- Email: [email protected]
Level of Hepatitis B Virus DNA in Inactive Carriers With Persistently Normal Levels of Alanine Aminotransferase
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2010;8:535–540
Level of Hepatitis B Virus DNA in Inactive Carriers With Persistently Normal Levels of Alanine Aminotransferase CHIA–MING CHU, YI–CHENG CHEN, DAR–IN TAI, and YUN–FAN LIAW Liver Research Unit, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan
BACKGROUND & AIMS: Little is known about the level of hepatitis B virus (HBV) DNA in individuals with chronic, inactive HBV infections. Patients who test positive for the antibody to hepatitis B e antigen (anti-HBe) and have normal levels of alanine aminotransferase for more than 10 years have a low risk of HBV reactivation and are considered to be inactive carriers. We investigated HBV DNA levels in inactive carriers and identified factors that correlated with this state among anti-HBe–positive carriers with HBV DNA levels of 104 copies/mL or greater (5.26 copies/mL ⫽ 1 IU/mL). METHODS: HBV DNA levels were assayed in 250 inactive carriers with persistently normal alanine aminotransferase levels for more than 10 years. Clinical and virologic features were compared between inactive carriers (with HBV DNA levels ⱖ104 copies/ mL) and age-matched patients with HBe antigen–negative chronic hepatitis (controls, n ⫽ 90). RESULTS: The median level of HBV DNA among inactive carriers was 3.70 log10 copies/mL (range, undetectable to 5.98 log10 copies/mL). Ninety (36%) had levels of 104 copies/mL or greater. Compared with control patients, significant differences of inactive carriers included sex (more female patients), lower HBV DNA levels, and lower prevalence of genotype C virus and the basal core promoter mutation T1762/A1764. The prevalence of the precore mutation A1896 was similar between groups. Multiple logistic regression analyses identified male sex, HBV DNA levels greater than 105 copies/mL, and the basal core promoter mutation as independent factors that correlated with active disease. CONCLUSIONS: Nearly 40% of inactive carriers had HBV DNA levels of 104 copies/mL or greater. Female sex, HBV DNA levels of 104 to 105 copies/mL, and wild-type basal core promoter correlated with inactive carrier state. Keywords: Chronic Hepatitis B; Sex; Viral Genotype; Viral Load; Viral Mutants.
T
he natural course of chronic hepatitis B virus (HBV) infection constitutes 3 chronologic phases: an initial immune tolerance phase during which the patients are positive for hepatitis B e antigen (HBeAg) and have normal alanine aminotransferase (ALT) levels, followed by an immune clearance phase during which the HBeAg–positive patients have increased ALT levels, and, finally, the inactive carrier state during which HBeAg seroconverts to its antibody (anti-HBe) and ALT levels normalize.1 The third phase may remain stably inactive in a lifetime. On the other hand, HBV also can reactivate either by reversion back to the previous HBeAg–positive phase or, much more frequently, by progression to HBeAg–negative chronic hepatitis.2,3
Among these phases, the inactive carrier state may be a retrospective–prospective diagnosis because inactive carriers show some propensity to reactivation. However, there is no single value of HBV DNA above which future reactivation is likely to occur and below which the disease is likely to be quiescent.4 A HBV DNA level threshold of 104 copies/mL has been used to discriminate active HBV infection from its inactive form.1,5–9 However, several studies have shown that between 7% and 55% of so-called inactive carriers with persistently normal ALT levels for variable duration had HBV DNA levels greater than 104 copies/mL,10 –13 and even in 2 studies from India and Taiwan, 35% and 32%, respectively, had levels greater than 105 copies/mL.12,13 According to the long-term observations of 1965 anti-HBe–positive hepatitis B surface antigen (HBsAg) carriers with normal ALT levels from Taiwan, reactivation of hepatitis B most often occurred during the first 5 to 10 years of follow-up evaluation and became extremely rare thereafter.14 However, in most previous studies the so-called inactive carriers had persistently normal ALT levels for a relatively short period of only 1 to 2 years; therefore, future reactivation can be anticipated in some of these carriers.15 Clearly, viral load status of the inactive carrier state needs to be defined further. Because reactivation of hepatitis B in anti-HBe–positive carriers became extremely rare 10 years after entry14 and virtually none of the anti-HBe–positive carriers with persistently normal ALT levels over 10 years died of liver disease,16 anti-HBe–positive carriers with persistently normal ALT levels over 10 years can be considered as real inactive carriers. Serum HBV DNA levels in such carriers may represent the viral load status of inactive carrier state. In this investigation, we first studied HBV DNA levels in 250 anti-HBe–positive HBsAg carriers with persistently normal ALT levels for more than 10 years. Our results showed that 90 (36%) of them had HBV DNA levels of 104 copies/mL or more, a level being used to indicate active hepatitis. To further identify factors that correlate with inactive carrier state among anti-HBe–positive carriers with HBV DNA levels of 104 copies/mL or more, we next compared the clinical and virologic features between inactive carriers with HBV DNA levels of 104 copies/mL or more and age-matched patients with HBeAg–negative chronic hepatitis.
Abbreviations used in this paper: ALT, alanine aminotransferase; anti-HBe, antibody against hepatitis B e antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus. © 2010 by the AGA Institute 1542-3565/$36.00 doi:10.1016/j.cgh.2010.03.006
536
CHU ET AL
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 6
Materials and Methods Inactive Carriers and Patients With Hepatitis B e Antigen–Negative Chronic Hepatitis Asymptomatic adults who were identified incidentally as HBsAg carriers during blood donation or health check-ups received regular clinic follow-up evaluation at the carrier clinic of Chang Gung Memorial Hospital in Taipei, Taiwan.14,17 The subjects were considered inactive carriers if they fulfilled the following criteria: (1) positive HBsAg, negative HBeAg, positive anti-HBe, and persistently normal ALT levels (ⱕ36 U/L) at least once every 6 to 12 months for at least 10 years until the last visit; (2) no evidence of cirrhosis or hepatocellular carcinoma based on clinical assessment and liver ultrasonographic findings18; (3) no concomitant infection with hepatitis C virus or hepatitis D virus; and (4) no antiviral or immunomodulatory therapy before enrollment. Patients who underwent HBsAg seroclearance during the follow-up period17 also were excluded. Patients were diagnosed as having HBeAg–negative chronic hepatitis if they were HBsAg positive, HBeAg negative, and anti-HBe positive, had persistently abnormal ALT levels more than twice the upper limit of normal for at least 2 years, and HBV DNA levels were 104 copies/mL or more. Patients who had concomitant infection with hepatitis C virus or hepatitis D virus, autoimmune or metabolic liver disease, who consumed alcohol or drugs that might be potential etiologic agents of hepatitis, or who had ever received antiviral or immunomodulatory therapy were excluded.
Methods HBsAg, HBeAg, anti-HBe, and antibody against hepatitis D virus were assayed using radioimmunoassay kits (Abbott Diagnostics, North Chicago, IL). Antibodies against hepatitis C virus were assayed using a second- or third-generation enzyme immunoassay (Abbott Diagnostics). Serum HBV DNA levels were assayed using the COBAS Amplicor HBV Monitor Test (Roche Diagnostics, Branchburg, NJ; lower limit of detection, 200 copies/mL). The conversion in IU/mL (1 IU is equivalent to 5.26 HBV DNA copies) was made according to the manufacturer’s instructions. HBV genotypes were determined using the polymerase chain reaction–restriction fragment length polymorphism of the surface gene of HBV, as previously described.19 Precore A1896 mutant was detected by amplification-created restriction site method, as described before.20 Basal core promoter genes were amplified by polymerase chain reaction, and nucleotide sequences of the amplified products were deter-
mined directly by using an automatic sequencer, as described by others.13
Statistical Analyses Data are presented as mean ⫾ standard deviation, median (range), or number (%). To compare characteristics between groups, either the chi-square test or the Fisher exact test was used to analyze categoric variables and the Student t test or the Mann–Whitney U nonparametric test was used to analyze continuous variables. HBV DNA levels were correlated with ALT levels or age of patients by using Spearman rank correlation. Univariate and multivariate logistic regression analyses were performed to identify the factors that correlated with active disease among anti-HBe–positive HBsAg carriers with HBV DNA levels of 104 copies/mL or more. Variables with P values less than .1 in the univariate models were tested in a multivariate setting. Significant associations identified in multivariate analysis are presented as odds ratio (95% confidence interval). Statistical procedures were performed using the SPSS statistical software (version 13.0; SPSS, Chicago, IL). P values less than .05 were considered significant.
Results Clinical and Demographic Features of Inactive Carriers From January 2007 to December 2007, there were 250 consecutive anti-HBe–positive HBsAg carriers who had had persistently normal ALT levels for at least 10 years who received regular follow-up evaluation at our hepatitis carrier clinic. They were considered inactive carriers and were enrolled in this study. The demographic and clinical data are summarized in Table 1. There were 84 men and 166 women. The mean age at baseline was 34.4 years (range, 18 – 64 y) and the mean age at enrollment was 50.6 years (range, 31– 81 y). The mean number of ALT measurements for each carrier was 28.4 (range, 12– 48) during a mean period of 16.1 years (range, 10 –25 y) before enrollment. The mean maximal ALT level before enrollment was 26.3 U/L (range, 9 –36 U/L). Maximal ALT levels were 19 U/L or less in 26 patients (10.4%), 20 to 30 U/L in 159 patients (63.6%), and 31 to 36 U/L in 65 patients (26%). ALT levels were significantly higher in male carriers than female carriers. A total of 52 male carriers and 23 female carriers had persistently normal ALT levels according to the strict criteria of ALT of 30 U/L or less in males and 19 U/L or less in females, as suggested by Prati et al.21
Table 1. Clinical and Demographic Data of Inactive Carriers Data
Total (n ⫽ 250)
Male (n ⫽ 84)
Female (n ⫽ 166)
Age at baseline, y Male:female ratio Duration of persistently normal ALT levels before enrollment, y Number of ALT determinations before enrollment Maximal ALT levels before enrollment, U/L ⱕ19 U/L 20–30 U/L 30–36 U/L Age at enrollment, y
34.4 ⫾ 8.8 84:166 16.1 ⫾ 4.7 28.4 ⫾ 8.9 26.3 ⫾ 5.6 26 (10) 159 (64) 65 (26) 50.6 ⫾ 9.6
35.6 ⫾ 9.3
33.9 ⫾ 8.6
.16
16.6 ⫾ 4.9 29.8 ⫾ 9.6 28.1 ⫾ 5.0 3 (4) 49 (58) 32 (38) 52.2 ⫾ 10.1
15.9 ⫾ 4.5 27.6 ⫾ 8.4 25.3 ⫾ 5.7 23 (14) 110 (66) 33 (20) 49.8 ⫾ 8.4
.27 .093 .0002 .012 .22 .0019 .069
NOTE. Data are given as mean ⫾ SD or number (%).
P
June 2010
HBV DNA LEVELS IN INACTIVE CARRIERS
537
Table 2. HBV DNA Levels in Inactive Carriers With Persistently Normal ALT Levels (ⱕ36 U/L) HBV DNA levels, log10 copies/mLa ⬍2.3 (undetectable) 2.3–2.99 3–3.99 4–4.99 5–5.99 Median (range)
Total (n ⫽ 250)
Males (n ⫽ 84)
Females (n ⫽ 166)
P
43 (17) 28 (11) 89 (36) 65 (26) 25 (10) 3.70 (⬍2.3 to 5.98)
9 (11) 12 (14) 30 (36) 25 (30) 8 (10) 3.73 (⬍2.3 to 5.79)
34 (20) 16 (10) 59 (36) 40 (24) 17 (10) 3.69 (⬍2.3 to 5.98)
.053 .16 .98 .33 .86 .40
NOTE. Data are given as number (%). a5.26 copies/mL ⫽ 1 IU/mL.
Hepatitis B Virus DNA Levels of Inactive Carriers As shown in Table 2, the median HBV DNA level in 250 inactive carriers was 3.70 (range, undetectable [⬍2.3] to 5.98) log10 copies/mL. Ninety carriers (36%) had levels of 104 copies/mL or more. HBV DNA levels showed no significant difference between males and females. There was also no significant correlation between HBV DNA levels and maximal ALT levels (P ⫽ .81) or age of patients at enrollment (P ⫽ .41). Among a subset of 75 inactive carriers with persistent ALT levels of 30 U/L or less for males and 19 U/L or less for females, the median HBV DNA level was 3.81 (range, undetectable [⬍2.3] to 5.45) with levels of 104 copies/mL or more in 32 (43%) of them (Table 3). Both figures were not different from those observed in the whole cohort of 250 inactive carriers (P ⫽ .25 and .29, respectively).
Comparison of Clinical and Virologic Features Between Inactive Carriers With Hepatitis B Virus DNA Levels of 104 Copies/ Milliliter or More and Patients With Hepatitis B e Antigen–Negative Chronic Hepatitis Clinical and virologic features of 90 inactive carriers with HBV DNA levels of 104 copies/mL or more were compared with 90 age-matched patients with HBeAg–negative chronic hepatitis who were selected randomly during the study period. As shown in Table 4, inactive carriers with HBV DNA levels of 104 copies/mL or more were significantly female predominant (P ⬍ .0001) and had significantly lower HBV DNA levels (P ⬍ .0001) when compared with the age-matched patients with HBeAg–negative chronic hepatitis. In addition, inactive carriers had a significantly lower prevalence of genotype C HBV (P ⫽ .0015) and the basal core promoter T1762/A1764 mutant (P ⬍
.0001) than did patients with HBeAg–negative chronic hepatitis, whereas the frequency of the precore A1896 mutant showed no significant difference between them (P ⫽ .84).
Predictive Factors for Active Infection in Anti-Hepatitis B e–Positive Carriers With Hepatitis B Virus DNA Levels Greater Than 104 Copies/Milliliter By using multiple logistic regression analysis, 3 independent factors were identified to be correlated significantly with active infection: male sex, serum HBV DNA levels greater than 105 copies/mL, and the presence of the basal core promoter T1762/A1764 mutant, whereas genotype C HBV was not found to be a predictor of active hepatitis (Table 5). The distribution of the 3 risk factors in inactive carriers with HBV DNA levels of 104 copies/mL or more and in patients with HBeAg–negative chronic hepatitis is shown in Table 6. Of the 90 inactive carriers, 73 (81%) had none or only one risk factor. On the contrary, 76 (84%) of 90 patients with HBeAg–negative chronic hepatitis had 2 or 3 risk factors. The prevalence of the number of the risk factors in inactive carriers with HBV DNA levels of 104 copies/mL or more and in patients with HBeAg– negative chronic hepatitis is shown in Figure 1. The odds ratios of active hepatitis increased with the presence of increasing number of the risk factors.
Discussion The male:female ratio in inactive carriers in this cohort was approximately 1:2 (Table 1). This ratio contrasts remarkably with the male predominance in patients with chronic hepatitis B, but is consistent with the observation that male carriers were significantly more likely to have reactivation of hepatitis B than female carriers.14,22
Table 3. HBV DNA Levels in Inactive Carriers With Persistently Normal ALT Levels (ⱕ30 U/L for Males and ⱕ19 U/L for Females) HBV DNA levels, log10 copies/mLa ⬍2.3 (undetectable) 2.3–2.99 3–3.99 4–4.99 5–5.99 Median (range) NOTE. Data are given as number (%). copies/mL ⫽ 1 IU/mL.
a5.26
Total (n ⫽ 75)
Males (n ⫽ 52)
Females (n ⫽ 23)
P
9 (12) 8 (7) 26 (35) 24 (32) 8 (11) 3.81 (⬍2.3 to 5.45)
6 (12) 6 (12) 15 (29) 20 (38) 5 (10) 3.95 (⬍2.3 to 5.45)
3 (13) 2 (9) 11 (48) 4 (17) 3 (13) 3.76 (⬍2.3 to 5.45)
.85 .71 .11 .071 .66 .72
538
CHU ET AL
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 6
Table 4. Comparison of Clinical and Virologic Features Between Inactive Carriers With HBV DNA Levels of 104 Copies/mL or More and Patients With HBeAg-Negative Chronic Hepatitis Data Age, y Male:female ratio HBV DNA levels, log10 copies/mLa 4–4.99 5–5.99 6–6.99 ⱖ7 Genotype B C Precore A1896 mutant Basal core promoter T1762/A1764 mutant
Inactive carriers (n ⫽ 90)
Chronic hepatitis (n ⫽ 90)
P
50.7 ⫾ 8.9 30:60 4.72 ⫾ 0.56 4.63 (4.01–5.98) 65 (72) 25 (28) 0 (0) 0 (0)
51.1 ⫾ 7.8 72:18 6.24 ⫾ 1.12 6.24 (4.09–9.18) 10 (11) 27 (30) 33 (37) 20 (22)
.75 ⬍.0001 ⬍.0001
80 (89) 10 (11) 75 (83) 13 (14)
67 (74) 23 (26) 74 (82) 39 (43)
.015 .84 ⬍.0001
NOTE. Data are given as mean ⫾ SD, median (range), or number (%). a5.26 copies/mL ⫽ 1 IU/mL.
This investigation showed that there was a wide range of HBV DNA levels in inactive carriers, ranging from less than 103 copies/mL in 28% to more than 105 copies/mL in 10% of them (Table 2). It should be noted that none of the inactive carriers in this cohort had HBV DNA levels more than 106 copies/mL. The median HBV DNA level in inactive carriers of our cohort was 3.70 (range, undetectable [⬍2.3] to 5.98) log10 copies/mL. This figure is comparable with the mean of 3.10 ⫾ 0.17 log10 copies/mL reported in a cohort of 29 inactive carriers in Hong Kong with persistently normal ALT levels for 41 to 92 months10 and the median of 3.52 (range, ⬍2.60 to 4.46) log10 copies/mL reported in a cohort of 62 inactive carriers in Greece with persistently normal ALT levels for at least 2 years.11 However, 36% of inactive carriers in our cohort had HBV DNA levels of 104 copies/mL or more, including 10% with levels of 105 to 106 copies/mL (Table 2), whereas only 7% of the Hong Kong cohort had levels greater than 104 copies/mL10 and none of the inactive carriers in the Hong Kong and Greece cohorts had levels greater Table 5. Factors Correlated With Active Hepatitis Among Anti-HBe–Positive Carriers With HBV DNA Levels of 104 Copies/mL or More: Multiple Logistic Regression Analysis Factors
Odds ratio (95% confidence interval)
P
than 105 copies/mL.10,11 It should be pointed out that our study enrolled a relatively large number of inactive carriers with a long duration of persistently normal ALT levels and the results more accurately should represent the viral load status of the inactive carrier state. Even if we adapted a strict criterion of normal ALT levels (ⱕ30 U/L in males and ⱕ19 U/L in females) as suggested by Prati et al,21 a similar proportion of inactive carriers still had HBV DNA levels of 104 copies/mL or more (Table 3). On the other hand, HBV DNA levels in inactive carriers of the present cohort were substantially lower than those of the 2 cohorts reported earlier from Taiwan and India. In a cohort of 414 Taiwanese carriers with persistently normal ALT levels for at least 2 years, the mean HBV DNA level was 4.7 ⫾ 1.5 log10 copies/mL with levels greater than 104 copies/mL in 52%, including 18% with levels of 105 to 106 copies/mL and 14% with levels greater than 106 copies/mL.12 In another cohort of 116 Indian carriers with persistently normal ALT levels for at least 1 year, the median HBV DNA level was 4.29 log10 copies/mL (range, 2.78 –9.20 log10 copies/mL) with levels greater than 105
Table 6. Distribution of the Risk Factors for Active Hepatitis in Inactive Carriers With HBV DNA Levels of 104 Copies/mL or More and Patients With HBeAgNegative Chronic Hepatitis Risk factors
Sex Female Male HBV DNA levelsa 104–105 copies/mL ⬎105 copies/mL Genotype B C Basal core promoter T1762/A1764 mutant No Yes a5.26
copies/mL ⫽ 1 IU/mL.
1 8.2 (3.4–20.0)
Male
HBV DNA levels ⬎105 copies/mLa
Basal core promoter mutation
Inactive carriers (n ⫽ 90)
Chronic hepatitis (n ⫽ 90)
⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹
⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹
⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹
39 16 16 2 6 8 3 0
1 3 6 4 38 2 8 28
⬍0.0001
1 21.5 (8.4–55.4)
⬍0.0001
1 1.8 (0.5–5.8)
0.34
1 3.5 (1.3–9.3)
0.011
⫺, absence; ⫹, presence. a5.26 copies/mL ⫽ 1 IU/mL.
June 2010
Figure 1. Frequency distribution of the number of risk factors (male sex, HBV DNA levels ⬎105 copies/mL [5.26 copies/mL ⫽ 1 IU mL], and the basal core promoter mutation). Among 90 inactive carriers with HBV DNA levels of 104 copies/mL or more (white bars), 39, 34, 17, and 0, respectively, had 0, 1, 2, and 3 risk factors. Among 90 patients with HBeAg-negative chronic hepatitis (black bars), 1, 13, 48, and 28, respectively, had 0, 1, 2, and 3 risk factors. The odds ratios for active hepatitis increased with the presence of an increasing number of risk factors.
copies/mL in 35%.13 The reason for the substantially high HBV DNA levels in these 2 cohorts remains unclear, but the duration of persistently normal ALT levels in these 2 studies was relatively short. A certain proportion of the carriers in these 2 cohorts will be likely to suffer future reactivation, as shown in a recent study from the same Indian group.23 Nearly 40% of inactive carriers in our cohort had HBV DNA levels of 104 copies/mL or more, albeit they had had persistently normal ALT levels for more than 10 years. These patients may be excluded from the diagnosis of inactive carriers using the currently accepted criteria.1,5–9 The possibility of ALT increase that was missed by regular follow-up evaluation still cannot be excluded and further follow-up evaluation also is warranted to exclude the possibility of future reactivation in these carriers. One major limitation of the present study was that there is no stored serum available for testing HBV DNA levels at baseline or before enrollment. Two previous studies have shown that viral levels remained stable and unchanged in inactive carriers with persistently normal ALT levels during a long-term follow-up evaluation of up to 92 months and 6 years, respectively,10,24 possibly owing to the absence of immune attack to the virus. Nevertheless, a high HBV DNA level of 104 copies/mL or more was found in a substantial proportion of inactive carriers with normal ALT levels over 10 years even if it was assayed only at enrollment. Another limitation of the present study was that there was no histologic examination or fibroscan to exclude significant liver disease in our inactive carriers. In a recent study from Greece, 97% of the 35 inactive carriers with persistently normal ALT levels for at least one year had minimal necroinflammation, whereas 83% of them had no or mild fibrosis.25 Inactive carriers enrolled in the present investigation had persistently normal ALT levels for at least 10 years during a close follow-up evaluation (mean of ALT measurements, 27.4; mean follow-up period, 15.7 y). They therefore are much more unlikely to have significant liver disease. Moreover, none of them had evidence of cirrhosis based on liver ultrasonographic findings, although this technique is somewhat limited with sensitivity and specificity in the 80% range.18
HBV DNA LEVELS IN INACTIVE CARRIERS
539
An additional original and important finding of the present study was that although a substantial proportion of inactive carriers had HBV DNA levels of 104 copies/mL or more, their clinical and virologic features differed remarkably from those with HBeAg-negative chronic hepatitis. As shown in Table 4, compared with age-matched patients with HBeAg-negative chronic hepatitis, inactive carriers with HBV DNA levels of 104 copies/mL or more were predominantly female, had significantly lower HBV DNA levels, and had significantly lower frequency of genotype C HBV and the basal core promoter T1762/A1764 mutant. Multiple logistic regression analysis identified 3 factors significantly predictive for active diseases among anti-HBe–positive carriers with HBV DNA levels of 104 copies/mL or more (Table 3). Patients with HBV DNA levels greater than 105 copies/mL were approximately 20 times more likely to have active hepatitis than those with levels of 104 to 105 copies/mL. Male carriers were 8 times more likely to suffer active hepatitis than female carriers. The reason for this genderrelated difference is unclear, but male predominance in HBeAgnegative chronic hepatitis was shown to be independent of HBV DNA levels or viral mutant. Another factor predictive of active hepatitis is the presence of the basal core promoter T1762/ A1764 mutant. Basal core promoter T1762/A1764 mutant has been shown to be related to the pathogenesis of hepatocellular carcinoma.26,27 The present results indicated that basal core promoter T1762/A1764 mutant plays a significant role in progressive liver disease as early as before the development of cirrhosis and hepatocellular carcinoma. Finally, although genotype C HBV correlated with reactivation of hepatitis B in the univariate analysis (Table 4), as shown in previous studies,19,22 it became nonsignificant in the multivariate analysis (Table 5). Notably, the odds ratios of active hepatitis increase with the presence of an increasing number of risk factors and are extraordinarily high if 2 or more risk factors are present (Table 6 and Figure 1). With regard to the management of anti-HBe–positive carriers with HBV DNA levels greater than 104 copies/mL and normal ALT levels, the guideline of the American Association for the Study of Liver Diseases did not have recommendations for this particular group of patients.5 The guideline of the Asian-Pacific Association for the Study of the Liver recommended biopsy for those older than age 40 and treatment if there is significant disease.1,6 The guideline of the European Association for the Study of the Liver and other experts recommended biopsy without consideration of patient age and treatment when there is significant inflammation or fibrosis.7–9 On the other hand, other experts suggested that these patients may not require immediate biopsy and treatment but need close follow-up evaluation because the majority of their HBeAgnegative carriers with persistently normal ALT levels and HBV DNA levels greater than 104 copies/mL had only minimal histologic lesions.25 The benefits of early initiation of treatment to prevent future reactivation and fibrosis, as suggested by some experts,28 should be balanced against the potential of unnecessary treatments for certain patients. On the basis of the present results, periodic follow-up evaluation should be adequate for females with HBV DNA levels of 104 to 105 copies/mL and wild-type basal core promoter. Liver biopsy and therapy should be considered in males, those with HBV DNA levels greater than 105 copies/mL, or those with basal core promoter T1762/A1764 mutant.
540
CHU ET AL
In conclusion, approximately 40% of inactive carriers with persistently normal ALT levels for more than 10 years have HBV DNA levels of 104 copies/mL or more. These data do not support the adaptation of HBV DNA level less than 104 copies/mL for diagnosis of inactive carriers and also argue against treatment of all anti-HBe–positive carries with HBV DNA levels of 104 copies/mL or more. Female sex, HBV DNA levels of 104 to 105 copies/mL, and the wild-type basal core promoter correlate with the inactive carrier state.
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 6
17.
18.
19.
References 1. Liaw YF, Chu CM. Hepatitis B virus infection. Lancet 2009;373: 582–592. 2. Hsu YS, Chien RN, Yeh CT, et al. Long-term outcome after spontaneous HBeAg seroconversion in patients with chronic hepatitis B. Hepatology 2002;35:1522–1527. 3. Chu CM, Hung SJ, Lin J, et al. Natural history of hepatitis B e antigen to antibody seroconversion in patients with normal serum aminotransferase levels. Am J Med 2004;116:829 – 834. 4. Feld JJ, Ayers M, El-Ashry D, et al. Hepatitis B virus DNA prediction rules for hepatitis B e antigen-negative chronic hepatitis B. Hepatology 2007;46:1057–1070. 5. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007;45: 507–539. 6. Liaw YF, Leung N, Kao JH, et al. Asian-Pacific consensus statement on management of chronic hepatitis B: a 2008 update. Hepatol Int 2008:2:263–283. 7. EASL. Clinical practice guidelines: management of chronic hepatitis B. European Association For The Study Of The Liver. J Hepatol 2009;50:227–242. 8. Gish RG, Locarnini SA. Chronic hepatitis B: current testing strategies. Clin Gastroenterol Hepatol 2006;4:666 – 676. 9. Keeffe EB, Dieterich DT, Han SH, et al. A treatment algorithm for the management of chronic hepatitis B virus infection in the United States: 2008 update. Clin Gastroenterol Hepatol 2008; 6:1315–1341. 10. Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection. Hepatology 2002;36:1408 –1415. 11. Manesis EK, Papatheodoridis GV, Sevastianos V, et al. Significance of hepatitis B viremia levels determined by a quantitative polymerase chain reaction assay in patients with hepatitis B e antigen-negative chronic hepatitis B virus infection. Am J Gastroenterol 2003;98:2261–2267. 12. Lin CL, Liao LY, Liu CJ, et al. Hepatitis B viral factors in HBeAgnegative carriers with persistently normal serum alanine aminotransferase levels. Hepatology 2007;45:1193–1198. 13. Kumar M, Sarin SK, Hissar S, et al. Virologic and histologic features of chronic hepatitis B virus-infected asymptomatic patients with persistently normal ALT. Gastroenterology 2008;134: 1376 –1384. 14. Chu CM, Liaw YF. Incidence and risk factors of progression to cirrhosis in inactive carriers of hepatitis B virus. Am J Gastroenterol 2009;104:1693–1699. 15. Papatheodoridis GV, Chrysanthos N, Hadziyannis E, et al. Longitudinal changes in serum HBV DNA levels and predictors of progression during the natural course of HBeAg-negative chronic hepatitis B virus infection. J Viral Hepat 2008;15:434 – 441. 16. Tai DI, Lin SM, Sheen IS, et al. Long-term outcome of hepatitis B e antigen-negative hepatitis B surface antigen carriers in relation
20.
21.
22.
23.
24.
25.
26.
27.
28.
to changes of alanine aminotransferase levels over time. Hepatology 2009;49:1859 –1867. Chu CM, Liaw YF. HBsAg seroclearance in asymptomatic carriers of high endemic areas: appreciably high rates during a long-term follow up. Hepatology 2007;45:1187–1192. Lin DY, Sheen IS, Chiu CT, et al. Ultrasonographic changes of early liver cirrhosis in chronic hepatitis B: a longitudinal study. J Clin Ultrasound 1993;21:303–308. Chu CM, Liaw YF. Genotype C hepatitis B virus infection is associated with a higher risk of reactivation of hepatitis B and progression to cirrhosis than genotype B: a longitudinal study of hepatitis B e antigen-positive patients with normal aminotransferase levels at baseline. J Hepatol 2005;43:411– 417. Chu CM, Yeh CT, Lee CS, et al. Precore stop mutant in HBeAgpositive patients with chronic hepatitis B: clinical characteristics and correlation with the course of HBeAg-to-anti-HBe seroconversion. J Clin Microbiol 2002;40:16 –21. Prati D, Taioli E, Zanella A, et al. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Intern Med 2002;137:1–10. Chu CM, Liaw YF. Predictive factors for reactivation of hepatitis B following hepatitis B e antigen seroconversion in chronic hepatitis B. Gastroenterology 2007;133:1458 –1465. Kumar M, Chauhan R, Gupta N, et al. Spontaneous increases in alanine aminotransferase levels in asymptomatic chronic hepatitis B virus-infected patients. Gastroenterology 2009;136:1272– 1280. Martinot-Peignoux M, Boyer N, Colombat M, et al. Serum hepatitis B virus DNA levels and liver histology in inactive HBsAg carriers. J Hepatol 2002;36:543–546. Papatheodoridis GV, Manesis EK, Manolakopoulos S, et al. Is there a meaningful serum hepatitis B virus DNA cutoff level for therapeutic decisions in hepatitis B e antigen-negative chronic hepatitis B virus infection? Hepatology 2008;48:1451–1459. Baptista M, Kramvis A, Kew MC. High prevalence of 1762(T) 1764(A) mutations in the basic core promoter of hepatitis B virus isolated from black Africans with hepatocellular carcinoma compared with asymptomatic carriers. Hepatology 1999;29:946 – 953. Kao JH, Chen PJ, Lai MY, et al. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology 2003;124:327–334. Lai CL, Yuen MF. The natural history and treatment of chronic hepatitis B: a critical evaluation of standard treatment criteria and end points. Ann Intern Med 2007;147:58 – 61.
Reprint requests Address requests for reprints to: Chia-Ming Chu, MD, Liver Research Unit, Chang Gung Memorial Hospital, 199, Tung Hwa North Road, Taipei 10591, Taiwan. e-mail: [email protected]; fax: (886) 3-3272236. Conflicts of interest The authors disclose the following: Yun-Fan Liaw has been involved in clinical trials or served as a global advisory board member of Roche, Bristol-Myers Squibb, GlaxoSmithKline, Novartis, and Gilead Sciences. The remaining authors disclose no conflicts. Funding This study was supported by a grant from the National Science Council of Taiwan (NSC97-2314-B-182-019-MY2).
Level of Hepatitis B Virus DNA in Inactive Carriers With Persistently Normal Levels of Alanine Aminotransferase CHIA–MING CHU, YI–CHENG CHEN, DAR–IN TAI, and YUN–FAN LIAW Liver Research Unit, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan
BACKGROUND & AIMS: Little is known about the level of hepatitis B virus (HBV) DNA in individuals with chronic, inactive HBV infections. Patients who test positive for the antibody to hepatitis B e antigen (anti-HBe) and have normal levels of alanine aminotransferase for more than 10 years have a low risk of HBV reactivation and are considered to be inactive carriers. We investigated HBV DNA levels in inactive carriers and identified factors that correlated with this state among anti-HBe–positive carriers with HBV DNA levels of 104 copies/mL or greater (5.26 copies/mL ⫽ 1 IU/mL). METHODS: HBV DNA levels were assayed in 250 inactive carriers with persistently normal alanine aminotransferase levels for more than 10 years. Clinical and virologic features were compared between inactive carriers (with HBV DNA levels ⱖ104 copies/ mL) and age-matched patients with HBe antigen–negative chronic hepatitis (controls, n ⫽ 90). RESULTS: The median level of HBV DNA among inactive carriers was 3.70 log10 copies/mL (range, undetectable to 5.98 log10 copies/mL). Ninety (36%) had levels of 104 copies/mL or greater. Compared with control patients, significant differences of inactive carriers included sex (more female patients), lower HBV DNA levels, and lower prevalence of genotype C virus and the basal core promoter mutation T1762/A1764. The prevalence of the precore mutation A1896 was similar between groups. Multiple logistic regression analyses identified male sex, HBV DNA levels greater than 105 copies/mL, and the basal core promoter mutation as independent factors that correlated with active disease. CONCLUSIONS: Nearly 40% of inactive carriers had HBV DNA levels of 104 copies/mL or greater. Female sex, HBV DNA levels of 104 to 105 copies/mL, and wild-type basal core promoter correlated with inactive carrier state. Keywords: Chronic Hepatitis B; Sex; Viral Genotype; Viral Load; Viral Mutants.
T
he natural course of chronic hepatitis B virus (HBV) infection constitutes 3 chronologic phases: an initial immune tolerance phase during which the patients are positive for hepatitis B e antigen (HBeAg) and have normal alanine aminotransferase (ALT) levels, followed by an immune clearance phase during which the HBeAg–positive patients have increased ALT levels, and, finally, the inactive carrier state during which HBeAg seroconverts to its antibody (anti-HBe) and ALT levels normalize.1 The third phase may remain stably inactive in a lifetime. On the other hand, HBV also can reactivate either by reversion back to the previous HBeAg–positive phase or, much more frequently, by progression to HBeAg–negative chronic hepatitis.2,3
Among these phases, the inactive carrier state may be a retrospective–prospective diagnosis because inactive carriers show some propensity to reactivation. However, there is no single value of HBV DNA above which future reactivation is likely to occur and below which the disease is likely to be quiescent.4 A HBV DNA level threshold of 104 copies/mL has been used to discriminate active HBV infection from its inactive form.1,5–9 However, several studies have shown that between 7% and 55% of so-called inactive carriers with persistently normal ALT levels for variable duration had HBV DNA levels greater than 104 copies/mL,10 –13 and even in 2 studies from India and Taiwan, 35% and 32%, respectively, had levels greater than 105 copies/mL.12,13 According to the long-term observations of 1965 anti-HBe–positive hepatitis B surface antigen (HBsAg) carriers with normal ALT levels from Taiwan, reactivation of hepatitis B most often occurred during the first 5 to 10 years of follow-up evaluation and became extremely rare thereafter.14 However, in most previous studies the so-called inactive carriers had persistently normal ALT levels for a relatively short period of only 1 to 2 years; therefore, future reactivation can be anticipated in some of these carriers.15 Clearly, viral load status of the inactive carrier state needs to be defined further. Because reactivation of hepatitis B in anti-HBe–positive carriers became extremely rare 10 years after entry14 and virtually none of the anti-HBe–positive carriers with persistently normal ALT levels over 10 years died of liver disease,16 anti-HBe–positive carriers with persistently normal ALT levels over 10 years can be considered as real inactive carriers. Serum HBV DNA levels in such carriers may represent the viral load status of inactive carrier state. In this investigation, we first studied HBV DNA levels in 250 anti-HBe–positive HBsAg carriers with persistently normal ALT levels for more than 10 years. Our results showed that 90 (36%) of them had HBV DNA levels of 104 copies/mL or more, a level being used to indicate active hepatitis. To further identify factors that correlate with inactive carrier state among anti-HBe–positive carriers with HBV DNA levels of 104 copies/mL or more, we next compared the clinical and virologic features between inactive carriers with HBV DNA levels of 104 copies/mL or more and age-matched patients with HBeAg–negative chronic hepatitis.
Abbreviations used in this paper: ALT, alanine aminotransferase; anti-HBe, antibody against hepatitis B e antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus. © 2010 by the AGA Institute 1542-3565/$36.00 doi:10.1016/j.cgh.2010.03.006
536
CHU ET AL
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 6
Materials and Methods Inactive Carriers and Patients With Hepatitis B e Antigen–Negative Chronic Hepatitis Asymptomatic adults who were identified incidentally as HBsAg carriers during blood donation or health check-ups received regular clinic follow-up evaluation at the carrier clinic of Chang Gung Memorial Hospital in Taipei, Taiwan.14,17 The subjects were considered inactive carriers if they fulfilled the following criteria: (1) positive HBsAg, negative HBeAg, positive anti-HBe, and persistently normal ALT levels (ⱕ36 U/L) at least once every 6 to 12 months for at least 10 years until the last visit; (2) no evidence of cirrhosis or hepatocellular carcinoma based on clinical assessment and liver ultrasonographic findings18; (3) no concomitant infection with hepatitis C virus or hepatitis D virus; and (4) no antiviral or immunomodulatory therapy before enrollment. Patients who underwent HBsAg seroclearance during the follow-up period17 also were excluded. Patients were diagnosed as having HBeAg–negative chronic hepatitis if they were HBsAg positive, HBeAg negative, and anti-HBe positive, had persistently abnormal ALT levels more than twice the upper limit of normal for at least 2 years, and HBV DNA levels were 104 copies/mL or more. Patients who had concomitant infection with hepatitis C virus or hepatitis D virus, autoimmune or metabolic liver disease, who consumed alcohol or drugs that might be potential etiologic agents of hepatitis, or who had ever received antiviral or immunomodulatory therapy were excluded.
Methods HBsAg, HBeAg, anti-HBe, and antibody against hepatitis D virus were assayed using radioimmunoassay kits (Abbott Diagnostics, North Chicago, IL). Antibodies against hepatitis C virus were assayed using a second- or third-generation enzyme immunoassay (Abbott Diagnostics). Serum HBV DNA levels were assayed using the COBAS Amplicor HBV Monitor Test (Roche Diagnostics, Branchburg, NJ; lower limit of detection, 200 copies/mL). The conversion in IU/mL (1 IU is equivalent to 5.26 HBV DNA copies) was made according to the manufacturer’s instructions. HBV genotypes were determined using the polymerase chain reaction–restriction fragment length polymorphism of the surface gene of HBV, as previously described.19 Precore A1896 mutant was detected by amplification-created restriction site method, as described before.20 Basal core promoter genes were amplified by polymerase chain reaction, and nucleotide sequences of the amplified products were deter-
mined directly by using an automatic sequencer, as described by others.13
Statistical Analyses Data are presented as mean ⫾ standard deviation, median (range), or number (%). To compare characteristics between groups, either the chi-square test or the Fisher exact test was used to analyze categoric variables and the Student t test or the Mann–Whitney U nonparametric test was used to analyze continuous variables. HBV DNA levels were correlated with ALT levels or age of patients by using Spearman rank correlation. Univariate and multivariate logistic regression analyses were performed to identify the factors that correlated with active disease among anti-HBe–positive HBsAg carriers with HBV DNA levels of 104 copies/mL or more. Variables with P values less than .1 in the univariate models were tested in a multivariate setting. Significant associations identified in multivariate analysis are presented as odds ratio (95% confidence interval). Statistical procedures were performed using the SPSS statistical software (version 13.0; SPSS, Chicago, IL). P values less than .05 were considered significant.
Results Clinical and Demographic Features of Inactive Carriers From January 2007 to December 2007, there were 250 consecutive anti-HBe–positive HBsAg carriers who had had persistently normal ALT levels for at least 10 years who received regular follow-up evaluation at our hepatitis carrier clinic. They were considered inactive carriers and were enrolled in this study. The demographic and clinical data are summarized in Table 1. There were 84 men and 166 women. The mean age at baseline was 34.4 years (range, 18 – 64 y) and the mean age at enrollment was 50.6 years (range, 31– 81 y). The mean number of ALT measurements for each carrier was 28.4 (range, 12– 48) during a mean period of 16.1 years (range, 10 –25 y) before enrollment. The mean maximal ALT level before enrollment was 26.3 U/L (range, 9 –36 U/L). Maximal ALT levels were 19 U/L or less in 26 patients (10.4%), 20 to 30 U/L in 159 patients (63.6%), and 31 to 36 U/L in 65 patients (26%). ALT levels were significantly higher in male carriers than female carriers. A total of 52 male carriers and 23 female carriers had persistently normal ALT levels according to the strict criteria of ALT of 30 U/L or less in males and 19 U/L or less in females, as suggested by Prati et al.21
Table 1. Clinical and Demographic Data of Inactive Carriers Data
Total (n ⫽ 250)
Male (n ⫽ 84)
Female (n ⫽ 166)
Age at baseline, y Male:female ratio Duration of persistently normal ALT levels before enrollment, y Number of ALT determinations before enrollment Maximal ALT levels before enrollment, U/L ⱕ19 U/L 20–30 U/L 30–36 U/L Age at enrollment, y
34.4 ⫾ 8.8 84:166 16.1 ⫾ 4.7 28.4 ⫾ 8.9 26.3 ⫾ 5.6 26 (10) 159 (64) 65 (26) 50.6 ⫾ 9.6
35.6 ⫾ 9.3
33.9 ⫾ 8.6
.16
16.6 ⫾ 4.9 29.8 ⫾ 9.6 28.1 ⫾ 5.0 3 (4) 49 (58) 32 (38) 52.2 ⫾ 10.1
15.9 ⫾ 4.5 27.6 ⫾ 8.4 25.3 ⫾ 5.7 23 (14) 110 (66) 33 (20) 49.8 ⫾ 8.4
.27 .093 .0002 .012 .22 .0019 .069
NOTE. Data are given as mean ⫾ SD or number (%).
P
June 2010
HBV DNA LEVELS IN INACTIVE CARRIERS
537
Table 2. HBV DNA Levels in Inactive Carriers With Persistently Normal ALT Levels (ⱕ36 U/L) HBV DNA levels, log10 copies/mLa ⬍2.3 (undetectable) 2.3–2.99 3–3.99 4–4.99 5–5.99 Median (range)
Total (n ⫽ 250)
Males (n ⫽ 84)
Females (n ⫽ 166)
P
43 (17) 28 (11) 89 (36) 65 (26) 25 (10) 3.70 (⬍2.3 to 5.98)
9 (11) 12 (14) 30 (36) 25 (30) 8 (10) 3.73 (⬍2.3 to 5.79)
34 (20) 16 (10) 59 (36) 40 (24) 17 (10) 3.69 (⬍2.3 to 5.98)
.053 .16 .98 .33 .86 .40
NOTE. Data are given as number (%). a5.26 copies/mL ⫽ 1 IU/mL.
Hepatitis B Virus DNA Levels of Inactive Carriers As shown in Table 2, the median HBV DNA level in 250 inactive carriers was 3.70 (range, undetectable [⬍2.3] to 5.98) log10 copies/mL. Ninety carriers (36%) had levels of 104 copies/mL or more. HBV DNA levels showed no significant difference between males and females. There was also no significant correlation between HBV DNA levels and maximal ALT levels (P ⫽ .81) or age of patients at enrollment (P ⫽ .41). Among a subset of 75 inactive carriers with persistent ALT levels of 30 U/L or less for males and 19 U/L or less for females, the median HBV DNA level was 3.81 (range, undetectable [⬍2.3] to 5.45) with levels of 104 copies/mL or more in 32 (43%) of them (Table 3). Both figures were not different from those observed in the whole cohort of 250 inactive carriers (P ⫽ .25 and .29, respectively).
Comparison of Clinical and Virologic Features Between Inactive Carriers With Hepatitis B Virus DNA Levels of 104 Copies/ Milliliter or More and Patients With Hepatitis B e Antigen–Negative Chronic Hepatitis Clinical and virologic features of 90 inactive carriers with HBV DNA levels of 104 copies/mL or more were compared with 90 age-matched patients with HBeAg–negative chronic hepatitis who were selected randomly during the study period. As shown in Table 4, inactive carriers with HBV DNA levels of 104 copies/mL or more were significantly female predominant (P ⬍ .0001) and had significantly lower HBV DNA levels (P ⬍ .0001) when compared with the age-matched patients with HBeAg–negative chronic hepatitis. In addition, inactive carriers had a significantly lower prevalence of genotype C HBV (P ⫽ .0015) and the basal core promoter T1762/A1764 mutant (P ⬍
.0001) than did patients with HBeAg–negative chronic hepatitis, whereas the frequency of the precore A1896 mutant showed no significant difference between them (P ⫽ .84).
Predictive Factors for Active Infection in Anti-Hepatitis B e–Positive Carriers With Hepatitis B Virus DNA Levels Greater Than 104 Copies/Milliliter By using multiple logistic regression analysis, 3 independent factors were identified to be correlated significantly with active infection: male sex, serum HBV DNA levels greater than 105 copies/mL, and the presence of the basal core promoter T1762/A1764 mutant, whereas genotype C HBV was not found to be a predictor of active hepatitis (Table 5). The distribution of the 3 risk factors in inactive carriers with HBV DNA levels of 104 copies/mL or more and in patients with HBeAg–negative chronic hepatitis is shown in Table 6. Of the 90 inactive carriers, 73 (81%) had none or only one risk factor. On the contrary, 76 (84%) of 90 patients with HBeAg–negative chronic hepatitis had 2 or 3 risk factors. The prevalence of the number of the risk factors in inactive carriers with HBV DNA levels of 104 copies/mL or more and in patients with HBeAg– negative chronic hepatitis is shown in Figure 1. The odds ratios of active hepatitis increased with the presence of increasing number of the risk factors.
Discussion The male:female ratio in inactive carriers in this cohort was approximately 1:2 (Table 1). This ratio contrasts remarkably with the male predominance in patients with chronic hepatitis B, but is consistent with the observation that male carriers were significantly more likely to have reactivation of hepatitis B than female carriers.14,22
Table 3. HBV DNA Levels in Inactive Carriers With Persistently Normal ALT Levels (ⱕ30 U/L for Males and ⱕ19 U/L for Females) HBV DNA levels, log10 copies/mLa ⬍2.3 (undetectable) 2.3–2.99 3–3.99 4–4.99 5–5.99 Median (range) NOTE. Data are given as number (%). copies/mL ⫽ 1 IU/mL.
a5.26
Total (n ⫽ 75)
Males (n ⫽ 52)
Females (n ⫽ 23)
P
9 (12) 8 (7) 26 (35) 24 (32) 8 (11) 3.81 (⬍2.3 to 5.45)
6 (12) 6 (12) 15 (29) 20 (38) 5 (10) 3.95 (⬍2.3 to 5.45)
3 (13) 2 (9) 11 (48) 4 (17) 3 (13) 3.76 (⬍2.3 to 5.45)
.85 .71 .11 .071 .66 .72
538
CHU ET AL
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 6
Table 4. Comparison of Clinical and Virologic Features Between Inactive Carriers With HBV DNA Levels of 104 Copies/mL or More and Patients With HBeAg-Negative Chronic Hepatitis Data Age, y Male:female ratio HBV DNA levels, log10 copies/mLa 4–4.99 5–5.99 6–6.99 ⱖ7 Genotype B C Precore A1896 mutant Basal core promoter T1762/A1764 mutant
Inactive carriers (n ⫽ 90)
Chronic hepatitis (n ⫽ 90)
P
50.7 ⫾ 8.9 30:60 4.72 ⫾ 0.56 4.63 (4.01–5.98) 65 (72) 25 (28) 0 (0) 0 (0)
51.1 ⫾ 7.8 72:18 6.24 ⫾ 1.12 6.24 (4.09–9.18) 10 (11) 27 (30) 33 (37) 20 (22)
.75 ⬍.0001 ⬍.0001
80 (89) 10 (11) 75 (83) 13 (14)
67 (74) 23 (26) 74 (82) 39 (43)
.015 .84 ⬍.0001
NOTE. Data are given as mean ⫾ SD, median (range), or number (%). a5.26 copies/mL ⫽ 1 IU/mL.
This investigation showed that there was a wide range of HBV DNA levels in inactive carriers, ranging from less than 103 copies/mL in 28% to more than 105 copies/mL in 10% of them (Table 2). It should be noted that none of the inactive carriers in this cohort had HBV DNA levels more than 106 copies/mL. The median HBV DNA level in inactive carriers of our cohort was 3.70 (range, undetectable [⬍2.3] to 5.98) log10 copies/mL. This figure is comparable with the mean of 3.10 ⫾ 0.17 log10 copies/mL reported in a cohort of 29 inactive carriers in Hong Kong with persistently normal ALT levels for 41 to 92 months10 and the median of 3.52 (range, ⬍2.60 to 4.46) log10 copies/mL reported in a cohort of 62 inactive carriers in Greece with persistently normal ALT levels for at least 2 years.11 However, 36% of inactive carriers in our cohort had HBV DNA levels of 104 copies/mL or more, including 10% with levels of 105 to 106 copies/mL (Table 2), whereas only 7% of the Hong Kong cohort had levels greater than 104 copies/mL10 and none of the inactive carriers in the Hong Kong and Greece cohorts had levels greater Table 5. Factors Correlated With Active Hepatitis Among Anti-HBe–Positive Carriers With HBV DNA Levels of 104 Copies/mL or More: Multiple Logistic Regression Analysis Factors
Odds ratio (95% confidence interval)
P
than 105 copies/mL.10,11 It should be pointed out that our study enrolled a relatively large number of inactive carriers with a long duration of persistently normal ALT levels and the results more accurately should represent the viral load status of the inactive carrier state. Even if we adapted a strict criterion of normal ALT levels (ⱕ30 U/L in males and ⱕ19 U/L in females) as suggested by Prati et al,21 a similar proportion of inactive carriers still had HBV DNA levels of 104 copies/mL or more (Table 3). On the other hand, HBV DNA levels in inactive carriers of the present cohort were substantially lower than those of the 2 cohorts reported earlier from Taiwan and India. In a cohort of 414 Taiwanese carriers with persistently normal ALT levels for at least 2 years, the mean HBV DNA level was 4.7 ⫾ 1.5 log10 copies/mL with levels greater than 104 copies/mL in 52%, including 18% with levels of 105 to 106 copies/mL and 14% with levels greater than 106 copies/mL.12 In another cohort of 116 Indian carriers with persistently normal ALT levels for at least 1 year, the median HBV DNA level was 4.29 log10 copies/mL (range, 2.78 –9.20 log10 copies/mL) with levels greater than 105
Table 6. Distribution of the Risk Factors for Active Hepatitis in Inactive Carriers With HBV DNA Levels of 104 Copies/mL or More and Patients With HBeAgNegative Chronic Hepatitis Risk factors
Sex Female Male HBV DNA levelsa 104–105 copies/mL ⬎105 copies/mL Genotype B C Basal core promoter T1762/A1764 mutant No Yes a5.26
copies/mL ⫽ 1 IU/mL.
1 8.2 (3.4–20.0)
Male
HBV DNA levels ⬎105 copies/mLa
Basal core promoter mutation
Inactive carriers (n ⫽ 90)
Chronic hepatitis (n ⫽ 90)
⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹
⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹
⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹
39 16 16 2 6 8 3 0
1 3 6 4 38 2 8 28
⬍0.0001
1 21.5 (8.4–55.4)
⬍0.0001
1 1.8 (0.5–5.8)
0.34
1 3.5 (1.3–9.3)
0.011
⫺, absence; ⫹, presence. a5.26 copies/mL ⫽ 1 IU/mL.
June 2010
Figure 1. Frequency distribution of the number of risk factors (male sex, HBV DNA levels ⬎105 copies/mL [5.26 copies/mL ⫽ 1 IU mL], and the basal core promoter mutation). Among 90 inactive carriers with HBV DNA levels of 104 copies/mL or more (white bars), 39, 34, 17, and 0, respectively, had 0, 1, 2, and 3 risk factors. Among 90 patients with HBeAg-negative chronic hepatitis (black bars), 1, 13, 48, and 28, respectively, had 0, 1, 2, and 3 risk factors. The odds ratios for active hepatitis increased with the presence of an increasing number of risk factors.
copies/mL in 35%.13 The reason for the substantially high HBV DNA levels in these 2 cohorts remains unclear, but the duration of persistently normal ALT levels in these 2 studies was relatively short. A certain proportion of the carriers in these 2 cohorts will be likely to suffer future reactivation, as shown in a recent study from the same Indian group.23 Nearly 40% of inactive carriers in our cohort had HBV DNA levels of 104 copies/mL or more, albeit they had had persistently normal ALT levels for more than 10 years. These patients may be excluded from the diagnosis of inactive carriers using the currently accepted criteria.1,5–9 The possibility of ALT increase that was missed by regular follow-up evaluation still cannot be excluded and further follow-up evaluation also is warranted to exclude the possibility of future reactivation in these carriers. One major limitation of the present study was that there is no stored serum available for testing HBV DNA levels at baseline or before enrollment. Two previous studies have shown that viral levels remained stable and unchanged in inactive carriers with persistently normal ALT levels during a long-term follow-up evaluation of up to 92 months and 6 years, respectively,10,24 possibly owing to the absence of immune attack to the virus. Nevertheless, a high HBV DNA level of 104 copies/mL or more was found in a substantial proportion of inactive carriers with normal ALT levels over 10 years even if it was assayed only at enrollment. Another limitation of the present study was that there was no histologic examination or fibroscan to exclude significant liver disease in our inactive carriers. In a recent study from Greece, 97% of the 35 inactive carriers with persistently normal ALT levels for at least one year had minimal necroinflammation, whereas 83% of them had no or mild fibrosis.25 Inactive carriers enrolled in the present investigation had persistently normal ALT levels for at least 10 years during a close follow-up evaluation (mean of ALT measurements, 27.4; mean follow-up period, 15.7 y). They therefore are much more unlikely to have significant liver disease. Moreover, none of them had evidence of cirrhosis based on liver ultrasonographic findings, although this technique is somewhat limited with sensitivity and specificity in the 80% range.18
HBV DNA LEVELS IN INACTIVE CARRIERS
539
An additional original and important finding of the present study was that although a substantial proportion of inactive carriers had HBV DNA levels of 104 copies/mL or more, their clinical and virologic features differed remarkably from those with HBeAg-negative chronic hepatitis. As shown in Table 4, compared with age-matched patients with HBeAg-negative chronic hepatitis, inactive carriers with HBV DNA levels of 104 copies/mL or more were predominantly female, had significantly lower HBV DNA levels, and had significantly lower frequency of genotype C HBV and the basal core promoter T1762/A1764 mutant. Multiple logistic regression analysis identified 3 factors significantly predictive for active diseases among anti-HBe–positive carriers with HBV DNA levels of 104 copies/mL or more (Table 3). Patients with HBV DNA levels greater than 105 copies/mL were approximately 20 times more likely to have active hepatitis than those with levels of 104 to 105 copies/mL. Male carriers were 8 times more likely to suffer active hepatitis than female carriers. The reason for this genderrelated difference is unclear, but male predominance in HBeAgnegative chronic hepatitis was shown to be independent of HBV DNA levels or viral mutant. Another factor predictive of active hepatitis is the presence of the basal core promoter T1762/ A1764 mutant. Basal core promoter T1762/A1764 mutant has been shown to be related to the pathogenesis of hepatocellular carcinoma.26,27 The present results indicated that basal core promoter T1762/A1764 mutant plays a significant role in progressive liver disease as early as before the development of cirrhosis and hepatocellular carcinoma. Finally, although genotype C HBV correlated with reactivation of hepatitis B in the univariate analysis (Table 4), as shown in previous studies,19,22 it became nonsignificant in the multivariate analysis (Table 5). Notably, the odds ratios of active hepatitis increase with the presence of an increasing number of risk factors and are extraordinarily high if 2 or more risk factors are present (Table 6 and Figure 1). With regard to the management of anti-HBe–positive carriers with HBV DNA levels greater than 104 copies/mL and normal ALT levels, the guideline of the American Association for the Study of Liver Diseases did not have recommendations for this particular group of patients.5 The guideline of the Asian-Pacific Association for the Study of the Liver recommended biopsy for those older than age 40 and treatment if there is significant disease.1,6 The guideline of the European Association for the Study of the Liver and other experts recommended biopsy without consideration of patient age and treatment when there is significant inflammation or fibrosis.7–9 On the other hand, other experts suggested that these patients may not require immediate biopsy and treatment but need close follow-up evaluation because the majority of their HBeAgnegative carriers with persistently normal ALT levels and HBV DNA levels greater than 104 copies/mL had only minimal histologic lesions.25 The benefits of early initiation of treatment to prevent future reactivation and fibrosis, as suggested by some experts,28 should be balanced against the potential of unnecessary treatments for certain patients. On the basis of the present results, periodic follow-up evaluation should be adequate for females with HBV DNA levels of 104 to 105 copies/mL and wild-type basal core promoter. Liver biopsy and therapy should be considered in males, those with HBV DNA levels greater than 105 copies/mL, or those with basal core promoter T1762/A1764 mutant.
540
CHU ET AL
In conclusion, approximately 40% of inactive carriers with persistently normal ALT levels for more than 10 years have HBV DNA levels of 104 copies/mL or more. These data do not support the adaptation of HBV DNA level less than 104 copies/mL for diagnosis of inactive carriers and also argue against treatment of all anti-HBe–positive carries with HBV DNA levels of 104 copies/mL or more. Female sex, HBV DNA levels of 104 to 105 copies/mL, and the wild-type basal core promoter correlate with the inactive carrier state.
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 8, No. 6
17.
18.
19.
References 1. Liaw YF, Chu CM. Hepatitis B virus infection. Lancet 2009;373: 582–592. 2. Hsu YS, Chien RN, Yeh CT, et al. Long-term outcome after spontaneous HBeAg seroconversion in patients with chronic hepatitis B. Hepatology 2002;35:1522–1527. 3. Chu CM, Hung SJ, Lin J, et al. Natural history of hepatitis B e antigen to antibody seroconversion in patients with normal serum aminotransferase levels. Am J Med 2004;116:829 – 834. 4. Feld JJ, Ayers M, El-Ashry D, et al. Hepatitis B virus DNA prediction rules for hepatitis B e antigen-negative chronic hepatitis B. Hepatology 2007;46:1057–1070. 5. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007;45: 507–539. 6. Liaw YF, Leung N, Kao JH, et al. Asian-Pacific consensus statement on management of chronic hepatitis B: a 2008 update. Hepatol Int 2008:2:263–283. 7. EASL. Clinical practice guidelines: management of chronic hepatitis B. European Association For The Study Of The Liver. J Hepatol 2009;50:227–242. 8. Gish RG, Locarnini SA. Chronic hepatitis B: current testing strategies. Clin Gastroenterol Hepatol 2006;4:666 – 676. 9. Keeffe EB, Dieterich DT, Han SH, et al. A treatment algorithm for the management of chronic hepatitis B virus infection in the United States: 2008 update. Clin Gastroenterol Hepatol 2008; 6:1315–1341. 10. Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different stages of chronic hepatitis B infection. Hepatology 2002;36:1408 –1415. 11. Manesis EK, Papatheodoridis GV, Sevastianos V, et al. Significance of hepatitis B viremia levels determined by a quantitative polymerase chain reaction assay in patients with hepatitis B e antigen-negative chronic hepatitis B virus infection. Am J Gastroenterol 2003;98:2261–2267. 12. Lin CL, Liao LY, Liu CJ, et al. Hepatitis B viral factors in HBeAgnegative carriers with persistently normal serum alanine aminotransferase levels. Hepatology 2007;45:1193–1198. 13. Kumar M, Sarin SK, Hissar S, et al. Virologic and histologic features of chronic hepatitis B virus-infected asymptomatic patients with persistently normal ALT. Gastroenterology 2008;134: 1376 –1384. 14. Chu CM, Liaw YF. Incidence and risk factors of progression to cirrhosis in inactive carriers of hepatitis B virus. Am J Gastroenterol 2009;104:1693–1699. 15. Papatheodoridis GV, Chrysanthos N, Hadziyannis E, et al. Longitudinal changes in serum HBV DNA levels and predictors of progression during the natural course of HBeAg-negative chronic hepatitis B virus infection. J Viral Hepat 2008;15:434 – 441. 16. Tai DI, Lin SM, Sheen IS, et al. Long-term outcome of hepatitis B e antigen-negative hepatitis B surface antigen carriers in relation
20.
21.
22.
23.
24.
25.
26.
27.
28.
to changes of alanine aminotransferase levels over time. Hepatology 2009;49:1859 –1867. Chu CM, Liaw YF. HBsAg seroclearance in asymptomatic carriers of high endemic areas: appreciably high rates during a long-term follow up. Hepatology 2007;45:1187–1192. Lin DY, Sheen IS, Chiu CT, et al. Ultrasonographic changes of early liver cirrhosis in chronic hepatitis B: a longitudinal study. J Clin Ultrasound 1993;21:303–308. Chu CM, Liaw YF. Genotype C hepatitis B virus infection is associated with a higher risk of reactivation of hepatitis B and progression to cirrhosis than genotype B: a longitudinal study of hepatitis B e antigen-positive patients with normal aminotransferase levels at baseline. J Hepatol 2005;43:411– 417. Chu CM, Yeh CT, Lee CS, et al. Precore stop mutant in HBeAgpositive patients with chronic hepatitis B: clinical characteristics and correlation with the course of HBeAg-to-anti-HBe seroconversion. J Clin Microbiol 2002;40:16 –21. Prati D, Taioli E, Zanella A, et al. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Intern Med 2002;137:1–10. Chu CM, Liaw YF. Predictive factors for reactivation of hepatitis B following hepatitis B e antigen seroconversion in chronic hepatitis B. Gastroenterology 2007;133:1458 –1465. Kumar M, Chauhan R, Gupta N, et al. Spontaneous increases in alanine aminotransferase levels in asymptomatic chronic hepatitis B virus-infected patients. Gastroenterology 2009;136:1272– 1280. Martinot-Peignoux M, Boyer N, Colombat M, et al. Serum hepatitis B virus DNA levels and liver histology in inactive HBsAg carriers. J Hepatol 2002;36:543–546. Papatheodoridis GV, Manesis EK, Manolakopoulos S, et al. Is there a meaningful serum hepatitis B virus DNA cutoff level for therapeutic decisions in hepatitis B e antigen-negative chronic hepatitis B virus infection? Hepatology 2008;48:1451–1459. Baptista M, Kramvis A, Kew MC. High prevalence of 1762(T) 1764(A) mutations in the basic core promoter of hepatitis B virus isolated from black Africans with hepatocellular carcinoma compared with asymptomatic carriers. Hepatology 1999;29:946 – 953. Kao JH, Chen PJ, Lai MY, et al. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology 2003;124:327–334. Lai CL, Yuen MF. The natural history and treatment of chronic hepatitis B: a critical evaluation of standard treatment criteria and end points. Ann Intern Med 2007;147:58 – 61.
Reprint requests Address requests for reprints to: Chia-Ming Chu, MD, Liver Research Unit, Chang Gung Memorial Hospital, 199, Tung Hwa North Road, Taipei 10591, Taiwan. e-mail: [email protected]; fax: (886) 3-3272236. Conflicts of interest The authors disclose the following: Yun-Fan Liaw has been involved in clinical trials or served as a global advisory board member of Roche, Bristol-Myers Squibb, GlaxoSmithKline, Novartis, and Gilead Sciences. The remaining authors disclose no conflicts. Funding This study was supported by a grant from the National Science Council of Taiwan (NSC97-2314-B-182-019-MY2).