Serologic Tests Used to
Diagnose Hepatitis C
Enzyme
Immunoassay
Anti-HCV is detected by enzyme immunoassay (EIA). The third-generation test
(EIA-3) used today is more sensitive and specific than previous ones. However,
as with all enzyme immunoassays, false-positive results are occasionally a problem
with the EIA-3. Additional or confirmatory testing is often helpful.
The best approach to confirm
the diagnosis of hepatitis C is to test for HCV RNA using a sensitive polymerase
chain reaction (PCR) assay. The presence of HCV RNA in serum indicates an active
infection. Testing for HCV RNA is also helpful in patients in whom EIA tests
for anti-HCV are unreliable. For instance, immunocompromised patients may test
negative for anti-HCV despite having HCV infection because they may not produce
enough antibodies for detection with EIA. Likewise, patients with acute hepatitis
may test negative for anti-HCV when the physician first tests. Antibody is present
in almost all patients by 1 month after onset of acute illness; thus, patients
with acute hepatitis who initially test negative may need followup testing.
In these situations, HCV RNA is usually present and confirms the diagnosis.
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Recombinant Immunoblot
Assay
Immunoblot assays are used to confirm anti-HCV reactivity, too. These tests
are also called "Western blots"; serum is incubated on nitrocellulose strips
on which four recombinant viral proteins are blotted. Color changes indicate
that antibodies are adhering to the proteins. An immunoblot is considered positive
if two or more proteins react and is considered indeterminate if only one positive
band is detected. In some clinical situations, confirmatory testing by immunoblotting
is helpful, such as for the person with anti-HCV detected by EIA who tests negative
for HCV RNA. The EIA anti-HCV reactivity could represent a false-positive reaction,
recovery from hepatitis C, or continued virus infection with levels of virus
too low to be detected (the last occurs only rarely when sensitive PCR assays
are used). If the immunoblot test for anti-HCV is positive, the patient has
most likely recovered from hepatitis C and has persistent antibody without virus.
If the immunoblot test is negative, the EIA result was probably a false positive.
Immunoblot tests are routine
in blood banks when an anti-HCV-positive sample is found by EIA. Immunoblot
assays are highly specific and valuable in verifying anti-HCV reactivity. Indeterminate
tests require further followup testing, including attempts to confirm the specificity
by repeat testing for HCV RNA.
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PCR Amplification
PCR amplification can detect low levels of HCV RNA in serum. Testing for HCV
RNA is a reliable way of demonstrating that hepatitis C infection is present
and is the most specific test for infection.
Testing for HCV RNA by
PCR is particularly useful when aminotransferases are normal or only slightly
elevated, when anti-HCV is not present, orwhen several causes of liver disease
are possible. This method also helps diagnose hepatitis C in people who are
immunosuppressed, have recently had an organ transplant, or have chronic renal
failure. At present, however, there are no PCR assays approved by the Food and
Drug Administration for general use, although commercial test systems are available.
Many commercial laboratories offer their own PCR assays, which are not subject
to strict independent quality controls. Thus, the reliability and specificity
of the PCR technique are not standardized. In addition, it is expensive and
prone to technical or laboratory error. When ordering HCV RNA testing by PCR,
the physician should use a high-quality laboratory willing to document standardization
of the test.
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Biochemical Indicators
of Hepatitis C Virus Infection
In chronic hepatitis C, increases in the alanine and aspartate aminotransferases
range from 0 to 20 times (but usually less than 5 times) the upper limit of
normal.
Alanine aminotransferase
levels are usually higher than aspartate aminotransferase levels, but that finding
may be reversed in patients who have cirrhosis.
Alkaline phosphatase and
gamma glutamyl transpeptidase are usually normal. If elevated, they may indicate
cirrhosis.
Rheumatoid factor and low
platelet and white blood cell counts are frequent in patients with cirrhosis,
providing clues to the presence of advanced disease.
The enzymes lactate dehydrogenase
and creatine kinase are usually normal.
Albumin levels and prothrombin
time are normal until late-stage disease.
Iron and ferritin levels
may be slightly elevated.
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Quantification of HCV
RNA in Serum
Several methods are available for measuring the titer or level of virus in serum,
which is an indirect assessment of viral load. These methods include a quantitative
PCR and a branched DNA (bDNA) test. Unfortunately, these assays are not standardized,
and different methods from different laboratories can provide different results
on the same specimen. In addition, serum levels of HCV RNA can vary spontaneously
by 3- to 10-fold over time. Nevertheless, when performed carefully, quantitative
assays provide important insights into the nature of hepatitis C. Viral load
does not correlate with the severity of the hepatitis or with a poor prognosis
(as it seems to in HIV infection); but viral load does correlate with the likelihood
of a response to antiviral therapy. Rates of response to a course of alpha interferon
and ribavirin are higher in patients with low levels of HCV RNA. There are several
definitions of a "low level" of HCV RNA, but the usual definition is below 2
million copies per milliliter (mL).
In addition, monitoring
viral load during the early phases of treatment may provide early information
on the likelihood of a response. Yet because of the shortcomings of the current
assays for HCV RNA level, these tests are not reliable guides to therapy. More
sensitive and reliable methods of quantitating HCV RNA in serum are needed.
Until that time, these tests should not be routinely used in practice.
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Genotyping and Serotyping
of HCV
There are 6 known genotypes and more than 50 subtypes of hepatitis C. The genotype
of infection is helpful in defining the epidemiology of hepatitis C. Knowing
the genotype or serotype (genotype-specific antibodies) of HCV is helpful in
making recommendations and counseling regarding therapy.
Patients with genotypes
2 and 3 are almost three times more likely to respond to therapy with alpha
interferon or the combination of alpha interferon and ribavirin. Furthermore,
when using combination therapy, the recommended duration of treatment depends
on the genotype. For patients with genotypes 2 and 3, a 24-week course of combination
treatment is adequate, whereas for patients with genotype 1, a 48-week course
is recommended. For these reasons, testing for HCV genotype is often clinically
helpful. Once the genotype is identified, it need not be tested again; genotypes
do not change during the course of infection.
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Normal Serum ALT Levels
Some patients with chronic hepatitis C have normal serum alanine aminotransferase
(ALT) levels, even when tested on multiple occasions. In this and other situations
in which the diagnosis of chronic hepatitis C may be questioned, the diagnosis
should be confirmed by testing for HCV RNA. The presence of HCV RNA indicates
that the patient has ongoing viral infection despite normal ALT levels.
Liver Biopsy
Liver biopsy is not necessary for diagnosis but is helpful for grading the severity
of disease and staging the degree of fibrosis and permanent architectural damage.
Hematoxylin and eosin stains and Masson's trichrome stain are used to grade
the amount of necrosis and inflammation and to stage the degree of fibrosis.
Specific immunohistochemical stains for HCV have not been developed for routine
use. Liver biopsy is also helpful in ruling out other causes of liver disease,
such as alcoholic liver injury or iron overload.
HCV causes the following
changes in liver tissue:
Necrosis and inflammation around the portal areas, so-called "piecemeal necrosis"
or "interface hepatitis."
Necrosis of hepatocytes
and focal inflammation in the liver parenchyma.
Inflammatory cells in the
portal areas ("portal inflammation").
Fibrosis, with early stages
being confined to the portal tracts, intermediate stages being expansion of
the portal tracts and bridging between portal areas or to the central area,
and late stages being frank cirrhosis characterized by architectural disruption
of the liver with fibrosis and regeneration.
Grading and staging of
hepatitis by assigning scores for severity are helpful in managing patients
with chronic hepatitis. The degree of inflammation and necrosis can be assessed
as none, minimal, mild, moderate, or severe. The degree of fibrosis can be similarly
assessed. Scoring systems are particularly helpful in clinical studies on chronic
hepatitis.
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Immunostaining
Immunostaining using polyclonal or monoclonal antibodies to detect HCV antigens
in the liver has been reported to be useful. However, these tests are not commercially
available, and, even in the hands of research investigators, immunostaining
detects HCV antigens in liver tissue in only 60 to 70 percent of patients with
chronic hepatitis C--largely in those with high levels of HCV in serum. This
test also requires special handling of liver tissue and thus is not appropriate
fo routine clinical use.
NIH Publication No. 99-4230
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