23

Serological and Molecular Detection of Viral

Infections
Linda E. Miller, PhD, MBCM(ASCP)SI, and Deborah Josko, PhD, MLT(ASCP)M, SM

LEARNING OUTCOMES
After finishing this chapter, you should be able to:
1. Describe the immune defenses that are important in protecting humans from viral infections.
2. Discuss mechanisms by which viruses can escape host defenses.
3. Correlate the presence of viral immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies with their
clinical significance in detecting current infections, congenital infections, or immunity to infections.
4. Discuss the role of molecular tests in diagnosing and monitoring patients with viral infections.
5. Differentiate between the different hepatitis viruses and their modes of transmission.
6. Correlate the various serological markers of hepatitis with their diagnostic significance.
7. Explain the laboratory methods that are most commonly used to screen for, confirm, or monitor hepatitis
virus infections.
8. Associate the following viruses with the specific diseases they cause: Epstein-Barr virus (EBV),
cytomegalovirus (CMV), varicella-zoster virus (VZV), rubella virus, rubeola virus, mumps virus, and the
human T-cell lymphotropic virus type I.
9. Discuss the laboratory methods used to diagnose and monitor infections with the preceding viruses.
10. Correlate the heterophile antibody and EBV-specific antibodies with their clinical significance and describe
the laboratory methods used to test for these antibodies.
CHAPTER OUTLINE
IMMUNE DEFENSES AGAINST VIRAL INFECTIONS
VIRAL ESCAPE MECHANISMS
LABORATORY TESTING FOR VIRAL INFECTIONS
HEPATITIS VIRUSES
Hepatitis A
Hepatitis E
Hepatitis B
Hepatitis D
Hepatitis C
HERPES VIRUS INFECTIONS
Epstein-Barr Virus (EBV)
Cytomegalovirus (CMV)
Varicella-Zoster Virus (VZV)
OTHER VIRAL INFECTIONS
Rubella
Rubeola
Mumps
Human T-Cell Lymphotropic Viruses
SUMMARY
CASE STUDIES
REVIEW QUESTIONS

Go to FADavis.com for the laboratory exercises that accompany this text.

 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 IgG antibodies indicates immunity to measles because of past infection or
immunization. Testing for IgG antibodies is therefore routinely performed on serum samples of individuals such as
health-care workers to determine their immune status.
Molecular methods to detect rubeola RNA can be used in cases in which serological tests are inconclusive or
inconsistent and can be used to genotype the virus in epidemiological studies. The preferred molecular technique is
RT-PCR, performed by traditional or qPCR methodologies. These assays are sensitive, can be performed on a
variety of clinical samples or on infected cell cultures, and can detect viral RNA within 3 days of rash appearance.

Mumps
The mumps virus, similar to rubeola, is a single-stranded RNA virus that belongs to the Paramyxoviridae family
(genus Rubulavirus). It is transmitted from person to person by infected respiratory droplets, saliva, and fomites
and replicates initially in the nasopharynx and regional lymph nodes. (Fomites are inanimate objects or substances
that can transmit infectious organisms.) Following an average incubation period of 14 to 18 days, the virus spreads
from the blood to various tissues, including the meninges of the brain, salivary glands, pancreas, testes, and
ovaries, and produces inflammation at those sites. Inflammation of the parotid glands, or parotitis, is the most
common clinical manifestation of mumps, occurring in 30% to 40% of cases (Fig. 23–11). The illness typically
resolves in 7 to 10 days and does not require therapy other than supportive treatment to alleviate the symptoms.
Mumps infection in pregnant women results in increased risk for fetal death when it occurs in the first trimester of
pregnancy, but it is not associated with congenital abnormalities.
The number of mumps cases in the United States has declined significantly since the introduction of a live,
attenuated mumps virus vaccine in 1967 and its routine use in childhood immunization schedules in 1977. The
vaccine is most commonly combined with the vaccines for rubella and measles (MMR) or is used in combination
with the rubella, measles, and varicella vaccines (MMRV). However, several outbreaks in the United States have
been reported in close-knit communities and on college campuses. From January 1 to December 29, 2018, 47
states and the District of Columbia reported 2,251 cases of mumps to the CDC.
FIGURE 23-11 Parotitis characteristic of mumps. Note the swollen neck region caused by an enlargement of the boy’s salivary glands.
(Courtesy of the Centers for Disease Control and Prevention, Public Health Image Library.)

The diagnosis of mumps is usually made on the basis of clinical symptoms, especially parotitis, and does not
require laboratory confirmation. However, laboratory testing is very useful in cases in which parotitis is absent or
when differentiation from other causes of parotitis is required. Culture of the mumps virus from clinical specimens
is considered to be the gold standard for laboratory confirmation of acute infection. Within the first few days of
illness, the mumps virus can be isolated from saliva, urine, cerebrospinal fluid, or swabs from the area around the
excretory duct of the parotid gland. The preferred specimens are a buccal swab or saliva from the buccal cavity
collected within 3 to 5 days of symptom onset. The specimen can then be used to inoculate cell lines such as
primary monkey kidney cells and Vero cells, which are grown in shell vial cultures and stained with fluorescein-
labeled monoclonal antibodies to identify mumps antigens. However, culture methods require experienced
personnel, and specialized reagents and are being increasingly replaced with molecular detection of viral nucleic
acid.
Standard and real-time RT-PCR methods have been developed to detect mumps virus RNA in specimens
collected from the buccal cavity, throat, cerebral spinal fluid, or urine of patients with a suspected mumps
infection. In many laboratories, RT-PCR is recommended as the primary diagnostic test for mumps because it is
more sensitive than serology. As with culture, buccal swabs collected early in the illness provide the best results,
and false-negative results are frequent in clinical samples collected after 1 week of symptom onset. Genotyping
may be performed to track transmission of the virus during mumps outbreaks.
When indicated, serological testing provides a simple means of confirming a mumps diagnosis, but it has some
important limitations. EIA and IFA antibody kits are used; however, most only measure IgG mumps virus
antibodies. ELISA is the most commonly used method to detect mumps antibodies because it is sensitive, specific,
cost effective, and readily performed by the routine clinical laboratory. Use of solid-phase IgM capture assays
reduces the incidence of false-positive results because of rheumatoid factor. Current or recent infection is indicated
by the presence of mumps-specific IgM antibody in a single serum sample or by at least a four-fold rise in specific
IgG antibody between two specimens collected during the acute and convalescent phases of illness. However,
acute IgG titers are often high, and a four-fold increase in titer may not be evident in the convalescent sample. IgM
antibodies can be detected within 3 to 4 days of illness and can persist for at least 8 to 12 weeks. However, a
negative IgM test does not rule out mumps because negative results can occur if the serum was collected too early
or too late. In addition, individuals who received any doses of the mumps vaccine tend to have lower or absent
IgM antibody. IgG antibodies become detectable within 7 to 10 days and persist for years. However, the presence
of mumps IgG antibodies does not necessarily correlate with the presence of neutralizing antibodies, which would
confer immunity to the virus.

Human T-Cell Lymphotropic Viruses

Human T-cell lymphotropic virus type I (HTLV-I) and human T-cell lymphotropic virus type II (HTLV-II)
are closely related retroviruses. Both viruses have three structural genes: gag, which codes for viral core proteins;
pol, which codes for viral enzymes; and env, which encodes proteins in the viral envelope; as well as a region
called pX, which encodes several regulatory proteins, including Tax and Rex. These viruses have RNA as their
nucleic acid and the enzyme reverse transcriptase, whose function is to transcribe the viral RNA into DNA. The
DNA then becomes integrated into the host cell’s genome as a provirus. The provirus can remain in a latent state
within infected cells for a prolonged period of time. Upon activation of the host cell, the provirus can proceed to
complete its replication cycle to produce more virions. However, HTLV-I and HTLV-II exist predominantly in the
proviral state and are spread directly to uninfected cells through a viral synapse. Additional copies of the viral
nucleic acid are produced when the infected host cells replicate.
The human T-cell lymphotropic viruses preferentially infect CD4+ T lymphocytes but can also infect CD8+ T
cells, dendritic cells, and macrophages. CD8+ CTLs effectively control the proliferation of virus-infected cells in
most individuals. However, inflammatory cytokines released during the immune response may contribute to the
pathogenesis of HTLV-associated diseases. In addition, researchers have reported that HTLV-I infection of CD4+ T
cells can increase the production of proinflammatory cytokines, impair production of Th1 cytokines necessary for
cell-mediated immunity, and induce differentiation of T regulatory (Treg) cells. The differentiated Treg cells can
facilitate viral persistence by suppressing the host’s immune response to the virus. HTLV-I also transforms CD4+
T lymphocytes into malignant cells in a small percentage of individuals through mechanisms mediated by the Tax
protein, resulting in increased cell proliferation and accumulation of harmful genetic mutations.
HTLV-I and HTLV-II can be transmitted by three major routes: bloodborne (mainly through transfusions
containing cellular components or through intravenous drug abuse), sexual contact (most commonly from men to
women), and mother to child (mainly through breastfeeding). HTLV-I infection is endemic in southwestern Japan,
the Caribbean islands, South and Central Africa, the Middle East, Romania, parts of South America, and Papua
New Guinea. Between 5 million and 20 million people are thought to be infected with HTLV-I worldwide.
Infections in the United States and Europe have resulted mainly from immigrants from endemic areas. HTLV-II
infections are highest in various Native Indian populations in the Americas, a few Pygmy tribes in Central Africa,
and intravenous drug abusers in North America and Europe.
HTLV-I is the cause of two diseases: adult T-cell leukemia/lymphoma (ATLL) and HTLV-associated
myelopathy/tropical spastic paraparesis (HAM/TSP). ATLL can be classified into four different subtypes based on
clinical manifestations: acute, T-cell non-Hodgkin’s lymphoma, chronic, and smoldering. Over half of patients
have the acute type, an aggressive variant with a median survival of 6 months. All four types of ATLL are
characterized by a monoclonal proliferation of mature T cells that express the surface markers CD3, CD4, and
CD25. The malignant cells have lobulated, “flower-shaped” nuclei that contain proviral HTLV-I nucleic acid. The
lifetime risk of HTLV-I carriers for developing ATLL is 3% to 5% and is highest in those who acquired the
infection perinatally. The disease typically appears after a latent period of 20 to 30 years following initial infection.
Individuals infected with HTLV-I also have a 4% lifetime risk of developing a progressive neurological disorder
called HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The risk is highest among those
who contracted the infection through sexual transmission. The disease is characterized by slowly progressive
weakness and stiffness of the legs, back pain, and urinary incontinence. HTLV-I has also been associated with a
variety of autoimmune and inflammatory disorders, including uveitis (intraocular inflammation of the eyes),
infective dermatitis, myositis (inflammation of the muscles), and arthropathy (inflammation of the joints);
however, a causal relationship of the virus with these conditions has not been established. It is unclear what factors
influence the development and types of clinical manifestations of HTLV-I infection, but differences in viral strains,
viral load, mode of transmission, HLA haplotypes, and immune responses mounted by the host may all play a role.
The association of HTLV-II infection with disease is unclear, and most individuals infected with the virus are
asymptomatic. However, there is evidence that HTLV-II may rarely be associated with a neurological disease that
is similar to HAM/TSP, as well as certain hematologic and dermatological diseases and increased incidence of
infections. In addition, a close link to cancer mortality in the United States has been identified. This evidence
suggests that more research is needed to understand the pathogenicity of the virus.
Serological testing plays an important role in detecting HTLV-I and HTLV-II infections because culture of the
viruses requires sophisticated techniques that cannot be performed in routine clinical laboratories. HTLV
antibodies develop 30 to 90 days after exposure to the virus and persist for life. Tests for HTLV-I and HTLV-II
antibodies are used to detect HTLV infections in individuals and to screen blood donors. The tests most commonly
used for screening are EIA, CLIA, and IFA methods that incorporate recombinant antigens or synthetic peptides
from both HTLV-I and HTLV-II. Particle agglutination tests have also been developed. Any sample producing a
reactive result in the initial screen is retested by the same method and subsequently tested by a confirmatory
method to reduce the incidence of false-positive results and to distinguish between HTLV-I and HTLV-II infection.
Commercially available Western blot assays are most commonly used for confirmation; line immunoassays
(LIAs) and IFA tests have also been developed. Western blot and LIA identify antibodies to specific HTLV
antigens. Specimens are considered positive if a particular band pattern representing antibodies to the gag and env
proteins of HTLV-I or HTLV-II is obtained. According to criteria published by the WHO, for example, a sample is
considered positive for HTLV-I antibodies if visible bands are produced for one of the env proteins (either gp46 or
gp62/68) and one of the gag proteins (either p19, p24, or p53). A major problem with the Western blot and LIA
methods is that indeterminate results can be obtained when a single band is observed or when multiple bands that
do not meet the criteria for positivity are seen. PCR can be performed to detect HTLV-I or HTLV-II DNA in
provirus-carrying peripheral blood mononuclear cells to clarify repeatedly indeterminate results. PCR methods can
also be used to monitor the proviral load in patients with HTLV-associated diseases during therapy and to
demonstrate the presence of the virus in cells from patients who are suspected of having HTLV-associated disease.

SUMMARY
• Viruses are obligate intracellular pathogens that can produce a wide range of diseases in humans.
• Viruses can exist as either free infectious virions or intracellular particles in infected host cells. These different
states require a combined effort of innate, humoral, and cell-mediated immune responses to successfully
defend the host against viral infections.
• Innate defenses against viruses include the skin and mucous membranes; IFNs to inhibit viral replication; and
NK cells, which release cytotoxic proteins that destroy virus-infected host cells.
• Antibodies directed against specific viral antigens can prevent the spread of viral infection by neutralizing a
virus and preventing it from binding to host cells, opsonizing a virus to make it more likely to be
phagocytized, activating complement-mediated mechanisms of destruction, and agglutinating viruses.
• Cell-mediated immunity is needed to eliminate intracellular viruses. Virus-specific CTLs bind to viral antigen
complexed with class I MHC on the surface of infected host cells and release cytotoxic proteins that cause the
cells to undergo apoptosis.
• Viruses have evolved in several ways to escape the host’s defenses. These include frequent genetic mutations
to produce new viral antigens; evading the action of IFNs, complement, or other components of the immune
system; or suppressing the immune system. Some viruses can establish a latent state by integrating their
nucleic acid into the host’s genome.
• Serological tests for viral antibodies can be used to indicate exposure to a viral pathogen. In general, the
presence of IgM indicates a current or recent infection or a congenital infection, if present in infant serum. The
presence of IgG antibodies indicates previous exposure to a virus or immunity because of vaccination.
• Culture, antigen detection, and molecular methods for viral nucleic acid can be used to directly identify viruses
in clinical samples. Molecular methods have become increasingly important in the diagnosis of viral infections
and can also be used to quantitate viral load to determine the effectiveness of antiviral therapy.
• The hepatitis viruses are those whose primary effect is inflammation of the liver. Hepatitis A and E are
transmitted mainly by the fecal–oral route, whereas hepatitis B, C, and D are transmitted primarily by the
parenteral route. Hepatitis B, C, and D can lead to chronic infections. Vaccines have been developed to prevent
hepatitis A, hepatitis B, and hepatitis E.
• Serological markers of hepatitis infections consist of virus-specific antibodies and antigens that are commonly
detected by automated immunoassays.
• IgM anti-HAV antibodies indicate current or recent hepatitis A infection, whereas IgG anti-HAV antibodies are
developed later in the infection and indicate immunity to hepatitis A. Likewise, IgM anti-HEV antibodies are
present during current or recent hepatitis E infection, and IgG anti-HEV antibodies indicate later infection and
immunity.
• Hepatitis B infection is indicated by the presence of the antigen HBsAg; HBeAg indicates high infectivity.
IgM antibodies to hepatitis B core antigen are present in acute hepatitis B, whereas IgG anti-HBc is present
during past or chronic hepatitis B infection. Antibodies to HBsAg (anti-HBs) indicate immunity and can be
produced because of past hepatitis B infection or immunization with the hepatitis B vaccine.
• The presence of anti-HCV indicates exposure to the hepatitis C virus but cannot distinguish between a current
and a past infection. Molecular tests for HCV RNA are used to confirm antibody-positive results; if positive,
they indicate a current infection. Molecular tests for hepatitis C are also used to quantitate viral load to
determine the effectiveness of antiviral therapy. A third application of molecular testing is genotyping of the
virus to guide decisions about therapy.
• Hepatitis D occurs as a super- or co-infection with hepatitis B and is indicated by antibodies to hepatitis D or
molecular tests to detect HDV RNA. Patients with co-infections are also positive for IgM-anti-HBc, whereas
patients with superinfections are positive for IgG-anti-HBc.
• The Epstein-Barr virus (EBV) is the cause of infectious mononucleosis (IM), Burkitt’s lymphoma, Hodgkin
disease, nasopharyngeal and gastric carcinomas, and lymphoproliferative disorders in immunosuppressed
individuals.
• Most patients with IM produce heterophile antibodies, which can react with antigens from bovine, horse, or
sheep RBCs. These antibodies are routinely screened for by the “Monospot” test, which is performed by rapid
immunochromatographic or agglutination methods to detect antibodies to bovine or horse erythrocyte
antigens.
• ELISA or IFA tests for EBV-specific antibodies are used to confirm a diagnosis of IM, detect heterophile-
negative cases of IM, and diagnose other EBV-associated diseases. Acute IM is indicated by the presence of
IgM anti-VCA and anti-EA-D, as well as the absence of anti-EBNA. Molecular tests are useful in detecting
EBV DNA in immunocompromised patients, who may not develop a good antibody response, and in
monitoring viral load in patients with EBV-related malignancies during therapy.
• Cytomegalovirus (CMV) infection is asymptomatic in most healthy individuals but may cause a
mononucleosis-like syndrome, disseminated infection in organ-transplant recipients and patients with
HIV/AIDS, and congenital abnormalities in infants born to infected mothers.
• CMV infection is best detected by molecular assays for CMV DNA, CMV antigenemia assays for pp65
antigen, or shell vial culture. Quantitative PCR is useful in determining the CMV DNA copy number in
immunocompromised hosts undergoing antiviral treatment. Serological assays for CMV antibody are most
helpful in documenting a past infection in potential blood and organ donors.
• Primary infection with varicella virus causes chickenpox (varicella), whereas reactivation of the virus in nerve
cells supplying the skin causes shingles (zoster). Diagnosis of current varicella virus infection is usually based
on clinical findings, but the detection of varicella virus DNA by PCR may be helpful in some clinical settings.
Serological methods, most commonly ELISA and CLIA, are used mainly to document immunity to varicella
virus.
• Vaccines have greatly reduced the incidence of three childhood infections: rubella, rubeola, and mumps.
Rubella infection is the cause of German measles and can result in severe congenital abnormalities if it occurs
during pregnancy. Rubeola viruses cause measles, a systemic infection that can cause complications in some
individuals. Mumps virus is the cause of mumps, whose classic feature is swelling of the parotid glands.
• Although the diagnosis of rubella, measles, and mumps is usually based on clinical findings, laboratory testing
may be helpful in confirmation. Current infections are indicated by the presence of IgM antibodies specific for
the appropriate virus or by a four-fold rise in virus-specific IgG antibodies in two separate specimens collected
during the acute and convalescent phases of disease. Testing for IgG antibodies is most commonly performed
to screen for immunity to these viruses. RT-PCR is a useful adjunct to serology in detecting viral RNA in
patients with inconclusive serology results, in epidemiological studies, and in the detection of congenital
rubella infections.
• The human T-cell lymphotropic viruses, HTLV-I and HTLV-II, are retroviruses that infect CD4 T lymphocytes.
HTLV-I is the cause of adult T-cell leukemia and lymphoma, HTLV-I-associated myelopathy/tropical spastic
paraparesis (HAM/TSP), and other inflammatory disorders. HTLV-II may be associated with a HAM/TSP-like
neurological disease as well as hematologic and skin disorders, but the disease associations of this virus are
unclear. EIA and CLIA tests are used routinely to screen blood donors for antibodies to HTLV-I and HTLV-II
and to detect exposure to HTLV in other individuals. Positive results are confirmed by Western blot or line
immunoassays. PCR for proviral DNA can be used to clarify indeterminate results and monitor viral load in
patients undergoing therapy.
Study Guide: Immune Defenses Against Viruses
IMMUNE DEFENSE MECHANISM OF ACTION
Innate Defenses First line of protection against viruses
Skin and mucous membranes serve as barriers to prevent viruses from invading the body
Type I IFNs inhibit viral replication
NK cells bind to virus-infected host cells in a manner that is not antigen-specific and destroy the
cells by releasing perforin and granzymes
Humoral Immunity Antibodies bind to cell-free viruses and prevent them from infecting host cells by neutralizing
their ability to bind to host cell receptors
Antibodies also play a key role in opsonization, ADCC, and complement-mediated lysis of
viruses
Cell-Mediated Immunity T lymphocytes attack viruses in their intracellular state
Th1 T helper cells produce cytokines that induce an anti-viral state and stimulate the
development of CTLs
CTLs bind specifically to viral antigens complexed with MHC class I on the surface of virus-
infected host cells and destroy the cells by releasing perforin and granzymes
ADCC = antibody-dependent cellular cytotoxicity; CTL = cytotoxic T lymphocytes; IFN = interferon; NK = natural killer.

Study Guide: Immune Escape Mechanisms Commonly Used by Viruses

VIRAL ESCAPE
MECHANISM EXAMPLES
Acquisition of genetic mutations that Influenza viruses, rhinoviruses, HIV
result in new viral antigens
Inhibition of immunologic components HCV blocks actions of IFNs; HSV inhibits C3b
Suppression of the immune system CMV and HIV reduce expression of class I MHC on the surface of virus-infected cells, reducing
their recognition by CTLs
HIV destroys infected CD4 Th cells
Establishment of a latent state CMV, VZV, and HIV integrate their nucleic acid into the host cell genome
CMV = cytomegalovirus; CTLs = cytotoxic T lymphocytes; HCV = hepatitis C virus; HIV = human immunodeficiency virus; HSV = herpes
simplex viruses; MHC = major histocompatibility complex; VZV = varicella zoster virus.

CASE STUDIES
1. A 25-year-old male had been experiencing flu-like symptoms, loss of appetite, nausea, and constipation for
2 weeks. His abdomen was tender, and his urine was dark in color. Initial testing revealed elevations in his
serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.
Questions
a. What laboratory tests should be used to screen this patient for viral hepatitis?
b. If the patient tested positive for hepatitis B, which tests should be used to monitor his condition?
c. If the patient were to develop chronic hepatitis B, which markers would be present in his serum?

2. A 5-pound infant was born with microcephaly, purpuric rash, low platelet count, cardiovascular defects, and
a cataract in the left eye. The infant’s mother recalled experiencing flu-like symptoms and a mild skin rash
early in her pregnancy. She had not sought medical attention at the time. The infant’s physician ordered tests
to investigate the cause of the newborn’s symptoms.
Questions
a. What virus is the most likely cause of the infant’s symptoms?
b. What laboratory tests would you suggest the doctor order for the mother to support your suggested
diagnosis?
c. What tests should be performed on the infant’s serum to support this diagnosis?
REVIEW QUESTIONS
1. The role of CTLs in immune responses against viruses is to
a. neutralize viral activity.
b. promote destruction of viruses by ADCC.
c. destroy virus-infected host cells.
d. attack free virions.

2. Viruses can escape immune defenses by

a. undergoing frequent genetic mutations.
b. suppressing the immune system.
c. integrating their nucleic acid into the host genome.
d. all of the above.

3. A patient who has developed immunity to a viral infection would be expected to have which of the
following serology results?
a. IgM +, IgG–
b. IgM–, IgG+
c. IgM+, IgG+
d. IgM–, IgG–

4. A newborn suspected of having a congenital viral infection should be tested for virus-specific antibodies of
which class?
a. IgM
b. IgG
c. IgA
d. IgE

5. Which of the following hepatitis viruses is transmitted by the fecal–oral route?

a. Hepatitis B
b. Hepatitis C
c. Hepatitis D
d. Hepatitis E

6. An individual with hepatomegaly, jaundice, and elevated liver enzymes has the following laboratory results:
IgM anti-HAV (negative), HBsAg (positive), IgM anti-HBc (positive), and anti-HCV (negative). These
findings support a diagnosis of
a. hepatitis A.
b. acute hepatitis B.
c. chronic hepatitis B.
d. hepatitis C.

7. The serum of an individual who received all doses of the hepatitis B vaccine should contain
a. anti-HBs.
b. anti-HBe.
c. anti-HBc.
d. anti-HDV.

8. Quantitative tests for HCV RNA are used to

 a. screen for hepatitis C.
b. determine the HCV genotype.
c. differentiate acute HCV infection from chronic HCV infection.
d. monitor hepatitis C patients on antiviral therapy.

9. In the laboratory, heterophile antibodies are routinely detected by their reaction with
a. B lymphocytes.
b. bovine erythrocyte antigens.
c. sheep erythrocyte antigens.
d. Epstein-Barr virus antigens.

10. The presence of IgM anti-rubella antibodies in the serum from an infant born with a rash suggests
a. a diagnosis of measles.
b. a diagnosis of German measles.
c. congenital infection with the rubella virus.
d. passive transfer of maternal antibodies to the infant’s serum.

11. A pregnant woman is exposed to a child with a rubella infection. She had no clinical symptoms but had a
rubella titer performed. Her antibody titer was 1:4. Three weeks later, the test was repeated, and her titer was
1:128. She still had no clinical symptoms. Was the laboratory finding indicative of rubella infection?
a. No, the titer must be greater than 256 to be significant.
b. No, the change in titer is not significant if no clinical signs are present.
c. Yes, a greater-than-four-fold rise in titer indicates early infection.
d. Yes, but clinical symptoms must also correlate with laboratory findings.

12. The cause of shingles is

a. cytomegalovirus.
b. rubella virus.
c. varicella-zoster virus.
d. HTLV-I.

13. The method of choice for detecting VZV infection in immunocompromised hosts is
a. serology to detect virus-specific IgM antibodies.
b. serology to detect virus-specific IgG antibodies.
c. viral culture.
d. (real-time) qPCR.

14. Which of the following is true regarding laboratory testing for mumps?
a. RT-PCR is recommended as the primary diagnostic test.
b. Serology is necessary for confirmation of a suspected clinical case.
c. IgM tests for mumps are highly specific.
d. An acute infection must be confirmed by a four-fold rise in IgG titer.

15. A positive result on a screening test for HTLV-I antibody should be

a. considered highly specific for HTLV-I infection.
b. followed by PCR.
c. confirmed by Western blot.
d. validated by viral culture.