信州医誌，58⑷：153〜162，2010

Comparison of Hepatitis B Virus DNA, RNA, and Core Related Antigen as
Predictors of Lamivudine Resistance in Patients with Chronic Hepatitis B
Akihiro M ATSUMOTO , Noboru M AKI , Kaname YOSHIZAWA
Takeji UMEMURA , Satoru JOSHITA and Eiji TANAKA ＊1） Department of Medicine, Gastroenterology, Shinshu University School of Medicine
2） R&D Division, Advanced Life Science Institute, Inc.

The clinical usefulness of hepatitis B virus (HBV)DNA, RNA, and core related antigen (HBcrAg)assays
for predicting the appearance of HBV DNA breakthrough was evaluated and compared in patients withchronic hepatitis B undergoing lamivudine therapy.Methods:Thirty six patients with chronic hepatitis B who
received lamivudine therapy for more than 1 year were enrolled. HBV RNA was measured simultaneouslywith HBV DNA (HBV RNA/DNA) using a real‑time detection polymerase chain reaction assay with apreceding step of reverse‑transcription. HBV DNA was measured by an HBV AM PLICOR monitor kit.
HBcrAg was measured using a chemiluminescence enzyme immunoassay. Results: Sixteen patients (44%)
developed HBV DNA breakthrough during the median observation period of 48.4 months (range 7.4‑87.8
months). Afterwards, HBV DNA breakthrough was prospected using the three parameters taken 6 months
after starting lamivudine therapy. The cut‑oﬀlevels for predictions were determined by receiver operating
characteristic curves, and were 2.6 log copies/ml for HBV DNA, 3.8 log U/ml for HBV RNA/DNA,and 4.0
log U/ml for HBcrAg.Sensitivity,speciﬁcity,and accuracyfor predicting HBV DNA breakthrough were 25%,
100%,and 67% respectively for HBV DNA.Similarly,they were 50%,90%,and 72% for HBV RNA/DNA,
and 100%, 40%, and 67% for HBcrAg. Conclusion : Our ﬁndings conﬁrm that HBV DNA is useful for
identifying patients who are at high risk for HBV breakthrough.HBcrAg is useful for isolating those who areat low risk, and HBV RNA/DNA showed predictive characteristics similar to HBV DNA with higher
sensitivity and the highest accuracy. Shinshu Med J 58 : 153 ―162, 2010
(Received for publication March 17, 2010;accepted in revised form May 7, 2010)
Key words:hepatitis B virus, HBV core related antigen, HBV RNA, lamivudine, breakthroughIntroduction

resistant viruses is a major drawback of lamivudine
therapy. Recently, new nucleoside and nu-

Lamivudine (LAM), a nucleoside analogue,

cleotide analogues have been developed, such as

inhibits the replication of hepatitis B virus (HBV),

adefovir dipiboxil and entecavir, which develop

reduces hepatitis,and improves histological ﬁndings

resistant viruses far less frequently thanof the liver during long‑term therapy. Lamivudine
treatment has also been reported to reduce the riskof complicating hepatocellular carcinoma . However, relapse of hepatitis due to the appearance ofAbbreviations:HBV, hepatitis B virus;HBcrAg,hepatitis
B virus core related antigen ;HBV RNA/DNA,hepatitis B
virus RNA and DNA ;LAM,lamivudine;YM DD,tyrosine‑
methionine‑aspartic acid‑aspartic acid ;HBsAg,hepatitis B
virus surface antigen ;HBeAg, hepatitis B virus e antigen ;Corresponding author:Eiji Tanaka

HBeAb, anti‑hepatitis B antibody; RTD‑PCR, real‑time

Department of M edicine, Shinshu University School ofdetection polymerase chain reaction assays;ROC, receiver

Medicine, 3‑1‑1, Asahi, Matsumoto‑city,

operating characteristic;cccDNA, covalently closed circu-

Nagano‑prefecture, 390‑8621, Japan

lar DNA.

No. 4, 2010153

M atsumoto・ Maki・ Yoshizawa et al.

lamivudine, but these analogues still face the prob-

surface antigen (HBsAg) for more than 6 months

lem of resistant viruses.

with liver histological ﬁndings consistent with

The concentration of HBV DNA in serum

chronic hepatitis. All patients had elevations indecreases and usually becomes undetectable duringserum alanine aminotransferase (ALT) levels, as

lamivudine administration, but can rapidly

well as detectable HBV DNA for at least 6 months.

increase when tyrosine‑methionine‑aspartic acid‑

Immediately prior to LAM administration,23 of theaspartic acid (YM DD)mutations induce lamivudine

patients were positive for HBV e antigen (HBeAg)

resistant strains. Thus, the measurement ofand 13 were positive for anti‑HBV e antibody

HBV DNA is useful for monitoring the anti‑viral(HBeAb) and negative for HBeAg. The HBV

eﬀects of lamivudine, but monitoring these eﬀects

genotype was C in all patients except three (twoby HBV DNA level alone is not satisfactorywere genotype B and one was F).Patients received

because lamivudine resistance occurs even in

100 mg doses of LAM daily for at least 12 months.

patients who show undetectable levels of serum

No patient underwent treatment with anyother anti‑

HBV DNA

viral agent,such as interferon,before or during the.

We previously described a HBV core‑relatedpresent study, and all patients were negative for

antigen (HBcrAg)assay which measures the total

hepatitis C and human immunodeﬁciency virus anti-

amount of protein encoded by the pre‑core and corebodies. Written informed consent was obtained

regions of the HBV genome, including core, e, andfrom each patient. This study was approved by the

p22cr

Ethics Committee of Shinshu University.

antigens. In those experiments, the serum

level of HBcrAg was shown to be useful for predict-Serum samples were collected from the start ofing the occurrence of lamivudine resistance in a

LAM therapy on a monthly basis and were stored

manner diﬀerent from serum levels of HBV DNA .

frozen at −20°
C or below until assayed.The occur-

We also reported that serum HBV RNA is detect-rence of LAM resistance was deﬁned as a rapid

able in a form incorporated into virus particles inincrease in serum HBV DNA with the appearance

patients with hepatitis B undergoing lamivudineof the YMDD mutations.Using these criteria,resis-

therapy,which could possibly represent a new viraltance appeared in 16 (44%)of the 36 patients. The

marker of diﬀerent signiﬁcance than that of HBV

median period from the start of LAM therapyto the

DNA in lamivudine therapy .

occurrence of resistance was 18.2 months, with aThus,in the present study,we sought to compare
the clinical usefulness of using HBV DNA, HBV

range of 7.4 to 57.7 months.
Routine laboratory tests

RNA, and HBcrAg to predict the occurrence ofSerological markers for HBV, including HBsAg,

lamivudine resistance, as detected by HBV DNA

HBeAg, and HBeAb, were tested using commer-

breakthrough in patients with chronic hepatitis B

cially available enzyme immunoassay kits (Abbott

receiving lamivudine therapy.

Japan Co.,Ltd.,Tokyo,Japan).Six HBV genotypes

Patients and Methods
Patients(A‑F) were evaluated according to the restriction
patterns of DNA fragments from the method reported by Mizokami et al . The YMDD motif, aA total of 36 patients with chronic hepatitis B

LAM ‑resistant mutation in the active site of HBV

who started LAM therapy at Shinshu University

polymerase, was detected using an enzyme‑linked

Hospital between July 2002 and February 2004 weremini‑sequence assay kit (HBV YM DD Mutation

enrolled in this study.They consisted of 28 men and

Detection Kit, Genome Science Laboratories Co.,

8 women and had a median age of 55 years (rangeLtd.,Tokyo,Japan) .Serum concentration of HBV

29 ‑80 years)at the commencement of LAM therapy.

DNA was determined using an AM PLICOR HBVChronic hepatitis B was deﬁned as positive for HBV

monitor kit (Roche, Tokyo, Japan), which had a154

信州医誌 Vol. 58

Prediction of YMDD mutation by HBV RNA and HBcrAg

quantitative range of 2.6 to 7.6 log copies/ml.

the mixture was transferred into a High Pure ﬁlter

HBV core‑related antigen assay
Serum HBcrAg

was measured using

tube ﬁtted with a collection tube.The ﬁlter tube wasacentrifuged for 1 min at 8,000 rpm in a standard

chemiluminescence enzyme immunoassay (CLEIA)

tabletop centrifuge at room temperature, thenas reported previously .In brief,100 μl aliquots ofattached to a new collection tube. An inhibitor

serum were mixed with pretreatment solution

removal buﬀer (5 M guanidine‑HCl, 20 mM Tris‑

containing 15% sodium dodecylsulfate. After incu-

HCl ［pH 6.6］ in ethanol)was added to the upperbation at 70°
C for 30 min, 50 μl pretreated serum

reservoir and the tube was centrifuged again for 1

was added to wells coated with monoclonal anti-

min at 8,000 rpm.After being washed with 250μl ofbodies against denatured HBV core and e antigens

wash buﬀer (20 mM NaCl, 2 mM Tris‑HCl ［pH(HB44, HB61, and HB114) and ﬁlled with 100 μl

7.5］ in ethanol), the ﬁlter was placed in a new

assay buﬀer.The mixture was then incubated for 2

collection tube and 50 μl of RNase‑and DNase‑free

hrs at room temperature.After washing with buﬀer,

water was added to elute the DNA and RNA.After

either alkaline phosphatase‑labeled HB50 mono-

centrifugation for 1 min at 8,000 rpm, the eluted

clonal antibody (speciﬁc for denatured HBV core

DNA and RNA was stored at −80°
C. Synthesis of

antigen) or a mixture of HB91 and HB110 mono-

cDNA was performed at 42°
C for 30 min in a 20 μl

clonal antibodies (speciﬁc for denatured HBV core

reaction mixture containing 10 μL of the extracted

and e antigens) were added to wells and incubated

DNA and RNA, 50 mM Tris‑HCl (pH 8.3), 75 mM

for 1 hr at room temperature.After washing again,

KCl,3 mM M gCl ,1 mM dNTP (1 mM each dATP,

CDP‑Star with Emerald Ⅱ (Applied Biosystems,

dGTP, dCTP and dTTP), 1 mM DTT, 100 nM

Bedford,M A)was added and plates were incubated‑reverse primer for the HBV surface gene (5ʼfor 20 min more at room temperature.The relative

GGTTGGTGAGTGATTGGAGGTT‑3ʼ
; nt. 345 tochemiluminescence intensity was measured, and324), 40 units of RNasin (TaKaRa, Kyoto, Japan),

HBcrAg concentrations were read by comparisonand 200 units of SuperScript Ⅱ RNase H‑ Reverse

to a standard curve generated with recombinant pro‑Transcriptase (Invitrogen,Carlsbad,CA).The reac-

hepatitis B e antigen (amino acids ‑10 to 183 of thetion mixture was inactivated by heating to 70°
C forprecore/core gene product). The concentration of

15 min, then cooled to ‑80°
C until real‑time detec-

HBcrAg was expressed as units/mland theimmune‑tion polymerase chain reaction (RTD‑PCR)assays.

reactivity of recombinant pro‑hepatitis B e antigen

A 4 μl aliquot of DNA and cDNA solution was used

at 10 fg/ml was deﬁned as 1 unit/ml. The lower

for RTD‑PCR,which was performed with the Lightdetection limit of this assay was set at 2 log units/

Cycler System (Roche Diagnostics) as reported

ml. Sera containing over 7 log units/ml of antigen

previously . The two primers and TaqMan probe

were diluted 10 or 100 fold in normal human serum

used were designed from a region of the HBVand measured again to obtain the end titer.

‑ACAACATCAGsurface gene:forward primer;5ʼ

HBV RNA/DNAGATTCCTAGGAC‑3ʼ(nt. 166 to 187), reverse

The High Pure Viral Nucleic Acid Kit (Roche

primer as stated above(nt.345 to 324),and TaqMan

Diagnostics) was used for isolation of HBV DNA‑FAM ‑CAGAGTCTAGACTCGTGGTGprobe; 5ʼ

and RNA from serum.Brieﬂy,200 μl of serum was

GACTTC‑TAM RA‑3ʼ(nt. 244 to 269). An HBVadded to 250 μL of freshly prepared working solu

genome (nt.20 to 1805)subcloned into a pUC vector

tion (6 M guanidine‑HCl,10 mM urea,10 mM Tris‑

was used as an internal standard. The lower detec-

HCl ［pH 4.4］ and 20% ［vol/vol］ Triton X‑100)tion limit for the HBV RNA/DNA assay was set at

supplemented with 20 μg of poly (A) carrier RNA

2.6 log copy/ml.

and 900 μg of Proteinase K.After incubation for 10
min at 72°
C, 100 μl of isopropanol was added andNo. 4, 2010

Statistical analysis
The proportion of each clinical factor was155

M atsumoto・ Maki・ Yoshizawa et al.

compared between the groups with or without HBVand no signiﬁcant diﬀerences were observed in any

DNA breakthrough using the χ and Fisherʼs exact

other characteristics, including ALT, HBV DNA,

probabilitytests,and group medians were compared

HBcrAg, and HBV RNA/DNA.

using the Mann‑Whitneyʼs U test. Correlations

Changes in serum HBV DNA, HBcrAg andbetween HBV RNA/DNA and HBV DNA or

HBV RNA/DNA

HBcrAg were tested using Spearmanʼ
s analysis.

Changes in serum levels of HBV DNA, HBcrAg,

The rates of HBV DNA breakthrough during LAM

and HBV RNA/DNA from baseline to 6 months oftreatment between higher and lower level groups oflamivudine therapy are shown in Fig.1.HBV DNA

HBV DNA, HBcrAg and HBV direct RT‑PCR

decreased rapidly and became undetectable within 6

were analyzed using the Kaplan‑M eier method,andmonths in all except 4 patients with HBV DNA

the diﬀerence in incidences was assessed with the

breakthrough.HBcrAg decreased more slowly thanlog‑rank test. Receiver operating characteristic

HBV DNA,and became undetectable onlyin 2(6%)(ROC)curves were used to decide each cut‑oﬀpointof the 36 patients at 6 months. HBV RNA/DNA

for predicting HBV DNA breakthrough. All tests

decreased faster than HBcrAg, but slower thanwere performed using the SPSS 10.0 J statistical

HBV DNA,and became undetectable at 6 months insoftware package(SPSS Inc.,Chicago,IL).P values

15 (42%)of the 36 patients.
Although HBV RNA/DNA was signiﬁcantly cor-less than 0.05 were considered signiﬁcant.

related with both HBV DNA (Fig.2A)and HBcrAg

Results(Fig. 2B) at the start of lamivudine therapy, thisComparison of characteristics between

association was lost at 6 months because over 90%

patients with and without HBV DNA break-of patients became undetectable for HBV DNA

through(Fig. 2C). On the other hand, HBV RNA/DNAHBV DNA breakthrough occurred in 16 (44.4%)

retained its correlation with HBcrAg at 6 monthsof 36 patients during the follow‑up period. The(Fig.2D).Although signiﬁcant,the HBcrAg to HBV

cumulative HBV DNA breakthrough incidence in

RNA/DNA ratio tended to be lower when compar-

all patients was 8.3% at 12 months, 27.8% at 24

ed to baseline.

months, 33.5% at 36 months, 40.2% at 48 months,

Prediction of occurrence of lamivudine resis-

and 48.2% at 60 months. The clinical characteris-tance

tics at baseline in the 16 patients with HBV DNA

The occurrence of lamivudine resistance was

breakthrough and 20 patients without are shown innext prospected using the levels of HBV DNA,

Table 1.The median follow‑up period did not diﬀer,

HBcrAg, and HBV RNA/DNA measured at 6

Table 1 Baseline characteristics of patients with and without HBV DNA breakthrough during lamivudine therapy

NumberAge (y.o.)Gender (male/female)
Follow‑up period (months)
HBeAg (＋/−)
HBeAb (＋/−)
ALT (IU/l)
HBV DNA (log copy/ml)
HBcrAg (log U/ml)
HBV RNA/DNA (log U/ml)

with breakthroughwithout breakthrough16
56.0 (29.5−64.0)
13/3
53.2 (21.5−84.0)

20
53.6 (41.0−79.5)
15/5
67.5 (45.8−89.5)

10/6
6/10

9 /11
11/9

60 (22−499)
6.7 (4.8−＞7.6)
5.9 (4.3−8.2)
6.2 (4.8−8.3)

119 (20−1816)
6.1 (3.9 −＞7.6)
5.8 (2.9 −8.7)
6.3 (4.4−8.8)

p

0.660
1.000
0.421
0.508
0.508
0.156
0.338
0.683
0.916

Data are expressed as median (range)
156

信州医誌 Vol. 58

Prediction of YMDD mutation by HBV RNA and HBcrAg

Fig.1 Changes in serum levels ofHBV DNA,HBcrAg,and HBV RNA/DNA from baseline to month 6.The
solid line indicates patients with HBV DNA breakthrough during the observation period, and thebroken line indicates patients without. The horizontal broken line indicates the lower detection limitof each assay.Cut‑oﬀvalues for predicting lamivudine resistance were 2.6 log copies/ml in HBV DNA(the same as the lower detection limit), 4.0 log U/ml in HBcrAg (solid line) and 3.8 log copies/ml in
HBV RNA/DNA (solid line). The upper detection limit of HBV DNA is also shown by a horizontal
broken line at 7.6 log copy/ml because 4 patients showed levels higher than the upper detection limit
at the baseline.

months after starting lamivudine therapy.The cut‑through was compared using a log‑rank testoﬀ values of HBV DNA (2.6 log copies/ml),

between two groups of patients divided by the cut‑

HBcrAg (4.0 log U/ml), and HBV RNA/DNA (3.8

oﬀvalues of HBV DNA (P＜0.0003), HBcrAg (P＝log copies/ml) for prediction of resistance was

0.0088), and HBV RNA/DNA (P＝0.0011) (Fig. 3).

determined by ROC analysis. The sensitivity,

All 4 patients whose HBV DNA levels were higher

speciﬁcity, and accuracy for predicting break-than the cut‑oﬀ showed lamivudine resistancethrough were 25%, 100%, and 67% respectively

within 24 months of the start of therapy. None offor HBV DNA. Similarly, they were 100%, 40%,the 9 patients whose HBcrAg levels were less thanand 67% for HBcrAg,and 50%,90%,and 72% for

the cut‑oﬀshowed lamivudine resistance during the

HBV RNA/DNA. The positive predictive values

follow‑up period. HBV RNA/DNA showed anfor predicting HBV DNA breakthrough by HBVintermediate character between HBV DNA andDNA, HBcrAg, and HBV RNA/DNA combined

HBcrAg in predicting the occurrence of HBV DNA

was 100%,57.1%,and 80.0% respectively.Similar-

breakthrough.

ly, the negative predictive values were 63.6%,
100%, and 62.5%.
The cumulative occurrence of HBV DNA breakNo. 4, 2010157

M atsumoto・ Maki・ Yoshizawa et al.

Fig.2 Correlation between the levels of the three parameters at baseline and at 6 months after starting
lamivudine administration :(A) between HBV DNA and HBV RNA/DNA at baseline, (B) between
HBcrAg and HBV RNA/DNA at baseline,(C)between HBV DNA and HBV RNA/DNA at 6 months,
and (D) between HBcrAg and HBV RNA/DNA at 6 months. Closed squares indicate patients withbreakthrough during the observation period, and open circles indicate patients without.

Discussion

RNA and second‑strand DNA synthesis then occurs
in the cytoplasm within viral capsids formed by the

HBV is an enveloped DNA virus containing a

HBV core protein. Because lamivudine inhibits

relaxed circular DNA genome that is converted

reverse transcription of pregenomic RNA,it direct-

into a covalently closed circular DNA (cccDNA)ly suppresses production of HBV virions,and serum

episome in the nucleus of infected cells which serves

HBV DNA levels decrease rapidly after the initia-

as a transcriptional template for the production oftion of lamivudine administration. On the otherviral RNA. Reverse transcription of pregenomic

hand,the amount of cccDNA decreases quite slowly

158

信州医誌 Vol. 58

Prediction of YMDD mutation by HBV RNA and HBcrAg

Fig.3 Cumulative occurrence of HBV DNA breakthrough between two groups of patients classiﬁed by theselected cut‑oﬀvalues of HBV DNA (2.6 log copy/ml),HBcrAg (4.0 log U/ml),and HBV RNA/DNA(3.8 copy/ml).

after commencement of nucleoside analogues.Intra-

binding to the encapsidation signal, epsilon, and ahepatic HBV cccDNA has been reported to besecondary structure on the pregenomic RNA,which

superior to serum HBV DNA in predicting virologic

is then complexed with coreproteins to form nucleo‑

response to nucleoside/nucleotide analogue ther-

capsids. The polymerase‑epsilon interaction is also

apies, such as lamivudine. However, the mea-

the ﬁrst step in initiating reverse transcription ofsurement of cccDNA seems ill‑suited for clinicalpregenomic RNA to yield the negative DNA strand

use because it requires a liver biopsy and compli-of the viral genome

cated measurements.The HBcrAg assay developed

esize that HBV particles containing pregenomic

by our research group has been shown to be useful

HBV RNA are produced rather than those contain-

for identifying patients who are at low risk ofing

developing lamivudine resistance during therapy ,Lamivudine inhibits reverse transcription ofas well as those who are at low risk of hepatitis

pregenomic RNA, suggesting that HBV particles

reactivation after cessation of lamivudine adminis-

containing HBV RNA are produced and may

tration. The serum HBcrAg levels were wellaccount for the majority of HBV particles .

correlated with intrahepatic cccDNA level after

Accordingly, it is also possible that serum HBV

HBV DNA became undetectable during anti‑viral

RNA reﬂects intrahepatic cccDNA and is useful fortherapy . Here, serum HBcrAg may reﬂect thepredicting the occurrence of lamivudine resistance.

intrahepatic cccDNA better than serum HBV DNA

Recently, Hatakeyama et al. reported that serum

under lamivudine therapy since lamivudine inhibits

HBV RNA was a predictor of early emergence ofthe synthesis of HBV DNA from pregenomic RNA

the YMDD mutant in patients treated withtranscribed from cccDNA, but does not inhibit

lamivudine .In a previous study,we demonstrated

synthesis of viral proteins which are translated

a method to measure serum HBV RNA only by

from viral mRNA directly.

eliminating HBV DNA. However, this method is. Therefore, we can hypoth-

HBV DNA during

lamivudine therapy.

M aturation of the HBV genome occurs in nucleo‑

prohibitively complicated for testing many samples

capsids.Viral polymerase initiates encapsidation bybecause it includes a digestion step with DNAase.

No. 4, 2010159

M atsumoto・ Maki・ Yoshizawa et al.

As such, HBV DNA and RNA were measured

wider range of patients.

simultaneously in the present study by eliminating

Lamivudine has already been eliminated fromthe digestion step. Because the proportion of HBV

ﬁrst line therapy in naive chronic hepatitis B

DNA to HBV RNA at 6 months was quite low,wepatients due to a higher incidence of developing

believe that the possibility of HBV RNA as a

resistant mutations than new antiviral agents,suchpredictor for lamivudine resistance could be tested.

as adefovir dipivoxil and entecavir. However, aThis study compared the abilities of HBV DNA,considerable number of patients who began

HBV RNA/DNA, and HBcrAg for predicting

lamivudine administration in the past still take thisoccurrence of lamivudine resistance. Lamivudine

treatment, so the present study may be valuable toresistance was monitored by HBV DNA break-such patients when theyconsider changing therapies

through because it is more sensitive than hepatitis

in the future.For example,lamivudine patients who

breakthrough in detecting

resistance. The

show low levels of HBV RNA/DNA or HBcrAg do

speciﬁcity of HBV DNA breakthrough was

not necessarily need to change their therapy to a

conﬁrmed by the existence of YMDD mutations.

new antiviral regimen.Additionally,since the main

HBV DNA appears useful for detecting patientsmechanisms of suppressing HBV replication are

who are at high risk of developing lamivudine resis-

similar among lamivudine, entecavir, and adefovirtance,but not for selecting patients who are at low

dipivoxil,it is possible that HBV DNA,HBV RNA/risk because the positive predictive value was asDNA, and HBcrAg are useful for monitoring thehigh as 100% and sensitivity as low as 25%.On theantiviral eﬀects of these drugs as well. However,contrary, HBcrAg was useful for identifying

further studies are required to determine whether

patients who are at low risk of lamivudine resis-

these three assays are indeed applicable to antiviraltance,but not for patients who are at high risk sinceagents other than lamivudine.

the negative predictive value was as high as 100%

In conclusion,monitoring HBV DNA is useful forand speciﬁcity as low as 40%. HBV DNA break-identifying patients with chronic hepatitis B underthrough did not occur until 60 months from the startlamivudine therapy who are at high risk ofof lamivudine therapy in 9 patients whose HBcrAg

lamivudine resistance, and measurement of

was less than 4.0 log U/ml at 6 months of therapy.

HBcrAg is useful for isolating those who are at low

The abilityof HBV RNA/DNA for predicting therisk of HBV DNA breakthrough. The predictive

occurrence of lamivudine resistance landed between

characteristics of HBV RNA/DNA are similar to

those of HBV DNA and HBcrAg ;both the positivethat of HBV DNA with higher sensitivity,and show(80%) and negative (63%) predictive values were

the highest accuracy.

intermediate. The accuracy (72%) of HBV RNA/

Acknowledgement

DNA was highest among the three parameters.
Thus, HBV RNA/DNA is presumed to have theThis research was supported in part bya researchpredictive characteristics of both HBV DNA andgrant on hepatitis from the Japanese M inistry of

HBcrAg, with the additional feature of including a

Health, Labor and Welfare (no. 19590757).

References
1) Liaw YF,Sung JJ,Chow WC,Farrell G,Lee CZ,Yuen H,Tanwandee T,Tao QM,Shue K,Keene ON,Dixon JS,
Gray DF, Sabbat J :Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med
351:1521‑1531, 2004
2) M atsumoto A,Tanaka E,Rokuhara A,Kiyosawa K,Kumada H,Omata M ,Okita K,Hayashi N,Okanoue T,Iino
S, Tanikawa K : Eﬃcacy of lamivudine for preventing hepatocellular carcinoma in chronic hepatitis B : A
multicenter retrospective study of 2,795 patients. Hepatology Research 32:173‑184, 2005160

信州医誌 Vol. 58

Prediction of YMDD mutation by HBV RNA and HBcrAg
3) Leung NW,Lai CL,Chang TT,Gran R,Lee CM,Ng KY,Lim SG,Wu PC,Dent JC,Edmundson S,Condreay LD,
Chien RN ;Asia Hepatitis Lamivudine Study Group : Extended lamivudine treatment in patients with chronic
hepatitis B enhances hepatitis B e antigen seroconversion rates:results after 3 years of therapy.Hepatology 33:
1527‑1532, 2001
4) Liaw YF, Chien RN, Yeh CT, Tsai SL, Chu CM : Acute exacerbation and hepatitis B virus clearance after
emergence of YMDD motif mutation during lamivudine therapy. Hepatology 30:567‑572, 1999
5) Kim JW, Lee HS, Woo GH, Yoon JH, Jang JJ, Chi JG, Kim CY :Fatal sub massive hepatic necrosis associatedwith tyrosine‑methionine‑aspartate‑aspartate‑ motif mutation of hepatitis B virus after long‑term lamivudine
therapy. Clin Infect Dis 33:403‑405, 2001
6) Villet S, Pichoud C, Villeneuve JP, Trepo C, Zoulim F :Selection of a multiple drug‑resistant hepatitis B virus
strain in a liver‑transplanted patient. Gastroenterology 131:1253‑1261, 2006
7) Yim HJ, Hussain M, Liu Y, Wong SN, Fung SK, Lok AS :Evolution of multi‑drug resistant hepatitis B virus
during sequential therapy. Hepatology 44:703‑712, 2006
8) Lee YS, Suh DJ, Lim YS, Jung SW, Kim KM , Lee HC, Chung YH, Yoo W, Kim SO :Increased risk of adefovir
resistance in patients with lamivudine‑resistant chronic hepatitis B after 48 weeks of adefovir dipivoxil monotherapy. Hepatology 43:1385‑1391, 2006
9) Dienstag JL,Goldin RD,Heathcote EJ,Hann HW,Woessner M,Stephenson SL,Gardner S,Gray DF,SchiﬀER :Histological outcome during long‑term lamivudine therapy. Gastroenterology 124:105‑117, 2003
10) Dienstag JL, Perrillo RP, SchiﬀER, Bartholomew M , Vicary C, Rubin M :A Preliminary trial of lamivudine forchronic hepatitis B infection. N Engl J M ed 333:1657‑1661, 1995
11) Doong SL,Tsai CH,Schinazi RF,Liotta DC,Cheng YC :Inhibition of the replication of hepatitis B virus in vitro‑dideoxy‑3ʼ
‑thiacytidine and related analogues. Proc Natl Acad Sci U S A 88:8495‑8499, 1991
by 2ʼ
,3ʼ
12) Lai CL, Chien RN, Leung NW, Chang TT, Guan R, Tai DI, Ng KY, Wu PC, Dent JC, Barber J, Stephenson SL,
Gray DF :A one‑year trial of lamivudine for chronic hepatitis B.Asia Hepatitis Lamivudine Study Group.N Engl
J Med 339 :61‑68, 1998
13) Liaw YF, Chien RN, Yeh CT, Tsai SL, Chu CM : Acute exacerbation and hepatitis B virus clearance after
emergence of YMDD motif mutation during lamivudine therapy. Hepatology 30:567‑572, 1999
14) Nafa S, Ahmed S, Tavan D, Pichoud C, Berby F, Stuyver L, Johnson M, M erle P, Abidi H, Trepo C, Zoulim F :Early detection of viral resistance by determination of hepatitis B virus polymerase mutations in patients treatedby lamivudine for chronic hepatitis B. Hepatology 32:1078‑1088, 2000
15) Yuen MF, Kato T, Mizokami M, Chan AO, Yuen JC, Yuan HJ, Wong DK, Sum SM, Ng IO, Fan ST, Lai CL :Clinical outcome and virologic proﬁles of severe hepatitis B exacerbation due to YMDD mutations.J Hepatol 39 :
850‑855, 2003
16) Kumashiro R,Kuwahara R,Ide T,Koga Y,Arinaga T,Hisamochi A,Ogata K,Tanaka K,Sata M :Subclones ofdrug‑resistant hepatitis B virus mutants and the outcome of breakthrough hepatitis in patients treated withlamivudine. Intervirology 46:350‑354, 2003
17) Suzuki F, Tsubota A, Arase Y, Suzuki Y, Akuta N, Hosaka T, Someya T, Kobayashi M , Saitoh S, Ikeda K,
Kobayashi M,M atsuda M,Satoh J,Takagi K,Kumada H :Eﬃcacy of lamivudine therapy and factors associatedwith emergence of resistance in chronic hepatitis B virus infection in Japan. Intervirology 46:182‑189, 2003
18) Zollner B,Schafer P,Feucht HH,Schroter M ,Petersen J,Laufs R :Correlation of hepatitis B virus load with lossof e antigen and emerging drug‑resistant variants during lamivudine therapy. J M ed Virol 65:659 ‑663, 2001
19) Rokuhara A, Tanaka E, Matsumoto A, Kimura T, Yamaura T, Orii K, Sun X, Yagi S, M aki N, Kiyosawa K :Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core‑related antigen ;a marker distinct
from viral DNA for monitoring lamivudine treatment. J Viral Hepat 10:324‑330, 2003
20) Kimura T, Ohno N, Terada N, Rokuhara A, M atsumoto A, Yagi S, Tanaka E, Kiyosawa K, Ohno S, Maki N :Hepatitis B virus DNA‑negative dane particles lack core protein but contain a 22‑kDa precore protein without C‑
terminal arginine‑rich domain. J Biol Chem 280:21713‑21719, 200521) Tanaka E,Matsumoto A,Suzuki F,Kobayashi M,M izokami M ,Tanaka Y,Okanoue T,Minami M,Chayama K,
No. 4, 2010161

M atsumoto・ Maki・ Yoshizawa et al.
Imamura M, Yatsuhashi H, Nagaoka S, Yotsuyanagi H, Kawata S, Kimura T, Maki N, Iino S, Kiyosawa K :M easurement of hepatitis B virus core‑related antigen is valuable for identifying patients who are at low risk oflamivudine resistance. Liver Int 26:90‑96, 2006
22) Rokuhara A, Matsumoto A, Tanaka E, Umemura T, Yoshizawa K, Kimura T, M aki N, Kiyosawa K :Hepatitis
B Virus RNA is Measurable in Serum and can be a New M arker for M onitoring Lamivudine Therapy. J
Gastroenterol 41:785‑790, 200623) M izokami M, Nakano T, Orito E, Tanaka Y, Sakugawa H, M ukaide M , Robertson BH : Hepatitis B virus
genotype assignment using restriction fragment length polymorphism patterns. FEBS Lett 450:66‑71, 1999
24) Kobayashi S,Shimada K,Suzuki H,Tanikawa K,Sata M :Development of a new method for detecting a mutationin the gene encoding hepatitis B virus reverse transcriptase active site(YM DD motif).Hepatol Res 17:31‑42,2000
25) Kimura T, Rokuhara A, SakamotoY, Yagi S, Tanaka E, Kiyosawa K, Maki N :Sensitive enzyme immunoassay
for hepatitis B virus core‑related antigens and their correlation to virus load. J Clin M icrobiol 40:439 ‑445,2002
26) Lee WM :Hepatitis B virus infection. N Engl J M ed 337:1733‑1745, 199727) M ason WS,Halpern MS,England JM,Seal G,Egan J,Coates L,Aldrich C,Summers J :Experimental transmissionof duck hepatitis B virus. Virology 131:375‑384, 1983
28) Summers J,Mason WS :Replication of the genome of a hepatitis B‑‑like virus by reverse transcription ofan RNA
intermediate. Cell 29 :403‑415, 1982
29) Tuttleman JS, Pourcel C, Summers J :Formation of the pool of covalently closed circular viral DNA in hepadnavirus‑infected cells. Cell 47:451‑460, 1986
30) Sung JJ, Wong ML, Bowden S, Liew CT, Hui AY, Wong VW, Leung NW, Locarnini S, Chan HL :Intrahepatic
hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy.Gastroenterology 128:1890‑1897, 200531) Shinkai N, Tanaka Y, Orito E, Ito K, Ohno T, Hirashima N, Hasegawa I, Sugauchi F, Ueda R, M izokami M :M easurement of hepatitis B virus core‑related antigen as predicting factor for relapse after cessation oflamivudine therapy for chronic hepatitis B virus infection. Hepatol Res 36:272‑276, 200632) M atsumoto A, Tanaka E, Minami M , Okanoue T, Yatsuhashi H,Nagaoka S,Suzuki F,Kobayashi M ,Chayama
K,Imamura M ,Yotsuyanagi H,Nakaoka S,Maki N,Kawata S,Kumada H,Iino S,Kiyosawa K :Low serum levelof hepatitis B virus core‑related antigen indicates unlikely reactivation of hepatitis after cessation of lamivudine
therapy. Hepatol Res 37:661‑666, 2007
33) Suzuki F, Miyakoshi H, Kobayashi M , Kumada H : Correlation between serum hepatitis B virus core‑related
antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients. J M ed Virol 81:27‑33,
200934) Hatakeyama T, Noguchi C, Hiraga N, Mori N, Tsuge M , Imamura M ,Takahashi S,Kawakami Y,Fujimoto Y,
Ochi H,Abe H,Maekawa T,Kawakami H,Yatsuji H,Aisaka Y,Kohno H,Aimitsu S,Chayama K :Serum HBV
RNA is a predictor of early emergence of the YMDD mutant in patients treated with lamivudine.Hepatology 45:1179 ‑1186, 2007（2010. 3.17 received；2010. 5. 7 accepted）

162

信州医誌 Vol. 58