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Coinfection of hepatitis C virus in patients with chronic hepatitis B infection
Copyright © Journalof Hepatology 1994
Journal of Hepatology 1994; 21: 159-166 Printed ha Demnark . All rights reserved Munk.~aard. Copenhagen
Journal of Hepatology ISSN 0168-8278
Coinfection of hepatitis C virus in patients with chronic hepatitis B infection Shinjiro Sato t, Shigetoshi F u j i y a m a ~, M o t o h i k o T a n a k a ~, K u n i o Yamasaki t, Izumi K u r a m o t o ~, Shin-ichi K a w a n o ~, T a t s u o Sato t, K y o s u k e M i z u n o 2 and Saneo N o n a k a 2 t Third Departnwnt o f btternal Medichle, Kutnanwto University School o f Medichte and :Chento-Sero-Therapeutic Research Institute, Kumamoto, Japan
(Received 2 November 1992)
Enzyme-linked immunosorbent assays for detecting antibodies against hepatitis C virus and the polymerase chain reaction were tested in 82 chronic hepatitis B surface antigen carriers for their accuracy in diagnosing patients coinfected with hepatitis B and C viruses. To clarify the role of each virus in chronic hepatitis, serologic assays against hepatitis B virus were also tested. Thirteen (14.9°/,,), 14 (17.1%) and 15 (18.3%) patients were anti-HCV positive using C100 (HCV1), JCC, and a second generation test (HCV2), respectively. HCV RNA was detected by polymerase chain reaction in 9 of 18 antiHCV-positive cases. Although HCV1 assays were not sufficient, either the JCC or HCV2 assay detected all polymerase chain reaction-positive cases. Fifteen of 18 specimens that were positive in at least one of the three ELISA were seronegative for the hepatitis B e antigen. As judged by HBV DNA potymerase activity, titers of hepatitis B surface antigen and immunoglobulin A antibody against hepatitis B core antigen (IgA anti-HBc), activity of hepatitis B virus replication and immune response against hepatitis B virus in patients with coinfection was decreased to the level of hepatitis B virus asymptomatic carriers. These results show that hepatitis C virus appears to be the primary cause of active hepatitis in most patients with hepatitis B and hepatitis C virus coinfection. © Journal of Hepatology. Key words: Enzyme-linked immunosorbent assay; IgA anti-HCV; Polymerase chain reaction
The major etiologic agents of blood-borne hepatitis are hepatitis B virus (HBV) and hepatitis C virus (HCV). These are two serious, prevalent diseases which cause chronic hepatitis, cirrhosis and hepatocellular carcinoma (1,2). Both viruses share a similar route of parenteral transmission, and some evidence suggests that both infections may also occur from vertical or sexual transmission (3,4). Similarities in the epidemiology of HCV and HBV infection were also noted in a report which showed a high prevalence of antibody to HCV (anti-HCV) in blood donors positive for antibody to hepatitis B core antigen (anti-HBc) (5), and in a trial of blood donors who were screened using anti-HBc as a surrogate marker for the prevention of posttransfusion non-A, non-B (NANB) hepatitis (6). Accordingly, coinfection with HCV might be expected to occur more frequently in persons seropositive for hepatitis B surface antigen (HBsAg).
Molecular cloning of HCV (7,8) led to the development of several types of immunoassays for detecting anti-HCV antibodies. C100 is an assay for detecting antibodies against a non-structural (NS) protein of HCV (HCV1) (9). New assay systems using HCV core antigen (JCC and a second generation test (HCV2)) are superior to HCVI in identifying HCV carriers (10,11), and are expected to reduce the incidence of posttransfusion hepatitis by HCV (12). However, positive results for anti-HCV with a single assay do not necessarily indicate HCV viremia since both false-positives and "memories" of prior transient infection (13) may occur in a substantial number of seropositive patients. One of the purposes of this study was to assess the prevalence of anti-HCV and circulating HCV RNA in patients with hepatitis B and assess the accuracy of these assays in diagnosing coinfection with HCV. A second purpose was to analyze the etiologic role of
Correspondence to: Shinjiro Sato, M.D., Third Department of Internal Medicine, Kumamoto University School of Medicine, 1-1, Honjo 1chome, Kumamoto 860, Japan.
160 each virus in ongoing liver disease. HCV associated markers indicating the activity of HCV-induced liver disease are currently unknown; however, the presence of HCV RNA in the sera may be a reliable marker of infectivity and active replication of HCV (14). In patients with chronic HBV infection, the disappearance of hepatitis B e antigen (HBeAg) and DNA polymerase (DNA-P) activity are used as common denominators of spontaneous remission of active hepatitis. However, seroconversion to detectable antibody against HBeAg (anti-HBe) does not always prevent the progression of liver disease (15). More accurate assays indicating the activity of HBV-induced liver disease are needed to determine the relative contribution of HBV in coinfected patients. Increased immunoglobulin A class anti-HBc (IgA antiHBc) in hepatitis B patients' sera implies an active immune response against HBV and, hence, HBV-induced liver injury (16). IgA anti-HBc may also be useful in monitoring disease activity, and the real value of IgA anti-HBc may be fully recognized in HBeAg-negative patients (17). In this report, we demonstrate the importance of IgA anti-HBc in HBsAg carriers who are simultaneously positive for anti-HCV. Moreover, the investigated assays may be of some value in assessing antiviral therapy against both viruses. Patients and Methods
Patients A total of 82 patients with persistent serum HBsAg were studied by ELISA for antibodies against HCV and for HCV RNA in the sera. None of the patients had serologic markers for acute hepatitis A virus, cytomegalovirus, or Epstein-Barr virus, or any other obvious cause of hepatitis (autoimmune disease, alcohol or drug hypersensitivity). There were 57 males and 25 females, with a mean age of 40.1 years (range 18 to 69). Twenty-nine patients were HBeAg-positive and 53 HBeAg-negative. Eighteen patients were asymptomatic HBV carriers with no evidence of hepatic disease (alanine aminotransferase (ALT) <24 International units per liter (IU/I)) without detectable markers of HBV replication (low level of DNA-P and had been seronegative for HBeAg for at least 2 years), 45 had chronic hepatitis, and 19 had cirrhosis including five with hepatocellular carcinoma (HCC). To validate serologic measures for HBV in patients positive for anti-HCV, nine asymptomatic HBV carriers and 17 anti-HCV-negative (anti-HCV(-)) patients with active hepatitis B and elevated ALT levels (>50 IU/I) after seroconversion to anti-HBe, were selected as controls. Serum samples were collected from June, 1989 to Au-
S. SATO et al. gust, 1991 and were stored at - 2 0 ° C or - 8 0 ° C until assayed.
Serologic methods of HCV detection Antibody against CI00 (anti-HCVl) was tested by the Ortho HCV Ab ELISA (Ortho Diagnostic Systems, Tokyo, Japan) (9). Antibody against JCC (anti-JCC) was tested by the JCC ELISA kit (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan). JCC is a recombinant protein corresponding to the putative core region of the HCV genome (10). Antibodies against multiple immune dominant proteins including NS and the core region of HCV (anti-HCV2) were tested by the Ortho HCV Ab ELISA second-generation kit Ortho Diagnostic Systems, Tokyo, Japan) (I 1). Reverse transcription-nested polymerase chain reaction ( R T-nested PCR) RNA was prepared from each 0.05 ml serum sample using the method of RNA isolation by acid guanidinumthiocyanate-phenol-chloroform extraction (18). Complementary DNA (cDNA) was prepared from RNA by reverse transcription. The cDNA was amplified twice by nested PCR for 35 cycles each (19) using primers corresponding to the 5' non-coding region (5'-NCR) (20) of the HCV genome. PCR products were subjected to electrophoresis on an agarose gel and stained with ethidium bromide for visualization. The sensitivity of PCR was assessed by serial testing of infectious sera derived from a patient with chronic hepatitis C, and sera diluted 1:105 had detectable HCV RNA. Further investigation was performed by the same method to controlled quantities of RNA prepared from plasmid vector which contained the 5'-NCR sequence of HCV (21). More than 40 copies of RNA could be detected by PCR. Serologic methods for HB V HBsAg was tested by Seroclit-HBs (The Chemo-SeroTherapeutic Research Institute, Kumamoto, Japan). The HBsAg titer was determined by testing serial two-fold dilutions of the serum against red cell samples in this kit. Results were expressed as the reciprocal of the highest serum dilution (2 °) that caused macroscopically visible agglutination. HBeAg and anti-HBe were analyzed by solid phase radioimmunoassay (Abbott Laboratories, North Chicago, IL, USA). DNA-P activity was measured by the method of Kaplan et al. (22), and levels >30 cpm were interpreted as positive. IgA anti-HBc was analyzed by IgA antibody capture ELISA. Polystyrene microtitre plates (Nunc-lmmune Plate Maxisorp; Nunc, Roskilde, Denmark) were coated with anti-human IgA (Dakopatts a/s, Glostrup, Den-
161
COINFECTION WITH HBV AND HCV
mark) at a 1 : 600 dilution with 0.15 M phosphate-buffered saline (PBS). Following overnight incubation at 4°C, the wells were washed three times with PBS containing 0.05°/,, (v/v) Tween-20 using an automatic plate washer. The wells were then coated with 3% (wt/v) bovine serum albumin (BSA) in PBS for 2 h at 37°C. After washing as before, 0.2 ml/well of sera at a 1:400 dilution with 3% BSA in PBS was added for 1 h at 37°C. After the first incubation, the plates were washed five times and 0.2 ml/well of HBcAg and peroxidase conjugated anti-HBc monoclonal antibody mixture (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan) was added for 1 h at 37°C. The plates were then washed five times before the addition of 0.2 ml/well of O-phenylenediamine (OPD) substrate solution (10 ml of 0.1 M citric acid-phosphate buffer and 10 ml of 0.2 M Na,_HPO4. 12H,O buffer, containing 8 mg of OPD and 4/.tl of 30% (v/v) H_,O2), and incubated for 30 min at room temperature. The reaction was terminated by the addition of 0.05 ml of 4 N H_,SO4. Optical density (OD) values were measured at 490-650 nm with a microplate reader (Molecular Devices Corp, City CA). The appropriate dilution of 1:400 was selected for preventing non-specific binding acc6rding to a dilution curve derived from a positive serum sample with anti-human lgA in plates coated or uncoated with IgA anti-HBc ELISA. Twenty serum samples collected from healthy blood donors were also tested with this ELISA as a negative control. The cut-off value was defined by the results of normal controls and values >0.03 were interpreted as being positive for IgA anti-HBc. Mean±S.E. of OD values was calculated from all samples in each group of patients with HBV infection.
clonal anti-human IgG/Fc, IgA/a, and IgM/p, respectively. OPD substrate was then added and OD measured. These fractions were also tested by IgA anti-HBc ELISA to confirm that the reaction occurred in the same fractions which, contained IgA. IgA class antibodies were well differentiated from other classes of immunoglobulins (Fig. 1). Given the molecular weight and chromatographic pattern of IgA, the first peak of high molecular size (Fr 66) was considered as polymeric IgA, and the second (Fr 77) as monomeric IgA (23). In the assay for IgA anti-HBc, serum samples from HBV carriers with chronic active hepatitis were reacted with the fractions containing primarily monomeric IgA. IgG class immunoglobulins did not react in this assay as evidenced by asymptomatic HBV carriers having high titers of lgG anti-HBc, all of whom were negative for IgA anti-HBc. To verify the IgA anti-HBc ELISA, 55 patients with anti-HCV-negative persistent HBV infection were tested. They included nine asymptomatic HBV carriers, seven with chronic persistent hepatitis, 21 with chronic active hepatitis, and 18 with cirrhosis. Histopathologic findings on liver biopsy were graded using standard criteria. IgA anti-HBc in patients with persistent HBV infection (by histologic classification) was evaluated as shown in Fig. 2.
Stat&tical analysis Differences in the mean value of each group were evaluated by the Wilcoxon rank-sum test. The difference in the proportion of patients with an abnormal value between two groups was evaluated by the Z2 method.
Characterization of lgA anti-HBc Characterization of the immunoglobulin class of the anti-HBc was performed in four samples collected from patients with chronic hepatitis B. Twenty microliters of sera were fractionated by high performance liquid chromatography (HPLC) in an LC-9A apparatus (Shimadzu, Kyoto, Japan) over G3000SWxc gel in two 7.8 mm I D × 3 0 cm columns (Tosoh, Tokyo, Japan) connected in tandem. Columns were eluted with phosphate-buffered saline (0.01 M, pH 7.2) at a flow rate of 0.6 ml/min, and 200/.tl fractions were collected. Before HPLC, sera were diluted 1:4 with phosphate-buffered saline and elution buffer, and filtered through 0.22 p Millipore filter paper. To identify the immunoglobulin class (IgG, IgA, or IgM), a sandwich ELISA was made separately. Microtiter plates were coated using polyclonal anti-human IgG/~, IgA/a, and IgM/p. Samples fractionated by HPLC were sandwiched by horseradish-peroxidase conjugated poly-
3.0
• ~ ,
IgA IgM lgA anfi-HBc
~ 2.0
-~ 1.0 O
0.0 50
60
70
80
90
1O0
110
Fraction Number
Fig. t. Elution curve of a serum sample from a patient with chronic
active hepatitis B. Closed circles, open circles and crossed bars represent IgM, IgA, and IgG types of immunoglobulins, respectively. Shaded areas represent the results for IgA anti-HBc ELISA. Void volume is indicated by the black arrow.
162
S. SATO et al.
10 0
E 0
=1
10"
©
10
-2
ASC
CPH
CAH
C
, : p < 0.001 Fig. 2. Immunoglobulin A antibody to hepatitis B core antigen (IgA anti-HBc) in asymptomatic carriers (ASC), patients with chronic persistent hepatitis (CPH), patients with chronic active hepatitis (CAH) and patients with cirrhosis (C). The mean optical density values_S.E, of all patients are given for each group (closed bars).
f
HCV2 •
: P C R (+)
0
: P C R (-)
no a n t i - H C V ( + ) cases in a s y m p t o m a t i c HBV carriers. All a n t i - H C V ( + ) patients had an elevated A L T or histology consistent with progression to cirrhosis. The relationship between HBeAg/anti-HBe findings and anti-HCV is shown in Table 2. Three patients who were HBeAg-positive were anti-HCV positive as measured by the HCVI assay and two of these were also positive by the HCV2 assay. However, all patients positive for anti-JCC had no measurable HBeAg. The results of these anti-HCV E L I S A and their cox'relation with the results o f RT-nested PCR are shown in Fig. 3. HCV R N A was detected in nine of the 18 antiH C V ( + ) specimens. All patients positive for HCV R N A ( P C R ( + ) ) had a positive result in multiple ELISA. HCV1 assay missed two P C R ( + ) cases, while JCC and HCV2 detected all P C R ( + ) cases. All cases positive in only one ELISA, and the remaining 64 a n t i - H C V ( - ) cases, were negative for HCV R N A ( P C R ( - ) ) . Table 3 presents the clinical and serologic characteristics and prevalence o f IgA anti-HBc in a s y m p t o m a t i c HBV carriers, a n t i - H C V ( + ) and a n t i - H C V ( - ) groups. The mean age of the P C R ( + ) group was higher than that o f others. The rates of H B V - D N A polymerase activity were significantly lower in a n t i - H C V ( + ) / P C R ( + ) cases than a n t i - H C V ( + ) / P C R ( - ) and a n t i - H C V ( - ) cases (0% vs 55.5% and 70.6%, respectively, p<0.01). Positivity in the IgA anti-HBc assay in the P C R ( + ) group was the same as in a s y m p t o m a t i c HBV carriers, and significantly lower than that in the a n t i - H C V ( + ) / P C R ( - ) group (p<0.025) and a n t i - H C V ( - ) group (p<0.025). N o differ-
TABLE 1 Prevalence of anti-HCV antibodies as measured in three ELISAs in patients with chronic hepatitis B virus infection
HCVI
JCC
Fig. 3. Venn diagram of anti-HCV ELISA and RT-nested PCR results in patients with hepatitis B. Each small circle represents a single patient. Closed and open circles denote patients with or without HCV RNA, respectively.
Diagnosis
No. of cases HCVI
JCC
ASC CH C HCC
18 45 14 5
0 10 2 1
Total (%)
82
13 (15.9'7,,) 14 (17.1"/.) 15 (18.YV,,)
0 9 4 1
HCV2 0 11 3 1
ASC; asymptomatic carrier. CH: chronic hepatitis. C: cirrhosis. HCC: hepatocellular carcinoma.
TABLE 2
Results The prevalence o f positive results indicating HCV infection in patients clinically diagnosed with chronic HBV is shown in Table 1. O f the 82 HBsAg carriers, 13 (15.9%),14 (17.1%), and 15 (18.3%) were positive for antiH C V I , anti-JCC, and anti-HCV2 respectively. Eighteen patients were positive in at least one assay. There were
Prevalence of anti-HCV antibodies in patients with chronic hepatitis B infection and variable HBe serologic status HBeAg/anti-HBe
No. of cases HCVI
JCC
HCV2
(+/-) (-/-) (-/+)
29 6 47
3 0 10
0 1 13
2 I 12
Total
82
13
14
15
COINFECTION WITH HBV AND HCV
163
TABLE 3 Clinical, serological characteristics and prevalence of immunoglobulin A antibody against hepatitis B core antigen in asymptomatic carriers, anti-HCV(+) and anti-HCV(-) groups Characteristics
ASC
n Male/female Mean age HBe antigen (+) (%) Serum ALT (Iu/I) DNA polymerase activity (cpm) IgA anti-HBc(+) (%) Cirrhosis (%) Blood transfusion (%)
Anti-HCV(+)
9 6/3 44 (18-69) 0% 17.1___1.4 1.7+_0.8 11.1% ND 22.2%
Anti-HCV(-)
PCR(+)
PCR(-)
9 7/2 47 (29-67) 0% 142.6+30.2 5.9--_2.5 11.1% 22.2% 22.2%
9 6/3 39 (20-55) 33.3% 161.9-+39.9 269.4 + - 167.9 77.7% 44.4% 33.3%
17 11/6 38 (20-59) 0% 135.1__.44.2 328.0-+ 147.0 70.6°,4, 17.7% 17.7%
ASC: asymptomatic carriers. PCR: polymerase chain reaction. Results are expressed as mean_S.E, or range in parentheses. ND; not determined.
ences were f o u n d in the severity o f h e p a t i c d a m a g e a n d
E L I S A . IgA a n t i - H B c was c o n s i s t e n t l y f o u n d in P C R ( - )
rate
p a t i e n t s , b u t in only a single P C R ( + )
of cirrhosis between
PCR(+)
and
anti-HCV(-)
groups.
p a t i e n t (Case 18
h a d b o t h IgA a n t i - H B c a n d H C V R N A ) . This p a t i e n t
T h e r e was a h i s t o r y o f b l o o d t r a n s f u s i o n in 27.7% o f
d e v e l o p e d e x a c e r b a t i o n o f his h e p a t i c injury with a n elev-
H B V c a r r i e r s with a n t i - H C V a n t i b o d y a n d in 22.2% o f
a t i o n in D N A - P activity a f t e r 1 y e a r ( d a t a n o t s h o w n ) .
P C R ( + ) . All a n t i - H C V ( + ) p a t i e n t s w i t h p e r s i s t e n t H B V
C a s e s 9 a n d 12 were a n t i - H C V positive in all t h r e e E L I -
i n f e c t i o n are s h o w n in T a b l e 4, with several clinical fea-
SA, b u t H C V R N A was n o t d e t e c t e d in e i t h e r p a t i e n t .
tures a n d
of HBV
T h e s e t w o p a t i e n t s h a d IgA a n t i - H B c , a n d o n e (Case 12)
n o t e d . T h e r e were n o p a t i e n t s with d r u g a b u s e o r a his-
h a d e l e v a t e d D N A - P activity. T h e s e p a t i e n t s h a d a his-
serologic or biochemical markers
tory o f t a t t o o s . T h e c u t - o f f i n d e x o f a n t i - H C V a n t i b o d y
t o r y o f i n t e r f e r o n t h e r a p y n o t e d in r e t r o s p e c t . A m o n g 18
in p a t i e n t s with p o s i t i v e results in a single a s s a y t e n d e d
o f the a n t i - H C V ( + ) cases, five h a d b e e r / t r e a t e d w i t h in-
to be l o w e r t h a n in p a t i e n t s f o u n d positive in m u l t i p l e
t e r f e r o n for at least 4 w e e k s several years before, b u t all
TABLE 4 Clinical and serologic characteristics of anti-HCV(+) patients with hepatitis B Case
Age
Sex
Diag
B-T
HBeAg
anti-HBe ALT
A. Patients negative for JCC 1 20 M CN 2 21 F CH 3 33 M CH 4 38 F HC
-
+ + + -
+
216 139 276 39
B: Patients positive for JCC 5 43 M C 6 54 F C 7 55 M HCC 8 46 M CH 9 43 M CH 10 43 F CH 11 41 F CH 12 41 M CH 13 49 M C 14 50 M CH 15 67 M CH 16 56 M CH 17 29 M CH 18 41 M C
+ + + + +
-
+ + + + + + + + + + + + +
49 119 77 244 111 128 48 68 75 55 48 184 249 252
DNA-P
HBsAg
IgA anti-HBc
HCVI
384 18 29 8
14 13 9 5
0.375 0.166 0.337 0.031
3.73 2.914 1.746 1.110
56 225 N.D. 3 37 15 0 1398 0 0 2 4 21 8
12 13 9 9 9 7 9 I1 5 9 3 3 12 6
0.029 0.443 0.007 0.001 0.088 0.005 0.002 0.081 0.001 0.005 0.002 0.004 0.019 0.206
0.182 0.080 0.793 6.220 4.684 5.726 0.284 5.843 6.220 0.076 5.031 7.424 6.451 5.841
JCC
HCV2
HCV RNA
0.286 0.256 0.422 0.231
2.917 1.875 0.125 0.138
-
1.036 2.633 3.169 3.656 4.282 8.300 9.072 9.834 9.875 10.024 10.057 10.117 10.120 10.280
0.039 2.872 5.636 6.000 5.987 6.220 6.200 6.038 6.150 5.974 6.067 6.236 6.190 6.104
+ + + + + + + + +
B-T; Blood-transfusion. HBsAg; Hepatitis B surface antigen titer (2"). ALT; alanine aminotransferase levels. DNA-P; DNA polymerase activity (cpm). PCR; polymerase chain reaction. M; male. F; female. CH; chronic hepatitis. C; cirrhosis. HCC; hepatocellular carcinoma. N.D.; not done. Titers of HBsAg are expressed in reciprocal of serial two-fold dilutions. IgA anti-HBc results are described in optical density values and results of anti-HCV ELISA are described by cut-off index (Cut-off index for positivity in the anti-HCV assay >1.000).
164
S. SATO et al.
had abnormal ALT levels, and two patients still had HCV RNA detected by PCR. The humoral anti-HCV responses in HBsAg carriers have persisted: however, changes in P C R ( - ) to PCR(+) were not observed in patients (Case 1, 2, 9 and 12) after 2 to 4 years of follow-up analysis. The relationship of HBV markers (titer of HBsAg, IgA anti-HBc) in asymptomatic HBV carriers, HCV(+)/ PCR(+), H C V ( + ) / P C R ( - ) , and a n t i - H C V ( - ) group patients is shown in Fig. 4 and 5. Statistically significant differences between HCV(+)/PCR(+) and anti-HCV(- ) groups were noted in HBsAg titer (/7<0.05) and to a greater extent in IgA anti-HBc (p<0.001).
=
I
15 • •
ooN
No
N N
N
ASC
anti-HCV(+) PCR(+)
ooo
anti-HCVl+) PCR(-) ,
•oo ~oooo
00
anti-HCV(-) : p < 0.05
Fig. 4. HBsAg titers in patients with hcpatitis B. Patient groups include HBeAg-negative asymptomatic carriers (ASC). seropositive for anti-HCV with or without HCV RNA (PCR(+) or PCR(-I) and anti-HCV(-) patients. Each dot represents a single patient.
101 I
-~ lo °
I
I
"N o N
u 10 |
T m
16~ ASC
anti-HCV(+) anti-HCV(+) anti-HCV(- ) PCR(+) PCR(-) , :p<0.01 * * : p < 0.001
Fig. 5. IgA anti-HBc in patients with hepatitis B. Patient groups are identical to those shown in Fig. 4. Each dot represents a single patient.
Discussion
In the present study, anti-HCV was detected in 18 (23%) of 82 HBsAg carriers, using three types of ELISA. Because all anti-HCV-positive patients had abnormal levels of ALT, their continuing hepatitis could have been caused by HBV and/or HCV, or even autoimmunity or hepatotoxic agents. Since hepatitis delta virus is infrequent in Japan, testing for this virus was not performed (24). It is important to determine if these anti-HCV(+)patients are actually coinfected with HBV and HCV, because HCV may prolong tile hepatic injury of HBV and progress to cirrhosis and HCC (25). In a previous study (21) several HCV-related antibodies were tested simultaneously in patients with various liver diseases, and almost all anti-HCV(+)-patients with NANB hepatitis were found to have HCV RNA in their blood. However, a recent study indicated that anti-JCC and HCV2 were also detected at relatively high rates in patients with other liver injuries and in blood donors, and the association between anti-HCV and the presence of HCV RNA was not as specific in these cases (12). The presence of patients positive for anti-HCV but negative for HCV RNA suggests that anti-HCV might be detected even in patients with transient HCV infection, similar to antibodies against HBcAg. RT-nested PCR assay might verify whether viable HCV is present in the serum of these patients. The results in a trial of combined assays for blood donor screening (12) indicate that multiple assays developed heterogeneously from different kinds of epitopes within the HCV genome might be used to replace RT-nested PCR to ascertain the presence or absence of viable HCV in serum, with considerable savings in time and effort. Further studies, however, are needed to confirm this evidence. In this study, nine patients who were positive in multiple anti-HCV ELISA also had HCV RNA in their sera. The presence of HCV RNA was closely associated with positive results in ELISA measuring HCVI and JCC assays. However, as presented here, none of the anti-HCV ELISA were effective as single or multiple assays, and therefore the PCR method was the single best predictor of actual HCV infection. In the serologic results associated with HBV, DNA-P activity was lower in anti-HCV(+) cases and undetectable in PCR(+) cases. H BsAg levels were similarly reduced in PCR(+) patients. Fong et al. (26) demonstrated identical results. This suggests that HBV replication is mild or moderate in coinfected patients. Although the proliferative activity of HBV could be determined by DNA-P activity (displaying the active replication of HBV) and HBsAg titer (representing the quantity of products), this did not always correlate with hepatic disease activity. In our
COINFECTION WITH HBV AND HCV study, lgA anti-HBc was used to predict the activity of HBV-induced liver disease. Ordinarily, IgA class immunoglobulins are associated with the recurrence of viral replication in persistent disease. Nomura et al. reported that IgA anti-HBc more accurately reflects hepatic injury resulting from HBV infection than other evidence of HBV replication, and is useful in identifying asymptomatic HBV carriers (16). The level of IgM anti-HBc also increased in parallel with IgA anti-HBc and was associated with acute exacerbations of persistent hepatitis B infection (27), but the correlation between IgA anti-HBc and hepatic injury is more reliable (17). In this study, the level of IgA anti-HBc was significantly different between asymptomatic HBV carriers, and patients with chronic persistent hepatitis, chronic active hepatitis, and cirrhosis. Mean OD values in patients with chronic active hepatitis and cirrhosis were higher than those with chronic persistent hepatitis. Asymptomatic HBV carriers were well separated from the other patients (Fig. 3). These results seem to indicate that this IgA antiHBc assay is reliable and applicable. In the anti-HCV(+) patients, lgA anti-HBc was rarely detected in HCV PCR(+) with mean values equivalent to asymptomatic HBV carriers. This suggests that the immune response against HBV was decreased to an equivalent degree to that in asymptomatic HBV carriers, and that the prolonged liver damage in these patients might not be dependent on HBV. However. in chronic hepatitis B patients with elevated levels of ALT and no detectable HCV RNA, IgA anti-HBc was consistently present. The serologic findings which show the replicative activity of each virus and the activity of HBV-induced liver disease seem to indicate that there are only a few patients with liver damage sustained by both viruses, because the viruses rarely proliferated concurrently in coinfected patients. In most HBV patients coinfected with HCV, HCV alone may be implicated in the pathogenesis and progression of hepatic damage (26, 28). Among a n t i - H C V ( - ) patients, abnormal ALT and DNA-P levels were found even after seroconversion to detectable anti-HBe. Regarding persistent abnormal levels of ALT and histopathologic progression in patients with persistent HBV infection after seroconversion to anti-HBe, precore mutant HBV appears to play an important role (29). These patients sometimes had acute exacerbations with elevated ALT levels, and DNA-P activity would probably have been detectable prior to ALT elevation. Activation of hepatitis by HBV alone, perhaps of a mutant virus in the precore region, appears to be the primary cause of continuing hepatic damage in these patients (30). In this study, a n t i - H C V ( - ) patients also had detectable IgA anti-HBc, which seems to be helpful
165 in monitoring tile disease activity in anti-HBe-positive HBsAg carrier patients. Nishioka et al. reported that a prior history of blood transfusion in Japanese patients was observed in 2.9% (7/ 242) and 5.5%, (6/110) of HBsAg positive/anti-HCV negative chronic hepatitis and cirrhosis, respectively (31). However, in the present study, 27.7°/, of HBsAg-carrier patients who were anti-HCV(+) had a history of blood transfusion and 22.2% of these patients were seropositive for HCV RNA. In addition, the mean age of PCR(+) cases was higher than other patient groups, and all PCR(+) patients had already become seropositive for anti-HBe. Based on the low prevalence of anti-HCV in children (32) and the lack of detectable HCV RNA in anti-HCV-negative persons who were tested in our family survey (unpublished observation of 88 relatives of patients with chronic hepatitis C), HCV vertical transmission appears to be extremely rare. Although the chronicity of HBsAg primarily depends on acquisition from mother to infant (33), occurrence of simultaneous infection with HCV in patients with HBsAg might represent a superinfection transmitted via parenteral exposure by means of blood transfusion or improper disposal of sharp instruments which contain infectious sera. Superinfection with HCV in anti-HBe-positive patients with HBV infection is probably the reason why HBV replication was decreased in these patients. Suppression of HBV replication by HCV may not occur as frequently in the patients in this study as previously reported (26,28). Although three patients with HBeAg and six patients without HBeAg were anti-HCV-positive, none had detectable HCV RNA in their serum. However, 77.8% had detectable IgA anti-HBc. If these anti-HCV results are not false positives or representative of prior infection, thus indicating actual coinfection (or superinfection) with HCV, they seem to indicate that HCV may lag behind the hepatitis caused by HBV. HCV may persist subclinically with no detectable levels of serum HCV RNA, arid then become apparent as HBV replication decreases with seroconversion to anti-HBe. However, this hypothesis has not been supported by 2 to 4 years follow up. Though we could not confirm the 9l vivo interference of each virus in patients with coinfection in this study, further prospective or retrospective studies (34) and PCR in the liver specimens from HCV RNA-negative patients positive for antiHCV in their serum may provide greater insight (35). The five patients suboptimally treated with interferon all had abnormal ALT levels, and two patients were still positive for HCV RNA. Recognition of the clinical course and virologic measures of coinfection are important in determining which virus is etiologically active and may affect decisions regarding interferon therapy. Assay of IgA
166
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a n t i - H B c m a y be o f p a r t i c u l a r value in m a k i n g tiffs decision.
18.
19.
References 1. Tiollais P, Pourcel C. Dejean A. The hepatitis B virus. Nature 1985; 317: 489-95. 2. Dienstag JL. Non-A, non-B hepatitis. 1. Recognition, epidemiology, and clinical features. Gastroenterology 1983; 85: 439-62. 3. Thaler MM, Park C-K, Landers DV, et al. Vertical transmission of hepatitis C virus. Lancet 1991: 338: 16-8. 4. Corona R, Prignano G, Mele A, et al. Heterosexual and homosexual transmission of hepatitis C virus: relation with hepatitis B virus and human immunodeficiency virus type I. Epidemiol Infect 1991: 107: 667-72. 5. Gaeta GB, Rapicetta M, Sardaro C. Chionne P, Costantino A, Giusti G. High prevalence of co-occurrence of anti-HCV and anti-HBc antibodies in chronic hepatitis patients from southern Italy. Ital J Gastroenterol 1990: 22: 350-1. 6. Kozoi DE, Holland PV, Ailing DW, et al. Antibody to hepatitis B core antigen as a paradoxical marker for non-A, non-B hepatitis agents in donated blood. Ann Intern Med 1986: 104: 488-95. 7. Choo Q-L, Kuo G, Welner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from bloodborne non-A, non-B viral hepatitis genome. Science 1989: 244: 359-62. 8. Maeno K, Kaminaka K, Sugimoto H. et al. A eDNA clone closely associated with non-A, non-B hepatitis. Nucleic Acids Res 1990: 18: 2685-9. 9. 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. 10. Chiba J, Ohba H, Matsuura Y, et al. Serodiagnosis of hepatitis C virus (HCV) infection with an HCV core protein molecularly expressed by recombinant baculovirus. Proc Natl Acad Sci USA 1991: 88: 4641-5. 11. Van der Poel CL, Cuypers HTM, Reesink HW, el al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991: 337: 317-9. 12. Fujiyama S, Kawano S. Sato S. et al. Evaluation of three hepatitis C virus-related antibodies CI00, KCL-163. JCC: test for screening blood donors. Dig Dis Sci 1992: 37: 1489-94. 13. Lenzi M, Johnson PJ, McFarlane IG, et al. Antibody to hepatitis C virus in autoimmune liver diseases: evidence for geographical heterogeneity. Lancet 1991; 338: 277-80. 14. Garson JA, Tedder RS, Briggs M, et al. Detection of hepatitis C viral sequences in blood donation by "'nested" polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. 15. Hoofnagle JH, Dusheiko GM, Seer LB, Jones EA. Wagoner JG, Bales ZB. Seroconversion from hepatitis e antigen to antibody in chronic type B hepatitis. Ann Intern Med 1981: 94: 744--8. 16. Nomura M, lmai M, Tsuda F, et al. lmmunoglobulin A antibody against hepatitis B core antigen in the acute and persistent infection with hepatitis B virus. Gastroenterology 1985: 89:
1109-13. 17. Luo K-X, Lu Q-S. Significance of various classes of anti-HBc
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in individuals with chronic asymptomatic HBV infection. Am J Gastroenterol 1989: 84: 160-3. Chomezynski P. Sacchi N. Single-step method of R N A isolation by acid guanidinium-thiocyanate-phenol-chloroformextraction. Anal Biochem 1987; 162: 156-9. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239: 487-91. Okamoto H, Okada S, Sugiyama Y, el al. Thc 5'-ternainal sequence of hepatitis C virus genome. Jpn J Exp Med 1990: 60: 167-77. Kawano S, Fujiyama S, Sato S. et al. Clinical evaluation of three anti-HCV ELISAs in patients with various liver diseases. Dig Dis Sci 1992: 37: 1268-74. Kaplan PW, Greenman RL. Gerin JL. DNA polymerase associated with human hepatitis B antigen. J Virol 1973: 12: 995-1005. Hikata M. Tachibana K, lmai M, et al. Immunoglobulin A antibody against hepatitis B core antigen of polymeric and monomeric forms, as well as of IgAI and IgA2 subclasses, in acute and chronic infection with hepatitis B virus. Hepatology 1986: 6: 652-7. Omata M, Yokosuka O, Mort J. ct al. Delta infection in Japan: a serological study. Jpn J Gastroenterol 1985: 82:1884-8 (in Japanese). Kaklamani E, Trichopoulos D, Tzonou A, et al. Hepatitis B and C viruses and their interaction in the origin of hcpatoccllular carcinoma. JAMA 1991: 265: 1974-6. Fong T-L, Di Bisceglie AM, 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. Koike K. lino S, Kurai K. Mitamura K, Endo Y. Oka H. lgM anti-HBc in anti-HBe positive chronic type B hepatitis with acute exacerbations. Hepatology 1987: 7: 573-6. Fiore G, Napoli N, Fera G, et al. Hepatitis C virus infection in anti-HBe-positive HBsAg carriers with chronic liver disease. Digestion 1991: 50: 121-6. Okamoto H, Yotsumoto S, Akahane Y, et al. Hepatitis B viruses with precore region defects prevail in persistently infected host along with seroconversion to the antibody against e antigen. J Virol 1990: 64: 1298-303. Moraleda G, Bartolome J, Molina J, Castillo I, Carreno V. Analysis of hepatitis B virus DNA, liver diseases and influence of antibody to hepatitis C virus in anti-HBc chronic carriers. Liver 1991: I1: 352-7. Nishioka K, Watanabe J, Furuta S. et al. Antibody to hepatitis C virus in acute hepatitis and chronic liver diseases in Japan. Liver 1991; 11: 65-70. Tanaka E. Kiyosawa K, Sodeyama T, et al. Prevalence of antibody to hepatitis C virus in Japanese schoolchildren: comparison with adult blood donors. Am J Trop Med Hyg 1992; 46: 460-4. Schweitzer IL, Mosley JW. Ashcavai M, Edwards VM, Overby LB. Factors influencing neonatal infection by hepatitis B virus. Gastroenterology 1973; 65: 277. Sheen IS, Liaw YF. Chu CM. Pao CC. Role of hepatitis C virus infection in spontaneous hepatitis B surface antigen clearance during chronic hepatitis B virus infection. J Infect Dis 1992: 165: 831-4. Lamas E, Baccarini P, Housset C, Kremsdorf D, Brcchot C. Detection of hepatitis C virus (HCV) RNA sequences in liver tissue by in sittt hybridization. J Hepatol 1992: 16: 219-23.
Journal of Hepatology 1994; 21: 159-166 Printed ha Demnark . All rights reserved Munk.~aard. Copenhagen
Journal of Hepatology ISSN 0168-8278
Coinfection of hepatitis C virus in patients with chronic hepatitis B infection Shinjiro Sato t, Shigetoshi F u j i y a m a ~, M o t o h i k o T a n a k a ~, K u n i o Yamasaki t, Izumi K u r a m o t o ~, Shin-ichi K a w a n o ~, T a t s u o Sato t, K y o s u k e M i z u n o 2 and Saneo N o n a k a 2 t Third Departnwnt o f btternal Medichle, Kutnanwto University School o f Medichte and :Chento-Sero-Therapeutic Research Institute, Kumamoto, Japan
(Received 2 November 1992)
Enzyme-linked immunosorbent assays for detecting antibodies against hepatitis C virus and the polymerase chain reaction were tested in 82 chronic hepatitis B surface antigen carriers for their accuracy in diagnosing patients coinfected with hepatitis B and C viruses. To clarify the role of each virus in chronic hepatitis, serologic assays against hepatitis B virus were also tested. Thirteen (14.9°/,,), 14 (17.1%) and 15 (18.3%) patients were anti-HCV positive using C100 (HCV1), JCC, and a second generation test (HCV2), respectively. HCV RNA was detected by polymerase chain reaction in 9 of 18 antiHCV-positive cases. Although HCV1 assays were not sufficient, either the JCC or HCV2 assay detected all polymerase chain reaction-positive cases. Fifteen of 18 specimens that were positive in at least one of the three ELISA were seronegative for the hepatitis B e antigen. As judged by HBV DNA potymerase activity, titers of hepatitis B surface antigen and immunoglobulin A antibody against hepatitis B core antigen (IgA anti-HBc), activity of hepatitis B virus replication and immune response against hepatitis B virus in patients with coinfection was decreased to the level of hepatitis B virus asymptomatic carriers. These results show that hepatitis C virus appears to be the primary cause of active hepatitis in most patients with hepatitis B and hepatitis C virus coinfection. © Journal of Hepatology. Key words: Enzyme-linked immunosorbent assay; IgA anti-HCV; Polymerase chain reaction
The major etiologic agents of blood-borne hepatitis are hepatitis B virus (HBV) and hepatitis C virus (HCV). These are two serious, prevalent diseases which cause chronic hepatitis, cirrhosis and hepatocellular carcinoma (1,2). Both viruses share a similar route of parenteral transmission, and some evidence suggests that both infections may also occur from vertical or sexual transmission (3,4). Similarities in the epidemiology of HCV and HBV infection were also noted in a report which showed a high prevalence of antibody to HCV (anti-HCV) in blood donors positive for antibody to hepatitis B core antigen (anti-HBc) (5), and in a trial of blood donors who were screened using anti-HBc as a surrogate marker for the prevention of posttransfusion non-A, non-B (NANB) hepatitis (6). Accordingly, coinfection with HCV might be expected to occur more frequently in persons seropositive for hepatitis B surface antigen (HBsAg).
Molecular cloning of HCV (7,8) led to the development of several types of immunoassays for detecting anti-HCV antibodies. C100 is an assay for detecting antibodies against a non-structural (NS) protein of HCV (HCV1) (9). New assay systems using HCV core antigen (JCC and a second generation test (HCV2)) are superior to HCVI in identifying HCV carriers (10,11), and are expected to reduce the incidence of posttransfusion hepatitis by HCV (12). However, positive results for anti-HCV with a single assay do not necessarily indicate HCV viremia since both false-positives and "memories" of prior transient infection (13) may occur in a substantial number of seropositive patients. One of the purposes of this study was to assess the prevalence of anti-HCV and circulating HCV RNA in patients with hepatitis B and assess the accuracy of these assays in diagnosing coinfection with HCV. A second purpose was to analyze the etiologic role of
Correspondence to: Shinjiro Sato, M.D., Third Department of Internal Medicine, Kumamoto University School of Medicine, 1-1, Honjo 1chome, Kumamoto 860, Japan.
160 each virus in ongoing liver disease. HCV associated markers indicating the activity of HCV-induced liver disease are currently unknown; however, the presence of HCV RNA in the sera may be a reliable marker of infectivity and active replication of HCV (14). In patients with chronic HBV infection, the disappearance of hepatitis B e antigen (HBeAg) and DNA polymerase (DNA-P) activity are used as common denominators of spontaneous remission of active hepatitis. However, seroconversion to detectable antibody against HBeAg (anti-HBe) does not always prevent the progression of liver disease (15). More accurate assays indicating the activity of HBV-induced liver disease are needed to determine the relative contribution of HBV in coinfected patients. Increased immunoglobulin A class anti-HBc (IgA antiHBc) in hepatitis B patients' sera implies an active immune response against HBV and, hence, HBV-induced liver injury (16). IgA anti-HBc may also be useful in monitoring disease activity, and the real value of IgA anti-HBc may be fully recognized in HBeAg-negative patients (17). In this report, we demonstrate the importance of IgA anti-HBc in HBsAg carriers who are simultaneously positive for anti-HCV. Moreover, the investigated assays may be of some value in assessing antiviral therapy against both viruses. Patients and Methods
Patients A total of 82 patients with persistent serum HBsAg were studied by ELISA for antibodies against HCV and for HCV RNA in the sera. None of the patients had serologic markers for acute hepatitis A virus, cytomegalovirus, or Epstein-Barr virus, or any other obvious cause of hepatitis (autoimmune disease, alcohol or drug hypersensitivity). There were 57 males and 25 females, with a mean age of 40.1 years (range 18 to 69). Twenty-nine patients were HBeAg-positive and 53 HBeAg-negative. Eighteen patients were asymptomatic HBV carriers with no evidence of hepatic disease (alanine aminotransferase (ALT) <24 International units per liter (IU/I)) without detectable markers of HBV replication (low level of DNA-P and had been seronegative for HBeAg for at least 2 years), 45 had chronic hepatitis, and 19 had cirrhosis including five with hepatocellular carcinoma (HCC). To validate serologic measures for HBV in patients positive for anti-HCV, nine asymptomatic HBV carriers and 17 anti-HCV-negative (anti-HCV(-)) patients with active hepatitis B and elevated ALT levels (>50 IU/I) after seroconversion to anti-HBe, were selected as controls. Serum samples were collected from June, 1989 to Au-
S. SATO et al. gust, 1991 and were stored at - 2 0 ° C or - 8 0 ° C until assayed.
Serologic methods of HCV detection Antibody against CI00 (anti-HCVl) was tested by the Ortho HCV Ab ELISA (Ortho Diagnostic Systems, Tokyo, Japan) (9). Antibody against JCC (anti-JCC) was tested by the JCC ELISA kit (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan). JCC is a recombinant protein corresponding to the putative core region of the HCV genome (10). Antibodies against multiple immune dominant proteins including NS and the core region of HCV (anti-HCV2) were tested by the Ortho HCV Ab ELISA second-generation kit Ortho Diagnostic Systems, Tokyo, Japan) (I 1). Reverse transcription-nested polymerase chain reaction ( R T-nested PCR) RNA was prepared from each 0.05 ml serum sample using the method of RNA isolation by acid guanidinumthiocyanate-phenol-chloroform extraction (18). Complementary DNA (cDNA) was prepared from RNA by reverse transcription. The cDNA was amplified twice by nested PCR for 35 cycles each (19) using primers corresponding to the 5' non-coding region (5'-NCR) (20) of the HCV genome. PCR products were subjected to electrophoresis on an agarose gel and stained with ethidium bromide for visualization. The sensitivity of PCR was assessed by serial testing of infectious sera derived from a patient with chronic hepatitis C, and sera diluted 1:105 had detectable HCV RNA. Further investigation was performed by the same method to controlled quantities of RNA prepared from plasmid vector which contained the 5'-NCR sequence of HCV (21). More than 40 copies of RNA could be detected by PCR. Serologic methods for HB V HBsAg was tested by Seroclit-HBs (The Chemo-SeroTherapeutic Research Institute, Kumamoto, Japan). The HBsAg titer was determined by testing serial two-fold dilutions of the serum against red cell samples in this kit. Results were expressed as the reciprocal of the highest serum dilution (2 °) that caused macroscopically visible agglutination. HBeAg and anti-HBe were analyzed by solid phase radioimmunoassay (Abbott Laboratories, North Chicago, IL, USA). DNA-P activity was measured by the method of Kaplan et al. (22), and levels >30 cpm were interpreted as positive. IgA anti-HBc was analyzed by IgA antibody capture ELISA. Polystyrene microtitre plates (Nunc-lmmune Plate Maxisorp; Nunc, Roskilde, Denmark) were coated with anti-human IgA (Dakopatts a/s, Glostrup, Den-
161
COINFECTION WITH HBV AND HCV
mark) at a 1 : 600 dilution with 0.15 M phosphate-buffered saline (PBS). Following overnight incubation at 4°C, the wells were washed three times with PBS containing 0.05°/,, (v/v) Tween-20 using an automatic plate washer. The wells were then coated with 3% (wt/v) bovine serum albumin (BSA) in PBS for 2 h at 37°C. After washing as before, 0.2 ml/well of sera at a 1:400 dilution with 3% BSA in PBS was added for 1 h at 37°C. After the first incubation, the plates were washed five times and 0.2 ml/well of HBcAg and peroxidase conjugated anti-HBc monoclonal antibody mixture (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan) was added for 1 h at 37°C. The plates were then washed five times before the addition of 0.2 ml/well of O-phenylenediamine (OPD) substrate solution (10 ml of 0.1 M citric acid-phosphate buffer and 10 ml of 0.2 M Na,_HPO4. 12H,O buffer, containing 8 mg of OPD and 4/.tl of 30% (v/v) H_,O2), and incubated for 30 min at room temperature. The reaction was terminated by the addition of 0.05 ml of 4 N H_,SO4. Optical density (OD) values were measured at 490-650 nm with a microplate reader (Molecular Devices Corp, City CA). The appropriate dilution of 1:400 was selected for preventing non-specific binding acc6rding to a dilution curve derived from a positive serum sample with anti-human lgA in plates coated or uncoated with IgA anti-HBc ELISA. Twenty serum samples collected from healthy blood donors were also tested with this ELISA as a negative control. The cut-off value was defined by the results of normal controls and values >0.03 were interpreted as being positive for IgA anti-HBc. Mean±S.E. of OD values was calculated from all samples in each group of patients with HBV infection.
clonal anti-human IgG/Fc, IgA/a, and IgM/p, respectively. OPD substrate was then added and OD measured. These fractions were also tested by IgA anti-HBc ELISA to confirm that the reaction occurred in the same fractions which, contained IgA. IgA class antibodies were well differentiated from other classes of immunoglobulins (Fig. 1). Given the molecular weight and chromatographic pattern of IgA, the first peak of high molecular size (Fr 66) was considered as polymeric IgA, and the second (Fr 77) as monomeric IgA (23). In the assay for IgA anti-HBc, serum samples from HBV carriers with chronic active hepatitis were reacted with the fractions containing primarily monomeric IgA. IgG class immunoglobulins did not react in this assay as evidenced by asymptomatic HBV carriers having high titers of lgG anti-HBc, all of whom were negative for IgA anti-HBc. To verify the IgA anti-HBc ELISA, 55 patients with anti-HCV-negative persistent HBV infection were tested. They included nine asymptomatic HBV carriers, seven with chronic persistent hepatitis, 21 with chronic active hepatitis, and 18 with cirrhosis. Histopathologic findings on liver biopsy were graded using standard criteria. IgA anti-HBc in patients with persistent HBV infection (by histologic classification) was evaluated as shown in Fig. 2.
Stat&tical analysis Differences in the mean value of each group were evaluated by the Wilcoxon rank-sum test. The difference in the proportion of patients with an abnormal value between two groups was evaluated by the Z2 method.
Characterization of lgA anti-HBc Characterization of the immunoglobulin class of the anti-HBc was performed in four samples collected from patients with chronic hepatitis B. Twenty microliters of sera were fractionated by high performance liquid chromatography (HPLC) in an LC-9A apparatus (Shimadzu, Kyoto, Japan) over G3000SWxc gel in two 7.8 mm I D × 3 0 cm columns (Tosoh, Tokyo, Japan) connected in tandem. Columns were eluted with phosphate-buffered saline (0.01 M, pH 7.2) at a flow rate of 0.6 ml/min, and 200/.tl fractions were collected. Before HPLC, sera were diluted 1:4 with phosphate-buffered saline and elution buffer, and filtered through 0.22 p Millipore filter paper. To identify the immunoglobulin class (IgG, IgA, or IgM), a sandwich ELISA was made separately. Microtiter plates were coated using polyclonal anti-human IgG/~, IgA/a, and IgM/p. Samples fractionated by HPLC were sandwiched by horseradish-peroxidase conjugated poly-
3.0
• ~ ,
IgA IgM lgA anfi-HBc
~ 2.0
-~ 1.0 O
0.0 50
60
70
80
90
1O0
110
Fraction Number
Fig. t. Elution curve of a serum sample from a patient with chronic
active hepatitis B. Closed circles, open circles and crossed bars represent IgM, IgA, and IgG types of immunoglobulins, respectively. Shaded areas represent the results for IgA anti-HBc ELISA. Void volume is indicated by the black arrow.
162
S. SATO et al.
10 0
E 0
=1
10"
©
10
-2
ASC
CPH
CAH
C
, : p < 0.001 Fig. 2. Immunoglobulin A antibody to hepatitis B core antigen (IgA anti-HBc) in asymptomatic carriers (ASC), patients with chronic persistent hepatitis (CPH), patients with chronic active hepatitis (CAH) and patients with cirrhosis (C). The mean optical density values_S.E, of all patients are given for each group (closed bars).
f
HCV2 •
: P C R (+)
0
: P C R (-)
no a n t i - H C V ( + ) cases in a s y m p t o m a t i c HBV carriers. All a n t i - H C V ( + ) patients had an elevated A L T or histology consistent with progression to cirrhosis. The relationship between HBeAg/anti-HBe findings and anti-HCV is shown in Table 2. Three patients who were HBeAg-positive were anti-HCV positive as measured by the HCVI assay and two of these were also positive by the HCV2 assay. However, all patients positive for anti-JCC had no measurable HBeAg. The results of these anti-HCV E L I S A and their cox'relation with the results o f RT-nested PCR are shown in Fig. 3. HCV R N A was detected in nine of the 18 antiH C V ( + ) specimens. All patients positive for HCV R N A ( P C R ( + ) ) had a positive result in multiple ELISA. HCV1 assay missed two P C R ( + ) cases, while JCC and HCV2 detected all P C R ( + ) cases. All cases positive in only one ELISA, and the remaining 64 a n t i - H C V ( - ) cases, were negative for HCV R N A ( P C R ( - ) ) . Table 3 presents the clinical and serologic characteristics and prevalence o f IgA anti-HBc in a s y m p t o m a t i c HBV carriers, a n t i - H C V ( + ) and a n t i - H C V ( - ) groups. The mean age of the P C R ( + ) group was higher than that o f others. The rates of H B V - D N A polymerase activity were significantly lower in a n t i - H C V ( + ) / P C R ( + ) cases than a n t i - H C V ( + ) / P C R ( - ) and a n t i - H C V ( - ) cases (0% vs 55.5% and 70.6%, respectively, p<0.01). Positivity in the IgA anti-HBc assay in the P C R ( + ) group was the same as in a s y m p t o m a t i c HBV carriers, and significantly lower than that in the a n t i - H C V ( + ) / P C R ( - ) group (p<0.025) and a n t i - H C V ( - ) group (p<0.025). N o differ-
TABLE 1 Prevalence of anti-HCV antibodies as measured in three ELISAs in patients with chronic hepatitis B virus infection
HCVI
JCC
Fig. 3. Venn diagram of anti-HCV ELISA and RT-nested PCR results in patients with hepatitis B. Each small circle represents a single patient. Closed and open circles denote patients with or without HCV RNA, respectively.
Diagnosis
No. of cases HCVI
JCC
ASC CH C HCC
18 45 14 5
0 10 2 1
Total (%)
82
13 (15.9'7,,) 14 (17.1"/.) 15 (18.YV,,)
0 9 4 1
HCV2 0 11 3 1
ASC; asymptomatic carrier. CH: chronic hepatitis. C: cirrhosis. HCC: hepatocellular carcinoma.
TABLE 2
Results The prevalence o f positive results indicating HCV infection in patients clinically diagnosed with chronic HBV is shown in Table 1. O f the 82 HBsAg carriers, 13 (15.9%),14 (17.1%), and 15 (18.3%) were positive for antiH C V I , anti-JCC, and anti-HCV2 respectively. Eighteen patients were positive in at least one assay. There were
Prevalence of anti-HCV antibodies in patients with chronic hepatitis B infection and variable HBe serologic status HBeAg/anti-HBe
No. of cases HCVI
JCC
HCV2
(+/-) (-/-) (-/+)
29 6 47
3 0 10
0 1 13
2 I 12
Total
82
13
14
15
COINFECTION WITH HBV AND HCV
163
TABLE 3 Clinical, serological characteristics and prevalence of immunoglobulin A antibody against hepatitis B core antigen in asymptomatic carriers, anti-HCV(+) and anti-HCV(-) groups Characteristics
ASC
n Male/female Mean age HBe antigen (+) (%) Serum ALT (Iu/I) DNA polymerase activity (cpm) IgA anti-HBc(+) (%) Cirrhosis (%) Blood transfusion (%)
Anti-HCV(+)
9 6/3 44 (18-69) 0% 17.1___1.4 1.7+_0.8 11.1% ND 22.2%
Anti-HCV(-)
PCR(+)
PCR(-)
9 7/2 47 (29-67) 0% 142.6+30.2 5.9--_2.5 11.1% 22.2% 22.2%
9 6/3 39 (20-55) 33.3% 161.9-+39.9 269.4 + - 167.9 77.7% 44.4% 33.3%
17 11/6 38 (20-59) 0% 135.1__.44.2 328.0-+ 147.0 70.6°,4, 17.7% 17.7%
ASC: asymptomatic carriers. PCR: polymerase chain reaction. Results are expressed as mean_S.E, or range in parentheses. ND; not determined.
ences were f o u n d in the severity o f h e p a t i c d a m a g e a n d
E L I S A . IgA a n t i - H B c was c o n s i s t e n t l y f o u n d in P C R ( - )
rate
p a t i e n t s , b u t in only a single P C R ( + )
of cirrhosis between
PCR(+)
and
anti-HCV(-)
groups.
p a t i e n t (Case 18
h a d b o t h IgA a n t i - H B c a n d H C V R N A ) . This p a t i e n t
T h e r e was a h i s t o r y o f b l o o d t r a n s f u s i o n in 27.7% o f
d e v e l o p e d e x a c e r b a t i o n o f his h e p a t i c injury with a n elev-
H B V c a r r i e r s with a n t i - H C V a n t i b o d y a n d in 22.2% o f
a t i o n in D N A - P activity a f t e r 1 y e a r ( d a t a n o t s h o w n ) .
P C R ( + ) . All a n t i - H C V ( + ) p a t i e n t s w i t h p e r s i s t e n t H B V
C a s e s 9 a n d 12 were a n t i - H C V positive in all t h r e e E L I -
i n f e c t i o n are s h o w n in T a b l e 4, with several clinical fea-
SA, b u t H C V R N A was n o t d e t e c t e d in e i t h e r p a t i e n t .
tures a n d
of HBV
T h e s e t w o p a t i e n t s h a d IgA a n t i - H B c , a n d o n e (Case 12)
n o t e d . T h e r e were n o p a t i e n t s with d r u g a b u s e o r a his-
h a d e l e v a t e d D N A - P activity. T h e s e p a t i e n t s h a d a his-
serologic or biochemical markers
tory o f t a t t o o s . T h e c u t - o f f i n d e x o f a n t i - H C V a n t i b o d y
t o r y o f i n t e r f e r o n t h e r a p y n o t e d in r e t r o s p e c t . A m o n g 18
in p a t i e n t s with p o s i t i v e results in a single a s s a y t e n d e d
o f the a n t i - H C V ( + ) cases, five h a d b e e r / t r e a t e d w i t h in-
to be l o w e r t h a n in p a t i e n t s f o u n d positive in m u l t i p l e
t e r f e r o n for at least 4 w e e k s several years before, b u t all
TABLE 4 Clinical and serologic characteristics of anti-HCV(+) patients with hepatitis B Case
Age
Sex
Diag
B-T
HBeAg
anti-HBe ALT
A. Patients negative for JCC 1 20 M CN 2 21 F CH 3 33 M CH 4 38 F HC
-
+ + + -
+
216 139 276 39
B: Patients positive for JCC 5 43 M C 6 54 F C 7 55 M HCC 8 46 M CH 9 43 M CH 10 43 F CH 11 41 F CH 12 41 M CH 13 49 M C 14 50 M CH 15 67 M CH 16 56 M CH 17 29 M CH 18 41 M C
+ + + + +
-
+ + + + + + + + + + + + +
49 119 77 244 111 128 48 68 75 55 48 184 249 252
DNA-P
HBsAg
IgA anti-HBc
HCVI
384 18 29 8
14 13 9 5
0.375 0.166 0.337 0.031
3.73 2.914 1.746 1.110
56 225 N.D. 3 37 15 0 1398 0 0 2 4 21 8
12 13 9 9 9 7 9 I1 5 9 3 3 12 6
0.029 0.443 0.007 0.001 0.088 0.005 0.002 0.081 0.001 0.005 0.002 0.004 0.019 0.206
0.182 0.080 0.793 6.220 4.684 5.726 0.284 5.843 6.220 0.076 5.031 7.424 6.451 5.841
JCC
HCV2
HCV RNA
0.286 0.256 0.422 0.231
2.917 1.875 0.125 0.138
-
1.036 2.633 3.169 3.656 4.282 8.300 9.072 9.834 9.875 10.024 10.057 10.117 10.120 10.280
0.039 2.872 5.636 6.000 5.987 6.220 6.200 6.038 6.150 5.974 6.067 6.236 6.190 6.104
+ + + + + + + + +
B-T; Blood-transfusion. HBsAg; Hepatitis B surface antigen titer (2"). ALT; alanine aminotransferase levels. DNA-P; DNA polymerase activity (cpm). PCR; polymerase chain reaction. M; male. F; female. CH; chronic hepatitis. C; cirrhosis. HCC; hepatocellular carcinoma. N.D.; not done. Titers of HBsAg are expressed in reciprocal of serial two-fold dilutions. IgA anti-HBc results are described in optical density values and results of anti-HCV ELISA are described by cut-off index (Cut-off index for positivity in the anti-HCV assay >1.000).
164
S. SATO et al.
had abnormal ALT levels, and two patients still had HCV RNA detected by PCR. The humoral anti-HCV responses in HBsAg carriers have persisted: however, changes in P C R ( - ) to PCR(+) were not observed in patients (Case 1, 2, 9 and 12) after 2 to 4 years of follow-up analysis. The relationship of HBV markers (titer of HBsAg, IgA anti-HBc) in asymptomatic HBV carriers, HCV(+)/ PCR(+), H C V ( + ) / P C R ( - ) , and a n t i - H C V ( - ) group patients is shown in Fig. 4 and 5. Statistically significant differences between HCV(+)/PCR(+) and anti-HCV(- ) groups were noted in HBsAg titer (/7<0.05) and to a greater extent in IgA anti-HBc (p<0.001).
=
I
15 • •
ooN
No
N N
N
ASC
anti-HCV(+) PCR(+)
ooo
anti-HCVl+) PCR(-) ,
•oo ~oooo
00
anti-HCV(-) : p < 0.05
Fig. 4. HBsAg titers in patients with hcpatitis B. Patient groups include HBeAg-negative asymptomatic carriers (ASC). seropositive for anti-HCV with or without HCV RNA (PCR(+) or PCR(-I) and anti-HCV(-) patients. Each dot represents a single patient.
101 I
-~ lo °
I
I
"N o N
u 10 |
T m
16~ ASC
anti-HCV(+) anti-HCV(+) anti-HCV(- ) PCR(+) PCR(-) , :p<0.01 * * : p < 0.001
Fig. 5. IgA anti-HBc in patients with hepatitis B. Patient groups are identical to those shown in Fig. 4. Each dot represents a single patient.
Discussion
In the present study, anti-HCV was detected in 18 (23%) of 82 HBsAg carriers, using three types of ELISA. Because all anti-HCV-positive patients had abnormal levels of ALT, their continuing hepatitis could have been caused by HBV and/or HCV, or even autoimmunity or hepatotoxic agents. Since hepatitis delta virus is infrequent in Japan, testing for this virus was not performed (24). It is important to determine if these anti-HCV(+)patients are actually coinfected with HBV and HCV, because HCV may prolong tile hepatic injury of HBV and progress to cirrhosis and HCC (25). In a previous study (21) several HCV-related antibodies were tested simultaneously in patients with various liver diseases, and almost all anti-HCV(+)-patients with NANB hepatitis were found to have HCV RNA in their blood. However, a recent study indicated that anti-JCC and HCV2 were also detected at relatively high rates in patients with other liver injuries and in blood donors, and the association between anti-HCV and the presence of HCV RNA was not as specific in these cases (12). The presence of patients positive for anti-HCV but negative for HCV RNA suggests that anti-HCV might be detected even in patients with transient HCV infection, similar to antibodies against HBcAg. RT-nested PCR assay might verify whether viable HCV is present in the serum of these patients. The results in a trial of combined assays for blood donor screening (12) indicate that multiple assays developed heterogeneously from different kinds of epitopes within the HCV genome might be used to replace RT-nested PCR to ascertain the presence or absence of viable HCV in serum, with considerable savings in time and effort. Further studies, however, are needed to confirm this evidence. In this study, nine patients who were positive in multiple anti-HCV ELISA also had HCV RNA in their sera. The presence of HCV RNA was closely associated with positive results in ELISA measuring HCVI and JCC assays. However, as presented here, none of the anti-HCV ELISA were effective as single or multiple assays, and therefore the PCR method was the single best predictor of actual HCV infection. In the serologic results associated with HBV, DNA-P activity was lower in anti-HCV(+) cases and undetectable in PCR(+) cases. H BsAg levels were similarly reduced in PCR(+) patients. Fong et al. (26) demonstrated identical results. This suggests that HBV replication is mild or moderate in coinfected patients. Although the proliferative activity of HBV could be determined by DNA-P activity (displaying the active replication of HBV) and HBsAg titer (representing the quantity of products), this did not always correlate with hepatic disease activity. In our
COINFECTION WITH HBV AND HCV study, lgA anti-HBc was used to predict the activity of HBV-induced liver disease. Ordinarily, IgA class immunoglobulins are associated with the recurrence of viral replication in persistent disease. Nomura et al. reported that IgA anti-HBc more accurately reflects hepatic injury resulting from HBV infection than other evidence of HBV replication, and is useful in identifying asymptomatic HBV carriers (16). The level of IgM anti-HBc also increased in parallel with IgA anti-HBc and was associated with acute exacerbations of persistent hepatitis B infection (27), but the correlation between IgA anti-HBc and hepatic injury is more reliable (17). In this study, the level of IgA anti-HBc was significantly different between asymptomatic HBV carriers, and patients with chronic persistent hepatitis, chronic active hepatitis, and cirrhosis. Mean OD values in patients with chronic active hepatitis and cirrhosis were higher than those with chronic persistent hepatitis. Asymptomatic HBV carriers were well separated from the other patients (Fig. 3). These results seem to indicate that this IgA antiHBc assay is reliable and applicable. In the anti-HCV(+) patients, lgA anti-HBc was rarely detected in HCV PCR(+) with mean values equivalent to asymptomatic HBV carriers. This suggests that the immune response against HBV was decreased to an equivalent degree to that in asymptomatic HBV carriers, and that the prolonged liver damage in these patients might not be dependent on HBV. However. in chronic hepatitis B patients with elevated levels of ALT and no detectable HCV RNA, IgA anti-HBc was consistently present. The serologic findings which show the replicative activity of each virus and the activity of HBV-induced liver disease seem to indicate that there are only a few patients with liver damage sustained by both viruses, because the viruses rarely proliferated concurrently in coinfected patients. In most HBV patients coinfected with HCV, HCV alone may be implicated in the pathogenesis and progression of hepatic damage (26, 28). Among a n t i - H C V ( - ) patients, abnormal ALT and DNA-P levels were found even after seroconversion to detectable anti-HBe. Regarding persistent abnormal levels of ALT and histopathologic progression in patients with persistent HBV infection after seroconversion to anti-HBe, precore mutant HBV appears to play an important role (29). These patients sometimes had acute exacerbations with elevated ALT levels, and DNA-P activity would probably have been detectable prior to ALT elevation. Activation of hepatitis by HBV alone, perhaps of a mutant virus in the precore region, appears to be the primary cause of continuing hepatic damage in these patients (30). In this study, a n t i - H C V ( - ) patients also had detectable IgA anti-HBc, which seems to be helpful
165 in monitoring tile disease activity in anti-HBe-positive HBsAg carrier patients. Nishioka et al. reported that a prior history of blood transfusion in Japanese patients was observed in 2.9% (7/ 242) and 5.5%, (6/110) of HBsAg positive/anti-HCV negative chronic hepatitis and cirrhosis, respectively (31). However, in the present study, 27.7°/, of HBsAg-carrier patients who were anti-HCV(+) had a history of blood transfusion and 22.2% of these patients were seropositive for HCV RNA. In addition, the mean age of PCR(+) cases was higher than other patient groups, and all PCR(+) patients had already become seropositive for anti-HBe. Based on the low prevalence of anti-HCV in children (32) and the lack of detectable HCV RNA in anti-HCV-negative persons who were tested in our family survey (unpublished observation of 88 relatives of patients with chronic hepatitis C), HCV vertical transmission appears to be extremely rare. Although the chronicity of HBsAg primarily depends on acquisition from mother to infant (33), occurrence of simultaneous infection with HCV in patients with HBsAg might represent a superinfection transmitted via parenteral exposure by means of blood transfusion or improper disposal of sharp instruments which contain infectious sera. Superinfection with HCV in anti-HBe-positive patients with HBV infection is probably the reason why HBV replication was decreased in these patients. Suppression of HBV replication by HCV may not occur as frequently in the patients in this study as previously reported (26,28). Although three patients with HBeAg and six patients without HBeAg were anti-HCV-positive, none had detectable HCV RNA in their serum. However, 77.8% had detectable IgA anti-HBc. If these anti-HCV results are not false positives or representative of prior infection, thus indicating actual coinfection (or superinfection) with HCV, they seem to indicate that HCV may lag behind the hepatitis caused by HBV. HCV may persist subclinically with no detectable levels of serum HCV RNA, arid then become apparent as HBV replication decreases with seroconversion to anti-HBe. However, this hypothesis has not been supported by 2 to 4 years follow up. Though we could not confirm the 9l vivo interference of each virus in patients with coinfection in this study, further prospective or retrospective studies (34) and PCR in the liver specimens from HCV RNA-negative patients positive for antiHCV in their serum may provide greater insight (35). The five patients suboptimally treated with interferon all had abnormal ALT levels, and two patients were still positive for HCV RNA. Recognition of the clinical course and virologic measures of coinfection are important in determining which virus is etiologically active and may affect decisions regarding interferon therapy. Assay of IgA
166
S. SATO et al.
a n t i - H B c m a y be o f p a r t i c u l a r value in m a k i n g tiffs decision.
18.
19.
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