Download Rapid Diagnosis of Viral Infections

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CE Update [molecular diagnostics | virology | histology | immunology | cytology]
Rapid Diagnosis of Viral Infections
Mike J. Loeffelholz, PhD, D(ABMM)
Virology & Molecular Diagnostics, CompuNet Clinical Laboratories
After reading this article, the reader should be able to identify the common rapid tests used to diagnose viral infections, and
describe how these tests vary with regard to sensitivity, specificity, ease of use, and time to results.
Molecular diagnostic exam 0201 questions and the corresponding answer form are located after the "Your Lab Focus" section, p. 307.
왘 Viruses that are commonly detected
왘
왘
using rapid test methods.
Rapid tests used to diagnose viral
infections.
Factors to consider when comparing
rapid tests.
The laboratory diagnosis of viral infections traditionally has been considered
a lengthy process, providing results that
may have minimal clinical impact. Specimens inoculated into cell culture tubes
typically require incubation for several
days prior to development of cytopathic
effect or detection by methods such as
hemadsorption. Culture tubes are often
observed for 2 weeks or longer before
reporting negative results. However, significant advances during the past 10 to 15
years in the development of viral diagnostic tests provide rapid and more useful
results. Cell culture incubation times have
been shortened significantly with the use
of centrifugation enhanced shell vials.
Methodologies that detect viral antigens
or nucleic acids directly in patient specimens can be completed in minutes or
hours. Decreased sensitivity and specificity may accompany improved time to
detection. The costs required to perform
newer, rapid tests versus traditional culture methods are additional
considerations. Rapid tests may have
unique specimen requirements. All of
these issues must be weighed carefully
when considering which tests to offer.
Respiratory Viruses
Rapid tests have had more impact on
the detection of respiratory viruses than any
other group of viruses. Indeed, studies have
demonstrated a number of positive
outcomes of rapid detection of respiratory
viruses in hospitalized patients, including
decreased length of hospitalization, patient
isolation, costs, and better use of
antibiotics.1,2 Respiratory viruses that are
frequently identified by both traditional and
rapid methods include influenza virus, respiratory syncytial virus (RSV), adenovirus,
and parainfluenza virus. All of these viruses
will grow in commonly available cell lines
in tube cultures and are usually detectable
after several days of incubation. However,
influenza and parainfluenza viruses may not
produce discernible cytopathic effect (CPE),
requiring blind hemadsorption or immunofluorescence staining of cells. Respiratory
syncytial virus is more labile than other respiratory viruses. Titers decrease rapidly during specimen transport, even at 4°C.
Culture is therefore a relatively insensitive
test for RSV. Non-culture methods perform
well in comparison.
Rapid non-culture tests used routinely
to detect respiratory viruses include direct
immunofluorescence assay (DFA) and antigen/enzyme assays employing solid membrane surfaces. Centrifugation-enhanced
shell vials provide rapid culture detection
of respiratory viruses. The DFA procedure
utilizes fluorochrome (usually fluorescein
isothiocyanate)-labeled monoclonal antibodies for the staining of viral antigens
within infected cells. Following excitation,
fluorochromes emit light at specific wavelengths. Fluorescein isothiocyanate appears
apple green when excited by ultraviolet
light. The DFA procedure can be
performed on a variety of respiratory specimens including swabs in viral transport
medium, nasal washes, and nasal aspirates.
Washes and aspirates are more optimal
than swabs because they usually contain a
greater number of epithelial cells from the
posterior nasopharynx. However, well-col-
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laboratorymedicine> april 2002> number 4> volume 33
lected swabs of the posterior nasopharynx
will usually yield an acceptable number of
epithelial cells, and swabs are often preferred by older patients and those who collect specimens. Specimens should be
centrifuged and washed with phosphatebuffered saline to remove material that may
affect fluorescence or interfere with adherence of cells to the slide.
Smears are prepared on slides by
placing a drop of washed, concentrated
specimen directly on the slide, or by cytocentrifuging the specimen onto the slide.
Cytocentrifugation produces a spot that
contains more cells, and is more easily
read than smears prepared by placing a
drop on the slide. Smears are allowed to
air dry completely, fixed in cold acetone,
then are stained with fluorochrome-labeled monoclonal antibodies according to
the manufacturers’ instructions. Antibody
reagents are either direct (primary antibody is also labeled with the
fluorochrome) or indirect (includes an
unlabeled primary antibody and a fluorochrome-labeled secondary antibody).
Since each antibody incubation step is
typically 30 minutes, direct reagents
shorten the assay time substantially. After
applying mounting fluid and a cover slip,
slides are examined using a fluorescent
microscope with the appropriate filter
matched to the fluorochrome.
The DFA procedure can be streamlined by the use of a single screening
reagent containing monoclonal antibodies
specific for several viruses. Specimens
exhibiting fluorescence are then stained
with individual antibodies to determine
the specific agent. The respiratory virus
screening approach has been further simplified by the use of dual fluorochromes
in a single reagent (Simulfluor, Chemicon
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M, Temecula, CA).3 This allows not only
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detection of multiple viruses within a single smear, but also identification, depending on which antibodies are labeled.
Drawbacks of DFA include the
requirement for a fluorescent microscope,
the subjective interpretation of fluorescence,
and the requirement for skilled technologists. Staining patterns of viral infected cells
must be differentiated from non-specific
staining. Fluorescence may be nuclear, cytoplasmic, or both, and may appear mottled
or more uniform. Control slides containing
viral-infected cells must be stained and read
alongside patient specimens.
An advantage of DFA is the short
assay time. The time required to wash a
specimen, apply it to a slide by cytocentrifugation, and to fix in acetone is under
1 hour. As stated earlier, staining time is
30 or 60 minutes, depending on whether
direct or indirect antibodies are used.
Shortening the staining time may result in
unsatisfactory staining. A skilled technologist can read a slide in several minutes
or less, for a total time to results of less
than 2 hours. Another advantage of DFA
is the ability to assess specimen quality.
Stained smears containing fewer than 20
total epithelial cells are generally considered to be inadequate. Negative DFA results from these specimens should be
reported only with a statement that an
insufficient number of cells might contribute to a false negative result.
While DFA is a relatively inexpensive
procedure, prices of DFA reagents may
vary considerably among vendors. Reagents
and other consumables are few with minimal volumes. Four major vendors of DFA
reagents are Chemicon, Bartels/Trinity
Biotech M (Wicklow, Ireland), DAKO M
(Carpinteria, CA), and Diagnostic Products
Corporation M (Los Angeles, CA). Fluorescence intensity and staining characteristics
will vary not only among reagents from
different vendors, but between direct and
indirect formats, and between reagents containing a single antibody versus screening
reagents containing several antibodies.
Medical technologists may develop a
comfort level with a particular reagent
and its staining characteristics, and therefore have difficulty in interpreting staining patterns of reagents from other
vendors or formats. Not all commercially
available, fluorochrome-labeled monoclonal antibodies are FDA-cleared for
direct specimen detection; some are
cleared only for culture confirmation.
The specificity of DFA is
consistently high. Sensitivity varies depending, in part, on the virus detected.4-6
Sensitivity is highest for RSV reagents
(equivalent to, or greater than that of culture) due to the lability of the organism
during specimen transport, and resultant
low sensitivity of culture, against which
DFA is compared. Sensitivity of DFA
reagents for other respiratory viruses is
generally less than that of culture.7 For
this reason, negative DFA results should
be confirmed with culture.
Rapid antigen/enzyme membrane tests
are commonly used for the detection of influenza virus and RSV. Five influenza A and
B membrane tests are currently commercially available in the United States. These
include the Directigen Flu A and Flu A+B
tests (Becton Dickinson M, Sparks, MD),
ZstatFlu (ZymeTx M, Oklahoma City, OK),
QuickVue (Quidel M, San Diego, CA), and
Flu OIA (Thermo Biostar M, Boulder, CO).
The Directigen tests are true EIAs, using
enzyme conjugated monoclonal antibodies
and substrate to generate a color in the presence of influenza virus antigen.8 The ZstatFlu test is an endogenous viral-encoded
enzyme assay that detects influenza virus
neuraminidase activity.9 The QuickVue test
is a lateral flow immunoassay.10 The Flu
OIA test is an optical immunoassay.11 Assay
times are all under 30 minutes. The Directigen, Flu OIA, and QuickVue tests can be
performed on multiple specimen types. The
ZstatFlu test can only be performed on
throat swabs using the swab provided in the
kit, which prevents reflexive testing or
adding other viral tests from a single specimen. There is currently 1 commercially
available rapid EIA test for RSV (Directigen, Becton Dickinson). The format of this
test is virtually identical to that of the Directigen influenza virus tests. In 4 published
studies, the sensitivity and specificity of the
RSV membrane test ranged from 71% to
86% and 76% to 91%, respectively.12-15
Advantages of all of these tests include
simplicity [the ZstatFlu and QuickVue influenza tests are classified as waived tests
laboratorymedicine> april 2002> number 4> volume 33
by the FDA under the Clinical Laboratory
Improvement Amendments (CLIA) of
1988], speed, and convenience for point-ofcare testing. However, speed and simplicity
come at a price which is decreased
performance. All of the antigen/enzyme
membrane assays are less specific than culture or DFA, resulting in decreased positive
predictive value outside of the influenza or
RSV “seasons” when disease prevalence is
low. Out-of-season positive test results
should be interpreted with caution and confirmed by culture. During the height of influenza or RSV season or when disease
prevalence is high, the predictive value of a
positive result is correspondingly higher.
The sensitivity of rapid membrane assays is
variable, depending in large part on the
specimen source (as with DFA, washes and
aspirates are generally better than
swabs).16-18 Most studies have shown that
membrane tests are less sensitive than culture.7,12,19,20 In spite of their relative inaccuracy, rapid membrane tests are performed
by many laboratories because their rapid
time to results offers greater potential impact on patient management.
The centrifugation-enhanced shell
vial assay is a rapid culture method for the
detection of respiratory and other viruses.21
In this modification of tube cultures, the
cell monolayer is grown on a coverslip in a
flat-bottom vial. Vials are centrifuged after
adding specimen to enhance viral infection
of the cells. Vials are incubated for 24 to 72
hours after which the coverslips are stained
with fluorochrome-labeled monoclonal
antibodies that are specific for early viral
antigens. Alternatively, a cell suspension
can be prepared from the coverslip and
dropped on a slide well. This substantially
reduces the amount of monoclonal antibody required but may potentially decrease
sensitivity since less of the cell monolayer
is stained and examined. To increase the
detection speed (but at greater cost), multiple vials are inoculated. A vial is stained
after 24 hours incubation, and the result is
reported if it is positive. If the 24-hour vial
is negative, other vial(s) are incubated an
additional 24 to 48 hours, and stained. Cell
lines used in shell vial assay for respiratory
viruses include primary monkey kidney,
Madin-Darby canine kidney, and others. In
a recent modification, a single coverslip
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containing a mixed monolayer of 2 cell
lines (mink lung and A549 human lung
carcinoma) supported the growth of all significant respiratory viruses (R-Mix, Diagnostic Hybrids M, Athens, OH).22
Advantages of the shell vial assay
include rapid time to results (though
not as rapid as non-culture methods),
sensitivity, and specificity. Evaluations
of the shell vial assay have
demonstrated variable sensitivity compared to conventional culture. Factors
affecting shell vial assay sensitivity include the cell line used, monolayer age,
incubation time, immunofluorescence
staining reagents, and others. Disadvantages of the shell vial assay include the
labor required to inoculate vials, and
(as with conventional tube culture) a
lack of standardization.
Due in part to the variety of rapid
methods available for detection of respiratory viruses, there has been less reliance on nucleic acid amplification
tests (NAATs) such as polymerase
chain reaction (PCR). This is in spite of
the greater sensitivity of NAATs, which
makes the point that sensitivity is not
the only factor to be considered when
implementing a diagnostic test. Nucleic
acid amplification tests have several
disadvantages relative to other rapid
non-culture tests: they are labor intensive; time to results is several hours;
there is limited availability of commercial or standardized tests; and none are
FDA approved. Real-time PCR using a
rapid hot air thermal cycler and simultaneous detection of fluorescent PCR
product reduces assay time to less than
2 hours (including sample preparation
time), but is not available in a standardized format. A commercially available
NAAT is the Hexaplex test (Prodesse
M, Waukesha, WI). This multiplex PCR
assay contains 7 different primer sets
specific for influenza virus types A and
B; parainfluenza virus types 1, 2, and 3;
and RSV subgroups A and B in a single
PCR reaction tube. Following amplification, PCR product is hybridized to
probes in different microtiter plate
wells and detected colorimetrically. The
Hexaplex test has been shown to be
both specific and highly sensitive.24
Herpesviruses
Among the herpesviruses, herpes
simplex virus (HSV), varicella-zoster
virus (VZV), and cytomegalovirus
(CMV) are commonly detected using
rapid methods. Herpes simplex virus
grows rapidly in cell culture.
Specimens with high viral titers (eg,
swabs of genital lesions) show characteristic CPE often after only 24 to 48
hours of incubation. However, nongenital and low titer genital specimens
may require incubation for several days
prior to development of CPE. For these
specimens, rapid HSV detection methods offer a distinct advantage. Varicella-zoster virus grows more slowly
than HSV, and CMV grows slower still.
Tube cultures for CMV detection are
usually observed for 4 weeks.
Additionally, tube cultures are generally an insensitive method for detection
of CMV, with the exception of urine
specimens from neonates. Rapid methods frequently used to detect HSV and
VZV include shell vial culture, DFA,
and NAATs. Rapid detection methods
used to detect CMV include shell vial
culture and NAATs.
The DFA procedure is commonly
used to detect HSV and VZV, particularly in dermal lesion specimens. Dual
fluorochrome reagents that distinguish
HSV and VZV in a single smear are
available (Simulfluor, Chemicon).25 Alternatively, 2 smears can be prepared
and stained separately with HSV- and
VZV-specific monoclonal antibodies.
These reagents can be obtained from
the same vendors that provide DFA
reagents for detection of respiratory
viruses. Reliable detection of HSV and
VZV in dermal lesions by DFA requires
the presence of cells, which are
obtained by vigorous swabbing of the
base of the lesion. The DFA procedure
is more sensitive than culture for the
detection of VZV,26 but less sensitive
for detection of HSV.
The shell vial assay is used routinely
for the detection of HSV, VZV, and CMV.
The procedure is similar to that used for
detection of respiratory viruses, except that
cell lines susceptible to infection by herpesviruses are used (eg, human diploid lung
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laboratorymedicine> april 2002> number 4> volume 33
fibroblasts). Shell vials are substantially
more sensitive than tube cultures for detection of CMV, and time-to-results is reduced
from weeks to 1 to 3 days. A modification
of the shell vial assay utilizes a monolayer
of genetically engineered baby hamster kidney cells containing an HSV-specific promoter and the E. coli lacZ gene [enzyme
linked virus inducible system (ELVIS), Diagnostic Hybrids M, Athens, Ohio]. Cells
infected with HSV type 1 or 2 produce
galactosidase, which turns a substrate (Xgal) blue. This system is specific for HSV. It
is rapid (16 to 24 hour incubation prior to
staining), results are easier to interpret than
fluorescence, and sensitivity is equivalent to
conventional tube culture.27
Nucleic acid amplification tests are
used more routinely to detect herpesviruses
than respiratory viruses. Nucleic acid amplification tests are the most reliable method
for detection of herpesviruses in cerebrospinal fluid (CSF) of persons with central
nervous system infections. As previously
discussed, NAATs are labor intensive and
technically demanding relative to other
rapid non-culture methods. There is currently 1 FDA-approved test for qualitative
detection of a herpesvirus (CMV). This is
the Hybrid Capture test (Digene M,
Gaithersburg, MD) which is based on signal
amplification to achieve adequate sensitivity.28 Specimens (peripheral white blood
cells) are lysed to release DNA, and combined with a CMV specific RNA probe.
The DNA:RNA hybrids are captured in
microtiter plate wells and detected with
multiple anti-RNA:DNA antibodies conjugated to alkaline phosphatase. Most NAATs
for herpesviruses are developed and validated in-house, resulting in little standardization among methods. A rapid PCR assay
(approximately 2 hours versus 4 to 6 hours
for conventional PCR) using rapid temperature cycling and simultaneous detection of
fluorescent PCR product has been used to
detect HSV and VZV in a variety of specimens.29,30 Nucleic acid amplification tests
are highly sensitive, and can also be very
specific when proper measures are taken to
avoid carryover contamination. A drawback
of qualitative NAATs for detection of CMV
is the questionable correlation of a positive
result with active disease. For this reason,
quantitative tests may have more utility.
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Enteroviruses
Enteroviruses are pathogens that cause
a myriad of clinical diseases. Most
enteroviruses can be detected in conventional cell culture, using susceptible cell
lines. The hardiness of these viruses makes
culture a reasonably sensitive method. Unlike HSV, enteroviruses can be reliably detected in CSF specimens using culture.
Because of typical slow development of
CPE in cell lines, NAATs have been used
for more rapid detection.31 There are no
commercially available, FDA-approved
NAATs for enteroviruses.
Another rapid method used for detection of enteroviruses is the shell vial
assay. Monolayers containing single cell
lines and mixed cell lines (E-Mix, Diagnostic Hybrids) have been described.32
Rotavirus
Rotavirus is an important cause of
acute gastroenteritis, primarily in young
children. Rotavirus cannot be grown in
cell lines commonly used in virology
laboratories, leaving antigen-detection
EIA (microtiter plate and membrane
formats) as the predominant method.
Because of high virus load in stool,
EIAs have reasonable sensitivity.33
Membrane EIAs are commercially
available (Meridian Diagnostics M,
Cincinnati, OH). These tests are rapid
and simple to perform. Latex agglutination is another rapid, commercially
available method for detection of rotavirus (Wampole Laboratories M,
Cranbury, NJ). Latex agglutination tests
are generally less sensitive than EIA.34
The PCR technique has been described
for the sensitive detection of
rotaviruses, but due to the availability
of easier, faster, commercial antigen
detection tests there is little need for
NAATs for routine diagnosis.
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Conclusions
Rapid methods are a significant
part of the diagnostic virology test
menu, greatly enhancing the impact of
diagnostic virology on patient management. The sensitivity and specificity of
rapid (faster than traditional cell culture) methods vary greatly depending
on the methodology. It is incumbent
upon the laboratory professional to be
aware of this variability and to communicate test limitations to clients.
18. Masters HB, Weber KO, Groothius JR, et al.
Comparison of nasopharyngeal washings and
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