- Email: [email protected]
Lamivudine and adefovir resistance in children and young adults with chronic hepatitis B
International Journal of Infectious Diseases 14 (2010) e236–e239
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Lamivudine and adefovir resistance in children and young adults with chronic hepatitis B Sezin Asik Akman a,*, Sukran Kose b, Oya Halicioglu c a
Department of Pediatric Gastroenterology, The Ministry of Health Tepecik Training and Research Hospital, Izmir, Turkey Department of Infectious Diseases and Clinical Microbiology, The Ministry of Health Tepecik Training and Research Hospital, Izmir, Turkey c Department of Pediatrics, The Ministry of Health Tepecik Training and Research Hospital, Izmir, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 October 2008 Received in revised form 16 April 2009 Accepted 20 April 2009
Objective: Long-term lamivudine (LAM) and adefovir (ADV) treatment has been found to induce the emergence of drug-resistant hepatitis B virus (HBV) in a significant number of patients with chronic hepatitis B (CHB) infection. The aim of our study was to evaluate the LAM and ADV mutations detected in our patient group. Materials and methods: Twenty-four patients diagnosed with CHB were enrolled in this study. The patient group consisted of those who had received 6 months of treatment with interferon-a and who did not response to this therapy. Patients were evaluated based on virologic and serologic response to therapy, and were classified as responders or non-responders. The treatment of non-responders continued with LAM (3 mg/kg/d, maximum 100 mg/d). Due to a lack of response to treatment, ADV (10 mg/g) was added to the treatment regimen of eight young adult patients. The mutations associated with HBV drug resistance were investigated using reverse hybridization methods and PCR. Results: The mutation studies indicated that 14 (58.4%) of the patients had resistance. Three patients developed ADV-associated mutations (A181T), one after 18 months of ADV; the other two had undergone 18 and 36 months of LAM therapy without ADV exposure. Although the average LAM treatment period of the patients with LAM resistance was longer than for those in whom no resistance was detected, no statistically significant difference was found. Conclusions: HBV treatment with nucleoside analogues results in the development of mutant strains, leading to drug resistance. Therefore genotypic resistance testing is important in planning and monitoring HBV treatment. ß 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Corresponding Editor: Mark Holodniy, California, USA Keywords: Lamivudine Adefovir Resistance Hepatitis B
1. Introduction Infection with the hepatitis B virus (HBV), which is known to affect approximately 400 million people, is a global health issue. HBV infection not only results in acute, fulminant, and chronic hepatitis, but it may also contribute to the development of hepatic cirrhosis and hepatocellular carcinoma in the long-term.1 The dominant viral strain in the majority of children diagnosed with chronic hepatitis B (CHB) is wild-type HBV during the normal development phase of the infection. Mutant viruses may be reported in the periods before and after hepatitis B e antigen (HBeAg) seroconversion in the long-term observation of patients. It is also possible that patients become infected primarily with the mutant strain of the virus. In addition, nucleoside analogs utilized in therapy are known to induce the formation of mutant strains.2
* Corresponding author. Tel.: +90 232 463 43 32; fax: +90 232 433 07 56. E-mail address: [email protected] (S.A. Akman).
The approved therapy options for childhood CHB infections are interferon-a (IFN-a) and lamivudine (LAM), which is a nucleoside analog. Currently, adefovir (ADV) may be added to the therapy options in children.3 The correct duration of LAM therapy, which has been evaluated safe in terms of side effects, is not yet clear. The safety and efficacy of LAM use for a duration of one year has been established in children aged 2 years. Favorable outcomes from LAM treatment of CHB in children are maintained for at least several years after completion of treatment. Up to 3–4 years of LAM treatment is safe in children.4 Considering the safety of LAM, it has been suggested that continuous therapy may be beneficial, particularly in patients who do not show HBeAg seroconversion.5 LAM therapy is discontinued in patients who do not respond to treatment and this results in an increase in HBV-DNA levels. The results of studies have shown that a sustained response with HBeAg to anti-hepatitis B e antibody (anti-HBe) seroconversion is rarely obtained, and hepatitis B surface antigen (HBsAg) loss is seldom seen.6 Another risk of the drug is the possibility of the development of drug-resistant mutations.7–10 Long-term LAM
1201-9712/$36.00 – see front matter ß 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2009.04.002
S.A. Akman et al. / International Journal of Infectious Diseases 14 (2010) e236–e239
therapy may therefore increase the likelihood of the development of resistance.5,7,11 Resistance to LAM is associated with mutations in the HBV polymerase gene. The most frequent mutation developing with LAM is the YMDD (tyrosine–methionine–aspartate–aspartate) mutation of the HBV polymerase gene, which is reported to be found in 19% of children subjected to therapy.2,12 These mutations usually affect the YMDD motif located in the C domain of the HBV reverse transcriptase (rt), by replacement of a methionine residue at position 204 with either valine or isoleucine.5,7 Another nucleoside analog which is used in the treatment of CHB in adulthood and childhood is ADV. ADV therapy which is administered orally like LAM, should be closely monitored in terms of long-term nephrotoxicity, and is known to be effective on viruses that are resistant to LAM. The frequency of resistance to ADV, which is known to be effective on HBeAg-negative and wildtype HBV infections, is low.3,13 As studies on mutation profiles in children and young adults are limited, the purpose of this study was to investigate drug resistance developing during the treatment process in patients with CHB. 2. Materials and methods This study investigated mutations that developed against nucleoside analogs in 24 patients who were diagnosed with CHB infection, who were undergoing LAM and ADV therapy as they had not responded to 6 months of IFN-a. Our study was performed prospectively between July 2005 and July 2007. Data recorded for this study were based on retrospective review of the medical records for the period 2003–2005. The baseline patient characteristics were measured prior to LAM therapy. Patients monitored in the pediatric gastroenterology and hepatology units and the infectious diseases and clinical microbiology departments, and (1) whose HBsAg and HBeAg were positive, (2) whose HBV-DNA level was over 1 105 copies/ml, (3) whose alanine aminotransferase (ALT) level was 1.5 times higher than normal for 6 months (upper limit 40 IU/l), and (4) who were reported with a moderate or acute effect on the liver on fine-needle biopsy (Knodell activity index >4 or fibrosis 2)14 were included in the study, in addition to patients (1) whose HBeAg was negative, (2) whose HBV-DNA level was over 1 104 copies/ml, and (3) who were reported to be acutely or moderately affected according to biopsy, as well as patients who were scheduled to be subjected to CHB infection treatment as their ALT level was found to be high. Exclusion criteria were: age less than 2 years, platelet count less than 150 109/l, white blood cell count less than 3 109/l, hemoglobin levels below 10 g/dl, existence of renal failure, hepatic decompensation or psychiatric disorders, any accompanying cause of chronic liver disease other than HBV, and central nervous system disease such as epilepsy. The participants of the study were not coinfected with hepatitis C virus, hepatitis D virus, or HIV. The patient group was made up of individuals who were administered IFN-a (IFN-a2b (Intron A), Schering Plough) therapy in the first phase (5 million units/m2, with a maximum dose 10 million units, given three times a week, subcutaneously for 6 months) and who did not respond to this therapy. Therapy efficacy measures included HBeAg loss or seroconversion, undetectable HBV-DNA, ALT normalization, and HBsAg loss or seroconversion. Patients whose ALT levels did not reach their normal values, whose HBV-DNA levels did not reduce, and who were not reported with HBeAg seroconversion were regarded irresponsive to treatment. These patients underwent LAM therapy (Zeffix, GlaxoSmithKline; 3 mg/kg/g, maximum 100 mg/g, oral). LAM therapy was continued until loss of HBeAg or seroconversion in patients who had not responded to the therapy. During LAM
e237
therapy, in the case of irresponsiveness or the loss of response to treatment, the current situation was to be evaluated in conjunction with the infectious diseases unit. We used the same criteria for response to IFN-a and LAM/ADV treatment: ‘responders’ were defined as those cases with undetectable HBV-DNA, ALT normalization, and HBeAg/HBsAg loss or seroconversion. If these parameters were not found, the patients were defined as ‘nonresponders’.In the case of non-responders, ADV (Hepsera, Er-Kim; 10 mg/g, oral) was added to the treatment schedule of eight patients who were over 18 years of age; ADV was not approved for use in children under the age of 18 in our country. The follow-up period for the patients was changed from 26 to 54 months. Adverse events were not determined during LAM and ADV therapy and there was no treatment discontinuation. During the course of antiviral therapy, cases were re-evaluated with complete blood count and ALT values monthly during the first two months, and after the third month they were re-evaluated with complete blood count and ALT values, and serologic studies once every three months. Quantitative HBV-DNA was studied once every six months. There was no patient who suffered from renal insufficiency, so dosing adjustments for both LAM and ADV were not required for renal insufficiency. Mutations that developed according to the nucleoside analog treatment administered in the 24 patients were investigated prospectively and their drug resistance was evaluated. Measurement of drug resistance was undertaken once at 20–24 months of LAM treatment. The mutation evaluations were carried out before ADV treatment in these patients. Upon obtaining written informed consent from the patients’ families in the case of those aged under 18 years and from the patients who were aged 18 years and over, 2 ml of blood was taken from each patient and sera were kept at under 20 8C before examination at the Infectious Diseases Clinical Microbiology Molecular Diagnosis Laboratory. Mutations in the HBV-DNA polymerase gene were determined using PCR and reverse hybridization. Drug resistance mutations were determined using the Innogenetics Inno-Lipa HBV DR assay according the manufacturer’s recommendations. The amplification reactions were performed as follows: the amplification reaction contained 1 ml each of 25 mmol/l specific primers (Inno-Lipa HBV DR, Innogenetics Ghent, Belgium), 1 ml 10 mmol/l dNTP mixture, 1 ml 25 mmol/l MgCl2, 0.4 ml Taq DNA polymerase (Qiagen, Inc., Hilden, Germany), and 5 ml 10 PCR buffer solution. The total volume was brought up to 50 ml using ddH2O. After amplification, the biotinylated DNA material generated from the HBV polymerase gene was hybridized with specific oligonucleotide probes immobilized as parallel lines on membrane-based strips. HBsAg, HBeAg, and anti-HBe antibodies were examined on the basis of the ELISA method (DiaSorin S.p.A. Saluggia, Italy). HBVDNA (Roche Molecular Systems, Inc, Branchburg, NJ, USA) was applied to determine the viral load. SPSS version 13.0 (SPSS Inc., Chicago, USA) was used for statistical analyses. Mean, median, standard deviation, minimum and maximum values were taken into account in the evaluation of viral load. A p-value of <0.05 was accepted as statistically significant. 3. Results The average age of the 24 patients monitored in the pediatric gastroenterology and infectious diseases polyclinics as a result of diagnosis with CHB was 12.9 4 years (median 12, range 6–20 years); 10 (41.7%) of these patients were female. Two of the patients were reported HBeAg-negative, anti-HBe-positive, and HBV-DNA-
S.A. Akman et al. / International Journal of Infectious Diseases 14 (2010) e236–e239
e238 Table 1 Patient characteristics.
was reported not to have any resistance, the difference was not statistically significant (p > 0.05).
Total number of patients (N) Age, yearsa Gender, M/F (n (%)) Age at diagnosis, yearsa Disease duration, yearsa ALT, IU/la HAIa LAM therapy, monthsa ADV therapy, monthsa,b Drug resistance, n (%)
24 12 (6–20), 12.9 4 14/10 (58.3/41.7) 6 (3–14), 6.9 2.9 6 (1–12), 5.9 2.9 96 (49–278), 105 43.2 9 (6–16), 9.5 3 32.5 (12–48), 32.3 8.3 23 (12–24), 22.2 5 14 (58.3)
M, male; F, female; ALT, alanine aminotransferase; HAI, hepatic activity index; LAM, lamivudine; ADV, adefovir; SD, standard deviation. a Results are median (range), mean SD. b ADV treatment was given to eight patients.
positive, and the average age of the 22 patients (91.7%) with positive HBsAg and HBeAg-positive was 12.5 3.9 years; their ALT level was 105 43.2 IU/l and the average of their viral load was 1 107 copies/ ml. HBeAg seroconversion developed in the last five months as a consequence of LAM therapy in five (20.8%) patients whose treatment duration was 32.3 8.3 months; eight cases whose ages were appropriate were administered ADV therapy. During LAM therapy, 22 (91.7%) patients had ALT normalization and 10 (41.7%) patients had decreased HBV-DNA levels. LAM + ADV combination therapy was used in eight patients in whom ALT normalization was determined in seven and HBVDNA clearance in five. The average duration of LAM therapy in previous applications in these eight patients was 36 9.1 months, while the duration of ADV therapy was found to be 22.2 5 months (Table 1). While no mutation was determined in 10 patients (41.6%), it was found that one patient out of three who were diagnosed with a mutation with regards to ADV, developed rt181T mutation following 18 months of ADV therapy; the other two developed mutations without ADV therapy. Among patients who were reported to have resistance to nucleoside analogs, eight patients were reported with rt80I mutation, six patients with rt204YMDD+YVDD mutation, five patients with rt180 M mutation, four patients with rt204YMDD+YIDD mutation, three patients with rt181T mutation, two patients with rt80 V mutation, and one patient was reported with rt173L, one with rt180FLL+FLM, and one with rt204YMDD+YVDD+YIDD mutations (Table 2). The presence of three or more mutant strains observed in 10 patients (71.4%) among a total of 14 patients was regarded as considerable. Although the average LAM therapy duration of the 12 patients having a single resistance to LAM was higher than in the group that
4. Discussion It is known that the use of nucleoside analogs in CHB infected patients increases the probability of mutant strain development in HBV. Mutations developing in HBV can be regarded as a protection method developed by the virus in order to create resistance to the immune system and/or to the treating agent, as seen in all societies in the world. Especially high mutation rates are determined in the polymerase gene of HBV. It is thought that HBV mutations play an important role in the natural development of HBV and contribute to the continuation of infections caused by viral genetic modifications.2,10,15 Nucleoside analogs such as LAM and ADV induce a decrease in serum HBV-DNA levels associated with normalization of serum ALT levels. But their sustained response with HBeAg to anti-HBe seroconversion is rarely seen, as in some other studies.6,9,13 In a multicenter study conducted by Jonas et al., which compared LAM therapy and placebo, the 19% mutation rate found in the group that had undergone LAM therapy may be accepted as an indicator of drug resistance that developed during treatment with nucleoside analogs. This study involved 191 children diagnosed with CHB; no mutation was observed in the HBV polymerase gene in the placebo group during the first year of treatment.8 On the other hand, in a study of LAM therapy in children of HBV carrier mothers, YMDD mutants developed in 34% of the LAM treated children after one year of therapy.16 As a result of a three-year LAM therapy in 429 patients, the frequency of YMDD mutation development per annum was calculated as 12.1%, 49.7%, and 70.5% in the first, second, and third years, respectively.17 The findings of our study are in parallel with the literature, as mutations were observed in 58.4% of the total 24 patients whose LAM therapy duration was 30.2 10.2 months. No statistically significant difference was determined between lengthening of the treatment and the development of mutations (p = 0.347), but it was reported that LAM therapy duration in patients with drug resistance was longer than in the group with no reported resistance (31.8 9.6 vs. 27.9 10.9). Adefovir is stated to be effective in LAM-resistant mutations. Genotypic resistance rates of 1%, 2%, 4%, and 4% were determined for each year of ADV therapy.18,19 There are also previous studies in which ADV resistance was found to be 0% at sixth months, 6.5% in the first year, and 24.6% at 18 months. The mutation development rate is lower when compared to LAM therapy and also occurs later. The mutation development rate in
Table 2 Detected mutations and treatment periods in subjects with chronic hepatitis B. No.
Case
LAM therapy (months)
ADV therapy (months)
Mutations
1 2 3 4 5 6 7 8 9 10 11 12 13 14
M.D. M.T. A.G. M.O. T.O. G.D. S.M. E.Y. Y.E. E.P. S.K. B.K. H.M. E.D.
48 36 36 24 36 24 48 24 36 36 36 24 20 18
18 24 12 18 12 -
rtL80 I, rtL180 M, rtM204I YMDD+YIDD rtM204I YMDD+YVDD rtL180 M, rtM204I YMDD+YVDD rtA181 T* rt180 FLL+FLM, rtM204I YMDD+YIDD rtL80 I rtL80 I, rtV173 L, rtL180 M, rtM204I YMDD+YVDD rtL80 I, rtM204I YMDD+YVDD rtL80 I, rtL180 M, rtM204I YMDD+YVDD+YIDD rtL80 V, rtL180 M, rtM204I YMDD+YVDD rtA181 T* rtL80 I, rtL180 M, rtM204I YMDD+YVDD rtL80 I, rtM204I YMDD+YIDD rtL80 I+V, rtM204I YMDD+YIDD, rtA181 T*
LAM, lamivudine; ADV, adefovir. * ADV-associated mutations.
S.A. Akman et al. / International Journal of Infectious Diseases 14 (2010) e236–e239
LAM-resistant patients during ADV therapy was found to be high. In a study conducted by Chen et al., the cumulative incidence of ADV resistance at months 6, 12, 18, and 24 was 0%, 6.5%, 24.6%, and 38.3%, respectively.20 The average ADV therapy duration for the eight patients who were aged between 18 and 20 years was determined to be 22.5 5 months. While the mutation rate with LAM therapy was 56.2%, the mutation rate with ADV therapy was 62.5%. In YMDD mutation, isoleucine or valine replaced methionine in the YMDD pattern of DNA polymerase dependent on RNA in the HBV-resistant strain. There are also other mutations in which methionine and serine are replaced in the YMDD pattern and leucine and methionine are replaced in the amino acid substitutions in codon 180 and codon 181.21,22 The mutation profile was heterogeneous in the patient group made up of 24 patients, all of whom were reported with genotype D. rt80 I/V, rt204 YMDD/ YVDD/YIDD, rt180 M/ FLL+FLM, and rt173 L mutations were observed in various combinations. In a study conducted in Turkey on the evaluation of LAM resistance before treatment in CHB patients, 7.8% of 77 patients with genotype D were reported with the tyrosine–methionine–aspartate–aspartate patterned mutation.23 In non-Turkish studies in which the relationship between genotypic characteristics and mutations has been investigated, no difference was observed in the YMDD mutation parameters.24 Recent clinical research associates the virological irresponsiveness developed during LAM or ADV therapy and the replacement of amino acids at position 181 of HBV polymerase. It has also been reported that this amino acid replacement induces the cross-resistance to LAM and ADV, and investigators have drawn attention to the importance of genotypic and phenotypic analysis of antiviral drug resistance.25 According to the study by Gaia et al., the existence of the rt181 mutation may be an indicator of HBV-DNA positivity and can be associated with irresponsiveness to treatment.26 In the study conducted by Lampertico et al., the rtA181 V/T mutation was observed in six of 145 patients before treatment for whom ADV therapy was planned because of irresponsiveness to LAM therapy, whereas three patients were reported with rtA181T in the course of treatment.19 On the other hand, within our patient group, three patients were reported with the rt181T mutation associated with ADV. It was found that one of these patients underwent ADV therapy for 18 months; however, the other two patients underwent LAM therapy for 18 or 36 months without undergoing ADV therapy. This situation was thought have arisen as a result of cross-resistance due to nucleoside analog treatments. Lamivudine resistance has been found to confer cross-resistance to some of these compounds and it is likely that resistance to other antivirals may also develop during prolonged use. Drug resistance therefore poses a major threat to nucleoside analogue-based therapies for chronic HBV infection.24,26 In our study, the rate of mutations that developed as a result of the use of nucleoside analogs in the course of treatment, resulting in drug resistance, was found to be 60%. As the development of mutant viruses affects the prognosis of the disease negatively by hindering the success of treatment, it is important to investigate these mutations. If mutant strains are to be determined, this will facilitate other treatments with no resistance development to viruses and with stable antiviral effects.
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Conflict of interest: No conflict of interest to declare. References 1. Baumert TF, Thimme R, von Weizsacker F. Pathogenesis of hepatitis B virus infection. World J Gastroenterol 2007;13:82–90. 2. Chang MH, Hepatitis. B virus mutation in children. Indian J Pediatr 2006;73: 803–7. 3. Jonas MM, Kelly D, Pollack H, Mizerski J, Sorbel J, Frederick D, et al. Safety, efficacy, and pharmacokinetics of adefovir dipivoxil in children and adolescents (age 2 to <18 years) with chronic hepatitis B. Hepatology 2008;47:1863–71. 4. Jonas MM, Little NR, Gardner SD, International Pediatric Lamivudine Investigator Group. Long-term lamivudine treatment of children with chronic hepatitis B: durability of therapeutic responses and safety. J Viral Hepat 2008;15:20–7. 5. Suzuki F, Tsubota A, Arase Y, Suzuki Y, Akuta N, Hosaka T, et al. Efficacy of lamivudine therapy and factors associated with emergence of resistance in chronic hepatitis B virus infection in Japan. Intervirology 2003;46:182–9. 6. Xu XW, Chen YG. Current therapy with nucleoside/nucleotide analogs for patients with chronic hepatitis B. Hepatobiliary Pancreat Dis Int 2006;5:350–9. 7. Sokal E. Drug treatment of pediatric chronic hepatitis B. Paediatr Drugs 2002;4:361–9. 8. Jonas MM, Kelly DA, Mizerski J, Badia IB, Areias JA, Schwarz KB, et al. Clinical trial of lamivudine in children with chronic hepatitis B. N Engl J Med 2002;346:1706–13. 9. Heller S, Valencia-Mayoral P. Treatment of viral hepatitis in children. Arch Med Res 2007;38:702–10. 10. Fung SK, Fontana RJ. Management of drug-resistant chronic hepatitis B. Clin Liver Dis 2006;10:275–302. 11. Delaney WE, Locarnini S, Shaw T. Resistance of hepatitis B virus to antiviral drugs: current aspects and directions for future investigation. Antivir Chem Chemother 2001;12:1–35. 12. Lok AS, Zoulim F, Locarnini S, Bartholomeusz A, Ghany MG, Pawlotsky JM, et al., Hepatitis B Virus Drug Resistance Working Group. Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management. Hepatology 2007;46:254–65. 13. Han SH. Natural course, therapeutic options and economic evaluation of therapies for chronic hepatitis B. Drugs 2006;66:1831–51. 14. Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981;1:431–5. 15. Kowalik-Mikolajewska B, Aniszewska M. [Lamivudine treatment of chronic hepatitis B in children after treatment with interferon alfa—evaluation of the effectiveness of prolonged therapy.]. Med Wieku Rozwoj 2004;8(4 Pt 1):901–10. 16. Ni YH, Huang FC, Wu TC, Kong MS, Jeng YM, Chen PJ, et al. Lamivudine treatment in maternally transmitted chronic hepatitis B virus infection patients. Pediatr Int 2005;47:372–7. 17. Yao GB, Cui ZY, Wang BE, Yao JL, Zeng MD. A 3-year clinical trial of lamivudine in treatment of patients with chronic hepatitis B. Hepatobiliary Pancreat Dis Int 2004;3:188–93. 18. Peters MG, Hann Hw H, Martin P, Heathcote EJ, Buggisch P, Rubin R, et al. Adefovir dipivoxil alone or in combination with lamivudine in patients with lamivudine-resistant chronic hepatitis B. Gastroenterology 2004;126:91–101. 19. Lampertico P, Vigano` M, Manenti E, Iavarone M, Sablon E, Colombo M. Low resistance to adefovir combined with lamivudine. A 3-year study of 145 lamivudine-resistant hepatitis B patients. Gastroenterology 2007;133:1445–51. 20. Chen CH, Wang JH, Lee CM, Hung CH, Hu TH, Wang JC, et al. Virological response and incidence of adefovir resistance in lamivudine-resistant patients treated with adefovir dipivoxil. Antivir Ther 2006;11:771–8. 21. Bozdayi AM, Eyigun CP, Turkyilmaz AR, Avci IY, Pahsa A, Yurdaydin C. A novel pattern (sW195a) in surface gene of HBV DNA due to YSDD (L180 M plus M204S) mutation selected during lamivudine therapy and successful treatment with adefovir dipivoxil. J Clin Virol 2004;31:76–7. 22. Melegari M, Scaglioni PP, Wands JR. Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. Hepatology 1998;27:628–33. 23. Tunc¸bilek S, Ko¨se S, Elaldi A, Akman S. Lamivudine resistance in untreated chronic hepatitis B patients in Turkey. Turk J Gastroenterol 2008;19:99–103. 24. Sun J, Wang Z, Ma S, Zeng G, Zhou Z, Luo K, et al. Clinical and virological characteristics of lamivudine resistance in chronic hepatitis B patients: a single center experience. J Med Virol 2005;75:391–8. 25. Villet S, Pichoud C, Billioud G, Barraud L, Durantel S, Tre´po C, et al. Impact of hepatitis B virus rtA181 V/T mutants on hepatitis B treatment failure. J Hepatol 2008;48:747–55. 26. Gaia S, Barbon V, Smedile A, Olivero A, Carenzi S, Lagget M, et al. Lamivudineresistant chronic hepatitis B: an observational study on adefovir in monotherapy or in combination with lamivudine. J Hepatol 2008;48:540–7.
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Lamivudine and adefovir resistance in children and young adults with chronic hepatitis B Sezin Asik Akman a,*, Sukran Kose b, Oya Halicioglu c a
Department of Pediatric Gastroenterology, The Ministry of Health Tepecik Training and Research Hospital, Izmir, Turkey Department of Infectious Diseases and Clinical Microbiology, The Ministry of Health Tepecik Training and Research Hospital, Izmir, Turkey c Department of Pediatrics, The Ministry of Health Tepecik Training and Research Hospital, Izmir, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 October 2008 Received in revised form 16 April 2009 Accepted 20 April 2009
Objective: Long-term lamivudine (LAM) and adefovir (ADV) treatment has been found to induce the emergence of drug-resistant hepatitis B virus (HBV) in a significant number of patients with chronic hepatitis B (CHB) infection. The aim of our study was to evaluate the LAM and ADV mutations detected in our patient group. Materials and methods: Twenty-four patients diagnosed with CHB were enrolled in this study. The patient group consisted of those who had received 6 months of treatment with interferon-a and who did not response to this therapy. Patients were evaluated based on virologic and serologic response to therapy, and were classified as responders or non-responders. The treatment of non-responders continued with LAM (3 mg/kg/d, maximum 100 mg/d). Due to a lack of response to treatment, ADV (10 mg/g) was added to the treatment regimen of eight young adult patients. The mutations associated with HBV drug resistance were investigated using reverse hybridization methods and PCR. Results: The mutation studies indicated that 14 (58.4%) of the patients had resistance. Three patients developed ADV-associated mutations (A181T), one after 18 months of ADV; the other two had undergone 18 and 36 months of LAM therapy without ADV exposure. Although the average LAM treatment period of the patients with LAM resistance was longer than for those in whom no resistance was detected, no statistically significant difference was found. Conclusions: HBV treatment with nucleoside analogues results in the development of mutant strains, leading to drug resistance. Therefore genotypic resistance testing is important in planning and monitoring HBV treatment. ß 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Corresponding Editor: Mark Holodniy, California, USA Keywords: Lamivudine Adefovir Resistance Hepatitis B
1. Introduction Infection with the hepatitis B virus (HBV), which is known to affect approximately 400 million people, is a global health issue. HBV infection not only results in acute, fulminant, and chronic hepatitis, but it may also contribute to the development of hepatic cirrhosis and hepatocellular carcinoma in the long-term.1 The dominant viral strain in the majority of children diagnosed with chronic hepatitis B (CHB) is wild-type HBV during the normal development phase of the infection. Mutant viruses may be reported in the periods before and after hepatitis B e antigen (HBeAg) seroconversion in the long-term observation of patients. It is also possible that patients become infected primarily with the mutant strain of the virus. In addition, nucleoside analogs utilized in therapy are known to induce the formation of mutant strains.2
* Corresponding author. Tel.: +90 232 463 43 32; fax: +90 232 433 07 56. E-mail address: [email protected] (S.A. Akman).
The approved therapy options for childhood CHB infections are interferon-a (IFN-a) and lamivudine (LAM), which is a nucleoside analog. Currently, adefovir (ADV) may be added to the therapy options in children.3 The correct duration of LAM therapy, which has been evaluated safe in terms of side effects, is not yet clear. The safety and efficacy of LAM use for a duration of one year has been established in children aged 2 years. Favorable outcomes from LAM treatment of CHB in children are maintained for at least several years after completion of treatment. Up to 3–4 years of LAM treatment is safe in children.4 Considering the safety of LAM, it has been suggested that continuous therapy may be beneficial, particularly in patients who do not show HBeAg seroconversion.5 LAM therapy is discontinued in patients who do not respond to treatment and this results in an increase in HBV-DNA levels. The results of studies have shown that a sustained response with HBeAg to anti-hepatitis B e antibody (anti-HBe) seroconversion is rarely obtained, and hepatitis B surface antigen (HBsAg) loss is seldom seen.6 Another risk of the drug is the possibility of the development of drug-resistant mutations.7–10 Long-term LAM
1201-9712/$36.00 – see front matter ß 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2009.04.002
S.A. Akman et al. / International Journal of Infectious Diseases 14 (2010) e236–e239
therapy may therefore increase the likelihood of the development of resistance.5,7,11 Resistance to LAM is associated with mutations in the HBV polymerase gene. The most frequent mutation developing with LAM is the YMDD (tyrosine–methionine–aspartate–aspartate) mutation of the HBV polymerase gene, which is reported to be found in 19% of children subjected to therapy.2,12 These mutations usually affect the YMDD motif located in the C domain of the HBV reverse transcriptase (rt), by replacement of a methionine residue at position 204 with either valine or isoleucine.5,7 Another nucleoside analog which is used in the treatment of CHB in adulthood and childhood is ADV. ADV therapy which is administered orally like LAM, should be closely monitored in terms of long-term nephrotoxicity, and is known to be effective on viruses that are resistant to LAM. The frequency of resistance to ADV, which is known to be effective on HBeAg-negative and wildtype HBV infections, is low.3,13 As studies on mutation profiles in children and young adults are limited, the purpose of this study was to investigate drug resistance developing during the treatment process in patients with CHB. 2. Materials and methods This study investigated mutations that developed against nucleoside analogs in 24 patients who were diagnosed with CHB infection, who were undergoing LAM and ADV therapy as they had not responded to 6 months of IFN-a. Our study was performed prospectively between July 2005 and July 2007. Data recorded for this study were based on retrospective review of the medical records for the period 2003–2005. The baseline patient characteristics were measured prior to LAM therapy. Patients monitored in the pediatric gastroenterology and hepatology units and the infectious diseases and clinical microbiology departments, and (1) whose HBsAg and HBeAg were positive, (2) whose HBV-DNA level was over 1 105 copies/ml, (3) whose alanine aminotransferase (ALT) level was 1.5 times higher than normal for 6 months (upper limit 40 IU/l), and (4) who were reported with a moderate or acute effect on the liver on fine-needle biopsy (Knodell activity index >4 or fibrosis 2)14 were included in the study, in addition to patients (1) whose HBeAg was negative, (2) whose HBV-DNA level was over 1 104 copies/ml, and (3) who were reported to be acutely or moderately affected according to biopsy, as well as patients who were scheduled to be subjected to CHB infection treatment as their ALT level was found to be high. Exclusion criteria were: age less than 2 years, platelet count less than 150 109/l, white blood cell count less than 3 109/l, hemoglobin levels below 10 g/dl, existence of renal failure, hepatic decompensation or psychiatric disorders, any accompanying cause of chronic liver disease other than HBV, and central nervous system disease such as epilepsy. The participants of the study were not coinfected with hepatitis C virus, hepatitis D virus, or HIV. The patient group was made up of individuals who were administered IFN-a (IFN-a2b (Intron A), Schering Plough) therapy in the first phase (5 million units/m2, with a maximum dose 10 million units, given three times a week, subcutaneously for 6 months) and who did not respond to this therapy. Therapy efficacy measures included HBeAg loss or seroconversion, undetectable HBV-DNA, ALT normalization, and HBsAg loss or seroconversion. Patients whose ALT levels did not reach their normal values, whose HBV-DNA levels did not reduce, and who were not reported with HBeAg seroconversion were regarded irresponsive to treatment. These patients underwent LAM therapy (Zeffix, GlaxoSmithKline; 3 mg/kg/g, maximum 100 mg/g, oral). LAM therapy was continued until loss of HBeAg or seroconversion in patients who had not responded to the therapy. During LAM
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therapy, in the case of irresponsiveness or the loss of response to treatment, the current situation was to be evaluated in conjunction with the infectious diseases unit. We used the same criteria for response to IFN-a and LAM/ADV treatment: ‘responders’ were defined as those cases with undetectable HBV-DNA, ALT normalization, and HBeAg/HBsAg loss or seroconversion. If these parameters were not found, the patients were defined as ‘nonresponders’.In the case of non-responders, ADV (Hepsera, Er-Kim; 10 mg/g, oral) was added to the treatment schedule of eight patients who were over 18 years of age; ADV was not approved for use in children under the age of 18 in our country. The follow-up period for the patients was changed from 26 to 54 months. Adverse events were not determined during LAM and ADV therapy and there was no treatment discontinuation. During the course of antiviral therapy, cases were re-evaluated with complete blood count and ALT values monthly during the first two months, and after the third month they were re-evaluated with complete blood count and ALT values, and serologic studies once every three months. Quantitative HBV-DNA was studied once every six months. There was no patient who suffered from renal insufficiency, so dosing adjustments for both LAM and ADV were not required for renal insufficiency. Mutations that developed according to the nucleoside analog treatment administered in the 24 patients were investigated prospectively and their drug resistance was evaluated. Measurement of drug resistance was undertaken once at 20–24 months of LAM treatment. The mutation evaluations were carried out before ADV treatment in these patients. Upon obtaining written informed consent from the patients’ families in the case of those aged under 18 years and from the patients who were aged 18 years and over, 2 ml of blood was taken from each patient and sera were kept at under 20 8C before examination at the Infectious Diseases Clinical Microbiology Molecular Diagnosis Laboratory. Mutations in the HBV-DNA polymerase gene were determined using PCR and reverse hybridization. Drug resistance mutations were determined using the Innogenetics Inno-Lipa HBV DR assay according the manufacturer’s recommendations. The amplification reactions were performed as follows: the amplification reaction contained 1 ml each of 25 mmol/l specific primers (Inno-Lipa HBV DR, Innogenetics Ghent, Belgium), 1 ml 10 mmol/l dNTP mixture, 1 ml 25 mmol/l MgCl2, 0.4 ml Taq DNA polymerase (Qiagen, Inc., Hilden, Germany), and 5 ml 10 PCR buffer solution. The total volume was brought up to 50 ml using ddH2O. After amplification, the biotinylated DNA material generated from the HBV polymerase gene was hybridized with specific oligonucleotide probes immobilized as parallel lines on membrane-based strips. HBsAg, HBeAg, and anti-HBe antibodies were examined on the basis of the ELISA method (DiaSorin S.p.A. Saluggia, Italy). HBVDNA (Roche Molecular Systems, Inc, Branchburg, NJ, USA) was applied to determine the viral load. SPSS version 13.0 (SPSS Inc., Chicago, USA) was used for statistical analyses. Mean, median, standard deviation, minimum and maximum values were taken into account in the evaluation of viral load. A p-value of <0.05 was accepted as statistically significant. 3. Results The average age of the 24 patients monitored in the pediatric gastroenterology and infectious diseases polyclinics as a result of diagnosis with CHB was 12.9 4 years (median 12, range 6–20 years); 10 (41.7%) of these patients were female. Two of the patients were reported HBeAg-negative, anti-HBe-positive, and HBV-DNA-
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e238 Table 1 Patient characteristics.
was reported not to have any resistance, the difference was not statistically significant (p > 0.05).
Total number of patients (N) Age, yearsa Gender, M/F (n (%)) Age at diagnosis, yearsa Disease duration, yearsa ALT, IU/la HAIa LAM therapy, monthsa ADV therapy, monthsa,b Drug resistance, n (%)
24 12 (6–20), 12.9 4 14/10 (58.3/41.7) 6 (3–14), 6.9 2.9 6 (1–12), 5.9 2.9 96 (49–278), 105 43.2 9 (6–16), 9.5 3 32.5 (12–48), 32.3 8.3 23 (12–24), 22.2 5 14 (58.3)
M, male; F, female; ALT, alanine aminotransferase; HAI, hepatic activity index; LAM, lamivudine; ADV, adefovir; SD, standard deviation. a Results are median (range), mean SD. b ADV treatment was given to eight patients.
positive, and the average age of the 22 patients (91.7%) with positive HBsAg and HBeAg-positive was 12.5 3.9 years; their ALT level was 105 43.2 IU/l and the average of their viral load was 1 107 copies/ ml. HBeAg seroconversion developed in the last five months as a consequence of LAM therapy in five (20.8%) patients whose treatment duration was 32.3 8.3 months; eight cases whose ages were appropriate were administered ADV therapy. During LAM therapy, 22 (91.7%) patients had ALT normalization and 10 (41.7%) patients had decreased HBV-DNA levels. LAM + ADV combination therapy was used in eight patients in whom ALT normalization was determined in seven and HBVDNA clearance in five. The average duration of LAM therapy in previous applications in these eight patients was 36 9.1 months, while the duration of ADV therapy was found to be 22.2 5 months (Table 1). While no mutation was determined in 10 patients (41.6%), it was found that one patient out of three who were diagnosed with a mutation with regards to ADV, developed rt181T mutation following 18 months of ADV therapy; the other two developed mutations without ADV therapy. Among patients who were reported to have resistance to nucleoside analogs, eight patients were reported with rt80I mutation, six patients with rt204YMDD+YVDD mutation, five patients with rt180 M mutation, four patients with rt204YMDD+YIDD mutation, three patients with rt181T mutation, two patients with rt80 V mutation, and one patient was reported with rt173L, one with rt180FLL+FLM, and one with rt204YMDD+YVDD+YIDD mutations (Table 2). The presence of three or more mutant strains observed in 10 patients (71.4%) among a total of 14 patients was regarded as considerable. Although the average LAM therapy duration of the 12 patients having a single resistance to LAM was higher than in the group that
4. Discussion It is known that the use of nucleoside analogs in CHB infected patients increases the probability of mutant strain development in HBV. Mutations developing in HBV can be regarded as a protection method developed by the virus in order to create resistance to the immune system and/or to the treating agent, as seen in all societies in the world. Especially high mutation rates are determined in the polymerase gene of HBV. It is thought that HBV mutations play an important role in the natural development of HBV and contribute to the continuation of infections caused by viral genetic modifications.2,10,15 Nucleoside analogs such as LAM and ADV induce a decrease in serum HBV-DNA levels associated with normalization of serum ALT levels. But their sustained response with HBeAg to anti-HBe seroconversion is rarely seen, as in some other studies.6,9,13 In a multicenter study conducted by Jonas et al., which compared LAM therapy and placebo, the 19% mutation rate found in the group that had undergone LAM therapy may be accepted as an indicator of drug resistance that developed during treatment with nucleoside analogs. This study involved 191 children diagnosed with CHB; no mutation was observed in the HBV polymerase gene in the placebo group during the first year of treatment.8 On the other hand, in a study of LAM therapy in children of HBV carrier mothers, YMDD mutants developed in 34% of the LAM treated children after one year of therapy.16 As a result of a three-year LAM therapy in 429 patients, the frequency of YMDD mutation development per annum was calculated as 12.1%, 49.7%, and 70.5% in the first, second, and third years, respectively.17 The findings of our study are in parallel with the literature, as mutations were observed in 58.4% of the total 24 patients whose LAM therapy duration was 30.2 10.2 months. No statistically significant difference was determined between lengthening of the treatment and the development of mutations (p = 0.347), but it was reported that LAM therapy duration in patients with drug resistance was longer than in the group with no reported resistance (31.8 9.6 vs. 27.9 10.9). Adefovir is stated to be effective in LAM-resistant mutations. Genotypic resistance rates of 1%, 2%, 4%, and 4% were determined for each year of ADV therapy.18,19 There are also previous studies in which ADV resistance was found to be 0% at sixth months, 6.5% in the first year, and 24.6% at 18 months. The mutation development rate is lower when compared to LAM therapy and also occurs later. The mutation development rate in
Table 2 Detected mutations and treatment periods in subjects with chronic hepatitis B. No.
Case
LAM therapy (months)
ADV therapy (months)
Mutations
1 2 3 4 5 6 7 8 9 10 11 12 13 14
M.D. M.T. A.G. M.O. T.O. G.D. S.M. E.Y. Y.E. E.P. S.K. B.K. H.M. E.D.
48 36 36 24 36 24 48 24 36 36 36 24 20 18
18 24 12 18 12 -
rtL80 I, rtL180 M, rtM204I YMDD+YIDD rtM204I YMDD+YVDD rtL180 M, rtM204I YMDD+YVDD rtA181 T* rt180 FLL+FLM, rtM204I YMDD+YIDD rtL80 I rtL80 I, rtV173 L, rtL180 M, rtM204I YMDD+YVDD rtL80 I, rtM204I YMDD+YVDD rtL80 I, rtL180 M, rtM204I YMDD+YVDD+YIDD rtL80 V, rtL180 M, rtM204I YMDD+YVDD rtA181 T* rtL80 I, rtL180 M, rtM204I YMDD+YVDD rtL80 I, rtM204I YMDD+YIDD rtL80 I+V, rtM204I YMDD+YIDD, rtA181 T*
LAM, lamivudine; ADV, adefovir. * ADV-associated mutations.
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LAM-resistant patients during ADV therapy was found to be high. In a study conducted by Chen et al., the cumulative incidence of ADV resistance at months 6, 12, 18, and 24 was 0%, 6.5%, 24.6%, and 38.3%, respectively.20 The average ADV therapy duration for the eight patients who were aged between 18 and 20 years was determined to be 22.5 5 months. While the mutation rate with LAM therapy was 56.2%, the mutation rate with ADV therapy was 62.5%. In YMDD mutation, isoleucine or valine replaced methionine in the YMDD pattern of DNA polymerase dependent on RNA in the HBV-resistant strain. There are also other mutations in which methionine and serine are replaced in the YMDD pattern and leucine and methionine are replaced in the amino acid substitutions in codon 180 and codon 181.21,22 The mutation profile was heterogeneous in the patient group made up of 24 patients, all of whom were reported with genotype D. rt80 I/V, rt204 YMDD/ YVDD/YIDD, rt180 M/ FLL+FLM, and rt173 L mutations were observed in various combinations. In a study conducted in Turkey on the evaluation of LAM resistance before treatment in CHB patients, 7.8% of 77 patients with genotype D were reported with the tyrosine–methionine–aspartate–aspartate patterned mutation.23 In non-Turkish studies in which the relationship between genotypic characteristics and mutations has been investigated, no difference was observed in the YMDD mutation parameters.24 Recent clinical research associates the virological irresponsiveness developed during LAM or ADV therapy and the replacement of amino acids at position 181 of HBV polymerase. It has also been reported that this amino acid replacement induces the cross-resistance to LAM and ADV, and investigators have drawn attention to the importance of genotypic and phenotypic analysis of antiviral drug resistance.25 According to the study by Gaia et al., the existence of the rt181 mutation may be an indicator of HBV-DNA positivity and can be associated with irresponsiveness to treatment.26 In the study conducted by Lampertico et al., the rtA181 V/T mutation was observed in six of 145 patients before treatment for whom ADV therapy was planned because of irresponsiveness to LAM therapy, whereas three patients were reported with rtA181T in the course of treatment.19 On the other hand, within our patient group, three patients were reported with the rt181T mutation associated with ADV. It was found that one of these patients underwent ADV therapy for 18 months; however, the other two patients underwent LAM therapy for 18 or 36 months without undergoing ADV therapy. This situation was thought have arisen as a result of cross-resistance due to nucleoside analog treatments. Lamivudine resistance has been found to confer cross-resistance to some of these compounds and it is likely that resistance to other antivirals may also develop during prolonged use. Drug resistance therefore poses a major threat to nucleoside analogue-based therapies for chronic HBV infection.24,26 In our study, the rate of mutations that developed as a result of the use of nucleoside analogs in the course of treatment, resulting in drug resistance, was found to be 60%. As the development of mutant viruses affects the prognosis of the disease negatively by hindering the success of treatment, it is important to investigate these mutations. If mutant strains are to be determined, this will facilitate other treatments with no resistance development to viruses and with stable antiviral effects.
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