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Hepatitis C virus antibody and hepatitis C virus replication in chronic hepatitis B patients
Journal of Hepatology 1994; 21:509-514 Prhlted #1 Denmark. ,,Ill rights reserved Munksgaard. Copenhagen
Copyright © Journalof Ht,patology 1994 Journal of Hepatology ISSN 0168-8278
Hepatitis C virus antibody and hepatitis C virus replication in chronic hepatitis B patients Kazuyoshi Ohkawa, N o r i o Hayashi, N o b u k a z u Yuki, Hideki Hagiwara, Michio Kato, Keiji Yamamoto, Hiroshi Eguchi, Hideyuki Fusamoto, M a n a b u Masuzawa and Takenobu K a m a d a First Department o1" Medichle, Osaka University School o f Medichle, Suita, Osaka and Department o f Gastroenterology, Osaka National Hospital, Osaka, Japan
(Received 1 April 1993)
We assessed hepatitis C virus infection in 156 chronic hepatitis B patients using second-generation hepatitis C virus antibody (anti-HCV). Active virus replication was further investigated in anti-HCV-positive cases by means of polymerase chain reaction assay for the detection of serum hepatitis C virus RNA. Anti-HCV prevalence was higher in patients negative for hepatitis B e antigen (HBeAg) (10/48, 21%) than in HBeAg-positive patients (10/108, 9%) (p<0.05), and the reactivity (cut-off index) in anti-HCV enzyme-linked immunosorbent assay of the positive cases was significantly higher in HBeAg-negative patients (4.1+0.1) than in -positive ones (3.6+0.6) (p<0.05). The prevalence of hepatitis C virus RNA in anti-HCV-positive cases was also higher in the HBeAg-negative group (9/10, 90%) than in the -positive group (3/10, 30%) (p<0.01). Viremia was found in association with high reactivity in anti-HCV ELISA (cut-off index >3.5) in both groups. Nine (90%) of 10 such cases were viremic in the HBeAg-negative group compared with three.(43%) of seven in the HBeAg-positive group (p<0.05). These results suggest that hepatitis C virus replication may be influenced by hepatitis B virus replicative states, indicating possible interference between hepatitis B and C viruses. © Journal of Hepatology. Key words." Chronic hepatitis B; Chronic hepatitis C; Hepatitis B virus; Hepatitis C virus; Hepatitis C virus antibody;
Hepatitis C virus RNA
Recently, the genome of a non-A, non-B hepatitis virus, designated hepatitis C virus (HCV), was cloned, and a specific assay for the detection of antibody to HCV (antiHCV) was developed (1,2). This first-generation antiHCV assay was based on a nonstructural HCV protein (C100-3 antigen). Seroepidemiological studies using this assay demonstrated that antibody to C100-3 antigen (anti-C100-3) was found in most chronic non-A, non-B hepatitis patients (3-7), and that HCV infection could result in advanced liver diseases, such as cirrhosis and hepatocellular carcinoma. The anti-Cl00-3 assay has also revealed possible coinfection with HCV and hepatitis B virus (HBV) in HBVendemic areas. Several reports from different geographical areas have shown that anti-C100-3 was found in approximately 10-15% of HBV carriers with various stages of
liver disease (6-9). In Italy and Greece, a surprisingly high prevalence of anti-C100-3 (54% and 72%, respectively) has also been reported in patients with HBV-associated hepatocellular carcinoma (4,10). However, the problems of sensitivity and specificity remain for this assay. It has been demonstrated that the HCV genome can be detected even in anti-C100-3-negative patients by reverse transcription-polymerase chain reaction (RT-PCR) (11). False-positive results are also a problem associated with the original anti-Cl00-3 assay (12). To resolve these problems, second-generation antiHCV assays, which detect antibodies to structural and nonstructural HCV proteins, were introduced, and have proved more sensitive and specific (13,14). In this study, we tried to make a more precise evaluation of coinfection with HBV and HCV using second-gener-
Correspondenceto: Norio Hayashi,M.D., First Department of Medicine,Osaka UniversitySchool of Medicine,Yamadaoka2-2, Suita 565, Japan.
510 ation anti-HCV assays and to further confirm active HCV replication by RT-PCR. The results were correlated with HBV replicative states and the interaction between the two viruses was investigated. Patients and Methods
Sera from 156 consecutive Japanese patients with chronic hepatitis B were used for this study. All patients had been positive for hepatitis B surface antigen (HBsAg) for more than 6 months. One hundred and eight patients had hepatitis B e antigen (HBeAg). Antibody to HBeAg (anti-HBe) was found in 35 (73%) of the remaining 48 patients. There were 111 men and 45 women, and the age ranged from 19 to 73 (mean 42.5) years. Histological diagnosis was available for 125 patients. Liver histology showed features of chronic persistent hepatitis in 18 cases, of chronic active hepatitis in 86 cases, and of cirrhosis in 21 cases. The remaining 31 patients had hepatocellular carcinoma (HCC) diagnosed by imaging procedures. Nineteen patients had had a previous blood transfusion. All serum samples were stored at -20°C for this study. HBsAg, HBeAg and anti-HBe were determined using commercially available enzyme immunoassays (Dainabot Co., Ltd., Tokyo, Japan). Serum HBV DNA was detected by a spot hybridization technique, as described by Scott et al. (15). HBV DNA levels were determined semi-quantitatively (graded as 1+ to 4+), according to the signal intensities of the autoradiography. Anti-HCV was first tested using a second-generation ELISA system (Ortho Diagnostic Systems Co., Ltd., Tokyo, Japan). This assay was performed according to the manufacturer's instructions. All assays were done in duplicate. To compare ELISA results of different samples, the ratio of sample values to the cut-off value (cut-off index) was calculated. As for ELISA-positive cases, a dot-blot assay (Matrix HCV, Dainabot Co., Ltd., Tokyo, Japan) was further performed as a confirmatory test. The dotblot assay included four recombinant HCV antigens, three antigens (core, NS3 and NS4) expressed in E. coil and one yeast-derived antigen (NS4). Reactivity to only one of the two NS4 antigens was interpreted as inconclusive for reactivity to the NS4 protein, and did not contribute to a positive interpretation. Reactivity to at least two distinct HCV proteins from the three HCV regions was considered positive, while reactivity to just one HCV protein was judged "single antigen reactive". Sera nonreactive for all HCV antigens were considered negative. Serum HCV RNA was tested for by the RT-PCR assay, as described previously (16,17). Primers were derived from the highly conserved 5'-noncoding region of the published sequence: anti-sense primer, 5'-CATGGTGCACGGTC-
K. OHKAWAet al. TACGAG-3', and sense primer, 5'-ACTCCACCATAGATCACTCC-3'. The PCR mixtures were amplified in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) for 40 cycles (94°C for 30 s; 55°C for 1 min; and 72°C for I min), followed by a 10-min final extension at 72°C. An aliquot of the PCR products was fractioned by agarose gel electrophoresis, transferred onto a nylon membrane, hybridized to a -a-'P-labeled HCV cDNA between the two primers, and autoradiographed. Great care was taken to prevent false-positive results, as recommended by Kwok & Higuchi (18). Using this assay, just a single copy of HCV cDNA could be detected. Statistical analysis was performed by the Z2 test and Student's t-test. Mann-Whitney's U test was also used for the group comparison of semi-quantitative HBV DNA levels. Results
Anti-HCV was found by ELISA in 24 (15°/,,) of the 156 patients with chronic HBV infection, and positivity was confirmed in 20 patients by the dot-blot assay. Of the remaining four ELISA-positive cases, two were judged negative and two were "single antigen reactive" by the dot-blot assay. The mean cut-off index (C.I.) in ELISA of these four cases was significantly lower than for the 20 dot-blotpositive cases (1.5_+0.6 vs. 3.8_+0.5, p<0.01). Serum HCV RNA was found in 12 (60%) of the 20 dot-blot-positive cases, but not in the four cases negative or "single antigen reactive" in the dot-blot assay (p<0.05). In this study, these four cases were excluded from the anti-HCV-positive group because of possible false-positive reactions of antiHCV ELISA, and the population of the 156 chronic hepatitis B patients was classified into 20 (13%) anti-HCV-positive and 136 (87°/,,) anti-HCV-negative cases. Table 1 shows the detection of HCV markers and patients' clinical characteristics in 156 chronic hepatitis B patients with various stages of the disease. The mean age was higher in patients with cirrhosis (49_+11 years) and patients with HCC (58+10 years) than in patients with chronic persistent hepatitis (37_+9 years) and chronic active hepatitis (36_+ I0 years) (p<0.01). A significant difference was also observed between patients with cirrhosis and patients with HCC (p<0.01). There were more males in the HCC group than in the group with chronic active hepatitis (p<0.05). More patients with HCC (29%) had received blood transfusions than those with chronic persistent hepatitis (0%) (p<0.05) and chronic active hepatitis (8%) (p<0.01). HBeAg was found less frequently in patients with HCC (29%) than in those with chronic persistent hepatitis (67%) and chronic active hepatitis (88%) (p<0.05 and p<0.01, respectively). HBeAg positivity was
HCV INFECTION IN CHRONIC HEPATITIS B
511
TABLE 1 Detection of concomitant HCV infection in chronic hepatitis B of various stages Patients
n
Agea (years)
Sex (M/F)
Previous blood transfusions
HBeAg positivity
Anti-HCV prevalence
HCV RNA prevalence in anti-HCV-positive cases
Chronic persistent hepatitis Chronic active hepatitis Cirrhosis Hcpatocellular carcinoma
18 86 21 31
37+9 b,c 36--10 bx 49+11 ~ 58_+10
14/4 55/31a 16/5 26/5
0d 7 (8%)c 3 (14%) 9 (29%)
12 (67%)dx 76 (88%,)bx 11 (52°/,,) 9 (29%)
0J'r 8 (9%) 5 (24%) 7 (23%)
4/8 (50%) 3/5 (60%) 5/7 (71%)
" Values are mean+_SD, b p<0.01 VS. cirrhosis. ': p<0.01 vs. hepatocellular carcinoma. 'j p<0.05 vs. hepatocellular carcinoma. "p<0.05 vs. chronic active hepatitis, r p<0.05 vs. cirrhosis. also less frequent in patients with cirrhosis (52%) than in patients with chronic active hepatitis (p<0.01). A significant difference was also seen between chronic persistent hepatitis and chronic active hepatitis (p<0.05). A n t i - H C V was found in eight (9%) o f the 86 patients with chronic active hepatitis, five (24%) o f the 21 patients with cirrhosis, and seven (23%) of the 31 patients with HCC, but not in the 18 patients with chronic persistent hepatitis. The prevalence o f a n t i - H C V was significantly higher in patients with cirrhosis and patients with H C C than in those with chronic persistent hepatitis (p<0.05). As for serum HCV R N A positivity in anti-HCV-positive cases, HCV R N A was detected in four (50%) o f the eight anti-HCV-positive patients with chronic active hepatitis, three (60%) o f the five patients with cirrhosis, and five (71%) o f the seven patients with HCC. N o significant difference was observed a m o n g the three groups. When a n t i - H C V .positivity was correlated with various clinical characteristics (age, sex, previous blood transfusions and HBeAg positivity) (Table 2), anti-HCV-positive patients were found to be significantly older than antiHCV-negative ones (52_+14 years vs. 41-+13 years, p<0.01). More anti-HCV-positive patients had received blood transfusions than anti-HCV-negative ones (40% vs. 8%, p<0.01). H B e A g prevalence was significantly lower in anti-HCV-positive patients than in -negative ones (50% vs. 72%, p<0.05). N o significant difference was observed in the sex ratio between the anti-HCV-positive and -negative groups. TABLE 2
Anti-HCVpositive (n=20)
Age (years)" 52---14 Sex (M/F) 14/6 Previous blood trans- 8 (40%) fusions HBeAg positivity 10 (50%) " Values are mean--+SD.
HBeAgpositive
Anti-HCVnegative (n= 136)
p-value
41 -+13 97/39 11 (8%)
p<0.01 NS p<0.01
98 (72%)
p<0.05
HBeAg. negative @@
&A
4
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Clinical characteristics of chronic hepatitis B patients positive and negative for HCV antibody Characteristics
We further investigated reactivity in anti-HCV E L I S A expressed as C.I. and serum HCV R N A positivity in relation to the presence o f HBeAg (Fig. 1). A n t i - H C V was found in 10 (9%) o f the 108 patients positive for HBeAg and in I0 (21%) of the 48 patients negative for it (p<0.05). In the HBeAg-negative group, all 10 anti-HCV-positive patients tested positive in E L I S A with C.I.>3.5. In the HBeAg-positive group, however, the C.I. o f E L I S A of the 10 anti-HCV-positive patients ranged from 2.2 to 4.3, and only seven (70%) patients tested positive in E L I S A with C.I.>3.5. A significant difference was observed in the C.I.
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Fig. I. Reactivity in second-generation anti-HCV assays and detection of serum HCV RNA in chronic hepatitis B patients in relation to HBeAg positivity. Reactivity in anti-HCV ELISA was expressed as cut-off index. A confirmatory dot-blot anti-HCV assay and testing for serum HCV RNA were performed on ELISA-positive cases. Open circles represent patients negative for anti-HCV by ELISA. Closed circles represent patients positive for anti-HCV by both ELISA and the dot-blot assay, and positive for HCV RNA. Closed triangles represent patients positive for anti-HCV by both ELISA and the dot-blot assay, but negative for HCV RNA. Open triangles represent patients positive for anti-HCV ELISA but negative or "single antigen reactive" in the dot-blot assay, who are also negative for HCV RNA.
512
K. O H K A W A et al.
of anti-HCV ELISA of positive cases between HBeAgpositive and -negative groups (3.6___0.6 vs. 4.1___0.1, p<0.05). Serum HCV RNA was found among patients positive for anti-HCV ELISA with C.I.>3.5, but not among patients positive for it with C.I.<3.5. Thus, the prevalence of serum HCV RNA in anti-HCV-positive cases was significantly higher in the HBeAg-negative group (9/10, 90%) than in the HBeAg-positive group (3/ 10, 30%) (p<0.01). As for patients with high reactivity (C.I.>3.5) in anti-HCV ELISA, the prevalence of serum HCV RNA was also higher in the HBeAg-negative group (9/10, 90%) than in the HBeAg-positive group (3/7, 43°/,,)
(p<0.05). As for anti-HCV-positive cases, serum HBV DNA levels were further determined to evaluate HBV replicative states more accurately. Serum HBV DNA was found in six (60°/,,) of the 10 HBeAg-positive cases, but not in the 10 HBeAg-negative cases (p<0.01). Serum HBV DNA levels semi-quantitated for the six positive cases were 1+ in two, 3+ in two and 4+ in two cases. When the detection of serum HCV RNA was correlated with serum HBV DNA positivity, serum HCV RNA was found in three (50%) of the six HBV DNA-positive cases compared with nine (64%) of the 14 HBV DNA-negative cases (not significant). Serum HBV DNA levels also did not differ significantly between serum HCV RNA-positive and -negative cases. We followed one HBeAg-positive chronic active hepatitis patient (age 29, male) for 4.2 years (Fig. 2). This case showed seroconversion to anti-HBe with the decline of serum HBV DNA levels at 1 year after a 4-week course of interferon-a therapy. A total of 30 megaunits (MU) of
Anti-HCV HBsAg HBeAE
Anti-HBe
+
t + +
H B V D N A H C V R N A
~
~
-
-4+3+
-
-
500
~-
400
-
300
-
200
-
100
-
0
+
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-
!
-
.-i-
+
-
chronic active hepatitis
(IU/l)
)
-
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-
Biopsy
ALT
]+][
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+
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IFN-~r
!
I J987
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1988
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-
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IZ
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I 1990
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/J
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IJ
I
Fig. 2. Detection of anti-HCV and serum HCV R N A in a chronic active hepatitis B patient who was followed for 4.2 years. Anti-HCV was persistently found by ELISA with C.I.>3.5, and also found by the dot-blot assay. Serum H C V R N A was not detected in the HBeAgpositive phase, but was detected after seroconversion to anti-HBe.
natural interferon-a (Sumiferon, Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan) was administered. He also cleared HBsAg during follow up. Anti-HCV was persistently found by ELISA with C.I.>3.5, and also found by the dot-blot assay during follow up. The patient did not have detectable levels of serum HCV RNA in the HBeAgpositive phase before interferon-a therapy, but became positive after seroconversion to anti-HBe. There had been no risk of infection with HCV during the follow-up period.
Discussion
Since the introduction of the first-generation anti-HCV assays, many chronic hepatitis B patients have also been shown to be infected with HCV, although the incidence differs among regions (4, 6-10). The prevalence of antiCI00-3 in these patients is much higher than in volunteer blood donors. However, the prevalence of HCV infection in HBV-endemic areas has not been fully reassessed using second-generation assays, which are more sensitive and specific. The relationship of HCV replicative states to HBV replication in HBV carriers also needs to be determined. The present study revealed that approximately 13% of Japanese patients with chronic hepatitis B tested positive in second-generation anti-HCV assays, which was quite similar to a previous report in our area based on a first-generation assay (7). In the present study, anti-HCV positivity showed a relationship to age and previous blood transfusions, indicating that parenteral transmission is also a major route of HCV infection in the chronic hepatitis B group. As for anti-HCV prevalence in various stages of chronic HBV infection, anti-HCV tended to be frequently detected in advanced liver diseases, such as cirrhosis and hepatocellular carcinoma, as has been indicated in a few previous reports using first-generation assays (7,9). Furthermore, active HCV replication was found in approximately 5070% of anti-HCV-positive cases irrespective of the disease stage. The clinical implications of frequent HCV markers in advanced liver diseases remain unclear. It may merely reflect increased risk of exposure to HCV in older patients with advanced liver diseases. Indeed, previous blood transfusions were more frequently observed in these cases. Nevertheless, coinfection with HCV in the course of chronic HBV infection may be a risk factor for more serious liver disease and the development of HCC, as is the case for chronic hepatitis C patients. Further studies must be performed to clarify this. The present study also clarified the interrelation of antiHCV reactivity, serum HCV RNA positivity and HBV replicative states. Serum HCV RNA was found in associ-
HCV INFECTION IN CHRONIC HEPATITIS B ation with high reactivity of anti-HCV, which has been reported for chronic non-A, non-B hepatitis patients (14). A n t i - H C V reactivity and serum H C V R N A positivity of anti-HCV-positive cases were also shown to differ considerably between HBeAg-positive and -negative groups. HBeAg-positive cases showed significantly lower reactivity of a n t i - H C V than HBeAg-negative patients. Furthermore, H C V R N A was detected less frequently in association with high reactivity o f a n t i - H C V in HBeAg-positive cases than in HBeAg-negative ones. These findings suggest that HCV replication may be suppressed in the HBeAg-positive phase, which may result in a decrease in a n t i - H C V reactivity. On the other hand, in the HBeAgnegative phase, most cases with a n t i - H C V seemed to have active HCV replication accompanied by high reactivity o f antibody. These findings indicate that the level of HCV replication may be influenced by the HBV replicative states. In chronic HBV infection, active HBV replication ceases with time, and patients with advanced liver diseases usually have low levels of HBV replication. In constrast with changes in HBV replicative levels, HCV replication might be suppressed in the early phase during active HBV replication, and enhanced in the iater phase. The longitudinal study in this work supports this hypothesis. In the present study, however, the prevalence o f serum HCV R N A in anti-HCV-positive cases did not differ significantly between serum HBV D N A - p o s i t i v e and -negative cases, although significant differences were observed between HBeAg-positive and -negative cases. This discrepancy may be because serum HBV D N A levels often change more markedly than H B e A g positivity in a short time. We suggest that HCV replication might be suppressed in the HBeAg-positive phase, when HBV replicative levels often fluctuate markedly, and enhanced in the HBeAg-negative phase, which reflects the persistent decrease in HBV replicative levels. So far, interference between HBV and H C V has been indicated by a few researchers. First-generation a n t i - H C V has been found more frequently in HBV carriers with lower levels of viral replication (9). In the present study based on second-generation assays, a n t i - H C V prevalence was also higher in HBeAg-negative patients than in -positive ones. It has also been suggested that the agent of nonA, non-B hepatitis may interfere with HBV replication (19), although controversy remains about suppression o f • HBV replication in HBV carriers superinfected with H C V (20). We cannot completely exclude the possibility that frequent occurrence o f results that are anti-HCV-positive but HCV RNA-negative in HBeAg-positive cases is due to false-positivity o f the a n t i - H C V assay, or just indicates
513
previous exposure to HCV, though a n t i - H C V positivity was confirmed using the dot-blot assay. However, the high prevalence o f these cases in the HBeAg-positive phase is difficult to explain. F u r t h e r quantitative analysis of HBV and HCV replicative states in prospective studies should offer a better understanding of this issue. Using second-generation a n t i - H C V assays and RT-PCR for detecting serum HCV R N A , HCV infection was shown to be involved in many chronic hepatitis B patients, indicating similar modes o f infection o f the two viruses. Our data also suggest possible interference between HBV and HCV, as has been indicated for other viruses.
References 1. Choo Q-L, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-62. 2. Kuo G, Choo Q-L, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989; 244: 362--4. 3. Bruix J, Barrera JM, Calvet X, et al. Prevalence of antibodies to hepatitis C virus in Spanish patients with hepatocellular carcinoma and hepatic cirrhosis. Lancet 1989; ii: 1004--6. 4. Colombo M, Kuo G, Choo Q-L, et al. Prevalence of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet 1989; ii: 1006-8. 5. Hopf U, M611er B, K~lther D, et al. Long-term follow-up of posttransfusion and sporadic chronic hepatitis nbn-A, non-B and frequency of circulating antibodies to hepatitis C virus (HCV). J Hepatol 1990; 10: 69-76. 6. Kiyosawa K, Sodeyama T, Tanaka E, et al. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990; 12: 671-5. 7. Yuki N, Hayashi N, Kasahara A, et al. Hepatitis B virus markers and antibodies to hepatitis C virus in Japanese patients with hepatocellular carcinoma. Dig Dis Sci 1992; 37: 65--72. 8. Fattovich G, Tagger A, Brollo L, et al. Hepatitis C virus infection in chronic hepatitis B virus carriers. J Infect Dis 1991; 163: 4002. 9. Fong T-L, Di BisceglieAM, Waggoner JG, Banks SM, Hoofnagle JH. The significance of antibody to hepatitis C virus in patients with chronic hepatitis B. Hepatology 1991; 14: 64-7. 10. Kaklamani E, Trichopoulos D, Tzonou A, et al. Hepatitis B and C viruses and their interaction in the origin of hepatocellular carcinoma. JAMA 1991; 265: 1974-6. 11. Weiner AJ, Kuo G, Bradley DW, et al. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet 1990; 335: 13. 12. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? Lancet 1990; 335: 754-7. 13. Yuki N, Hayashi N, Hagiwara H, et al. Serodiagnosis of chronic hepatitis C in Japan by second-generation recombinant immunoblot assay. J Hepatol 1993; 17: 170-4. 14. Yuki N, Hayashi N, Hagiwara H, eta|. Improved serodiagnosis of chronic hepatitis C in Japan by a second-generation enzymelinked immunosorbent assay. J Med Virol 1992; 37: 237-40. 15. Scott J, Hadchouel M, Hery C, Yvart J, Tiollais P, Brechot C. Detection of hepatitis B virus DNA in serum by a simple spot hybridization technique: comparison with results for other viral markers. Hepatology 1983; 3: 279-84.
514 16. Hagiwara H, Hayashi N, Mita E, et al. Detection of hepatitis C virus RNA in chronic non-A, non-B liver disease. Gastroenterology 1992: 102: 692~.. 17. Hagiwara H, Hayashi N, Mita E, et al. Detection of hepatitis C virus RNA in serum of patients with chronic hepatitis C treated with interferon-a. Hepatology 1992: 15: 37-41. 18, Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989: 339: 237-8.
K. OHKAWA et al. 19. Tsiquaye KN, Portmann B. Tovey G, et al. Non-A, non-B hepatitis in persistent carriers of hepatitis B virus. J Med Virol 1983: 1 I : 179-89. 20. Wang JT. Wang T-H, Sheu J-C, Lin J-T, Wang C-Y, Chen D-S. Posttransfusion hepatitis revisited by hepatitis C antibody assays and polymerase chain reaction. Gastroenterology 1992; 103: 60916.
Copyright © Journalof Ht,patology 1994 Journal of Hepatology ISSN 0168-8278
Hepatitis C virus antibody and hepatitis C virus replication in chronic hepatitis B patients Kazuyoshi Ohkawa, N o r i o Hayashi, N o b u k a z u Yuki, Hideki Hagiwara, Michio Kato, Keiji Yamamoto, Hiroshi Eguchi, Hideyuki Fusamoto, M a n a b u Masuzawa and Takenobu K a m a d a First Department o1" Medichle, Osaka University School o f Medichle, Suita, Osaka and Department o f Gastroenterology, Osaka National Hospital, Osaka, Japan
(Received 1 April 1993)
We assessed hepatitis C virus infection in 156 chronic hepatitis B patients using second-generation hepatitis C virus antibody (anti-HCV). Active virus replication was further investigated in anti-HCV-positive cases by means of polymerase chain reaction assay for the detection of serum hepatitis C virus RNA. Anti-HCV prevalence was higher in patients negative for hepatitis B e antigen (HBeAg) (10/48, 21%) than in HBeAg-positive patients (10/108, 9%) (p<0.05), and the reactivity (cut-off index) in anti-HCV enzyme-linked immunosorbent assay of the positive cases was significantly higher in HBeAg-negative patients (4.1+0.1) than in -positive ones (3.6+0.6) (p<0.05). The prevalence of hepatitis C virus RNA in anti-HCV-positive cases was also higher in the HBeAg-negative group (9/10, 90%) than in the -positive group (3/10, 30%) (p<0.01). Viremia was found in association with high reactivity in anti-HCV ELISA (cut-off index >3.5) in both groups. Nine (90%) of 10 such cases were viremic in the HBeAg-negative group compared with three.(43%) of seven in the HBeAg-positive group (p<0.05). These results suggest that hepatitis C virus replication may be influenced by hepatitis B virus replicative states, indicating possible interference between hepatitis B and C viruses. © Journal of Hepatology. Key words." Chronic hepatitis B; Chronic hepatitis C; Hepatitis B virus; Hepatitis C virus; Hepatitis C virus antibody;
Hepatitis C virus RNA
Recently, the genome of a non-A, non-B hepatitis virus, designated hepatitis C virus (HCV), was cloned, and a specific assay for the detection of antibody to HCV (antiHCV) was developed (1,2). This first-generation antiHCV assay was based on a nonstructural HCV protein (C100-3 antigen). Seroepidemiological studies using this assay demonstrated that antibody to C100-3 antigen (anti-C100-3) was found in most chronic non-A, non-B hepatitis patients (3-7), and that HCV infection could result in advanced liver diseases, such as cirrhosis and hepatocellular carcinoma. The anti-Cl00-3 assay has also revealed possible coinfection with HCV and hepatitis B virus (HBV) in HBVendemic areas. Several reports from different geographical areas have shown that anti-C100-3 was found in approximately 10-15% of HBV carriers with various stages of
liver disease (6-9). In Italy and Greece, a surprisingly high prevalence of anti-C100-3 (54% and 72%, respectively) has also been reported in patients with HBV-associated hepatocellular carcinoma (4,10). However, the problems of sensitivity and specificity remain for this assay. It has been demonstrated that the HCV genome can be detected even in anti-C100-3-negative patients by reverse transcription-polymerase chain reaction (RT-PCR) (11). False-positive results are also a problem associated with the original anti-Cl00-3 assay (12). To resolve these problems, second-generation antiHCV assays, which detect antibodies to structural and nonstructural HCV proteins, were introduced, and have proved more sensitive and specific (13,14). In this study, we tried to make a more precise evaluation of coinfection with HBV and HCV using second-gener-
Correspondenceto: Norio Hayashi,M.D., First Department of Medicine,Osaka UniversitySchool of Medicine,Yamadaoka2-2, Suita 565, Japan.
510 ation anti-HCV assays and to further confirm active HCV replication by RT-PCR. The results were correlated with HBV replicative states and the interaction between the two viruses was investigated. Patients and Methods
Sera from 156 consecutive Japanese patients with chronic hepatitis B were used for this study. All patients had been positive for hepatitis B surface antigen (HBsAg) for more than 6 months. One hundred and eight patients had hepatitis B e antigen (HBeAg). Antibody to HBeAg (anti-HBe) was found in 35 (73%) of the remaining 48 patients. There were 111 men and 45 women, and the age ranged from 19 to 73 (mean 42.5) years. Histological diagnosis was available for 125 patients. Liver histology showed features of chronic persistent hepatitis in 18 cases, of chronic active hepatitis in 86 cases, and of cirrhosis in 21 cases. The remaining 31 patients had hepatocellular carcinoma (HCC) diagnosed by imaging procedures. Nineteen patients had had a previous blood transfusion. All serum samples were stored at -20°C for this study. HBsAg, HBeAg and anti-HBe were determined using commercially available enzyme immunoassays (Dainabot Co., Ltd., Tokyo, Japan). Serum HBV DNA was detected by a spot hybridization technique, as described by Scott et al. (15). HBV DNA levels were determined semi-quantitatively (graded as 1+ to 4+), according to the signal intensities of the autoradiography. Anti-HCV was first tested using a second-generation ELISA system (Ortho Diagnostic Systems Co., Ltd., Tokyo, Japan). This assay was performed according to the manufacturer's instructions. All assays were done in duplicate. To compare ELISA results of different samples, the ratio of sample values to the cut-off value (cut-off index) was calculated. As for ELISA-positive cases, a dot-blot assay (Matrix HCV, Dainabot Co., Ltd., Tokyo, Japan) was further performed as a confirmatory test. The dotblot assay included four recombinant HCV antigens, three antigens (core, NS3 and NS4) expressed in E. coil and one yeast-derived antigen (NS4). Reactivity to only one of the two NS4 antigens was interpreted as inconclusive for reactivity to the NS4 protein, and did not contribute to a positive interpretation. Reactivity to at least two distinct HCV proteins from the three HCV regions was considered positive, while reactivity to just one HCV protein was judged "single antigen reactive". Sera nonreactive for all HCV antigens were considered negative. Serum HCV RNA was tested for by the RT-PCR assay, as described previously (16,17). Primers were derived from the highly conserved 5'-noncoding region of the published sequence: anti-sense primer, 5'-CATGGTGCACGGTC-
K. OHKAWAet al. TACGAG-3', and sense primer, 5'-ACTCCACCATAGATCACTCC-3'. The PCR mixtures were amplified in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) for 40 cycles (94°C for 30 s; 55°C for 1 min; and 72°C for I min), followed by a 10-min final extension at 72°C. An aliquot of the PCR products was fractioned by agarose gel electrophoresis, transferred onto a nylon membrane, hybridized to a -a-'P-labeled HCV cDNA between the two primers, and autoradiographed. Great care was taken to prevent false-positive results, as recommended by Kwok & Higuchi (18). Using this assay, just a single copy of HCV cDNA could be detected. Statistical analysis was performed by the Z2 test and Student's t-test. Mann-Whitney's U test was also used for the group comparison of semi-quantitative HBV DNA levels. Results
Anti-HCV was found by ELISA in 24 (15°/,,) of the 156 patients with chronic HBV infection, and positivity was confirmed in 20 patients by the dot-blot assay. Of the remaining four ELISA-positive cases, two were judged negative and two were "single antigen reactive" by the dot-blot assay. The mean cut-off index (C.I.) in ELISA of these four cases was significantly lower than for the 20 dot-blotpositive cases (1.5_+0.6 vs. 3.8_+0.5, p<0.01). Serum HCV RNA was found in 12 (60%) of the 20 dot-blot-positive cases, but not in the four cases negative or "single antigen reactive" in the dot-blot assay (p<0.05). In this study, these four cases were excluded from the anti-HCV-positive group because of possible false-positive reactions of antiHCV ELISA, and the population of the 156 chronic hepatitis B patients was classified into 20 (13%) anti-HCV-positive and 136 (87°/,,) anti-HCV-negative cases. Table 1 shows the detection of HCV markers and patients' clinical characteristics in 156 chronic hepatitis B patients with various stages of the disease. The mean age was higher in patients with cirrhosis (49_+11 years) and patients with HCC (58+10 years) than in patients with chronic persistent hepatitis (37_+9 years) and chronic active hepatitis (36_+ I0 years) (p<0.01). A significant difference was also observed between patients with cirrhosis and patients with HCC (p<0.01). There were more males in the HCC group than in the group with chronic active hepatitis (p<0.05). More patients with HCC (29%) had received blood transfusions than those with chronic persistent hepatitis (0%) (p<0.05) and chronic active hepatitis (8%) (p<0.01). HBeAg was found less frequently in patients with HCC (29%) than in those with chronic persistent hepatitis (67%) and chronic active hepatitis (88%) (p<0.05 and p<0.01, respectively). HBeAg positivity was
HCV INFECTION IN CHRONIC HEPATITIS B
511
TABLE 1 Detection of concomitant HCV infection in chronic hepatitis B of various stages Patients
n
Agea (years)
Sex (M/F)
Previous blood transfusions
HBeAg positivity
Anti-HCV prevalence
HCV RNA prevalence in anti-HCV-positive cases
Chronic persistent hepatitis Chronic active hepatitis Cirrhosis Hcpatocellular carcinoma
18 86 21 31
37+9 b,c 36--10 bx 49+11 ~ 58_+10
14/4 55/31a 16/5 26/5
0d 7 (8%)c 3 (14%) 9 (29%)
12 (67%)dx 76 (88%,)bx 11 (52°/,,) 9 (29%)
0J'r 8 (9%) 5 (24%) 7 (23%)
4/8 (50%) 3/5 (60%) 5/7 (71%)
" Values are mean+_SD, b p<0.01 VS. cirrhosis. ': p<0.01 vs. hepatocellular carcinoma. 'j p<0.05 vs. hepatocellular carcinoma. "p<0.05 vs. chronic active hepatitis, r p<0.05 vs. cirrhosis. also less frequent in patients with cirrhosis (52%) than in patients with chronic active hepatitis (p<0.01). A significant difference was also seen between chronic persistent hepatitis and chronic active hepatitis (p<0.05). A n t i - H C V was found in eight (9%) o f the 86 patients with chronic active hepatitis, five (24%) o f the 21 patients with cirrhosis, and seven (23%) of the 31 patients with HCC, but not in the 18 patients with chronic persistent hepatitis. The prevalence o f a n t i - H C V was significantly higher in patients with cirrhosis and patients with H C C than in those with chronic persistent hepatitis (p<0.05). As for serum HCV R N A positivity in anti-HCV-positive cases, HCV R N A was detected in four (50%) o f the eight anti-HCV-positive patients with chronic active hepatitis, three (60%) o f the five patients with cirrhosis, and five (71%) o f the seven patients with HCC. N o significant difference was observed a m o n g the three groups. When a n t i - H C V .positivity was correlated with various clinical characteristics (age, sex, previous blood transfusions and HBeAg positivity) (Table 2), anti-HCV-positive patients were found to be significantly older than antiHCV-negative ones (52_+14 years vs. 41-+13 years, p<0.01). More anti-HCV-positive patients had received blood transfusions than anti-HCV-negative ones (40% vs. 8%, p<0.01). H B e A g prevalence was significantly lower in anti-HCV-positive patients than in -negative ones (50% vs. 72%, p<0.05). N o significant difference was observed in the sex ratio between the anti-HCV-positive and -negative groups. TABLE 2
Anti-HCVpositive (n=20)
Age (years)" 52---14 Sex (M/F) 14/6 Previous blood trans- 8 (40%) fusions HBeAg positivity 10 (50%) " Values are mean--+SD.
HBeAgpositive
Anti-HCVnegative (n= 136)
p-value
41 -+13 97/39 11 (8%)
p<0.01 NS p<0.01
98 (72%)
p<0.05
HBeAg. negative @@
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Clinical characteristics of chronic hepatitis B patients positive and negative for HCV antibody Characteristics
We further investigated reactivity in anti-HCV E L I S A expressed as C.I. and serum HCV R N A positivity in relation to the presence o f HBeAg (Fig. 1). A n t i - H C V was found in 10 (9%) o f the 108 patients positive for HBeAg and in I0 (21%) of the 48 patients negative for it (p<0.05). In the HBeAg-negative group, all 10 anti-HCV-positive patients tested positive in E L I S A with C.I.>3.5. In the HBeAg-positive group, however, the C.I. o f E L I S A of the 10 anti-HCV-positive patients ranged from 2.2 to 4.3, and only seven (70%) patients tested positive in E L I S A with C.I.>3.5. A significant difference was observed in the C.I.
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Fig. I. Reactivity in second-generation anti-HCV assays and detection of serum HCV RNA in chronic hepatitis B patients in relation to HBeAg positivity. Reactivity in anti-HCV ELISA was expressed as cut-off index. A confirmatory dot-blot anti-HCV assay and testing for serum HCV RNA were performed on ELISA-positive cases. Open circles represent patients negative for anti-HCV by ELISA. Closed circles represent patients positive for anti-HCV by both ELISA and the dot-blot assay, and positive for HCV RNA. Closed triangles represent patients positive for anti-HCV by both ELISA and the dot-blot assay, but negative for HCV RNA. Open triangles represent patients positive for anti-HCV ELISA but negative or "single antigen reactive" in the dot-blot assay, who are also negative for HCV RNA.
512
K. O H K A W A et al.
of anti-HCV ELISA of positive cases between HBeAgpositive and -negative groups (3.6___0.6 vs. 4.1___0.1, p<0.05). Serum HCV RNA was found among patients positive for anti-HCV ELISA with C.I.>3.5, but not among patients positive for it with C.I.<3.5. Thus, the prevalence of serum HCV RNA in anti-HCV-positive cases was significantly higher in the HBeAg-negative group (9/10, 90%) than in the HBeAg-positive group (3/ 10, 30%) (p<0.01). As for patients with high reactivity (C.I.>3.5) in anti-HCV ELISA, the prevalence of serum HCV RNA was also higher in the HBeAg-negative group (9/10, 90%) than in the HBeAg-positive group (3/7, 43°/,,)
(p<0.05). As for anti-HCV-positive cases, serum HBV DNA levels were further determined to evaluate HBV replicative states more accurately. Serum HBV DNA was found in six (60°/,,) of the 10 HBeAg-positive cases, but not in the 10 HBeAg-negative cases (p<0.01). Serum HBV DNA levels semi-quantitated for the six positive cases were 1+ in two, 3+ in two and 4+ in two cases. When the detection of serum HCV RNA was correlated with serum HBV DNA positivity, serum HCV RNA was found in three (50%) of the six HBV DNA-positive cases compared with nine (64%) of the 14 HBV DNA-negative cases (not significant). Serum HBV DNA levels also did not differ significantly between serum HCV RNA-positive and -negative cases. We followed one HBeAg-positive chronic active hepatitis patient (age 29, male) for 4.2 years (Fig. 2). This case showed seroconversion to anti-HBe with the decline of serum HBV DNA levels at 1 year after a 4-week course of interferon-a therapy. A total of 30 megaunits (MU) of
Anti-HCV HBsAg HBeAE
Anti-HBe
+
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H B V D N A H C V R N A
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~-
400
-
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200
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-
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Fig. 2. Detection of anti-HCV and serum HCV R N A in a chronic active hepatitis B patient who was followed for 4.2 years. Anti-HCV was persistently found by ELISA with C.I.>3.5, and also found by the dot-blot assay. Serum H C V R N A was not detected in the HBeAgpositive phase, but was detected after seroconversion to anti-HBe.
natural interferon-a (Sumiferon, Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan) was administered. He also cleared HBsAg during follow up. Anti-HCV was persistently found by ELISA with C.I.>3.5, and also found by the dot-blot assay during follow up. The patient did not have detectable levels of serum HCV RNA in the HBeAgpositive phase before interferon-a therapy, but became positive after seroconversion to anti-HBe. There had been no risk of infection with HCV during the follow-up period.
Discussion
Since the introduction of the first-generation anti-HCV assays, many chronic hepatitis B patients have also been shown to be infected with HCV, although the incidence differs among regions (4, 6-10). The prevalence of antiCI00-3 in these patients is much higher than in volunteer blood donors. However, the prevalence of HCV infection in HBV-endemic areas has not been fully reassessed using second-generation assays, which are more sensitive and specific. The relationship of HCV replicative states to HBV replication in HBV carriers also needs to be determined. The present study revealed that approximately 13% of Japanese patients with chronic hepatitis B tested positive in second-generation anti-HCV assays, which was quite similar to a previous report in our area based on a first-generation assay (7). In the present study, anti-HCV positivity showed a relationship to age and previous blood transfusions, indicating that parenteral transmission is also a major route of HCV infection in the chronic hepatitis B group. As for anti-HCV prevalence in various stages of chronic HBV infection, anti-HCV tended to be frequently detected in advanced liver diseases, such as cirrhosis and hepatocellular carcinoma, as has been indicated in a few previous reports using first-generation assays (7,9). Furthermore, active HCV replication was found in approximately 5070% of anti-HCV-positive cases irrespective of the disease stage. The clinical implications of frequent HCV markers in advanced liver diseases remain unclear. It may merely reflect increased risk of exposure to HCV in older patients with advanced liver diseases. Indeed, previous blood transfusions were more frequently observed in these cases. Nevertheless, coinfection with HCV in the course of chronic HBV infection may be a risk factor for more serious liver disease and the development of HCC, as is the case for chronic hepatitis C patients. Further studies must be performed to clarify this. The present study also clarified the interrelation of antiHCV reactivity, serum HCV RNA positivity and HBV replicative states. Serum HCV RNA was found in associ-
HCV INFECTION IN CHRONIC HEPATITIS B ation with high reactivity of anti-HCV, which has been reported for chronic non-A, non-B hepatitis patients (14). A n t i - H C V reactivity and serum H C V R N A positivity of anti-HCV-positive cases were also shown to differ considerably between HBeAg-positive and -negative groups. HBeAg-positive cases showed significantly lower reactivity of a n t i - H C V than HBeAg-negative patients. Furthermore, H C V R N A was detected less frequently in association with high reactivity o f a n t i - H C V in HBeAg-positive cases than in HBeAg-negative ones. These findings suggest that HCV replication may be suppressed in the HBeAg-positive phase, which may result in a decrease in a n t i - H C V reactivity. On the other hand, in the HBeAgnegative phase, most cases with a n t i - H C V seemed to have active HCV replication accompanied by high reactivity o f antibody. These findings indicate that the level of HCV replication may be influenced by the HBV replicative states. In chronic HBV infection, active HBV replication ceases with time, and patients with advanced liver diseases usually have low levels of HBV replication. In constrast with changes in HBV replicative levels, HCV replication might be suppressed in the early phase during active HBV replication, and enhanced in the iater phase. The longitudinal study in this work supports this hypothesis. In the present study, however, the prevalence o f serum HCV R N A in anti-HCV-positive cases did not differ significantly between serum HBV D N A - p o s i t i v e and -negative cases, although significant differences were observed between HBeAg-positive and -negative cases. This discrepancy may be because serum HBV D N A levels often change more markedly than H B e A g positivity in a short time. We suggest that HCV replication might be suppressed in the HBeAg-positive phase, when HBV replicative levels often fluctuate markedly, and enhanced in the HBeAg-negative phase, which reflects the persistent decrease in HBV replicative levels. So far, interference between HBV and H C V has been indicated by a few researchers. First-generation a n t i - H C V has been found more frequently in HBV carriers with lower levels of viral replication (9). In the present study based on second-generation assays, a n t i - H C V prevalence was also higher in HBeAg-negative patients than in -positive ones. It has also been suggested that the agent of nonA, non-B hepatitis may interfere with HBV replication (19), although controversy remains about suppression o f • HBV replication in HBV carriers superinfected with H C V (20). We cannot completely exclude the possibility that frequent occurrence o f results that are anti-HCV-positive but HCV RNA-negative in HBeAg-positive cases is due to false-positivity o f the a n t i - H C V assay, or just indicates
513
previous exposure to HCV, though a n t i - H C V positivity was confirmed using the dot-blot assay. However, the high prevalence o f these cases in the HBeAg-positive phase is difficult to explain. F u r t h e r quantitative analysis of HBV and HCV replicative states in prospective studies should offer a better understanding of this issue. Using second-generation a n t i - H C V assays and RT-PCR for detecting serum HCV R N A , HCV infection was shown to be involved in many chronic hepatitis B patients, indicating similar modes o f infection o f the two viruses. Our data also suggest possible interference between HBV and HCV, as has been indicated for other viruses.
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514 16. Hagiwara H, Hayashi N, Mita E, et al. Detection of hepatitis C virus RNA in chronic non-A, non-B liver disease. Gastroenterology 1992: 102: 692~.. 17. Hagiwara H, Hayashi N, Mita E, et al. Detection of hepatitis C virus RNA in serum of patients with chronic hepatitis C treated with interferon-a. Hepatology 1992: 15: 37-41. 18, Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989: 339: 237-8.
K. OHKAWA et al. 19. Tsiquaye KN, Portmann B. Tovey G, et al. Non-A, non-B hepatitis in persistent carriers of hepatitis B virus. J Med Virol 1983: 1 I : 179-89. 20. Wang JT. Wang T-H, Sheu J-C, Lin J-T, Wang C-Y, Chen D-S. Posttransfusion hepatitis revisited by hepatitis C antibody assays and polymerase chain reaction. Gastroenterology 1992; 103: 60916.