Hepatitis B Virus Genotype
Test Summary

Clinical Background

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The goals of chronic hepatitis B (CHB) treatment are:
•	sustained suppression of HBV replication and remission of liver disease, defined as normalized ALT levels,
•	undetectable HBV DNA in a non-amplification assay,
•	loss of HBV e antigen (HBeAg), and
•	improved liver histology.1

Until recently, interferon-alfa and lamivudine have been the only drugs FDA-approved for treatment of CHB, yielding response rates of about 20% to 35% overall and >50% in patients with ALT levels >5 times the upper limit of normal.^2,3 Adefovir dipivoxil (ADV) is now also approved as a first-line treatment for CHB.

Lamivudine provides sustained response in patients with loss of HBeAg, but those who do not become HBeAg-negative tend to relapse after therapy is withdrawn. Continued lamivudine treatment in patients without HBeAg loss after 1 year may still yield clinical benefit, however, including loss of HBeAg, loss of HBV DNA, and biochemical and histologic improvement.1 In some cases, withdrawing lamivudine without initiating another antiviral agent such as ADV can lead to acute exacerbation of hepatitis.⁴ However, long-term lamivudine therapy selects for resistance-associated mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HBV DNA polymerase; these mutations arise in about 16% to 32% of patients at 1 year of treatment⁵ and in about 57% by 3 years.⁶

ADV shows clinical efficacy against lamivudine-resistant and wild-type strains of HBV. Although continued treatment for up to 60 weeks appeared not to select for resistant variants,⁷ ADV resistance associated with the rtN236T or rtA181V mutations arises in about 2% of patients by year 2 and 3.9% by year 3.^8,9 ADV-resistant strains appear to remain susceptible to lamivudine.⁸

Resistance should be assessed every 3 to 6 months in patients receiving lamivudine.¹⁰ Because of adefovir's lower rate of resistance, adefovir-treated patients should be monitored for resistance every 6 months after the first year of therapy. Resistance can be clinically detected by a 1-log10 increase in serum HBV DNA level from the patient's lowest on-treatment level, occurring on 2 sequential occasions, and can be confirmed by genotyping.

In addition to detecting mutations associated with resistance to antiviral drugs (lamivudine, ADV, and famciclovir) resistance, this assay also assesses HBV genotype (A-G) and mutations in the precore (pre-C) and basal core promoter (BCP) regions. HBV genotypes A-G are classified according to sequence variations in the HBV "S" gene and have distinct geographic distributions. Mutations at nucleotide 1896 of the pre-C region abolishes production of the HBeAg, while mutations at nucleotide 1762 and 1764 of the BCP region may play a role in HBeAg clearance. The pre-C and BCP mutations may also influence response to interferon treatment in HBeAg-positive patients.¹¹

Individuals Suitable for Testing

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•	Patients receiving lamivudine or ADV for treatment of CHB
•	Patients being considered for antiviral therapy

Specimen Requirements

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•	1 mL frozen potassium EDTA plasma (PPT white-top tube); 0.3 mL minimum. Plasma should be separated and frozen within 2 hours of collection.
•	Alternatively, submit 1 mL (0.3 mL minimum) frozen serum in a sterile, plastic, screw-capped, aliquot tube.

Method

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•	Amplification of relevant portions of the HBV genome by polymerase chain reaction with HBV-specific primers
•	Analysis of sequence data for mutations at codons 180, 181, 204, 207, and 236 of the polymerase gene, nucleotide 1896 of the pre-C region, and nucleotides 1762 and 1764 of the BCP region
•	HBV genotype determination via computer-aided alignment and phylogenetic analysis of theamplified portion of the S gene
•	Analytical sensitivity: 1,000 HBV copies/mL plasma; 500 HBV copies/mL serum
•	Analytical specificity: no known cross-reaction with other blood-borne pathogens
•	CPT Codes*: 83891, 83901 x2, 83894, 83904 x4, 83912

Interpretive Information

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The rtL180M, rtM204V/I and rtA181T mutations decrease sensitivity to lamivudine, whereas the rt236 and rtA181V mutations reduce sensitivity to ADV. The rtL180M and rtV207I mutations appear to decrease sensitivity to famciclovir. The effect of these mutations on the clinical course in patients treated with lamivudine is variable. Some patients develop severe exacerbation of hepatitis or, rarely, decompensated hepatitis, suggesting the need for careful monitoring when these mutations are identified.7 However, many patients with resistant virus experience clinical benefit from continued therapy, including reduced ALT and HBV DNA levels, decreased hepatic damage, and HBeAg seroconversion.6 Individuals with lamivudine-resistant variants appear to respond well to ADV, and patients with an ADV resistance-associated mutation have responded to lamivudine. The influence of HBV genotype on disease course and treatment response is not fully understood. Genotype C may be associated with more severe liver disease than genotype B,12,13 although contradictory findings have been reported.10 In addition, genotypes A and B may respond better than genotypes C and D to interferon treatment.14 Although their effects are not yet well established, mutations in the BCP and pre-C regions may influence disease course and response to interferon. BCP mutations at nucleotides 1762 and 1764 are associated with low levels of HBeAg and may predict a more favorable response to interferon treatment. The G1896A pre-C variant, which abrogates HBeAg formation, has been associated with lower rates of interferon response. Patients with mutations in either region can return to high levels of HBV viremia after loss of HBeAg.10

Nomenclature for HBV Resistance-Associated Mutations15 Table

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	Former Numbering System Genotype								Standardized System
	A		B, C, F		D		E, G		All genotypes
	L528M		L526M		L515M		L525M		rtL180M
	A529T/V		A527T/V		A515T/V		A526T/V		rtA181T/V
	M552V/I		M550V/I		M539V/I		M549V/I		rtM204V/I
	V555I		V553I		V542I		V552I		rtV207I
	N584T		N582T		N571T		N581T		rtN236T

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Online Test Information for Your Patients

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•	Hepatitis B
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* The CPT codes provided are based on AMA guidelines and are for informational purposes only. CPT coding is the sole responsibility of the billing party. Please direct any questions regarding coding to the payor being billed.

References

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1 Lok AS, McMahon BJ; Practice Guidelines Committee, American Association for the Study of Liver Diseases. Chronic hepatitis B. Hepatology. 2001;34:1225-1241. 2 Lok AS, Heathcote EJ, Hoofnagle JH. Management of hepatitis B: 2000-summary of a workshop. Gastroenterology. 2001;120:1828-1853. 3 Malik A, Lee W. Chronic hepatitis B virus infection: treatment strategies for the next millennium. Ann Intern Med. 2000;132:723-731. 4 Honkoop P, de Man RA, Niesters HG, et al. Acute exacerbation of chronic hepatitis B virus infection after withdrawal of lamivudine therapy. Hepatology. 2000;32:635-639. 5 GlaxoSmithKline. Epivir-HBV Product Information. August 2001. Available at http://us.gsk.com/products/assets/us_epivir_hbv.pdf. Accessed May 8, 2002. 6 Leung NW, Lai CL, Chang TT, et al; on behalf of the Asia Hepatitis Lamivudine Study Group. Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: results after 3 years of therapy. Hepatology. 2001;33:1527-1532. 7 Yang H, Westland CE, Delaney WE 4th, et al. Resistance surveillance in chronic hepatitis B patients treated with adefovir dipivoxil for up to 60 weeks. Hepatology. 2002;36:464-473. 8 Angus P, Vaughan R, Xiong S, et al. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology. 2003;125:292-297. 9 Xiong S, Qi X, Snow A, et al. Long-term incidence of adefovir dipivoxil resistance in chronic hepatitis B patients after 144 weeks of therapy. Abstract 57 (oral). Presented at the 39th Annual Meeting of the European Association for the Study of Liver Disease (EASL). April 14-18, 2004. Berlin, Germany. 10 Keeffe EB, Dieterich DT, Han SH, et al. A treatment algorithm for the management of chronic hepatitis B virus infection in the United States. Clin Gastroenterol Hepatol. 2004;2:87-106. 11 Conjeevaram HS, Lok AS. Management of chronic hepatitis. Br J Hepatol. 2003;38(Suppl):S90-S103. 12 Kao JH, Chen PJ, Lai MY, et al. Genotypes and clinical phenotypes of hepatitis B virus in patients with chronic hepatitis B virus infection. J Clin Microbiol. 2002;40:1207-1209. 13 Lindh M, Hannoun C, Dhillon AP, et al. Core promoter mutations and genotypes in relation to viral replication and liver damage in East Asian hepatitis B virus carriers. J Infect Dis. 1999;179:775-782. 14 Kao JH, Wu NH, Chen PJ, et al. Hepatitis B genotypes and the response to interferon therapy. J Hepatol. 2000;33:998-1002. 15 Stuyver LJ, Locarnini SA, Lok A, et al. Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region. Hepatology. 2001;33:751-757.

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