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Response to Hepatitis C Therapy
Can Last for Years
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".......The present study with prolonged follow-up of >5
years showed that the clearance of liver HCV RNA was sustained up to 12
years after therapy for all 15 patients. Neither positive strands nor
negative strands were found in any liver biopsy specimens. Sustained
reductions in HCV core antibody titers at a constant rate further
corroborated complete HCV eradication. One report available in the
literature showed that liver HCV RNA was not found in five SVRs 10--11
years after therapy [Lau et al., 1998]. Collectively, these findings
suggest that HCV seroclearance at 6 months after IFN therapy withdrawal
would usually imply virological cure......"
".......All SVRs showed sustained serum HCV RNA clearance during
follow-up, but hepatocellular carcinoma (HCC) developed in 4 (11%) SVRs......Patients
6 and 14 had cirrhosis before treatment, whereas bridging fibrosis was
observed in patients 7 and 19....." (note from Jules Levin: my
understanding is that HCC rates appear to be higher in China; delaying
HCV therapy until a patient has advanced liver disease increases risk
for developing liver cancer despite achieving sustained virologic
response).
".......the present study showed that the SVRs were completely free of
occult HCV and HBV up to a decade after therapy withdrawal. When the
long-term liver histological outcomes were evaluated, the SVRs showed
improvement of the necroinflammatory grade and regression of the
fibrosis stage. Nevertheless, mild liver inflammation persisted in the
majority, the reason for which remains unclear. Intrahepatic
inflammatory response triggered by HCV infection may take a very long
time to cease and may continue in the absence of occult viral
infections. It was also demonstrated that HCC, a late complication after
sustained virological response, occurs without occult HCV or HBV
infection. All HCC patients had an advanced stage of liver fibrosis
before treatment, and it is conceivable that longstanding HCV-related
liver injury had initiated the carcinogenesis process although
persistent low-grade liver inflammation after IFN therapy may have
exerted some influence on the subsequent course......"
"......improvement of serum ALT elevation without HCV seroclearance
should be considered another favorable response to IFN therapy.....
Although further studies with a larger number of patients are necessary,
control of biochemical disease (ALT/AST) activity to near-normal levels
may also confer favorable long-term histological outcomes..... The
present study revealed that the 5-year risk of biochemical flare-up
showing ALT fluctuations of >2 ULN was 41%...... It was shown that
occult HBV was not present in the livers of BRs and did play no role in
the clinical course. Occult HBV is known to be common and exert
virulence in chronic hepatitis C patients, but its clinical relevance
after IFN therapy may be less significant. The flare-up was easily
controlled by retreatment with IFN. Paired liver biopsies showed that
necroinflammation was ameliorated and that the fibrosis stage remained
unchanged....."
NEW YORK (Reuters Health) - A sustained response to successful treatment
with interferon for hepatitis C virus (HCV) infection can persist for up
to 12 years, according to a report in the November issue of the Journal
of Medical Virology.
Previous studies have shown remission for up to 5 years after successful
treatment with interferon, the authors explain, but HCV eradication from
the liver has not been well validated in longer follow-up. Dr. Natsuko
Tsuda from Osaka National Hospital, Osaka, Japan and colleagues
monitored 38 patients who had a virological response to interferon
therapy, defined as clearance of HCV from the blood after 6 months.
Thirty-seven patients who had a biochemical response were also
monitored. These patients were defined as having normal liver function
test results after 6 months of therapy, but detectable levels of virus
in the blood.
The subjects were followed for 4.4 years to 12 years after interferon
therapy. All sustained virological responders remained persistently
negative for HCV in the blood during the entire follow-up period, the
authors report. The biochemical responders continued to have HCV
detected in the blood and nearly half (46 percent) experienced flared
ups. Four sustained virological responders (but no biochemical
responders) developed liver cancer (between 6 months and 5.5 years after
treatment), the report indicates. However, all four patients had
advanced liver disease before treatment. HCV was not detected in biopsy
samples from 15 sustained virological responders taken 5.9 to 12.5 years
after pretreatment biopsies, the researchers note. However, all but one
of the 15 biochemical responders who had repeat biopsies had HCV
detected in the liver. In evaluations of liver tissue samples, all
measures improved significantly in the sustained virological responders,
whereas only partial improvements were observed the biochemical
responders. In both groups, evidence of at least mild inflammation
remained in the biopsy tissue samples after treatment.
The results suggest that clearance of HCV by 6 months after interferon
therapy indicates a "virological cure," the authors conclude. "Although
further studies with a larger number of patients are necessary, control
of biochemical disease activity to near-normal levels may also confer
favorable long-term...outcomes," they add.
SOURCE: Journal of Medical Virology, November 2004.
Long-term clinical and virological outcomes of chronic hepatitis C after
successful interferon therapy
Journal of Medical Virology
Volume 74, Issue 3
Natsuko Tsuda 1, Nobukazu Yuki 1 *, Kiyoshi Mochizuki 2, Takayuki
Nagaoka 3, Masatoshi Yamashiro 3, Masao Omura 3, Kazumasa Hikiji 3,
Michio Kato 1 1Department of Gastroenterology, Osaka National Hospital,
Osaka, Japan 2Department of Internal Medicine and Therapeutics, Osaka
University Graduate School of Medicine, Suita, Japan 3SRL, Inc., Hino,
Japan
ABSTRACT/SUMMARY
Clinical relevance of occult hepatitis C virus (HCV) and/or hepatitis B
virus (HBV) infection(s) remains uncertain years after interferon (IFN)
therapy for chronic hepatitis C. By 1993, 38 sustained virological
responders (SVRs) showing HCV RNA clearance at 6 months post-treatment
and 37 biochemical responders (BRs) with end-of-treatment alanine
aminotransferase (ALT) normalization and subsequent 6-month
stabilization within 2 × the upper limit of normal (ULN) were enrolled.
They were monitored for 4.4-12 years (median 6.8), then 15 SVRs and 15
BRs underwent paired liver biopsies. Biopsy samples were tested for
positive and negative HCV RNA strands, and HBV DNA surface and X
sequences.
All SVRs showed sustained serum HCV RNA clearance during follow-up, but
hepatocellular carcinoma (HCC) developed in 4 (11%) SVRs.
On paired liver biopsies, histological improvement was significant, but
mild inflammation persisted in 87% of SVRs. Nonetheless, no HCV RNA
sequence was amplified from liver tissues, and HBV DNA sequences were
found in only one SVR. The liver fibrosis score also improved in the
SVRs (median 4, range 3--6 before treatment vs. median 3, range 2--6 at
the end; P1/40.007) and was unchanged in the BRs (median 3, range 3--4
before treatment vs. median 3, range 3--6 at the end; P1/40.480). The
mean change was 0.08 U/year (95% CI 0.03 to 0.13) for the SVRs compared
with 0.01 U/year (0.04 to 0.07) for the BRs (P1/40.066).
As for BRs, biochemical flare-up of >2 × ULN occurred at a 5-year risk
of 41% (95% CI 24.7-56.4). The event was unpredictable but controllable
by retreatment in 70%. Liver tissues after follow-up contained positive
and negative HCV RNA strands, but no HBV DNA sequence was amplified.
All SVRs were persistently negative for serum HCV RNA and retained
normal ALT levels during the entire follow-up periods. On the other
hand, HCV viremia persisted in all BRs, and biochemical flare-up defined
as ALT fluctuations of >2 ULN occurred in 17 (46%) patients. Only two
patients showed persistently normal ALT levels.
These results suggest that SVRs, albeit free of occult HCV and/or HBV
infection(s) over a decade, retain mild liver inflammation and the risk
of HCC. Occult HBV was also shown uninvolved in flare-up during
follow-up of BRs.
HCC CASES
During follow-up after IFN therapy, HCC developed in four SVRs but not
in the BRs (11 [95% CI 4--24] vs. 0% [0--9]; P1/40.115). Two small HCCs
of 1.5 and 1 cm were found in patient 6 at month 1, and solitary small
HCC of 2 cmdeveloped in patients 7, 14, and 19 at year 3.8--5.5.
Patients 6 and 14 had cirrhosis before treatment, whereas bridging
fibrosis was observed in patients 7 and 19. Moderately differentiated
HCC was resected for patients 14 and 19. The surrounding non-tumor liver
tissue of patient 19 only showed slight portal inflammation with no
fibrosis. The remaining two patients were treated by transcatheter
arterial embolization (TAE) and percutaneous ethanol injection therapy (PEIT).
Recurrence occurred in patient 14 but was controlled by the combination
therapy.
At the end of follow-up, 15 patients with sustained virological
response, including three HCC cases, provided written informed consent
to liver biopsies. Paired biopsies were performed at a median of 7.3
years (range 5.9--12.5) after pre-treatment biopsies. Of the four HCC
cases with sustained virological response, the two noncirrhotic patients
showed further alleviation of liver fibrosis. Patients 7 and 19
developed HCC at 4.6 and 5.5 years posttreatment and underwent final
liver biopsies at 5.5 and 6.2 years post-treatment, respectively. Ishak
scores improved in both patient 7 (inflammatory grade 8 and fibrosis
stage 4 before treatment vs. 0 and 3, respectively, at the end) and
patient 19 (inflammatory grade 5 and fibrosis stage 3 before treatment
vs. 0 and 1, respectively, at the end).
AUTHOR DISCUSSION
Previous studies have shownthat 91--95% of theSVRs with no serum HCV RNA
at 6 months post-treatment had no detectable liver HCV RNA 1--2 years
after therapy [Reichard et al., 1995; Shindo et al., 1995], whereas
Marcellin et al. [1997] demonstrated that HCV eradication from the liver
was sustained up to 5 years after therapy in all such patients examined.
Studies on liver HCV with a longer follow-up period could shed further
light on this issue but are very limited. The present study with
prolonged follow-up of >5 years showed that the clearance of liver HCV
RNA was sustained up to 12 years after therapy for all 15 patients.
Neither positive strands nor negative strands were found in any liver
biopsy specimens. Sustained reductions in HCV core antibody titers at a
constant rate further corroborated complete HCV eradication. One report
available in the literature showed that liver HCV RNA was not found in
five SVRs 10--11 years after therapy [Lau et al., 1998]. Collectively,
these findings suggest that HCV seroclearance at 6 months after IFN
therapy withdrawal would usually imply virological cure.
We further investigated occult HBV infection in the liver tissue from
chronic hepatitis C patients treated with IFN. Approximately half of our
patients had serological evidence of previous HBV exposure, but HBV DNA
was not found in the liver tissue irrespective of HCV clearance. In our
local region, occult HBV infection is frequently found in the livers of
untreated HCV-related chronic liver disease patients [Tamori et al.,
1999]. Further studies may be necessary to examine the possible
influence of IFN therapy on concomitant HBV.
Thus, the present study showed that the SVRs were completely free of
occult HCV and HBV up to a decade after therapy withdrawal. When the
long-term liver histological outcomes were evaluated, the SVRs showed
improvement of the necroinflammatory grade and regression of the
fibrosis stage. Nevertheless, mild liver inflammation persisted in the
majority, the reason for which remains unclear. Intrahepatic
inflammatory response triggered by HCV infection may take a very long
time to cease and may continue in the absence of occult viral
infections. It was also demonstrated that HCC, a late complication after
sustained virological response, occurs without occult HCV or HBV
infection. All HCC patients had an advanced stage of liver fibrosis
before treatment, and it is conceivable that longstanding HCV-related
liver injury had initiated the carcinogenesis process although
persistent low-grade liver inflammation after IFN therapy may have
exerted some influence on the subsequent course.
Even using IFN alone or in combination therapy of IFN plus ribavirin
leads to at least half of the chronic hepatitis C patients showing no
sustained virological response [McHutchison et al., 1998; Poynard et
al., 1998]. Under these circumstances, improvement of serum ALT
elevation without HCV seroclearance should be considered another
favorable response to IFN therapy. At present, the definition of a
biochemical response with long-term clinical benefits remains
controversial. A recent European study showed that the best ALT
threshold predicting significant liver injury is about two times the ULN
[Pradat et al., 2002]. In the second part of the study, the long-term
clinical course of a biochemical response defined as end-of-treatment
ALT normalization followed by near-normal ALT levels of 2 ULN during the
subsequent 6 months was studied. The BRs were more likely to be older
and have low-grade necroinflammatory reaction as compared with the SVRs,
thus indicating that failure in HCV clearance may be attributable to
inefficient host immune responses to HCV-infected hepatocytes.
The present study revealed that the 5-year risk of biochemical flare-up
showing ALT fluctuations of >2 ULN was 41% (95% CI 24.7--56.4) but that
the flare-up could not be predicted by any clinical, virological, and
histological characteristics. However, the type of IFN therapy was not
uniform, and quantitative data on HCV RNA levels during IFN therapy were
not available. There remains a possibility that these factors may have
had relevance to the clinical course of BRs.
It was shown that occult HBV was not present in the livers of BRs and
did play no role in the clinical course. Occult HBV is known to be
common and exert virulence in chronic hepatitis C patients, but its
clinical relevance after IFN therapy may be less significant. The
flare-up was easily controlled by retreatment with IFN. Paired liver
biopsies showed that necroinflammation was ameliorated and that the
fibrosis stage remained unchanged. These observations are compatible
with a few studies on the histological outcomes of a biochemical
response defined as sustainedALT normalization [Bruno et al., 2001;
Shindo et al., 2001]. Although further studies with a larger number of
patients are necessary, control of biochemical disease activity to
near-normal levels may also confer favorable long-term histological
outcomes.
INTRODUCTION
Interferon (IFN) has been used for the treatment of chronic hepatitis C
for a decade. Previous studies have shown that sustained virological
responders (SVRs)who were negative for serum hepatitis C virus (HCV) RNA
6 months after treatment were likely to remain in virological and
biochemical remission with histological amelioration [Marcellin et al.,
1997; Shiratori et al., 2000]. Low risk of hepatocellular carcinoma (HCC)
can also be expected in such patients [Imai et al., 1998; Yoshida et
al., 1999]. However, late biochemical relapse after a sustained
virological response has been observed [Reichard et al., 1995; Marcellin
et al., 1997], and HCC can develop as a late complication. Under these
circumstances, questions arise about whether SVRs are completely free of
occult HCVand/or hepatitis B virus (HBV) infection(s). HCV eradication
from the liver has not been well validated based on long-term follow-up
of more than 5 years after treatment. It is possible that HCV infection
may persist in the liver after spontaneous circulating HCV clearance [Haydon
et al., 1998; Dries et al., 1999; Sugiyasu et al., 2003]. Recently,
occult HBV coinfection and its possible virulence have been shown for
chronic hepatitis C patients [Cacciola et al., 1999; Tamori et al.,
1999]. However, little is known about the clinical relevance of occult
hepatitis B after IFN-therapy. To address these issues, patients with a
sustained virological response were monitored over 4.4--12 years after
IFN therapy, and the long-term virological outcomes were investigated by
detecting liver HCV and HBV sequences. We also studied the clinical
relevance of occult HBV infection in patients who were monitored after a
biochemical response.
RESULTS
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Baseline clinical features are shown in Table I. The SVRs had lower
pre-treatment viral loads than the BRs (median <0.5, range <0.5--8.6 vs.
median <0.5, range <0.5--15.6 Meq/ml;P1/40.031) and were more frequently
infected with genotype 2 (53 vs. 30%; P1/40.043).
Patients with sustained virological response showed more marked
piecemeal necrosis expressed as the Ishak score (median 3, range 0--4
vs. median 1, range 0--4; P1/40.049) and were treated with a higher
total IFN dose (median 480, range 174--936 vs. median 396, range 36--810
MU; P1/40.030). In multivariate logistic-regression analysis, however, a
sustained virological response was associated with marked piecemeal
necrosis of 3 points (odds ratio 3.8 [95% CI 1.3--11.3], P1/40.017) and
the age of <50 years (odds ratio 3.8 [1.2--11.8], P1/40.020). As for the
BRs, baseline clinical features were similar between the 10 patients
with sustained ALT normalization during the 6-month post-treatment
period and the remaining 27 patients with end-of-treatment ALT
normalization and subsequent slight ALT fluctuations of 2 ULN.
The SVRs were monitored over 4.4--12 years (median 6.2) after therapy
withdrawal, and the BRs over 4.8-- 9.6 years (median 7.3). All SVRs were
persistently negative for serum HCV RNA and retained normal ALT levels
during the entire follow-up periods.
On the other hand, HCV viremia persisted in all BRs, and biochemical
flare-up defined as ALT fluctuations of >2 ULN occurred in 17 (46%)
patients. Only two patients showed persistently normal ALT levels. The
flare-up rates at 1, 2, 3, 4, and 5 years were 11, 22, 30, 32, and 41%,
respectively. None of the clinical, virological, and histological
characteristics of the patients (listed in Table I) were predictive of
the flare-up. The flare-up rates did not differ between patients with
sustained ALT normalization during 6 months post-treatment and those
without it. Ten of the 17 flare-up cases were retreated with IFN at a
median of 6 years (range 3.3--7.3) after the end of initial treatment.
The total IFN dose ranged from 168 to 1,476 MU (median 759), and seven
patients were retreated with a higher dose. Pre-retreatment viral loads
ranged between <0.5 and 31.0 Meq/ml (median 4.5), and genotypes 1 and 2
were found in six and four patients, respectively. Six patients, three
of whom had genotype 1 and high viral loads (>5 Meq/ml), achieved a
biochemical response again. A sustained virological response was
achieved by another patient.
During follow-up after IFN therapy, HCC developed in four SVRs but not
in the BRs (11 [95% CI 4--24] vs. 0% [0--9]; P1/40.115). Two small HCCs
of 1.5 and 1 cm were found in patient 6 at month 1, and solitary small
HCC of 2 cmdeveloped in patients 7, 14, and 19 at year 3.8--5.5.
Patients 6 and 14 had cirrhosis before treatment, whereas bridging
fibrosis was observed in patients 7 and 19. Moderately differentiated
HCC was resected for patients 14 and 19. The surrounding non-tumor liver
tissue of patient 19 only showed slight portal inflammation with no
fibrosis. The remaining two patients were treated by transcatheter
arterial embolization (TAE) and percutaneous ethanol injection therapy (PEIT).
Recurrence occurred in patient 14 but was controlled by the combination
therapy.
At the end of follow-up, 15 patients with sustained virological
response, including three HCC cases, provided written informed consent
to liver biopsies. Paired biopsies were performed at a median of 7.3
years (range 5.9--12.5) after pre-treatment biopsies. Paired biopsies
were also performed 6.2--9.8 years apart (median 8.3) on 15 BRs
including seven retreated cases. Positive and negative HCV RNA strands
were not found in the livers of the 15 SVRs. The log2 titer of the HCV
core antibody also decreased in each patient (median 7.2, range 2.8--8.3
before treatment vs. median 3.7, range negative to 5.0 at the end;
P1/40.001), and the mean log2 change was estimated at 0.46/year (95% CI
0.38 to 0.55). In contrast, all BRs had both HCV RNA strands in the
liver except patient 62 who had achieved sustained virological response
after retreatment.
Occult HBV in the liver was further studied for the 30 chronic hepatitis
C patients after IFN therapy. Paired liver and serum samples were
subjected to HBV DNA PCR to amplify the surface and X regions. HBV DNA
sequences were not found in any serum samples, but one non-HCC SVR with
previous HBV coinfection (patient 24) had the two HBV genomic regions in
the liver. The other 29 patients were persistently negative for HBsAg.
Anti-HBc and/or anti-HBs were found in 13 patients, but none had liver
HBV DNA.
The total necroinflammatory score improved in the SVRs (median 7, range
4--11 before treatment vs. median 1, range 0--4 at the end; P1/40.001)
and in the BRs (median 8, range 3--11 before treatment vs. median 5,
range 1--8 at the end; P1/40.003). The mean change was 0.74 U/year (95%
CI 0.52 to 0.96) for the SVRs compared with 0.30 U/year ( 0.14 to 0.46)
for the BRs (P1/40.003). The SVRs showed improvement of each category of
necroinflammation, whereas improvement was significant with respect to
piecemeal necrosis and focal lytic necrosis in the BRs. However, in the
absence of occult HCV and HBV infections, 13 (87%) of the SVRs retained
low grade of necroinflammation, especially portal inflammation in the
late convalescent phase.
The liver fibrosis score also improved in the SVRs (median 4, range 3--6
before treatment vs. median 3, range 2--6 at the end; P1/40.007) and was
unchanged in the BRs (median 3, range 3--4 before treatment vs. median
3, range 3--6 at the end; P1/40.480). The mean change was 0.08 U/year
(95% CI 0.03 to 0.13) for the SVRs compared with 0.01 U/year (0.04 to
0.07) for the BRs (P1/40.066). Of the four HCC cases with sustained
virological response, the two noncirrhotic patients showed further
alleviation of liver fibrosis. Patients 7 and 19 developed HCC at 4.6
and 5.5 years posttreatment and underwent final liver biopsies at 5.5
and 6.2 years post-treatment, respectively. Ishak scores improved in
both patient 7 (inflammatory grade 8 and fibrosis stage 4 before
treatment vs. 0 and 3, respectively, at the end) and patient 19
(inflammatory grade 5 and fibrosis stage 3 before treatment vs. 0 and 1,
respectively, at the end).
MATERIALS AND METHODS
Patients
By 1993, a total of 38 chronic hepatitis C patients showed a sustained
virological response to IFN therapy, and 37 patients showed a
biochemical response. A sustained virological response was defined as
clearance of circulating HCV RNA at 6 months post-treatment, whereas a
biochemical response was defined as end-of treatment alanine
aminotransferase (ALT) normalization and subsequent 6-month ALT
stabilization within 2 the upper limit of normal (ULN) (33 IU/L) in the
absence of serum HCV RNA clearance. They comprised 53 men and 22 women
and ranged in pre-treatment age from 22 to 66 years (median 52). Before
treatment, all patients had elevated serum ALT activity for at least 6
months and tested positive forHCVantibody and serum HCV RNA. After liver
biopsies, they were treated with IFN in various dose regimens for 6--62
weeks (median 24). Natural IFN-a (Sumiferon, Sumitomo Pharm Co., Osaka
or OIF, Otsuka Pharm Co., Tokyo, Japan), IFNa2a (Roferon-A, Nippon Roche
K.K., Tokyo, Japan) or IFN-a2b (Intron A, Schering-Plough Co., Osaka,
Japan) was given by intramuscular injection to 34, 26, and 6 patients,
respectively. The remaining nine patients were treated with intravenous
injection of IFN-b (Feron, Toray Co., Tokyo, Japan). The total IFN dose
ranged from 36 to 936 MU (median 432). Serum ALT activity and HCV RNA
were monitored monthly during the 6-month post-treatment period. Of the
37 biochemical responders (BRs), 10 patients showed sustained ALT
normalization during 6 months post-treatment, whereas the remaining 27
patients showed end-of-treatment ALT normalization and subsequent slight
ALT fluctuations within 2 ULN.
These 75 consecutive patients were enrolled and followed for 4.4--12
years (median 6.8) after therapy withdrawal. SVRs were tested for serum
ALT activity and HCV RNA at least every 6 months. For BRs, serum ALT
activity was monitored monthly, and serum HCV RNA at least every 6
months. All patients underwent abdominal ultrasonography every 6 months.
If HCC was suspected, they had additional examinations by dynamic
computed tomography, magnetic resonance imaging, arteriography, and
ultrasonography-guided tumor biopsy, as deemed necessary. All but three
patients had no apparent cause of hepatocellular injury other than HCV
during the entire follow-up period. They were persistently negative for
hepatitis B surface antigen (HBsAg). They had no history of
administration of hepatotoxic drugs or alcohol abuse (>10 g/day) and
showed no evidence of autoimmune liver disease. Of the remaining three
patients, two SVRs (patients 9 and 24) had HBsAg before treatment but
were cleared of it at 3.1 and 5.4 years after treatment, respectively.
One BR (patient 39) was persistently positive for HBsAg. At the end of
the follow-up, 15 SVRs and 15 BRs had paired liver biopsies 5.9--12.5
years apart (median 7.7). This study was approved by the local Research
Ethics Committee in accordance with the 1975 Declaration of Helsinki.
All patients provided written informed consent.
Virological Tests
Serum HCV RNA was detected by reverse transcription-polymerase chain
reaction (RT-PCR) [Hagiwara et al., 1993] and quantified using a
branched DNA (bDNA) assay (Quantiplex HCV-RNA, Chiron Corporation,
Emeryville, CA). The detection limit of the RT-PCR method was 300 copies
of syntheticHCVRNA/ml serum. HCV RNA-positive serum samples were
subjected to a serological genotyping assay (Immucheck-HCV Gr,
International Reagent Corporation, Kobe, Japan). Quantification of HCV
core antibody was performed using a commercially available
kit(HCVCore-Ab IRMA, Ortho Diagnostic Systems Co., Ltd., Tokyo, Japan).
Serum samples were tested for HBsAg, antibody to HBsAg (anti-HBs) and
antibody to hepatitis B core antigen (anti-HBc) with radioimmunoassays
(Abbott Laboratories, North Chicago, IL). SerumHBVDNAwas detected by
real-time detection PCR based on Taq Man chemistry as previously
reported [Abe et al., 1999]. The HBV surface and X regions were
amplified using set 2 primers and set 3 primers, respectively. For each
primer set, the PCR sensitivity was 10 copies of synthetic HBV DNA per
reaction, and the detection limit of serum HBV DNA was 200 copies/ml
serum.
Percutaneous needle liver biopsies were performed using 14-gauge Tru-Cut
needles at the follow-up end, and biopsy specimens sufficient for
histological and virological evaluation were obtained. Liver specimens
for PCR testing were frozen immediately and then stored at 808C.
Positive and negative HCV RNA strands in the liver were independently
amplified by specific RT semi-nested PCR as described elsewhere [Tomimatsu
et al., 1997]. Using synthetic HCV RNA strands, the PCR sensitivity was
10 copies per reaction. For the detection ofHBVDNA, totalDNAwas
extracted from liver tissue using a commercially available kit (SMI test
EX R and D, Sumitomo Metal Industries, Tokyo, Japan). Purified total
hepatic DNA was resuspended in 500 ml of distilled water. A 25-ml
aliquot of DNA solution was subjected to the real-time detection PCR. In
preliminary experiments, liver biopsy specimens from 12 chronic
hepatitis B patients, who had been clear of serum HBsAg for 1.3--15.3
years (median 4.9), were tested to evaluate the ability to detect occult
HBV infection. Liver HBV DNA was successfully amplified in each patient,
thus indicating that the real-time detection PCR method used was
practically sensitive enough to detect occult HBV in liver tissues. To
avoid contamination in all PCR assays, the contamination avoidance
measures [Kwok and Higuchi, 1989] were strictly applied throughout, and
positive and negative controls were used.
Histological Evaluation
Liver biopsy specimens for histological evaluation were fixed in
formalin and embedded in paraffin for routine staining with hematoxylin--eosin.
All specimens were examined by the same experienced pathologist, who was
unaware of the biochemical, serological, and virological data. Biopsy
specimens were semiquantitatively evaluated by the modified histological
activity index [Ishak et al., 1995]. The histological outcome of IFN
therapy was assessed by comparing the pretreatment biopsy specimenwith
the last biopsy specimen obtained after treatment. Using the
inflammatory grade and fibrosis staging scores of the paired biopsy
samples, the yearly progression or regression was calculated as the
change in the scores divided by time (years) between biopsies.
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