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Assay Sheet

Test ID

6100 Hepatitis B Virus Antiviral Resistance Line Probe Assay

CPT Code

83891, 83893, 83896 (x32), 83898, 83912

Clinical Utility

Extended treatment of chronic hepatitis B with lamivudine, adefovir, telbivudine, and emtricitabine can result in the development of antiviral resistance. This process begins with a mutation in the viral genome that becomes selectively amplified by the pressure exerted by antiviral therapy. Assessment of resistance-associated mutations may be utilized to differentiate between non-adherence and drug-resistance. Moreover, early detection permits prompt initiation of therapy adjustment, and improved clinical outcomes.

Procedure

Extraction of nucleic acid from plasma or serum followed by amplification of portions of the HBV polymerase gene. Biotinylated PCR products are hybridized to immobilized oligonucleotide probes specific to the polymorphisms in the HBV polymerase gene which have been shown to confer resistance to lamivudine, adefovir, telbivudine, and emtricitabine.

Note: It is recommended that 6100 Hepatitis B Antiviral Resistance be ordered with 1100 Hepatitis B (HBV) Real-time qPCR. HBV Antiviral Resistance is performed following confirmation of adequate viral load to obtain a genotyping result. If the HBV viral load is less than 1,000 IU/ml, antiviral resistance testing may not be successful.

Specimens

Plasma: 5 to 7 ml collected in EDTA, ACD A or PPT. Minimum specimen requirement is 2ml plasma. Separate plasma from cells by centrifugation. Transfer plasma to screw-cap tube for shipment. If specimen collected in PPT tube, the entire tube can be shipped following centrifugation. Specimen may be shipped ambient or frozen.

Serum: 5 to 7 ml collected in red-top or SST. Minimum specimen requirement is 2ml serum. Separate serum from cells by centrifugation. Transfer serum to screw-cap tube for shipment. If specimen was collected in SST, the entire tube can be shipped following centrifugation. Specimen may be shipped ambient or frozen.

Causes for Rejection

Specimen types other than plasma or serum, whole blood frozen, or specimens containing HBV DNA levels too low to allow for antiviral drug resistance testing. 

Call ViraCor at 800-305-5198 if specimen is greater than 96 hrs old.

Reference values

Mutations in the HBV polymerase gene will be reported as detected/not detected.  Interpretation of gene mutations and association with antiviral resistance, including lamivudine, adefovir, telbivudine, and emtricitabine  will be provided with the report as indicated by the table below.

HBV Reportable Drug Resistance Mutations

Turnaround Time

Assay performed twice weekly on Mondays and Wednesdays with results reported Tuesdays and Thursdays.

Shipping

Ship Monday through Friday. Friday shipments must be labeled for Saturday delivery. All specimens must be labeled with patient's name and collection date. Multiple tests can be run on one specimen. Ship specimens FedEx Priority Overnight® to:

ViraCor Laboratories, 1001 NW Technology Dr, Lee's Summit, MO 64086

References

1. Niesters HG, Honkoop P, Haagsma EB, de Man RA, Schalm SW, Osterhaus AD. Identification of more than one mutation in the hepatitis B virus polymerase gene arising during prolonged lamivudine treatment. J Infect Dis. 1998;177:1382-5.

2. Delaney WE 4th, Yang H, Westland CE, Das K, Arnold E, Gibbs CS, Miller MD, Xiong S. The hepatitis B virus polymerase mutation rtV173L is selected during lamivudine therapy and enhances viral replication in vitro. J Virol. 2003;77:11833-41.

3. Cooley L, Ayres A, Bartholomeusz A, Lewin S, Crowe S, Mijch A, Locarnini S, Sasadeusz J. Prevalence and characterization of lamivudine-resistant hepatitis B virus mutations in HIV-HBV co-infected individuals. AIDS. 2003 25;17:1649-57.

4. Angus P, Vaughan R, Xiong S, Yang H, Delaney W, Gibbs C, Brosgart C, Colledge D, Edwards R, Ayres A, Bartholomeusz A, Locarnini S. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology. 2003;125:292-7.

5. Stuyver LJ, Locarnini SA, Lok A, Richman DD, Carman WF, Dienstag JL, Schinazi RF. Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region. Hepatology. 2001;33:751-7. Review.

6. Fung SK, Andreone P, Han SH, Rajender Reddy K, Regev A, Keeffe EB,  Hussain M, Cursaro C, Richtmyer P, Marrero JA, Lok AS. Adefovir-resistant hepatitis B can be associated with viral rebound and hepatic decompensation. J Hepatol. 2005;43:937-43. 

7. Sheldon J, Camino N, Rodes B, Bartholomeusz A, Kuiper M, Tacke F, Nunez M, Mauss S, Lutz T, Klausen G, Locarnini S, Soriano V. Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir. Antivir Ther. 2005;10:727-34.

8. Tenney DJ, Levine SM, Rose RE, Walsh AW, Weinheimer SP, Discotto L, Plym M, Pokornowski K, Yu CF, Angus P, Ayres A, Bartholomeusz A, Sievert W, Thompson G, Warner N, Locarnini S, Colonno RJ. Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to Lamivudine. Antimicrob Agents Chemother. 2004;48:3498-507.

9. Lai CL, Leung N, Teo EK, Tong M, Wong F, Hann HW, Han S, Poynard T, Myers M, Chao G, Lloyd D, Brown NA; Telbivudine Phase II Investigator Group. A 1-year trial of telbivudine, lamivudine, and the combination in patients with hepatitis B e antigen-positive chronic hepatitis B. Gastroenterology. 2005;129:528-36.

10. Yang H, Qi X, Sabogal A, Miller M, Xiong S, Delaney WE 4th. Cross-resistance testing of next-generation nucleoside and nucleotide analogues against lamivudine-resistant HBV. Antivir Ther. 2005;10(5):625-33.

11. Ogata N, Fujii K, Takigawa S, Nomoto M, Ichida T, Asakura H. Novel patterns of amino acid mutations in the hepatitis B virus polymerase in association with resistance to lamivudine therapy in japanese patients with chronic hepatitis B. J Med Virol.1999;59:270-6.

12. Tenney DJ, Levine SM, Rose RE, Walsh AW, Weinheimer SP, Discotto L, et al.  Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to Lamivudine. Antimicrob. Agents Chemother. 2004;48:3498-507.

13. Sheldon J, Camino N, Rodes B, Bartholomeusz A, Kuiper M, Tacke F, et al.  Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir. Antivir Ther. 2005;10:727-34.

14. Schildgen O, Sirma H, Funk A, Olutu C, Wend UC, Hartmann H, et al.  Variant of hepatitis B virus with primary resistance to adefovir. N Engl J Med. 2006:27;354:1807-12.

Pathogen Overview

ABOUT HEPATITIS B VIRUS

Hepatitis B virus (HBV) is a small, hepatotropic DNA virus. It is the prototype member of a family of closely related viruses called Hepadnaviradae. HBV is the only member of the Hepadnaviradae family that infects humans. The HBV genome is composed of a relaxed, circular, partially double-stranded DNA molecule. There are 6 HBV genotypes: A through F.

HEPATITIS B VIRUS CLINICAL MANIFESTATIONS

HBV infection may be symptomatic or asymptomatic, though more likely to be symptomatic in young children. More than 95% of HBV infections in adults are self-limiting; the virus is cleared about 6 months post-infection and the patient is immune to reinfection. Symptoms of acute infection can include jaundice, fatigue, abdominal pain, loss of appetite, nausea, vomiting, and joint pain.

Hepatitis B is transmitted by direct contact with the blood or body fluids of an infected person; e.g. having sex or sharing needles with an infected person. Infants can be infected as they pass through the birth canal of an infected mother, where they are exposed to large amounts of the virus. Hepatitis B is not spread through food, water, or by casual contact. However, the virus can be spread by sharing personal items that might have small amounts of blood present, such as toothbrushes or razors. High risk populations include people who practice unsafe sex, drug abusers, and hospital workers who have exposure to blood products.

HBV Vaccine: In 1982, a hepatitis B vaccine was made available. The number of new infections per year has declined from an average of about 260,000 in the 1980s to about 78,000 in 2001. This great decline has occurred in children and adolescents as a result of the vaccine. The HBV vaccine is now routine in the immunization schedule of infants and children.

Chronic HBV Infections: About 5% of adults will not resolve the infection and go on to become a chronic carrier. Conversely, chronic infections occur in 90% of infants infected at birth, 30% if infected at age 1 to 5 years and 6% if infected after age 5. Chronic infection is more common in infants due to their poorly developed cellular immune response. There are an estimated 1.25 million chronically infected Americans of whom 20 to 30% acquired their infection in childhood.

The clinical course of individuals who are chronically infected is highly variable, ranging from asymptomatic to a small subset with severe chronic active hepatitis, who have a 5 year survival rate of less than 50%. The reasons that some develop a chronic infection remain poorly understood. Evidence suggests that variation in host immune response is a critical factor. Individuals with overt deficits in cell-mediated immunity, such as transplant patients and HIV patients, are more likely to become chronic carriers than those with a fully competent immune response.

About 30% of chronic carriers will have no signs or symptoms. The asymptomatic carriers are the primary reservoir of infection; it is from these individuals that the spread of HBV to susceptible hosts occurs. Adults are more likely than children to be symptomatic. Symptoms can include jaundice, fatigue, abdominal pain, loss of appetite, nausea, vomiting, and joint pain. Some patients will remain in generally good health but have persistent or recurrent elevations in liver enzymes (AST/ALT) without jaundice. Persistent mild hepatomegaly is not unusual, and splenomegaly is occasionally present. Unfortunately, a significant number of chronically-infected individuals will develop cirrhosis, hepatic failure, or hepatocellular carcinoma.

Just a decade ago, HBV infection was considered a contraindication to liver transplant because of frequent viral recurrences and the development of associated liver disease, which led to graft failure and death. Antiviral therapies have diminished the risk of HBV recurrence and led to improvements in patient and graft survival. HBV infection after a liver transplant can be classified into 2 groups; patients who have HBV recurrence who were transplanted because of HBV end-stage liver disease and patients who develop HBV after the transplant from their donors or from a community acquired infection.

HEPATITIS B VIRUS LABORATORY DIAGNOSIS

HBV infection can be diagnosed and monitored with a number of antibody and antigen assays. There are a wide variety of laboratory assays available to differentiate acute from chronic infection, to assess the immune status of an individual, or to evaluate infectivity and prognosis. There are a number of excellent reviews in the primary literature to assist in understanding the immune response to HBV.

Due to the highly sensitive and specific nature of quantitative, real-time polymerase chain reaction (PCR), it has become the standard of care for assessment of viral burden (viral load) in a patient undergoing treatment for HBV. HBV often achieves titers of greater than 10e9 IU/ml; for this reason, it is important that the quantitative, real-time PCR assay employed to follow the patient has an extremely wide assay range. The ViraCor HBV assay has a range of 5 to 200,000,000 IU/ml.

HEPATITIS B VIRUS TREATMENT

Treatment of hepatitis B has evolved from isolation and bed rest in the 1950s to development of antiviral therapies in the 1990s. The ideal goal of treatment is to eradicate HBV, but this may not be achievable. The indications for treatment include evidence of viral replication and abnormal ALT levels. Patient age, severity of liver disease, likelihood of response, and the potential for adverse events must be weighed before treatment is initiated. There are a variety of treatment options available for HBV chronic infections, with the understanding of treatment rapidly evolving at this time. For an excellent review of treatment options, the Hepatitis B Foundation website is recommended: http://www.hepb.org/treatment/aasld_guidelines.htm .

Selected References

Centers for Disease Control and Prevention. http://www.cdc.gov

Hepatitis B Foundation. http://www.hepb.org

Knipe D, Howley P. Fields Virology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2006.

PAO-05-0707 PCR tests are performed pursuant to a license agreement with Roche Molecular Systems, Inc.