GASTROENTEROLOGY 1993;104:877-883

Quantitative Analysis of Hepatitis C Virus RNA in Serum During

Interferon Alfa Therapy

HIDEKI HAGIWARA, NORIO HAYASHI, EIJI MITA, TETSUO TAKEHARA, AKINORI KASAHARA,
HIDEYUKI FUSAMOTO, and TAKENOBU KAMADA
First Department of Medicine, Osaka University Medical School, Fukushima, Osaka, Japan

Bac&round: Interferon alfa is effective for controlling tients are unclear. Recently, an assay was developed to
disease activity in chronic hepatitis C. However, many detect HCV RNA as a marker of viral replication by
responders suffer relapse after cessation of therapy. combining reverse transcription of the RNA and sub-
In the present study, the serum concentration of hepati- sequent amplification of the complementary DNA
tis C virus RNA was correlated with a sustained re-
(cDNA) bY P o lYmerase chain reaction (RT-PCR).‘5-‘9
sponse to interferon therapy. Methods: Fifty-three pa- Evaluation of HCV RNA in serum has shown that
tients with chronic hepatitis C received a 26-week
therapy with interferon alfa is beneficial in chronic
course of interferon alfa. Hepatitis C virus RNA was
hepatitis C because of its suppressive effects on HCV
quantitated in serum at the beginning and end of treat-
ment using a competitive assay that combined reverse replication. l9 In the present study, we quantified the
transcription and polymerase chain reaction. Results: amount of HCV RNA in serum by a competitive RT-
Inlong-term responders, whosealanineaminotransfer- PCR assay based on coamplification of target HCV
ase levels remained within the normal range during the RNA in samples with synthetic mutant HCV RNA in
24 weeks after therapy, the titer of viral RNA (logarith- 53 patients with chronic hepatitis C and correlated the
mic transformed copy numbers per milliliter of serum) serum concentration of HCV RNA with the response
before therapy (7.1 + 1.2) was significantly lower (P < to interferon alfa therapy. We also identified the pre-
0.001) than that of short-term responders (8.3 f 0.5) dictive factor for beneficial response to interferon alfa
who had a relapse within the 24 weeks after therapy by multivariate analysis.
and nonresponders (8.1 + 0.4). Multivariate multiple
logistic regression showed that the titer of viral RNA
before therapy was the strongest independent predic-
Materials and Methods
tor of a sustalned response to interferon-alfa therapy Patients and Interferon Treatment
(P = 0.002). Conclusions: The titer of hepatitis C virus
RNA is the most important factor influencing the sus- The subjects were 60 patients (46 men and 14
tained response to interferon treatment. women) who had elevated serum ALT levels (at least two
times the upper limit of the normal range) for at least 6
months and who had been histologically proven to have
esting for an antibody to hepatitis C virus (HCV)
chronic hepatitis. All patients had anti-HCV (anti-ClOO-3)
T has shown that HCV is the etiological agent for
as detected by the Ortho Diagnostics (Tokyo, Japan) en-
most cases of non-A, non-B hepatitis,le5 which can zyme-linked immunosorbent assay, and all were negative for
lead to serious consequences including liver cirrhosis hepatitis B surface antigen. Patients with evidence of other
and hepatocellular carcinoma.3T4p6Several investigators forms of liver disease and decompensated liver disease were
have reported that interferon alfa therapy is effective excluded. The patients’ ages ranged from 20 to 66 years
for decreasing serum alanine aminotransferase (ALT) (mean, 48.8 years). Eighteen patients had a history of blood
levels and improving liver histology in chronic hepati- transfusion and 3 a history of acute hepatitis. Biopsy speci-
tis C.7-12 However, at least half of the patients re- mens were obtained before entry to the study and graded by
sponding to interferon alfa subsequently have a relapse a pathologist according to the numerical scoring system of
after therapy is stopped; achieving a sustained response Knodell et al.”
is difficult even in patients given prolonged treatment.
In hepatitis B virus (HBV) infections, factors in- Abbreviationsusedin this paper: 2,5-AS, 2’,8-oligoedenylate syn-
fluencing response to interferon therapy have been de- thetase; CR-L, long-term complete response group: CR-S, short-
scribed.‘3*‘4 In treating patients with chronic hepatitis term complete responsegroup;MU, mllllon unlts; NR, no response;
PCR, polymerasechain reaction; RT, reversetranscription.
C, the relationship between a beneficial response to 0 1993 by the American GastroenterologlcalAssoclatlon
interferon alfa and the initial characteristics of the pa- 00 16-5085/93/$3.00
878 HAGIWARA ET AL. GASTROENTEROLOGY Vol. 104, No. 3

After written informed consent had been obtained, the the sequence that lay between but did not include the RT-
patients were randomly allocated into two groups. Thirty PCR primers (5’-GAGTGTCGTGCAGCCTCCAGGTCC-
patients were assigned to a treatment group given 3 million 3’).
units (MU) of natural interferon-alfa (human lymphoblas-
toid interferon; Sumitomo Pharmaceuticals Co., Osaka, Ja- Detection of HCV RNA in Serum
pan) by intramuscular injection every day for 14 days and HCV RNA was detected by the RT-PCR assay with
then three times a week for 24 weeks (3 MU-treated group). minor modifications as previously described.5s’9 In the modi-
The remaining 30 patients were assigned to another treat- fication for RNA extraction, 100 PL of serum was mixed
ment group given 6 MU of natural interferon-alfa by intra- with 600 FL of guanidinium homogenization buffer, con-
muscular injection every day for 14 days and then three taining 4 mol/L guanidinium thiocyanate, 0.1 mol/L Tris-
times a week for 24 weeks (6 MU-treated group). Twenty- HCl (pH 7.5), 0.5% sodium lauryl sarcosinate, 0.3 mol/L
six patients receiving 3 MU of interferon-alfa and 27 pa- sodium acetate (pH 6), and 1% fi-mercaptoethanol. RNA
tients receiving 6 MU completed the therapy and were in- was precipitated with absolute ethanol overnight at -80°C
cluded in the following analyses. Comparison of the before reverse transcription. Sequences for the RT-PCR
characteristics of the patients at entry between the 3 MU- primers were changed from 20-mer oligonucleotides to 25-
treated group and the 6 MU-treated group are shown in mer oligonucleotides (BKP-7 and BKP-8). We tested nine
Table 1. There were no significant differences between the serum samples, two negative control sera, and one sample of
selected clinical and biochemical features of these two 200 PL of distilled water in each batch. The negative control
groups. sera were chosen from samples of individuals not expected
to have HCV infection. All samples were assayed at least
Testing for 2’,5’-Oligoadenylate Synthetase two times with reproducible results. The threshold of the
Serum level of 2’,5’-oligoadenylate synthetase (2,5- RT-PCR assay was analyzed using synthetic mutant HCV
AS), an interferon-inducible enzyme that activates intracel- RNA (described below) as the template. Serial dilutions of
lular ribonucleases to cleave viral mRNA, was determined mutant HCV RNA were added to 100 PL of negative con-
by radioimmunoassay (Eiken Co., Tokyo, Japan; normal < trol sera after mixing with guanidinium homogenization
100 pmol/dL). buffer. These samples were tested according to the proce-
dure of the RT-PCR assay, and the detection limit was de-
Oligonucleotides termined by Southern blot hybridization. We could detect
Oligonucleotide primers were derived from the 30 copies of synthetic HCV RNA per sample constantly
5’-terminal sequence of HCV isolated in Japan.” The after 40 cycles of amplification and 5 copies of synthetic
first primer used for reverse transcriptase reaction and HCV RNA under the best condition.
amplification was BKP-8 (5’-ATGGTGCACGGTCTA-
Quantification of HCV RNA in Serum
CGAGACCTCC-3’; antisense), and the second primer for
amplification was BKP-7 (5’-CACTCCCCTGTGAGG- HCV RNA in serum was quantified by a competitive
AACTACTGTC-3’; sense). The sequence of mutagenic RT-PCR assay according to the method of Becker-Andre
primer for site-directed mutagenesis was derived from an and Hahlbrock22 using synthetic mutant HCV RNA (Figure
HCV cDNA clone M64219 (5’-GAGCCATAGAATTCT- 1).
GCGGAAC-3’) and contained two bases mismatched at po- Site-directed mutagenesis and in vitro transcrip-
sitions 11 (GA) and 12 (G:T). The hybridization probe was tion. An HCV cDNA clone M642 was cloned into the Ml 3
vector system (Toyobo Co. Ltd., Osaka, Japan). A mutant
strand having a novel restriction endonuclease (EcoRI) site
was generated by annealing the 22-mer mutagenic primer
Table 1. Clinical and Biochemical Characteristics
followed by extension with T7 DNA polymerase. The mu-
of 53 Patients With Chronic Hepatitis C Before
Interferon Therapy tated HCV cDNA was cloned into a vector (pBluescript II;
Toyobo Co. Ltd.), digested with EcoRV, and served as tem-
3 MU-treated 6 MU-treated plates for T7 RNA polymerase to generate sense transcripts.
Clinical features (n = 26) (n = 27)
The templates were digested with ribonuclease-free deoxyri-
Female/male 9/17 5/22 bonuclease. The mutant RNA transcripts were purified by
Age [yr (mean f SD)] 49.3 + 6.6 47.4 + 11.8 phenol-chloroform extraction and ethanol precipitation.
History of blood transfusion 11 6
RNA preparation and RT-PCR assay. For extraction
AST [IU/L (mean k SD)] 112.6 f 77.4 89.5 f 74.6
ALT [/U/L (mean -r- SD)] 179.5 f 117.5 124.7 f 79.6 of RNA, 5-50 PL of serum sample was mixed with 400 PL
2,5-AS [pmo//dL (mean f SD)] 162.2 + 153.9 169.7 f 192.9 of guanidinium homogenization buffer. After addition of 10
Histology pg of transfer RNA (Sigma Chemical Co., St. Louis, MO)
CPH 5 6 and different amounts of mutant HCV RNA, the solution
CAH 21 21
was extracted twice with phenol-chloroform, and then 2.5
CPH, chronic persistent hepatitis; CAH, chronic active hepatitis. volumes of absolute ethanol was added to the aqueous
March 1993 IFN THERAPY AND QUANTITATION OF HCV RNA 879

mixture of HCV RNA in sample / mutant HCV RNA rank test and Wilcoxon rank sum test were used. Multivar-
iate multiple logistic regression was used to analyze the con-
ROverSO
tranccription
I
tribution of the patients’ initial characteristics to the re-
HCV cDNA / mutant HCV cDNA sponse to interferon therapy.

Polymerase chain reaction Results

I (EcoRI) Analysis of PCR Products
ampllfiod :
A
DNA fragment PCR products derived from target HCV RNA
in sera were 306 base pairs (bp) long. Digestion with
EcoRl dlgostlon

I EcoRI selectively cuts PCR products derived from mu-

-- tam HCV RNA into two fragments of 10% and 198-
--
306bp 108-bp 198-bp bp long, respectively. A representative result of com-
petitive RT-PCR assay is shown in Figure 2. The top
Electrophoresis

I
band represents nondigested DNA fragment derived
HCV RNA 306~bp - - from target HCV RNA in serum, and the lower two
- - lQ8-bp
mutant
bands represent &RI-digested DNA fragments de-
HCV RNA
- + 108-bp 7 rived from mutant HCV RNA. We determined the
amount of HCV RNA in serum by comparing the sig-
Figure 1. Outline of quantitative analysis of HCV RNA by a competi- nal intensities of the upper two bands on an agarose
tive RT-PCR assay. Different amounts of synthetic mutated HCV RNA
were amplified in the same tube with target HCV RNA in serum sam-
gel. Target HCV RNA of >104.5 copies could be
ples. After digestion with EcoRl and electrophoresis on an agarose quantified after 30 cycles of amplification, and HCV
gel, amplified DNA fragments were separated to three bands and RNA >300 molecules could be quantified after 40 cy-
viewed under an ultraviolet lamp.
cles of amplification directly on an agarose gel. Hybrid-
ization was necessary to quantify ~300 target HCV
RNA. The hybridization probe detected only 5’-up-
phase. This mixture was left at -80°C overnight, and then stream amplified DNA fragments (306-bp and 10%
the RNA was pelleted by centrifugation (20 minutes, bp). The amounts of HCV RNA varied from lo5 to
18,5OOg, 4’C). The pellet was washed with 70% ethanol, 1 09.5 copies/ml serum before interferon administra-
dried, and dissolved in 9 FL of distilled water. The RNA
tion (Figure 3).
solution was heated at 65°C for 10 minutes with 20 pmol of
antisense primer and cooled at room temperature. The re-
verse transcriptase reaction and 30 or 40 cycles of PCR were
performed according to a method described elsewhere.‘,”
Analysis of the PCR products. A 30-yL portion of
each PCR product was purified by ethanol precipitation and
treated with 32 units of EcoRI (Toyobo Co. Ltd.) according
to the manufacturer’s instructions. After digestion, 10 ktL of
solution was analyzed by electrophoresis on a 2.5% agarose
gel containing 0.5 /rg/mL ethidium bromide. The gels were
viewed and photographed on an ultraviolet transillumina-
tor. To quantify small amounts of HCV RNA, the separated
DNA fragments were blotted onto a nylon membrane, hy-
bridized to a 32P-end-labeled HCV cDNA, and autoradio-
graphed. The titer of circulating HCV RNA was defined as
log,, (copy numbers of HCV RNA per milliliter of serum). Figure 2. A representative result of competitive RT-PCR assay. Mix-
The titer of samples in which target HCV RNA could not be ture of 50 pL of serum and different amounts of mutated HCV RNA
were tested. After 30 cycles of amplification and EcoRl digestion, the
detected was evaluated as 0.
products were analyzed by electrophoresis in a 2.5% agarose gel. The

Statistical Analysis top band (306-bp) represents amplified DNA fragments derived from
target HCV RNA, and the lower two bands (108-bpand 198-bp) repre-
The x2 test or Fisher’s Exact Test was used for statis- sent those from the competitor. At the equivalent point of signal in-
tensities between the upper two bands, the amounts of target HCV
tical analysis of comparison between group frequencies.
RNA are the same as the copy numbers of the competitor. The arrow
Where appropriate, laboratory features and titers of HCV indicates the transitional point of titration (5 X lo6 copies/50 uL
RNA were compared by Student’s t test and paired t test. serum equivalent to 10’ copies/ml serum). Numbers are the copies
When data were not normally distributed, Wilcoxon signed of synthetic mutated HCV RNA.
 880 HAGIWARA ET AL. GASTROENTEROLOGY Vol. 104, No. 3

sponse was observed in 9 (34.6%) of the 26 patients

receiving 3 MU of interferon and in 9 (33.3%) of the
0
27 patients receiving 6 MU. The third was the no re-
A 00
sponse (NR) group of 23 patients, whose ALT levels
Ll.....“.‘. ??oeoAA did not decrease during therapy or fluctuated persis-
AAAA AAAAAA tently. Thirteen (50%) of the 3 MU-treated patients
0OO.A AOOOAA
and 10 (37.0%) of the 6 MU-treated patients were
AAA 00
nonresponders.
@A 0
Changes in HCV RNA in Serum During and
0
After Therapy
0 00
HCV RNA in serum was detected in all patients
before therapy and became undetectable in 6 (23.1%)
0
of the 26 patients in the 3 MU-treated group and in 13
(48.1%) of the 27 patients in the 6 MU-treated group
at the end of therapy. During the following 24 weeks,
HCV RNA remained undetectable in 2 (7.7%) patients
in the 3 MU-treated group and 7 (25.9%) patients in
3 - MU - treated 6 - MU - treated the 6 MU-treated one. The titer of HCV RNA signifi-
(n = 26) (n = 27) cantly decreased (P < 0.001) during therapy in both
Figure 3. Amounts of circulating HCV RNA in 53 patients with chronic
groups, and the decrease was greater in patients receiv-
hepatitis C before interferon therapy. 0, long-term complete re- ing 6 MU of interferon than in those receiving 3 MU
sponders with sustained disappearance of HCV RNA; A, long-term (Table 2).
responders without sustained disappearance of HCV RNA; 0, short-
We correlated the changes in HCV RNA in serum
term complete responders; A, nonresponders.
with responses to interferon therapy (Table 3). HCV
RNA became undetectable in 10 (83.3%) of 12 long-
term responders and in 9 (50%) of 18 short-term re-
Changes in ALT Levels During and After
sponders at the end of therapy. Although sustained
Therapy
loss of HCV RNA was observed in 9 (75%) of the 12
The ALT levels had decreased to the normal patients with long-term response, HCV RNA reap-
range at the end of therapy in 12 (46.2%) of 26 patients peared in all patients who had a relapse after therapy.
of the 3 MU-treated group and 16 (59.3%) of 27 pa- In complete responders (groups CR-L and CR-S), the
tients of the 6 MU-treated group. However, 9 patients titer of HCV RNA was significantly decreased during
of the 3 MU-treated and 9 of the 6 MU-treated had a therapy (P < 0.001). In nonresponders, HCV RNA
relapse within 24 weeks after therapy was stopped. was continuously detected but the decrease of HCV
The 53 patients treated with interferon alfa were RNA titers during therapy was significant (P < 0.005).
categorized into three groups based on changes in The titer before interferon administration was signifi-
serum ALT levels. The first was the long-term com-
plete response (CR-L) group of 12 patients, whose
ALT levels decreased during therapy and remained Table 2. Positive Rate and the Titer of HCV RNA During
within the normal range during the 24 weeks after and After Interferon Therapy
therapy. The CR-L group included 2 patients whose
No. of positive results/total
ALT levels gradually declined but slightly increased at
the end of therapy and became normal after interferon Before End of 24 wk after
administration was stopped. Long-term complete re- Group therapy therapy therapy

sponse was achieved in 4 (15.4%) of 26 patients treated 3 MU-treated 26/26 20/26 24/26
with 3 MU of interferon and in 8 (29.6%) of 27 pa- (8.1 f 0.7) (4.9 + 3.2pb
6 MU-treated 27/27 14/27 20/27
tients treated with 6 MU. The second was the short- (7.8 + 0.9) (2.9 + 3.2)8
term complete response (CR-S) group of 18 patients,
NOTE. Numbers in parentheses are titers of HCV RNA in serum
whose ALT levels decreased to the normal range dur-
(mean f SD).
ing therapy but then increased to abnormal levels dur- ‘P < 0.001 vs. before therapy.
ing the following 24 weeks. Short-term complete re- bP < 0.05 vs. 6 MU-treated group.
March 1993 IFN THERAPY AND QUANTITATION OF HCV RNA 881

Table
3. Positive Rate and the Titer of HCV RNA According blood transfusion, initial aspartate aminotransferase
to ALT Response to Interferon Therapy
(AST), ALT, 2,5-A& and titer of HCV RNA) were
No. of positive results/total selected as independent variables in a stepwise regres-
sion (significance level for entry into the model =
Before End of 24 wk after
therapy
0.15) with complete response to interferon alfa treat-
Group therapy therapy
ment as the dependent variable. The continuous vari-
CR-L 12/12 2/12= 3/12b
ables were not transformed to categories. Only the ini-
(7.1 f 1.2)afb (0.8 f 1.6)
CR-S 18/18 9/18 18/18 tial titer of HCV RNA was estimated to be
(8.3 ? 0.5) (2.3 + 2.7) independently predictive of a long-term complete re-
NR 23/23 23/23 23/23
sponse (P = 0.002; odds ratio, 1.794; 95% confidence
(8.1 -+ 0.4)d (6.8 * 1.7)
intervals, 1.902-19.001). However, it was not found to
NOTE. Numbers in parentheses are titers of HCV RNA in serum be independently predictive of a complete response
(mean f SD).
aP < 0.001 vs. end of therapy.
including the CR-L and CR-S groups.
bP < 0.001 vs. CR-S and NR groups.
‘P < 0.001 vs. NR group. Discussion
dP -c 0.005 vs. end of therapy.
Interferon alfa has been used in the treatment of
eP < 0.001 vs. CR-L and CR-S groups.
non-A, non-B hepatitis, and its efficacy was estab-
lished in two randomized control trials assessing inter-
cantly lower in the CR-L group than in the
(P< 0.001) feron and placebo for effects on ALT levels and liver
CR-S and NR groups. The titer at the end of therapy histology. **’ In chronic HBV infections, serum AST
was significantly higher in the NR group (P< 0.001) level sex,13 baseline HBV DNA, known duration of
than in the CR-L and CR-S groups. The 5 patients hepa;itis, sexual orientation, and serum ALT level14
with HCV RNA titers less than 7 achieved sustained have been reported to have important effects on the
disappearance of circulating HCV RNA. However, of response rate to interferon therapy. Although pro-
the 48 patients with HCV RNA titers of 7 or more, longed therapy with higher doses of interferon alfa
four (8.3%) patients showed sustained loss of HCV seem to be necessary to achieve sustained remission of
RNA (Figure 3). chronic hepatitis C,7*8,12predictive factors for benefi-
cial response to interferon therapy have remained un-
Changes in Serum 2,5-AS Activity clear.
We tested serum 2,5-AS activity at the begin- In our study, ALT levels declined to the normal
ning and the fourth week of therapy. The 2,5-AS activ- range during therapy in 28 (52.8%) of the 53 patients.
ity at the fourth week of therapy (3 MU-treated, However, 18 patients subsequently had a relapse
296.8 + 223.9; 6 MU-treated, 300.8 + 189.8) was sig- within 24 weeks after therapy. Sustained loss of circu-
nificantly higher than that at the beginning
(P< 0.01) lating HCV RNA was observed in 9 patients of the
of therapy in both treatment groups. There was no CR-L group but in none of the other groups. In the
significant difference of 2,5-AS activity at the fourth remaining 3 patients of the CR-L group, HCV RNA
week of therapy between these two treatment groups. was detected at 24 weeks after the therapy, whereas
We correlated the 2,5-AS activities with responses to serum ALT levels remained within the normal range.
interferon therapy. The initial 2,5-AS activities of the These 3 patients may have a chance of subsequent re-
CR-L, CR-S, and NR groups were 186.5 k 152.3, lapse. Thus, HCV seemed to have been successfully
129.7 + 162.4, and 155.3 f 133.1, respectively. The eradicated in 9 (17.0%) of the 53 patients who received
2,5-AS activities at the fourth week of therapy (CR-L, prolonged interferon therapy.
300.8 -+ 170.8; CR-S, 349.4 + 218.2; NR, 269.3 -I- We quantified HCV RNA in the serum of patients
206.1) were significantly higher (P< 0.01) than those treated with interferon alfa and tried to correlate the
at the beginning of treatment in all groups. However, amount with the response to interferon. Methods
those at the beginning and the fourth week of therapy based on PCR assay have been reported to be useful for
were not significantly different among the CR-L, CR- quantifying small amounts of RNA,22-24 and we used a
S, and NR groups. competitive RT-PCR assay with synthetic mutant
HCV RNA as a competitor according to the methods
Multivariate Analysis
of Becker-Andre and Hahlbrock.22 The advantage of
Several variables (doses of interferon alfa, age, our method is that the quantification is independent of
sex, pretreatment total histological index, history of the many variables that affect RNA purification, re-
882 HAGIWARA ET AL. GASTROENTEROLOGY Vol. 104, No. 3

verse transcription, and cDNA amplification. How- hepatocellular carcinoma: analysis by detection of antibody to
ever, in our assay, the target RNA might have been hepatitis C virus. Hepatology 1990; 12:67 l-675.
5. Hagiwara H, Hayashi N, Mita E, Hiramatsu N, Ueda K, Takehara
overestimated because of (1) signal intensities of dif- T, Yuki N, Kasahara A, Fusamoto H, Kamada T. Detection of
ferent sizes of DNA fragments (306-bp and 198-bp) hepatitis C virus RNA in chronic non-A, non-B liver disease. Gas-
were compared on the agarose gel and (2) heterodu- troenterology 1992; 102:692-694.
6. Yuki N, Hayashi N, Kasahara A, Hagiwara H, Katayama K, Fusa-
plex formation could have occurred. We found that
mot0 H, Kamada T. Hepatitis B virus markers and antibodies to
overestimation influencing quantification on an aga- hepatitis C virus in Japanese patients with hepatocellular carci-
rose gel was a minimal problem when the numbers of noma. Dig Dis Sci 1992;37:65-72.
7. Hoofnagle JH, Mullen KD, Jones DB, Rustgi V, Di Bisceglie A,
amplification cycles are appropriate, depending on the
Peters M, Waggoner JG, Park Y, Jones EA. Treatment of chronic
initial amounts of target HCV RNA (data not shown). non-A, non-B hepatitis with recombinant human alpha interferon.
We previously evaluated HCV RNA in serial serum A preliminary report. N Engl J Med 1986;3 15: 1575- 1578.
samples during interferon therapy by an RT-PCR assay 8. Davis GL, Balart LA, Schiff ER, Lindsay K, Bodenheimer HC,
Perrillo RP, Carey W, Jacobson IM, Payne J, DienstagJL, VanThiel
and found that HCV RNA disappeared before the DH, Tanburro C, Lefkowitch J, Albrecht J, Meschievitz C, Ortego
ALT levels became normal in cases responding to in- TJ, Gibas A. The hepatitis interventional therapy group. Treat-
terferon and that this viral marker reappeared in pa- ment of chronic hepatitis C with recombinant interferon alfa. A
multicenter randomized, controlled trial. N Engl J Med
tients who had a relapse before the increase in ALT
1989;321:1501-1506.
level.‘” Quantification of the circulating HCV RNA 9. Di Bisceglie AM, Martin P, Kassianides C, Lisker-Melman M,
showed that the decreasing effect of interferon alfa on Murray L, Waggoner J, Goodman Z, Banks SM, Hoofnagle JH.
Recombinant interferon alfa therapy for chronic hepatitis C. A
serum concentration of viral particles was greater in
randomized, double-blind, placebo-controlled trial. N Engl J Med
the 6 MU-treated group than the 3 MU-treated group 1989;321:1506-1510.
and that the titer of HCV RNA significantly decreased 10. Saez-Royuela F, Porres JC, Moreno A, Castilio I, Martinez G, Ga-
in not only responders (groups CR-L and CR-S) but liana F, Carreno V. High doses of recombinant a-interferon or
y-interferon for chronic hepatitis C: a randomized, controlled trial.
also nonresponders (NR group). These results sug- Hepatology 199 1; 13:327-33 1.
gested that interferon alfa is effective for suppressing 11. Marcellin P, Boyer N, Giostra E, Degott C, Courouce AM, Degos F,
replication of HCV and that higher doses are better for Coppere H, Cales P, Couzigou P, Benhamou JP. Recombinant
human a-interferon in patients with chronic non-A, non-B hepati-
controlling disease activity. Furthermore, the titer of
tis: a multicenter randomized controlled trial from France. Hepa-
HCV RNA in serum before interferon treatment was tology 199 1; 13:393-397.
significantly lower in the CR-L group than the CR-S 12. Causse X, Godinot H, Chevallier M, Chossegros P, Zoulim F, Ou-
zan D, Heyraud JP, Fontanges T, Albrecht J, Meschievitz C, Trepo
group and the NR group. Multivariate multiple logis-
C. Comparison of 1 or 3 MU of interferon alfa-2b and placebo in
tic regression also showed that the patients with low patients with chronic non-A, non-B hepatitis. Gastroenterology
initial titers of HCV RNA are most likely to achieve a 1991;101:497-502.
sustained response to interferon alfa therapy. 13. Hoofnagle JH, Peters M, Mullen KD, Jones DB, Rustgi V, Di Bi-
sceglie A, Hallahan C, Park Y, Meschievitz C, Jones EA. Random-
Based on these findings, we conclude that the initial ized, controlled trial of recombinant human a-interferon in pa-
titer of HCV RNA is the most important factor affect- tients with chronic hepatitis 6. Gastroenterology 1988;
ing the sustained response to interferon therapy. 95:1318-1325.
14. Perrillo RP, Schiff ER, Davis GL, Bodenheimer HC, Lindsay K,
References Payne J. Dienstag JL, O’Brien C, Tamburro C, Jacobson IM, Sam-
pliner R, Feit D, Lefkowitch J, Kuhns M, Meschievitz C, Sanghvi B,
Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Albrecht J, Gibas A. The hepatitis interventional therapy group. A
Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, randomized, controlled trial of interferon alfa-2b, alone and follow-
Bonino F, Colombo M, Lee WS, Kuo C, Berger K, Shuster JR, ing predonisone withdrawal, in the treatment of chronic hepatitis
Overby LR, Bradley DW, Houghton M. An assay for circulating B. N Engl J Med 1990;323:295-301.
antibodies to a major etiologic virus of human non-A, non-B hepa- 15. Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C,
titis. Science 1989;244:362-364. Rosenblatt J, Choo QL. Houghton M. Detection of hepatitis C viral
Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo sequence in non-A, non-B hepatitis. Lancet 1990;335: l-3.
QL, Kuo G. Detection of antibody to hepatitis C virus in prospec- 16. Ulrich PP, Romeo JM, Lane PK, Kelly I, Daniel IJ, Vyas GN. Detec-
tively followed transfusion recipients with acute and chronic non- tion, semiquantitation, and genetic variation in hepatitis C virus
A, non-B hepatitis. N Engl J Med 1989;321: 1494-1500. sequence amplified from the plasma of blood donors with ele-
Hopf U, Moller B, Kuther D, Stemerowicz R, Lobeck H, Ludtke- vated alanine aminotransferase. J Clin Invest 1990;86: 1609-
Handjery A, Walter E, Blum HE, Roggendorf M, Deinhardt F. 1614.
Long-term follow-up of posttransfusion and sporadic chronic hep- 17. Kato N, Yokosuka 0, Omata M, Hosoda K, Ohto M. Detection of
atitis non-A, non-B and frequency of circulating antibodies to hep- hepatitis C virus ribonucleic acid in the serum by amplification
atitis C virus (HCV). J Hepatol 1990; 10:69-76. with polymerase chain reaction. J Clin Invest 1990;86: 1764-
Kiyosawa K, Sodeyama T, Tanaka E, Gibo Y, Yoshizawa K, Na- 1767.
kano Y, Furuta S, Akahane Y, Nishioka K, Purcell RH, Alter HJ. 18. Takehara T, Hayashi N, Mita E, Hagiwara H, Ueda K, Katayama K,
Interrelationship of blood transfusion, non-A, non-B hepatitis and Kasahara A, Fusamoto H, Kamada T. Detection of minus strand
March 1993 IFN THERAPY AND QUANTITATION OF HCV RNA 883

of hepatitis C virus RNA by reverse transcription and polymerase PCR aided transcript titration assay (PATTY). Nucleic Acids Res
chain reaction. Implication for hepatitis C virus replication in in- 1989; 17:9437-9446.
fected tissue. Hepatology 1992; 15:387-390. 23. Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the
19. Hagiwara H, Hayashi N, Mita E, Ueda K, Takahara T, Kasahara A, polymerase chain reaction. Proc Natl Acad Sci USA
Fusamoto H, Kamada T. Detection of hepatitis C virus RNA in 1989;86:97 17-972 1.
serum of patients with chronic hepatitis C treated with interferon- 24. Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine
a. Hepatology 1992; 15:37-4 1. mRNA and DNA: detection and quantitation by competitive poly-
20. Knodell RG, lshak KG, Black WC, Chen TS, Craig R, Kaplowitz N, merase chain reaction. Proc Natl Acad Sci USA 1990;87:2725-
Kiernan TW, Wollman J. Formulation and application of a numeri- 2729.
cal scoring system for assessing histological activity in asymp-
tomatic chronic active hepatitis. Hepatology 199 1; I:43 l-435.
Received April 24, 1992. Accepted October 6, 1992.
21. Takamizawa A, Mori C, Fuke I, Manabe S, Murakami S, Fujita J, Address requests for reprints to: Norio Hayashi, M.D., First Depatt-
Onishi E, Andoh T, Yoshida I, Okayama H. Structure and organiza- ment of Medicine, Osaka University Medical School, Fukushima l-l-
tion of the hepatitis C virus genome isolated from human carriers. 50, Fukushima-ku, Osaka 553, Japan.
J Virol 1991;65:1105-1113. Supported by a Grant-in-Aid from the Ministry of Education,
22. Becker-Andre M, Hahlbrock K. Absolute mRNA quantification us- Science and Culture, Japan, and the research group of Intractable
ing the polymerase chain reaction (PCR). A novel approach by a Hepatitis sponsored by the Ministry of Health and Welfare of Japan.