iral hepatitis is a major cause of morbidity and

mortality worldwide. The Centers for Disease

Control and Prevention estimated that there
were nearly 200,000 new cases of viral hepatitis
in the United States in 2001.
1
Although many cases are
subclinical or present with only flu-like symptoms, viral
hepatitis has a significant impact on society. There are
5 hepatitis viruses that have their principal site of injury
in the liver: hepatitis A, B, C, D, and E. All the major
hepatotropic viruses can cause acute viral hepatitis, but
only hepatitis B (with or without co-infection with hep-
atitis D) and hepatitis C cause chronic liver disease.
Chronic infection can lead to cirrhosis and even hepato-
cellular carcinoma.
Over the past several decades, significant advances
have been made in our understanding of hepatotropic
viruses, which has led to the development of various
serologic and molecular tests that have made it possi-
ble to accurately diagnose the specific type of viral hep-
atitis (Table 1). A firm understanding of these tests and
serologic markers will lead to the accurate diagnosis
and appropriate management. This review will focus
on the serologic and molecular tests available, rational
ordering of the tests, and their interpretation in the
diagnosis of viral hepatitis.
HEPATITIS A
Epidemiology
Hepatitis A virus (HAV), an RNA-containing virus
that is transmitted exclusively through the fecal-oral
route, is considered to be the most common cause of
acute viral hepatitis. Worldwide, it is estimated to infect
1.4 million people annually, most prominently in
South America, Africa, and Asia. The Centers for
Disease Control and Prevention estimated 90,000
symptomatic cases of HAV infection and 180,000
asymptomatic infections in the United States in 1997.
2
Accurate diagnosis is not only important for the care of
the infected individual but also for close contacts who
may be candidates for immunoglobulin prophylaxis.
3
Laboratory Testing for HAV
After infection, the incubation period for HAV can
range from 15 to 49 days, averaging 30 days.
4
The gold
standard for the diagnosis of acute HAV is the detection
IgM anti-HAV in the serum by radioimmunoassay or
enzyme-linked immunoassay (EIA). No confirmation
test is necessary if there is a positive IgM anti-HAV result
in conjunction with elevated transaminases and clinical
symptoms consistent with acute hepatitis. IgM anti-HAV
is invariably detected at the presentation of acute HAV.
IgG anti-HAV also may be detectable during an acute
HAV infection. Other methods of documenting HAV in-
fection exist, such as detecting HAV RNA by reverse tran-
scriptase polymerase chain reaction (RT-PCR) or direct
HAV antigen detection in stool, but these are expensive,
impractical, and have no routine clinical application.
IgM anti-HAV is detectable in the serum for 4 to
6 months after infection, although an infection that is
relapsing can have a positive IgM anti-HAV for up to
12 months before resolution.
5
HAV infection does not
cause chronic liver disease. The presence of IgG anti-
HAV is indicative of immunity. Anti-HAV IgG becomes
detectable after vaccination or natural infection. HAV
vaccination provides immunity for an estimated 10 to
20 years,
6
whereas natural infection provides lifelong
immunity.
HEPATITIS B
Epidemiology and Viral Structure
Hepatitis B virus (HBV) is the ninth leading cause
of death worldwide, with estimates of more than 350
million carriers worldwide.
7
In the United States, ap-
proximately 1 to 1.25 million people are chronically
infected.
HBV is a complex double-shelled DNA virus. The
V
Dr. Kim is a medical resident, Department of Medicine, VA Greater Los Angeles
Healthcare System, Los Angeles, CA. Dr. Saab is assistant professor of medicine
and surgery, University of California, Los Angeles.
www.turner- white.com Hospital Physician September 2004 15
C l i n i c a l R e v i e w A r t i c l e
Interpretation of Laboratory Tests for
Diagnosing Viral Hepatitis
Andrew I. Kim, MD
Sammy Saab, MD, MPH
outer membrane contains hepatitis B surface antigen
(HBsAg), while the inner core of the virus contains
hepatitis B core antigen (HBcAg) and hepatitis B e
antigen (HBeAg). Tests for these specific particles and
their corresponding antibodies aid in the diagnostic
evaluation of an HBV-infected patient.
HBV Virology and Viral Markers
The incubation period for HBV can vary from ap-
proximately 2 to 6 months. HBsAg is detectable by
enzyme-linked immunosorbent assay (ELISA) approxi-
mately 6 weeks after infection and usually is present
when an infected person is symptomatic from HBV.
With successful clearance of the virus, HBsAg should
begin to disappear and become undetectable by about
4 to 6 months. The disappearance of HBsAg is followed
by the appearance of antibody to HBsAg (anti-HBs), a
neutralizing antibody that confers immunity. Occasion-
ally, the appearance of anti-HBs may trail the disappear-
ance of HBsAg by several weeks to months. During this
window period, both HBsAg and anti -HBs may be
below the level of detection; if this occurs, the sole
markers of recent HBV infection during this time are
antibodies against the HBV core antigen, IgM and IgG
anti-HBc. Like HBsAg, IgM anti-HBc is detectable at
the onset of clinical symptoms. Although both anti-HBc
IgM and IgG rise together, levels of IgM anti-HBc usual-
ly will decrease while IgG remains increased. The pres-
ence of IgM anti-HBc is suggestive of a recent infection
(< 6 months). However, IgM anti-HBc may become
detectable during an exacerbation of chronic hepatitis
as well. Thus, differentiating an acute HBV infection
from a reactivation of chronic HBV using the presence
of IgM anti-HBc may be difficult.
8
After resolution of an
HBV infection, both anti-HBc and anti-HBs will be pre-
sent. Vaccination leads to the production of only anti-
HBs. The titers of anti-HBs may fall below the level of
detection over time.
Although uncommon, it is possible to see both
HBsAg and anti-HBs present simultaneously. The pres-
ence of both may suggest either resolving acute HBV
infection or the presence of non-neutralizing antibod-
ies, indicating a chronic carrier state.
9
There are sever-
al explanations for an isolated anti-HBc IgG, which are
discussed in Table 2 along with the interpretation of
other serologic outcomes for the diagnosis of HBV in-
fection.
Laboratory Testing for Chronic HBV
Fewer than 10% of all HBV-infected patients develop
chronic HBV. Patients who tend to be at greater risk for
chronic disease are the very young (up to 90% in new-
borns)
10,11
and the immunocompromised, including
hemodialysis patients.
12,13
Chronic HBV is suggested
by the presence of HBsAg in the serum for at least
6 months. After it has been determined that HBV is
chronic, the level of viral activity or replication is assessed
by testing for HBeAg and HBV DNA in the serum using
ELISA. Although HBeAg and HBV DNA become appar-
ent immediately after the incubation period along with
HBsAg, these tests are not routinely ordered at that time
unless there is evidence of chronic HBV infection. The
presence of HBeAg in the serum correlates with HBV
DNA synthesis and thus is a marker of viral replication
and infectivity.
14
The two types of chronic HBV infection are the
active and inactive forms. Active chronic HBV is char-
acterized by HBeAg positivity, HBV DNA exceeding
10
5
copies/mL, elevated transaminases, and liver biop-
sy findings showing chronic hepatitis. Inactive chronic
HBV is characterized by seroconversion from HBeAg
to antibody for HBeAg (anti-HBe), HBV DNA below
10
5
copies/mL, normal transaminases, and the ab-
sence of significant hepatitis on liver biopsy.
11
Both
active and inactive HBV infection will continue to
exhibit detectable HBsAg.
HEPATITIS C
Epidemiology and Natural History
Currently, approximately 3 to 4 million individuals
in the United States are infected with hepatitis C virus
(HCV), an RNA virus.
15
There are 6 different geno-
types of HCV. Genotype 1 accounts for 70% to 75% of
HCV infections in the United States and is associated
with a lower rate of response to treatment.
16
Symptoms of acute HCV infection usually are mild
and nonspecific. The incubation period for HCV is 14 to
160 days. HCV typically is diagnosed in the chronic
phase. Approximately 80% of infected individuals devel-
op chronic hepatitis. Patients with chronic hepatitis are
16 Hospital Physician September 2004 www.turner- white.com
K i m & S a a b : D i a g n o s i n g V i r a l H e p a t i t i s : p p . 1 5 1 9
Table 1. Serological Markers for Diagnosing Acute Viral
Hepatitis
Hepatitis Virus Markers of Infection
A IgM anti -HAV
B IgM anti -HBc, HBsAg
C Anti -HCV, HCV RNA
D IgM anti -HDV
E IgM anti -HEV
Anti -HBc = antibody to hepatitis B virus core antigen; HAV = hepati-
tis A virus; HBsAg = hepatitis B surface antigen; HDV = hepatitis D
virus; HEV = hepatitis E virus.
at risk of cirrhosis and subsequently hepatocellular carci-
noma. HCV is currently the leading cause for liver trans-
plantation.
Laboratory Testing for HCV
Both serologic and molecular tests are available for
the diagnosis and management of HCV.
17
Screening
for HCV is performed using EIA.
18,19
Both its sensitivity
and specificity for the diagnosis of HCV are greater
than 99%.
20,21
Positive EIA results can be confirmed
with a recombinant immunoblot assay (RIBA). Note
that anti-HCV is not neutralizing and thus does not
confer immunity.
Molecular tests utilizing RT-PCR to detect HCV
RNA are extremely sensitive tools for the diagnosis and
management of HCV. HCV RNA can be detected in
the serum within 1 to 3 weeks of exposure and at the
onset of symptoms.
16
Both quantitative and qualitative
assays exist. Qualitative testing can detect as few as 100
HCV RNA copies/mL, and the results are read as
either positive or negative. Quantitative assays provide
an estimate of viral load. However, the HCV viral load
neither correlates with severity of disease nor helps in
predicting the prognosis. Nevertheless, low levels of
viremia may correlate with a better probability of re-
sponse to antiviral therapy.
22
Besides estimating the
probability of achieving a therapeutic response to anti-
viral therapy, RT-PCR testing also may be useful in diag-
nosing seronegative patients who are immunosup-
pressed when there is a high suspicion of disease
23
and
assessing treatment efficacy during therapy.
24
Historically, confirmation testing was done using
RIBA after a positive EIA. More recently, RT-PCR test-
ing has become the confirmatory test of choice. An al-
gorithm of tests used in the diagnosis of HCV is shown
in the Figure.
HEPATITIS D
Natural History
Hepatitis D virus (HDV), also called the delta virus,
is a defective RNA virus that is unable to cause disease
K i m & S a a b : D i a g n o s i n g V i r a l H e p a t i t i s : p p . 1 5 1 9
www.turner- white.com Hospital Physician September 2004 17
Table 2. HBV Diagnostic Tests and Interpretation
HBsAg IgM Anti -HBc IgG Anti -HBc Anti -HBs Interpretation
+ + +
+ +
+ +
+
+ +
+
Anti -HBc = antibody to hepatis B core antigen; anti -HBs = antibody to HBsAg; HBsAg = hepatitis B surface antigen; HBV = hepatitis B virus.
Adapted from Centers for Disease Control and Prevention. Disease burden from hepatitis A, B, and C in the United States. National Center for
Infectious Diseases Viral Hepatitis Surveillance. Available at www.cdc.gov/ncidod/diseases/hepatitis/resource/dz_burden02.htm. Accessed 4 Aug
2004; and Saab S, Martin P. Tests for acute and chronic viral hepatitis. Finding your way through the alphabet soup of infection and superinfec-
tion. Postgrad Med 2000;107:1236, 12930.
Acute HBV infection
Chronic HBV infection
Past exposure with resolution and immunity
Immunity from vaccination
Window period
4 Interpretations:
Remote HBV infection and anti -HBs no longer detectable or
Recent recovery from acute infection or
Undetectable level of HBsAg in chronically infected patient or
False positive
Figure. Hepatitis C infection diagnostic algorithm. EIA =
enzyme-linked immunoassay; HCV RNA = hepatitis C virus
RNA detection with polymerase chain reaction; RIBA = re-
combinant immunoblot assay. *HCV EIA can give a false nega-
tive result in patients who are immunosuppressed. If the clini-
cal suspicion is high, an HCV RNA test should be performed.
HCV infection unlikely*
Confirms infection
EIA
+
+
+

False-positive EIA
Prior infection or
HCV RNA below level
of detection
HCV RNA
RIBA
on its own without the presence of a concurrent HBV
infection. The areas with the highest endemic rates are
the Middle East, Mediterranean countries, and certain
parts of South America.
2527
HDV is uncommon in
Western Europe, North America, and East Asia. HDV
can occur as a co-infection during an acute HBV infec-
tion or as a superinfection in chronic HBV carriers.
HDV infections also have been noted in liver trans-
plant patients with a prior history of HBV before overt
reinfection of the graft with HBV.
28
In co-infection,
severe and fulminant hepatitis can develop. In superin-
fection, there may be acute worsening of liver disease,
but the greatest risk is the development of chronic liver
disease with rapid progression to cirrhosis.
29,30
Laboratory Testing
The delta antigen (HDV Ag) becomes present in
the body during the late incubation period of acute
infection, which lasts 21 to 45 days. The presence of
HDV Ag is followed by anti-HDV IgM and IgG. IgM
anti-HDV lasts about 10 to 20 days when the infection
is acute and self-limited but may persist longer if the
infection becomes chronic. Anti-HDV, like anti-HCV, is
not neutralizing and does not confer immunity. RT-
PCR used to detect serum HDV RNA is the most reli-
able test, with a near 100% sensitivity.
31
IgM anti-HDV
does not help distinguish between co-infection and
superinfection. This distinction can be made by sero-
logic HBV testing, specifically, the presence or absence
of IgM anti-HBc. Patients with co-infection will be posi-
tive for IgM anti -HDV and IgM anti -HBc, whereas
patients with superinfection will be positive for IgM
anti-HDV and negative for IgM anti-HBc. Another clue
pointing toward co- infection would be a biphasic
aminotransferase elevation in the face of positive IgM
anti -HBc, with one peak resulting from the HBV-
induced liver injury and the other peak from the HDV
individual liver injury.
HEPATITIS E
Hepatitis E virus (HEV), an RNA virus transmitted
through the fecal-oral route, is an uncommon cause of
acute hepatitis in the United States. Like HAV, HEV
does not cause chronic liver disease. Most cases occur in
underdeveloped countries, with the highest incidence
in Africa, Asia, the Middle East, and Latin America. It
should be considered in a traveler returning from an
area where HEV is endemic.
32
The incubation period is
40 days on average and ranges from 15 to 60 days. Both
anti-HEV IgM and IgG are detectable at the onset of ill-
ness, but the sensitivities with current available tests are
only 53% and 87%, respectively, with specificities of
99% and 92%, respectively.
33
Levels of IgM anti-HEV
may decline rapidly, whereas IgG anti-HEV may remain
elevated although declining in titer. Detection of HEV
RNA by RT-PCR is not widely available. HEV infection
should be suspected in patients with a travel history to
areas endemic for HEV along with symptoms and labo-
ratory findings consistent with acute hepatitis, who are
also negative for HAV, HBV, HCV, cytomegalovirus, and
Epstein-Barr virus on serologic testing.
CONCLUSION
Viral hepatitis remains a major cause of morbidity
and mortality worldwide. It is virtually impossible to
clinically differentiate among the various causes of viral
hepatitis; accurate diagnosis can only be achieved with
serologic and molecular testing; these techniques have
revolutionized our ability to diagnose the offending
pathogen. It is crucial to have a firm understanding of
the serologic markers that characterize the viral hepati-
tides at various stages of the disease process. Knowledge
of the strengths and limitations of these tests allows for
rational ordering and interpretation. Ongoing research
of the major hepatotropic viruses, along with further
development of better diagnostic techniques and treat-
ments of viral hepatitis, will continue to aid in the care
of patients. HP
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