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A real-time quantitative polymerase chain reaction method for hepatitis B virus in patients with chronic hepatitis B treated with lamivudine
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2003 by Am. Coll. of Gastroenterology Published by Elsevier Inc.
Vol. 98, No. 9, 2003 ISSN 0002-9270/03/$30.00 doi:10.1016/S0002-9270(03)00554-9
A Real-Time Quantitative Polymerase Chain Reaction Method for Hepatitis B Virus in Patients With Chronic Hepatitis B Treated With Lamivudine Tatsuya Ide, M.D., Ryukichi Kumashiro, M.D., Yuriko Koga, M.D., Eisuke Tanaka, M.D., Teruko Hino, M.D., Akiko Hisamochi, M.D., Shiro Murashima, M.D., Kei Ogata, M.D., Kazuo Tanaka, M.D., Reiichiro Kuwahara, M.D., and Michio Sata, M.D. Second Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan
OBJECTIVES: During treatment of chronic hepatitis B with lamivudine, changes in the level of hepatitis B virus (HBV) DNA were investigated using a real-time polymerase chain reaction (PCR) method with a detection limit of 1.7 log copies/ml (50 copies/ml) to clarify its clinical significance, particularly the association between HBV DNA levels and the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) mutants. METHODS: Twenty-four patients who had received lamivudine therapy for ⬎1 yr were studied. HBV DNA levels were determined using transcription-mediated amplification for sera with ⬎3.7 log genome equivalents/ml, the Roche Monitor kit for sera with ⱖ2.6 log copies/ml, and real-time PCR for sera with ⬍ 2.6 log copies/ml (the detection limit was 1.7 log copies/ml). Patients were classified into three groups according to the minimal HBV DNA level attained during lamivudine therapy: the ⬍1.7 log copies/ml group (eight patients), the 1.7–2.5 log copies/ml group (five patients), and the ⱖ2.6 log copies/ml group (11 patients). RESULTS: Pretreatment HBV DNA levels were significantly lower in the ⬍1.7 copies/ml group than in the other two groups (p ⬍ 0.05). Neither the emergence of YMDD mutants nor a virological breakthrough of serum HBV DNA was observed in any of the eight patients in the ⬍1.7 copies/ml group. In contrast, in the 1.7–2.5 copies/ml and ⱖ2.6 copies/ml groups, virological breakthroughs resulting from the emergence of YMDD mutants were observed in two of five patients and in all 11 patients, respectively (p ⬍ 0.001). Virological breakthroughs were observed at a mean of 49.6 ⫾ 18.4 wk in 11 the patients in the ⱖ2.6 copies/ml group and at wk 107 and 115 in two patients in the 1.7–2.5 copies/ml group. CONCLUSIONS: The real-time PCR method is useful for predicting the emergence of YMDD mutants and the estimated time of their emergence. (Am J Gastroenterol 2003;98: 2048 –2051. © 2003 by Am. Coll. of Gastroenterology)
INTRODUCTION Lamivudine has been widely used in the treatment of chronic hepatitis B. When it is administered to hepatitis B
patients, a decrease or disappearance in serum hepatitis B virus (HBV) DNA and hepatitis Be (Hbe) antigen is observed and is associated with a decrease in ALT. In previous studies (1–3), the HBV DNA levels during treatment with lamivudine were measured mainly using the solution hybridization assay (Abbott Laboratories, North Chicago, IL) and branched DNA signal amplification technology (b-DNA assay). In these methods, serum HBV DNA fell to undetectable levels at least once during therapy in ⬎89% of the patients receiving lamivudine (1–3). Recently, we reported that a transcription-mediated amplification (TMA) assay, which had approximately 100-fold higher sensitivity (103.7 genome equivalents/ml) than other methods, is more useful for clarifying the changes in the HBV DNA levels (4). In addition, to detect much lower levels of HBV DNA, a quantitative real-time polymer chain reaction (PCR) methods has been developed. This new method combines the Roche Monitor kit and real-time PCR. The minimum detection limits of the Roche Monitor kit were 102.6copies/ml and those of the new real-time PCR were 101.7 copies/ml. After extended lamivudine therapy, tyrosine-methionineaspartate-aspartate (YMDD) mutation has been identified in some patients (5, 6). Some studies have suggested that monitoring the virus load can predict the later emergence of YMDD mutants (7–10). However, there has been no report investigating the association between the virus load and the emergence of YMDD mutants using a method that can detect ⬍102 copies/ml. In addition, the observation periods of patients in the above mentioned were ⬍2 yr (7–9) and the numbers of patients were small (10). The aim of this study was to determine whether this real-time PCR method is useful for predicting the emergence of YMDD mutants.
MATERIALS AND METHODS We studied 24 patients with HBV related-chronic liver diseases, who had received lamivudine therapy for ⬎12 months between March, 1995, and January, 2001 (19 men and five women, mean age 48.8 ⫾ 11.1 yr). Of the 24 patients, 14 had chronic hepatitis, nine cirrhosis, and one
AJG – September, 2003
hepatocellular carcinoma complicating cirrhosis. The daily dosage of lamivudine ranged from 75 to 300 mg, and dosages varied among the patients; therefore mean dosages were calculated for each patient. A liver function test was performed once per month. HBV DNA, hepatitis Be antigen (HBeAg), and anti-HBe antibody were measured every 1–3 months. The HBV DNA level was measured using the TMA assay (Chugai Diagnostic Science, Tokyo, Japan). The measurement range of the TMA assay was 103.7–108.7 genome equivalents/ml (3.7– 8.7 log genome equivalents [LGE]/ml). At the point at which the levels of HBV DNA were ⬍3.7 LGE, they were measured using a Roche Monitor kit (Roche Diagnostics, Tokyo, Japan) using serum stored at –20°C. The detection limit of the Monitor kit was 102.6 copies/ml (2.6 log copies/ml) and the measurement range of the kit was 102.6–107.6 copies/ml (2.6 –7.6 log copies/ml). There was a good correlation between the TMA assay and the Monitor kit (r ⫽ 0.969, y ⫽ 0.9203x ⫹ 0.5049; unpublished data). In addition, in serum with HBV DNA levels lower than 2.6 log copies/ml, the levels were measured using our new real-time PCR assay. The real-time PCR was performed as follows: PCR products obtained by the Roche Monitor kit were purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Tokyo, Japan). The oligonucleotide sequences of the primers were: HBVnested-F(5'-CTTTDGGRCATGGACATTGA-3'; D ⫽ A, G, or T; R ⫽ A or G), and HBV-nested-R (5'-ACTCCACAGWAGCTCCAAATTCTTTA-3'; W ⫽ A or T). The PCR reaction was performed with FastStart Taq DNA polymerase (Roche Diagnostics) and SYBR Green I (BioWhittaker Molecular Applications, Rockland, ME) using an ABI PRISM 7700 Sequence Detection System. The PCR cycle consisted of an initial denaturing step at 95°C for 12 min, followed by 30 amplification cycles at 95°C for 15 s, 50°C for 30 s, and 65°C for 30 s. To quantify the loads of HBV DNA, we used external standards containing 0, 1.25, 2.5, 10, 102, 103, 104, and 106 copies. Comparison of the cycle threshold value to a calibration curve allows the absolute quantification of unknown viral loads of DNA. The detection limit of this real-time PCR assay was 1.7 log copies/ml and the measurement range of this assay was 101.7–102.5 copies/ml (1.7–2.5 log copies/ml). The coefficient of variation in the inner assay (within-run reproducibility; n ⫽ 3) was 1.4% at a mean of 2.5 log copies/ml. To establish the linearity of HBV DNA quantification of real-time PCR, three positive samples estimated to contain 2.5 log copies/ml were diluted in HBV negative human serum to 2.2, 1.9, 1.6 log copies/ml (expected values). The results were 2.5 ⫾ 0.03, 2.2 ⫾ 0.17, 1.9 ⫾ 0.12, and 1.7 ⫾ 0.30 log copies/ml, respectively. A linear relationship was observed between the expected values and the results (r ⫽ 0.996). The present findings were shown in LGE (log genome equivalents)/ml in the TMA assay and log copies/ml in the Roche Monitor assay and the real-time PCR assay. A virological breakthrough of serum HBV DNA was defined by a ⬎10-
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Table 1. Patients’ Background Before Therapy and Emergence of YMDD Mutants During Therapy According to the Minimal Levels of HBV DNA Minimal Level of DNA (log copies/ml) ⱖ2.6
2.5–1.7
⬍1.7
p Value*
No. of patients 11 5 8 HBV DNA (LGE/ 8.3 ⫾ 0.6 7.4 ⫾ 1.2 7.3 ⫾ 0.8 ⬍0.05† ml) ALT (IU/L) 170 ⫾ 118 130 ⫾ 100 196 ⫾ 101 0.29† Dose (mg/day) 146 ⫾ 56 124 ⫾ 22 133 ⫾ 38 0.46† Treatment period 90 ⫾ 40 104 ⫾ 39 89 ⫾ 35 0.83† (wk) HBeAg ⫹/⫺ 10/1 4/1 7/1 0.7‡ Cirrhosis ⫹/⫺ 5/6 1/4 4/4 0.44‡ YMDD mutant 11 (100%) 2 (40%) 0 (0%) ⬍0.001‡ Time of reincreased 49.6 ⫾ 18.4 107, 115 HBV DNA (wk) No. of patients n ⫽ 11 n⫽2 n⫽0 * ⱖ2.6 and 2.5–1.7 vs ⬍ 1.7. † Mann-Whitney U test. ‡ Fisher’s exact probability test.
fold increase from the minimal level of DNA. HBeAg, and anti-HBe antibody were detected by enzyme immunoassay (AxSYM, HBe, anti-HBe; Dainabot, Tokyo, Japan). HBeAg assay findings were expressed as ⫹(S/N ⬎5.0), ⫾ (2.1 ⬍S/N ⱕ5.0), and ⫺(S/N ⬍2.1). Anti-HBe antibody assay findings were expressed as ⫹ (% inhibition ⬎70%), ⫾ (50 ⬍% inhibition ⱕ 70), and ⫺ (% inhibition ⱕ50%). The YMDD mutant strain was measured 6 –12 months after the initiation of treatment using SMITEST HBV-YMDD motif ELMA (Genome Science Laboratories, Fukushima, Japan) (11). Values are reported as the mean ⫾ SEM and were analyzed by the Mann-Whitney U test and the Fisher’s exact probability test. A p value ⬍ 0.05 was considered to be significant
RESULTS Background Characteristics of the Patients The patients were classified into three groups according to the minimal HBV DNA level attained during lamivudine therapy: 1) the ⬍1.7 log copies/ml group (eight patients); 2) the 1.7–2.5 log copies/ml group (five patients); and 3) the ⱖ2.6 log copies group (11 patients). Background characteristics of the patients were compared among these three groups (Table 1). The mean duration of administration was 88.5 ⫾ 34.8 wk (52–172 wk). Ten of the 24 patients were treated for ⬎2 yr. In seven patients, the therapy was discontinued because of phase III trials (four patients), seroconversion (one patient), and refusal to continue therapy (two patients). The remaining 17 patients are still being treated. The HBV DNA level before treatment was significantly lower in the ⬍1.7 copies/ml group than in the other two groups (p ⬍ 0.05). However, there were no differences in
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Ide et al.
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HBV DNA was 107 and 115 wk. The HBV DNA of the patients who did not develop YMDD mutants did not increase during therapy (Fig. 1B). In eight patients of the ⬍ 1.7 copies/ml group, the mean time at ⬍1.7 log copies/ml was 57 ⫾ 22 wk. Changes in HBe Antigen A total of 22 patients were HBe antigen positive before treatment. Of the 11 patients comprising the ⱖ2.6 copies/ml group during therapy, only two (9%) converted to HBe antigen negative status. Of the four patients in the 1.7–2.5 copies/ml group, three (75%) patients were converted to HBe antigen negative status. Of the seven patients in the ⬍1.7 copies/ml group, all patients became HBe antigen negative.
DISCUSSION
Figure 1. Kinetics of HBV DNA levels during lamivudine therapy. (A) Patients who developed YMDD mutants. (B) Patients who did not develop YMDD mutants. The units of HBV DNA level are LGE/ml for ⱖ3.7 and log copies/ml for ⬍3.7 LGE/ml, 2.6 log copies/ml and 1.7 log copies/ml were the detection limits of the TMA assay, the Roche Monitor kit, and real-time PCR, respectively.
the ALT levels, dose, treatment period, HBeAg status, or presence of cirrhosis. Emergence of YMDD Mutants During Therapy YMDD mutants were found in 13 of the 24 patients (54.1%). Of the 13 patients with YMDD mutant HBV, seven patients (53.8%) exhibited a substitution of isoleucine for methionine (YIDD), and four patients (30.8%) exhibited a substitution of valine for methionine (YVDD). The remaining two cases (15.4%) were of a mixed type (YIDD ⫹ YVDD). The emergence of YMDD mutants was compared among the three groups (Table 1). The rate of emergence was 100% (11 of 11) in the ⱖ2.6 group, 40% (two of five) in the 1.7–2.5 copies/ml group and 0% (none of eight) in the ⬍1.7 copies/ml group. The rate was significantly higher in the ⱖ2.6 copies/ml group and the 1.7–2.5 copies/ml group than in the ⬍1.7 copies/ml group (p ⬍ 0.001). Kinetics of HBV DNA Levels During Lamivudine Therapy The kinetics of HBV DNA levels during lamivudine therapy are shown in Figure 1. The HBV DNA of the patients who developed YMDD mutants did not decrease to ⬍1.7 log copies/ml (Fig. 1A). The mean time at virological breakthrough of HBV DNA in the ⱖ2.6 copies/ml group was 50 ⫾ 18 wk (Table 1). However, in two patients of the 1.7–2.5 copies/ml group, the time at virological breakthrough of
This study investigated the association between HBV DNA levels and the emergence of YMDD mutants during lamivudine therapy, by observing changes in the level of HBV DNA using a highly sensitive HBV DNA assay. Many studies have reported sensitive HBV DNA assay systems with detection limits of approximately 1.0 –3.0 log copies/ml (7–10, 12–15). The highest sensitivity ever reported was 1.0 log copies/ml (12), but this assay system was assessed in only one patient. Two studies reported a sensitivity of 2.3 log copies/ml (13, 14) but one of these studies examined changes in the DNA levels for 100 days in the early period of treatment, and the other study examined the levels of HBV DNA only after 6 months of lamivudine therapy. Several studies investigated the association between DNA levels and YMDD mutants (7–10), but the detection limits for HBV DNA were 2.6 –3.0 log copies/ml. Such a detection sensitivity would not have yielded results comparable to those obtained in this study. In the present study, the Roche Monitor kit was not used at all time points because TMA, which can measure higher levels of the virus, was more suitable for measurement when the virus levels were high, such as they were before treatment. The TMA assay also had a strong positive correlation with the Roche Monitor kit. Thus, we considered that it would not significantly affect the results of this study. Analysis of the association between HBV DNA levels and the emergence of YMDD mutants showed that an increase in the level of HBV DNA, because of YMDD mutations was observed after an average of 1 yr of treatment in all patients in the ⱖ2.6 copies/ml group. In two of five patients in the 1.7–2.5 copies/ml group, YMDD mutants were found after within about 2 yr (107 and 115 wk), which was about 1 yr later than in the patients in the ⱖ2.6 copies/ml group. No YMDD mutants were found in the remaining three patients, in whom lamivudine had been discontinued at wk 52, 96, and 100, and who could not be monitored further for YMDD mutants. Although four of the eight patients in the ⬍1.7 copies/ml group had been treated for
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⬎2 yr, no YMDD mutants were found and no virological breakthrough occurred. These results indicate that a greater decrease in HBV DNA levels after lamivudine administration is associated with a later appearance of YMDD mutants. Further follow-up is needed to determine whether YMDD mutants seem in the future in the ⬍1.7 copies/ml group. Also, it may be necessary to consider using other antiviral agents concomitantly in patients whose HVB DNA levels did not decrease to ⬍1.7 log copies/ml. In conclusion, we consider the real-time PCR method to be useful for predicting the emergence of YMDD mutants and the estimated time of their emergence.
Real-Time Quantitative PCR for Chronic Hepatitis B
4.
5. 6. 7.
8.
ACKNOWLEDGMENTS The authors thank Norihiko Takahashi and Makio Oka, Otsuka Assay Laboratories, Otsuka Life Science Initiative, Otsuka Pharmaceutical, Ltd., Tokushima, Japan, for their technical assistance. Reprint requests and correspondence: Tatsuya Ide, M.D., Second Department of Internal Medicine, Kurume University School of Medicine, 67, Asahi-machi, Kurume-shi, Fukuoka-ken 8300011, Japan. Received Aug. 16, 2002; accepted Jan. 28, 2003.
9.
10. 11.
12.
REFERENCES 1. Lai CL, Chien RN, Leung NW, et al. A one-year trial of lamivudine for chronic hepatitis B. N Engl J Med 1998;339: 61–8. 2. Dienstag J, Schiff E, Wright T, et al. Lamivudine treatment for one year in previously untreated US hepatitis B patients: Histological improvement and hepatitis B e-antigen (HBe AG) seroconversion. Gastroenterology 1998;114:A1235. 3. Schiff E, Karayalcin S, Grimm I, et al. A placebo controlled study of lamivudine and interferon alpha-2b in patients with
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chronic hepatitis B who previously failed interferon therapy. Hepatology 1998;28:388A. Ide T, Kumashiro R, Hino T, et al. Transcription-mediated amplification is more useful in the follow-up of patients with chronic hepatitis B treated with lamivudine. Hepatol Res 2001;21:76 –84. Ling R, Mutimer D, Ahmed M, et al. Selection of mutations in the hepatitis B virus polymerase during therapy of transplant recipients with lamivudine. Hepatology 1996;24:711–3. Tipples GA, Ma MM, Fischer KP, et al. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996;24:714 –7. Gauthier J, Bourne EJ, Lutz MW, et al. Quantification of hepatitis B viremia and emergence of YMDD variants in patients with chronic hepatitis B treated with lamivudine. J Infect Dis 1999;180:1757–62. Puchhammer-Sto¨ ckl E, Mandl CW, Kletzmayr J, et al. Monitoring the virus load can predict the emergence of drugresistant hepatitis B virus strains in renal transplantation patients during lamivudine therapy. J Infect Dis 2000;181: 2063–6. Buti M, Sa´ nchez F, Cotrina M, et al. Quantitative hepatitis B virus DNA testing for the early prediction of the maintenance of response during lamivudine therapy in patients with chronic hepatitis B. J Infect Dis 2001;183:1277–80. Jardi R, Rodrigues F, Buti M, et al. Quantitative detection of hepatitis B virus DNA in serum by a new rapid real-time fluorescence PCR assay. J viral Hepat 2001;8:465–71. Kobayashi S, Shimada K, Suzuki H, et al. Development of a new method for detecting a mutation in the gene encoding hepatitis B virus reverse transcriptase active site (YMDD motif). Hepatol Res 2000;17:31–42. Abe A, Inoue K, Tanaka T, et al. Quantitation of hepatitis B virus genomic DNA by real-time detection PCR. J Clin Microbiol 1999;37:2899 –903. Brechtbuehl K, Whalley SA, Dusheiko GM, et al. A rapid real-time quantitative polymerase chain reaction for hepatitis B virus. J Virol Methods 2001;93:105–13. Lewin SR, Ribeiro RM, Walters T, et al. Analysis of hepatitis B load decline under potent therapy: Complex decay profiles observed. Hepatology 2001;34:1012–20. Yuen MF, Sablon E, Hui CK, et al. Factors associated with hepatitis B virus DNA breakthrough in patients receiving prolonged lamivudine therapy. Hepatology 2001;34:785–91.
Vol. 98, No. 9, 2003 ISSN 0002-9270/03/$30.00 doi:10.1016/S0002-9270(03)00554-9
A Real-Time Quantitative Polymerase Chain Reaction Method for Hepatitis B Virus in Patients With Chronic Hepatitis B Treated With Lamivudine Tatsuya Ide, M.D., Ryukichi Kumashiro, M.D., Yuriko Koga, M.D., Eisuke Tanaka, M.D., Teruko Hino, M.D., Akiko Hisamochi, M.D., Shiro Murashima, M.D., Kei Ogata, M.D., Kazuo Tanaka, M.D., Reiichiro Kuwahara, M.D., and Michio Sata, M.D. Second Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan
OBJECTIVES: During treatment of chronic hepatitis B with lamivudine, changes in the level of hepatitis B virus (HBV) DNA were investigated using a real-time polymerase chain reaction (PCR) method with a detection limit of 1.7 log copies/ml (50 copies/ml) to clarify its clinical significance, particularly the association between HBV DNA levels and the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) mutants. METHODS: Twenty-four patients who had received lamivudine therapy for ⬎1 yr were studied. HBV DNA levels were determined using transcription-mediated amplification for sera with ⬎3.7 log genome equivalents/ml, the Roche Monitor kit for sera with ⱖ2.6 log copies/ml, and real-time PCR for sera with ⬍ 2.6 log copies/ml (the detection limit was 1.7 log copies/ml). Patients were classified into three groups according to the minimal HBV DNA level attained during lamivudine therapy: the ⬍1.7 log copies/ml group (eight patients), the 1.7–2.5 log copies/ml group (five patients), and the ⱖ2.6 log copies/ml group (11 patients). RESULTS: Pretreatment HBV DNA levels were significantly lower in the ⬍1.7 copies/ml group than in the other two groups (p ⬍ 0.05). Neither the emergence of YMDD mutants nor a virological breakthrough of serum HBV DNA was observed in any of the eight patients in the ⬍1.7 copies/ml group. In contrast, in the 1.7–2.5 copies/ml and ⱖ2.6 copies/ml groups, virological breakthroughs resulting from the emergence of YMDD mutants were observed in two of five patients and in all 11 patients, respectively (p ⬍ 0.001). Virological breakthroughs were observed at a mean of 49.6 ⫾ 18.4 wk in 11 the patients in the ⱖ2.6 copies/ml group and at wk 107 and 115 in two patients in the 1.7–2.5 copies/ml group. CONCLUSIONS: The real-time PCR method is useful for predicting the emergence of YMDD mutants and the estimated time of their emergence. (Am J Gastroenterol 2003;98: 2048 –2051. © 2003 by Am. Coll. of Gastroenterology)
INTRODUCTION Lamivudine has been widely used in the treatment of chronic hepatitis B. When it is administered to hepatitis B
patients, a decrease or disappearance in serum hepatitis B virus (HBV) DNA and hepatitis Be (Hbe) antigen is observed and is associated with a decrease in ALT. In previous studies (1–3), the HBV DNA levels during treatment with lamivudine were measured mainly using the solution hybridization assay (Abbott Laboratories, North Chicago, IL) and branched DNA signal amplification technology (b-DNA assay). In these methods, serum HBV DNA fell to undetectable levels at least once during therapy in ⬎89% of the patients receiving lamivudine (1–3). Recently, we reported that a transcription-mediated amplification (TMA) assay, which had approximately 100-fold higher sensitivity (103.7 genome equivalents/ml) than other methods, is more useful for clarifying the changes in the HBV DNA levels (4). In addition, to detect much lower levels of HBV DNA, a quantitative real-time polymer chain reaction (PCR) methods has been developed. This new method combines the Roche Monitor kit and real-time PCR. The minimum detection limits of the Roche Monitor kit were 102.6copies/ml and those of the new real-time PCR were 101.7 copies/ml. After extended lamivudine therapy, tyrosine-methionineaspartate-aspartate (YMDD) mutation has been identified in some patients (5, 6). Some studies have suggested that monitoring the virus load can predict the later emergence of YMDD mutants (7–10). However, there has been no report investigating the association between the virus load and the emergence of YMDD mutants using a method that can detect ⬍102 copies/ml. In addition, the observation periods of patients in the above mentioned were ⬍2 yr (7–9) and the numbers of patients were small (10). The aim of this study was to determine whether this real-time PCR method is useful for predicting the emergence of YMDD mutants.
MATERIALS AND METHODS We studied 24 patients with HBV related-chronic liver diseases, who had received lamivudine therapy for ⬎12 months between March, 1995, and January, 2001 (19 men and five women, mean age 48.8 ⫾ 11.1 yr). Of the 24 patients, 14 had chronic hepatitis, nine cirrhosis, and one
AJG – September, 2003
hepatocellular carcinoma complicating cirrhosis. The daily dosage of lamivudine ranged from 75 to 300 mg, and dosages varied among the patients; therefore mean dosages were calculated for each patient. A liver function test was performed once per month. HBV DNA, hepatitis Be antigen (HBeAg), and anti-HBe antibody were measured every 1–3 months. The HBV DNA level was measured using the TMA assay (Chugai Diagnostic Science, Tokyo, Japan). The measurement range of the TMA assay was 103.7–108.7 genome equivalents/ml (3.7– 8.7 log genome equivalents [LGE]/ml). At the point at which the levels of HBV DNA were ⬍3.7 LGE, they were measured using a Roche Monitor kit (Roche Diagnostics, Tokyo, Japan) using serum stored at –20°C. The detection limit of the Monitor kit was 102.6 copies/ml (2.6 log copies/ml) and the measurement range of the kit was 102.6–107.6 copies/ml (2.6 –7.6 log copies/ml). There was a good correlation between the TMA assay and the Monitor kit (r ⫽ 0.969, y ⫽ 0.9203x ⫹ 0.5049; unpublished data). In addition, in serum with HBV DNA levels lower than 2.6 log copies/ml, the levels were measured using our new real-time PCR assay. The real-time PCR was performed as follows: PCR products obtained by the Roche Monitor kit were purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Tokyo, Japan). The oligonucleotide sequences of the primers were: HBVnested-F(5'-CTTTDGGRCATGGACATTGA-3'; D ⫽ A, G, or T; R ⫽ A or G), and HBV-nested-R (5'-ACTCCACAGWAGCTCCAAATTCTTTA-3'; W ⫽ A or T). The PCR reaction was performed with FastStart Taq DNA polymerase (Roche Diagnostics) and SYBR Green I (BioWhittaker Molecular Applications, Rockland, ME) using an ABI PRISM 7700 Sequence Detection System. The PCR cycle consisted of an initial denaturing step at 95°C for 12 min, followed by 30 amplification cycles at 95°C for 15 s, 50°C for 30 s, and 65°C for 30 s. To quantify the loads of HBV DNA, we used external standards containing 0, 1.25, 2.5, 10, 102, 103, 104, and 106 copies. Comparison of the cycle threshold value to a calibration curve allows the absolute quantification of unknown viral loads of DNA. The detection limit of this real-time PCR assay was 1.7 log copies/ml and the measurement range of this assay was 101.7–102.5 copies/ml (1.7–2.5 log copies/ml). The coefficient of variation in the inner assay (within-run reproducibility; n ⫽ 3) was 1.4% at a mean of 2.5 log copies/ml. To establish the linearity of HBV DNA quantification of real-time PCR, three positive samples estimated to contain 2.5 log copies/ml were diluted in HBV negative human serum to 2.2, 1.9, 1.6 log copies/ml (expected values). The results were 2.5 ⫾ 0.03, 2.2 ⫾ 0.17, 1.9 ⫾ 0.12, and 1.7 ⫾ 0.30 log copies/ml, respectively. A linear relationship was observed between the expected values and the results (r ⫽ 0.996). The present findings were shown in LGE (log genome equivalents)/ml in the TMA assay and log copies/ml in the Roche Monitor assay and the real-time PCR assay. A virological breakthrough of serum HBV DNA was defined by a ⬎10-
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Table 1. Patients’ Background Before Therapy and Emergence of YMDD Mutants During Therapy According to the Minimal Levels of HBV DNA Minimal Level of DNA (log copies/ml) ⱖ2.6
2.5–1.7
⬍1.7
p Value*
No. of patients 11 5 8 HBV DNA (LGE/ 8.3 ⫾ 0.6 7.4 ⫾ 1.2 7.3 ⫾ 0.8 ⬍0.05† ml) ALT (IU/L) 170 ⫾ 118 130 ⫾ 100 196 ⫾ 101 0.29† Dose (mg/day) 146 ⫾ 56 124 ⫾ 22 133 ⫾ 38 0.46† Treatment period 90 ⫾ 40 104 ⫾ 39 89 ⫾ 35 0.83† (wk) HBeAg ⫹/⫺ 10/1 4/1 7/1 0.7‡ Cirrhosis ⫹/⫺ 5/6 1/4 4/4 0.44‡ YMDD mutant 11 (100%) 2 (40%) 0 (0%) ⬍0.001‡ Time of reincreased 49.6 ⫾ 18.4 107, 115 HBV DNA (wk) No. of patients n ⫽ 11 n⫽2 n⫽0 * ⱖ2.6 and 2.5–1.7 vs ⬍ 1.7. † Mann-Whitney U test. ‡ Fisher’s exact probability test.
fold increase from the minimal level of DNA. HBeAg, and anti-HBe antibody were detected by enzyme immunoassay (AxSYM, HBe, anti-HBe; Dainabot, Tokyo, Japan). HBeAg assay findings were expressed as ⫹(S/N ⬎5.0), ⫾ (2.1 ⬍S/N ⱕ5.0), and ⫺(S/N ⬍2.1). Anti-HBe antibody assay findings were expressed as ⫹ (% inhibition ⬎70%), ⫾ (50 ⬍% inhibition ⱕ 70), and ⫺ (% inhibition ⱕ50%). The YMDD mutant strain was measured 6 –12 months after the initiation of treatment using SMITEST HBV-YMDD motif ELMA (Genome Science Laboratories, Fukushima, Japan) (11). Values are reported as the mean ⫾ SEM and were analyzed by the Mann-Whitney U test and the Fisher’s exact probability test. A p value ⬍ 0.05 was considered to be significant
RESULTS Background Characteristics of the Patients The patients were classified into three groups according to the minimal HBV DNA level attained during lamivudine therapy: 1) the ⬍1.7 log copies/ml group (eight patients); 2) the 1.7–2.5 log copies/ml group (five patients); and 3) the ⱖ2.6 log copies group (11 patients). Background characteristics of the patients were compared among these three groups (Table 1). The mean duration of administration was 88.5 ⫾ 34.8 wk (52–172 wk). Ten of the 24 patients were treated for ⬎2 yr. In seven patients, the therapy was discontinued because of phase III trials (four patients), seroconversion (one patient), and refusal to continue therapy (two patients). The remaining 17 patients are still being treated. The HBV DNA level before treatment was significantly lower in the ⬍1.7 copies/ml group than in the other two groups (p ⬍ 0.05). However, there were no differences in
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HBV DNA was 107 and 115 wk. The HBV DNA of the patients who did not develop YMDD mutants did not increase during therapy (Fig. 1B). In eight patients of the ⬍ 1.7 copies/ml group, the mean time at ⬍1.7 log copies/ml was 57 ⫾ 22 wk. Changes in HBe Antigen A total of 22 patients were HBe antigen positive before treatment. Of the 11 patients comprising the ⱖ2.6 copies/ml group during therapy, only two (9%) converted to HBe antigen negative status. Of the four patients in the 1.7–2.5 copies/ml group, three (75%) patients were converted to HBe antigen negative status. Of the seven patients in the ⬍1.7 copies/ml group, all patients became HBe antigen negative.
DISCUSSION
Figure 1. Kinetics of HBV DNA levels during lamivudine therapy. (A) Patients who developed YMDD mutants. (B) Patients who did not develop YMDD mutants. The units of HBV DNA level are LGE/ml for ⱖ3.7 and log copies/ml for ⬍3.7 LGE/ml, 2.6 log copies/ml and 1.7 log copies/ml were the detection limits of the TMA assay, the Roche Monitor kit, and real-time PCR, respectively.
the ALT levels, dose, treatment period, HBeAg status, or presence of cirrhosis. Emergence of YMDD Mutants During Therapy YMDD mutants were found in 13 of the 24 patients (54.1%). Of the 13 patients with YMDD mutant HBV, seven patients (53.8%) exhibited a substitution of isoleucine for methionine (YIDD), and four patients (30.8%) exhibited a substitution of valine for methionine (YVDD). The remaining two cases (15.4%) were of a mixed type (YIDD ⫹ YVDD). The emergence of YMDD mutants was compared among the three groups (Table 1). The rate of emergence was 100% (11 of 11) in the ⱖ2.6 group, 40% (two of five) in the 1.7–2.5 copies/ml group and 0% (none of eight) in the ⬍1.7 copies/ml group. The rate was significantly higher in the ⱖ2.6 copies/ml group and the 1.7–2.5 copies/ml group than in the ⬍1.7 copies/ml group (p ⬍ 0.001). Kinetics of HBV DNA Levels During Lamivudine Therapy The kinetics of HBV DNA levels during lamivudine therapy are shown in Figure 1. The HBV DNA of the patients who developed YMDD mutants did not decrease to ⬍1.7 log copies/ml (Fig. 1A). The mean time at virological breakthrough of HBV DNA in the ⱖ2.6 copies/ml group was 50 ⫾ 18 wk (Table 1). However, in two patients of the 1.7–2.5 copies/ml group, the time at virological breakthrough of
This study investigated the association between HBV DNA levels and the emergence of YMDD mutants during lamivudine therapy, by observing changes in the level of HBV DNA using a highly sensitive HBV DNA assay. Many studies have reported sensitive HBV DNA assay systems with detection limits of approximately 1.0 –3.0 log copies/ml (7–10, 12–15). The highest sensitivity ever reported was 1.0 log copies/ml (12), but this assay system was assessed in only one patient. Two studies reported a sensitivity of 2.3 log copies/ml (13, 14) but one of these studies examined changes in the DNA levels for 100 days in the early period of treatment, and the other study examined the levels of HBV DNA only after 6 months of lamivudine therapy. Several studies investigated the association between DNA levels and YMDD mutants (7–10), but the detection limits for HBV DNA were 2.6 –3.0 log copies/ml. Such a detection sensitivity would not have yielded results comparable to those obtained in this study. In the present study, the Roche Monitor kit was not used at all time points because TMA, which can measure higher levels of the virus, was more suitable for measurement when the virus levels were high, such as they were before treatment. The TMA assay also had a strong positive correlation with the Roche Monitor kit. Thus, we considered that it would not significantly affect the results of this study. Analysis of the association between HBV DNA levels and the emergence of YMDD mutants showed that an increase in the level of HBV DNA, because of YMDD mutations was observed after an average of 1 yr of treatment in all patients in the ⱖ2.6 copies/ml group. In two of five patients in the 1.7–2.5 copies/ml group, YMDD mutants were found after within about 2 yr (107 and 115 wk), which was about 1 yr later than in the patients in the ⱖ2.6 copies/ml group. No YMDD mutants were found in the remaining three patients, in whom lamivudine had been discontinued at wk 52, 96, and 100, and who could not be monitored further for YMDD mutants. Although four of the eight patients in the ⬍1.7 copies/ml group had been treated for
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⬎2 yr, no YMDD mutants were found and no virological breakthrough occurred. These results indicate that a greater decrease in HBV DNA levels after lamivudine administration is associated with a later appearance of YMDD mutants. Further follow-up is needed to determine whether YMDD mutants seem in the future in the ⬍1.7 copies/ml group. Also, it may be necessary to consider using other antiviral agents concomitantly in patients whose HVB DNA levels did not decrease to ⬍1.7 log copies/ml. In conclusion, we consider the real-time PCR method to be useful for predicting the emergence of YMDD mutants and the estimated time of their emergence.
Real-Time Quantitative PCR for Chronic Hepatitis B
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ACKNOWLEDGMENTS The authors thank Norihiko Takahashi and Makio Oka, Otsuka Assay Laboratories, Otsuka Life Science Initiative, Otsuka Pharmaceutical, Ltd., Tokushima, Japan, for their technical assistance. Reprint requests and correspondence: Tatsuya Ide, M.D., Second Department of Internal Medicine, Kurume University School of Medicine, 67, Asahi-machi, Kurume-shi, Fukuoka-ken 8300011, Japan. Received Aug. 16, 2002; accepted Jan. 28, 2003.
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