Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection.
ABSTRACT Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.
- SourceAvailable from: chinagene.cn[Show abstract] [Hide abstract]
ABSTRACT: BMPR-IB gene increases ovulation rate and litter size as a major gene in Chinese Merino, because of 746 A to G mutation. The aim of this study was to make oligonucleotide chips to determine single nucleotide polymorphism (SNP) in BMPR-IB gene. The oligonucleotide chips were manufactured by using six sequence-specific oligonucleotide probes derived from polymorphic regions in the mutation and spotting the probes by microarrayer onto the aldehyde modified glass slides. The 746 A to G mutation was detected with ovine blood in reaction cells of chips. The results were identified by designed the Arraydoctor 2.0 software, in accordance with those of restriction fragment length polymorphisms (RFLP). The results showed the oligonucleotide chips are a parallel, accurate and effective way for screening prolificacy in molecular-assisted selection(MAS)of sheep.Hereditas (Beijing) 09/2007; 29(8):957-62.
- [Show abstract] [Hide abstract]
ABSTRACT: The early emergence of lamivudine (3TC)-resistant tyrosine-methionine-aspartate-aspartate (YMDD) mutants has been reported during 3TC therapy in patients with chronic hepatitis B (CHB) in hepatitis B virus (HBV)-endemic areas; however, its clinical impact during long-term 3TC therapy is unknown. This study was performed to investigate the impact of the early emergence of YMDD mutants 3 months after the initiation of treatment on the outcomes of long-term 3TC therapy in HBV e antigen (HBeAg)-positive CHB. We analysed YMDD genotypes in consecutive samples from 30 patients with HBeAg positive CHB throughout 3TC treatment using both restriction fragment length polymorphism and mass spectrometric assays. Long-term outcome was compared between patients who had YMDD mutations detected at 3 months and those who had no mutations. YMDD mutation was detected in 16 (53.3%) out of 30 patients at 3 months and only the rtM2041 mutation was found. Cumulative HBeAg loss rates at 3 years was 12.5% and 57.4% in patients who had the rtM2041 mutant and wild-type virus at 3 months, respectively (P=0.010). Cumulative viral breakthrough rates at 3 years was 75.0% and 14.3% in patients who had the rtM204I mutant and wild-type virus at 3 months, respectively (P=0.002). Logistic regression revealed that YMDD mutation at 3 months was significantly related to viral breakthrough within 24 months (P=0.003). In conclusion, early detection for HBV YMDD mutation at 3 months may be useful to predict the long-term outcomes of 3TC therapy in patients with HBeAg-positive CHB in HBV-endemic areas.Antiviral therapy 02/2006; 11(4):447-55. · 3.07 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: To determine the distribution of viral genotypes for primary or acquired lamivudine resistance. A total of 283 patients with chronic hepatitis B virus (HBV) infection (245 patients with chronic hepatitis B and 38 inactive hepatitis B surface antigen carriers) were included in the study. The HBV genotype was determined by using quantitative real-time polymerase chain reaction and sequence analysis, and tyrosine-methionine-aspartate-aspartate (YMDD) motif mutations were determined using the reverse transcriptase hybridization method. Lamivudine resistance was determined in a total of 25 (10.7%) chronic hepatitis B patients. Eight subjects (4%) had primary resistance to lamivudine, and 17 (53.1%) had secondary resistance to lamivudine. Genotype D, which was isolated from 267 of the patients with chronic HBV infection, was the dominant genotype in Turkey. Identification of YMDD motif mutations should have a positive impact on the selection of proper antiviral medication for patients, even for those who are nucleoside naïve.World Journal of Gastroenterology 12/2011; 17(45):4987-92. · 2.55 Impact Factor
Lamivudine, a nucleoside analogue, has been known to be
an effective antiviral agent for hepatitis B virus (HBV) infec-
tion.The effectiveness of lamivudine therapy is indicated by
serum HBV DNA clearance and the seroconversion of HBeAg
to anti-HBe. According to follow-up reports of the multi-
center study, seroconversion rates increased with the dura-
tion of treatment from 17% at 1 yr to 27% and 40% at 2
and 3 yr, respectively (1, 2). However, the emergence of viral
mutants resistant to lamivudine is the main concern with
the treatment. The resistance begins to occur after 8 months
of treatment (3, 4) and the resistance rates increase with the
duration of therapy from 17% at 1 yr to 39% and 57% at 2
and 3 yr, respectively (2). The lamivudine-resistant mutants
affect the YMDD motif of the catalytic domain of the HBV
DNA polymerase. The most common mutants have a change
at codon 552 from M to V (M552V) or from M to I (M552I).
Although the mutations of YMDD motif are thought to be
secondary to lamivudine use, it is considered that the pre-
treatmentmutants already exist in endemic regions like Korea
(5). Oligonucleotide chip technology is a very useful tool for
the rapid and accurate identification of pathogenic microbes
and the detection of drug-resistant and point mutations. We
developed an oligonucleotide chip for the detection of mutants
resistant to lamivudine. This study aimed to determine the
prevalence of natural mutants of YMDD motif by oligonu-
cleotidechip technology in patients with chronic HBV infec-
tion who had never received treatment of any antiviral agent
MATERIALS AND METHODS
This study consisted of 40 patients with chronic HBV
infection who had never received any antiviral medication
including lamivudine. Their average age was 29 yr (from
13 to 57 yr), and there were 38 males and 2 females. Thir-
ty-four patients had chronic hepatitis and 6 had cirrhosis.
HBV DNA purification and target DNA preparation
As soon as blood samples were collected they were refrig-
erated for one hour and left to coagulate, which then were
Jeong Heo, Mong Cho, Hyung Hoi Kim*,
Young Min Shin, Hyun Jung Jang
Hee Kyung Park
Gwang Ha Kim, Dae Hwan Kang,
Geun Am Song, Ung Suk Yang
�, Cheol Min Kim
Department of Internal Medicine, and *Department of
Laboratory Medicine, Pusan National University
College of Medicine; �Department of Microbiology,
Pusan National University College of Natural Science;
�Department of Biochemistry, Pusan National
University College of Medicine, Busan, Korea
Address for correspondence
Mong Cho, M.D.
Department of Internal Medicine, Pusan National
University College of Medicine, 1-10 Ami-dong,
Seo-gu, Busan 602-739, Korea
Tel : +82.51-240-7215, Fax : +82.51-244-8180
E-mail : email@example.com
J Korean Med Sci 2004; 19: 541-6
Copyright � The Korean Academy
of Medical Sciences
Detection of YMDD Motif Mutants by Oligonucleotide Chips in
Lamivudine-Untreated Patients with Chronic Hepatitis B Virus Infection
Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral
agent for the treatment of patients with chronic hepatitis B virus (HBV) infection.
However, the YMDD motif mutation of HBV polymerase resistant to lamivudine
occurs very frequently after long term therapy. We developed an oligonucleotide
chip for the detection of YMDD motif mutants resistant to lamivudine and investi-
gated the prevalence of the mutants in patients with chronic HBV infection who
had not been treated by lamivudine before. Forty patients who had not been treat-
ed with lamivudine were included in this study. Serum samples were tested by the
oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and
M552I. Samples were confirmed by restriction fragment length polymorphism
(RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide
chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cir-
rhosis). The results were in accordance with those of RFLP. YMDD motif mutants
occur as natural genome variabilities in patients with chronic HBV infection who
had not been treated with lamivudine before. Oligonucleotide chip technology is a
reliable and useful diagnostic tool for the detection of mutants resistant to antiviral
therapy in chronic HBV infection.
Key Words : Hepatitis, Viral; Hepatitis B, Chronic; Lamivudine; Polymorphism (Genetics); YMDD Motif
Mutants; Oligonucleotide Array Sequence Analysis
Received : 11 November 2003
Accepted : 1 April 2004
J. Heo, M. Cho, H.H. Kim, et al.
centrifuged at 3,000 rpm for 5 min to separate the sera. These
sera were stored at -70℃, and HBV DNA was extracted by
alkaline methods. Eight L of 1 M NaOH and 72 L of fresh
defrosted serum were placed in a sterilized 1.5 mL test tube,
which was shaken for 10 sec and then left at 37℃ for one
hour. In this 80 L mixture of serum and NaOH, 0.1 MHCl
of the same amount was added and shaken for 10 sec, which
was then centrifuged at 12,000 rpm for 10 min. The super-
natant was transferred to a new test tube and storedat -20℃.
Two L of the DNA isolated from this process was used for
each polymerase chain reaction (PCR).
Amplification of the target DNA to detect YMDD-mutant
HBV was prepared by two biotin-modified primers, HBF2-
biotin and HBR-biotin (Table 1). The primers and probes
used in this study were oligonucleotide manufactured by the
Perkin-Elmer DNA synthesizer (BioBasic, Ontario, Canada).
PCR reactants consisted of 500 mM KCl, 100 mM Tris HCl
(pH 9.0), 1% Triton X-100, 0.2 mM dNTP (dATP, dGTP,
dTTP and dCTP), 1.5 mM MgCl2, 20 pmol primer and 1 U
Taq DNA polymerase (QIAGEN, CA, U.S.A.). This mix-
turewas constituted in total to 25 L by adding 2 L of tem-
plate DNA. It was left to react at 95℃ for 3 min, enough to
denature, followed by 30 cycles of reactions at 95℃ for 1
min, 50℃ for another minute, and at 72℃ for 1 min. The
last cycle was extended for 10 min at 72℃. After these reac-
tions, this PCR product was verified to be 180 bp by elec-
trophoresis with 2% agarose gel, stained with ethidium bro-
mideand visualized on a gel documentation system (gel doc
2000, Bio-Rad, CA, U.S.A.).
The design of oligonucleotide DNA chip
One probe (HB552-WM) was to detect wild-type YMDD
motif and four kinds of probes (HB552-MV, HB552-MI1,
HB552-MI2 and HB552-MI3) were to detect YMDD mutant
on the bases of the HBV DNA polymerase sequence (Table
1). The 5 -end of each oligonucleotide probe was aminated
to increase the efficacy of the immobilization of the oligonu-
cleotideto a glass slide. The probes were diluted to 50 pmol
with 3×SCC (300 mM NaCl, 30 mM Na-citrate, pH 7.0)
solution. These probes were printed in duplex arrays as shown
in Fig. 1, and were adhered to the silylated glass slide (Cel
Associates, Texas, U.S.A.) using the Microarrayer (PLXSYS
7500 SQXL Microarryer, Cartesian Technologies, CA, U.S.A.).
To get rid of unattached probes on the surface, the glass
slide was washed with 0.2% SDS and then with distilled
water twice, followed by sodium borohydride solution (300
mL of PBS, 100 mL of ethanol and 1 g of Na2BH4), and finally
washed out with boiling distilled water. The slide was washed
using 0.2% SDS again and then using distilled water at room
The detection of the mutants using the oligonucleotide
The biotin-labeled target DNA was boiled for 10 min and
annealed at 4℃for 10 min to separate the two strands. Hybri-
dization of 2 L of biotin-labeled ssDNA samples to the oligo-
nucleotide chip was performed in hybridization solution, 3 L
of 20×SSPE, 1.35 L of 22.2 M formamide, 0.5 L of bovine
serum albumin (BSA) and 0.1 L of salmon sperm DNA
containing 0.06 L of diluted Cy3-labeled streptavidin (Amer-
sham Pharmacia Biotech, NJ, U.S.A.). Each sampling was
placed on the slide, the cover slip being applied carefully so
that no air bubble was trapped inside, and was left for reac-
tionat 40℃for 30 min. After removing the cover slip using
2×SSC, the slide was washed out using 2×SSC and 0.2×
SCC solution in this order. The GenePix 4000A (Axon Instru-
ments, CA, U.S.A.), non-confocal laser scanner was used for
oligonucleotide chip analysis (543 nm for excitation of Cy3).
The variable sequences are in bold and italic print. W means wild type.
M means mutant type.
*Characters following W or M mean amino acid type encoded by codon
at mutation region. V, valine; I, isoleucine.
Primer and probe*Sequence
Table 1. Primers and probes used in YMDD motif mutant detec-
Fig. 1. This schematic illustration shows the probe layout for the
17 mer oligonucleotide sequences noted in Table 1 and the inter-
pretative results of oligonucleotide chips for YMDD motif mutation
detection. The oligonucleotide probes are patterned as duplex
arrays. ① Positive is mixture of HB552-WM, HB552-MV, HB552-
MI1, HB552-MI2 and HB552-MI3 probes (Table 1), ② 522WM,
③ 552MI3, ④ 552MI2, ⑤ 552MI1 and ⑥ 552MV indicates HB552-
WM, HB552-MV, HB552-MI1, HB552-MI2 and HB552-MI3 probes,
WildYIDD (I1) YIDD (I2) YIDD (I3)YVDD
Duplex spotted probes
YMDD Motif Mutants in Untreated Patients with Chronic HBV Infection
Restriction fragment length polymorphism (RFLP) of
YIDD (I3) mutant
To confirm the results of the oligonucleotide chip experi-
ment, the partial polymerase gene containing YMDD was
analyzed with the restriction fragment length polymorphism
(RFLP) method and the PCR direct-sequencing.
A PCR reactant solution, which consisted of 500 mM KCl,
100 mM Tris HCl (pH 9.0), 1% Triton X-100, 0.2 mM
dNTP (dATP, dGTP, dTTP and dCTP), 1.5 mM MgCl2,
10 pmol primer (BF108 and BR112, Table 2) (4) and 1 U
Taq DNA polymerase (QIAGEN, CA, U.S.A.), was added
to 2 L of HBV DNA purified by the alkaline method to
make it 25 L in total. This mixture solution was left to dena-
tureat 94℃ for 4 min, followed by 35 cycles of 1 min at 94
℃, 1 min at 58℃ and 1 min and 30 sec at 72℃. For the
last cycle, it was left for 10 min at 72℃. The second PCR
was performed under the same circumstances as the first PCR
using 1 L of the first PCR product, YNSspI and BR109
primer. This product was electrophoresed with 2% agarose
gel to confirm that the PCR product was about 160 bp. Six
L of this confirmed product was added to 5 units of the
restriction enzyme SspI (TaKaRa, Shiga, Japan) and SspI buffer
and left for reaction at 37℃ for 3 hr, which was again con-
firmed by electrophoresis with 4% agarose gel.
RFLP of YVDD mutant
As described above, the PCR was performed with BF107
and TMPpu10I primers (the first PCR) and BF111 and TMA-
paLI primers (the second PCR) (Table 2). This product was
electrophoresed with 2% agarose gel to confirm that the PCR
product was about 190 bp. Six L of this confirmed product
was added to 5 units of the restriction enzyme ApaLI (TaKaRa,
Shiga, Japan) and ApaLI buffer and left for reaction at 37℃
Primer Sequence Direction
5 -TTTCCCCCACTGTTTGGCTTTCA- 711-740
TMPpu10I 5 -CAGACTTGGCCCCCAATACCA-
BF 111 5 -TCGGACGGAAACTGCACTTG-3
TMApaLI 5 -CAGACTTGGCCCCCAATACCA-
Table 2. Primers used in sequencing and enzyme digestion
Fig. 2. Sensitivity and specificity of the detection of the YMDD
mutants using oligonucleotide chip. (A) Detection limit of the
YMDD mutants by oligonucleotide chip, (B) Detection capaci-
ty of mixture of wild-type and YMDD motif mutants. YMDD:
YVDD represents ratios of YMDD and YVDD; YMDD:YIDD3
represents ratios of YMDD and YIDD3.
J. Heo, M. Cho, H.H. Kim, et al.
for 3 hr, which was again confirmed by electrophoresis with
4% agarose gel.
Sequencing and sequence analysis
PCR was performed with BF105 and BR112 primers
(Table 2) under the same condition as target DNA prepara-
tion. The sequence of PCR products was determined with
BF111 and BR109 primers, and using the ABI prism BigDye
Terminator cycle sequencing Ready Reaction Kit (Applied
Biosystem Inc., CA, U.S.A.) in the automated BaseStation-
51 DNA fragment analyzer (MJ Research, CA, U.S.A.).
Sensitivity and specificity of oligonucleotide chip
After serially diluting samples (106-103copies/mL) with
known HBV DNA concentrations by the Cobas Amplicor
HBV Monitor testTM‚ (Roche Diagnostics Systems, Meylan,
France), the detection limit of the DNA chip was evaluated.
The detection limit obtained by a DNA chip assay was 104
copies/mL of serum by the first PCR (Fig. 2A) and 103copies/
mL by nested PCR (data not shown). We constructed plas-
mids containing a copy of the YMDD, YVDD and YIDD
of the polymerase gene of HBV. The YMDD, YVDD and
YIDD plasmids (cloned HBV DNA) were used to deter-
minethe capacity of the DNA chip to detect wild-type HBV
and YMDD motif mutant in a single sample. We could detect
the YMDD motif mutant with the DNA chip when the
mutant was mixed with a ten-fold-higher and -lower amount
of wild-type HBV (Fig. 2B).
Detection of YMDD Mutants by oligonucleotide chip
Thirty seven patients out of 40 had the wild-type YMDD
mutants, and 3 of them (7.5%) had the mixed type of YMDD
and YIDD (I3). Among 3 patients with any mutation (24th,
27th and 37th samples), 2 had chronic hepatitis and one had
liver cirrhosis (Table 3, Fig. 3).
Detection of YMDD mutants by RFLP and direct sequencing
When the codon 552 of the second PCR product by the
TMApaLI primer was GTG (GTGCAC), there was no band
seen, and was 30 bp shorter by the limiting enzyme ApaLI,
throughout the samples. When the codon 552 of the second
PCR product using the YNSspI primer was ATT (AATATT),
about 30 bp were cut by the limiting enzyme SspI, and this
shortened band was confirmed by electrophoresis with 4%
agarose gel. Out of the total samples, shortened bands were
observed in sample Nos. 24, 27 and 37, which were identi-
cal to YIDD mutants (ATT) detected by the oligonucleotide
chip. No shortened band was seen in the other samples (Fig. 4).
The PCR direct-sequencing method was unable to prove
YMDD mutation, which was confirmed by the oligonucleo-
tide chip (Fig. 5).
The YMDD motif mutant of the polymerase gene is fre-
quently found in patients who used lamivudine on a long
term basis, and it is usually reported to emerge after 8 months
of therapy (2, 6, 7). The selection of a mutation in the YMDD
motif is known to occur several months earlier than the phe-
notypic resistance, however the information about how early
the genotypic resistance occurs and what causes the resistance
is limited due to the small number of subjects and differences
in detection methods (3, 4, 8). Hence the rate of hepatitis B
resistance to lamivudine might increase further when more
sensitive tests are used to detect the small amount of YMDD
mutants among the wild type HBV, which would impact
upon detection and monitoring the resistance.
This study demonstrated by oligonucleotide chip technolo-
Patient No. Age (yr) Sex ALT (IU/L) HBeAg Anti-HBe HBV DNA (copies/mL) Diagnosis
Table 3. Clinical features of patients with YMDD motif mutation (YMDD+YIDD) at the time of sampling
Fig. 3. Scanned images of oligonucleotide chip (A) wild type
[YMDD], (B), (C), (D) YMDD motif mutation [YMDD+YIDD(I3)],
slides of No. 24, 27 and 37, respectively.
gy that the YMDD mutants exist naturally in the patients
with chronic hepatitis B infection who had not received lami-
YMDD mutants have a significantly low replication abili-
ty. It is thought that naturally occurring YMDD mutants
occupy very small portion of total HBV, and very sensitive
investigative tools are required to prove it. Oligonucleotide
chip technology is a very useful tool for the rapid and accu-
rate identification of pathogenic microbes and the detection
of drug-resistant and point mutations. This method has such
sensitivity that it can find rifampin-resistant tuberculosis
resultant from point mutation in amounts of less than 1%
(9). Oligonucleotide chip technology has been expected to
be clinically applicable to detect minor variants in viral quasi-
species and to increase our knowledge of spontaneous viral
genome variability and selection of mutants by antiviral thera-
py(10). We developed the oligonucleotide chip for the detec-
tion of mutants resistant to lamivudine. In the experiment
using reference sequences generated by site-directed muta-
genesis, we confirmed the sensitivity and specificity of probes.
The oligonucleotide chips could specifically detect mutations
in the YMDD motif (codon 552) of the HBV DNA poly-
merase.The chips included three types of probe (five species):
wild-probe (YMDD), valine-probe (YVDD) and isoleucine-
probes (YIDD1, YIDD2 and YIDD3). We could accurately
detect YMDD motif mutants of YMDD, YVDD, YIDD2
and YIDD3 types in sera from chronic HBV-infected patients.
These chip results were in accordance with results from RFLP,
sequence analysis and allele-specific PCR. We could detect
easily as little as 10% of coexisting mutant viruses in wild
The oligonucleotide chip method detected 3 YIDD mutants
in the 40 patients included in this study. Only ATT (YIDD3)
was detected in all three samples. This chip result was con-
sistent with that of the RFLP analysis. The mutants were
not detected by direct-sequencing and this is reasonable when
taking into consideration that naturally occurring YMDD
mutants are a minor portion of the total HBV and that the
standard base sequence determinant method requires more
than 25% of mutants among HBV.
Our study supports that oligonucleotide chip technology
may have an important role as a reliable and useful diagnos-
tic tool for the early detection of mutants resistant to lamivu-
1. Liaw YF, Leung NW, Chang TT, Guan R, Tai DI, Ng KY, Chien
RN, Dent J, Roman L, Edmundson S, Lai CL. Effects of extended
lamivudine therapy in Asian patients with chronic hepatitis B. Asia
Hepatitis Lamivudine Study Group. Gastroenterology 2000; 119:
2. Leung NW, Lai CL, Chang TT, Guan R, Lee CM, Ng KY, Lim SG,
YMDD Motif Mutants in Untreated Patients with Chronic HBV Infection
Fig. 4. (A) YVDD RFLP anal-
ysis in 4% agarose gel does
not show any band digest-
ed by the restriction enzyme
ApaLI. M: 100 bp ladder.
(B) YIDD RFLP analysis in
4% agarose gel shows DNA
bands (arrows) digested by
the restriction enzyme SspI
in 3 YIDD (I3) mutants (sam-
ple Nos. 24, 27 and 37). M:
100 bp ladder.
M 1 2 3 . . . . .
23 M 24 27
. . . . .
Digested DNA bands by SspI
No digested band by ApaLI
Fig. 5. The sequences of 290 bp DNA including codon 552 rep-
resent only the wild-type (ATG) in the samples with YIDD mutants
(No. 24, 27 and 37).
Wu PC, Dent JC, Edmundson S, Condreay LD, Chien RN. On behalf
of the 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. Hep-
atology 2001; 33: 1527-32.
3. Lau DT, Khokhar MF, Doo E, Ghany MG, Herion D, Park Y, Klein-
er DE, Schmid P, Condreay LD, Gauthier J, Kuhns MC, Liang TJ,
Hoofnagle JH. Long- term therapy of chronic hepatitis B with lamivu-
dine.Hepatology 2000; 32: 828-34.
4. Chayama K, Suzuki Y, Kobayashi M, Kobayashi M, Tsubota A,
Hashimoto M, Miyano Y, Koike H, Kobayashi M, Koida I, Arase
Y, Saitoh S, Murashima N, Ikeda K, Kumada H. Emergence and
takeover of YMDD motif mutant hepatitis B virus during long-term
lamivudine therapy and re-takeover by wild type after cessation of
therapy. Hepatology 1998; 27: 1711-6.
5. Paik YH, Chung HY, Ryu WS, Lee KS, Lee JS, Kim JH, Lee CK,
Chon CY, Moon YM, Han KH. Emergence of YMDD motif mutant
of hepatitis B virus during short-term lamivudine therapy in South
Korea. J Hepatol 2001; 35: 92-8.
6. Perrillo R, Rakela J, Dienstag J, Levy G, Martin P, Wright T, Cald-
well S, Schiff E, Gish R, Villeneuve JP, Farr G, Anschuetz G, Crowther
L, Brown N. Multicenter study of lamivudine therapy for hepatitis B
after liver transplantation. Lamivudine Transplant Group. Hepatol-
ogy 1999; 29: 1581-6.
7. Liaw YF, Chien RN, Yeh CT, Tsai SL, Chu CM. Acute exacerba-
tion and hepatitis B virus clearance after emergence of YMDD motif
mutation during lamivudine therapy. Hepatology 1999; 30: 567-72.
8. Nafa S, Ahmed S, Tavan D, Pichoud C, Berby F, Stuyver L, John-
son M, Merle P, Abidi H, Trepo C, Zoulim F. Early detection of
viral resistance by determination of hepatitis B virus polymerase
mutations in patients treated by lamivudine for chronic hepatitis B.
Hepatology 2000; 32: 1078-88.
9. Mikhailovich V, Lapa S, Gryadunov D, Sobolev A, Strizhkov B,
Chernyh N, Skotnikova O, Irtuganova O, Moroz A, Litvinov V, Vla-
dimirskii M, Perelman M, Chernousova L, Erokhin V, Zasedatelev
A, Mirzabekov A. Identification of rifampin-resistant Mycobacteri-
um tuberculosis strains by hybridization, PCR, and lipase detection
reaction on oligonucleotide microchips. J Clin Microbiol 2001; 39:
10. Zoulim F. Detection of hepatitis B virus resistance to antivirals. J
Clin Virol 2001; 21: 243-53.
J. Heo, M. Cho, H.H. Kim, et al.