Journal of Hepatology, 1992; 16:184-189 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0168-8278/92/$05.00

184

HEPAT 01152

Serum hepatitis C virus RNA and hepatitis B virus D N A in non-A, non-B post-transfusional and sporadic chronic hepatitis Colette P o r c h o n a, D i n a K r e m s d o r P , Stanislas Pol b, Franqoise L u n e l - F a b i a n n i d, Franqoise Driss c, Pierre O p o l o n ~, Pierre Berthelot b and Christian Br6chot a'b'e "INSERM U-75, CHU Necker, Paris, bUnitE d'HEpatologie, INSERM U-99, H6pital La~nnec, Paris, CBanque du Sang. H6pital LaYnnec, Paris, dSert,iced 'HEpato-GastroentErologie, H6pital Pitik-SalpEtriEre. Paris and Clnstitut Pasteur, Hybridotest. Paris, France (Received 20 September 1991)

The sera of 36 French patients with post-transfusional and sporadic non-A, non-B chronic hepatitis were investigated, for HBV and HCV infections using a combination of serological and polymerase chain reaction (PCR) assays. AntiHCV was detected in 75% (27/36) of the patients by ELISA1 and/or RIBA2 tests. HCV-RNA sequences were found in 75% (27/36) of the sera by a single step PCR, using a set of primers located in the 5' non-coding region. Altogether, 89% (32/36) of the patients were found positive with serological and/or molecular tests. Among the positive patients, 68% (22/32) were found positive for both anti-HCV and HCV-RNA, 16% (5/32) and 16% (5/32) were found positive for either anti-HCV or HCV-RNA, respectively. HBV-DNA sequences were detected in two patients associated to the HCV viraemia. This study confirms the extremely high prevalence of HCV infection in NANB chronic hepatitis in France. It also shows possible co-infection by HCV and HBV in hepatitis.

Key words." HCV-RNA; HBV-DNA; PCR assay; NANB chronichepatitis

Hepatitis C virus (HCV) is a positive-stranded RNA of about IO kb, distantly related to the Flavi- and Pestiviruses (1,2). cDNAs derived from infected chimpanzee and human serum samples have been cloned, allowing a comparison of distinct viral isolates in different geographical areas (I,3-10). Almost complete nucleic sequences of American and Japanese HCV strains have been reported (5-7) as well as partial sequences from European HCV strains (8-10). The HCV genome encodes a large polyprotein precursor, which is processed in structural (nucleocapsid and envelope) and nonstructural proteins (2,7). A first ELISAI test has been developed which includes a polypeptide from the nonstructural domain of the HCV genome (C100-3) synthetized in yeast, and used to capture circulating viral antibodies (11). With this test, anti-HCV antibodies were detected in the serum of 60-80% of patients with posttransfusional or sporadic chronic non-A, non-B (NANB) hepatitis (11-14).

Recently, a new serological test, the Recombinant ImmunoBlot Assay 2 (RIBA2), has been introduced (I 5). This test allows the detection of antibodies directed against different structural (C22: capsid antigen) and non-structural (CI00.3:NS3-NS4 domain, 5.1.1.; NS4 domain, C33c: NS2-NS3) antigens. For the moment, however, there are several limitations to the interpretation of results; since seroconversion after acute infection is often delayed and there is no test to measure HCV antigenemia (16,17), detection of viral RNA in serum is of primary importance for the estimation of viral multiplication. However, due to the low number of viral particles in serum, conventional hybridization technics generally fail to identify HCV-RNA. Several groups have therefore provided evidence that polymerase chain reaction (PCR) is useful in the evaluation of HCV viraemia (18-25), and can be used to detect serum HCV-RNA at the early stages of acute HCV infection, before seroconversion (17,24). In addition, PCR allows the detection

Correspondence to: Colette Porchon, CHU Necker, INSERM U-75, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.

HCV AND HBV 1N NANB CHRONIC HEPATITIS of HCV-RNA in certain sera which are negative by current anti-HCV assays (18-25). The nested PCR procedure has been used by several laboratories to increase sensitivity and specificity, and to avoid the time-consuming hybridization step (17,21,24). However, nested PCR dramatically increases the risk of contamination already inherent in PCR itself. Persistent HBV infections might also be involved in the development of some NANB chronic hepatitis and HCC. Indeed HBV-DNA sequences have been shown in HBsAg-negative individuals with chronic liver diseases (26-31). The association of HBV-DNA and anti-HCV has been reported (31) in the serum of subjects with NANB chronic hepatitis. However, it is not clear whether or not this reflects active viral multiplication of both viruses. We therefore performed a study of post-transfusional and sporadic chronic NANB hepatitis in patients in France. Sera were tested for anti-HCV and HBV serological markers as well as HCV-RNA and HBV-DNA with PCR.

Patients and Methods

Patients Thirty-six serum samples from patients with histologically proven chronic NANB hepatitis (19 posttransfusional, 12 sporadic, 5 intravenous drug users (IVDU)) were investigated. Alcoholic liver disease and hemochromatosis were excluded. None of the patients had any marker of auto immunity. Serological assays Antibodies against HCV polypeptides were determined by ELISAI and RIBA2 tests (Ortho Diagnostics Systems). HBsAg, anti-HBs and anti-HBc were determined using a standard RIA (Abbott Lab.). HCV and HBV nucleic acids extraction HBV and HCV viral genomes were extracted from sera as follows: 200 gl of serum were diluted 1:5 with 50 mM Tris-HCi (pH 8.0), 1 mM EDTA, 100 mM NaCI, 0.5% SDS and I mg/ml of proteinase K, and digested at 37°C for 1 h. Proteins were removed by one extraction with Tris-EDTA (pH 8.0) saturated phenol, two with phenol/chloroform (3:1) and one with chloroform. 0.2 M Na-acetate and 20 gg of glycogen were added to the aqueous phase, and the nucleic acids were precipitated by addition of 2 volumes of ice-cold absolute ethanol. After one night at -20°C, the nucleic acids were pelleted,

185 air-dried for 1 h and resuspended in 20 p.l of DEPCtreated water. PC R amplification HCV-RNA was detected by reverse transcription followed by PCR (RT-PCR). Nested PCR was not used in this study due to the extremely high risk of contamination. Primers located in the highly conserved 5' noncoding region of HCV genome were used. HCV-RNA was transcribed to cDNA by M-mlv reverse transcriptase (Gibco-BRL) with an antisense primer SRI from the 5' non-coding region (5'TGCACGGTCTACGAGACCTC3') corresponding to the position -22 to -3 of the prototype HCV-cDNA nucleotide sequence (6). Briefly, a mixture containing 10 I~1of a 1:50 diluted RNA solution, 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 10 mM DTT, 3 mM MgC12, 0.25 mM of each dNTPs (Pharmacia), 20 units (U) of ribonuclease inhibitor (Pharmacia), 15 pmol of antisense primer SRI, in a final volume of 25 p_l, was incubated at 65°C for 10 min. 200 U of M-mlv reverse transcriptase were added and the mixture was incubated at 37°C for 45 min. The PCR was carried out in a DNA thermal cycle (PerkinElmer, Cetus): 100 gl of reaction mixture containing 5 gl of cDNA sample, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2 m M MgCI2, 100 gg/ml of gelatin, 10 laM of antisense primer SRI and sense primer SFI (5'GCCATGGCGTTAGTATGAGT3': nucleotides -259 to -240), 400 gM dNTPs, and 2 U of Thermus aquaticus DNA polymerase (Perkin-Elmer, Cetus) were overlaid with 100 gl of mineral oil. After an initial cycle of 94°C for 5 min, 55°C for 1 min, 72°C for ! min, 38 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min were carried out, ending by 1 cycle of 94°C for 1 min, 55°C for 1 min and 10 min at 72°C for elongation. For HBV-DNA amplification, PCR was performed under the same conditions as HCV amplification with primers located in the S-region: sense primer MD 06 (5'TTCCTATGGGAGTGGGCC3') corresponding to the nucleotides 637-654 of the ayw subtype and antisense primer MD 13'(5'CCGCTGTTACCAATTTTCTT3'), nucleotides 796-815 (ayw subtype) (32). Ten gl of each reaction were applied to 1.5% agarose gel in Tris-borate buffer (pH 8.0). Bands were visualized by ethidium bromide staining and by Southern blot hybridization using a a2p-labelled 40-mer oligonucleotide sequence probe: 5'ACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGAT3', (nucleotides -177 to -138) for HCV amplification; 5'TTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGT3', (nucleotides 694-737) for HBV amplification.

186

C. PORCHON et al. Results

The membranes (Genescreen plus, N E N Res. Products, Du Pont) were washed twice with 2 × SSC/I% SDS at 20°C for 15 min, with 0.2 × SSC/0.1% SDS at 55°C for 10 min, and exposed to Kodak XAR film overnight at -80°C.

Viral markers in serum Table ! shows the clinical and histological status of the 36 patients with chronic N A N B hepatitis. Twentytwo of the 36 patients (61%) were anti-HCV-positive with ELISAI and 27/36 (75%) with RIBA2 tests. Eleven of the sera positive by the RIBA2 test reacted with the 4 antigens included in the test (C100.3, 5.1.1, C33c, C22). A m o n g the 14 sera anti-HCV-negative by ELISAI, 5 were anti-HCV-positive in the RIBA2 test, and reacted with only 2 or 3 epitopes. The presence of HBV serological markers was also investigated. All 36 individuals were HBsAg-negative.

Control of spec(ficity

Due to the risk of contamination, the following precautions were used: the different steps in the PCR procedure were performed in separate rooms. Only disposable reagents were used. Controls included sera from 8 blood donors who were HBV- and HCV-negative. These sera were extracted and analyzed in the same experiments and each sample was tested at least twice.

TABLE

1

Clinical,

serological

Patients

Risk factor ~

and

PCR

results

Sex

Histology liver b

HCV

HBV HBsAG

~-HBs

~-HBc

DNA

anti-HCV

anti-HCV

ELISAI

RIBA2

RNA

~

c

CI00.3

CI00.3

5.1.1

C33c

C22

-

-

+

++

+++

+ + + +

++

+

-

-

+

-

_

++

++

+

-

+

++

-

+++

+

-

-

-

+

-

+++

+++

-

-

-

-

+

+ + + +

+ + + +

+ + + +

+ + + +

+

CAH

-

-

-

+

+ + + +

+++

+++

+ + + +

-

+++

I

SPO

F

CAH

-

+

2

SPO

F

CAH

-

3

PT

M

CAH

-

-

4

PT

M

CAH

-

-

-

5

PT

M

CAH

-

-

6

PT

F

CAH

-

7

PT

F

CAH

8

PT

F

-

9

PT

F

CA H

.

.

.

.

+

10

PT

M

CAH

.

.

.

.

_

11

PT

M

CAH

.

.

.

.

12

PT

M

CAH+C

-

+

+

13

SPO

F

CAH

-

-

-

+

+++

+ + + +

+ + + +

+

_

_

+++

-

+

+

-

+ + + +

++

+

+

-

_

+ + + +

+++

+

14

SPO

M

CAH+C

-

-

+

+

+ + + +

+ + + +

+ + + +

+ + + +

15

SPO

F

CAH+C

-

-

-

_

_

_

++

+++

-

16

PT

M

CAH

-

-

-

+

+++

+++

+ + + +

+ + + +

+

17

PT

M

CAH+C

-

-

-

+

-

_

+ + + +

+++

+

18

PT

F

CAH+C

-

+

+

+

+ + + +

+ + + +

+ + + +

+ + + +

+

-

+ + + +

+++

+

19

SPO

F

CAH

-

+

+

20

PT

F

CAH

-

-

-

21

SPO

M

CAH

.

22

1VDU

M

CAH

-

-

23

SPO SPO

M M

CAH CAH+C

-

+

24

-

25

IVDU

M

CAH

.

26

PT

M

CAH+C

-

+

27

PT

M

CAH

.

28

PT

M

CAH

.

29

SPO

M

CAH+C

-

+

-

30

PT

M

CAH

-

-

-

31

IVDU

F

CAH

-

32

SPO

M

CAH+C

33

PT

M

CAH+C

34

IVDU

F

CAH

-

.

.

.

+ .

.

.

.

.

.

.

.

.

.

-

+ + + +

-

++

+++

+

-

_

_

+++

+++

-

+

-

+

+ + + +

+ + + +

+ + + +

+ + + +

+

-

+

+++

+++

+ + + +

+++

+

+

+++

+

+

+

++

+

+

-

+

+

++

+++

+++

+

+

+

++

+++

+ + + +

+ + + +

+

+

-

+

-

++

++

+++

+

+

+

-

+

+ + + +

+ + + +

+

-

-

-

_

-

-

-

+ + + +

+ + + +

+++

+++

+

+ +

CAH

+

+

-

CAH

-

+

-

Results

PT

active

were

considered

=

post-transfusional:

hepatitis: as positive

C

=

IVDU

=

intravenous

+++

-

drug

_

user.

cirrhosis.

when

+

+

M

chronic

.

-

M

sporadic;

+

+

IVDU

=

. .

SPO =

. .

35

bCAH

. .

36 J SPO

++ .

the serum

reacted

with

at least two

different

HCV

epitpopes.

+

187

HCV AND HBV IN NANB CHRONIC HEPATITIS TABLE 3

Five patients were positive for anti-HBs and anti-HBc and 7 for anti-HBs or anti-HBc only. Eleven patients showed both HCV and HBV serological markers: antiHBs and anti-HBc (5 pts.), isolated anti-HBs (2 pts.) or anti-HBc (4 pts.). Finally, only 1 of the 9 anti-HCVnegative subjects had anti-HBc compared to 11/27 of the anti-HCV-positive subjects who scored positive for anti-HBs and/or anti-HBc.

HBV serological and DNA results Anti-HBs and/or anti-HBc + 1~

+

I" HBV-DNA

-

11

23

12/36

24/36

"Also + for HCV-RNA detection.

Detection of HC V and HB V genomes HCV sequences were amplified by PCR with primers located in the 5' non-coding region. This region was used as a target for amplification due to its high sequence conservation (3,5-7,33). The PCR products of 259 bp were observed in agarose gel after BET staining (Fig. 1). The specificity of amplification was confirmed by hybridization with an oligonucleotide probe (Fig. 1). HCV-RNA sequences were detected in 27/36 of the samples tested including 5 anti-HCV-negative patients (Table 2). Among the 9 HCV-RNA-negative samples, 5 were anti-HCV-positive. The presence of HBV-DNA sequences was investigated by PCR using a set of primers located in the 3' end of the S-gene (Table 3). Two samples scored positive. It should be noted that one of these samples was negative for both anti-HBs

and anti-HBc. The 2 HBV-DNA-positive samples were also positive for HCV-RNA.

Discussion

This study confirms the extremely high prevalence of HCV infection in NANB chronic hepatitis in France. It also illustrates the efficiency of primers located in the 5' non-coding domain of HCV sequence, even though a single PCR test was used for HCV-RNA identification. Finally the possible co-infection of HCV and HBV in NANB hepatitis is also shown and thus may shed some light on their respective roles. In the present series, anti-HCV were found by RIBA2 assay in 75% (27/36) of patients with NANB chronic active hepatitis (CAH). Figures were identical for CAH related to transfusion (14/19), drug abuse (4/5) or sporadic (9/12) forms. This observation supports all reports from different geographic areas (I 1-14). As also expected, RIBA2 allowed an increase in sensitivity, due to the combination of several viral epitopes. HCV multiplication was demonstrated in most (27/36: 75%) of the antiHCV-positive individuals. It is important to note that

TABLE 2 HCV serological and RNA results Anti-HCV + HCV-RNA

+

22

5

5 27/36

4 9/36

RIBA

A

b.p.

2

M O

--

--

+

+

--

--

+

+

+

T--

T--

27

29

20

33

32

19

30

259.-

B 06

6 0 6 6

Fig. 1. Ethidium bromide staining (A) and blot hybridization (B) of HCV amplification. Patients' sera with NANB hepatitis (patients 27, 29, 20, 33, 32, 19, 30 in Table 1) and sera of two healthy donors (T-) were subjected to PCR amplification using primers located in the 5' non-coding region of HCV genome. Water control was also included in the experiment. The specific band of 259 bp was visualized by BET staining (A) in 1.5% agarose slab gel and/or after blot hybridization (B) with a specific oligonucleotide HCV probe. Results ot the RIBA2 test are indicated on the top. Mq~=Phi X174 Haelll molecular weight.

188 this sensitive PCR test was developed using a single pair of primers located in the 5' non-coding HCV region. This avoided repetition of experiments due to the use of combined sets of primers in the non-structural less conserved parts of the viral genome (24). It is also likely that the 'classical' nested PCR should be avoided for large-scale studies, due to the high risk of contamination. Further studies will now attempt to provide a semiquantitative analysis of HCV-RNA in order to compare HCV viraemia pattern and liver disease activity. Among the positive patients for serological and/or molecular tests (32/36), 68% (22/32) were found positive for both the detection of anti-HCV and HCV-RNA, 16% (5/32) and 16% (5/32) were found positive only for the detection of either anti-HCV antibodies or for HCVRNA. A discrepancy between serology and PCR results has been observed by other groups in patients with chronic or acute N A N B hepatitis (18-25). These results may be due to a low immune response to HCV antigens in some patients or to amino acid variability of viral epitopes. On the other hand they might also reflect the presence of minute amounts of viral RNA in the specimens or the persistence of HCV antibodies without HCV viraemia. HBV-DNA sequences were found in 2/36 HBsAgnegative patients. This result supports previous reports, where HBV-DNA was found in about 10% of the subjects with N A N B CAH (26). The lower prevalence observed in our study is probably due to the inclusion of a large number of post-transfusional N A N B CAH cases, cases which were previously shown to have the lowest rate of H B V - D N A positivity (27). Furthermore, in previous studies the prevalence of positive results for liver HBV-DNA was also higher than in serum (26,31) but this was not tested in this series. Serum H B V - D N A forms have been shown to be contained in infectious particles and their nucleotide sequence has been deter-

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Acknowledgements C.P. is a recipient of a fellowship from Diagnostic Pasteur, Centre National de Transfusion Sanguine and Pasteur Fondation. This work was supported by Diagnostic Pasteur, CNTS, Pasteur Fondation, INSERM, CEE, ARC, LNC, CNAM.

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Serum hepatitis C virus RNA and hepatitis B virus DNA in non-A, non-B post-transfusional and sporadic chronic hepatitis.

The sera of 36 French patients with post-transfusional and sporadic non-A, non-B chronic hepatitis were investigated, for HBV and HCV infections using...
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