An overview of current practice in hepatitis C testing.

Hepatocyte injury, commonly encountered in the practice of' medicine, can be caused by a number of diseases such as hepatitis, autoimmune disorders, and congenital or acquired disorders of metabolism, or by exposure to alcohol, chemicals, I and drugs. The most common cause of liver injury worldwide is infection with viruses that primarily infect the liver, often termed hepatitis viruses. Viral infections are the most common cause of acute hepatitis. The range of responsible viruses is very broad, but those of greatest importance are the hepatitis viruses A, B, C, D, and E. The viral hepatitis "alphabet" is still growing. In 1996, novel RNA viruses were identified from the sera of patients with liver disease by two research groups. These possible agents have been named hepatitis GB virus type C (GBV-C) and hepatitis G virus (HGV), respectively. (1) These viruses are distinguished from each other by their morphology, modes of transmission, and propensity for development of chronic infections. The A and E viruses are transmitted via the fecal-oral route and cause acute hepatitis very rarely with any long-term complications, while the B, C, and D viruses are transmitted by exchange of body fluids; major methods of transmission include serum, sexual intercourse, illegal drug injections, and transmission from mother to infant (usually occurring during delivery), and are associated with development of chronic hepatitis infections, which might, ultimately, lead to cirrhosis (scarring) of the liver, hepatocellular carcinoma, liver failure, and death.

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Viral hepatitis is a major public-health problem, affecting people of all ages, races, and ethnicities. The World Health Organization (WHO) estimates that over 2 billion people alive today have been infected with the hepatitis B virus (HBV) at some point in their lives, and about 350 million of these remain chronically infected. In addition to this, available data indicate that approximately 3% of the world's population is infected with hepatitis C virus (HCV), giving an estimated total of 170 million people. With all of these viruses, the highest prevalence is among the people in Asia, Africa, South America, and eastern, central, and southern Europe. The actual number is hard to obtain because many people are not aware that they are infected and are not clinically ill.

In the United States, chronic liver disease and cirrhosis is the 12th leading cause of death among adults; deaths from cirrhosis are predicted to increase 360% by 2028 due to cases developing from chronic hepatitis-C infection. Hepatocellular-carcinoma or cancer incidence has doubled in the past 20 years and is expected to rise another 68% over the next decade from cancers developing in hepatitis-C-infected individuals. (2) Due to the extensive implementation of HBV vaccine and well-established protocols, the infection and complications of HBV have gone down tremendously. HBV vaccine is considered the first vaccine against cancer. It is estimated that 1.25 million Americans have chronic HBV infection. Although prototype vaccines that induce antibodies to HCV-envelope proteins have been developed, currently, hepatitis-C vaccination is not feasible practically. Genotype and quasispecies viral heterogeneity, as well as rapid evasion of neutralizing antibodies by this rapidly mutating virus, conspire to render HCV a difficult target for immunopropbylaxis with a vaccine. An estimated 3.9 million Americans have been infected with HCV, and 2.7 million people have chronic infection; HCV is the most common reason for liver transplantation, making HCV infection the most common chronic (long-term) blood-borne viral infection in the United States(2).

Liver disease is often clinically silent until late in its course. For this reason, laboratory tests are usually needed for recognition and characterization of the type of liver injury present. This is a field that combines different principles of laboratory medicine, such as clinical chemistry, immunology, virology, and molecular diagnostics. We will focus our discussion on the current practice of HCV testing. Serologic and nucleic-acid-based tests (NATs) are required to document exposure to and presence of the virus, and are also used to monitor treatment of infected individuals.

Hepatitis C virus (HCV)

HCV is an RNA (ribonucleic) virus of the flaviviridae family. The structure of the HCV consists of a core of genetic material (the RNA), surrounded by an icosahedral protective shell of protein, and further encased in a lipid (fatty) envelope of cellular origin. Two viral envelope glycoproteins, E1 and E2, are embedded in the lipid envelope. (1)

Clinical and laboratory features of hepatitis-C infection

Diagnosing acute hepatitis-C infection with certainty can be difficult, primarily because more than 70% of patients do not have symptoms associated with the acute infection. (3) It is even more challenging due to 1ack of a reliable and specific IgM-based serologic test, and potential overlapping laboratory findings with acute and chronic hepatitis-C infection (elevated alanine transaminase of ALT levels, positive serum HCV RNA, and anti-HCV lgG antibodies).

Overall, approximately 25% of all patients with acute HCV present with jaundice, and 10% to 20% develop gastrointestinal symptoms (nausea, vomiting, or abdominal pain).(3). On average, when symptoms do occur, they typically manifest six to eight weeks after exposure (range five to 12 weeks) and last for two to 12 weeks (3) In many cases, laboratory abnormalities may provide the initial clue to suggest a diagnosis of acute HCV infection. Rising ALT levels are typically observed approximately 40 to 50 days after infection. (4) Mean peak ALT values have tended to range between 400 lU/Lto 1,000 IU/L. Serum bilirubin levels may also be elevated, but they do not typically exceed 12 mg/dL. Occasionally, acute hepatitis C can manifest as a severe illness, but no cases of acute liver failure have been reported in the United States.

Overall, approximately 70% to 80% of individuals infected with hepatitis C will progress to persistent infection and about 20% to 30% will spontaneously resolve the infection. Serum HCV levels generally peak within six to 10 weeks of infection, regardless of eventual progression to chronic or resolved infection. In most individuals, anti-HCV antibodies appear seven to eight weeks after exposure. Notably, up to 3% of patients with chronic hepatitis C may never seroconvert. (4) Among those individuals who have spontaneous resolution of the HCV, it typically occurs within one year after infection. Both the presence of jaundice at the time of initial infection and a rapid decline in viral load during the first four to eight weeks after infection (4) correspond with sustained viral clearance. Acute hepatitis C can be diagnosed with a high level of certainty when the following three criteria are met:

* the patient reports recent risk factors for acquiring HCV;

* laboratory studies show positive HCV RNA levels, an elevated ALT level, and a positive anti-HCV-antibody test; and

* laboratory studies obtained within the prior 12 months demonstrate negative HCV viremia, normal serum hepatic aminotransferase levels, and negative HCV antibodies.

In the absence of historical data, several studies of acute HCV have relied on a stricter biochemical criterion of an ALT level greater than 10 or 20 times normal. (3,4) The presence of HCV RNA without detectable antibody response may also suggest acute hepatitis C, but, as previously noted, some individuals with chronic hepatitis C may never seroconvert. People who have recovered from a prior HCV infection often continue to make HCV-specific antibodies for decades. Consequently, their screening HCV EIA will remain positive even though they do not have ongoing infection.

Treatment recommendations for hepatitis C

The goal of therapy for hepatitis C is to achieve a sustained virologic response, defined as undetectable HCV RNA at least six months after cessation of therapy. Despite controversial findings among studies in the literature, the American Association for the Study of Liver Disease (AASLD) 2004 has generated interim recommendations, stating sufficient data exist to support serious consideration for treatment in most instances for patients with acute hepatitis C, after waiting two to four months for possible spontaneous clearance and assuming no contraindication for therapy exists. In addition, the AASLD has issued interim recommendations regarding length of therapy and possible therapeutic regimens. (5)

The length of treatment for acute hepatitis C has varied, depending on the response rates patients with acute hepatitis-C infection. HCV has six major genotypes and 50 subtypes. The prognosis is closely related to the genotype. Genotype 1 causes 70% to 75% of infections in the United States and is characterized by a lower rate of response to treatment (50% cure rate). Genotypes 2 and 3 have a much better response to treatment (80% cure rate). It is estimated that 70% to 80% of adults who are infected with HCV go on to have chronic infection, which is defined as the presence of HCV RNA in the blood for more than six months. Chronic infection is promoted by a high rate of viral mutation, lack of a vigorous host T-cell response, and replication in hepatocytes without cytotoxicity.

Infants born to HCV-infected mothers have passively acquired maternal antibody for up to 18 months after birth and, therefore, should only be screened using an HCV-antibody screening test after 18 months of age. (6) If earlier diagnosis is required, a qualitative polymerase chain reaction (PCR) can be performed at three months of age (6); however, a positive result does not mean the child will be chronically infected and false-positive results occur.

Currently, there is no safe and effective intervention known to prevent perinatal transmission of HCV. An effective hepatitis C vaccine has not been developed, and the drugs used most commonly to treat hepatitis C in both children and adults, interferon and ribavirin, are not recommended for use in pregnancy. There are no Food and Drug Administration-(FDA-) licensed therapies for children younger than 18 years of age. Therapy, however, may be indicated in select cases. Consultation with a pediatric specialist with experience in treating HCV infections in children is warranted.

Screening for HCV infection--flow chart

Because testing for the presence of HCV is complicated, and false-positive and false-negative results occur, an algorithm for the use of laboratory tests to diagnose patients with hepatitis C was developed by the Centers for Disease Control and Prevention (CDC) in 2003 (7). The HCV-antibody screen that we offer at our medical center gives us the ability to separate probable false-positive reactions from true-positive reactions by means of a signal-to-cutoff ratio.

1) Positive screens that have a low signal-to-cutoff ratio will be automatically reflexed to a recombinant immunoblot assay, or RIBA, confirmatory test before reporting the antibody screen.

2) If the RIBA is negative, the patient will be considered not infected with HCV, and the screen will be considered to be a false positive.

2a) If the RIBA is positive, which should happen rarely with a low signal-to-cut-off ratio, the physician will be called and blood obtained for a HCV quantitative PCR and, if positive, reflex to genotype, if possible and clinically appropriate.

3a) If the PCR quantitative test is positive, the genotype will be done and the patient considered to have active infection with hepatitis C and recommended for evaluation by a specialist in hepatitis-C disease.

3b) If the PCR is negative, then the patient may be one of the 20% to 30% who has spontaneous cure of the disease, in which case he will be positive for HCV antibodies but negative for active disease by the PCR results (no viral RNA detected). A comment will be in the report that a single negative PCR result does not rule out active infection, and a second PCR (qualitative test) is recommended within a month.

4) If the HCV antibody screen has a high signal-to-cutoff ratio, this indicates the patient has a greater than 95% chance of truly being infected. The physician will be called and blood obtained for the supplemental tests (HCV quantitative PCR) to confirm the antibody screen.

5) If the quantitative PCR is positive, the specimen will automatically have a genotype done, and the patient will be considered to have active infection with HCV and recommended for evaluation by a specialist in hepatitis-C disease.

5a) If the quantitative PCR is negative, the specimen will automatically be reflexed to a RIBA confirmatory test.

6) If RIBA is negative, the screen result would be reported as negative, and the patient considered not infected with hepatitis C (false-positive antibody screen).

6a) If the RIBA is positive, then the patient is positive for hepatitis-C antibodies and a single negative PCR result does not rule out active infection. The recommendation is a second PCR (qualitative) in a month to confirm spontaneous cure.

HCV-antibody screening test--EIA

The algorithm for diagnosing HCV infection begins with detection of antibodies to HCV using an FDA-approved enzyme immunoassay (EIA). In order to increase sensitivity, each reagent bead is coated with multiple HCV antigens (recombinant HCV polypeptides representing core [c22p], c200 [NS3 and NS4], and NS5 sequences). Indeed, the sensitivity is almost 100% in healthy adults with chronic HCV infection. There are two situations in which the HCV EIA test can be falsely negative. After exposure to HCV, it takes six to eight weeks to develop HCV IgG antibodies. (4, 5) Thus, the HCV EIA can fail to detect acute HCV infection. In addition, immunosuppressed persons, including those with relatively advanced HIV infection, can have a false-negative HCV EIA test. (5) In all other cases, the HCV EIA serves as an outstanding screening test for HCV infection because of its high sensitivity and relatively low price. The specificity of the HCV EIA is good.

There are two different strategies for confirming a positive HCV EIA. When a patient has a positive HCV EIA test, the primary objective is to confirm the EIA results by RIBA and also to determine whether they have chronic hepatitis C, a goal most expeditiously achieved by testing for HCV RNA.

HCV-antibody confirmation test--RIBA

RIBA contains the same HCV antigens, as do the EIA tests, separately on a strip along with superoxide dismutase (SOD) to detect non-specific antibodies to yeast proteins (recombinant HCV antigens are typically derived using yeast as the vector). A positive RIBA is defined as reactivity against two or more HCV antigens from different regions of the genome, without reactivity to SOD. Reactivity to a single HCV antigen or to a multiband with reactivity to SOD is considered indeterminate. Reactivity to none of the HCV antigen or to SOD only is considered negative. The advantage of the confirmatory anti-HCV test is that it can be performed on the same serum or plasma sample that was collected for the screening anti-HCV assay.

A positive RIBA result is interpreted as anti-HCV positive but does not distinguish between current or past infection; further HCV RNA testing is required to diagnose active infection. A negative RIBA result indicates a false-positive screening-test result. Several studies have demonstrated that HCV RNA is not detected in these sera, and HCV was not transmitted from blood units that were found to be EIA positive, RIBA negative. Thus, patients with EIA-positive, RIBA-negative sera do not require further testing and are considered "true negative;" and these patients are considered uninfected, although similar results may occur early after infection (prior to seroconversion) or in some chronically infected patients; additional NAT testing is appropriate if there is high clinical suspicion.

RIBA has lower sensitivity but higher specificity than the EIA method, therefore serving a perfect purpose for a confirmatory test. HCV-antibody result should not be reported until the initial EIA result is confirmed by the subsequent RIBA test. When HCV infection is not suspected, this approach is very helpful because the patient can be counseled accordingly, generally to his great relief.

HCV RNA assays

There are several HCV RNA assays that have been approved by the FDA for detecting chronic HCV infection. Some tests merely determine the presence or absence of HCV RNA; these tests are often referred to as qualitative tests, and the lower limits of detection of approximately 50 IU/mL (100 RNA copies/mL). Other assays can determine the quantity of HCV RNA; these are referred to as quantitative tests. The lower limit of detection is 200 IU/mL (500 RNA copies/mL). In general, qualitative assays are more sensitive, but this is not universal. Results from different methods cannot be directly compared because different standards are used. A WHO international standard for HCV RNA for NAT testing is now available (8) and is being introduced to use by kit manufacturers.

HCV RNA is very susceptible to degradation by the high activities of RNase present in blood; therefore, serum specimens for HCV RNA should be centrifuged as soon as possible after clot formation. EDTA or sodium-citrate plasma are preferred specimens for HCV RNA tests. Heparinized plasma is inhibitory for many NAT assays, and serum specimens provide suboptimal stability unless serum is frozen soon after specimen collection. If centrifugation is performed immediately, less than 10% of HCV RNA is lost, even if the plasma or serum is not separated from the formed elements for up to six hours (9). If a serum-separator tube is used, specimens are stable after centrifugation for up to 24 hours. (9) Short-term (< 7 days) storage of serum or plasma at 4[degree]C is acceptable. Once frozen, samples are stable through at least three freeze-thaw cycles. (9)

The quantitative assays, however, were configured to detect HCV RNA in more than 95% of persons with chronic HCV infection. Since they almost always are "positive" in persons with chronic hepatitis C--and because they also provide information used to predict treatment response--many clinicians prefer to use quantitative tests to confirm a positive HCV EIA in a person suspected of having HCV infection. A positive NAT not only confirms infection, it indicates active HCV infection; however, if the NAT is negative, then the positive screening HCV EIA still needs to be confirmed with RIBA. The added analytic sensitivity of the qualitative assays is probably important in monitoring response to treatment since persons who still have trace amounts of HCV RNA detected at the end of treatment will very likely relapse. (10)

If HCV RNA is used to confirm a positive EIA result instead of RIBA, there are some issues to consider. Because there are rare instances in which HCV RNA is intermittently undetectable in persons with chronic HCV infection, a negative RNA test result in a person with a positive screening HCV ETA needs to be repeated several months later. This is one of the reasons that HCV RNA testing is inferior to RIBA for persons who have a low pre-test probability of HCV infection. On the other hand, EIV-and RIBA- positive sera usually contain HCV RNA. The EIA-positive, RIBA-indeterminate sera may also contain HCV RNA, especially if the reactivity was to core or NS3 antigens. When EIA- and RIBA-reactive sera do not contain HCV RNA, it still represents prior HCV infection that has resolved.

Genomic amplification and sequencing, followed by sequence comparison and phylogenetic-tree construction is the reference method for genotype determination. (11) A variety of genotype-screening assays have been described, including PCR using genotype-specific primers, restriction fragment-length polymorphism of amplified sequences, and a commercially available line-probe assay. (11) These methods compare favorably with the reference method for determining HCV genotype.

Other tests

Liver-function tests and liver biopsies are not sensitive or specific enough to be used for screening. They may be the initial test that triggers testing for hepatitis C, and they are used as adjunct tests by specialists in treatment of hepatitis C who are caring for patients with the disease.

References

(1.) Kazuhiko N, Khin MW, San SO, et al. Molecular characteristic-based epidemiology of hepatitis B, C, and E viruses and GB virus C/hepatitis G virus in Myanmar. J Clin Microbiol.2000;39(4):1536-l539.

(2.) National Center for Health Statistics. National vital statistics reports. 2008;56(10).

(3.) Centers for Disease Control and Prevention (CDC). Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR Recomm Rep. 1998;47(RR-l9):1-39.

(4.0 Bowen DG, Walker CM. Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature. 2005;436:946-952.

(5.) Strader DB, Wright T, Thomas DL, et al. American Association for the Study of Liver Diseases. Diagnosis, management, and treatment of hepatitis C. Hepatology. 2004;39:1147-1171.

(6.) Mast AF, Hwang L-Y,Seto D, et al. Perinatal hepatitis C virus transmission: maternal risk factors and optimal timing of diagnosis. Hepatology. 1999;30:499A.

(7.) Alter MJ, Kuhnert WL, Finelli L. Guidelines for laboratory testing and result reporting of antibody to hepatitis C virus. MMWR Recomm Rep. 2003;52:1-16.

(8.) Saldanha J, Lelie N, Heath A. Establishment of the first international standard for nucleic acid amplification technology assays for HCV RNA. Vox Sanguinis. 1999;76:149-158.

(9.) Davis GL, Lau JY, Urdea MS, et al. Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification assay: definition of optimal conditions for specimen collection and clinical application in interferon-treated patients. Hepatology. 1994;19:1337-1341.

(10.) Sarrazin C, Teuber G, Kokka R, et al. Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays. Hepatology. 2000;32:818-823.

(11.) Forns X, Bukh J. Methods for determining the hepatitis C virus genotype. Veral Hepatitis. 1998;4:1-19.

CONTINUING EDUCATION

To earn CEUs, see current test at www.mlo-online.com under the CE Tests tab.

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. review the classification of viral hepatitis.

2. describe clinical features of HCV.

3. identify laboratory-screening tests to recognize HCV infections.

4. describe recommendations for treatment strategies.

5. describe methodology for the HCV tests by EIA, RIBA, and-RNA arrays.

Shu-Ling Liang, PhD, is an instructor in pathology at the Harvard Medical School, and an assistant director of Clinical Chemistry in the Department of Pathology at Beth Israel Deaconess Medical Center in Boston.

By Shu-Ling Liang, PhD