CASE REPORT
Nonresponse to 18-month Lamivudine Monotherapy in Chronic Hepatitis B Patients with Dual Genotype B and C Infection and Acute Exacerbation Ming-Jen Sheu, Ching-Yih Lin, Chi-Shu Sun, Hsing-Tao Kuo, Lok-Beng Koay, Chuan Lee, Jyh-Jou Chen,1 Ling-Yu Tang,2 Sun-Lung Tsai2* Molecular epidemiologic studies have indicated the possible existence of mixed infection of different hepatitis B virus (HBV) genotypes in chronic hepatitis B (CH-B) carriers, but the effect of dual HBV genotype B and C infection on the efficacy of lamivudine therapy remains unclear. We report four CH-B patients with dual HBV genotype B and C infection and acute exacerbation who received lamivudine monotherapy forr about 18 months. None of them had achieved a sustained response at the end of the 18-month trial off treatment. [J Formos Med Assoc 2006;105(7):588–593] Key Words: acute exacerbation, chronic hepatitis B, dual genotype infection, hepatitis B virus, lamivudine therapy, sustained response
1, 2, 3 and 4 years of treatment, respectively.4,9,11,12 Importantly, resistance to lamivudine therapy also increased in frequency with continuation of therapy, with detection rates of 17%, 40%, 55% and 67% after 1, 2, 3 and 4 years of treatment, respectively.11,12 One mechanism responsible for the low w sustained response (SR) rate to lamivudine therapy may be the development of mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the reverse transcriptase domain of HBV V DNA polymerase. However, some patients withoutt YMDD mutation still responded poorly to the therapy. Pretreatment alanine aminotransferase (ALT) levels, infection with HBV genotype B, age less than 36 years and additional lamivudine
Lamivudine has become a main therapeutic option for treating hepatitis B virus (HBV) infection.1–5 Its antiviral effects against HBV have been established both in vitro and in vivo.6–8 Clinical trials revealed that lamivudine is effective in reducing HBV replication and preventing the progression of chronic liver disease.4,5,9,10 Although complete suppression of viral replication, as determined by negative serum HBV DNA, may be achieved in most patients, clearance of hepatitis B virus e antigen (HBeAg) with antibody (anti-HBe) seroconversion occurred only in a minority of patients.1,3,5 In Asian patients, HBeAg seroconversion with loss of HBV DNA as detected by hybridization techniques occurred in 16%, 27%, 40% and 47% after
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Division of Hepatogastroenterology, Department of Internal Medicine, Chi Mei Medical Center, 1Department of Internal Medicine, Chi Mei Hospital Liouying, and 2Liver Research Unit, Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan. Received: June 17, 2005 Revised: July 28, 2005 Accepted: September 13, 2005
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*Correspondence to: Dr Sun-Lung Tsai, Liver Research Unit, Department of Medical Research, Chi Mei Medical Center, 901, Chung Hwa Road, Yung Kang City, Tainan 710, Taiwan. E-mail:
[email protected]
J Formos Med Assoc | 2006 • Vol 105 • No 7
Lamivudine therapy in dual HBV genotype infection f
treatment over 8 months have been shown to be important determinants of SR to lamivudine therapy.12,13 At least eight genotypes of HBV have been distinguished based on a divergence over the entire genomic sequence of > 8% and designated A to H.14–16 These genotypes have distinct geographical distribution. Molecular epidemiologic studies have indicated the existence of mixed infection of different HBV genotypes in chronic hepatitis B (CH-B) carriers.17,18 A high prevalence of mixed genotype infections has been reported in HBVinfected intravenous drug users.19 It has also been shown that in HBV genotypes B and C co-infected patients, recombination between different HBV genotypes are not unusual.20 The impact of recombination on HBV evolution and the clinical significance of mixed genotype infection remain to be elucidated. Here, we report four CH-B patients with dual HBV genotype B and C infection who received lamivudine monotherapy for 18 months. None of them achieved a sustained response during the 18-month trial of treatment.
Methods
normal (200 U/L).21,22 Before the treatment, all patients met the following criteria: presence off hepatitis B surface antigen (HBsAg) and HBeAg in serum for at least 6 months; with AE of CH-B, i.e., ALT > 200 U/L; seropositivity for HBV DNA on att least two occasions 1 month apart before entry; and undetectable YMDD mutants. None of the patients had concurrent hepatitis C virus (HCV), hepatitis delta virus (HDV) or human immunodeficiency virus (HIV) infection. Oral lamivudine (Zeffix; GlaxoWellcome, Greenford, UK) 100 mg/day was administered for 18 months as recommended by the National Health Insurance Program of Taiwan (NHIT) and the cost of medication was fully covered byy the NHIT. SR to the treatment was defined as a sustained loss of HBeAg with seroconversion to anti-HBe, together with the disappearance off serum HBV DNA (virological response), and with the normalization of serum ALT levels (biochemical response). A transient normalization off serum ALT levels followed by a relapse eitherr after the end of treatment or during lamivudine therapy was defined as partial response (PR). Nonresponse (NR) was defined as the lack off either a biochemical or a virological response to the therapy.
Case identification A total of 175 consecutive CH-B patients positive for HBeAg with acute exacerbation (AE) participated in a nationwide therapeutic trial of 18month lamivudine therapy in Chi Mei Medical Center (CMMC, Tainan, Taiwan). The initial 40 cases were recruited from the outpatient clinics of eight physicians at CMMC. All these patients completed the 18-month treatment course and post therapy follow-up for more than 6 months. Four of the patients with nonresponse to therapy were found to be co-infected with HBV genotypes B and C by INNO-LiPA HBV genotyping (Innogenetics NV, Belgium). This report describes the clinical courses of these patients during the 18-month trial of lamivudine. AE was defined as an abrupt increase of serum ALT levels (upper limit of normal, 40 U/L) to a level greater than five times the upper limit of J Formos Med Assoc | 2006 • Vol 105 • No 7
Follow-up Patients with AE were treated with oral lamivudine and followed up weekly or biweekly. Hospital care was recommended for patients with severe AE as it may be associated with hepatic decompensation or failure.16 After episodes of AE, patients were followed up monthly with continuing lamivudine therapy. Routine follow-up studies were performed including clinical assessment, conventional liver biochemical tests, and virological assays. All biochemical tests were carried out in the clinical pathology laboratories of CMMC with routine automated techniques.
Virological assays HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HDV, antiHIV-1, and anti-HIV-2 were assayed using commercially available ELISA kits (Abbott Laboratories, 589
M.J. Sheu, et al
North Chicago, IL, USA; Ortho Diagnostic Systems, Raritan, NJ, USA; and Sanofi Diagnostic Pasteur, Marnes-la Coquette, France). Serum IgM antiHBc was assayed using a microparticle enzyme immunoassay kit (AxSYM CORE-M MTM, Abbott Laboratories). Serum antibodies to hepatitis C virus were assayed using a third-generation enzyme immunoassay kit (AxSYM HCV, Version 3.0, Abbott Laboratories). Serum HBV DNA was quantified using a hybrid capture assay (Digene Hybrid Capture II HBVDNA Test, Digene Corp., Gaithersburg, MD, USA). The detection limit of HBV DNA by this assay is 0.5 pg/mL or 1.4 × 105 copies/mL.
HBV genotyping and YMDD mutation assay HBV genotyping was performed by INNOLiPA HBV genotyping, according to the manufacturer’s instructions. Although line probe assay was shown to be more sensitive than polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in identifying HBV genotype,19 the line probe assay INNO-LiPA kit can only detect the seven HBV genotypes A–G. We therefore conducted PCR-RFLP assay in parallel as mutual confirmation of the HBV genotypes A–G and to detect other possible mixed genotype infections. In brief, primers (sense, 5’-GTGGTGGACTTCTCTCAATTTTC(256–278); anti-sense, 5’-CGGTA(A/T) AAAGGGACTCA(A/C)GAT(796–776)) were used to amplify S gene from patients’ serum and the geneclean DNA of PCR products was digested with restriction enzymes TSP 509 I and Hinf I.23,24 YMDD mutants were also detected using the INNO-LiPA HBV DR kit, and confirmed by DNA sequencing of PCR products from each serum sample using the CEQ2000 DNA Analysis System (Beckman Coulter, Inc., Fullerton, CA, USA). To avoid false positive results, instructions to prevent cross contamination were strictly followed. In addition, an aliquot of HBV-positive serum containing 108 copies/mL of HBV DNA was serially diluted and used as the standard for HBV DNA quantitation and for determination of the sensitivity of HBV genotyping. The sensitivity of these two assays approximated 100 copies of HBV DNA. 590
Results Figure 1 shows the clinical courses of the fourr patients infected with dual HBV genotype B and C viruses. Although transient biochemical responses were achieved in cases 2 and 3, their ALT levels relapsed eventually. HBV genotyping of the fourr patients compared with six consecutive patients with single HBV genotype infection is shown in Figure 2. No patient with dual HBV genotype infection achieved HBeAg clearance or seroconversion, or loss of serum HBV DNA as determined byy hybrid capture assay during the 18 months off lamivudine therapy. Case 4 also developed YMDD mutation with appearance of YIDD mutant virus after 18 months of lamivudine treatment (data nott shown). Post therapy normalization of serum ALT T levels or loss of HBV DNA was not detected att follow-up for more than 6 months after the end of treatment (data not shown in Figure 1).
Discussion HBV infection is a global challenge with an estimated worldwide distribution of 350 million chronic carriers.25 All of these patients are at risk k of developing adverse sequelae, including chronic hepatitis, liver cirrhosis, and hepatocellularr carcinoma (HCC). It is implicated that HBV genotypes may influence the clinical outcomes of CH-B patients. The clinical significance of different HBV genotypes in patients with both acute and chronic HBV infections has become increasingly recognized. Forr example, genotype C is more commonly associated with the progression of liver diseases, whereas genotype B may also be associated with the development of HCC in young noncirrhoticc patients.26–28 In addition, genotype C has a higher frequency of core promoter mutation and a lower response rate to interferon therapy compared to genotype B.29,30 Genotypes C and D are associated with a lower response rate to interferon therapy compared with genotypes B and A.29 A correlation between HBV genotype and response J Formos Med Assoc | 2006 • Vol 105 • No 7
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Figure i 1. Clinical courses off the four f patients infected f with dual HBV genotype B and C: (A) Case 1: 39-year-old male; (B) Case 2: 29-yearold male; (C) Case 3: 35-year-old male; (D) Case 4: 33-year-old male. Time points (3, 3, 2 and 2 for cases 1, 2, 3 and 4, respectively) for detection of YMDD variants in serum by INNO-LiPA HBV DR kit (data not shown) are indicated on the figures. 3-TC = lamivudine; M = YMDD wildtype virus; I = YIDD mutant virus. 1
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Figure i 2. INNO-LiPA HBV genotyping. The typing results were confirmed f by using polymerase chain reaction-restriction fragment f length polymorphism assay of the surface gene of HBV DNA fragment (541 bp; nt 256–796).
to lamivudine or adefovir dipivoxil therapy has not been demonstrated.31,32 Several studies in Asia, all involving small patient numbers and varying durations of lamivudine treatment, showed that HBeAg seroconversion and breakthrough infection J Formos Med Assoc | 2006 • Vol 105 • No 7
occurred in similar proportions of patients with genotypes B and C.12,13,31,32 Kato et al explored the mechanism by which HBV genotype G, which has two stop codons in 33 the precore region, maintains HBeAg production. c 591
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They found all four patients with HBV genotype G, who were HBeAg positive, were also co-infected with HBV genotype A. The clinical significance of mixed HBV genotype infection remains unclear. All four CH-B patients with dual HBV genotype B and C infection and with AE did not respond to an 18-month course of lamivudine monotherapy. Thus, a longer duration of monotherapy may be required or combination with other treatment modalities should be considered. Effective treatment of these patients, however, may require tremendous medical expenditure, and further analysis of cost vs. benefit of treatment modalities is required to determine the indicated course of treatment for CH-B patients with dual genotype B and C infection and AE.
Acknowledgments This work was supported by the Chi Mei Foundation, Tainan, Taiwan, grants number CMFHR9427 to M.J. Sheu and CMFHT9102 to S.L. Tsai.
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