Anti-HCV-antibody to hepatitis C virus.
ALT-Liver enzyme released from liver cells that are injured, eg. by virus, alcohol, fat, drug, etc.
RIBA-2 Supplemental test to detect antibody to hepatitis C virus.
Indeterminate means 1 of 4 antigens positive.
HCV-RNA test by polymerase chain reaction (PCR) determines whether the virus is multiplying.

Testing and Diagnosis

How can I get tested?

Antibody Tests

PCR

Liver Function Tests

GENOTYPE

LIVER BIOPSY

Questions to ask your health care provider about testing.

How can I get tested?

There are several tests that your health care provider will do to diagnose hepatitis C, monitor the condition of your liver, and determine if you should consider treatment. Understanding the tests and what the results mean will help you become an active partner in managing your illness.

Antibody Tests
The first test usually done for hepatitis C is an antibody test such as an EIA. A reactive (positive) result means that, at some time in your life, you were exposed to hepatitis C and your body produced antibodies to fight off the virus. If the EIA test is reactive (positive), a second antibody test called the RIBA (which is more accurate) may be used to confirm the result. Most people who test reactive (positive) to both antibody tests are chronic carriers, meaning they carry the virus in their blood and can pass the virus on to others. However, some people (about 15-25%) who have a reactive (positive) result can clear the virus on their own without treatment. Further testing should be done to determine if you are chronically infected.

PCR
A PCR is a viral load test that detects the presence of hepatitis C in the blood. If the PCR is positive, you are infected with the hepatitis C virus, and are probably a chronic carrier. If you undergo treatment for hepatitis C, this test helps to monitor whether the medicines are working.

Liver Function Tests
Liver function tests (LFTs) help your health care provider determine whether your liver is working properly. These blood tests measure the levels of enzymes and other substances in your liver. When the liver is inflamed or damaged, certain enzymes will be released or the level of some substances will change. Some common LFTs include albumin, total protein, the enzymes ALT and AST, alkaline phosphatase, and bilirubin. Learn what your numbers mean and talk to your health care provider about how often the tests should be done.

Genotype
Genotype refers to the particular type of hepatitis C. There are at least six genotypes of hepatitis C. Most people (75%) in the U.S. have genotype 1. Genotypes 2 and 3 are the next most common. If you are infected with one genotype it is possible to become infected with another type. Therefore, it is important not to expose yourself to the blood of others. If you decide to be treated for your hepatitis C infection, knowing your genotype will help determine how you may respond to treatment, and how long your treatment will be. Your health care provider can find out your genotype by testing your blood.

Liver Biopsy
Your health care provider may want to do a liver biopsy. To do a biopsy, the provider takes a small piece of your liver to check for inflammation and scarring. It is a way for your health care provider to help you decide if and when you should begin treatment and what type of treatment you should receive. A biopsy is the only way to truly know the stage of liver disease. As with any medical procedure, there is a small risk associated with a biopsy, so be sure to ask your health care provider about the risks and what to expect before you decide to have a biopsy.

Questions to ask your health care provider about testing.
The following questions relate to routine and one-time liver tests:

What is my hepatitis C viral load? (If you have already gotten a viral load test)

What are the test results?
How often should I have my viral load checked?
May I have a copy of the test results for my records?

What is my hepatitis C genotype? (This is a one-time liver test)

(If you have already gotten a genotype test) What are the test results?
How does my genotype affect my illness and possible treatment?
May I have a copy of the test results for my records?

What are my liver function test levels?
(Liver function tests are ALT/AST, ALP, and SGTP, bilirubin, albumin, and prothrombin time)

(If you have already gotten liver function tests) What are the test results? How do they compare with normal levels?
How often should I have liver function tests done?
May I have a copy of the test results for my records?

Do you recommend I have a liver biopsy?

If yes, why? If no, why not?
What is involved in getting a biopsy?
What are the risks?
How is the procedure performed?
How long does the procedure take?
What experience do you have, or does the doctor performing the procedure have in doing liver biopsies? (The more experience they have, the better.)
If a liver biopsy shows that I have fibrosis or cirrhosis (scarring), how does that affect my treatment options?
(If you have already gotten a biopsy) What are the results of my liver biopsy and what does it mean? Will the result affect my treatment?
May I have a copy of the biopsy report for my records?

What does it all mean? (Interpreting Liver Function Tests)

Special Considerations in Interpreting Liver Function Tests.

Author/s: David E. Johnston
Issue: April 15, 1999

A number of pitfalls can be encountered in the interpretation of common blood liver function tests. These tests can be normal in patients with chronic hepatitis or cirrhosis. The normal range for aminotransferase levels is slightly higher in males, nonwhites and obese persons. Severe alcoholic hepatitis is sometimes confused with cholecystitis or cholangitis. Conversely, patients who present soon after passing common bile duct stones can be misdiagnosed with acute hepatitis because aminotransferase levels often rise immediately, but alkaline phosphatase and g-glutamyltransferase levels do not become elevated for several days. Asymptomatic patients with isolated, mild elevation of either the unconjugated bilirubin or the g-glutamyltransferase value usually do not have liver disease and generally do not require extensive evaluation. Overall hepatic function can be assessed by applying the values for albumin, bilirubin and prothrombin time in the modified Child-Turcotte grading system.

The commonly used liver function tests (LFTs) primarily assess liver injury rather than hepatic function. Indeed, these blood tests may reflect problems arising outside the liver, such as hemolysis (elevated bilirubin level) or bone disease (elevated alkaline phosphatase [AP] level).

Abnormal LFTs often, but not always, indicate that something is wrong with the liver, and they can provide clues to the nature of the problem. However, normal LFTs do not always mean that the liver is normal. Patients with cirrhosis and bleeding esophageal varices can have normal LFTs. Of the routine LFTs, only serum albumin, bilirubin and prothrombin time (PT) provide useful information on how well the liver is functioning.

The general subject of LFTs1,2 and the differential diagnosis of abnormal LFTs in asymptomatic patients3-5 have been well reviewed. This article discusses some common pitfalls in the interpretation of LFTs. Hints for interpreting these tests are presented in Table 1.

Markers of Hepatocellular Injury

The most commonly used markers of hepatocyte injury are aspartate aminotransferase (AST, formerly serum glutamic-oxaloacetic transaminase [SGOT]) and alanine aminotransferase (ALT, formerly serum glutamate-pyruvate transaminase [SGPT]). While ALT is cytosolic, AST has both cytosolic and mitochondrial forms.

Hepatocyte necrosis in acute hepatitis, toxic injury or ischemic injury results in the leakage of enzymes into the circulation. However, in chronic liver diseases such as hepatitis C and cirrhosis, the serum ALT level correlates only moderately well with liver inflammation. In hepatitis C, liver cell death occurs by apoptosis (programmed cell death) as well as by necrosis. Hepatocytes dying by apoptosis presumably synthesize less AST and ALT as they wither away. This probably explains why at least one third of patients infected with hepatitis C virus have persistently normal serum ALT levels despite the presence of inflammation on liver biopsy.6,7 Patients with cirrhosis often have normal or only slightly elevated serum AST and ALT levels. Thus, AST and ALT lack some sensitivity in detecting chronic liver injury. Of course, AST and ALT levels tend to be higher in cirrhotic patients with continuing inflammation or necrosis than in those without continuing liver injury.

As markers of hepatocellular injury, AST and ALT also lack some specificity because they are found in skeletal muscle. Levels of these aminotransferases can rise to several times normal after severe muscular exertion or other muscle injury, as in polymyositis,8 or in the presence of hypothyroidism, which can cause mild muscle injury and the release of aminotransferases. In fact, AST and ALT were once used in the diagnosis of myocardial infarction.

Slight AST or ALT elevations (within 1.5 times the upper limits of normal) do not

necessarily indicate liver disease. Part of this ambiguity has to do with the fact that unlike the values in many other biochemical tests, serum AST and ALT levels do not follow a normal bell-shaped distribution in the population.9 Instead, AST and ALT values have a skewed distribution characterized by a long "tail" at the high end of the scale (Figure 1).5 For example, the mean values for ALT are very similar from one population to another, but the degree to which the distribution is skewed varies by gender and ethnicity. The ALT distributions in males and nonwhites (i.e., blacks and Hispanics) tend to have a larger tail at the high end, so that more values fall above the upper limits of normal set for the average population.10,11

AST and ALT values are higher in obese patients, probably because these persons commonly have fatty livers.12 ALT levels have been noted to decline with weight loss.13 Depending on the physician's point of view, the upper limits of normal for AST and ALT levels could be set higher for more obese persons.

Rare individuals have chronically elevated AST levels because of a defect in clearance of the enzyme from the circulation.14 For both AST and ALT, the average values and upper limits of normal in patients undergoing renal dialysis are about one half of those found in the general population.15 Mild elevations of ALT or AST in asymptomatic patients can be evaluated efficiently by considering alcohol abuse, hepatitis B, hepatitis C and several other possible diagnoses (Table 2).5

Various liver diseases are associated with typical ranges of AST and ALT levels (Figure 2). ALT levels often rise to several thousand units per liter in patients with acute viral hepatitis. The highest ALT levels-often more than 10,000 U per L-are usually found in patients with acute toxic injury subsequent to, for example, acetaminophen overdose or acute ischemic insult to the liver. AST and ALT levels usually fall rapidly after an acute insult.

Lactate dehydrogenase (LDH) is less specific than AST and ALT as a marker of hepatocyte injury. However, it is worth noting that LDH is disproportionately elevated after an ischemic liver injury.16

It is especially important to remember that in patients with acute alcoholic hepatitis, the serum AST level is almost never greater than 500 U per L and the serum ALT value is almost never greater than 300 U per L. The reasons for these limits on AST and ALT elevations are not well understood. In typical viral or toxic liver injury, the serum ALT level rises more than the AST value, reflecting the relative amounts of these enzymes in hepatocytes. However, in alcoholic hepatitis, the ratio of AST to ALT is greater than 1 in 90 percent of patients and is usually greater than 2.17 The higher the AST-to-ALT ratio, the greater the likelihood that alcohol is contributing to the abnormal LFTs. In the absence of alcohol intake, an increased AST-to-ALT ratio is often found in patients with cirrhosis.

The elevated AST-to-ALT ratio in alcoholic liver disease results in part from the depletion of vitamin B6 (pyridoxine) in chronic alcoholics.18 ALT and AST both use pyridoxine as a coenzyme, but the synthesis of ALT is more strongly inhibited by pyridoxine deficiency than is the synthesis of AST. Alcohol also causes mitochondrial injury, which releases the mitochondrial isoenzyme of AST.

Patients with alcoholic hepatitis can present with jaundice, abdominal pain, fever and a minimally elevated AST value, thereby leading to a misdiagnosis of cholecystitis. This is a potentially fatal mistake given the high surgical mortality rate in patients with alcoholic hepatitis.19

Markers of Cholestasis

Cholestasis (lack of bile flow) results from the blockage of bile ducts or from a disease that impairs bile formation in the liver itself. AP and g- glutamyltransferase (GGT) levels typically rise to several times the normal level after several days of bile duct obstruction or intrahepatic cholestasis. The highest liver AP elevations-often greater than 1,000 U per L, or more than six times the normal value-are found in diffuse infiltrative diseases of the liver such as infiltrating tumors and fungal infections.

Diagnostic confusion can occur when a patient presents within a few hours after acute bile duct obstruction from a gallstone. In this situation, AST and ALT levels often reach 500 U per L or more in the first hours and then decline, whereas AP and GGT levels can take several days to rise.

Both AP and GGT levels are elevated in about 90 percent of patients with cholestasis.20 The elevation of GGT alone, with no other LFT abnormalities, often results from enzyme induction by alcohol or aromatic medications in the absence of liver disease. The GGT level is often elevated in persons who take three or more alcoholic drinks (45 g of ethanol or more) per day.21 Thus, GGT is a useful marker for immoderate alcohol intake. Phenobarbital, phenytoin (Dilantin) and other aromatic drugs typically cause GGT elevations of about twice normal. A mildly elevated GGT level is a typical finding in patients taking anticonvulsants and by itself does not necessarily indicate liver disease.22,23

Serum AP originates mostly from liver and bone, which produce slightly different forms of the enzyme. The serum AP level rises during the third trimester of pregnancy because of a form of the enzyme produced in the placenta. When serum AP originates from bone, clues to bone disease are often present, such as recent fracture, bone pain or Paget's disease of the bone (often found in the elderly). Like the GGT value, the AP level can become mildly elevated in patients who are taking phenytoin.22,23

If the origin of an elevated serum AP level is in doubt, the isoenzymes of AP can be separated by electrophoresis. However, this process is expensive and usually unnecessary because an elevated liver AP value is usually accompanied by an elevated GGT level, an elevated 5[acute accent]-nucleotidase level and other LFT abnormalities.

In one study,24 isolated AP elevations were evaluated in an unselected group of patients at a Veterans Affairs hospital. Most mild AP elevations (less than 1.5 times normal) resolved within six months, and almost all greater elevations had an evident cause that was found on routine clinical evaluation.

Persistently elevated liver AP values in asymptomatic patients, especially women, can be caused by primary biliary cirrhosis, which is a chronic inflammatory disorder of the small bile ducts. Serum antimitochondrial antibody is positive in almost all of these patients.

Indicators of How Well the Liver Functions

Bilirubin

Bilirubin results from the enzymatic breakdown of heme. Unconjugated bilirubin is conjugated with glucuronic acid in hepatocytes to increase its water solubility and is then rapidly transported into bile. The serum conjugated bilirubin level does not become elevated until the liver has lost at least one half of its excretory capacity. Thus, a patient could have obstruction of either the left or right hepatic duct without a rise in the bilirubin level.

Because the secretion of conjugated bilirubin into bile is very rapid in comparison with the conjugation step, healthy persons have almost no detectable conjugated bilirubin in their blood. Liver disease mainly impairs the secretion of conjugated bilirubin into bile. As a result, conjugated bilirubin is rapidly filtered into the urine, where it can be detected by a dipstick test. The finding of bilirubin in urine is a particularly sensitive indicator of the presence of an increased serum conjugated bilirubin level.

In many healthy persons, the serum unconjugated bilirubin is mildly elevated to a concentration of 2 to 3 mg per dL (34 to 51 [micro sign]mol per L) or slightly higher, especially after a 24-hour fast. If this is the only LFT abnormality and the conjugated bilirubin level and complete blood count are normal, the diagnosis is usually assumed to be Gilbert syndrome, and no further evaluation is required. Gilbert syndrome was recently shown to be related to a variety of partial defects in uridine diphosphate-glucuronosyl transferase, the enzyme that conjugates bilirubin.25

Mild hemolysis, such as that caused by hereditary spherocytosis and other disorders, can also result in elevated unconjugated bilirubin values, but hemolysis is not usually present if the hematocrit and blood smear are normal. The presence of hemolysis can be confirmed by testing other markers, such as haptoglobin, or by measuring the reticulocyte count.

Severe defects in bilirubin transport and conjugation can lead to markedly elevated unconjugated bilirubin levels, which can cause serious neurologic damage (kernicterus) in infants. However, no serious form of liver disease in adults causes elevation of unconjugated bilirubin levels in the blood without also causing elevation of conjugated bilirubin values.

When a patient has prolonged, severe biliary obstruction followed by the restoration of bile flow, the serum bilirubin level often declines rapidly for several days and then slowly returns to normal over a period of weeks. The slow phase of bilirubin clearance results from the presence of delta-bilirubin, a form of bilirubin chemically attached to serum albumin.26 Because albumin has a half-life of three weeks, delta-bilirubin clears much more slowly than bilirubin-glucuronide. Clinical laboratories can measure delta-bilirubin concentrations, but such measurements are usually unnecessary if the physician is aware of the delta-bilirubin phenomenon.

Albumin

Although the serum albumin level can serve as an index of liver synthetic capacity, several factors make albumin concentrations difficult to interpret.27 The liver can synthesize albumin at twice the healthy basal rate and thus partially compensate for decreased synthetic capacity or increased albumin losses. Albumin has a plasma half-life of three weeks; therefore, serum albumin concentrations change slowly in response to alterations in synthesis. Furthermore, because two thirds of the amount of body albumin is located in the extravascular, extracellular space, changes in distribution can alter the serum concentration.

In practice, patients with low serum albumin concentrations and no other LFT abnormalities are likely to have a nonhepatic cause for low albumin, such as proteinuria or an acute or chronic inflammatory state. Albumin synthesis is immediately and severely depressed in inflammatory states such as burns, trauma and sepsis, and it is commonly depressed in patients with active rheumatic disorders or severe end-stage malnutrition. In addition, normal albumin values are lower in pregnancy.

Prothrombin time

The liver synthesizes blood clotting factors II, V, VII, IX and X. The prothrombin time (PT) does not become abnormal until more than 80 percent of liver synthetic capacity is lost. This makes PT a relatively insensitive marker of liver dysfunction. However, abnormal PT prolongation may be a sign of serious liver dysfunction. Because factor VII has a short half-life of only about six hours, it is sensitive to rapid changes in liver synthetic function. Thus, PT is very useful for following liver function in patients with acute liver failure.

An elevated PT can result from a vitamin K deficiency. This deficiency usually occurs in patients with chronic cholestasis or fat malabsorption from disease of the pancreas or small bowel. A trial of vitamin K injections (e.g., 5 mg per day administered subcutaneously for three days) is the most practical way to exclude vitamin K deficiency in such patients. The PT should improve within a few days.

Blood ammonia

Measurement of the blood ammonia concentration is not always useful in patients with known or suspected hepatic encephalopathy. Ammonia contributes to hepatic encephalopathy; however, ammonia concentrations are much higher in the brain than in the blood and therefore do not correlate well.28 Furthermore, ammonia is not the only waste product responsible for encephalopathy. Thus, blood ammonia concentrations show only a mediocre correlation with the level of mental status in patients with liver disease. It is not unusual for the blood ammonia concentration to be normal in a patient who is in a coma from hepatic encephalopathy.

Blood ammonia levels are best measured in arterial blood because venous concentrations can be elevated as a result of muscle metabolism of amino acids. Blood ammonia concentrations are most useful in evaluating patients with stupor or coma of unknown origin. It is not necessary to evaluate blood ammonia levels routinely in patients with known chronic liver disease who are responding to therapy as expected.

Grading Liver Function by Child-Turcotte Class

In communicating among themselves, many physicians use the Child-Turcotte class as modified by Pugh, often termed the "Child class," to convey information about overall liver function and prognosis (Table 3).29 This grading system can be used to predict overall life expectancy and surgical mortality in patients with cirrhosis and other liver diseases.30

For elective general abdominal surgery, perioperative mortality is in the neighborhood of several percent for patients who fall into the Child class A, 10 to 20 percent for those in class B and approximately 50 percent for those in class C.31 These percentages must be balanced by prognostic considerations when transplantation becomes an option. The presence of cirrhosis by itself is not an indication for liver transplantation, and transplantation is rarely performed in patients who fall into Child class A. For example, the 10-year survival rate is as high as 80 percent in patients with hepatitis C and cirrhosis who have Child class A liver function and no variceal bleeding.32 However, once patients with any type of liver disease fall into the Child-Turcotte class B or class C category, survival is significantly reduced and transplantation should be considered.

REFERENCES

1.Kaplan MM. Laboratory tests. In: Schiff L, Schiff ER, eds. Diseases of the liver. 7th ed. Philadelphia: Lippincott, 1993:108-44.

2.Kamath PS. Clinical approach to the patient with abnormal liver function test results. Mayo Clin Proc 1996;71:1089-94.

Noninvasive Markers of Fibrosis for Longitudinal Assessment of Fibrosis in Chronic Liver Disease: Are They Ready for Prime Time? EDITORIAL

The American Journal of Gastroenterology
September 2005

Paul J. Thuluvath, M.D., F.R.C.P.1, and Karen L. Krok, M.D.1
1Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

….. the current published evidence suggests that FibroTest is not yet ready for prime time either to diagnose the severity of fibrosis or for longitudinal assessment of fibrosis since 15-20% are likely to be misdiagnosed by these markers….

The study published that this Editorial refers to follows the Editorial below.

SUMMARY
Over the past decade, there has been a renewed enthusiasm to develop noninvasive serum markers or tests to assess the presence and severity of fibrosis in chronic liver disease. Although a single marker or test has lacked the necessary accuracy to predict fibrosis, different combinations of these markers or tests have shown encouraging results. However, interlaboratory variability and inconsistent results with liver diseases of varying etiologies have made it difficult to assess the reliability of these markers in clinical practice. In this issue of the Journal, Poynard and colleagues describe the "histological" response to lamivudine in patients with chronic HBV over a 24-month period using surrogate serum biomarkers (FibroTest-ActiTest) without corroborating histological data. Investigators found improvement in fibrosis and inflammation in 85% and 91%, respectively, despite the emergence of YMDD mutation in 41.5% of patients. The higher improvement rates reported in this study should be interpreted with caution for a number of reasons including the absence of data on virological response rates, corroboratory histological data, and data on the validity of FibroTest to evaluate fibrosis in a longitudinal manner. Although FibroTest has been studied extensively by the authors of the current study, to date there are only few independent studies. In addition to significant interlaboratory variations, these studies have shown that significant fibrosis could be missed, or conversely significant fibrosis diagnosed in the absence of minimal or no fibrosis in about 15-20% of patients. We may be approaching a time when serum biomarkers may become an integral part of the assessment of patients with chronic liver disease, but published evidence suggests that these markers are not yet ready for prime time.

ARTICLE TEXT

The assessment of the presence and severity of liver fibrosis is of paramount importance in determining treatment strategies, response to treatment, prognosis, and the potential risk for complications in patients with chronic liver disease. Liver biopsy, the gold standard for assessing the severity of necroinflammatory activity and fibrosis, is invasive, and even in expert hands, it is associated with rare but serious complications including bleeding, pneumothorax, and perforation of colon or gallbladder (1, 2). It is also not practical or cost-effective to perform serial biopsies within a short time interval to assess treatment-related response. In addition to the sampling error that is inherently associated with percutaneous liver biopsies, there is an intra- and interobserver variability that may range from 15% to 33% in determining the fibrosis staging (3-5). Nevertheless, liver biopsy has remained the "gold standard" to assess the severity of inflammation and fibrosis in patients with chronic liver disease.

In the past decade, many investigators have proposed noninvasive tests to replace liver biopsy using either a single biochemical marker or a combination of tests. An ideal noninvasive marker for the evaluation of liver fibrosis should accurately predict the presence or absence of significant fibrosis (high sensitivity, specificity, positive, and negative predictive value [NPV]). In addition, it should be readily available and reproducible with low interlaboratory variability and with applicability in liver disease of various etiologies. Although liver biopsy does not fulfill many of these criteria, it has remained the "gold standard" mainly because of the absence of better alternatives. Recently, there has been a renewed interest in noninvasive markers of fibrosis because of evolving novel therapies for hepatitis C and B (6-9). While some investigators have focused on a combination of laboratory tests such as reversal of aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio, or AST/platelet ratio index (APRI), others have been searching for more novel markers of fibrosis and inflammation (8-12). Despite the common pathway for fibrosis, an imbalance between synthesis, and degradation of extra cellular matrix by activated stellate cells, there are currently no liver-specific markers that could accurately reflect liver fibrosis in liver disease of different etiologies. Many serum markers of matrix degradation products have been studied previously, either as a single marker or in combination, including N-terminal propeptide of type III collagen (PIIINP), collagen type-IV, hyaluronic acid, prolyl hydroxylase, laminin, matrix metalloproteinase-1 (MMP-1), and tissue inhibitors of metalloproteinase-1 (TIMP-1) and metalloproteinase-2 (TIMP-2). Many of these tests are not widely available, and when critically evaluated, they have shown disease-specific variations and inconsistencies suggesting that they are nonspecific. Many of these markers are nonspecifically elevated in the presence of inflammation or expressed in others tissues including skin, blood vessels, bone, and kidney. Other markers that have been studied include alpha2-macroglobulin, haptoglobins, and apolipoprotein A1. Increased serum levels of alpha2-macroglobulin, a protease inhibitor expressed by activated stellate cells, may relate to increased fibrosis. Haptoglobin levels may be decreased in liver fibrosis because of its association with profibrotic cytokine TGF-beta1, and apolipoprotein A1 levels may be decreased because of its binding affinity with extracellular matrix. Other tests that merits further independent corroboration include YKL-40 (a growth factor for fibroblasts that is expressed in the human liver), transient elastography, 13C-caffeine breath test, and DNA sequenced-based serum protein glycomics (10-13). The recent trend has been to use a combination of these tests to improve accuracy of predicting fibrosis in a consistent and reproducible manner (6-9).

The prospective multicenter study reported in this issue of American Journal of Gastroenterology is a longitudinal assessment of FibroTest and ActiTest (FT-AT) in 283 patients with chronic hepatitis B being treated with lamivudine (14). The biochemical tests were done in a central laboratory, but liver biopsy was done only at baseline. They found a statistically significant decrease in both fibrosis and inflammation as measured by FT-AT at 24 months irrespective of the presence of YMDD mutation. Interestingly, there was an immediate improvement at 6 month with a plateau between 6-12 months and then further improvement at 24 months. Lamivudine had previously been shown to significantly improve the histological features both for necroinflammatory activity and fibrosis. In the current study, 85% showed improvement in fibrosis and 91% showed improvement in inflammation at 24 months. It is reassuring to note the continued improvement in both fibrosis and inflammatory scores despite the emergence of YMDD mutation in 41.5% of patients. The higher improvement rates reported in this study should be interpreted with caution for a number of reasons including the absence of data on virological response rates, no corroboratory histological data, and a paucity of previously published data on the validity of FibroTest to evaluate fibrosis in a longitudinal manner.

The same investigators have published many previous articles on the validity of this proprietary test score (FibroTest) that uses five biochemical components including alpha2-macroglobulin, haptoglobin, apolipoprotein A1, gamma-glutamyl transpeptidase (GGT), and total bilirubin. ActiTest includes the same components plus serum ALT levels. The score is computed (using a complex formula) by entering patient's age and sex (older age and male sex were shown to be associated with more fibrosis in previous studies) along with the five components into a proprietary program (available for a fee) available at a website (http://www.biopredictive.com) and this composite value is used to determine the presence or absence of significant fibrosis. Poynard and colleagues have previously reported that the FibroTest score has a NPV (absence of fibrosis) of 100% and positive predictive value (presence of significant fibrosis) of 91% in patients with appropriate cut-off scores (6). This would be a remarkable development if it could be verified independently in a consistent fashion in liver disease of varying etiology. However, when the same investigators compared the test scores using laboratory results from eight centers with the reference laboratory, the interlaboratory agreement for fibrosis stage using this test score was very poor with kappa (kappa statistics) ranging from 0.32 to 0.94 with a mean 0.6 (15). In the current study, authors do not comment on the reproducibility of the tests in their reference laboratory over an unspecified period of time. Only a few studies have independently assessed FibroTest outside the current study group (12, 16). In an independent study, Rossi et al. found that 21% of patients who were predicted to have significant fibrosis by the FibroTest scores had only minimal fibrosis by histology and conversely, 18% of patients who were not supposed to have fibrosis by the test score had significant fibrosis (16). When FibroTest was studied in patients with chronic HBV in a previous study by Poynard's group, a FibroTest scores <= 0.20 had a NPV of 92% to exclude F2-F4 fibrosis, but the specificity was only 52% (17). The discrepancy in the published results could be explained by the inadequate length or sampling error of liver biopsy specimens, and inconsistencies in the measurement of biochemical markers (5, 15, 16, 18). Nevertheless, the current published evidence suggests that FibroTest is not yet ready for prime time either to diagnose the severity of fibrosis or for longitudinal assessment of fibrosis since 15-20% are likely to be misdiagnosed by these markers (15, 16, 18, 19).

Readers may find it disturbing to note that the first author of the study has a capital interest in Biopredictive, the company that markets FT-AT at their website for a fee, and another author is an employee of Biopredictive. Appropriately, most publications on the validity of the FT-AT have come from the same authors who have a research interest to develop noninvasive markers of fibrosis. The authors, rightfully so, have declared their conflict of interest. Another disturbing aspect of this article is that the authors are withholding important information on virological response rates perhaps for a later publication. This important piece of information is critical to interpret the results of the study.

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17.         Myers RP, Tainturier MH, Ratziu V, et al.. Prediction of liver histological lesions with biochemical markers in patients with chronic hepatitis B. J Hepatol 2003;39: 222-30.
18.         Rosenthall-Allieri MA, Peritore ML, Tran A, et al.. Analytical variability of the Fibrotest proteins. Clin Biochem 2005;38: 473-8.
19.         Bissell DM. Assessing fibrosis without a liver biopsy: Are we there yet? Gastroenterology 2004;127: 1847-9.

Longitudinal Assessment of Histology Surrogate Markers (FibroTest-ActiTest) During Lamivudine Therapy in Patients with Chronic Hepatitis B Infection

The American Journal of Gastroenterology
Volume 100 Issue 9 Page 1970 - September 2005

Thierry Poynard, M.D., Ph.D.1, Fabien Zoulim, M.D., Ph.D.2, Vlad Ratziu, M.D., Ph.D.1, Françoise Degos, M.D., Ph.D.3, Francoise Imbert-Bismut, Ph.D.4, Paul Deny, M.D.5, Paul Landais, M.D.6, Abdelkader El Hasnaoui, M.D.7, Alain Slama, M.D.7, Patrick Blin, Ph.D.8, Vincent Thibault, M.D.9, Parviz Parvaz, M.D.10, Mona Munteanu, M.D.11, and Christian Trepo, M.D.2

ABSTRACT
OBJECTIVES: The noninvasive serum markers, FibroTest-ActiTest (FT-AT), are an alternative to liver biopsy in patients with chronic hepatitis C and B. The aim was to use these markers in a prospective study of patients treated with lamivudine in order to assess the impact of treatment, as well as the factors associated with fibrosis progression.

METHODS: Two hundred and ninety-eight patients were included in a prospective longitudinal study in 50 hospitals across France. FT-AT were measured at baseline, and then after 6, 12, and 24 months of lamivudine 100-mg treatment. Epidemiological, clinical, and virologic characteristics were analyzed by univariate and multivariate analysis.

RESULTS: Two hundred and eighty-three patients were included for analysis. The accuracy of FT-AT versus biopsy was validated with the area under the ROC curve, 0.77 (SE = 0.03) for bridging fibrosis and 0.75 (SE = 0.06) for severe activity (A3). At baseline, bridging fibrosis (METAVIR stages F2-F3-F4) was highly associated (p< 0.001) in multivariate analysis with male gender and age and marginally associated with anti-HBe presence (p= 0.05) and non-Asian ethnic origin (p= 0.046). Lamivudine treatment had a very significant impact overall. FT decreased significantly from 0.51 at baseline to 0.37 at 24 months (p< 0.001), and 85% of patients had improvement at 24 months. AT also decreased significantly from 0.56 to 0.13 (p< 0.0001), and 91% of patients had improvement at 24 months. A three-phase kinetics was observed for both fibrosis and activity; there was a marked improvement during the first 6 months, followed by a plateau between 6 and 12 months, and another improvement between 12 and 24 months. The occurrence of a YMDD variant does not entirely explain these three-phase variations. The first phase impact on fibrosis rates was higher in Asian patients (p= 0.01) and in patients younger than 40 yr (p< 0.001).

CONCLUSIONS: In patients with chronic hepatitis B, a 24-month course of lamivudine treatment leads to a significant decrease in necroinflammatory grades and fibrosis stages as assessed by noninvasive markers, with the occurrence of a three-phase kinetics. FT-AT should be useful in the noninvasive follow-up of lamivudine treatment.

AUTHOR DISCUSSION
In this large multicenter cohort study of patients with chronic hepatitis B starting lamivudine therapy, we used noninvasive markers to estimate the histological impact of the first 24 months of treatment.

The use of biomarkers has many advantages and few disadvantages. The main advantage is the simplicity of the estimation procedure. Such simplicity would be unachievable using the classical estimate of four liver biopsies in 24 months. The detailed study by Dienstag et al. was possible only on a small sample of 63 patients who had already been included in a randomized trial, and used only three estimates (35). Another advantage of biomarkers is the reduced variability related to time differences. There is much more time variability for biopsy estimates than for biomarker estimates. In previous studies, which described 3 yr of treatment between the baseline biopsy and the end of follow-up biopsy, the exact interval was often actually 24 wk longer, which, therefore, included time without treatment (20, 35). This time variability is probably more significant for necroinflammatory activity than for fibrosis, as the variation in grades is generally more rapid than the variation in stages.

One disadvantage of biomarkers is that they are indirect markers of histological features and have 20-30% of discordance with biopsy. However, prospective studies in chronic hepatitis C have demonstrated that many of these discordances were in fact due to sampling error from biopsies that were too small (17, 20, 25). The diagnostic values of FT-AT observed in the present multicenter study were similar (AUROC = 0.74-0.77) to those observed in patients contaminated with HCV (10-26) and in the first validation study in patients contaminated with HBV (27).

The present study with biomarkers confirms previous findings made with biopsy on a greater number of patients but also presents new findings. In patients treated with lamivudine for 2 yr, there was an improvement of histological features both for necroinflammatory activity and fibrosis. Compared with Dienstag et al.'s study (35) and using a definition of improvement as a decrease in 0.01 units for FT or AT, we observed a higher improvement rate, probably because biomarkers are more sensitive than biopsy. If results were expressed in worsening percentages, the overall results for activity were similar: 11% for Dienstag at 3 yr versus 9% at 2 yr in the present study (35). The results were also similar for fibrosis impact. In patients with baseline bridging fibrosis, Dienstag et al. observed a 30% improvement rate (12 out of 19 patients with a drop of HAI score from 3 to 0 or 1) (35). Here, we observed a 94% FT improvement rate, including 31 patients (32%) with a drop of FT values of 0.30, which is equivalent to a 2 histological stage improvement. In cirrhotic patients, significant fibrosis regression was observed in 32%, confirming the reversibility of cirrhosis. This confirms previous studies using liver biopsy in a smaller group of HBV patients (35), and FT in HCV treated patients, without the risk of biopsy sampling error (20).

It was also confirmed that there had been an overall clear and significant histological benefit in patients with YMDD-variant HBV at 2 yr, without any case of dramatic worsening. However, the histological benefit was reduced after prolonged YMDD-variant HBV. We observed that the occurrence of a YMDD-variant significantly increased the mean AT compared to patients without a YMDD-variant; this occurred as early as the 6 month follow-up and during 2 yr of treatment. This early difference in AT at 6 months was not expected, as only 8% of patients already had a detectable YMDD-variant at this date. AT is, therefore, a sensitive marker which could be useful for the management of patients treated by lamivudine. FT values were not significantly higher in patients with a YMDD-variant, but results were close to those observed in Dienstag et al.'s study (35).

The new findings from this study were first, the kinetics of the histological impact of lamivudine. There was, both for fibrosis and activity estimates, a first phase of rapid improvement in the first 6 months of treatment, followed by a second phase with a plateau between 6 and 12 months and a third phase with another improvement between 12 and 24 months.

The first phase was particularly marked in patients with baseline bridging fibrosis who had two significant independent favorable prognostic factors: younger age and Asian ethnicity. This observation suggests that long-term management (duration of treatment and follow-up) of patients with HBV could be different in patients according to age and ethnic origin.

Contrary to expectations, the plateau observed in the second phase could not be entirely explained by the occurrence of a YMDD-variant, as the plateau was also observed in patients without a YMDD-variant. The only differences were a small increase of FT-AT between 6 and 12 months in patients with a YMDD-variant, which was not observed in patients without a YMDD-variant. We have no clear explanation for this plateau in patients without a YMDD-variant. The presence of an occult variant could be one explanation, as could be an immune response.

Weaknesses of the present study were the lack of intermediate estimates and a longer follow-up. We have assessed biomarkers at four different times only, baseline, 6, 12 and 24 months. We speculate that there is perhaps a plateau between 6 and 12 months in the antifibrotic efficacy of lamivudine. Other scenario are, however, possible such as rapid early improvement, followed by more steady improvement in inflammation and, particularly, fibrosis scores. The three phase pattern should be validated in another study to eliminate an artefact. However, these findings suggest that lamivudine treatment should not be stopped too early in patients with high risk factors such as non-Asian patients or those older than 40 yr.

Another original finding is the difference in the lamivudine impact according to baseline liver injury. In patients with baseline bridging fibrosis, the impact of treatment was already significant at 6 months, a point that encourages starting treatment very early in these patients. In patients without bridging fibrosis at baseline, the effect on fibrosis was much slower, with a significant decrease observed only at 24 months. For necroinflammatory activity, the impact of treatment was also very rapid for patients with moderate or severe activity at baseline. In patients without activity or with minimal activity, the impact was much slower, with a significant decrease only observed at 24 months. This observation is interesting, as it does not support the classical view of the nonefficacy of treatment in patients with low ALT, which are considered to lack a strong spontaneous immune response. Although delayed, an improvement of necroinflammatory features was observed. However, the risk-benefit of treatment in patients without significant activity at baseline should be discussed, as the benefit in terms of necroinflammatory features was only observed in patients without a YMDD-variant.

Factors Associated with Baseline Fibrosis

This study has demonstrated for the first time in a large number of subjects that sophisticated viral characteristics, such as HBV genotype or precore mutations, were marginally associated with bridging fibrosis compared with gender and age. Most of the significance observed in univariate analysis was no longer seen when adjusted for age and sex. It is, therefore, mandatory that studies assessing the prognostic value of molecular characteristics take into account age and gender. The present study was not perfect, as it is very difficult to assess the duration of contamination in our population with mixed ethnic origin. One advantage was that in contrast with the Phase III trials, which imposed rigorous inclusion and exclusion criteria, our study recruited a consecutive sample of patients with hepatitis B infections fulfilling broad inclusion criteria, thus representative of patients presenting at specialist hepatology departments in France.

Symptoms Observed During Treatment

In terms of clinical outcome, all patients responded to lamivudine treatment with a progressive decrease in symptoms of liver disease and ALT levels during the study period, HBe-seroconversion, and reduction of HBV DNA viral load detected by PCR. This desirable therapeutic response was observed in both patients with and without HBV polymerase mutations. There were no differences in the rate of disappearance of symptoms or in the incidence of new symptoms according to the presence or absence of severe activity or bridging necrosis estimated by biopsy or FT-AT (data not shown).

ALT flares accompanied by hepatic decompensation (i.e., jaundice, liver failure) have been observed at the time of lamivudine resistance and viral breakthrough (43). These cases were mainly described in patients with severe liver disease at baseline and/or with a precore mutant infection. In our cohort study, no cases of acute exacerbation and liver disease decompensation were observed, at least during the study period.

In conclusion, our cohort study provides new information on the histological impact of lamivudine in patients with chronic hepatitis B. This began with a rapid improvement in patients with baseline bridging fibrosis during the first 6 months, particularly in Asian and young patients, followed by a plateau between the 6th and 12th month, and another phase of improvement during the second year of treatment. The emergence of a YMDD-variant was associated with higher necroinflammatory activity at 2 yr. The use of FT-AT every 6 months could be a considerable help for making treatment decisions in patients treated with lamivudine. Future protocols assessing the histological impact of other drugs, alone or in combination, should make use of these noninvasive markers.

INTRODUCTION
Chronic hepatitis B virus (HBV) infection affects 350 million individuals globally. Approximately 15-40% may develop serious complications, including end-stage liver disease and hepatocellular carcinoma (1). Patients with significant hepatic inflammation and fibrosis are at the highest risk of these complications (2). Prior to considering antiviral treatment, current guidelines recommend liver biopsy (3). This procedure provides important information regarding the severity of necroinflammatory activity and fibrosis, features potentially useful for predicting treatment response and prognosis. Unfortunately, liver biopsy is invasive (4), costly, and limited by sampling error and poor intra- and interobserver concordance (5-7).

Considering these limitations and patient reluctance to undergo liver biopsy, noninvasive predictors of histology have been studied in the last 4 yr, particularly in patients with chronic hepatitis C (8). Aspartate (AST) and alanine amino transferase (ALT) are widely used for assessing hepatitis activity, but the ideal cut-offs are unclear (9). We developed a panel of biochemical markers, FibroTest-ActiTest (FT-AT), which have repeatedly demonstrated high predictive values for fibrosis and necroinflammatory histological activity in patients with chronic hepatitis C (10-26). FT-AT was also validated in patients with chronic hepatitis B in a single study (27).

The introduction of lamivudine in 1998 represented a significant advance in the treatment of chronic hepatitis B infections (28). This drug is a nucleoside analog that prevents replication of the HBV by inhibiting the viral RNA-dependent DNA polymerase enzyme. Lamivudine has been shown to stimulate viral clearance and improve clinical status in several randomized clinical trials and to provide sustained benefit over periods up to 4 yr (1, 28, 29). The efficacy of lamivudine in the treatment of hepatitis B is, however, compromised by the development of viral resistance (30). This is due to the selection of HBV mutants containing mutations in the YMDD motif of the hepatitis B polymerase. These viral strains are present as minor species in the pretreatment viral quasi-species and thus become the dominant species due to selection pressure during drug treatment. However, these polymerase mutants have reduced viability due to impaired catalytic activity and are replaced by the wild-type virus if lamivudine treatment is stopped (31).

The histological consequences of YMDD mutations are not well established. Only a few prospective studies have been undertaken using liver biopsy to determine the progression of fibrosis and activity in patients treated with lamivudine mostly at 1 year (28, 32-34). Two studies, including one study on 63 patients (35), have investigated treatment longer than 1 year, being the histological consequences of HBV mutants resistant to lamivudine by repeated estimates of histological features during 3 yr of treatment (35, 36).

The aim of this study was to use noninvasive markers, instead of biopsy, in a prospective study of patients treated with lamivudine in order to assess the dynamic impact of treatment on fibrosis and necroinflammatory histological activity while taking risk factors, particularly the occurrence of YMDD mutations, into account.

METHODS
Subject Selection
This prospective longitudinal study was carried out in 50 hospital hepato-gastroenterology or internal medicine departments across France.

Patients were to be included in the study if they were over the age of 18 yr and presented with active chronic hepatitis B infection that the investigator decided to treat with lamivudine. Exclusion criteria included previous exposure to lamivudine, dialysis, HIV infection, immunosuppressant chemotherapy, or organ graft recipients. In addition, lamivudine was only provided if the conditions set out in the official prescribing information for the drug were fulfilled, notably with respect to pregnancy.

Study Design
Following initiation of treatment with lamivudine, patients were followed up for a total of 24 months. A blood sample was taken for virologic characterization and for biochemical markers at baseline, 6, 12, and 24 months.

At the baseline visit, data were recorded on the sociodemographic characteristics of the patient, alcohol consumption, risk factors for hepatitis B infections (intravenous drug use, at-risk sexual practices, travel to countries where hepatitis B is endemic, familial or perinatal contamination), other viral infections including hepatitis C and D, duration of hepatitis B infection, symptom presentation, and results of previous serological tests of liver biopsies and previous treatments for hepatitis B.

Estimates of Liver Injury
We used the previously validated FT-AT (Biopredictive, Paris, France; FibroSURE LabCorp, Burlington, NC) (10-26). FT-AT is a noninvasive blood test that combines the quantitative results of six serum biochemical markers (alpha2-macroglobulin, haptoglobin, gamma glutamyl transpeptidase (GGT), total bilirubin, apolipoprotein A1, and ALT) with patients' age and gender in a patented artificial intelligence algorithm (USPTO 6631330) in order to generate a measure of fibrosis and necroinflammatory activity in the liver. FT-AT is a continuous linear biochemical assessment of fibrosis stage and necroinflammatory activity grade. It provides a numerical quantitative estimate of liver fibrosis, ranging from 0.00 to 1.00, corresponding to the well-established METAVIR scoring system (37) of stages F0-F4 and of grades A0-A3. The same conversions that had been established in HCV patients were used. Corresponding stages and grades were calculated from median scores and 95% confidence intervals (CIs) were observed in 1,270 HCV patients and 300 healthy blood donors (22, 24, 25). Among the 300 controls, the median FT value (±SE) was 0.08 ± 0.004 (95%, 0.23) and the median AT value was 0.07 ± 0.004 (95%, 0.26). Among the 1270 HCV-infected patients, the FT conversion was 0.000-0.2100 for F0; 0.2101-0.2700 for F0-F1; 0.2701-0.3100 for F1; 0.3101-0.4800 for F1-F2; 0.4801-0.5800 for F2; 0.5801-0.7200 for F3; 0.7201-0.7400 for F3-F4; and 0.7401-1.00 for F4. The AT conversion was 0.00-0.1700 for A0; 0.1701-0.2900 for A0-A1; 0.2901-0.3600 for A1; 0.3601-0.5200 for A1-A2; 0.5201-0.6000 for A2; 0.6001-0.6200 for A2-A3; and 0.6201-1.00 for A3.

Samples stored at -80°C were centralized in the reference laboratory at Pitié Salpêtrière Hospital. GGT, ALT, and total bilirubin were measured by Hitachi 917 Analyzer and Roche Diagnostics reagents (both Mannheim, Germany). alpha2-Macroglobulin, apolipoprotein A1, and haptoglobin were measured using a Modular analyzer (BNII, Dade Behring; Marburg, Germany). All coefficients of variation assays were lower than 6%.

Liver biopsies were fixed, paraffin-embedded, and stained with at least hematoxylin-eosin-safran, and Masson's trichrome or picrosirius red for collagen. At each center pathologists analyzed in a blinded fashion the biopsies using the METAVIR classification (37). Fibrosis was staged from F0 to F4: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis with few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis. Necroinflammatory activity was graded from A0 to A3: A0, no activity; A1, mild activity; A2, moderate activity; and A3, severe activity.

Virology
Blood samples taken at each study visit were subjected to virological analysis in a single center (INSERM 271, Lyon) without knowledge of the clinical data. Blood samples were analyzed for viral genome centrally in a blinded fashion with respect to the clinical data. Viral DNA was extracted from serum using the QIAmp DNA Blood Mini Kit (QIAGEN, Courtaboeuf, France). Baseline HBV DNA was quantified using the Versant branched DNA kit version 3.0 (Bayer, France) with a lower limit of detection of 357 IU/ml (1 IU/ml represents 5.6 copies/ml). The HBV polymerase gene was sequenced following amplification by polymerase chain reaction (PCR) using appropriate oligonucleotide primers. The lower limit of detection of the PCR assay was 70 IU/ml (data not shown). The sequencing reaction was performed using labeled nested primers (CY5.5-POL3M and CY5.0-P4M), CY5/CY5.5-Dye Primer Kit, Long-Read TOWER sequencer, and Opengene System software (Visible Genetics, Evry, France). INNO-LiPA genotyping and Precore mutant detection kits were used, and the blot reactions were performed using AUTOLiPA kits on nitrocellulose strips.

Statistical Analysis
We performed statistical analyses on all patients with at least one sample and in patients with all four samples (sensitivity analysis). Categorical variables were compared with the chi2 test or Fisher's exact test (in cases with less than five theoretical subjects per cell), quantitative variables with the Mann-Whitney test, and ANOVA for repeated measures used Bonferroni and Tukey-Kramer multiple comparison tests (38). Multivariate analysis used logistic regression analysis. Diagnostic value was assessed by the area under the receiver operating characteristics curve (AUROC). A sensitivity analysis was performed according to the duration between biopsy and baseline serum, using the median as a cutoff. Three methods were used in the statistical comparison of liver injury dynamics: the difference between FT-AT values, the percentage of patients with FT-AT improvement, and the fibrosis rate per year as previously used for HCV modeling (39-41). A sensitivity analysis included date of birth to calculate the fibrosis progression before treatment in order to exclude bias due to inaccurate date of infection (42). As we had already observed a strong interaction between baseline fibrosis and treatment impact in a previous analysis of treatment impact in patients with chronic hepatitis C, we stratified the analysis according to the baseline fibrosis stage (bridging vs non bridging fibrosis) (20). All data were centrally analyzed using BMDP statistical software (38).

RESULTS
Patients Included
Between October 1999 and March 2001, 298 subjects were included. Fifteen (5.1%) were excluded from the analysis due to the absence of follow-up data in 11 and protocol violation in 4. In all, 274 subjects (96.8% of the analyzed population of 283 subjects and 91.9% of the included population) completed the 24-month course of treatment. A total of 916 FT were assessed in the analyzed population; 258 were done at baseline, 229 at 6 months, 227 at 12 months, and 202 at 24 months (Fig. 1). In the remaining patients (no different from included patients, data not shown), serum was not available for FT assessments.

At the time of inclusion, 55.1% of the population was treatment naive; the rest had received interferon alpha predominantly. Only 20 patients had received previous nucleoside analog antiviral drugs, principally vidarabine (12 patients) and/or famciclovir (10 patients).

At the time when biochemical markers were obtained, liver biopsy results were available in 214 subjects, at a median of 5.2 months before inclusion in the study. At the time of biopsy, 66.7% of patients had bridging fibrosis (F2-F3-F4), including 18.7% cirrhosis F4. At the time of inclusion, as estimated by biochemical markers, 57.8% of patients had bridging fibrosis, including 25.2% cirrhosis (Table 1).

Hepatitis B virus DNA was quantified at baseline in all patients. At inclusion, basic core promoter (BCP) and precore mutants were identified in the majority of subjects (81.0%). There was a slight preponderance of promoter mutations compared to stop codon mutations, with both mutations being observed in 33.6% of subjects. A total of 119 patients (42%) were HBeAg-positive and 164 (58%) were HBeAg-negative. Genotype D was the most frequent genotype, followed by genotype A. Genotypes F and G were very rare.

Validation of Biochemical Markers
Using the previous biopsy as "a gold standard," the AUROC for the diagnosis of bridging fibrosis (METAVIR stages F2-F3-F4) was 0.77 (SE = 0.03); for many septa (F3) or cirrhosis (F4), 0.74 (SE = 0.03); for the diagnosis of moderate or severe activity (METAVIR grades A2, A3), 0.62 (SE = 0.04); and for severe activity (A3), 0.75 (SE = 0.06). When AUROCs were compared according to the duration between biopsy and biomarkers measurements (less vs more than 4 months), there was no significant difference for FT (for F2-F3-F4): 0.76 (0.05) versus 0.79 (0.05) (p= 0.53) and a significance for AT (for A2A3): 0.69 (0.06) versus 0.52 (0.06) p= 0.04.

Six patients were at high risk of having false positive results for baseline FT due to possible hemolysis (haptoglobin <0.11 g/l); 1 patient was at risk of having a false negative result due to acute inflammation (A2M 6.22 g/l, haptoglobin 1.95 g/l).

Factors Associated with Fibrosis at Baseline
In univariate analysis, bridging fibrosis (METAVIR stages F2-F3-F4) was associated with age, male gender, BMI, alcohol consumption, baseline activity grade, anti-HBe presence in the serum, and genotypes A, D, E (Table 2).

In multivariate analysis, baseline bridging fibrosis was highly associated (p< 0.001) with male gender (odds ratio (OR) = 0.14, 95% CI 0.06-0.32) and age (OR = 0.93, CI = 0.91-0.96), and marginally associated with anti-HBe presence (OR = 0.53, CI = 0.28-1.00; p= 0.05) and non-Asian ethnic origin (OR = 2.31, CI = 1.01-5.30; p= 0.046).

Evolution of Fibrosis and Histological Activity During Treatment
The values of FT-AT before and during treatment are shown in Table 3. Lamivudine treatment had a very significant impact overall. FT decreased significantly from 0.51 at baseline to 0.37 at 24 months (p< 0.001), and 85% of patients had an improvement at 24 months. AT also decreased significantly from 0.56 to 0.13 (p< 0.0001), and 91% of patients had an improvement at 24 months. The mean fibrosis progression rate was 0.15 FT units per year (0.02; n = 254) before treatment, -0.12 (0.02; n = 217) during the first 6 months, 0.04 (0.02; n = 187) between 6 and 12 months, and -0.09 (0.02; n = 166) between 12 and 24 months (the progression rates before treatment and between 6 and 12 months were significantly different vs all) (Table 4). A total of 44 patients had at baseline a cirrhosis estimated by FT and a second FT assessment at 24 months; 95% (42/44) had an improvement of FT at 24 months, including a significant regression of fibrosis greater than 0.30 in 32% (14/44).

The impact of treatment on fibrosis was greater during the first 6 months, with a plateau between 6 and 12 months and a greater impact between 12 and 24 months. The impact of treatment on necroinflammatory activity was marked at 6 months (87% improved), with a plateau between 6 and 12 months and another impact between 12 and 24 months.

Factors Associated with Lamivudine Impact
The impact was greater in patients with bridging fibrosis at baseline. The significant impact of treatment was observed both in patients with and without an occurrence of YMDD mutation.

The impact on AT was lower at 12 and 24 months in patients with YMDD mutation than in patients without (Table 3).

During the course of the study, YMDD mutations were observed in 114 patients. This corresponds to an incidence rate of 41.5% (95% CI limits 35.6% and 47.3%). The time course for the emergence of mutations was linear, with 17 (6%) occurring in the first 6 months, 47 (17%) between 6 and 12 months, and 51 (18%) between 12 and 24 months. The FT changes were from 0.41 ± 0.07 to 0.33 ± 0.07 (NS) in the group of mutation occurrence in the first 6 months; from 0.59 ± 0.04 to 0.43 ± 0.05 (p< 0.0001) in the group of mutation occurrence between 6 and 12 months; from 0.46 ± 0.04 to 0.36 ± 0.04 (p< 0.0001) in the group of mutation occurrence after 12 months. In 44 patients with baseline cirrhosis, the only 2 patients with worsening FTs were those with YMDD-variant HBV (2/20); there was a significant FT reduction in 25% of these patients (5/20).

The AT changes were from 0.53 ± 0.10 to 0.10 ± 0.03 (p= 0.04) in the group of mutation occurrence in the first 6 months; from 0.59 ± 0.04 to 0.20 ± 0.03 (p< 0.0001) in the group of mutation occurrence between 6 and 12 months; from 0.55 ± 0.04 to 0.19 ± 0.04 (p< 0.0001) in the group of mutation occurrence after 12 months.

The fibrosis progression rates during the three phases according to the baseline fibrosis and YMDD mutation are shown in Table 4. In the group of patients with baseline bridging fibrosis, very significant differences in the kinetics of the first 12 months of treatment were observed in Asian patients, who had a greater decrease in fibrosis rate during the first month compared with non-Asian patients (p= 0.01) (Fig. 2A), and patients younger than 40 yr (p< 0.001) (Fig. 2B), both still significant in multivariate analysis (p= 0.03 and p= 0.04, respectively). Kinetics were not different according to gender or anti-HBe status (data not shown).

Sensitivity Analyses
The same results were obtained when the population of patients with all completed FT or AT at the four follow-up dates was used (Table 3 panel B). When patients were stratified according to stage or grade as estimated by liver biopsy instead of FT-AT, the results were similar (data not shown). When baseline fibrosis progression rates were calculated according to the date of birth instead of the date of contamination, results were similar (data not shown).

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Prometheus Laboratories Introduces Non-Invasive Test for the Detection of Liver Fibrosis

Prometheus Laboratories Introduces FIBROSpect(SM) II
Tuesday January 20, 2004 5:16 pm ET

- A New Improved Non-Invasive Test for the Detection of Liver Fibrosis -

SAN DIEGO, Calif., Jan. 20 /PRNewswire/ -- Prometheus Laboratories Inc., a specialty pharmaceutical company, announced today the introduction of FIBROSpect II, a unique non-invasive diagnostic panel to aid in the detection of liver fibrosis in patients with chronic hepatitis C. This simple blood test helps physicians differentiate the presence of no/mild from significant liver fibrosis without the pain, anxiety and risk associated with liver biopsy.

(Logo: http://www.newscom.com/cgi-bin/prnh/20030416/PROMETHEUSLOGO )

"In chronic hepatitis C infection, liver biopsy has been the favored approach to evaluate the extent of liver fibrosis and help guide treatment decisions; however, it is expensive, associated with possible complications, and limited by sampling error and observer variability," stated Dr. F. Fred Poordad, Associate Director, Hepatology and Liver Transplant at Cedars Sinai Medical Center. "Non-invasive methods to aid in assessing liver disease severity, such as FIBROSpect II, provide additional diagnostic options."

FIBROSpect II, an enhanced version of the original FIBROSpect test, eliminates indeterminate test results that were occasionally reported with the first generation test, and improves the ability to accurately differentiate patients with and without significant liver fibrosis. FIBROSpect II is based on three extracellular matrix remodeling proteins utilizing a new algorithm and unique index, thereby providing physicians with even more clinically useful information.

According to the most recent estimates by the Centers for Disease Control and Prevention, 3.9 million people are currently infected with hepatitis C in the U.S., with 2.7 million being chronically infected. Worldwide, over 170 million are infected. Chronic hepatitis C varies in its cause and outcome. At one end of the spectrum are patients who have no sign of liver disease and for whom the overall prognosis may be good. At the other end of the spectrum are patients with chronic hepatitis C and advanced fibrosis that may ultimately develop end-stage liver disease. The major consequence of liver disease is the progression to fibrosis and cirrhosis, which can lead to liver cancer or the need for a liver transplant. Therefore, early and accurate diagnoses and staging are critical for proper patient management.

Prometheus Laboratories Inc. is a specialty pharmaceutical company committed to developing new ways to help physicians individualize patient care. The Company focuses on the treatment, diagnosis and detection of gastrointestinal, autoimmune and inflammatory diseases and disorders. The Company's strategy includes the marketing and delivery of pharmaceutical products complemented by its proprietary, high-value diagnostic testing services. By integrating these therapeutic, diagnostic and treatment monitoring services, Prometheus addresses the full continuum of care, thereby providing physicians with a comprehensive solution to treat chronic diseases. Prometheus' corporate offices are located in San Diego, California. Additional information about Prometheus Laboratories Inc. can be found at www.prometheuslabs.com .

LabCorp® Announces U.S. Launch Of Exclusive Liver Fibrosis Assay HCV Fibrosure™

Noninvasive Blood Test Provides Alternative to Liver Biopsy for Assessing Status of Hepatitis C Patients

Burlington, NC, March 17, 2004 - Laboratory Corporation of America® Holdings (LabCorp®) (NYSE: LH) today announced the availability of HCV FIBROSURE™, a noninvasive blood test for assessing liver status in hepatitis C virus (HCV) patients. Developed by leading hepatologists at the Pitie-Salpetriere Hospital and BioPredictive in France, HCV FIBROSURE™ is only available in the United States through LabCorp.

HCV FIBROSURE™ provides an easily accessible alternative to liver biopsy, which physicians use to assess liver fibrosis and necroinflammatory activity in HCV patients. While liver biopsy has long been considered the gold standard to monitor the status of HCV and determine therapy options, it is an invasive procedure that carries a risk of serious complications. HCV FIBROSURE™ uses a combination of six serum biochemical markers plus age and gender in a patented algorithm to determine the degree of liver fibrosis and the level of ongoing necroinflammatory activity. The test, which has been clinically available in Europe for the past two years, has been shown in several studies to enable quantitative, reproducible assessment of fibrogenic and necrotic activity in the liver of HCV patients.

"The launch of this important new test once again validates LabCorp's strategy of creating a world-class national laboratory with the best and broadest array of diagnostic testing services," said Myla P. Lai-Goldman, M.D., executive vice president, chief scientific officer and medical director at LabCorp. "Our focus on bringing forth innovative new technologies and tests, coupled with our scientific expertise and national scope, helps us broadly deliver vital new tools like BioPredictive's liver fibrosis assay to U.S. physicians managing HCV patients."

BioPredictive is currently researching clinical use of this test for other disease populations, including hepatitis B, HIV-HCV, and alcoholic and non-alcoholic steato hepatitis (NASH). "We anticipate that HCV FIBROSURE™ will prove to be just the first in a family of innovative, noninvasive diagnostic testing products aimed at hepatitis and non-hepatitis-related conditions," said Dr. Thierry Poynard, a world-renowned hepatologist, head of Hepato-Gastrotroenterology department in Pitie-Salpetriere Hospital in Paris, and researcher and founder of BioPredictive. "We look forward to continuing our relationship with LabCorp and building upon their expertise in the world of hepatitis testing for future products."

HCV FIBROSURE™ is recommended for use to assess liver status following a diagnosis of HCV, as a baseline determination of liver status before initiating HCV therapy, as post-treatment assessment of liver status six months after therapy completion, and for noninvasive assessment of liver status in patients at risk of complications from a liver biopsy. The blood sample for HCV FIBROSURE™ can be collected in minutes and results can be returned to the physician within days. The test uses six biochemical markers that are routine and considered standard of care in the United States.

About BioPredictive

Founded at Paris University in 2002, BioPredictive is focused on the study, design and development of medically important biological tests. At the center of the company's scientific efforts is the desire to improve disease management by replacing invasive strategies with noninvasive alternatives. BioPredictive has developed two noninvasive tests for chronic liver disease - FibroTest and ActiTest. FibroTest is a biochemical marker of liver fibrosis and ActiTest is a biochemical marker of inflammation and necrosis of the liver. BioPredictive licenses the FibroTest and ActiTest technology from Assistance Public-HTMpitaux de Paris (AP-HP). The company performs more than 2,000 tests per month, and services 150 private and 12 public hospital laboratories in France, Switzerland, Portugal, Morocco and Mexico. To learn more about BioPredictive, visit the company Web site at: www.BioPredictive.com.

FibroTest and ActiTest are available in the U.S. exclusively through Laboratory Corporation of America® Holdings (LabCorp) under the name HCV FIBROSURE™.

About LabCorp

Laboratory Corporation of America® Holdings is a pioneer in commercializing new diagnostic technologies and the first in its industry to embrace genomic testing. With annual revenues of $2.9 billion in 2003, approximately 23,000 employees nationwide, and more than 220,000 clients, LabCorp offers over 4,400 clinical assays ranging from blood analyses to HIV and genomic testing. LabCorp combines its expertise in innovative clinical testing technology with its Centers of Excellence: The Center for Molecular Biology and Pathology, in Research Triangle Park, NC; National Genetics Institute, Inc. in Los Angeles, CA; ViroMed Laboratories, Inc. based in Minneapolis, MN; The Center for Esoteric Testing in Burlington, NC; and DIANON Systems, Inc. based in Stratford, CT. LabCorp clients include physicians, government agencies, managed care organizations, hospitals, clinical labs, and pharmaceutical companies. To learn more about our growing organization, visit our Web site at: www.LabCorp.com.

Each of the above forward-looking statements is subject to change based on various important factors, including without limitation, competitive actions in the marketplace and adverse actions of governmental and other third-party payors. Actual results could differ materially from those suggested by these forward-looking statements. Further information on potential factors that could affect LabCorp's financial results is included in the Company's Form 10-K for the year ended December 31, 2003, and subsequent SEC filings.

For questions or comments about this site, please contact the LabCorp Webmaster.

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Blood Tests Provide Alternative to Liver Biopsy for Assessing Status of Liver Disease in HCV Patients

Oneida TheraDiagnostics Ltd has launched two non-invasive blood tests for assessing the extent of liver disease in patients infected with hepatitis C virus (HCV). Developed by hepatologists at the Pitie-Salpetriere Hospital and BioPredictive in France, the two tests are called FibroTest and ActiTest.

The new tests provide easily accessible alternatives to liver biopsy, which is currently used to assess liver fibrosis and necroinflammatory activity in these patients. The tests do not replace the use of liver biopsy, however, which may be the appropriate diagnostic in many cases. Whether to use these new blood tests instead of a liver biopsy should be discussed between each individual patient and his/her primary care physician or liver specialist.

Although liver biopsy has long been considered the gold standard for monitoring the status of HCV and to determine therapy options, it is an invasive procedure that carries some risk of serious complications. The new assays use a combination of six serum biochemical markers, plus age and gender data, in a patented algorithm to determine the degree of liver fibrosis and the level of ongoing necroinflammatory activity.

The tests have been available in parts of Europe for the past two years and have recently been launched in the USA. Each test has been clinically validated in HCV patients to enable quantitative, reproducible assessment of fibrogenic and necrotic activity in the livers of HCV patients.

Oneida TheraDiagnostics Ltd. is a recently established company based in the UK offering molecular diagnostic services for clinical studies with particular emphasis on viral disease.

“Our intention is to establish a world-class portfolio of molecular diagnostics that will facilitate individual patient management and clinical trial studies performed in the UK and Ireland. The tests launched today are very well validated and address one of the major challenges in patients with chronic viral hepatitis. They will be the cornerstone of our viral hepatitis diagnostic service,” said Berwyn Clarke, CEO at Oneida.

FibroTest and ActiTest may be recommended by physicians for use in assessing liver status following a diagnosis of HCV. This provides a baseline determination of liver status before the initiation of HCV therapy and a post-treatment assessment of liver status six months after the completion of therapy.

The tests can also be applied for the non-invasive assessment of liver status in patients at risk of complications from a liver biopsy. The blood sample for the tests can be collected in minutes and results normally returned to the doctor within days.

10/11/04

Source
Oneida TheraDiagnostics. www.oneidathd.co.uk “Validated blood tests provide new alternative to liver biopsy for assessing status of liver disease in hepatitis C patients.” Press Release. October 8, 2004.

Index to Hepatitis C News Articles by Topic [ A -- Z ]

Internet Conference Report
39th EASL - April 14 - 18, 2004, Berlin Germany

Biochemical Markers (Fibrotest) Reliably Predict Extensive Fibrosis and Cirrhosis in Non Alcoholic Fatty Liver Disease (NAFLD)

The aim of the current study was to assess the predictive value of Fibrotest (FT) a biochemical marker of fibrosis for the diagnosis of fibrosis stage in patients with non-alcoholic fatty liver disease (NAFLD).

The study participants consisted of 89 patients with steatosis on ultrasound, abnormal transaminases and no other causes of liver disease. Fibrosis was staged: F0= none; F1=mild (perisinusoidal or portal); F2=moderate (portal+perisinusoidal and/or bridging); F3F4 =severe (extensive bridging fibrosis or cirrhosis). 300 prospectively included blood donors (BD) were used as controls.

Results

Severe fibrosis was present in 11%, moderate or severe in 45%. The mean FT (se) value was 0.09 (0.01) in BD; 0.19 (0.04) in F0 (n=18); 0.21 (0.03) in F1 (n=31); 0.33 (0.03) in F2 (n=30); 0.62 (0.05) in F3F4 (n=10) (p<0.05 between F3F4 and F0,F1,F2, and between F2 and F0, F1 and all stages vs BD).

For the diagnosis of severe fibrosis, AUROC= 0.94 (0.05); accuracy rate= 90%, kappa= 0.51 (0.11); Spearman correlation= 0.50 (p<0.001). An FT cutoff of 0.60 had a 95% PPV for severe fibrosis (Sp 97%, Se 60%). An FT cutoff of 0.30 had a 100% NPV for severe fibrosis (Sp=73%, Se 100%). Sensitivity analyses found an improvement of F2F3F4 AUROC for a biopsy size longer than 15mm as compared to a size smaller than 15 mm, 0.84 (0.07) vs. 0.63 (0.20) (p=0.04).

The authors conclude, “Fibrotest, reliably predicts extensive fibrosis and cirrhosis in NAFLD. This should improve the identification of the population at risk of significant liver injury and reduce the number of unnecessary liver biopsies.”

04/30/04

Reference
V Ratziu and others. DIAGNOSTIC VALUE OF BIOCHEMICAL MARKERS (FIBROTEST) FOR THE PREDICTION OF LIVER FIBROSIS IN PATIENTS WITH NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD). Abstract 597. 39^th EASL. April 14-18, 2004. Berlin, Germany.

www.hivandhepatitis.com

Internet Conference Report
39th EASL - April 14 - 18, 2004, Berlin Germany

A New, Sensitive, Real Time PCR-based Assay for Quantification of HCV RNA from Roche Diagnostics

Management of therapy of patients with hepatitis C is based on qualitative and quantitative measurement of HCV-RNA by different assays. A new real-time RT-PCR based assay (COBAS TaqMan, Roche Diagnostics, Pleasanton, USA) was designed for highly sensitive quantification of HCV-RNA thereby covering the range of qualitative and quantitative assays in one test.

COBAS TaqMan a single tube, single enzyme, real-time RT-PCR-based assay was evaluated on the basis of reference panels (AcroMetrix, clinical), and clinical samples (n=100) for testing of sensitivity, specificity, linearity, intra-, inter-assay variability (6-18 repeats) and comparability with COBAS Amplicor Monitor 2.0.

HCV-RNA extraction was performed with the high-pure-system (HPS, Roche Diagnostics).

The lower detection limit of the COBAS TaqMan was 100% and 72% at 10 and 5 IU/mL, respectively. The specificity was 99% with equal amplification of HCV genotypes 1-6.

The mean intra- and inter-assay variability within the range of 7 Mill. to 1000 IU/mL was between a SD of 0.076 and 0.096 log compared to a SD of 0.107 and 0.254 log by the COBAS Amplicor assay.

The linearity tests showed a regression coefficient of 0.99 between 7 Mill. and 30 IU/ml.

Comparison of HCV RNA quantification by COBAS TaqMan and COBAS Amplicor showed a high concordance with a correlation coefficient of 0.95.

In conclusion the authors state, “The new COBAS TaqMan assay is a highly sensitive, precise, and linear assay for HCV RNA quantification and has the potential to be used instead of previous qualitative and quantitative measurements before, during and after antiviral therapy.”

Saarland University Clinic, Homburg.

04/30/04

Reference
C Sarrazin and others. EVALUATION OF A NEW, SENSITIVE, REAL TIME PCR BASED ASSAY FOR QUANTIFICATION OF HCV RNA. Abstract 511. 39^th EASL. April 14-18, 2004. Berlin, Germany.

www.hivandhepatitis.com

Quest Diagnostics Announces Availability of HEPTIMAX Ultra-Sensitive Quantitative Hepatitis C Virus Test

TETERBORO, N.J., July 23 /PRNewswire/ -- Quest Diagnostics Incorporated (NYSE: DGX - news),

The nation's leading provider of gene-based medical testing, information and services, announced the availability of a new ultra-sensitive viral load test for hepatitis C virus (HCV) that is approximately 10 times more sensitive than any other commercially available test. The test detects the level of hepatitis C virus based on an innovative application of transcription mediated amplification (TMA) technology to HCV testing. Quest Diagnostics developed the test and is the first laboratory in the world to offer it.

The new offering, called the HEPTIMAX(TM) viral load test, is capable of detecting minute quantities of hepatitis C virus down to as few as 5 International Units (IUs) per milliliter (ml). The ability to detect minute quantities of virus is useful to physicians in monitoring the effectiveness of various treatments for hepatitis C in patients. The HEPTIMAX(TM) viral load test not only delivers the maximum sensitivity but also offers the maximum range (5 IUs/ml to 8.3 million IUs/ml).

This proprietary test is ordered by physicians to monitor and confirm hepatitis C viral infection and to demonstrate post-treatment resolution of the infection. Because the HEPTIMAX(TM) viral load test combines the new TMA technology with traditional quantitative viral load testing using branched DNA technology, it simplifies patient management by allowing physicians to order just one test to cover the complete range of possible viral load values from 5 IUs to 8.3 million IUs per ml.

``The improved sensitivity of the HEPTIMAX(TM) viral load test is particularly relevant to physicians with the advent of new forms of hepatitis C therapeutics,'' said Jorge Leon, Ph.D., Vice President for Applied Genomics at Quest Diagnostics. ``New combination therapies utilizing sustained-action forms of interferon have shown better treatment responses in clinical trials that translate to lower levels of virus in patients.''

``Preliminary studies comparing the HEPTIMAX(TM) viral load test to other technologies and laboratories confirm it has superior sensitivity,'' said Dr. Peter Heseltine, Medical Director of Infectious Diseases for Quest Diagnostics. ``We are planning additional studies with other outside investigators to confirm and publish our findings.''

A study published in the October 2000 issue of the journal ``Hepatology'' utilizing a hepatitis C qualitative version of the TMA test, showed it to be a better predictor of end-of-treatment resolution than the other methods.

The new test was developed by Quest Diagnostics at Nichols Institute and is offered exclusively through Quest Diagnostics' national laboratory network. The highly sensitive HEPTIMAX(TM) HCV assay utilizes reagents from Bayer Diagnostics, based on proprietary TMA technology made available through Bayer's exclusive agreement with Gen-Probe, San Diego, CA.

The HEPTIMAX(TM) viral load test is the latest addition to Quest Diagnostics' comprehensive test menu for hepatitis C disease management. Quest Diagnostics provides a complete selection of tests for initial diagnosis, treatment monitoring and managing HCV infected patients. The menu also features the DupliType(TM) HCV genotype test, which helps physicians establish the appropriate duration of HCV therapy. Quest Diagnostics introduced the HCV DupliType(TM) test to provide subtyping for a broader range of hepatitis C viral isolates than was previously available using other technologies.

An estimated 4 million people are infected with hepatitis C virus in the United States, and approximately 130,000 patients currently receive treatment. Physicians monitor viral loads in infected patients to test the effectiveness of various types of combination therapies.

Quest Diagnostics' gene-based testing focuses on infectious disease, oncology and heritable conditions, and helps physicians target individual treatment regimes, monitor resistance to therapies and predict predisposition to various genetic conditions. Quest Diagnostics is a leading innovator in genomics testing, through its research and development center and esoteric testing laboratory, the world-renowned Nichols Institute, as well as through alliances with leading academic and commercial technology developers.

About Quest Diagnostics

Quest Diagnostics is the nation's leading provider of diagnostic testing, information and services with annual revenues of $3.4 billion in 2000. The company's diagnostic testing yields information that enables health care professionals and consumers to make better decisions to improve health. Quest Diagnostics offers patients and physicians the broadest access to diagnostic testing services through its national network of approximately 30 full-service laboratories, 150 rapid response laboratories and more than 1,300 patient service centers, where specimens are collected. Quest Diagnostics is the leading provider of esoteric testing, including gene-based testing, and is the leader in routine medical testing, drugs of abuse testing, and non-hospital-based anatomic pathology testing. Through partnerships with pharmaceutical, biotechnology and information technology companies, Quest Diagnostics provides support to help speed the development of health care insights and new therapeutics. Additional company information can be found on the Internet at: http://www.questdiagnostics.com.

The statements in this press release which are not historical facts or information may be forward-looking statements. These forward-looking statements involve risks and uncertainties that could cause the outcome to be materially different. Certain of these risks and uncertainties are described in the Quest Diagnostics Incorporated 2000 Form 10-K and subsequent filings.

The HEPTIMAX viral load test and DupliType HCV genotype test are trademarks of Quest Diagnostics Incorporated.

SOURCE: Quest Diagnostics Incorporated

Understanding Log Changes in your Viral Load

( What is a 2 log Increase or Decrease ?)

An easy way to figure out log changes is to either drop the last "0"or add "0"to the

original number. Drop one "0" for one log drop and two "00" for a two log drop.

Changes in viral load are often reported as logarithmic or "log changes." This mathematical term denotes a change in the value of what is being measured by a factor of 10. For example, if the baseline viral load by PCR were 20,000 copies/ml plasma, then a 1 log increase equals a 10-fold (10 times) increase or 200,000 copies/ml plasma. A 2 log increase equals 2,000,000 copies/ml plasma, or a 100-fold increase.

Using the same starting point of 20,000 copies/ml plasma, a 1 log decrease means that the viral load has dropped to 2,000 copies/ml. A 2 log decrease equals a viral load of 200 copies/ml plasma. An easy way to figure out log changes is to either drop the last "0"or add "0"to the original number.

Any change of less than one-half log is considered insignificant. More simply, if the viral load measurement has not tripled or dropped to one-third of its previous level, the difference might be unimportant. For example, if the baseline viral load were 20,000 copies, a rise to 60,000 or a fall to 7,000 copies might just be the result of transient changes. Repeat testing of a single specimen may give two quite different results and natural biological day-to-day variability of samples from the same person may cause measurements to vary slightly. Researchers believe that clinical decisions made on the basis of changes in viral load ideally should be based on measurements taken 2-3 weeks apart.
http://vhaaidsinfo.cio.med.va.gov/aidsinfo/newsletters/phe/phe6.htm

The Importance of Laboratory Test Results in Hepatitis C Infection

David Bernstein, MD, FACP, FACG
Director of Hepatology
North Shore University Hospital
Associate Professor of Medicine
SUNY-Downstate School of Medicine

Most people with hepatitis C feel well and have no specific findings on physical examination that would lead a health care provider to suspect liver disease. Even the vast majority of people with liver disease that has advanced to cirrhosis have a normal physical examination. Therefore, the evaluation and treatment of liver disease, in particular hepatitis C, places a large emphasis on laboratory tests results to diagnose, stage and predict and evaluate response to therapy.

The liver has several general functions and it is often called both the body’s manufacturing center and its filtering plant. Blood tests used to evaluate the liver can be divided into those representing liver cell damage, cholestasis or liver function. The serum aminotransaminases, alanine aminotransferase (ALT or SGPT) and aspartate aminotransferase (AST or SGOT) are part of most automated blood chemistry panels. Elevation of these enzymes is caused by damage to the hepatocyte or liver cell. The degree of elevation may be important in acute disease but is unimportant in chronic disease. The most common causes of elevated aminotransaminases are fatty liver, viral hepatitis, medication induced hepatitis, autoimmune hepatitis and alcoholic liver disease. The tests are a reflection of cell damage and death but are not liver function tests. Although many patients and physicians refer to these tests as “liver function tests”, this term is incorrect and they do not reflect the liver’s ability to either synthesize or metabolize various chemicals. Therefore, an abnormality in these tests does not mean that the liver is not functioning. In fact, the vast majority of patients with elevated aminotransaminases, regardless of degree, have normal liver function.

Cholestatic liver disease is any condition leading to the obstruction of bile ducts in either the liver or biliary tree. Elevation of the enzymes alkaline phosphatase and gamma-glutamyl transpeptidase are indicative of this type of disease. Conditions that commonly lead to the elevation of these enzymes include primary biliary cirrhosis, primary sclerosing cholangitis and gallstone disease.

Bilirubin is the final breakdown product of heme, the majority of which comes from hemoglobin. Bilirubin can be elevated in many liver-related and non-liver- related conditions and it may be elevated in conditions which lead to liver cell damage and cholestasis. The level of serum bilirubin is not a sensitive indicator of liver function and it may not accurately reflect the degree of liver damage.

Albumin and blood clotting factors are proteins made in the liver. Blood tests such as the serum albumin and prothrombin time are measures of these proteins. As these tests evaluate the functional integrity of the liver, they can be correctly called “liver function tests”. Abnormalities of these tests are of concern and are indicative of extensive liver damage.

The most common laboratory abnormality seen in chronic hepatitis C infection is an isolated, elevated alanine aminotransferase (ALT) although as many as 60% of hepatitis C infected patients will have a normal ALT level. The level of serum ALT elevation does not correlate with histological disease and may be normal in any stage of chronic hepatitis C. Therefore, patients with minimal ALT elevations should be evaluated for the presence of chronic hepatitis. In advanced disease, an increase in alkaline phosphatase and total bilirubin as well as thrombocytopenia (low platelets) may be seen.

In the patient with risk factors for hepatitis C or an abnormal ALT, the most practical method of diagnosing HCV infection is by obtaining a second generation enzyme linked immunosorbent assay (EIA) antibody to hepatitis C (anti-HCV). False-positive results may occur at a rate of 10-20% and are usually seen in the presence of autoimmune disease, hypergammaglobulinemia and low-risk blood donors.False negative results may occur in immunosuppressed patients, including people infected with the human immunodeficiency virus. In early infection, anti-HCV testing may be negative, as antibodies may not develop until 4-6 weeks after exposure. Unfortunately, a positive hepatitis C antibody does not distinguish acute from chronic disease or active from past infection nor is it a sign of immunity or protection. Therefore, a positive EIA anti-HCV test is a marker that hepatitis C may be present and it must be followed by confirmatory viral load testing.

The recombinant immunoblot assay (RIBA) is another type of antibody test with limited utility. As with EIA antibody tests, it does not distinguish between acute or chronic disease or between past and active infection. Therefore, it adds little to the care of a patient with a positive hepatitis C antibody by the EIA method and known risk factors except extra expense. The NIH consensus conference on hepatitis C has recommended that it be used as a confirmatory test in patients without known risk factors who test positive for the EIA anti-HCV to eliminate the possibility of a false positive EIA. RIBA testing is not influenced by the presence of autoimmune disease or hypergammaglobulinemia. In clinical practice, this test has little if any utility and, except for rare exceptions, it should not be obtained.

Confirmatory tests for the presence of hepatitis C infection are those tests that determine the presence of hepatitis C viral particles (HCV-RNA) in the blood. A positive HCV-RNA in the serum confirms the diagnosis of active hepatitis C. This type of viral testing may be either qualitative or quantitative. Qualitative testing is more sensitive and specific than quantitative testing and results are reported as either positive or negative. Quantitative testing reports on the actual measured amount of viral particles in the serum and the viral levels are usually expressed as thousands or millions of international units. Of note, quantitative viral testing may be falsely negative if viral levels are below the lower limit of detection of the assay being used. Therefore, qualitative HCV-RNA testing is used for diagnosis while quantitative testing should be reserved for use during treatment. It is important to note that the level of virus does not correlate with prognosis, underlying liver histology or how ill a person feels. Therefore, a patient with a viral level of 3,000,000 international units does not have a worse prognosis nor is the person any sicker than someone with a viral count of 200,000 international units. Small fluctuations in HCV-RNA level are equally unimportant. A patient whose viral level has decreased from five to one million international units has shown no significant change in viral level and should not be rejoicing. The converse is also true and an elevation from one to five million international units should not lead a patient to be upset. It is important to understand the relative lack of importance of viral level in the untreated hepatitis C patient. Misconceptions about viral levels often lead to tremendous angst among patients who insist on comparing numbers in the waiting room, are upset by the initial level of viremia or feel falsely relieved or upset with small changes in HCV-RNA viral load. Recently, however, it has been suggested that in the population co-infected with hepatitis C and HIV, a higher hepatitis C viral load is associated with a more rapid progression to advanced disease. As regards viral level and its significance, the co-infected patient appears to behave differently that the HCV mono-infected person. Quantitative viral load testing should not be repeated yearly or more often as it adds little to the care of the untreated patient other than increased expense and anxiety. These tests, however, should be followed serially in someone undergoing anti-viral therapy, as the goal of therapy is the loss of detectable serum HCV-RNA.

Several other liver tests are frequently obtained in patients with hepatitis C. The serum alpha-fetoprotein is a marker of liver cancer but it may be mildly elevated in patients with chronic hepatitis C in the absence of liver cancer. If it is elevated, this test should be followed closely. Autoimmune markers may be present in as many as 25% patients with hepatitis C without the presence of autoimmune disease. These markers include an anti-nuclear antibody, smooth muscle antibody, anti-mitochondrial antibody or anti-thyroid antibodies. The presence of these antibodies does not appear to influence disease progression. Patients in whom autoimmune disease is suspected should be adequately evaluated before the presence of autoantibodies is attributed to HCV infection.

The adequate interpretation of laboratory test results is very important to understand the evaluation of hepatitis C infection. Unfortunately, in the majority of cases, these blood tests are unable to accurately predict current disease stage or possible disease progression. Therefore, despite all these advanced tests, the performance of a liver biopsy cannot be emphasized enough as this is the best test to accurately stage the disease and predict disease progression.

Third Wave Launches Invader(R) HCV Genotyping Reagents At Clinical Virology Symposium
Monday April 26, 7:31 am ET

MADISON, Wis., April 26 /PRNewswire-FirstCall/ -- Third Wave Technologies Inc. (Nasdaq: TWTI - News) today announced the launch of its Invader® Hepatitis C Virus (HCV) genotyping reagents at the 20th Annual Clinical Virology Symposium in Clearwater Beach, Fla. In addition, scientists from the Cleveland Clinic Foundation and Mayo Clinic are presenting data from separate collaborative studies regarding Third Wave's Invader® HCV genotyping reagents at this meeting. These study results detail the Invader® platform's ability to provide faster and more accurate results when compared to other commercially available HCV genotyping methods and technologies. The study abstracts and the posters that are being presented at the symposium are available in PDF format at http://www.twt.com/pressreleases/2004/April_26_2004.html.

The Invader® HCV genotyping product, based on the company's proprietary Invader® technology platform, enables clinical laboratories to develop assays that identify all six genotypes of the HCV virus, a major predictor of disease progression and response to HCV treatment. Viral pathogens such as HCV have a number of specific genetic variations, known as genotypes. While all of these strains are grouped into the larger HCV category, the identification of the specific genotype of a virus is essential to maximizing treatment and disease management. Once the correct genetic make-up of the virus is identified, an appropriate therapeutic course of action can be specifically tailored.

The HCV genotyping market in the United States and Europe is currently estimated to be more than $50 million and is growing rapidly. Third Wave's reagents incorporate all of the performance benefits associated with the Invader® technology platform. Use of the Invader® platform in manual assay setups provides results faster than other commercially available technologies, but more importantly, the high-throughput and ease-of-use advantages of the company's proprietary Invader® platform enable laboratories to automate the HCV genotyping process easily, reducing hands-on time, while decreasing the time to result.

"The Invader® HCV genotyping reagents are just the latest innovation to emerge from our robust pipeline," said John Puisis, president and chief operating officer of Third Wave. "This product provides us with a superior molecular solution for entry into an additional market segment. As we continue to accelerate high-value products from pipeline to market, we are further establishing our position in the molecular diagnostic industry."

About HCV

Hepatitis C virus (HVC) is a positive, single-stranded RNA virus that is a member of the Flaviviridae family. Almost 4 million Americans, or 1.8 percent of the U.S. population and 170 million individuals worldwide, are anti-HCV antibody-positive, indicating ongoing or previous infection with the virus. At least 75 percent of patients with acute hepatitis C ultimately develop chronic infection, and most of those patients have accompanying chronic liver disease. HCV is the leading cause of chronic liver disease in the United States and accounts for approximately 15 percent of acute viral hepatitis cases and up to 50 percent of cases of cirrhosis, end-stage liver disease and liver cancer. HCV infection causes an estimated 10,000 to 12,000 deaths annually in the United States.

About Third Wave Technologies

Third Wave Technologies is a leader in the development and marketing of molecular diagnostics for a variety of DNA and RNA analysis applications, providing physicians and researchers with superior tools to diagnose and treat disease. Third Wave's Invader® technology provides the company's customers with exceptional accuracy, scalability and ease of use. The company offers a number of clinical products based on its Invader® technology for genetic testing related to multiple disease areas. For more information about Third Wave and its products, please visit the company's website at www.twt.com.

All statements in this news release that are not historical are forward-looking statements within the meaning of the Securities Exchange Act of 1934 as amended. Such forward-looking statements are subject to factors that could cause actual results to differ materially for Third Wave from those projected. Those factors include risks and uncertainties relating to technological approaches of Third Wave and its competitors, product development, manufacturing, market acceptance, cost and pricing of Third Wave products, dependence on collaborative partners and commercial customers, successful performance under collaborative and commercial agreements, competition, the strength of the Third Wave intellectual property, the intellectual property of others and other risk factors identified in the documents Third Wave has filed, or will file, with the Securities and Exchange Commission. Copies of the Third Wave filings with the SEC may be obtained from the SEC Internet site at www.sec.gov. Third Wave expressly disclaims any obligation or undertaking to release publicly any updates or revisions to any forward-looking statements contained herein to reflect any change in Third Wave's expectations with regard thereto or any change in events, conditions, or circumstances on which any such statements are based. Third Wave Technologies, Invader and the Third Wave logo are trademarks of Third Wave Technologies, Inc.

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