920 The Immunoassay Handbook
Epstein-Barr Virus (EBV)
ETIOLOGIC AGENT AND CLINICAL
Epstein-Barr virus (EBV) was discovered in 1964 and is a
member of the herpesvirus family. Over 95% of the world’s
population has been infected with EBV, making it the most
ubiquitous virus known. Most people are infected as infants
and children in whom the infection may cause a mild sore
throat and minor fever for a few days. Transmission of the
virus requires direct contact with a person shedding virus,
which can be found in saliva. If an individual escapes child-
hood without getting infected (generally only in developed
countries), the primary infection as an adolescent or adult
can be more severe. Infectious mononucleosis (IM) was
ﬁrst recognized as a distinct clinical condition in 1888, but
it was not until 1969 that EBV was identiﬁed as the cause.
About one third of primary infections in adults result in
IM, with clinically apparent IM occurring with a frequency
of about 45 per 100,000. Symptoms include fever, malaise,
swollen lymph nodes, and sometimes splenomegaly. The
situation is very different in individuals who have genetic
or acquired immunosuppression (transplant recipients,
AIDS, X-linked lymphoproliferative syndrome). Some
patients who are apparently immunocompetent may
develop chronic EBV infections, a condition associated
with high viral loads, clonal expansion of EBV-infected T
or Natural killer (NK) cells, and increased morbidity.
EBV is closely associated with several malignancies.
Burkitt lymphoma occurs in about 10 per 100,000 chil-
dren in equatorial Africa and New Guinea. Nasopharyngeal
carcinoma (NPC) constitutes 20% of all cancers in south-
east China. Following receipt of an organ graft, patients
may develop post-transplant lymphoproliferative disor-
der (PTLD), an aggressive neoplasm that almost always
harbors EBV DNA within the neoplastic lymphocytes, and
it is often fatal if not recognized and treated promptly.
DIAGNOSIS AND ASSAY TECHNOLOGY
IM must be distinguished from other more serious condi-
tions that produce similar symptoms. These conditions
include streptococcal tonsillitis, infectious hepatitis, CMV
mononucleosis, Lyme disease, toxoplasmosis, and chronic
lymphatic diseases such as Hodgkin’s disease, lymphoma,
Traditionally, a ﬁrst step in the diagnosis of IM is hema-
tology. Lymphocytosis occurs with an increase in lympho-
cytes and monocytes to 50% or more and atypical
lymphocytes represent 10–20% of the lymphocyte and
In 1932, Paul and Bunnell discovered that heterophile
antibodies in serum from IM patients caused sheep red
blood cells to agglutinate. Inclusion of an absorption step
using guinea pig kidney a few years later improved speci-
ﬁcity. Even though heterophile antibodies have nothing
to do with EBV, the cause of IM, heterophile antibody
tests remain the principal method to diagnose IM and per-
haps the earliest serological test to ﬁnd routine use in the
near-patient setting. Numerous versions of heterophile
antibody tests are available commercially, most using
agglutination of red cells as the end point. Latex aggluti-
nation versions and a few ELISAs are also available.
Analysis of the proteins produced by EBV has led to a
variety of serological tests for the antibodies produced at
various stages of the infection (see Figure 2 in the chapter
DETECTION OF ANTIBODIES RELEVANT TO INFECTIOUS DIS-
EASE). These tests can be used to differentiate acute disease
from prior infection. Tests based on indirect ﬂuorescence
(IFA) have been largely replaced by ELISAs in most labo-
ratories; these tests use recombinant antigens and offer
better reproducibility as well as automation.
It is now recognized that the heterophile antibody test
is indicative of acute disease, but sensitivity is 60–70% at
best. Detection of IgM antibody to the Viral Capsid Anti-
gen (VCA) is the most useful test for acute infection, and
the IgG antibody for VCA is also seen within a few weeks
of the onset of clinical symptoms and typically persists for
life. On the other hand, the IgG antibody against Epstein-
Barr Nuclear Antigen-1 (EBNA-1) is not seen until 2–3
months after infection and is indicative of convalescence
or prior disease. Antibodies against early antigen (EA)
have also been measured; it is proposed that re-appearance
of IgG antibodies against EA may indicate reinfection.
Evaluation of ELISA tests and heterophile antibody meth-
ods have been reported in numerous studies. One evaluation
in 2000 of 12 commercial tests demonstrated discordance for
individual antibody results for numerous samples, although it
is generally possible to distinguish acute from past infection
when a panel of multiple tests is used. Sensitivity of the indi-
vidual tests were >95%. In a 2010 evaluation of three meth-
ods used for IgM testing, with immunoblot as a reference
method, sensitivity of 84–89% and speciﬁcity of 96–98% was
reported. In the last decade, automated multiplex tests have
been established; the availability of multiple antibody results
simpliﬁes the interpretation of a panel of IgM and IgG tests.
In situations where the clinical status is unclear, avidity test-
ing of anti-VCA IgG or anti-EBNA-1 IgG is often helpful.
In areas where nasopharyngeal carcinoma is common, the
measurement of IgA antibodies to VCA and EBNA-1 has
been very helpful to establish that diagnosis.
Virus detection is difﬁcult and rarely done and has little
diagnostic value. Because previously infected people can
periodically shed virus, it is impossible to determine from
the presence of virus alone whether the patient’s symptoms
are due to a primary EBV infection or another cause. PCR
testing is important for immunocompromised patients but
is not used for routine screening. Large changes in viral load
may be indicative of reactivation. Also, PCR testing can be
used to detect EBV in cerebrospinal ﬂuid (CSF), and diag-
nose and monitor patients with PTLD.
Berth, M. and Bosmans, E. Comparison of three automated immunoassay methods
for the determination of Epstein-Barr virus-specific immunoglobulin M. Clin.
Vaccine Immunol. 17, 559–563 (2010).
Bruu, A.L., Hjetland, R., Holter, E., Mortensen, L., Natås, O., Petterson, W., Skar,
A.G., Skarpaas, T., Tjade, T. and Asjø, B. Evaluation of 12 commercial tests for
detection of Epstein-Barr virus-specific and heterophile antibodies. Clin.
Diagn. Lab. Immunol. 7, 451–456 (2000).
Germi, R., Lupo, J., Semenova, T., Larrat, S., Magnat, N., Grossi, L., Seigneurin,
J.M. and Morand, P. Comparison of commercial extraction systems and PCR
assays for quantification of Epstein-Barr virus DNA load in whole blood.
J. Clin. Microbiol. 50, 1384–1389 (2012).
921CHAPTER 9.19 Viral Diseases
Gulley, M.L. and Tang, W. Using Epstein-Barr viral load assays to diagnose,
monitor, and prevent post-transplant lymphoproliferative disorder. Clin.
Microbiol. Rev. 23, 350–366 (2010).
Hess, R.D. Routine Epstein-Barr virus diagnostics from the laboratory perspective:
still challenging after 35 years. J. Clin. Microbiol. 42, 3381–3387 (2004).
Kimura, H., Hoshino, Y., Kanegane, H., Tsuge, I., Okamura, T., Kawa, K. and
Morishima, T. Clinical and virologic characteristics of chronic active Epstein-
Barr virus infection. Blood 98, 280–286 (2011).
Klutts, J.S., Ford, B.A., Perez, N.R. and Gronowski, A.M. Evidence-based
approach for interpretation of Epstein-Barr virus serological patterns. J. Clin.
Microbiol. 47, 3204–3210 (2009).
Odumade, O.A., Hogquist, K.A. and Balfour, Jr. H.H. Progress and problems in
understanding and managing primary Epstein-Barr virus infections. Clin.
Microbiol. Rev. 24, 193–209 (2011).
Okano, M., Thiele, G.M., Davis, J.R., Grierson, H.L. and Purtilo, D.T. Epstein-
Barr virus and human diseases: recent advances in diagnosis. Clin. Microbiol.
Rev. 1, 300–312 (1988).
Paramita, D.K., Fachiroh, J., Haryana, S.M. and Middeldorp, J.M. Two-step
Epstein-Barr virus immunoglobulin A enzyme-linked immunosorbent assay
system for serological screening and confirmation of nasopharyngeal carci-
noma. Clin. Vaccine Immunol. 16, 706–711 (2009).
Paul, J.R. and Bunnell, W.W. The presence of heterophile antibodies in infectious
mononucleosis. Am. J. Med. Sci. 183, 90–104 (1932).
Rea, T.D., Ashley, R.L., Russo, J.E. and Buchwald, D.S. A systematic study of
Epstein-Barr virus serologic assays following acute infection. Am. J. Clin.
Pathol. 117, 156–161 (2002).
Vilibic-Cavlek, T., Ljubin-Sternak, S., Kos, L. and Mlinaric-Galinovic, G. The
role of IgG avidity determination in diagnosis of Epstein-Barr virus infection
in immunocompetent and immunocompromised patients. Acta Microbiol.
Immunol. Hung. 58, 351–357 (2011).
Herpes Simplex Virus (HSV)
ETIOLOGIC AGENT AND PATHOGENESIS
Herpes simplex virus-1 (HSV-1) and HSV-2 are two of the
eight known members of the human herpesvirus family. The
DNAs of HSV-1 and HSV-2 have considerable homology,
and most of the polypeptides expressed by one are also
expressed by the other. The identiﬁcation of speciﬁc glyco-
protein G (gG) sequences in the 1990s permitted serological
differentiation of these two viruses. At one time HSV-1 was
considered essentially an orolabial infection and HSV-2 was
considered a genital infection but these distinctions are less
useful today. HSV-1 infection rates rise steadily through
childhood and more than 90% of the population has acquired
the infection by adulthood. HSV-2 infection rates are closer
to 25% but recurrence is much more common. Prior HSV-1
infection does not offer protection against HSV-2.
HSV-2 is acquired through direct contact with virus shed
from another person. Infections acquired in the absence of
HSV-1 antibody are usually more severe. The onset of
symptoms occur 7–10 days after exposure and the symp-
toms generally last 7–14 days. Reactivation can be triggered
by stress, hormone changes, trauma to the lesion site etc.
Asymptomatic genital infections are a signiﬁcant problem.
In addition, 2–5% of all infected individuals are shedding
virus asymptomatically at any given time. It is estimated
that 70% of HSV-2 transmission occurs during periods of
The most serious consequence of genital herpes infection
is to the newborn at the time of delivery. Untreated neonatal
herpes has a very high mortality rate (80%) with less than
10% of the survivors developing normally. If the risk for
infection is known from the history of the mother, and infec-
tion is identiﬁed early and treated promptly with acyclovir,
the prognosis for the newborn is signiﬁcantly improved.
HSV testing is also required for investigations of central
nervous system (CNS) infections, where all herpes viruses
may be involved but most commonly HSV or varicella
zoster virus (VZV). Herpes simplex encephalitis is the most
common cause of sporadic fatal encephalitis in the West-
ern world; more than 90% of the cases are due to HSV-1.
DIAGNOSIS AND ASSAY TECHNOLOGY
Cell culture is the historic ‘gold standard’ for HSV diagnosis,
with high sensitivity and speciﬁcity. However, turnaround
time even with shell vial assays is 2–3 days.
Immunoassays were developed to provide faster turn-
around times and increased automation. Tests that do not
distinguish HSV-1 and HSV-2 were developed in the
1990s and are still offered commercially, but the use of
type-speciﬁc methods has been recommended by all
experts for the past 10 years. The advantage of HSV
immunology testing is the ability to detect past infection,
in the absence of visible lesions or other symptoms. IgM
tests have been offered but do not add much to sensitivity,
since the IgG response occurs rapidly and persists indeﬁ-
nitely. Point-of-care devices have also been introduced for
HSV-2 testing. Multiplex bead-based tests are available to
support simultaneous detection and differentiation of
HSV-1 and HSV-2. Western blot testing has been widely
used as a reference method for IgG testing and shows good
concordance with current methods.
PCR tests were developed in the early 2000s for diagno-
sis of CNS infections, and also for HSV detection from
swabs from lesions, because of the rapid turnaround time
compared to culture, as well as high sensitivity and speci-
ﬁcity. This method is now available for the diagnosis of
active infection at a variety of sites, and commercial kits are
Ashley, R.L. Performance and use of HSV type-specific serology test kits. Herpes
9, 38–45 (2002).
Binnicker, M.J., Jespersen, D.J. and Harring, J.A. Evaluation of three multiplex
flow immunoassays compared to an enzyme immunoassay for the detection and
differentiation of IgG class antibodies to herpes simplex virus types 1 and 2.
Clin. Vaccine Immunol. 17, 253–257 (2010).
Boivin, G. Diagnosis of herpesvirus infections of the central nervous system. Herpes
11 (Suppl 2), 48A–56A (2004).
Kimberlin, D.W. Neonatal herpes simplex infection. Clin. Microbiol. Rev. 17, 1–13
Laderman, E.I., Whitworth, E., Dumaual, E., Jones, M., Hudak, A., Hogrefe, W.,
Carney, J. and Groen, J. Rapid, sensitive, and specific lateral-flow immuno-
chromatographic point-of-care device for detection of herpes simplex virus
type 2-specific immunoglobulin G antibodies in serum and whole blood. Clin.
Vaccine Immunol. 15, 159–163 (2008).
Roett, M.A., Mayor, M.T. and Uduhiri, K.A. Diagnosis and management of genital
ulcers. Am. Fam. Physician. 85, 254–262 (2012).
Strick, L. and Wald, A. Type-specific testing for herpes simplex virus. Expert Rev.
Mol. Diagn. 4, 443–453 (2004).
Strick, L.B. and Wald, A. Diagnostics for herpes simplex virus: is PCR the new gold
standard? Mol. Diagn. Ther. 10, 17–28 (2006).
ETIOLOGIC AGENT AND PATHOGENESIS
The four serotypes of dengue virus are members of the Fla-
viviridae family. They have type-common epitopes present
on the viral glycoproteins, making accurate serologic diag-
nosis difﬁcult. Infection with one serotype does provide
lifelong immunity to reinfection with that same serotype,
922 The Immunoassay Handbook
but provides only partial cross-protective immunity to the
other three serotypes.
The dengue viruses can be transmitted to humans via a
number of mosquito species in the genus Aedes. The prin-
cipal vector is Aedes aegypti, a small, highly domesticated
tropical mosquito that lays its eggs in artiﬁcial containers
found in and around homes. If the geographic distribution
of the secondary vector Aedes albopictus is included, nearly
two thirds of the world’s population is at risk for infection.
The mobility of the human population has led to the
introduction of new serotypes into new regions of the world.
During the 1960s, for example, only dengue virus types 2
and 3 were present in America. Dengue type 1 was intro-
duced in 1977, it rapidly spread and became endemic in the
region. These hyperendemic regions, where multiple strains
of dengue virus are circulating, have led to the emergence
and spread of dengue hemorrhagic fever (DHF).
Uncomplicated, classic dengue fever develops 3–14 days
after being bitten by an infected mosquito. Primary dengue
infections are always self-limiting and the disease resolves
within two weeks of onset. Dengue fever is primarily
observed in older children and adults.
DHF and dengue shock syndrome (DSS), in con-
trast, are predominantly infections seen in children less
than 15 years of age. The acute phase of DHF is almost
indistinguishable from the acute phase of dengue fever, or
the acute phase of any number of illnesses common in
dengue endemic regions. In contrast to dengue fever,
DHF and DSS can progress rapidly, resulting in death
within 8–24h of onset of symptoms. With early recogni-
tion of infection and aggressive supportive therapy, par-
ticularly with ﬂuid and electrolyte replacement, starting
with saline then on to plasma and plasma expanders in
more severe cases, the fatality rate can be reduced to less
than 1%. Once the shock is overcome, recovery is rapid,
usually within 2–3 days.
The majority of cases of DHF appear to be the result of
a secondary infection with a dengue serotype different
than that which caused the primary dengue fever infec-
tion. While protective antibody was made to the ﬁrst virus,
cross-reactive antibodies do not neutralize other strains of
dengue, thus permitting infection of the second strain.
The cross-reactive antibody binds to the virus enhancing
uptake into leukocytes through normal Fc receptor bind-
ing and uptake functions. This phenomenon is called the
Dengue Antibody-Dependent Enhancement.
DIAGNOSIS AND ASSAY TECHNOLOGY
Traditional serologic methods of hemagglutination inhi-
bition (HI), complement ﬁxation, and neutralization have
been largely supplanted by a variety of enzyme immunoas-
say (ELISA) formats. One of the most widely used formats
is an IgM capture ELISA. In primary infection, IgM is
present in 80% of patients’ sera by day 5 of illness, increas-
ing to 93% by day 10 and 99% by day 20. For this assay,
IgM from the patient sera is captured on the solid support
by anti-IgM antibodies coated to the support. Once cap-
tured, dengue antigen is added with enzyme-labeled
monoclonal antibody to the antigen.
ELISAs to detect IgG have also been developed and are
best used to assess previous exposure to dengue virus. IgG
testing alone is not useful for assessing acute disease unless
both an acute and a convalescent serum sample are avail-
able, to demonstrate a signiﬁcant increase in antibody titer.
Ratios of IgM over IgG are often used to differentiate pri-
mary and secondary dengue infection. IgG avidity tests can
also be used to distinguish active from past infection, but
this approach is not widely performed.
In addition to the standard microtiter formats, rapid
immunochromatographic assays have been developed to
simplify testing in poor resource settings. For this for-
mat, a drop of patient serum or blood migrates along a
nitrocellulose strip and the IgG and IgM are captured on
separate bands where anti-human IgG or anti-IgM anti-
bodies have been applied. Diagnosis of a primary dengue
infection is made if a band appears for the IgM line, but
not the IgG line. Secondary dengue infection is deﬁned
by the presence of IgG with or without IgM. There are
certain limitations to this approach. The sample during
primary infection must be late enough after infection to
have detectable IgM, but early enough not to have detect-
able IgG, a window of about 5–10 days. The presence of
IgG must also be viewed cautiously. The IgG is indica-
tive of previous exposure to dengue virus, but its pres-
ence does not necessarily prove that the set of symptoms
the patient is presenting with is a secondary dengue
In the past decade, tests for the NS1 antigen of dengue
have been introduced. This antigen, present at high con-
centrations and readily detected at the initial onset of fever,
can be used to diagnose dengue more quickly than other
protein markers. Both sandwich ELISA and strip tests are
available to detect NS1. Some studies have suggested that
NS1 antigen is the most sensitive marker of disease, with
up to 92% sensitivity. However, after the early acute phase,
NS1 concentration decreases and the test is less useful for
the detection of acute Dengue fever. A combination of
NS1 and IgM detection has therefore been recommended
to cover a wider range of patients, especially when the time
since disease onset may not be known. Use of NS1, IgM,
and IgG testing together may also be useful in differentiat-
ing acute secondary infection. Reverse transcriptase-
polymerase chain reaction (RT-PCR) methods are
available but have not been as sensitive as immunological
tests in published evaluations and they are not as easily
performed in endemic regions.
Blacksell, S.D., Jarman, R.G., Gibbons, R.V., Tanganuchitcharnchai, A., Mammen,
Jr. M.P., Nisalak, A., Kalayanarooj, S., Bailey, M.S., Premaratna, R., de Silva,
H.J., Day, N.P. and Lalloo, D.G. Comparison of seven commercial antigen and
antibody enzyme-linked immunosorbent assays for detection of acute dengue
infection. Clin. Vaccine Immunol. 19, 804–810 (2012).
Chua, K.B., Mustafa, B., Abdul Wahab, A.H., Chem, Y.K., Khairul, A.H.,
Kumarasamy, V., Mariam, M., Nurhasmimi, H. and Abdul Rasid, K. A com-
parative evaluation of dengue diagnostic tests based on single acute serum
samples for laboratory confirmation of acute dengue. Malays. J. Pathol. 33,
Gubler, D.J. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 11, 480–
Guzman, M.G., Halstead, S.B., Artsob, H., Buchy, P., Farrar, J., Gubler, D.J.,
Hunsperger, E., Kroeger, A., Margolis, H.S., Martínez, E., Nathan, M.B.,
Pelegrino, J.L., Simmons, C., Yoksan, S. and Peeling, R.W. Dengue: a continu-
ing global threat, Nat. Rev. Microbiol. 8 (Suppl) (2010). S7–16.
Halstead, S. Pathogenesis of dengue: challenges to molecular biology. Science 239,
923CHAPTER 9.19 Viral Diseases
Peeling, R.W., Artsob, H., Pelegrino, J.L., Buchy, P., Cardosa, M.J., Devi, S.,
Enria, D.A., Farrar, J., Gubler, D.J., Guzman, M.G., Halstead, S.B.,
Hunsperger, E., Kliks, S., Margolis, H.S., Nathanson, C.M., Nguyen, V.C.,
Rizzo, N., Vázquez, S. and Yoksan, S. Evaluation of diagnostic tests: dengue.
Nat. Rev. Microbiol. 8 (Suppl), S30–S38 (2010).
Whitehorn, J. and Simmons, C.P. The pathogenesis of dengue. Vaccine 23, 7221–
ETIOLOGIC AGENT AND PATHOGENESIS
Rubella virus (also known as German measles) is the sole
member of the genus Rubivirus within the Togaviridae
family. Although experimental infections can be induced
in a variety of laboratory animals, man is the only known
natural reservoir for rubella virus.
Postnatal primary infections, acquired by inhalation of
aerosols, are nearly always mild. Within a week after
exposure, virus can be found in both the blood and naso-
pharynx; the latter being responsible for the spread of the
virus from one individual to another. Symptoms of pri-
mary rubella infection may include a rash (the hallmark
of clinical rubella), low-grade fever, lymphadenopathy,
sore throat, conjunctivitis, and arthralgia. Joint involve-
ment is very common in adults, particularly among
women. Serious sequelae are rare but CNS involvement
and thrombocytopenia have been reported.
Primary rubella infection during pregnancy can be devas-
tating. Viremia associated with primary infection leads to
infection of the placenta and fetus, which can lead to fetal
death. Those infants surviving fetal infection may exhibit
one or more of a variety of symptoms known collectively as
congenital rubella syndrome (CRS), which include low
birth weight, deafness, eye disease, mental retardation, car-
diac abnormalities, hepatomegaly, splenomegaly, and throm-
bocytopenia. Virus may be isolated from virtually every
organ and is shed in urine and nasopharyngeal secretions; a
few infants will continue to shed virus for well over a year.
Infants born without apparent symptoms may develop
late-onset disease, months to years later, which includes
hearing loss, mental retardation, retinopathy, and diabetes
mellitus. The risk of CRS or late-onset disease is as high as
85% if infection occurs during the ﬁrst trimester, falls to
10–24% during the 13th to 16th weeks and approaches 0%
beyond the 20th week of gestation. The introduction of a
vaccine in 1969 has greatly reduced the risk of rubella dur-
ing pregnancy in developed countries, but it is remains a
signiﬁcant problem in other areas.
DIAGNOSIS AND ASSAY TECHNOLOGY
Serology plays a critical role in the diagnosis of rubella for
two main reasons. First, 30% of primary infections are
subclinical; and second, other rash-inducing illnesses may
confuse a diagnosis of acute, primary rubella infection.
Infection with parvovirus B19 is frequently impossible to
distinguish from rubella; it is nonteratogenic but is associ-
ated with a high incidence of miscarriage. HHV 6 can also
cause rash and fever in children. In many parts of the trop-
ics, alpha viruses and ﬂaviviruses, including Dengue, may
induce rubella-like illness. A primary immune response,
consisting both of IgG and IgM antibodies, can be detected
at about the same time as the rash. IgM antibody usually
falls to undetectable or very low levels within 4–6 months;
however, in some cases, it may remain at detectable levels
for a year or more. IgG antibody may eventually decline to
a low level but lasts indeﬁnitely.
Diagnosis of rubella infection and the determination of
an individual’s immune status are accomplished with sero-
logical assays detecting antibody in serum samples. Hem-
agglutination inhibition (HAI) has largely been replaced
by passive enzyme-immunoassays. Some ELISAs provide
quantitative results expressed in international units (IU) of
antibody per mL referenced to the World Health Organi-
zation International Standard for Anti-Rubella Serum.
Use of the appropriate assay provides data required in
the three principal areas of rubella serodiagnosis: immune
status screening, detection of recent primary infection, and
diagnosis of congenital rubella syndrome.
Immune Status Screening
It is generally agreed that levels of antibody detectable by
HAI, about 10–15IU/mL, are protective. Antibodies at or
above these levels are sufﬁcient to protect an individual from
clinical reinfection; levels of antibody below 10–15IU/mL
may not prevent clinical rubella after exposure. CRS in
babies born to mothers with detectable antibody prior to
conception has been reported but is very rare.
Today IgG tests are widely used to establish immune
status. Sensitivity is typically 99%. Speciﬁcity is harder to
deﬁne, since low levels that were below the detection limit
of HAI are observed, with considerable variability, by the
more sensitive modern methods.
Serodiagnosis of Recent Primary Infection
Historically, primary infection was established using two
serum samples. The ﬁrst sample was drawn from the patient
during the acute stage of the disease and a second, the con-
valescent sample, about 10–14 days later. A fourfold, or
greater, rise in HAI titer indicated recent exposure. IgG-
speciﬁc ELISAs available today may be used in a similar
manner if the equivalent of a fourfold rise in HAI titer for
that assay has been deﬁned. However, paired sera testing
only differentiates primary from secondary rubella infec-
tions if the acute sample is seronegative and the convalescent
sample is positive, demonstrating seroconversion.
Primary infection induces both an IgG and a signiﬁcant
IgM antibody response. The secondary immune response
induced by secondary rubella infection is characterized by
rising IgG antibody in the absence of signiﬁcant IgM anti-
body. The presence of clinically signiﬁcant levels of IgM
antibody is serodiagnostic evidence of recent, primary
rubella infection. Because of the relatively short duration
of IgM antibody, the time at which a sample is drawn after
exposure is critical. IgG avidity has also been reported as
an aid to differentiate primary and secondary infections
and is available in a commercial kit.
Serodiagnosis of Congenital
Maternal IgM antibody induced by primary rubella infec-
tion does not cross the intact placenta as does maternal
924 The Immunoassay Handbook
IgG. The infected fetus develops IgM antibody to the
virus and detection of that antibody in the neonate aids in
the diagnosis of CRS. A sample should be drawn from the
neonate as soon as possible following parturition. Com-
parison of IgG antibody in the neonate at the time of birth
and 6 months later also aids in the diagnosis of CRS. A
signiﬁcant drop in antibody over that time interval sug-
gests decreasing maternal antibody in the absence of peri-
natal or prenatal infection. Pre- or perinatal infection
should be suspected if stable or increasing IgG antibody
levels are detected.
Little data are available comparing Rubella RNA detec-
tion by PCR with IgM detection, since outbreaks are rare.
One 2008 study in Peru reported that IgM testing is more
sensitive for samples collected at the time of rash onset but
gave equivalent performance after 3–4 days. Since virus
levels in blood drop rapidly after rash onset, PCR may be
less useful to establish this diagnosis.
Abernathy, E., Cabezas, C., Sun, H., Zheng, Q., Chen, M.H., Castillo-Solorzano, C.,
Ortiz, A.C., Osores, F., Oliveira, L., Whittembury, A., Andrus, J.K., Helfand, R.F.
and Icenogle, J. Confirmation of rubella within 4 days of rash onset: comparison
of rubella virus RNA detection in oral fluid with immunoglobulin M detection in
serum or oral fluid. J. Clin. Microbiol. 47, 182–188 (2009).
Banatvala, J.E. and Brown, D.W. Rubella. Lancet 363, 1127–1137 (2004).
Dimech, W., Panagiotopoulos, L., Francis, B., Laven, N., Marler, J., Dickeson, D.,
Panayotou, T., Wilson, K., Wootten, R. and Dax, E.M. Evaluation of eight
anti-rubella virus immunoglobulin g immunoassays that report results in inter-
national units per milliliter. J. Clin. Microbiol. 46, 1955–1960 (2008).
Morice, A., Ulloa-Gutierrez, R. and Avila-Agüero, M.L. Congenital rubella syn-
drome: progress and future challenges. Expert Rev. Vaccines 8, 323–331 (2009).
Vauloup-Fellous, C., Ursulet-Diser, J. and Grangeot-Keros, L. Development of a
rapid and convenient method for determination of rubella virus-specific immu-
noglobulin G avidity. Clin. Vaccine Immunol. 14, 1416–1419 (2007).
Mumps, measles, and varicella-zoster virus infections are
typically clinical diagnoses and laboratory tests are rarely
required. Although at one time nearly eradicated, mumps
and measles outbreaks now regularly occur in developed
countries, as many families choose to decline vaccination.
IgG testing is important to establish immunity, especially for
healthcare workers; ELISAs and multiplex methods (which
also measure rubella) are widely available for this purpose.
IgM testing is available by ELISA but cannot be used for
screening since the incidence of these diseases is so low and
the positive predictive value is poor. IgM can be useful in
tracking an outbreak and IgG avidity testing has been used to
identify vaccination failures. PCR testing is also available for
identifying active infections but is not widely employed.
Arvin, A.M. Varicella-zoster virus. Clin. Microbiol. Rev. 9, 361–381 (1996).
Hviid, A., Rubin, S. and Mühlemann, K. Mumps. Lancet 371, 932–944 (2008).
Mosquera, M.M., de Ory, F., Gallardo, V., Cuenca, L., Morales, M., Sánchez-Yedra,
W., Cabezas, T., Hernández, J.M. and Echevarría, J.E. Evaluation of diagnostic
markers for measles virus infection in the context of an outbreak in Spain. J. Clin.
Microbiol. 43, 5117–5121 (2005).
Moss, W.J. and Griffin, D.E. Measles. Lancet 379, 153–164 (2012).
Park, D.W., Nam, M.H., Kim, J.Y., Kim, H.J., Sohn, J.W., Cho, Y., Song, K.J. and
Kim, M.J. Mumps outbreak in a highly vaccinated school population: assess-
ment of secondary vaccine failure using IgG avidity measurements. Vaccine 25,
Tipples, G. and Hiebert, J. Detection of measles, mumps, and rubella viruses.
Methods Mol. Biol. 665, 183–193 (2011).
Human T-Cell Leukemia
Human T-cell leukemia virus (HTLV-1) was the ﬁrst
human retrovirus discovered, and shortly thereafter,
HTLV-2 was described. It is estimated that 20million
people worldwide are infected with HTLV-1, although
90% are asymptomatic. HTLV-1 infection can result in
subclinical immunosuppression such that infected individ-
uals are at increased risk for development of opportunistic
coinfections, including but not limited to strongyloidiasis
and tuberculosis. Approximately 5% of infected individu-
als will go on to develop HTLV-1 associated manifesta-
tions, typically following an incubation period of 10–40
years. Adult T-cell leukemia/lymphoma is an aggressive
lymphoma of the skin and most viscera, including the liver,
spleen, and lymph nodes. The other major clinical mani-
festation is HTLV-1 associated myelopathy (HAM) or
tropical spastic paraparesis (TSP) which is a chronic
neurological disorder due to progressive demyelination in
the spinal cord. HTLV-1 uveitis and dermatological con-
ditions have also been reported. HTLV-2 causes hairy
T-cell leukemia, although this is rare.
Japan, Africa, Central and South America, and the
Caribbean islands are the areas with the highest prevalence
of HTLV-1. The most common routes of HTLV-1 trans-
mission are sexual transmission, intravenous drug abuse,
and blood transfusion. Mother to child transmission via
breastfeeding is also considered an important route of
The risk of HTLV-1 transmission during blood trans-
fusion varies with the prevalence of the virus in the speciﬁc
blood donor population, but prevention of transfusion-
associated HTLV-1 transmission has been a major focus
of public health efforts. Programs throughout the world
have been initiated to screen blood donors to prevent
transmission of HTLV-1 via blood transfusion. The Cen-
ters for Disease Control and Prevention recommended
screening for HTLV-1 in 1988, and several countries fol-
lowed with similar recommendations in subsequent years.
In the United States, all blood donors have been routinely
screened for both HTLV-1 and HTLV-2 antibodies since
1997. As a result of widespread donor screening, the resid-
ual risk of transfusion-associated HTLV-1 infection in low
prevalence countries is now minimal.
The most common screening tests for HTLV are
enzyme immunoassays (EIA) and particle agglutination
(PA) assays performed on serum or plasma. Typically the
EIA assays detect HTLV-1 and -2, while the PA is
HTLV-1 speciﬁc. PA assays are somewhat more subjective
as the interpretation is a visual interpretation by the test
operator. In addition, there are no FDA licensed PA assays,
so this test is not used in the United States. Modern EIA
assays use either HTLV-1 and -2 infected cell lysates or
recombinant antigens, and have excellent sensitivity and
925CHAPTER 9.19 Viral Diseases
speciﬁcity. Analysis of commercially available screening
assays for HTLV-1 and HTLV-2 suggests that sensitivity
ranges from 98 to 100% and speciﬁcity ranges from 90 to
100%. A positive screening test should be followed with a
more speciﬁc conﬁrmation test; the assays most commonly
used for this purpose are western blot or immunoﬂuores-
cence assays. These assays are also used to differentiate
HTLV-1 from HTLV-2. The most commonly used ver-
sion of the western blot is a “second generation” western
blot. Specimens that react with p19gag, r21e, and recom-
binant gp461 are typical for HTLV-1, and specimens that
are positive for reactivity with p24 gag, r21e, and gp46II
are consistent with HTLV-2 infection. Specimens without
reactivity to any of the western blot antigens are consid-
ered to be negative for HTLV, consistent with a false-
positive EIA screening assay.
If indeterminate results are obtained during conﬁrma-
tory testing, these are most commonly due to the individual
being in the “window period” early in infection before fully
seroconverting, or a false-positive screening EIA result due
to a non-speciﬁc reaction of the patient’s serum to viral
antigens. These indeterminate results are typically resolved
using PCR for the detection of HTLV-1 proviral DNA.
Andersson, S., Thorstensson, R., Ramirez, K.G., Krook, A., von Sydow, M., Dians,
F. and Biberfeld, G. Comparative evaluation of 14 immunoassays for detection
of antibodies to the human T-lymphotropic virus types I and II using panels of
sera from Sweden and West Africa. Transfusion 39, 845–851 (1999).
Goncalves, D.U., Proiette, F.A., Ramos Ribas, J.O.G., Grossi, M., Pinheiro, S.R.,
Guedies, A.C. and Carneiro-Proiettei, A.B.F. Epidemiology, Treatment, and
Prevention of Human T-Cell Leukemia Virus 1-associated diseases. Clin.
Micro. Rev. 23, 577–589 (2010).
Guidance for Industry Donor Screening for Antibodies to HTLV-2 http://www.fda.gov/
Information/Guidances/Blood/UCM170916.pdf (1997) .
Qiu, X., Hodges, S., Lukaszewka, T., Hino, S., Arai, H., Yamaguchi, J., Swanson,
P., Schochetman, G. and Davare, S.G. Evaluation of a new, fully automated
immunoassay for detection of HTLV-1 and HTLV-2 antibodies. J. Med. Virol.
80, 484–493 (2008).
Thorstensson, R., Albert, J. and Anderson, S. Strategies for diagnosis of HTLV-1
and -2. Transfusion 42, 780–791 (2002).
Watanabe, T. Current Status of HTLV infection. Int. J. Hematol. 94, 430–434
Parvovirus B19 is a single-stranded, nonenveloped Eryth-
rovirus. The virus is so named because of its capacity to
autonomously replicate in erythroid precursor cells. B19
is found worldwide and has been associated with a late
winter, spring, and early summer seasonality. The virus
is spread from person to person by droplets containing
viral particles which are produced during a cough or a
Parvovirus B19 disease manifests in several different
ways depending on the immune status of the host. The
most common manifestation of disease is erythema infec-
tosum (EI) or Fifth’s Disease which primarily affects
immunocompetent school-aged children. Parvovirus B19
also causes EI in adults but has also been associated with
joint manifestations in these patients. Only a minority of
adults experiences the facial exanthema (rash) seen in chil-
dren. Parvovirus can also cause transient aplastic crisis in
patients with low erythrocyte counts. This can lead to a
potentially life-threatening drop in hemoglobin. Immuno-
compromised patients lacking functional humoral immu-
nity can be persistently infected with parvovirus B19,
leading to bone marrow suppression and chronic anemia.
Lastly, parvovirus can cause fetal hydrops when nonim-
mune pregnant women get their primary infection in the
ﬁrst or second trimester.
Molecular and serological methodologies exist for diag-
nosing parvovirus infection, and their respective utilities are
acid ampliﬁcation techniques can be either qualitative or
quantitative and detect viral DNA. Serologic tests detect
B19-speciﬁc IgM and IgG antibody directed against viral
capsid antigen. In the United States, these assays are most
commonly EIA, but IFA and western blot assays do exist.
PCR is useful in patients with aplastic crisis because
high levels of circulating virus are usually present. How-
ever, PCR is rarely positive in children with EI and serol-
ogy is the preferred method of diagnosis. EI patients will
generally have detectable IgM within 7–10 days of expo-
sure, which remains detectable for 2–3 months but can
sometimes be found out to 6 months postinfection. There-
fore, presence of IgM is suggestive but not absolutely diag-
nostic for acute infection. Demonstrating a ≥4-fold rise
in IgG titer can also be used to diagnose acute infection.
In immunocompromised patients with chronic infection
and sustained erythropenia, parvovirus IgM and IgG are
typically not detectable and PCR is preferred. It is impor-
tant to note that levels of viremia can be low in these
patients so PCR sensitivity varies.
The most important role for parvovirus serology is in
screening pregnant women. Parvovirus naive women who
acquire their primary parvovirus B19 infection have about
a 30% risk of vertical transmission to the baby. The risk
appears to be greatest if the infection occurs during the
ﬁrst or second trimester. Pregnant women who are exposed
develop signs and symptoms of parvovirus B19 infection,
and IgG and IgM tests should be carried out for evidence
of parvovirus infection. IgG positive, IgM negative results
suggest past infection and that the mother is immune to
future parvovirus infection. IgG negative, IgM positive
may suggest acute infection or a falsely positive IgM result.
During acute infection, there is only a short window of
time when IgM is positive and IgG is negative. Thus, this
is a relatively rare ﬁnding. Pregnant women should have
repeat titers drawn in 1–2 weeks, and if IgG seroconver-
sion occurs, this is suggestive of an acute infection. If both
IgG and IgM are negative, the IgM was likely a false-
positive. Nucleic acid ampliﬁcation assays performed on
amniotic ﬂuid are also occasionally employed in this situa-
tion to help resolve serological ﬁndings.
Dijkmans, A.C., de Jong, E.P., Dijkmans, B.A., Loprior, E., Vossen, A., Walther,
F.J. and Oepkes, D. Parvovirus B19 in pregnancy: prenatal diagnosis and man-
agement of fetal complications. Curr. Opin. Obstet. Gynecol. 24, 95–101 (2012).
De Jong, E.P., de Haan, T.R., Kroes, A.C., Beersma, M.R., Oepkes, D. and
Wlahter, F.J. Parvovirus B19 infection in pregnancy. J. Clin. Virol. 36, 1–7
De Jong, E.P., Wlather, F.J., Kroes, A.C. and Oepkes, D. Parvovirus B19 infection
in pregnancy: new insights and management. Prenat. Diagn. 31, 419–425 (2011).
926 The Immunoassay Handbook
West Nile Virus
West Nile Virus (WNV) is a member of the Japanese
encephalitis serogroup in the family Flaviviridae. It is an
enveloped, single-stranded, positive-sense RNA virus. WNV
is transmitted primarily between birds and mosquitoes, and
while it can infect a wide variety of bird species, the Passeri-
formes (which includes crows, blackbirds, ﬁnches, and spar-
rows amongst others) appear to be a particularly important
reservoir for disease. In contrast to birds, infection in humans
results in a much lower viremia and therefore, disease is not
normally transmitted from humans.
Based on epidemiological surveys, most infections are
asymptomatic in humans. The incubation period for WNV
disease is 2–14 days, after which time symptomatic indi-
viduals will develop some or all of the following symptoms:
high fever with headache, myalgias, arthralgias, and eye-
pain. Symptoms present acutely and may also include a
rash, hepatomegaly, and splenomegaly. Patients who will
go on to develop neurologic symptoms, experience a febrile
prodrome period of 1–7 days prior to onset of neurologic
symptoms. WNV neurologic symptoms can manifest as
either encephalitis (~66%) or meningitis (~33%).
WNV infection is primarily diagnosed using serologic
methods. Direct detection via PCR of blood or cerebrospi-
nal ﬂuid is usually negative because viral loads are low by the
time patients develop symptoms. One study estimated that
PCR-based detection only captured 55% of infections. The
diagnostic test of choice is the detection of WNV-speciﬁc
IgM antibody in either the serum or CSF. IgM detected in
the CSF is diagnostic for neurologic infection. Many
patients will have positive serology at the time of admission
(~50%) and nearly all will be positive 7 days post-admission.
The preferred method of detecting West Nile speciﬁc IgM
is an IgM-speciﬁc ELISA otherwise referred to as the MAC
ELISA (MAC: IgM Antibody Capture). This assay is more
than 95% sensitive if done on samples collected between 7
and 10 days after the onset of illness. In cases of suspected
encephalitis it is important to analyze both the serum and
CSF, as CSF IgM antibodies may develop earlier. Although
sensitivity for infection is very high, IgM antibodies to
WNV can linger in the serum for up 16 months, which lim-
its speciﬁcity. This assay can cross-react with other ﬂavivi-
ruses and so the CDC recommends that positive results be
conﬁrmed with a neutralization test. The plaque reduction
and neutralization test (PRNT) is a speciﬁc test that allows
for identiﬁcation of virus speciﬁcity.
Solomon, T., Ooi, M.H., Beasley, D.W. and Mallewa, M. West Nile encephalitis.
BMJ 326, 865–869 (2003).
Centers for Disease Control and Prevention. Epidemic/Epizootic West Nile Virus in the
United States: Guidelines for Surveillance, Prevention and Control. (2003) 3rd Revision.
De Filette, M., Ulbert, S., Diamond, M. and Sanders, N.N. Recent progress in
West Nile virus diagnosis and vaccination. Vet. Res. 43 (2012).
Rotavirus is a double capsid and double-stranded segmented
RNA virus in the family Reoviridae, with at least 7 distinct
antigenic groups (A through G). Group A are the major
cause of rotavirus diarrhea worldwide. Infection with rotavi-
rus causes nonbloody diarrhea, often preceded or accompa-
nied by fevers, and symptoms generally last 3–8 days.
Rehydration therapy may be required for patients with
severe infection. The most common patient population
affected is children less than 5 years of age. While mortality
due to Rotavirus is very low in developed nations, in devel-
oping countries Rotavirus is a major cause of mortality. The
route of infection is fecal–oral; infection typically results
from direct or indirect contact with infected people. Rota-
virus is present at a high titer in stools of infected patients
and can persist in the stool for as long as 21 days after the
onset of symptoms in immunocompetent hosts (longer in
immunocompromised). Rotavirus can be found on toys and
hard surfaces in day care centers; spread within families and
institutions is common. In temperate climates, Rotavirus is
most prevalent during the cooler months. Seasonal varia-
tion is less pronounced in tropical climates. No speciﬁc
antiviral therapy exists for rotavirus. Fluid replacement can
be given to prevent dehydration, especially in infants.
The monovalent rotavirus vaccine (RV1; Rotarix) and
pentavalent rotavirus vaccine (RV5; RotaTeq) have been
approved for use in many countries. Widespread vaccina-
tion has dramatically altered the epidemiology of rotavirus
in areas where vaccination has occurred. Prior to the vac-
cine, virtually all children were infected by 3 years of age.
Historically, electron microscopy was used to identify
Rotavirus in stool. Today EIA and latex agglutination
assays for the detection of rotavirus antigen in stool are
commercially available and commonly used for detecting
rotavirus infection. In addition to improved sensitivity
(close to 100%) these immunoassays are much easier to
perform than electron microscopy, result in improved
turnaround time, and are considered the Rotavirus diag-
nostic test of choice. The speciﬁcity of these assays is not
perfect (80–99%) and Rotavirus does exhibit seasonal vari-
ation in temperate countries, with a typical winter–spring
peak. Therefore, the positive predictive value of Rotavirus
EIA testing drops dramatically when these tests are per-
formed on stool samples outside the Rotavirus season.
This is important for clinical laboratories to consider when
designing testing algorithms for gastroenteritis. In addi-
tion, Rotavirus EIA assays can remain positive for many
days following resolution of symptoms.
Nucleic acid ampliﬁcation tests are also in development
for Rotavirus diagnostics, and some labs are using labora-
tory-developed tests for this purpose, sometimes as part of a
multiplex panel to detect multiple agents of gastroenteritis
Bernstein, D.P. Rotavirus overview. Pediatr. Infect. Dis. J. 28, S50–S53 (2009).
Bodo, R., Guenter, E., Horst, M.A., Baumeister, G. and Kuhn, J.E. Evaluation of
two enzyme immunoassays for detection of Human Rotaviruses in fecal speci-
mens. J. Clin. Microbiol. 39, 4532–4534 (2001).
Dennehy, P.H. Effects of vaccine on rotavirus disease in the pediatric population.
Curr. Opin. Pediatr. 24, 76–84 (2012).
Greenberg, H.B. and Estes, M.K. Rotaviruses: from pathogenesis to vaccination.
Gastroenteriol. 136, 1939–1951 (2009).
Kenswick, B.H., Hejkalk, T.W., DuPonta, H.L. and Pickeringa, L.K. Evaluation of
a commercial enzyme immunoassay kit for rotavirus detection. Diag. Microbiol.
Infect. Dis. 1, 111–115 (1983).
927CHAPTER 9.19 Viral Diseases
Wolffs, P.F., Bruggeman, C.A., vanWell, G.T. and van Loo, I.H. Replacing tradi-
tional diagnostics of fecal viral pathogens by a comprehensive panel of real-
time PCRs. J. Clin. Microbiol. 49, 1926–1931 (2011).
Yolken, R.H. and Leister, F. Rapid Multiple-Determinant Enzyme Immunoassay
for the detection of Human Rotavirus. J. Infect. Dis. 146, 43–46 (1982).
Human adenoviruses are members of the Adenoviridae
family. Adenoviruses are large, nonenveloped icosahedral
viruses that are 70–90nm in diameter. Each particle con-
sists of a single linear, double-stranded DNA molecule.
There are seven recognized species (A through G) based
on immunologic and genetic properties. Within each spe-
cies there are many serotypes, and about half of the known
serotypes cause disease. Adenovirus infections are com-
mon and ubiquitous, and this virus can result in a wide
variety of clinical manifestations, including gastroenteritis,
ocular infections, and, less commonly, infections of the
urinary tract, CNS, and liver. Adenovirus is an important
cause of respiratory infections in both immunocompro-
mised and immunocompetent individuals. The speciﬁc
manifestations and clinical course of adenovirus infection
vary depending on the age and immune status of the
patient. In immunocompromised hosts (such as stem cell
transplant or solid organ transplant patients) disseminated
adenovirus can result in serious systemic infections with
disease manifestations including hemorrhagic cystitis,
pneumonia, hepatitis, viremia, and disseminated disease.
Adenoviruses are highly resistant to inactivation by chemi-
cal and physical treatment; most serotypes are stable at
35°C for a week, room temperature for several weeks, and
for several months at 4°C.
It has been estimated that adenoviruses account for
5–15% of diarrheal illnesses in children; speciﬁcally, ade-
noviruses 40 and 41 are an important cause of gastroen-
teritis in children less than 2 years of age. The stool is
usually watery and nonbloody, lacks fecal leukocytes, and
diarrhea lasts a mean of 10 days. Vomiting, mild fever, and
abdominal pain may also be present. The disease is typi-
cally self-limiting and resolves without sequelae. Although
the gold standard for adenovirus detection is usually con-
sidered to be viral culture, the enteric adenoviruses (40 and
41) do not grow well in culture. Therefore, if a laboratory
conﬁrmation of adenovirus gastroenteritis is required, EIA
for adenovirus 40/41 antigen detection can be used to
detect this virus directly in stool samples; these assays are
commercially available. Compared to electron micros-
copy, the sensitivity and speciﬁcity of these assays is
thought to be >90% and >97%, respectively. In immuno-
competent individuals, viral shedding is not typically pro-
longed after symptoms resolve and a positive adenovirus
EIA is suggestive of acute infection.
“Generic” lateral-ﬂow immunoassays that are thought
to detect most types of adenovirus are also commercially
available; these tests can be performed on ocular swabs,
respiratory secretions, and urine. The rapid turnaround
time and ease of use makes this assay format an attractive
option, but the performance characteristics of these assays
are suboptimal. Namely, these assays lack sensitivity, with
values ranging from 55 to 85% reported. The sensitivity of
these tests is degraded even further if ocular swabs are
placed into viral transport medium prior to analysis.
Therefore these assays have not been widely adopted.
Typical signs and symptoms of adenoviral respiratory
tract infection include fever, nasal congestion, cervical ade-
nopathy, pharyngitis, and cough. Direct ﬂuorescent anti-
body (DFA) reagents are widely used for the detection of
adenoviral antigens in respiratory specimens, such as nasal
washes, nasal aspirates, nasopharyngeal swabs, and bron-
chial alveolar lavage, and tracheal aspirates. The reported
sensitivity for DFA of adenovirus in respiratory specimens
is approximately 40–60% compared to viral culture, which
is lower than the DFA sensitivity for most other respiratory
viruses. Sensitivity is higher in specimens collected from
pediatric patients (who typically shed higher concentrations
of virus) compared to specimens from adults.
PCR is emerging as an additional diagnostic mode for
adenovirus gastroenteritis, respiratory infections, and dis-
seminated adenoviral disease, but challenges exist in
designing PCR assays that are efﬁcient at ampliﬁcation of
all serotypes that could be implicated in infection from a
particular specimen type. In addition, quantitative adeno-
virus PCR can be used in disseminated infections in
immunocompromised patients to assess viral load and
response to therapy. It is likely in the future that PCR will
be the mainstay of adenovirus detection in both stool and
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edn, (eds Pickering, L.K., Baker, C.J., Kimberlin, D.W. and Long, S.S.),
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August, M.J. and Warford, A.L. Evaluation of a commercial monoclonal antibody
for detection of adenovirus antigen. J. Clin. Microbiol. 25, 2233–2253 (1987).
Damen, M., Minnaar, R. and Glasius, P. Real-time PCR with an internal control
for detection of all known human adenovirus serotypes. J. Clin. Microbiol. 46,
Herrmann, J.E., Perron-Henry, D.M. and Blacklow, N.R. Antigen detection with
monoclonal antibodies for the diagnosis of adenovirus gastroenteritis. J. Infect.
Dis 155, 1167–1171 (1987).
LaSala, P.R., Bufton, K.K., Ismail, N. and Smith, M.B. Prospective comparison of
R-mix shell vial system with direct antigen tests and conventional culture for
respiratory virus detection. J. Clin. Virol 38, 210–216 (2007).
Levent, F., Greer, J.M., Snider, M. and Demmler-Harrison, G.J. Performance of a
new immunochromatographic assay for detection of adenoviruses in children.
J. Clin. Virol. 44, 173 (2009).
Mahafazah, A.M. and Landry, M.L. Evaluation of immunofluorescent reagents,
centrifugation, and conventional cultures for the diagnosis of adenovirus infec-
tion. Diagn. Microbiol. Infect. Dis. 12, 407–411 (1989).
Russell, W.C. Update on adenovirus and its vectors. J. Gen. Virol. 81, 2573–2604
Christenson, M.L. Human viral gastroenteritis. Clin. Micro. Rev. 2, 51–89 (1989).
Sambursky, R., Tauber, S., Schirra, F., Kozich, K., Davidson, R. and Cohen, E.J.
The RPS adeno detector for diagnosing adenoviral conjunctivitis. Ophthalmology
113, 1758–1764 (2006).