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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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