THE ROLE OF THE CLINICAL MICROBIOLOGY

LABORATORY 0891-5520/01 $15.00 + .OO

LABORATORY DIAGNOSIS OF
VIRAL HEPATITIS
Donna M. Wolk, PhD, MHA, MT; Mary F. Jones, BS (MT),
and Jon E. Rosenblatt, MD

The diagnosis of viral hepatitis involves epidemiological, clinical,

and laboratory findings. Typical results are described in all these areas
for each of the types of viral hepatitis. The clinician, however, must
remember that atypical presentations commonly occur and that a diag-
nosis should not hinge on any single epidemiological, clinical, or labora-
tory finding. In particular, laboratory tests vary depending on many
factors including when during the disease course the specimen was
obtained, how it was handled, and whether the most appropriate tests
were ordered. False-positive and negative results occur for many rea-
sons, some known and some unknown. Performance characteristics dif-
fer for different method versions of the same diagnostic test. Both
serologic and molecular assays are useful in the diagnosis of viral
hepatitis. This article will attempt to describe the usefulness of these
various assays and point out critical factors and problems in interpreting
their results.

HEPATITIS A VIRUS

Hepatitis A virus (HAV) is an RNA virus of the Picovnaviridae family.

It is transmitted by the fecal/oral route, usually either by ingestion of
contaminated food or water or by person-to-person contact. Individuals

From the Molecular Diagnostic Laboratories, Southern Arizona VA Health Care System
(DMW); Department of Pathology, University of Arizona, Tucson, Arizona (DMW);
Department of Laboratory Medicine and Pathology, Division of Clinical Microbiology
(MFJ, JER), Mayo Clinic and Mayo Medical School, Rochester, Minnesota

INFECTIOUS DISEASE CLINICS OF NORTH AMERICA

VOLUME 15 * NUMBER 4 * DECEMBER 2001 1109

1110 WOLK et a1

at risk for acquiring HAV infection include those close to an infected

person, male homosexuals, travelers to parts of the world where HAV
is endemic, children in day care centers, and institutionalized popula-
tions. HAV is responsible for approximately 50% of acute hepatitis cases
in the United States. Person-to-person contact during community-wide
outbreaks accounts for most HAV infections in the United States9 In-
creased use of effective HAV vaccines will undoubtedly lead to further
decreases in the incidence of HAV infection in industrialized countries,
although the vaccines are not widely available in developing parts of
the world

Serologic Assays

When symptoms occur in a patient with HAV infection, they are

indistinguishable from other types of hepatitis. The use of serologic
assays (primarily enzyme-linked immunoassays [EIA])makes it possible
to more accurately diagnose HAV infection (Fig. 1). By the time symp-
toms develop (mean of 30 days after infection, range of 15 to 50 days),
immunoglobulin M (IgM) class antibody directed against HAV (IgM
anti-HAV) is usually detectable. As the patient convalesces, IgM anti-
body levels decline, and by 6 to 7 months post infection, 86% will
become undetectable.21Immunoglobulin G (IgG) class antibody becomes
detectable during the acute phase of the infection, persists for the life of
the patient, and is a reliable indicator of immunity to HAV i n f e ~ t i o n . ~ ~
Commercially produced, Food and Drug Administration (FDA)-
approved assays are available for the qualitative detection of IgM anti-

Buration
-
Incubation
(15-45 Days) II Early Acute
(0-14Days)
Acute
(3-6Months)
Recovery &
Lifelong
Immunity

.-c
Y
0
-Y-
Total
anti-HAV
e
ICI
c
al
U
c
0

-P
U

-
Y

E
rn
al

Time After Exposure to HAV

Figure 1. Hepatitis A diagnostic profile. Anti-HAV IgM = IgM antibody to HAV. (Courtesy
of Abbott Laboratories, Abbott Park, 111.)
 LABORATORY DIAGNOSIS OF VIRAL HEPATITIS 1111

HAV and for HAV total antibodies (IgG and IgM). Use of the IgM anti-
HAV assay can establish the diagnosis of acute or current infection with
HAV. A positive test for HAV total antibody may indicate either current
or past infection. The total antibody assay is useful for screening large
numbers of patients in an outbreak, or for epidemiological studies. It
is also useful for determining immune status either prior to or after
vaccinati~n.~~ At this time, there are no FDA-approved quantitative
assays to measure HAV antibody; however, there are some ”research use
only” (RUO) assays available that may assist in determining the efficacy
of vaccination.

Molecular Assays

Molecular methods for detection of HAV are available2I but not

performed routinely in clinical laboratories.

HEPATITIS E VIRUS

Another type of enterically transmitted hepatitis is hepatitis E

(HEV). HEY a single-stranded RNA Calicivirus, is endemic in many
underdeveloped countries of the world.2 It is food- and waterborne and
is spread by the fecal-oral route. In the United States, HEV is seen
primarily in persons who have traveled to areas of the world where the
virus is endemic, particularly Mexico. The infection is usually mild and
self limiting, although it can be severe in pregnant women. There is no
vaccine for prevention of HEV infection.

Serologic Assays

There are no FDA-approved commercially available assays for the

diagnosis of HEV infection in the United States. There are, however,
several EIAs available for research use to detect antibody to HEV in
serum (Abbott Laboratories, Abbott Park, Ill., Genelabs Technologies,
Redwood City, Calif.), and the Centers for Disease Control will also test
for HEV antib0dy.2~Both IgG and IgM versions of these assays can be
performed, but their performance characteristics have not been exten-
sively documented.

Molecular Assays

Neither commercially produced nor ”home brew” molecular assays

for detection of HEV are available in the United States; however, HEV
RNA has been detected in serums using reverse transcriptase-polymer-
ase chain reaction (RT-PCR). This methodology was used to describe a
1112 WOLK et a1

unique strain of HEV (HEV US-1) from the first known case of HEV
infection in a patient who had not traveled outside the continental
United States.30

HEPATITIS B VIRUS

Hepatitis B virus (HBV) is a DNA virus of the family Hepadnaviridae.

It may be transmitted through blood and other body fluids, sexual
exposure, or perinatally, from mother to infant. Groups at risk for HBV
infection include intravenous drug users, sexual partners of infected
persons, children born to infected mothers, health care workers exposed
to infected blood and/or body fluids, and patients in developing coun-
tries who may receive contaminated blood or be exposed to inadequately
sterilized needles and syringes.
Most cases of HBV infection are mild and self limited; however,
approximately 10% of infected adults and 90% of infected infants will
develop chronic HBV infection.* In the United States, approximately 1
million people have chronic HBV infection, and 15% to 25% of these
will die prematurely of cirrhosis or hepatocellular carcinoma.

Serologic Assays

Commercially produced, FDA-approved serologic assays (Abbott

Laboratories, DiaSorin, Ortho Diagnostics, Genetic Systems, and others),
primarily EIAs, are available to diagnose HBV infection, distinguish
between acute and chronic infection, and determine immunity. Table 1
is an interpretive guide to the results of HBV profiles.
Hepatitis B surface antigen (HbsAg) is the first detectable marker
of HBV infection, and the laboratory tests used to detect it are the
primary screening assays for diagnosis of active HBV infection in pa-
tients and blood and organ donors. HbsAg can be detected as early as l
to 2 weeks and as late as 11 to 12 weeks after infection (mean of 7
weeks) (Fig. 2). As symptoms develop and/or liver enzyme levels rise
in the bloodstream, HBsAg titers rise, and as symptoms resolve, HBsAg
titers fall and liver enzymes return to normal. The patient is considered
infected with HBV and potentially infectious if HBsAg is detected. The
presence of HBsAg longer than 20 weeks after primary infection indi-
cates patients are likely to remain positive indefinitely (Fig. 3). This
persistence may be associated with active hepatitis or an asymptomatic
carrier state. Laboratory testing for HBsAg includes confirmation of the
positive result by neutralization studies.
Hepatitis B e antigen (HBeAg) is present during the acute phase of
HBV infection and is usually detectable at approximately the same time
as HbsAg (Figs. 2 and 3). It is a reflection of viral replication, and its
presence indicates infectivity. Antibody to HBeAg (anti-HBe) is pro-
duced as the infection resolves. It replaces HBeAg and may persist for 1
Table 1. INTERPRETATION OF HEPATITIS B PROFILES
Test*

Anti-HBc IgM
HBsAg Anti-HBs (total) Anti-HBc HBeAg Anti-HBe Interpretation

+- - + + + - Acute hepatitis B virus (HBV) infection

- + + Acute HBV infection (window period)
+ - + - + - Chronic HBV infection, active
replicating virus
+ - + - - + Chronic persistent HBV infection,
nonreplicating virus
- + + - - + Past HBV infection with recovery
and/or immunity
- + - Successful HBV vaccination
- - - Susceptible to HBV infection

"See text for explanation of abbreviations

1114 WOLK et a1

Duration (4-12 weeks) (2 weeks-3 months)

Figure 2. Hepatitis B diagnostic profile. (Courtesy of Abbott Laboratories, Abbott Park, 111.)

to 2 years after resolution of HBV infection. HBeAg and anti-HBe are

not usually detectable at the same time. The use of tests for HBeAg and
anti-HBe may be helpful in recognizing chronic persistent hepatitis.
Patients who have persistence of HBeAg longer than 10 weeks are likely
to be chronically infected.
Two tests are available for the detection of antibody to hepatitis B
core (HBc) (Figs. 2 and 3). The first, anti-HBc, is a total antibody (IgG
and IgM) assay that is indicative of current or past infection, but does
not distinguish between them. It develops early in infection and may be
the only serological marker of HBV infection in a time period where
HBsAg is disappearing and prior to the development anti-HBs. It is a
lifelong marker, but its presence does not denote immunity to HBV.
Additionally, it may be the only positive test in chronic infection when
HBsAg is below detectable levels and antibody to hepatitis B surface
antigen has not been produced. Anti-HBc IgM is produced during the
acute phase of the infection and is detectable for approximately 6 months.
The presence of IgM is an indicator of recent or current infection.
Immunity to HBV is determined by detecting antibody to hepatitis
B surface antigen (anti-HBs). In the case of self-limited primary HBV
infections, anti-HBs appears 4 to 12 weeks after exposure and persists
for 6 months to 6 years (Fig. 2). It does not appear until HBsAg has
become undetectable (if it ever was detectable), and its presence indi-
cates recovery and immunity. Anti-HBs is also the antibody that is
produced as a result of vaccination. Two types of assays are available
for anti-HBs testing. The most widely used assays are qualitative deter-
minations of the presence of antibody. In some instances, it may be
useful to have a quantitative measurement of antibody to determine
immune status or vaccine efficacy (associated with protective levels of
anti-HBs of >10 IU). This is particularly important in the case of vacci-
nated health care workers, some of whom may require additional immu-
nizations beyond the primary series of three to achieve adequate protec-
tive levels of anti-HBs.2O
 LABORATORY DIAGNOSIS OF VIRAL HEPATITIS 1115

Duration Incubation Acute Infection Chronic Infection

(4 to 12 weeks) (6 months) (years)

-
E
0
L
a Anti-HBc total
L
Y
c
al
u
C
s
->a
Q)

L
4
0)
z
Time

Duration Incubation Acute Infection Chronic infection

(4 to12 weeks) (6 months) (years)

-
-
E
0
U
e
U
E Anti-HBc total
s
E
0
U
->
al
Y
a
4

2
Time

Figure 3. A. Chronic Hepatitis B virus, no seroconversion. €2. Chronic Hepatitis 6 virus

infection, late seroconversion. (Courtesy of Abbott Laboratories, Abbott Park, Ill.)

Molecular Assays

There are several commercial and non-commercial molecular assays

available for detection of HBV in serum. Presence of HBV measured by
these tests indicates persistence of HBV infection. The specific clinical
usefulness of molecular assays is currently being determined. They can
reduce the HBV seroconversion “window period” (time to initial detec-
tion of HBsAg) by 25 days34 and, therefore, may be more sensitive
detectors of virus in early infections and have a role in screening blood
donors (Nucleic Acid Testing “AT]). They may be of help in resolving
equivocal serological results, are sensitive indicators of persistent infec-
tion, and, undoubtedly, will be important in monitoring responses to
1116 WOLK et a1

anti-viral therapy. Currently available methods include the hybrid cap-

ture assay (HCA), branched DNA (bDNA), liquid hybridization, nucleic
acid cross-linking assay, and PCR. To date, no standardized HBV DNA
assay exists, so results may vary widely from laboratory to laboratory.
There are few data to determine the desirable lower limits of detection
for HBV DNA measurement~,1~ and detection limits for each type of
assay vary.

Specific Molecular Assays

RNA.DNA Hybrid Capture Assay. The Digenea HBV Test, Hybrid

Capture@IT, (Digene Corporation, Gaithersburg, Md.) is a signal ampli-
fication and hybridization method that can detect and quantify two
subtypes of HBV from serum. Target DNA hybridizes with an RNA
probe mix, and the RNA:DNA hybrids are captured onto a microplate
with a hybrid-specific antibody. Antibodies coated onto the microplate
are conjugated to an enzyme that cleaves a chemiluminescent substrate
to allow signal detection. The assay is as sensitive as the bDNA assay
for quantitation of HBV8,27and detects as few as 142 X 0.103 copies/mL
of serum. An ”ultrasensitive” modification of this assay is also available
with a sensitivity of 5 X lo3 copies/mL.
Quantiplex HBV-DNA Assay. The branched DNA @DNA) assay is
a sandwich nucleic acid hybridization assay (Bayer Diagnostics, Em-
eryville, Calif.) and is used for quantitative measurement of HBV DNA.32
The assay detects as few as 700 X lo3 copies/mL of serum.
Nucleic Acid Based Crosslinking Assay. In the nucleic acid-based
crosslinking assay (NAXCOR, Menlo Park, Calif., http: / /www.naxcor
.corn), crosslinking nucleotides (XLnt“) are integrated into the backbone
of nucleic acid probes. Once a probe is bound, the XLnt’” is activated
by ultraviolet light, irreversibly linked to viral DNA target, captured on
magnetic beads, and coupled with an antibody-enzyme conjugate, which
catalyzes a fluorescent reaction to produce signal. In a recent study, the
NAXCOR assay compared favorably with the bDNA assay for quantita-
tion of HBV.8,24Further studies need to be performed to assess the
clinical usefulness of this assay for detection of HBV.

Other Commercial and “Home-Brew” Methods

The COBAS Amplicor HBV Monitor ’“ test (Roche Diagnostics, Indi-

anapolis) is an automated competitive PCR system that has a sensitivity
of 200 copies of HBV DNA/mL.22The COBAS system can also be used
for molecular detection of other viruses. ”Home-brew” dot hybridiza-
tion, in situ hybridization, and other PCR methods exist for detection
of HBV, but none are commercially available. PCR amplification meth-
ods can be extremely sensitive (100 to 1000 copies/mL serum) and
are being evaluated to screen blood donations for detection of HBV
 LABORATORY DIAGNOSIS OF VIRAL HEPATITIS 1117

DNA (NAT). Interpretation however, can be difficult, because there

is no standardization for these assays.19 In general, HBV DNA PCR
methodology is more sensitive than the quantitative assays, but lacks
clinical predictive value. Whichever molecular method is used, patients
should be followed using the same method to gain some measure of test
result consistency.

HEPATITIS D VIRUS

Hepatitis D virus or the delta agent (HDV) is a defective RNA virus.

It depends on a helper function of HBV to replicate and cause infection.
It occurs as either a coinfection or superinfection with HBV.26A coinfec-
tion occurs in persons infected with both HBV and HDV at the same
time, and the acute infection may be more severe than with HBV infec-
tion alone. A superinfection occurs in chronic carriers of HBV. These
patients have a greater incidence of chronic liver disease and cirrhosis
(70% to 80%) than those chronically infected with HBV alone (15% to
30%). Because of its relationship with HBV, HDV is transmitted the same
ways (exposure to infected blood and sexual transmission), and risk
groups are similar.

Serologic Assays

Antibody to HDV (anti-HDV) can be detected by an EIA. This is

the test of choice to determine HDV infection, although it does not
discriminate between acute, chronic, or resolved infections (Fig. 4). Re-
search assays are available for HDV antigen; however, the antigen is
transient in serum at best.6

Molecular Assays

Hepatitis D virus RNA can be detected in serum using PCR and

hybridization technology. Its presence indicates active viral replication.
It is transiently found in acute delta hepatitis and persistently present
in chronic infection. This assay is available only in research laboratories.

HEPATITIS C VIRUS

Hepatitis C virus (HCV) is an RNA flavivirus believed to be

transmitted only through blood. HCV is primarily transmitted paren-
terally by intravenous drug users, but needle-stick injury, contaminated
medical equipment, and blood spills are also potential sources of trans-
mission. Sexual and maternal-fetal transmission also occur. Many
1118 WOLK et a1

r-
~~~~~

Duration Acute Infection Years

(2-6Weeks)

anti-HBc Total
-c
E
0
I 1
Y

Y
c
- II
' 1

8
c
0
I
HBsAg
anti-HDV
U

-f
Y
I
0
d
HDAg
P r

Figure 4. A. Hepatitis D superinfection of a chronic Hepatitis B virus. B. Hepatitis D

coinfection. (Courtesy of Abbott Laboratories, Abbott Park, 111.)

HCV victims became infected through blood transfusions in the 1970s

and 1980s, but because of the implementation of blood donor screening
assays, by 1996 the rate of posttransfusion HCV infection had declined
to 0.1%.3The most unique characteristic of HCV is its ability to persist
in the host. Although 70% to 80% of acute infections are asymptomatic,
70% to 80% of HCV-infected patients go on to develop chronic infec-
tions. There are serious long-term consequences with chronic HCV
infection. Epidemiological studies show that within 20 years after de-
velopment of chronic HCV infection, 20% to 30% of patients will have
cirrhosis, and 1%to 5% will have hepatocellular car~inoma.~
 LABORATORY DIAGNOSIS OF VIRAL HEPATITIS 1119

There are six major genotypes of HCV and many subtypes.14Geno-

types vary in their worldwide distribution and sequence variation,
with some differing from each other by as much as 33% over the entire
genome. In addition, HCV exhibits a high rate of spontaneous mutation
and often produces discrete quasispecies that vary from one individual
to the next.7 Depending on the HCV genotype, disease outcome, and
rates of response to therapy, results of laboratory tests (including sero-
logic and molecular assays) may ~ary.lO,~~ This must be kept in mind
when interpreting the results of the assays described.

Serologic Assays

The only commercially available, FDA-approved tests to aid in the

diagnosis of HCV infection are antibody detection assays.16 A second-
generation EIA is the screening assay of choice for HCV antibody (anti-
HCV) (Fig. 5), followed by a supplemental assay depending on the
presence or absence of risk factors. In a low-risk population, such as
blood donors, a second, more specific, antibody assay, the HCV 3.0 Strip
Recombinant Immunoblot Assay (RIBA, Chiron Corporation, Emery-
ville, Calif.), is recommended. In the case of blood donors, the FDA
mandates the use of the RIBA as the supplemental assay. Persons with
risk factors for HCV exposure should be tested by a molecular method
(qualitative HCV RNA PCR) as the supplemental assay. Because of the
long window period between HCV exposure and the development of
detectable antibody (20 to 150 days, mean of 50 days), antibody screen-
ing may not be useful for diagnosing new HCV infection^.^^ In addition,
because anti-HCV can be a lifelong marker (which does not indicate
immunity), antibody testing alone cannot distinguish between acute,
chronic, or resolved infections. Molecular testing is necessary to deter-
mine whether an infection is ongoing or resolved.
The RIBA uses recombinant HCV encoded antigens (c33c, NS5) and
synthetic HCV encoded peptides (clOOp, 5-1-lp, c22p) as bands-on test
strips to detect antibody in patient serum. The RIBA is considered
positive when bands corresponding to at least two antigens are detected.
An indeterminate result is obtained when only one band is detected.
Indeterminate RIBA results may occur in recently infected persons who
have not completely seroconverted, in a chronically infected person, or
the result may be false p ~ s i t i v e . ~

Molecular Assays

Because HCV RNA can be detected in serum within 1 to 2 weeks

after acute infection (compared with a "window period" of 20 to 150 days
for serology), molecular assays provide clinicians and blood banks with
the first diagnostic sign of acute HCV infection. Detection is also possible
in patients unable to mount an antibody response because of age or
 Duration Incubation Acute Infection Chronic infection
(2-26weeks) (6 months) Nears)

-e
t
L
0

L
c
sc
0

-g!
W

Y
(D
d

P
Time

Duration Incubation Acute Infection Years

(2-26weeks) (6months)

-e
L
E
0

U
E
sc
8
->
U
(D
-.
Q

Q
a
Time
B
Figure 5. A. Serological profile in majority of patients with hepatitis C.8.Serological profile
in some patients with hepatitis C. (Courtesy of Abbott Laboratories, Abbott Park, Ill.)

immune status. In addition, molecular testing is recommended for con-

firmatory testing of high-risk patients with positive anti-HCV EIA serol-
ogy results or to resolve indeterminate EIA results? This algorithm
approach to patient testing is depicted in Figure 6 .
Although not all are FDA-approved, several molecular methods are
commonly used in clinical practice and provide essential tools for the
diagnosis of active acute HCV infection. Most of the molecular diagnos-
tic assays detect the relatively conserved 5’ untranslated region (5’ UTR)
of HCV. In RT-PCR, viral RNA is extracted and reverse-transcribed
into double-stranded complementary DNA (cDNA), which can then
be amplified by various target amplification methods. Transcription-
mediated amplification (TMA) produces ssRNA molecules. Detection of
products is generally performed by hybridization to specific probes. Of
note is that qualitative RNA assays may sometimes be more sensitive
 LABORATORY DIAGNOSIS OF VIRAL HEPATITIS 1121

Positive

Low risk High risk

(blood donor) (patients)

HCV RNA by PCR

Positive'

inderminate'

Figure 6. Hepatitis C infection diagnostic algorithm. a. A negative test result does not
exclude the possibility of exposure to or infection with HCV. Negative results in this assay
in individuals with prior exposure to HCV may be caused by antibody levels below the limit
of detection of this assay or lack of reactivity to the HCV antigens used in this assay.
Patients with recent infections with HCV may have false-negative results because of the
time for seroconversion (an average of 8 to 9 weeks). If HCV infection is suspected,
qualitative RNA testing for viral nucleic acid is recommended. b. Indicates ongoing or
current infection with HCV. Genotype and viral load information may be useful for evaluation
of response to antiviral treatment regimens. The serum specimen will be held in the
laboratory for 3 months in the event that these tests are required. c. A negative test does
not exclude the possibility of exposure to or infection with HCV, and repeat qualitative HCV
RNA testing using a new serum specimen should be considered. d. Indicates false-positive
anti-HCV enzyme immunoassay. e. Indicates presence of anti-HCV caused by past or
current infection. f. An indeterminate test result indicates that anti-HCV may or may not be
present and that a decision as to whether HCV infection exists cannot be made. All
individuals who are indeterminate should be retested over a period of 6 to 12 months to
ascertain whether increased reactivity has occurred. RlBA = recombinant immunoblot
assay, PCR = polymerase chain reaction. (Courtesy of Mayo Communique 254, 2000)

than quantitative HCV assays. For example, for some quantitative

assays, the lower limit of detection is actually higher than the qualitative
assays (100 copies/mL), so it is important to always choose the most
sensitive method for initial detection of HCV RNA. Assays and units of
measure need to be standardized so that the sensitivities of different
assays can be easily compared. Results of some assays are reported in
copies/mL, others in IU/mL, and still others in megaequivalents/mL.
IU/mL values may be up to 0.8 log less than values in copies/mL.
Conversion tables must be used in comparing results of one assay with
those of another. It is preferable to use the same assay throughout the
monitoring period.
Qualitative Molecular Assays
Reverse Transcriptase Polymerase Chain Reaction. Reverse tran-
scriptase polymerase chain reaction (RT-PCR), with primers specific for
the 5' untranslated (5'UTR) region of HCV genotype, is the most com-
mon molecular method for the detection of HCV. The most sensitive
HCV RNA PCR can detect less than 100 copies of HCV RNA per
milliliter.l1 Despite availability, most of the commercial methods are not
yet FDA-approved and are still subject to variability; however, several
commercial assays may provide the methodology necessary to provide
more standardized qualitative HCV testing. Important specimen collec-
tion and storage parameters need to be considered. EDTA and sodium
citrated plasma are the preferred specimens for HCV PCR. Specimens
must be centrifuged promptly to prevent falsely lowered results. Serum
is also an acceptable specimen for PCR if serum is centrifuged immedi-
ately after clot formation and frozen. Refrigerated (4°C) short-term stor-
age of serum or plasma is also acceptable.
The Amplicor'" HCV 2.0 (Roche Diagnostics, Indianapolis) is a
standardized RT-PCR for qualitative (positive or negative results re-
ported) detection of HCV RNA in serum. The assay reports a lower
limit of detection at 100 copies/mL and equal sensitivity for detection
of all known genotypes of HCV with a reported specificity of 97% to
99%.16This method is RT-PCR targeting the 5' UTR region. Both positive
and negative controls are included in the test kit, as are enzymes to
minimize the possibility of carryover contamination. A modification of
the original Amplicor'" test is also available for use with the automated
COBAS instrument (COBAS Amplicor Hepatitis C Virus Test versions
2.0), so that both amplification and detection of amplification products
are performed with measures of specimen inhibition and sample pro-
cessing efficiency included. The lower limit of sensitivity of this assay is
approximately 100 copies of HCV RNA/mL of blood.12This system is
the one most commonly used in ongoing evaluations of blood bank
screening of pooled donor units for HCV (Nucleic Acid Testing or NAT).
A comparison of the COBAS Amplicor HCV assay with the manual
Amplicor HCV assay proves that the two methods are equivalent for
the detection of active infection, with nearly identical concordance and
accuracy and similar sensitivities, 100% and 9870, respectively.'
Transcription Mediated Amplification. Transcription mediated am-
plification (TMA) is a new technology developed by Gen-Probe (San
Diego) and Bayer (Emeryville, Calif.) that may have important implica-
tions for the diagnosis of HCV.28The target for this assay is also the
5'UTR. Although not a PCR method, it is subject to the same precautions
and limitations. The VERSANT'" HCV RNA Qualitative TMA assay is
available for investigational use only, and test performance characteris-
tics have not been fully established. The qualitative TMA testing for
HCV is available in the United States as a service of the Bayer Reference
Testing Laboratory (Emeryville, Calif.). This assay may provide a more
sensitive detection method than RT-PCR. The company reports the detec-
 LABORATORY DIAGNOSIS OF VIRAL HEPATITIS 1123

tion of fewer than 50 HCV copies/mL and less than 5 HCV IU/mL.
This sensitivity allows early identification of virus replication and is also
useful to verify viral clearance. Specificity is reported to be greater
than 99.5%. The test incorporates contamination prevention systems and
internal controls to maximize reproducibility and minimize operator-
dependent variability. In a study in which 47 patient samples had tested
negative for HCV with commercial PCR assays, the VERSANT'" HCV
Qualitative RNA Assay showed that 36% of patient samples were posi-
tive with the new TMA test. After being tested with the conventional
PCR test, all of these patients had relapsed after treatment was ~topped.'~

QuantitativeMolecular Assays (Viral Load)

Quantitation of HCV RNA is useful for monitoring and defining

the end of treatment for HCV infections. They are not the preferred tests
for initial diagnosis of HCV infection. Several studies also report that
HCV RNA levels correlate with disease severity. As with qualitative RT-
PCR, there has also been a lack of standardization and reproducibility
for quantitative home-brew RT-PCR, which prevents direct comparison
of results with tests performed in different laboratories. In addition,
there are issues related to the limited dynamic ranges for the quantita-
tive assays.
The two most commonly used methods used to determine HCV
viral load are quantitative RT-PCR and the branched DNA @DNA)
assay; however, HCV Super Quant'" RT-PCR assay (performed as a
reference test by the National Genetics Institute, Culver City, Calif.) is
one of the most sensitive methods for quantitation of HCV and able to
detect as few as 50 to 100 copies/mL.
The Amplicor HCV Monitor'" quantitative RT-PCR test (Roche Di-
agnostics, Indianapolis) is not recommended for diagnosing HCV infec-
tion, because it is not considered to be as sensitive as the qualitative
methods.16 It should be used to monitor response to antiviral therapy.
Positive RNA controls and internal RNA standards, essential to quantita-
tive methods, are included in these assays. A version of this assay has
been converted to the semiautomated COBAS format, resulting in re-
duced setup time and improved reprod~cibi1ity.l~ This version has a
lower detection limit of 600 IU/mL, and the quantitative range extends
to 8.5 X lo5 IU/mL. It is also equally efficient in the quantification of
HCV genotypes 1 and 2.
The Quantiplex HCV RNA Assay 2.0 branched DNA assay (Bayer
Diagnostics, Emeryville, Calif .) is a standardized signal amplification
method. This is a hybridization technique that uses capture probes and
primers to detect and quantify RNA in serum or plasma. The results of
this assay are reported in megaequivalents of HCV RNA per mL, and
the dynamic range is from 0.2 megaequivalents per mL to 120 megaequi-
valents per mL (3.2 X lo4 to 1.9 x lo7 IU/mL). The sensitivity is similar
to the Roche Monitor'" s y ~ t e m , and
' ~ therefore it is not recommended
1124 WOLK et a1

to confirm or exclude the diagnosis of HCV, because qualitative RT-PCR

is a more sensitive method for detection.ls
Bayer recently released a new version of its quantitative HCV RNA
assay. The VERSANT'" HCV RNA 3.0 Assay @DNA)is available world-
wide for RUO (not for use for diagnostic procedures), and is designed
to quantitate HCV RNA over a broad range of values. The new version
of the bDNA assay is now available in a semiautomated format and has
a dynamic range of 2.5 X lo3 to 4.0 X lo7 copies/mL (5.21 X lo2 to 8.3
x lo6 IU/mL).I3 With assay results expressed in both copies/mL and
IU/mL, the clinician can compare results generated with the VERSANT
assay directly with results from other nucleic acid assays.

SUMMARY

Both serologic and molecular assays are useful in the diagnosis of

viral hepatitis. They may detect early infections before other signs of
disease appear, differentiate acute from chronic infections, and detect
persistence of viremia or verify development of immunity. Molecular
assays may also be used to monitor responses to antiviral therapy, and
in the future, be a primary method to screen blood and organ donors
(NAT). EIA serologies are used to diagnose acute HAV infections or
establish immune status. Similar immunoassays are used to detect HBV
infections, verify persistence of antigenemia and degree of infectivity,
and indicate immunity (including the response to vaccination). HBV
molecular assays can shorten the diagnostic window period, verify per-
sistence of viremia, including monitoring response to antiviral therapy,
and be useful in NAT screening of donors. Molecular assays play a
major role in HCV diagnosis where serologic tests can document past or
present infection but cannot differentiate one from the other. A variety
of molecular tests can be used as sensitive (and early) detectors of
viremia (and serve as confirmatory tests for positive serologies and as
donor NAT methods), document its persistence as an indicator of chronic
infection, and monitor responses to antiviral therapy. Both qualitative
and quantitative molecular assays are available, and their efficient use
requires familiarity with the sensitivity and dynamic ranges of each
method.

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Address reprint requests to

Jon E. Rosenblatt, MD
Mayo Clinic
Department of Laboratory Medicine and Pathology
Division of Clinical Microbiology
200 First St., SW
Rochester, MN 55905

e-mail: rosenblatt.jon@mayo.edu