739

SPECIAL SECTION: MEDICAL MICROBIOLOGY

L. Barth Reller and Melvin P. Weinstein, Section Editors

Diagnostic Virology

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Gregory A. Storch Departments of Pediatrics, Medicine, and Molecular Microbiology,
Washington University School of Medicine, St. Louis, Missouri

Diagnostic virology has now entered the mainstream of medical practice. Multiple methods
are used for the laboratory diagnosis of viral infections, including viral culture, antigen de-
tection, nucleic acid detection, and serology. The role of culture is diminishing as new im-
munologic and molecular tests are developed that provide more rapid results and are able to
detect a larger number of viruses. This review provides specific recommendations for the
diagnostic approach to clinically important viral infections.

Diagnostic virology is rapidly moving into the mainstream
of clinical medicine as a result of the convergence of several
independent developments. First, dramatic progress in antiviral
therapeutics has increased the need for specific viral diagnoses.
Second, technological developments, particularly in the area of
nucleic acid chemistry, have provided important new tools for
viral diagnosis. Third, the number of patients at risk for op-
portunistic viral infections has expanded greatly as a result of
the HIV/AIDS epidemic. Finally, modern management of HIV
infection and hepatitis C is providing a new paradigm for the
integration of molecular techniques into management of
chronic viral infections. These developments are not only in-
creasing the use of diagnostic virology but are reshaping the
field. The purpose of this article is to review the field of di-
agnostic virology at the beginning of the 21st century, to provide
guidance about current use of the tools of diagnostic virology,
and to provide a glimpse of important future developments.
sive (and in some cases potentially toxic) has created an obvious
need for specific viral diagnosis.
In some cases, establishing a specific viral diagnosis limits
other diagnostic procedures and may allow discontinuation of
antibiotic therapy [1]. Likewise, in some cases, confirmation of
a specific viral diagnosis helps in determining prognosis. A re-
cent study of diagnostic testing for respiratory syncytial virus
(RSV) documented that physicians usually believed that rapid
test results influenced their management of cases [2]. Rapid
RSV testing has also been used as the basis for patient place-
ment to limit nosocomial transmission [3]. Finally, viral diag-
nosis may be important for public health purposes. For ex-
ample, laboratory documentation of cases of rubella or rubeola
can set in motion extensive vaccination campaigns.
Methods Used in Diagnostic Virology

Viral isolation and a number of methods for detection of viral

Rationale for Specific Viral Diagnosis
Historically, diagnostic virology has had to justify its use.
The reasons have been that traditional viral diagnostic tech-
niques, especially culture, are slow, expensive, and often pe-
ripheral to clinical decision-making, particularly when no ther-
apeutic agents are available. The availability of antiviral
therapeutic agents such as acyclovir, ganciclovir, foscarnet, ci-
dofovir, antiretroviral drugs, neuraminidase inhibitors, and
IFN-a that are effective for specific viral infections but expen-
Received 21 June 2000; electronically published 4 October 2000.
Reprints or correspondence: Dr. Gregory A. Storch, Dept. of Pediatrics,
Washington University School of Medicine at St. Louis Children’s Hospital,
One Children’s Place, St. Louis, MO 63110 (storch@kids.wustl.edu).
Clinical Infectious Diseases 2000; 31:739–51
q 2000 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2000/3103-0018$03.00
antigens, nucleic acids, and antibodies (serology) are the core rep-
ertoire of techniques used for the laboratory diagnosis of viral
infections, although some other techniques are also occasionally
used (table 1). Viral isolation by means of cell culture is virtually
always performed in designated virology laboratories. The other
methods may be performed in those laboratories as well but may
also be performed in diverse laboratory sections such as general
microbiology, serology, blood bank, clinical chemistry, pathology,
or molecular virology. The trend for viral diagnostic testing to be
done outside of traditional virology laboratories is likely to accel-
erate as rapid diagnostic techniques based on immunologic and
nucleic acid methodologies increasingly replace viral culture.
Cell culture. The modern era of diagnostic virology dates to
the first descriptions of viral isolation in cell culture by Weller and
Enders in 1948 [4] and Enders et al. in 1949 [5]. Indeed, the need
for cell culture techniques is the raison d’être for virology labo-
ratories as entities separate from other general clinical microbiology
laboratories. While the relative importance of viral isolation as a
diagnostic method is rapidly diminishing, it still remains necessary
740
Table 1. Techniques used in diagnostic virology.
Cell culture
Antigen detection
Fluorescent antibody staining
Immunoperoxidase antibody staining
Enzyme immunoassay
Nucleic acid detection
Polymerase chain reaction
Other nucleic acid amplification methods
Electron microscopy
Cytology
Histology
Immunohistochemistry
In situ hybridization
Serology
because it is the only technique capable of providing a viable isolate
that can be used for further characterization, such as with phe-
notypic antiviral susceptibility testing. An additional advantage is
that in contrast to most antigen and nucleic acid detection methods,
viral culture allows detection of multiple viruses, not all of which
may have been suspected at the time the culture was ordered.
Because no one cell culture type can support the growth of all
medically relevant viruses, virology laboratories must maintain sev-
eral different cell culture types. The minimum requirements are a
primary monkey kidney cell line, used for the isolation of respi-
ratory and enteroviruses, and a human fibroblast line, used for the
isolation of cytomegalovirus (CMV), varicella-zoster virus (VZV),
and rhinoviruses. A continuous human epithelial cell line such as
HEp-2 is required for the isolation of RSV. Which cell lines are
used for a specific specimen is determined by the information com-
municated from the ordering physician to the laboratory and by
knowledge of the specimens usually isolated from a given specimen
type. For example, CMV is the main virus isolated from urine
specimens, and therefore any viral culture of urine must at least
involve inoculation onto human fibroblast cells to allow CMV
isolation.
Growth of viruses in cell culture is usually detected by visualizing
morphological changes in the cells, known as cytopathic effect
(CPE). The characteristics of the CPE are often sufficiently dis-
tinctive to allow the laboratory to be suspicious of which virus is
responsible. When necessary, confirmation can be achieved by
scraping the infected cells from the walls of the tube or vessel in
which they are growing and preparing a fluorescent antibody stain
with use of monoclonal antibodies specific for the virus or viruses
thought to be responsible for the CPE. Cell cultures are typically
viewed microscopically to detect CPE every 1–2 days for the first
week of incubation. The time required to detect CPE varies from
1–2 days after inoculation for herpes simplex virus (HSV) to 1–3
weeks for CMV.
The shell vial culture method, first developed for CMV [6, 7],
dramatically decreases the time required for detection of viruses in
cell culture. The method involves centrifugation of the specimen
onto the cell culture monolayer and incubation for 1–2 days, fol-
lowed by fluorescent antibody staining of the cell culture, regardless
of whether CPE is visible. In addition to detection of CMV, shell
vial cultures have also been used to speed the detection of HSV,
VZV, respiratory viruses, and the enteroviruses.
Another modification of traditional cell culture involves the use
of genetically engineered cell lines. In these systems, genes are trans-
Storch CID 2000;31 (September)
fected into indicator cell lines to direct insertion of viral receptors
on the surface of the cell and/or to direct expression of promoters
that respond to a specific viral protein present in the specimen.
Activation of the promoter triggers a reporter enzyme such as b-
galactosidase that acts on a substrate to indicate the presence of
the virus being sought. This approach has been most widely used
for HSV [8] and HIV [9].
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The virus for which culture remains most uniquely useful is HSV.
Cell culture is also sometimes applied to the detection of CMV,
VZV, adenovirus, RSV, influenza and parainfluenza viruses, rhi-
novirus, and the enteroviruses. It can also be used to detect measles,
rubella, and mumps viruses, although those diseases are currently
very unusual in the United States. For all of the viruses mentioned,
the rapid tests described below are gradually replacing viral culture.
Antigen detection. Methods of antigen detection include fluo-
rescent antibody (FA) staining, immunoperoxidase staining, and
EIA. Of these, FA staining is the most widely used in diagnostic
virology. Rapid viral diagnosis by means of FA staining was first
described by Liu in 1956 [10] for detection of influenza and was
pioneered for numerous viruses by Gardner and McQuillan [11].
The method was widely adopted by clinical laboratories during the
1980s, particularly for detection of respiratory viruses. The com-
mercial availability of specific monoclonal antibodies was crucial.
FA staining methods continue to be improved through the use of
cytocentrifugation to prepare specimens [12, 13] and simultaneous
staining with multiple different antibodies labeled with different
fluorescent labels [14].
Antigen detection methods are particularly useful for viruses that
grow slowly or are labile, making recovery in culture difficult. The
most important targets have been RSV, influenza and parainfluenza
viruses, and adenovirus in respiratory specimens; HSV and VZV
in cutaneous specimens; rotavirus in stool specimens; and CMV
and hepatitis B virus (surface antigen) in blood specimens. Viruses
such as the enteroviruses and the rhinoviruses that have extensive
antigenic heterogeneity and lack cross-reacting antigens are not
suitable for antigen-detection techniques. The advantages of an-
tigen-detection techniques are rapidity (results can be available
within hours of receipt of the specimen into the laboratory) and
lack of requirement for viral viability in the specimen, allowing
greater flexibility in the handling and transport of specimens.
Nucleic acid detection. The development of PCR analysis in
1985 [15] made possible the diagnosis of viral infection through
sensitive detection of specific viral nucleic acids. Any virus can
potentially be detected in this way, and applications of PCR anal-
ysis and other nucleic acid amplification techniques continue to be
developed. There is little doubt that during the next decade, ap-
plications of nucleic acid detection techniques will drastically re-
shape the field of diagnostic virology. By inclusion of a step em-
ploying the enzyme reverse transcriptase (RT), PCR analysis can
be adapted to detect viral RNA. Viral load assays for HIV and
hepatitis C virus (HCV) are examples of quantitative nucleic acid
detection techniques. Multiplex techniques permit the simultaneous
detection of more than one virus or even of a virus and a different
class of pathogen. For example, a multiplex PCR analysis that
detects the DNA of Epstein-Barr virus (EBV) and Toxoplasma
gondii has been used for the diagnosis of mass lesions in the brains
of patients with AIDS [16].
Nucleic acid amplification assays can be classified as target am-
CID 2000;31 (September) Diagnostic Virology
plification assays or signal amplification assays. Examples of target
amplification assays in addition to PCR include the ligase chain
reaction, which has been used for detection of sexually transmitted
disease agents [17], and the transcription-mediated amplification
assay [18]. In signal amplification assays, the target itself is not
amplified; rather, amplification is of a chemical signal used to detect
hybridization of a probe with the target nucleic acid. Examples
include the branched chain DNA (bDNA) assay [19] and the hybrid
capture assay [20]. Signal amplification assays are less sensitive than
target amplification assays but are also less prone to yield false-
positive results due to contamination of reactions with laboratory-
amplified nucleic acid.
Recent modifications of PCR analysis called “real-time” PCR
are a dramatic development that may potentially greatly expand
the applicability of PCR analysis for diagnosis of viral infections.
In these assays, a fluorescent signal is generated as PCR takes place.
The assays are run on highly specialized automated instruments
that include optical systems to excite the fluorescent dyes and detect
fluorescent emissions. Examples of real-time PCR instruments in-
clude the Light Cycler (Roche Diagnostics, Mannheim, Germany)
and the Prism 7700, which utilizes “Taqman” technology (Perkin
Elmer, Foster City, CA). The combination of amplification and
signal detection markedly reduces the time required for nucleic acid
detection and greatly reduces the likelihood of contamination of
reactions with laboratory-amplified nucleic acid, since there is no
need to open the tubes in which PCR has taken place. For example,
PCR reactions run on the Light Cycler can be completed in 30
minutes.
Despite virtually unlimited potential applications, availability of
diagnostic nucleic acid amplification assays remains limited because
few such assays are currently licensed by the US Food and Drug
Administration (FDA). The only assays currently licensed and
available to laboratories in kit form are the Amplicor HIV-1 Mon-
itor assay for HIV RNA (Roche Molecular Systems, Indianapolis),
the Nuclisens assay for CMV pp65 RNA (Organon-Teknika, Dur-
ham, NC), and hybrid capture assays for CMV and human pap-
illomavirus (HPV; Digene Corporation, Beltsville, MD).
Many laboratories have developed their own “home-brew” PCR
assays. Implementation and use of these assays are regulated by
the Clinical Laboratories Improvement Amendments of 1988
(CLIA ’88). Availability of home-brew assays is largely limited to
selected university laboratories whose personnel have the necessary
skills and interest and to large commercial reference laboratories.
The quality and performance of these assays vary widely. Inter-
laboratory certification programs to help standardize these assays
are only beginning to become available.
Serology. The diagnosis of viral infections by detection of spe-
cific antiviral antibodies is a traditional method whose clinical util-
ity is limited by the need for comparison of acute and convalescent
antibody titers. However, detection of virus-specific IgM antibodies
allows a diagnosis to be made from a single specimen. Viruses for
which detection of virus-specific IgM antibodies are useful include
EBV (IgM antibodies to the viral capsid antigen); CMV; hepatitis
A virus; hepatitis B virus (IgM antibodies to the hepatitis B core
antigen); parvovirus B19; measles, rubella, and mumps viruses; and
the arboviruses such as St. Louis encephalitis virus.
Another area of utility of serological methods is for certain
chronic infections such as with HIV or HCV, in which the presence
741
of any antiviral antibodies is always (HIV) or usually (HCV) in-
dicative of current infection. Finally, serology is uniquely useful
for defining specific antiviral immunity. Viruses for which definition
of immune status by serology is useful include VZV, CMV, EBV,
HSV, measles and rubella viruses, parvovirus B19, hepatitis A (total
antibodies), and hepatitis B (antibodies to the hepatitis B surface
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antigen).
Approach to the Diagnosis of Specific Viral Infections
Mucocutaneous infections. Methods used for the labora-
tory diagnosis of mucocutaneous infections caused by viruses
are shown in table 2. The most common reason for viral di-
agnosis of mucocutaneous infections is the presence of vesicular
or ulcerative lesions, which may be caused by HSV or VZV
and occasionally by enteroviruses. When laboratory diagnosis
is required, culture and FA staining are the procedures of
choice. The sensitivity of culture for detecting HSV in genital
lesions has been estimated to be 80% overall [21] and is higher
for vesicular or pustular lesions than for crusted lesions [22].
Cultures for HSV usually become positive within 1–3 days.
VZV is a more labile virus than HSV, and cultures are both
less sensitive and slower, typically requiring 4–10 days for
detection.
FA staining of a specimen obtained by scraping the base of
a lesion with a scalpel blade or rubbing it vigorously with a
polyester or rayon swab is more sensitive for detecting VZV
than is culture [23] and should be considered the procedure of
choice. FA staining for HSV can also be done effectively with
use of the same specimen, especially if cytocentrifugation is used
to process the specimen in the laboratory [12]. A recent study
showed that PCR analysis was more sensitive than viral culture
for detection of HSV in cutaneous lesions, suggesting the po-
tential for future application of PCR analysis for routine de-
tection of HSV [24].
Other common viral infections of the skin or mucous mem-
branes are caused by HPVs and poxviruses (e.g., molluscum
contagiosum and orf). These viruses are not cultured in clinical
laboratories. The role of HPV testing of specimens from the
female genital tract to detect high-risk HPV types that are as-
sociated with carcinoma of the cervix is not yet established,
although it is likely that it will be used in the future in specific
situations, such as for assessment of the cervical cancer risk of
women with equivocal Papanicolaou smears [25].
When specific viral diagnosis is required, HPV infections can
Table 2. Laboratory diagnosis of viral infections of the skin and
mucous membranes.
Virus Method(s)
Herpes simplex Culture, fluorescent antibody staining
Varicella zoster Fluorescent antibody staining, culture
Enterovirus Culture
Human papilloma Hybrid capture assay, PCR analysis, histology, cytology
Poxvirus Histology, electron microscopy
742
Table 3. Laboratory diagnosis of viral infections of the respiratory
tract.
Virus Antigen detection Culture
Respiratory syncytial Yes Yes
Influenza Yes Yes
Parainfluenza Yes Yes
Adenovirus Yes Yes
Rhinovirus No Yes
Coronavirus No No
be diagnosed by DNA detection methods such as the hybrid
capture assay [26] and PCR analysis [27] or by visualization of
characteristic cytological or histologic changes. The hybrid cap-
ture assay, but not PCR analysis, is currently licensed by the
FDA for HPV detection. Because of the different cervical can-
cer risks conferred by different papillomavirus types, useful
tests will have to discriminate between high-risk and low-risk
types. Both the hybrid capture assay and PCR analysis have
this capability.
Respiratory infections. Methods available for the laboratory
diagnosis of viral respiratory infections are shown in table 3.
Specific diagnosis of viral respiratory infections is widely done
for pediatric patients and is increasingly done for adult patients
as well. The most widely employed methods are viral culture and
antigen detection by FA staining or EIA. Suitable specimens
include nasopharyngeal aspirates, washings, or swabs; bronchial
washings; and bronchoalveolar lavage fluid. Antigen detection
tests performed directly on the specimens have the greatest clin-
ical utility because of the potential for rapid availability of results.
Antigen detection procedures are available for RSV, the parain-
fluenza and influenza viruses, and adenoviruses, but not for rhi-
noviruses or coronaviruses. All of these viruses except the co-
ronaviruses can be grown in cell culture.
Antigen detection techniques are more sensitive than culture
for RSV but less sensitive for the other respiratory viruses [25].
The sensitivity of FA staining for influenza and parainfluenza
in comparison with culture varies widely. Many laboratories
report sensitivities of ∼80%, although some laboratories report
sensitivities approaching 100% [11]. The sensitivity of FA stain-
ing is enhanced when the laboratory uses cytocentrifugation
for specimen preparation [13].
Several commercial assays are available for rapid detection
of influenza virus. These assays are interesting because they are
similar to widely used rapid tests for group A streptococci and
can be performed by individuals without specialized expertise
in virology, a circumstance which potentially makes rapid in-
fluenza diagnosis widely available outside of virology labora-
tories. The Directigen assay (Becton Dickinson, Cockeysville,
MD) is an EIA that can be used to detect influenza A virus.
An enhanced version, currently in development, can also detect
influenza B virus. The Directigen assay has a sensitivity of ∼80%
in relation to culture [28, 29].
FluOIA (Biostar, Boulder, Colorado) is an optical immu-
noassay that detects but does not distinguish between influenza
Storch CID 2000;31 (September)
A and B viruses. This assay had a sensitivity comparable to
that of culture in one evaluation [30]. Zstatflu (ZymeTx,
Oklahoma City, OK) is based on detection of influenza neu-
raminidase activity and also detects but does not distinguish
between influenza A and B viruses. This assay had a sensitivity
(in comparison with culture) of 76% for influenza A virus and
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46% for influenza B virus in one evaluation, on the basis of
analysis of nasal wash and nasal aspirate specimens [31]. It
should be noted that this assay is currently licensed only for
use with throat swab specimens. A rapid test called QuickVue
influenza test (Quidel, San Diego) is licensed by the FDA for
use on nasal swabs, aspirates, or washes but has not yet been
evaluated in any peer-reviewed publications. The performance
of all of these assays may be affected by the specimen used for
testing. A recent study of adult volunteers with experimental
influenza A infection showed that influenza virus titers were
highest in nasal aspirate specimens, followed by nasopharyn-
geal swabs and throat swabs [32].
CNS infection. For diagnostic purposes, it is useful to di-
vide viral infections of the CNS into three categories: acute
meningitis in immunologically normal hosts, acute encephalitis
occurring in immunologically normal hosts, and opportunistic
infections occurring in immunocompromised individuals. In
each category, nucleic acid amplification–based tests are rapidly
becoming the most important means of laboratory diagnosis.
Tests used for the laboratory diagnosis of acute meningitis and
encephalitis are shown in table 4.
Currently, the most common viral causes of acute meningitis
are the enteroviruses and HSV type 2 (HSV-2). A recent report
suggests that VZV may also be an important cause, sometimes
producing meningitis in the absence of cutaneous lesions [33].
For the detection of enteroviruses, RT-PCR performed on CSF
Table 4. Laboratory diagnosis of viral meningitis and encephalitis.
a
Virus Method(s)
HSV PCR analysis
VZV PCR analysis
Enterovirus RT-PCR analysis (preferred), culture (CSF is
the preferred specimen for culture; culture
of nasopharynx, throat, and stool are
more sensitive but less specific)
Arbovirus Serology (tests for virus-specific IgM pre-
ferred) on CSF and serum
Colorado tick fever PCR analysis (whole blood), serology (for
Colorado tick fever virus–specific IgM)
b
Rabies RT-PCR analysis (saliva, skin), FA staining
(skin), culture (saliva), serology (CSF and
serum)
Mumps Serology (for mumps-specific IgM), culture
(CSF, saliva, urine)
Measles (subacute sclerosing
panencephalitis) Serology
Lymphocytic choriomeningitis Serology (serum), culture (CSF and blood)
NOTE. FA, fluorescent antibody; HSV, herpes simplex virus; RT, reverse
transcriptase; VZV, varicella-zoster virus.
a
Performed on CSF unless another specimen is specified.
b
Consult public health laboratories whenever diagnosis of rabies is considered;
testing is carried out in public health laboratories.
CID 2000;31 (September) Diagnostic Virology 743

Table 5. Laboratory diagnosis by PCR analysis of opportunistic HSV infection of the CNS. We also recommend that a negative
viral infections of the CNS. result can be used as the basis for discontinuing therapy with
PCR analysis acyclovir if the clinical suspicion for that diagnosis is low. How-
Sensitivity, Specificity, ever, when the clinical suspicion is high, acyclovir should be
Virus Syndrome % % continued even when the HSV PCR analysis is negative, be-
Epstein-Barr Primary CNS lymphoma 97 98 cause the sensitivity of the test is !100%. In these cases, it may

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Cytomegalovirus Encephalitis, radiculomyelitis 82 98
be useful to repeat the PCR analysis on a specimen obtained
Varicella-zoster Encephalitis, myelitis NA NA
JC Progressive multifocal 72 99 1–2 days later and to consider other diagnoses that may mimic
leukoencephalopathy HSV encephalitis [47].
Herpes simplex Meningoencephalitis 100 99.5
The arboviruses are also important causes of encephalitis in
NOTE. Data are from [51]. NA, not available. normal hosts. The most important viruses causing human in-
fection in the United States in recent years are the California
(LaCrosse), St. Louis, eastern equine, western equine, Vene-
is considerably more sensitive than viral culture [34, 35]. Primers
zuelan equine, and West Nile viruses. These infections are best
that amplify a highly conserved segment of the 50 nontranslated
diagnosed by serology, especially with tests that detect virus-
region of the enterovirus genome detect 60 of the 67 enterovirus
specific IgM antibodies. Both serum and CSF should be tested
serotypes [36], including those serotypes most commonly as-
whenever possible. The sensitivity of these tests approaches
sociated with meningitis [36, 37]. Rapid nucleic acid amplifi-
100% by the 10th day of illness [48]. Culture and nucleic acid
cation techniques such as RT-PCR analysis to detect entero-
amplification techniques are less useful than serology for di-
viruses have the potential for decreasing necessary antibiotic
agnosing these infections because of the transient and low levels
usage, especially for patients who have been pretreated with
of viremia that are characteristic. State public health labora-
antibiotics (for whom withdrawal of antibiotics on the basis of
tories are currently implementing diagnostic testing for West
negative bacterial cultures of CSF and blood might otherwise
Nile virus. Acute-phase serum and CSF specimens should be
be done reluctantly) and for patients with atypical clinical fea-
submitted to the state public health laboratory when this di-
tures. Even in cases with typical clinical features, rapid testing
agnosis is considered. Use of an RT-PCR assay has been de-
may be useful for shortening hospitalization and decreasing
scribed but was not positive in all cases [49]. Colorado tick
medical costs [1, 38, 39]. Unfortunately, no such enterovirus
fever is also often considered along with the arboviral diseases,
nucleic acid amplification assay has yet been licensed by the
and is an important cause of CNS infection in parts of the
FDA, and accordingly these tests are not yet widely available.
western United States. The causative virus is a coltivirus, which
In adult patients, HSV meningitis, usually caused by HSV-
causes infection of circulating erythrocytes and can be detected
2, is a common cause of aseptic meningitis. This illness occurs
by culture, FA staining, or PCR analysis performed on a blood
with or without concomitant genital lesions and can have re-
clot [50].
current episodes. While primary infections may yield a positive
Testing for rabies, which is done in public health laboratories,
culture of CSF, recurrent cases are always culture-negative.
should be considered for patients with a history of exposure
PCR analysis has been very useful in permitting rapid recog-
or with encephalitis of unknown etiology. Early in the illness,
nition of both primary and recurrent cases [40, 41]. Likewise,
the diagnosis is based on detection of rabies antigens or nucleic
PCR analysis for VZV can be instrumental in identifying cases
acids in saliva, CSF, or skin obtained by biopsy from the back
of acute meningitis caused by VZV, which can occur as a com-
of the neck. After the 8th day of illness, the diagnosis can also
plication of either varicella or zoster and can occur with or
be made by detection of rabies antibodies in serum or CSF.
without cutaneous lesions [33, 42].
Rabies antibodies can also be present in serum but not CSF
HSV-1 is the most common cause of sporadic encephalitis
as a result of preexposure immunization. Consultation with
in the United States. Early recognition of this entity is extremely
public health authorities is recommended whenever the diag-
important because early antiviral therapy can reduce morbidity
nosis of rabies is considered.
and mortality, which can reach 70% among untreated cases
The most important viruses causing infection of the CNS in
[43]. The results of PCR analysis performed on CSF correlate
immunocompromised patients are CMV, EBV, VZV, and JC
very closely with the results of synthesis of HSV antibodies
from brain biopsy and/or intrathecal specimens [44, 45]. In a
recent meta-analysis that compared CSF PCR analysis to one Table 6. Laboratory diagnosis of viral gastroenteritis.
of these reference procedures, the sensitivity of PCR analysis a
Virus Preferred test method(s)
was 96% and the specificity was 99% [46]. For this reason, CSF
Rotavirus EIA
PCR analysis is now widely accepted as a substitute for analysis Adenovirus (enteric serotypes) EIA
of a brain biopsy specimen for diagnosis of HSV encephalitis. Calicivirus Reverse transcriptase PCR analysis
On the basis of a decision-analysis model, we recommend ac- Astrovirus EIA, reverse transcriptase PCR analysis
a
cepting a positive CSF PCR analysis result as diagnostic of All performed on stool.
744 Storch CID 2000;31 (September)

Table 7. Preferred test methods for diagnosis of cytomegalovirus used for the laboratory diagnosis of viral gastroenteritis are
infection.
shown in table 6.
a
Diagnostic need Preferred test(s) Rotavirus is the most important cause of gastroenteritis in
Immune status Serology (IgG) young children. Because large amounts of rotavirus are shed
Congenital infection, postpartum Culture of urine in the stool during acute rotavirus infection, antigen detection
Congenital infection, in utero Culture or PCR analysis of amniotic fluid
Mononucleosis, normal host Serology (IgM) tests performed on stool specimens are very sensitive for es-

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Systemic infection, pp65 Antigenemia assay, hybrid capture tablishing the presence of this virus. A number of rotavirus
immunosuppressed host assay, NASBA analysis, PCR analysis, antigen assays have been licensed by the FDA and are widely
culture
Initiation of preemptive therapy pp65 Antigenemia assay, PCR analysis, available. EIAs are generally more sensitive than agglutination-
hybrid capture assay, NASBA analysis based assays [56].
Monitoring response to therapy Quantitative PCR analysis, pp65 antigene- Enteric adenoviruses are specific adenovirus serotypes (es-
mia assay, quantitative hybrid capture
assay pecially 40 and 41) that cause gastroenteritis, and unlike the
b
Retinitis PCR analysis of vitreous fluid serotypes that cause respiratory infections, do not grow in the
Gastrointestinal disease Histology, immunohistochemistry cell cultures typically used in diagnostic virology laboratories.
Pneumonitis Histology, immunohistochemistry
CNS disease PCR analysis of CSF An FDA-licensed EIA called Adenoclone (Meridian Diagnos-
tics, Cincinnati) is available for detection of these viruses in
NOTE. NASBA, nucleic acid sequence–based amplification.
a
Performed on blood unless otherwise indicated. stool specimens. The caliciviruses such as Norwalk agent are
b
Most cases are diagnosed on the basis of clinical findings. the most common viral causes of outbreaks of gastroenteritis.
These infections are mild and usually do not require hospital-
ization. Specific diagnosis is most important in the context of
polyomavirus. The neurological syndromes associated with
an outbreak. RT-PCR assays have been developed to detect
each of these viruses in immunocompromised patients and the
caliciviruses in stool [57] and are becoming available in many
reported sensitivity and specificity of PCR analysis for diag-
state health department laboratories. Astroviruses can be de-
nosis are shown in table 5 [51]. CMV is an important cause of
tected in stool specimens by EIA and by RT-PCR assay [58].
encephalitis and radiculomyelitis in patients with advanced
Currently, these assays are not commercially available, and di-
AIDS. The virus can only rarely be cultured from CSF in these
agnostic specimens must be referred to research laboratories
cases but is detectable by PCR analysis [52]. Quantitation of
interested in this virus.
the level of CMV DNA is useful to distinguish between clini-
CMV. Viral culture and serological testing were the tra-
cally significant CMV CNS infection and cases with asymp-
ditional methods used to diagnose CMV infection, but both
tomatic or presymptomatic infection [53]. EBV causes primary
are being increasingly replaced by a confusing array of alter-
CNS lymphoma in patients with AIDS, and the detection of
native methods. The reason for seeking replacements is that
EBV DNA by PCR analysis performed on CSF from AIDS
neither culture nor serology had adequate sensitivity and spec-
patients with contrast-enhancing mass lesions visualized by
ificity for defining clinically significant infection. Newer direct
neuroimaging techniques correlates closely with the presence of
detection methods include the pp65 antigenemia assay per-
CNS lymphoma [54]. PCR analysis for JC virus is useful for
formed on peripheral blood leukocytes [59] and a variety of
diagnosing progressive multifocal leukoencephalopathy, al-
nucleic acid detection methods, including PCR analysis, the
though the sensitivity in most laboratories is !100% [51]. The
hybrid capture assay [60], and nucleic acid sequence–based am-
use of VZV PCR for diagnosis of VZV-associated myelitis and
plification (NASBA) [61]. The latter is unique in that it detects
encephalitis is complicated by the observation that VZV DNA
CMV RNA, possibly a more specific indicator of active infec-
is sometimes detectable in CSF from patients with acute zoster
tion than CMV DNA. Eventually, quantitative nucleic acid
who do not have neurological manifestations [55]. PCR analysis
amplification assays are likely to become the definitive tests for
may be more useful for the diagnosis of VZV infections of the
CMV because of a growing body of information that supports
CNS occurring in the absence of cutaneous lesions (zoster sine
herpete).
Gastrointestinal tract infection. Four viruses or groups of Table 8. Epstein-Barr virus (EBV) antibody profiles.
viruses are currently recognized as important causes of gastro- Antibody profile
enteritis: rotaviruses, enteric adenoviruses, caliciviruses, and as- EBV status VCA-G VCA-M EA-D EA-R EBNA
troviruses. None of these are cultured in clinical laboratories, Infectious mononucleosis 1 1 1 2 2
and consequently, viral culture has no role in the laboratory Convalescent mononucleosis 1 2 1/2 1 1
evaluation of the patient with diarrhea. These viruses were dis- Past infection 1 2 2 2 1
Susceptible 2 2 2 2 2
covered by electron microscopy of fecal material. Although
negative-staining transmission electron microscopy can be used NOTE. EA-D, diffuse component of early antigen; EA-R, restricted com-
ponent of early antigen; EBNA, Epstein-Barr nuclear antigen; VCA-G, IgG to
as a diagnostic test, newer, more convenient assays have re- viral capsid antigen; VCA-M, IgM to viral capsid antigen; 1, positive; 2, neg-
placed electron microscopy in all but a few laboratories. Tests ative; 1/2, variable.
CID 2000;31 (September) Diagnostic Virology 745

Table 9. Diagnostic tests for viral hepatitis.

Virus, feature tested for Significance, use, and comments
Hepatitis A
IgM Useful for diagnosis of acute infection
Total antibody Measures all antibodies against hepatitis A virus. Presence indicates past or cur-
rent infection, or immunization. Used to evaluate HAV immune status
Hepatitis B

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Surface antigen Presence indicates current infection and can represent either acute infection or
chronic carriage
Surface antibody Presence indicates past infection or immunization. Occasionally positive in individ-
uals who are also positive for hepatitis B surface antigen
Total core antibody Measures all antibodies against hepatitis B core antigen. Presence indicates past or
current infection. Does not become positive as a result of immunization
Core antibody Positive in recent infection. Occasionally positive at low levels in chronic carriers
e antigen Presence indicates active current infection and correlates with infectivity
Antibody to e antigen Presence indicates less active current infection with lower infectivity
DNA Quantitative; measures level of replication
Hepatitis C
Antibody An EIA. Presence of antibody indicates current or past infection; false-
positives are common, especially in a low-risk population such as blood donors
Antibody A radioimmunobinding assay. Presence of antibody indicates current or past infec-
tion; confirms a positive EIA, especially in low-risk populations
RNA Presence indicates current infection; confirms a positive EIA and indicates a re-
sponse to therapy
Genotype Multiple methods are available, including line-probe assay, EIA and nucleotide se-
quencing; genotype affects response to interferon therapy
Hepatitis D
Antibody Presence indicates current or recent infection
Hepatitis E
a
IgM Useful for diagnosis of current infection
a
IgG Presence indicates past or current infection; defines hepatitis E virus immune status
a
Available only on a research basis and in some specialized referral laboratories.

a relationship between the level of CMV in blood and the like-
lihood of present or future symptomatic disease [62–66].
The choice of test begins with the purpose of the testing.
Important considerations include the clinical syndrome under
investigation and whether the patient is immunologically nor-
mal or immunocompromised. Test methods for different di-
agnostic needs related to CMV are shown in table 7. In general,
active infection is best diagnosed by direct tests for the presence
of the virus, although an exception is the diagnosis of CMV
mononucleosis in otherwise normal hosts, in which testing for
CMV-specific IgM antibodies is useful [67]. A caveat in using
this test is that for some patients with acute EBV infection,
tests are false-positive for CMV-specific IgM antibodies [67],
suggesting that testing for acute EBV infection should be per-
formed simultaneously. Congenital infection can be conven-
iently diagnosed by urine culture, which must be performed
within the first week of life to distinguish congenital infection
acquired in utero from neonatal infection occurred during or
shortly after birth.
For the diagnosis of systemic CMV infection in immuno-
compromised patients, the pp65 antigenemia assay [59] prob-
ably has the best combination of sensitivity and specificity
among widely available test methods. An additional advantage
is that it provides a quantitative estimate of the level of CMV
viremia. Qualitative PCR analysis performed on peripheral
blood leukocytes is too sensitive for the diagnosis of clinically
significant systemic infection, having a low positive predictive
value [68]. PCR analysis performed on plasma may be more
specific [69], but further clinical evaluation is required. Local-
ized CMV infection such as pneumonitis or gastrointestinal
tract disease is best diagnosed by histologic examination of
biopsy tissue, supplemented when necessary with
immunohistochemistry.
Preemptive therapy is now a widely used approach for the
control of CMV after solid organ or bone marrow transplan-
tation. This approach is based upon the use of a laboratory
marker that signals the onset of CMV infection before signif-
icant clinical manifestations have occurred. For this purpose,
leukocyte or plasma PCR analysis, the pp65 antigenemia assay,
and the hybrid capture assay are all appropriate. PCR analysis
is preferred for bone marrow transplantation patients because
of its greater sensitivity [70, 71]. In AIDS patients who are
asymptomatic, detection of CMV by any of a variety of meth-
ods signifies an increased risk of future symptomatic disease
[72], but such a finding is not currently used as an indication
for starting therapy with anti-CMV drugs.
Epstein-Barr virus. EBV is the cause of most cases of in-
fectious mononucleosis. It is also associated with primary CNS
lymphoma in patients with AIDS and posttransplantation lym-
phoproliferative syndrome in recipients of solid organ and bone
marrow transplants. The diagnostic approach differs for each
of these clinical syndromes. Because cultures for EBV are too
slow and cumbersome to be carried out in clinical laboratories,
other diagnostic methods must be used. Serological methods
746
are used to diagnose infectious mononucleosis and other man-
ifestations in immunologically normal individuals, while mo-
lecular methods are used in immunocompromised individuals.
The diagnosis of infectious mononucleosis can be made in
most cases on the basis of characteristic clinical findings, the
presence of atypical lymphocytes, and the demonstration of
heterophile antibodies, usually done with a rapid slide test.
EBV-specific serological tests are reserved for young children,
especially those !4 years old, who may fail to develop heter-
ophile antibodies with acute EBV infection [73], and for adults
suspected of having “heterophile-negative” EBV infection.
Most EBV antibody panels include tests for IgG and IgM an-
tibodies to the viral capsid antigen (VCA) and antibodies to
early antigen (EA) and nuclear antigen (EBNA) [74]. Some
laboratories also distinguish between diffuse and restricted
components of EA because the presence of antibodies to the
diffuse component is more specific for recent infection.
Testing for IgM anti-VCA is very useful in the diagnosis of
acute EBV infection because these antibodies are typically de-
tectable for only a few months after infection. Testing for IgG
VCA is useful in defining EBV immune status, since these an-
tibodies are detectable throughout the life of the individual.
Antibodies to EA arise within a few weeks of infection, but
they are not detected in all patients with acute infectious mon-
onucleosis. Antibodies to the restricted component of an early
antigen may persist for several years after infection [75]. An-
tinuclear antibodies arise later after infection than antibodies
to EA, and they persist for life.
Thus, measurement of antibodies to EA (especially the dif-
fuse component) and antinuclear antibodies is occasionally use-
ful for determining the timing of infection. Table 8 summarizes
the expected pattern of these antibodies in various EBV-related
disease states. EBV serological testing should not be used for
the evaluation of chronic fatigue because the relationship be-
tween EBV and this syndrome has not been validated.
Serology is much less useful in the diagnosis of certain specific
manifestations of EBV infection that occur in immunocom-
promised individuals. In patients with AIDS, EBV is highly
associated with primary CNS lymphoma, and EBV PCR anal-
ysis performed on CSF is useful for establishing this diagnosis
[54]. EBV is also associated with lymphoproliferative disease
after solid organ or bone marrow transplantation. Quantitative
PCR testing of peripheral blood leukocytes may be useful for
the early recognition of posttransplantation lymphoprolifera-
tive disorder, particularly in high-risk patients such as pediatric
transplant recipients, and can also be useful for monitoring
response to therapy [76]. Quantitative testing is required be-
cause many transplant recipients have EBV detectable in pe-
ripheral blood mononuclear cells after transplantation, and
measurement of the level of DNA is required to distinguish
those with posttransplantation lymphoproliferative disorder.
Viral hepatitis. Five organisms, hepatitis viruses A–E, are
currently known to cause hepatitis. All are either difficult or
Storch CID 2000;31 (September)
impossible to culture, and hence all current laboratory testing
involves detection of specific antigens, antibodies, or nucleic
acids. Although no molecular tests are currently licensed by
the FDA, molecular testing for HCV is widely used by clinicians
caring for patients with HCV, and molecular testing for hep-
atitis B virus is coming into wider use as well. Blood banks are
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currently using molecular tests to screen blood units for HCV
on an experimental basis. Table 9 shows diagnostic tests for
viral hepatitis and their specific uses.
HIV and other human retroviruses. Several tests for HIV
have become part of the mainstream of modern medical prac-
tice. The indications for use of these tests are summarized in
table 10. In most circumstances, the laboratory diagnosis of
HIV is based on a positive EIA performed on serum or plasma.
HIV EIAs are widely available and have sensitivity and spec-
ificity 199.9% [77]. However, false-positives still make up a sub-
stantial percentage of all positives when these tests are used on
low-risk populations, such as blood donors. For this reason,
all first-time-positive HIV EIAs should be confirmed with an
additional test, usually a western blot. Since it takes ∼22 days
after infection for HIV antibodies to become detectable [78],
the HIV EIA may be falsely negative during the first few weeks
after infection. HIV RNA is detectable in plasma before the
HIV EIA becomes positive and can be an indicator of acute
HIV infection. It is important to keep in mind that false-positive
HIV RNA tests have occurred [79], and the diagnosis of HIV
should not be based solely on a viral load assay.
Rapid tests for HIV antibodies are now available and make
it possible to perform HIV antibody testing within minutes.
One such test, called SUDS (Murex Diagnostics, Norcross,
GA), has been licensed by the FDA. Rapid testing is recom-
mended by the US Public Health Service for situations in which
individuals in need of testing are at high risk of not returning
for a separate visit to receive test results [80]. Tests to measure
HIV antibodies in oral transudate [81] and in urine [82] perform
well and have also been licensed by the FDA.
Antibody assays cannot be used to diagnose HIV infection
in infants born to HIV-infected mothers because of transpla-
cental passage of antibodies, regardless of the infection status
of the baby. HIV DNA PCR testing is now widely used for
Table 10. Laboratory testing for HIV.
Use Test(s)
Routine diagnosis EIA/western blot
Diagnosis of acute infection Plasma HIV RNA analysis
a
Diagnosis of congenital infection HIV DNA PCR analysis, plasma HIV
a
RNA analysis
Prognosis Plasma HIV RNA analysis
Response to therapy Plasma HIV RNA analysis
Blood donor screening EIA/western blot, p24 antigen assay,
plasma HIV RNA analysis
Resistance to antiretroviral drugs Genotypic or phenotypic susceptibility
assay
a
A positive test for plasma HIV RNA should not be used as the sole basis
for diagnosis, because there have been false-positive test results [79].
CID 2000;31 (September) Diagnostic Virology 747

this purpose and allows determination of HIV infection status Table 11. Laboratory diagnosis of miscellaneous viral infections.
within the first few months of life [83]. Assays for HIV RNA Virus; indication for testing Test(s)
(see below) can also be used [84]. Measles; acute infection Measles IgM antibody, culture
Assays for plasma HIV RNA, often referred to as viral load Rubella
Acute infection Rubella IgM antibody, culture
assays, are now an essential component of the management of
Congenital infection, in utero Culture or reverse transcriptase PCR
care for patients infected with HIV [85]. The level of HIV RNA analysis of amniotic fluid

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predicts the rate of progression and can be used to monitor the Congenital infection, postpartum
Mumps; acute infection
response to therapy. The only assay currently licensed by the Parvovirus
FDA is the Roche Amplicor HIV-1 Monitor (Roche Diagnos- Acute infection
tics, Indianapolis), which is an RT-PCR assay. An “ultrasen- Chronic infection
Aplastic crisis
sitive” modification of the assay has a detection limit of 40 Congenital infection, in utero
copies per mL of plasma. Other test methods, including Congenital infection, postpartum
branched-chain DNA and NASBA, are also under develop- HHV-6; roseola
ment. It should be noted that the Roche assay has lower sen-
sitivity for detection of some subtypes of HIV-1 that occur BK; hemorrhagic cystitis,
predominantly outside of the United States and does not detect nephropathy
Rubella IgM antibody, culture
Mumps IgM antibody, culture
Parvovirus B19 IgM
PCR analysis of serum
PCR analysis of serum
PCR analysis of amniotic fluid
Parvovirus IgM antibody
PCR analysis of leukocytes in the
absence of detectable human her-
pesvirus 6 IgG antibody
PCR analysis of urine (hemorrhagic
cystitis) or plasma (nephropathy)

HIV-2. NOTE. HHV-6, human herpesvirus 6.

HIV drug resistance is currently evaluated by either geno-
typic or phenotypic methods. Most genotypic testing is per-
formed by amplification of the RT and protease genes by RT-
PCR, followed by nucleotide sequencing. Codons that have
been associated with drug resistance are analyzed [86]. A dis-
advantage of genotypic testing is that minority viral popula-
tions, which constitute !20%–30% of the total population, are
not detected. In addition, the potential interactions among mul-
tiple different mutations are not well understood.
Phenotypic testing is performed by amplifying the RT and
protease genes and transferring them into a viable retroviral
vector, which is then grown in the presence of varying concen-
trations of the drugs to be tested [87, 88]. The advantage of
phenotypic testing is that it provides an “in vivo” view of the
behavior of the patient’s virus. This is particularly helpful when
multiple mutations are present. Several studies have shown that
genotypic susceptibility testing improves the management of
HIV [89, 90]. Studies to evaluate the impact of phenotypic
testing are under way.
HIV tests vary in their ability to detect HIV-2 in addition to
HIV-1. EIAs used by blood banks all detect HIV-2, but some
other commercial assays detect only about two-thirds of indi-
viduals with HIV-2 infection [91]. Testing for human T-cell
leukemia virus types I and II is analogous to that for HIV.
Commercial EIAs are widely available, since testing of donated
blood is required. Confirmatory assays are available through
reference laboratories.
Miscellaneous viral infections. The laboratory approach to
a number of other viral infections is summarized in table 11.
None of these infections is diagnosed primarily by culture. Vi-
rus-specific IgM antibody assays are the preferred method for
rapid diagnosis of measles, rubella, mumps, and parvovirus
B19. Some laboratories are also skilled in using immunoflu-
orescence of nasopharyngeal secretions to provide a rapid di-
agnosis of measles [92]. Virus-specific IgG antibody assays are
used to determine past infection and specific immune status.
PCR analysis performed on serum is used to diagnose aplastic
crisis caused by parvovirus B19. Because this manifestation
occurs early after infection, parvovirus IgM titers may not be
positive at the time of illness, although parvovirus IgM and
IgG antibodies will become detectable within a few days. Serum
PCR analysis is also used to diagnose chronic parvovirus B19
infection, which may cause chronic RBC aplasia in immuno-
compromised patients [93]. Many patients with chronic par-
vovirus infection do not have a diagnostic serological response.
BK virus is a polyomavirus that can cause hemorrhagic cys-
titis in bone marrow transplant recipients. The virus is difficult
to grow in the laboratory, but PCR analysis performed on urine
readily allows detection of BK virus. A recent study reported
a correlation between the presence of BK virus in plasma of
kidney transplant recipients and clinically significant nephro-
pathy [94].
The diagnosis of human herpesvirus (HHV) types 6–8 re-
mains difficult for clinical laboratories. None of these viruses
can be grown in typical, hospital-based, diagnostic virology
laboratories. The presence of virus-specific IgM antibodies can
be used to diagnose acute HHV-6 or HHV-7 infection in young
children with roseola. An alternative is to demonstrate by PCR
analysis the presence of HHV-6 or HHV-7 DNA in peripheral
blood leukocytes from a patient who does not have virus-spe-
cific antibodies [95]. The diagnosis of HHV-6 and HHV-7 in-
fection in immunocompromised patients is currently problem-
atic because serological testing may be nonspecific in this
setting, and PCR analysis of peripheral blood leukocytes does
not distinguish those patients who have clinically significant
illness attributable to the virus from those who have asymp-
tomatic reactivation. The clinical utility of laboratory diagnosis
of HHV-8 infections is undetermined.
The diagnosis of unusual viral infections, especially those
acquired overseas, requires consultation with public health lab-
oratories, usually at the level of the state health department.
748
Laboratory diagnosis of hantavirus infection can be achieved
by detection of hantavirus-specific IgM antibodies [96] or by
detection of hantavirus RNA by means of RT-PCR analysis
performed on peripheral blood leukocytes [97]. Some state pub-
lic health laboratories perform hantavirus tests, and others for-
ward specimens to the Centers for Disease Control and Pre-
vention. Dengue can also be diagnosed either by detection of
dengue-specific IgM antibodies, which typically become de-
tectable within a few days following defervescence, or by de-
tection of dengue RNA in peripheral blood leukocytes by RT-
PCR analysis [98].
Other exotic viral infections, including Lassa, Marburg, and
Ebola virus infections, yellow fever, Congo-Crimean hemor-
rhagic fever, and Rift Valley fever are also diagnosed by means
of IgM serology, PCR analysis (usually performed on blood),
and culture. Serum and whole blood samples are usually re-
quired. Throat swabs, urine specimens, and CSF specimens may
also be useful, depending upon the infection. Public health lab-
oratories should be consulted regarding the choice of specimen,
specimen collection, and transport.
Antiviral Susceptibility Testing
With the increasing use of antiviral drugs, antiviral drug re-
sistance has become a clinical problem. However, the field of
antiviral susceptibility testing is relatively undeveloped, testing
is not widely available, and testing methods are not standard-
ized. The viruses other than HIV for which antiviral suscep-
tibility testing has greatest clinical relevance are HSV and CMV.
The methodological considerations for detecting resistance are
different for these 2 viruses.
For HSV, phenotypic testing with a culture-based method is
preferred. The reasons for this are that HSV grows rapidly in
the laboratory, making culture-based testing practical; in addi-
tion, whereas most resistance to acyclovir is related to changes
in viral thymidine kinase, the genetic basis for the changes is
diverse, complicating the application of genotypic methods [99].
The most widely used culture-based method is the plaque-re-
duction assay. The results of this assay are usually expressed as
the 50% inhibitory concentration, or the concentration of drug
required to produce a 50% decrease in the number of plaques
that appear in culture, in comparison with the number that ap-
pear in the absence of drug. This test requires 1–2 weeks to
perform and can provide data for a range of drugs, including
acyclovir, foscarnet, and cidofovir. Other culture-based test meth-
ods include the Hybriwix assay (Diagnostic Hybrids, Athens,
OH), which measures the effects of drugs on synthesis of HSV
DNA [100], and the ELVIS assay (Diagnostic Hybrids, Athens,
OH), which uses a genetically engineered cell line to compare
viral growth in the presence and absence of drugs [101].
For CMV, culture-based phenotypic susceptibility testing is
impractical because of the slow and fastidious growth of the
virus. Approximately 80% of CMV resistance to ganciclovir is
Storch CID 2000;31 (September)
the result of a limited number of mutations in a specific gene
designated UL97, and these mutations can be detected by a
PCR-based assay [102–104]. This assay can be carried out either
on an isolate recovered from a patient or directly in a patient
specimen if the level of virus is sufficiently high to allow effective
PCR amplification.
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The other source of ganciclovir resistance is mutations in the
DNA polymerase gene [104]. The only method currently avail-
able to detect these mutations is nucleotide sequencing. Se-
quencing can be carried out either on a viral isolate or directly
from a clinical specimen with a sufficiently high level of virus.
CMV resistance detection is performed only in a limited num-
ber of university and commercial reference laboratories, which
have special interests in CMV.
Acknowledgments
I am grateful to Max Arens, Richard Buller, William Mulford, and
the technologists who work in the Virology Laboratory at St. Louis
Children’s Hospital for their ongoing collaboration with me in the day-
to-day work of diagnostic virology.
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