes viruses HHV-6, HHV-7, and HHV-8, the latter being associated with Kaposi sarcoma. This section presents the clinical manifestations and laboratory diagnosis of some of these viruses. Epstein-Barr Virus (EBV) The EBV is ubiquitous in nature and causes a wide spectrum of diseases, including infectious mononucleosis (IM), lymphoproliferative disorders, and several malignancies. EBV infections most commonly result from intimate contact with salivary secretions from an infected individual. Although transmission of the virus can occur by other means, including blood transfusions, bone marrow and solid-organ transplants, sexual contact, and perinatal exposure, these routes appear to be much less frequent. In developing nations of the world and lower socioeconomic groups living under poor sanitation, EBV infections usually occur during early childhood, whereas in industrialized nations with higher hygiene standards, infections are typically delayed until adolescence or adulthood. However, by adulthood (age 40), almost 100% of individuals have been infected, as evidenced by the presence of EBV antibodies in their serum. Initial infection with EBV is believed to occur in the oropharynx, where the virus primarily infects epithelial cells and B lymphocytes. EBV binds to β1 integrins on the surface of the epithelial cells, which take up the virus by endocytosis. Inside the oropharyngeal epithelial cells, EBV enters a lytic cycle, characterized by viral replication, lysis of host cells, and release of infectious virions, until the acute infection is resolved. The virions spread to adjacent structures, including the salivary glands and tonsils. There, EBV infects B lymphocytes, which spread the virus throughout the lymphoreticular system. EBV enters the B cells by binding to surface CD21, which is also the receptor for the C3d component of complement. The virus-infected B cells become polyclonally activated, proliferating and secreting several antibodies, including EBV-specific antibodies; heterophile antibodies; and autoantibodies such as cold agglutinins, rheumatoid factor, and antinuclear antibodies. In healthy individuals, this process is kept in check by the immune response of NK cells and specific CTLs. However, EBV can persist in the body indefinitely in a small percentage of memory B cells, where it establishes a latent infection. In the latent state, EBV nucleic acid exists as episomal DNA outside of the chromosomes; in these cases, active viral replication does not occur. Periodic reactivation results in re-entry of the virus into the lytic cycle, with viral shedding into the saliva and genital secretions, even in healthy, asymptomatic individuals. Several antigens have been identified in EBV-infected cells that are associated with different phases of the viral infection. Antibodies to these antigens have become an important diagnostic tool. Antigens produced during the initial stages of viral replication in the lytic cycle are known as the early antigens (EAs). These antigens can be further classified into two groups based on their location within the cells: EA-D, which has a diffuse distribution in the nucleus and cytoplasm, and EA-R, which is restricted to the cytoplasm only. The late antigens of EBV are those that appear during the period of the lytic cycle following viral DNA synthesis. They include the viral capsid antigens (VCAs) in the protein capsid and the membrane antigens in the viral envelope. Antigens appearing during the latent phase include the EBV nuclear antigen (EBNA) proteins, EBNA-1, EBNA-2, EBNA-3 (or -3a), EBNA-4 (or -3b), EBNA-5 (or -LP), and EBNA-6 (or -3c), and the latent membrane proteins (LMPs), LMP-1, LMP-2A, and LMP-2B (Table 23–2). Table 23-2 Epstein-Barr Virus Antigens EARLY ACUTE PHASE LATE PHASE LATENT PHASE EA-R (early antigen VCA (viral capsid antigen) EBNA (EBV nuclear antigens): EBNA-1, EBNA-2, EBNA-3 (3a), EBNA-4 (3b), restricted) MA (membrane antigen) EBNA-5 (LP), EBNA-6 (3c) EA-D (early antigen diffuse) Latent membrane proteins (LMP-1, LMP-2A, LMP-2B) The clinical manifestations of EBV vary with the host’s age and immune status. Infections in infants and young children are generally asymptomatic or mild, whereas primary infections in healthy adolescents or adults commonly result in IM. More than half of patients with IM present with three classic symptoms: fever, lymphadenopathy, and sore throat. Symptoms usually last for 2 to 4 weeks, but fatigue, myalgias, and need for sleep can persist for months. Treatment is mainly directed at alleviating symptoms. Although the associated symptoms are essential in diagnosing IM, they can also be caused by many other infectious agents, so laboratory testing plays an important role in differentiating IM from other infections. Characteristic laboratory findings in patients with IM include an absolute lymphocytosis of greater than 50% of the total leukocytes and at least 20% atypical lymphocytes (Fig. 23–8). The atypical lymphocytes are predominantly activated cytotoxic T cells that are responding to the viral infection. Serological findings include the presence of a heterophile antibody and antibodies to certain EBV antigens. By definition, heterophile antibodies are antibodies that are capable of reacting with similar antigens from two or more unrelated species. The heterophile antibodies associated with IM are IgM antibodies produced because of polyclonal B-cell activation and are capable of reacting with horse red blood cells (RBCs), sheep RBCs, and bovine RBCs. These antibodies are produced by 40% of patients with IM during the first week of clinical illness and by 80% to 90% of patients by the third week. They disappear in most patients by 3 months after the onset of symptoms but can be detected in some patients for 1 to 2 years. Because the heterophile antibody is present in most patients during the acute phase of illness, testing for this antibody has been typically performed to screen for IM in patients who present with symptoms of the disease. For many years, the heterophile antibody of IM was detected by a rapid slide agglutination method called the “Monospot.” In this test, serum premixed with guinea pig kidney antigen was still capable of agglutinating horse RBCs, whereas serum premixed with beef erythrocyte antigen could not agglutinate horse RBCs because the heterophile antibody was absorbed during the first step. The test was used to distinguish the heterophile antibody of IM from heterophile antibodies produced in other conditions, which had different reactivity. The antibody could then be titered by incubating serial dilutions of the patient’s serum with sheep RBCs in the Paul–Bunnell test (see the Lab Exercise on DavisPius). FIGURE 23-8 Atypical lymphocytes from a patient with infectious mononucleosis. Note the variation in size, nucleancytoplasmic ratio, and chromatin coarseness. (From Harmening D. Clinical Hematology and Fundamentals of Hemostasis. 5th ed. Philadelphia, PA: F. A. Davis; 2009.) Today, these methods have been replaced by more sensitive, rapid agglutination tests or immunochromatographic assays using purified bovine RBC extract as the antigen. Although screening tests for the heterophile antibody are ideal for point-of-care testing, they are not as sensitive or specific as tests for antibodies to EBV, the direct cause of IM. Negative heterophile antibody results occur in about 10% of adult patients with IM and up to 50% of children younger than 4 years old. False-positive results, although uncommon, can occur in patients with lymphoma, viral hepatitis, malaria, and autoimmune disease or can be caused by errors in result interpretation. Testing for EBV-specific antibodies can be performed to aid in the diagnosis of IM, especially in patients with a negative heterophile antibody screen, or to determine if individuals have had a past exposure to EBV. These antibodies can be detected by indirect immunofluorescence assays (IFAs) using EBV-infected cells, blot techniques, enzyme-linked immunosorbent assay (ELISA) or CLIA using recombinant or synthetic EBV proteins, or flow cytometric microbead immunoassays. Although all of these methods have a high level of sensitivity (95% to 99%), IFA tests have a higher level of specificity and are considered the “gold standard” of EBV serology methods. However, many laboratories prefer ELISA or CLIA tests because they are automated and easier to interpret. Table 23-3 Serological Responses of Patients With Epstein-Barr Virus-Associated Diseases ANTI-VCA ANTI-EA HETEROPHILE ANTI- ANTIBODY CONDITION IgM IgG IgA EA-D EA-R EBNA (IgM) Uninfected — — — — — — — Acute IM + ++ ± + — — ± Convalescent IM — + — — ± + ± Past infection IM — + — — — + — Chronic active infection IM — +++ ± + ++ ± — Post-transplant lymphoproliferative — ++ ± + + ± — disease Burkitt’s lymphoma — +++ — ± ++ + — Nasopharyngeal carcinoma — +++ + ++ ± + — EA-D = early antigen—diffuse; EA-R = early antigen—restricted; EBNA = EBV nuclear antigen; IM = infectious mononucleosis; VCA = viral capsid antigen. Adapted from Straus SE, et al. Epstein-Barr virus infections: biology, pathogenesis, and management. Ann Intern Med. 1993;118:45, with permission. IgM antibody to the VCA is the most useful marker for acute IM because it usually appears at the onset of clinical symptoms and disappears by 3 months. IgG anti-VCA is also present at the onset of IM but persists for life and can thus indicate a past infection. Antibodies to EA-D are also seen during acute IM, whereas anti-EBNA appears during convalescence. Thus, acute primary infection is typically indicated by the presence of IgM anti- VCA and anti-EA-D, as well as the absence of anti-EBNA. A summary of serological responses during acute, convalescent, and post-IM is shown in Table 23–3. Some individuals develop chronic active EBV infection, with severe, often life-threatening IM-associated symptoms that persist or recur for more than 6 months after the acute illness. In addition, EBV can sometimes integrate its DNA into the genome of the cells it infects and transform them into cancer cells. Therefore, EBV has been associated with several malignancies, both hematologic (e.g., Burkitt’s lymphoma and Hodgkin disease) and nonhematologic (e.g., nasopharyngeal carcinoma and gastric carcinoma). EBV can also cause lymphoproliferative disorders in immunocompromised patients, including central nervous system (CNS) lymphomas in patients with AIDS, X-linked lymphoproliferative disease in males with a rare genetic mutation, and post-transplant lymphoproliferative disorders (PTLD) in patients who have received hematopoietic stem cell or solid-organ transplants. These disorders result from the inability of immunosuppressed patients to control primary EBV infection, leading to massive polyclonal expansion of the EBV-infected B cells and life-threatening illness with a high rate of mortality. EBV-associated malignancies can be diagnosed with the help of serology tests for EBV antibodies and molecular methods to detect EBV DNA in blood and tissue samples. Typical patterns of EBV antibodies seen in some of these disorders are shown in Table 23–3. Molecular tests may be more reliable than serology in immunocompromised patients, who may not demonstrate a good humoral response. Quantitative real-time PCR is useful in monitoring viral load in transplant patients; a high or steadily increasing EBV viral load indicates the need to decrease immunosuppressive treatment and administer antiviral therapy. Detection of EBV-encoded RNA transcripts (EBERs) by in situ hybridization is the method of choice for detecting EBV in tumor tissue. Cytomegalovirus (CMV) CMV is a ubiquitous virus with worldwide distribution. The prevalence of CMV ranges from 40% to 100%, depending on the population, and increases with age; however, crowded living conditions and poor personal hygiene facilitate spread earlier in life. Transmission of the virus can occur in a variety of ways. CMV is spread through close, prolonged contact with infectious body secretions; intimate sexual contact; blood transfusions; solid-organ transplants; and perinatal exposure from infected mother to infant. The virus has been isolated in many body fluids, including saliva, urine, stool, vaginal and cervical secretions, semen, breast milk, and blood. Primary, or initial, infections in healthy individuals are usually asymptomatic. However, some people experience a self-limiting, heterophile antibody-negative IM-like illness with fever, myalgias, and fatigue. A small number of immunocompetent individuals who have other underlying disorders may develop severe CMV disease, which most commonly involves the gastrointestinal tract, CNS, and hematologic abnormalities. An immune response against CMV is stimulated, but the virus persists in a latent state in monocytes, dendritic cells, myeloid progenitor cells, and peripheral blood leukocytes. It may be reactivated at a later time in the individual’s life. The clinical consequences of CMV infection are much more serious in the immunocompromised host, most notably organ-transplant recipients and patients with HIV/AIDS. CMV is the most important infectious agent associated with organ transplantation, with infections resulting from reactivation of CMV in the recipient or transmission of CMV from the donor organ. CMV infection of a previously unexposed recipient is associated with increased risk for allograft failure or graft-versus-host disease (GVHD) and poses a high risk for a variety of syndromes, such as fever and leukopenia, hepatitis, pneumonia, gastrointestinal complications, CNS dysfunction, and retinitis. Although combination antiretroviral therapy has reduced the incidence of CMV-related illness in patients with HIV infection, CMV remains a major opportunistic pathogen in patients with low CD4 T-cell counts. Various measures can be undertaken to reduce the risk for CMV transmission and treat CMV infection in the immunocompromised host. Serological testing can be performed to identify CMV-positive donors so that transplantation of their organs into CMV-negative recipients can be avoided. If a CMV infection has been established in a transplant patient, immunosuppressive treatment should be reduced to the lowest dose possible. In addition, a variety of antiviral drugs are currently used to treat CMV infection and may, in some instances, be given prophylactically (i.e., before organ transplantation). Researchers are also investigating a vaccine design that involves the production of specific CMV antigens using genetic technologies. CMV is also the most common cause of congenital infections, occurring in 0.3% to 2.3% of all neonates. Transmission of the virus may occur through the placenta, by passage of the infant through an infected birth canal, or by postnatal contact with breast milk or other maternal secretions. About 10% to 15% of infants with congenital CMV infection are symptomatic at birth. Mothers who acquire primary CMV infection during their pregnancy have a significantly higher risk of giving birth to a symptomatic or severely affected infant than do women in whom CMV was reactivated during pregnancy. Symptomatic infants present with a multitude of symptoms that reflect platelet dysfunction and CNS involvement. Ten percent of infants who are asymptomatic at birth progressively develop sensorineural hearing loss. Several laboratory methods have been developed to detect CMV infection; the tests recommended for use depend on the clinical situation. Assays for direct detection of the virus, such as viral culture, identification of CMV antigens, and molecular tests for CMV DNA, are necessary to detect a current CMV infection in individuals who are immunocompromised or in neonates suspected of being congenitally infected with CMV. Serology is most beneficial in determining a past exposure to the virus, for example, in pregnant women or in patients in need of a transplant. Isolation of the virus in culture is the traditional method of direct viral detection. In this method, human fibroblast cell lines are inoculated with CMV-infected specimens, most commonly urine, respiratory secretions, or anticoagulated whole blood. The presence of the virus is indicated by characteristic cytopathic effects (CPEs) that produce enlarged, rounded, refractile cells. Although conventional culture provides definitive results when positive, it is limited because CPEs do not appear until a few days to several weeks after inoculation, depending on the viral titer. Implementation of the rapid centrifugation-enhanced (shell vial) method has reduced the time of detection to within 24 hours after inoculation. In this assay, infected cells are grown on coverslips in shell vials and incubated with fluorescent-labeled monoclonal antibodies to CMV antigens produced early in the replication cycle. Fluorescent staining will appear in the nuclei of positive cells. A widely used method for direct identification of CMV has been the CMV antigenemia assay, which uses immunocytochemical or immunofluorescent staining to detect the CMV lower-matrix protein pp65 in infected leukocytes from peripheral blood or cerebral spinal fluid. Following lysis of erythrocytes in the sample, the leukocytes are fixed onto a microscope slide, permeabilized, and stained with labeled monoclonal anti-pp65. Fluorescence appears in the nuclei of the infected cells, which can be counted to give quantitative results. The test can be completed in 2 to 4 hours, allowing for more rapid diagnosis and treatment of CMV infection in organ- transplant patients and individuals infected with HIV. Although the antigenemia assay and shell vial culture methods are sensitive, specific, and rapid, they are labor- intensive and require personnel with expertise in performing and interpreting these tests. For these reasons, they are progressively being replaced with molecular methods that detect CMV DNA or mRNA. Real-time PCR is the most widely used molecular method because it is sensitive, simple to perform, and can provide quantitative results. PCR amplification of CMV DNA has been extremely useful for detecting CMV infections in HIV-infected hosts and establishing the diagnosis of CMV infection in transplant recipients. PCR also provides a more sensitive alternative to culture in diagnosing congenital CMV infections. Identification of CMV or CMV DNA in amniotic fluid after the 20th week of gestation is considered the gold standard for confirmation of fetal infection. Neonatal infection is established by detecting CMV or CMV DNA in the urine of the infant during the first 10 days of life. Quantitative PCR, which detects the CMV copy number in the peripheral blood, is used to monitor the effectiveness of antiviral treatment in immunocompromised hosts and to identify patients at risk for developing disseminated CMV disease. In addition, increasing CMV DNA levels over time can be helpful in distinguishing an active infection from asymptomatic or latent infections. Although serology tests for CMV have been commercially available for many years, their clinical utility is limited. The serology methods performed most commonly are semi- or fully automated EIAs that use microtiter plates or microparticle systems. Assays for CMV IgG are most useful in documenting a past CMV infection and determining if an individual is at risk for future infection. For example, screening of blood and organ donors for CMV IgG is performed to identify those donors who are CMV-positive so that the risk of post-transfusion/post- transplant primary CMV infection in seronegative recipients can be reduced. In addition, screening of pregnant women for CMV IgG can determine if they have been exposed to the infection in the past or if they are susceptible to primary infection. In the latter case, the women could be educated on measures to reduce their chances of exposure while pregnant. Although a single positive CMV IgG result indicates past exposure to the virus, conversion from a negative antibody result to a positive antibody result over time indicates a recent CMV infection. However, serial assays for CMV IgG are not routinely performed. Assays for IgM CMV antibodies have been developed but are limited in value because of the potential for false-negative results in newborns and immunocompromised patients and for false-positive results caused by other infections or the presence of rheumatoid factor. In addition, IgM antibodies may not necessarily indicate primary CMV infection because they can also be produced because of CMV reactivation and may persist for up to 18 months. Serological methods that distinguish CMV antibody avidity appear to be more useful in distinguishing a past exposure from a current primary infection. Low-avidity IgG antibodies indicate a recent infection, whereas high-avidity IgG antibodies reflect a past exposure because the avidity of the antibody increases during the course of the immune response. The presence of both IgM and low- avidity IgG antibodies can help identify pregnant women who have contracted a primary CMV infection. Because of the limitations of serology testing, direct methods of detecting CMV infection are essential. Connections Rheumatoid Factor Recall that rheumatoid factor (RF) is an antibody (usually of the IgM class) that is directed against the Fc portion of IgG. RF can cause a false-positive result in some IgM assays because it binds to IgG antibodies in the patient serum that are directed against the viral antigen bound to the solid phase (see Chapter 11). Varicella-Zoster Virus (VZV) VZV is the cause of two distinct diseases: varicella, more commonly known as chickenpox, and herpes zoster, also known as shingles. The virus is transmitted primarily by inhalation of infected respiratory secretions or aerosols from skin lesions associated with the infection. Transplacental transmission to the fetus may also occur. Primary infection with VZV results in varicella, a highly contagious illness characterized by a blister-like rash with intense itching and fever. Historically, the majority of varicella cases have occurred during childhood. In a typical infection, vesicular lesions first appear on the face and trunk and then spread to other areas of the body (Fig. 23–9). The illness is usually mild and self-limiting in healthy children; however, in some cases, it may produce complications, the most common of which are secondary bacterial skin infections caused by scratching of the lesions. CNS involvement may occur in some cases but does not usually require hospitalization. Primary infections in adults, neonates, or pregnant women tend to be more severe, with a larger number of lesions and a greater chance of developing other complications such as pneumonia. Varicella infection in pregnant women may also cause premature labor or congenital malformations if the infection is acquired during the first trimester of pregnancy or may cause severe neonatal infection if transmission of the virus occurs around the time of delivery. Infections in immunocompromised patients are likely to result in disseminated disease, with extensive skin rash, neurological conditions (e.g., encephalitis), and other complications, including pneumonia, hepatitis, and nephritis. FIGURE 23-9 Vesicular lesions characteristic of chickenpox. These blisterlike lesions have a pus-filled center. (Courtesy of the Centers for Disease Control and Prevention, Public Health Image Library.) During the course of primary infection, VZV is thought to travel from the skin lesions and the blood to sensory neurons, where it deposits its DNA and establishes a lifelong latent state in the dorsal root, autonomic, and cranial ganglia. The host’s T-cell–mediated immune response is believed to keep the virus under control during this time. Reactivation of VZV, with active viral replication, occurs in 15% to 30% of persons with a history of varicella infection. The number of cases increases with age or the development of an immunocompromised condition, probably because of decreased cell-mediated immunity. During reactivation, the virus moves down the sensory nerve to the dermatome supplied by that nerve, resulting in eruption of a painful vesicular rash known as herpes zoster, or shingles, in the affected area. The rash may persist for weeks to months and is more severe in immunocompromised and elderly individuals. A significant number of patients with herpes zoster develop complications, the most common being postherpetic neuralgia, characterized by debilitating pain that persists for weeks, months, or even years after resolution of the infection. Life-threatening complications, such as herpes ophthalmicus, that lead to blindness, pneumonia, and visceral involvement are more common in immunosuppressed persons. Implementation of a vaccine consisting of a strain of live, attenuated varicella virus in 1995 has resulted in a significant decline in the incidence of chickenpox and its associated complications in the United States. In 2005, a vaccine was licensed for use in healthy children that combines the varicella vaccine with that for measles, mumps, and rubella. In addition, a single-agent live, attenuated VZV vaccine was licensed in 2006 for the prevention of herpes zoster in persons aged 60 or older, presumably by boosting T-cell immunity to the virus. Because these vaccines all contain a live agent, they are not recommended for use in immunocompromised persons. GlaxoSmithKline developed a recombinant (genetically engineered) zoster vaccine for shingles, and the FDA licensed the vaccine in 2017. This vaccine is preferred over the live, attenuated vaccine. The CDC recommends healthy adults over the age of 50 receive two doses of the recombinant vaccine 2 to 6 months apart to protect against shingles. Diagnosis of varicella and herpes zoster is usually based on identifying the characteristic vesicular lesions associated with the infection. Laboratory testing is most important in the diagnosis of atypical cases, such as those in which the rash is absent or delayed, and in immunocompromised patients with disseminated disease. Definitive diagnosis is based on identifying VSV or one of its products in skin lesions, vesicular fluids, or tissue. Older methods of identification involved cell culture and microscopy, but these have significant disadvantages. Culture of the virus and observation of characteristic CPE can be performed in several cell lines but is time consuming (4 days to 2 weeks) and may not yield productive results if clinical specimens do not contain sufficient amounts of the infectious virus. Microscopic detection of multinucleated giant cells called Tzanck cells in stained smears made from material from the vesicles allowed for rapid identification of the virus, but this procedure could not distinguish between VZV and HSV. Direct immunofluorescence staining of scrapings from vesicular lesions with monoclonal antibodies directed against VZV antigens provides a rapid, but more sensitive and specific means of detecting the virus. Today, qPCR for VZV DNA is the laboratory method of choice for diagnosing varicella zoster infection because it is highly accurate, sensitive, and rapid. Quantitative PCR is also useful in monitoring the response of immunocompromised patients to antiviral drugs. PCR can be performed on a variety of samples, including vesicular fluid or scabs, skin swabs, throat swabs, cerebrospinal fluid, blood, saliva, and tissues from biopsies or autopsies. Serology testing is of limited use in detecting current infections because accurate detection requires demonstration of a four-fold rise in antibody titer between acute and convalescent samples, a process that takes 2 to 4 weeks to perform. In addition, testing for VZV IgM is not performed routinely for several reasons: IgM antibodies to VZV may not be detectable until the convalescent stage of illness, they cannot distinguish between primary and reactivated infection, and they may not be free of IgG antibodies when serum is processed for testing. In certain cases, IgG avidity assays may be used to differentiate between recent and past infection. Serology is most useful in determining if immunity to VZV is present in certain individuals, such as health-care workers, pregnant women, and patients about to undergo organ transplantation. Therefore, most serology tests detect total VZV antibody, which consists primarily of IgG. Several methods have been developed for this purpose. The most sensitive and reliable method of detecting VZV antibody is a fluorescent test called fluorescent antibody to membrane antigen (FAMA) that detects antibody to the envelope glycoproteins of the virus. Although FAMA is considered to be the reference method for VZV antibody, it requires live, virus-infected cells and is not suitable for large-scale routine testing. The most commonly used method to detect VZV antibodies in the clinical laboratory is the ELISA because it is automated, provides objective results, and does not require viral culture. Although older ELISA methods that use a whole antigen extract are less sensitive than FAMA, a newer ELISA that detects antibody to a highly purified VZV envelope glycoprotein has been shown to have a high level of sensitivity. Despite this improvement, false-positive results can occur because the method can detect low levels of antibodies that do not confer long-term protection to varicella. Other Viral Infections Rubella The rubella virus is a single-stranded, enveloped RNA virus of the genus Rubivirus, belonging to the family Togaviridae. It is transmitted through respiratory droplets or through transplacental infection of the fetus during pregnancy. This virus is the cause of the typically benign, self-limited disease that is also known as German measles or 3- day measles. Before widespread use of the rubella vaccine, this was mainly a disease of young children. However, today it occurs most often in young, unvaccinated adults. Following an incubation period of 12 to 23 days, the virus replicates in the upper respiratory tract and cervical lymph nodes, then travels to the bloodstream. It produces a characteristic erythematous, maculopapular rash, which appears first on the face, then spreads to the trunk and extremities, and usually resolves in 3 to 5 days. In adolescents and adults, this is usually preceded by a prodrome of low-grade fever, malaise, swollen glands, and upper respiratory infection lasting 1 to 5 days. However, up to 50% of rubella infections are asymptomatic. The infection usually resolves without complications, and no specific treatment is available. A significant number of infected adult women experience arthralgias and arthritis, but chronic arthritis is rare. Rubella infection during pregnancy may have severe consequences, including miscarriage, stillbirth, or congenital rubella syndrome (CRS). The likelihood of severe consequences increases when infection occurs earlier in the pregnancy, especially during the first trimester. Infants born with CRS may present with several abnormalities, the most common of which are deafness; eye defects, including cataracts and glaucoma; cardiac abnormalities; mental retardation; liver and spleen damage; and motor disabilities. In mild cases, symptoms may not be recognized until months to years after birth. Scientists developed a vaccine consisting of live, attenuated rubella virus with the primary goal of preventing infection of pregnant women by reducing dissemination of the virus in the population as a whole. The vaccine is part of the routine immunization schedule in infants and children and is usually given in combination with vaccines for measles and mumps (measles/mumps/rubella [MMR] vaccine) and sometimes with varicella (MMRV). Following licensure of the vaccine in 1969, the number of rubella infections and cases of CRS in the United States has dropped dramatically, with only limited outbreaks occurring, mostly among unvaccinated young immigrants to this country. However, rubella and CRS are still important health problems in parts of the world where routine immunization against the virus is not established. Laboratory testing is helpful in confirming suspected cases of German measles because its symptoms may mimic those of other viral infections. It is essential in the diagnosis of CRS and in the determination of immune status in other individuals. Laboratory diagnosis of rubella infection can be accomplished through culture of the virus, demonstration of viral RNA, or detection of virus-specific antibodies. Rubella virus can be grown in a variety of cultured cells inoculated with throat swabs, nasopharyngeal secretions, or other clinical specimens and can be detected in almost all infected infants at the time of birth. However, viral growth is slow and may not produce characteristic CPE upon primary isolation, requiring at least two successive subpassages. In the absence of CPE, viral nucleic acid can be identified by RT-PCR, or viral proteins can be detected by IFA or EIA. Because culture is time consuming and labor intensive, it is increasingly being replaced by molecular methods that are more practical to perform in the clinical laboratory and provide more timely results. The most widely used molecular method is RT-PCR. RT-PCR is a highly sensitive and specific aid in prenatal or postnatal diagnosis and can be used to detect rubella RNA in a variety of clinical samples, including chorionic villi, placenta, amniotic fluid, fetal blood, lens tissue, products of conception, pharyngeal swabs, spinal fluid, or brain tissue. Serology tests are the most common means of confirming a rubella diagnosis because they are rapid, cost effective, and practical in clinical laboratory settings. Several methods have been developed to detect rubella antibodies, including hemagglutination inhibition (HI), latex agglutination, and immunoassays. Although HI was once the standard technique for measuring rubella antibodies, the most commonly used method today is the ELISA because of its sensitivity, specificity, ease of performance, and adaptability to automation. More specific solid- phase capture ELISAs can be used to detect IgM rubella antibodies. Automated chemiluminescence assays and a multiplex bead immunoassay that can simultaneously detect measles, mumps, rubella, and varicella are also available and demonstrate comparable performance with ELISAs. Primary rubella infection is indicated either by the presence of rubella-specific IgM antibodies or by a four-fold or greater rise in rubella-specific IgG antibody titers between acute and convalescent samples collected at least 10 to 14 days apart. The timing of serum collection is important because IgM antibodies to rubella do not appear in many patients until about 5 days after the onset of the rash, whereas IgG antibodies may not be detectable until 8 days after the rash. Only about 50% of patients are positive for IgM antibodies on the day that the rash appears; thus, a false-negative result can occur if the sample is obtained too early. False-positive results can also occur. Although IgM antibodies generally decline by 4 to 6 weeks, they may persist in low levels for a year or more in some cases. False-positive rubella IgM results have also been observed in individuals with parvovirus infections, enterovirus infections, heterophile antibodies, or rheumatoid factor. It is therefore recommended that positive IgM results, particularly in pregnant women, be confirmed by a more specific test, such as an EIA that measures the avidity of rubella IgG antibodies, to distinguish between recent and past rubella infections. In these assays, low antibody avidity indicates a recent infection (with a high risk for CRS), whereas high antibody avidity is seen in past infections, reflecting the normal change in avidity during the course of an immune response. Laboratory diagnosis of congenital rubella infection begins with serological evaluation of the mother’s antibodies and measurement of rubella-specific IgM antibodies in fetal blood, cord blood, or neonatal serum, depending on the age of the fetus or infant. To enhance the reliability of a CRS diagnosis, any positive IgM results should be confirmed by viral culture, RT-PCR–amplification of rubella nucleic acid, or demonstration of persistently high titers of rubella IgG antibodies after 3 to 6 months of age. Serology tests are also used to screen for immunity to rubella in populations such as pregnant women or health- care workers. IgG antibodies provide immunity and persist for life. Rubella-specific IgG antibodies are produced because of natural infection or immunization. An antibody level of 10 to 15 IU/mL is considered to be protective. Rubeola The rubeola virus is a single-stranded RNA virus belonging to the genus Morbillivirus in the Paramyxoviridae family. It is a highly contagious infection that is spread by direct contact with aerosolized droplets from the respiratory secretions of infected individuals. After initial infection of the epithelial cells in the upper respiratory tract, rubeola virus is disseminated through the blood to multiple sites in the body, such as the skin, lymph nodes, and liver. Rubeola virus infection is the cause of the disease commonly known as measles. Following an incubation period of about 10 to 12 days, the virus produces prodromal symptoms of fever, cough, coryza (runny nose), and conjunctivitis, which last 2 to 4 days. During the prodromal period, characteristic areas known as Koplik spots appear on the mucous membranes of the inner cheeks or lips; these appear as gray-to-white lesions against a bright red background and persist for several days. The typical rash of measles appears about 14 days after exposure to the virus and is characterized by an erythematous, maculopapular eruption that begins on the hairline, then spreads to the face and neck and gradually moves down the body to the trunk, arms, hands, legs, and feet (Fig. 23–10). The rash usually lasts 5 to 6 days. FIGURE 23-10 Characteristic rash of measles appearing on the face of a boy. (Courtesy of the Centers for Disease Control and Prevention, Public Health Image Library.) Measles is a systemic infection that can result in complications, including diarrhea, otitis media, croup, bronchitis, pneumonia, and encephalitis. Rarely, a fatal degenerative disease of the CNS, called subacute sclerosing panencephalitis (SSPE), can result from persistent replication of measles virus in the brain. Measles infection during pregnancy can result in a higher risk of premature labor, spontaneous abortion, or low birth weight. The incidence of measles has been greatly reduced in developed nations of the world since the introduction of a live, attenuated measles virus vaccine in 1968. A vaccine consisting of killed rubeola virus was originally licensed in 1963 but was ultimately ineffective because recipients developed a case of atypical measles if they were subsequently infected with the measles virus. The newer vaccine is used in the routine immunization schedule of infants and children, either in combination with rubella and mumps (MMR) or in combination with rubella, mumps, and varicella (MMRV). The recommended administration of the vaccine is in two doses, the first between the ages of 12 and 15 months and the second between ages 4 and 6. Administration of the first dose before the age of 12 months may result in vaccine failure because the presence of maternal antibodies can interfere with the infant’s immune response. The vaccine was considered to be so successful that the CDC and WHO declared measles to be eliminated from the United States in the year 2000 and from the Americas in 2002. However, measles continues to be a global concern, and most cases in the United States and other industrialized nations are brought in by unvaccinated individuals from other countries. Measles outbreaks have occurred in recent years in the United States because some people in the population refuse to become vaccinated or have their children vaccinated on the basis of religious reasons or unfounded fears of vaccine associations with disorders such as autism. According to the CDC, in 2018, 349 individual cases of measles were reported in 26 states and in the District of Columbia. This was the second largest documented number of cases reported since 2000 (first largest number of cases was 667 in 2014). These cases are linked to unvaccinated individuals traveling to the United States, as well as communities of unvaccinated people living in close proximity in the United States. The diagnosis of measles has typically been based on clinical presentation of the patient. However, the success of the U.S. immunization program in reducing the number of measles cases has decreased the ability of some physicians to recognize the clinical features of measles. In addition, atypical presentations of measles can occur in individuals who received the earlier form of the measles vaccine, who have low antibody titers, or who are immunocompromised. Laboratory tests are therefore of value in ensuring rapid, accurate diagnosis of sporadic cases; in addition, they are important for epidemiological surveillance and control of community outbreaks. Isolation of rubeola virus in conventional cell cultures is technically difficult and slow and is not generally performed in the routine diagnosis of measles, but it may be useful in epidemiological surveillance of measles virus strains. The optimal time to recover measles virus from nasopharyngeal aspirates, throat swabs, or blood is from the prodrome period of 3 to 4 days after rash onset. The virus may be isolated from urine up to 1 week after the appearance of the rash. Serological testing provides the most practical and reliable means of confirming a measles diagnosis. In conjunction with clinical symptoms, a diagnosis of measles is indicated by the presence of rubeola-specific IgM antibodies or by a fourfold rise in the rubeola-specific IgG antibody titer between serum samples collected soon after the onset of rash and 10 to 30 days later. SSPE is associated with extremely high titers of rubeola antibodies. IgM antibodies are preferentially detected by an IgM capture ELISA method, which is highly sensitive and has a low incidence of false-positive results. IgM antibodies are detectable by 3 to 4 days after appearance of symptoms and persist for 1 to 2 months. Samples collected before 72 hours may yield false-negative results, and repeat testing is recommended in that situation. A variety of methods have been developed to detect IgG rubeola antibodies, but the most commonly used is ELISA. IgG antibodies become detectable 7 to 10 days after the onset of symptoms and persist for life. The presence of rubeola-specific

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