C. Brechot

Hepatitis B virus DNA in mononuclear blood cells: A frequent event in hepatitis B surface antigen-positive and -negative patients with acute and chronic liver disease

We have investigated 38 hepatitis B surface antigen (HBsAg)-positive and 34-negative patients with acute and chronic liver disease for the presence of hepatitis B virus (HBV) DNA in peripheral mononuclear blood cells. Among the HBsAg-positive subjects HBV DNA was detected in the mononuclear cells of asymptomatic HBV carriers (2/6), patients with acute hepatitis (8/8), chronic active hepatitis (18/21), and with hepatocellular carcinoma (2/3); the viral DNA sequences were also identified in the mononuclear cells of patients with HBsAg-negative acute hepatitis (2/3), chronic active hepatitis (5/15) and hepatocellular carcinoma (5/16), some of these showing no evidence of HBV by conventional serological markers. By contrast HBV DNA was not detected after resolution of the acute viral infection. For 7 patients different mononuclear cell-enriched subpopulations were assayed and the viral DNA was observed in T lymphocytes (both OKT4+ and OKT8+ enriched subsets) and/or in B enriched lymphocytes; the restriction DNA patterns showed in some patients a genetic organisation of the viral DNA similar to those observed in the liver (including free monomeric and oligomeric HBV DNA and results consistent with integrated viral sequences); however, no HBV DNA replicative forms were detected. These results show that the hepatitis B virus infection of mononuclear blood cells (including lymphoid cells) is a frequent event at all stages of the viral infection which might be related to immunological abnormalities observed in HBV carriers; in addition the mononuclear blood cells analysis may provide an insight to the liver cells status.

Hepatitis C viremia and anti-HCV antibodies in alcoholics.

We determined serum hepatitis C status using a RIBA2 kit and a sensitive PCR procedure in 62 chronic alcoholics, 36 of whom had anti-HCV antibodies (Ab) detectable in an ELISA1 assay. Anti-HCV antibodies were detected in 22 patients using RIBA2. HCV RNA was detected by means of PCR in 18 patients who were RIBA2 positive and in none who were RIBA2 negative. Liver biopsies, available for 12 HCV RNA-positive patients, revealed histological features of purely alcohol-related lesions in seven and mixed alcohol-viral lesions in five. These results indicate that HCV replication is maintained in most alcoholics who score positive for anti-HCV Ab in the RIBA2 test, and that HCV viremia can be associated with histological features typical of alcoholic liver disease.

Hepatitis B virus multiplication in the absence of usual serological markers: A study of 146 chronic alcoholics

The possible role of HBV infection in the progression of alcoholic liver disease remains debated. However, serum HBV markers in alcoholics, although present with a high frequency, mainly consist of anti-HBs and/or anti-HBc antibodies. In order to detect an HBV multiplication that could be missed by the usual markers, we looked for HBV-DNA in the serum of 146 chronic alcoholics; the results were compared with those of the usual serological HBV markers. Sixty-eight of the 146 patients could be studied for HBV-DNA both in the liver and the serum. The 146 alcoholics were divided in 48 with normal liver function (group I); 67 with non-cirrhotic alcoholic liver disease (group II); 31 with alcoholic cirrhosis (group III). Among the 146 patients, 17 had a viral multiplication reflected by serum positive HBV-DNA, as against none of 100 healthy controls (P less than 0.01). Six of the 17 had a normal liver function (6/48 = 12.5%), 7 were of group II (7/67 = 10.4%) and 4 had cirrhosis (4/31 = 12.9%). Serum HBV-DNA was associated with HBsAg in 3 occasions; in addition serum HBV-DNA was also present in 5 HBsAg-negative patients with anti-HBc and/or anti-HBs and even in 9 without any usual HBV marker. The overall prevalence of HBV markers in the 146 patients went from 30.8% to 37.0% when serum HBV-DNA was taken into account; it was similar in the 3 groups studied. Eight patients, of the 68 studied, were liver HBV-DNA-positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis B vaccination in chronic alcoholics

Given the possible role of hepatitis B virus in the occurrence of hepatocellular carcinoma and the high prevalence of HBV infection in alcoholics, we attempted to prevent HBV infection in alcoholic patients with or without cirrhosis. Among 32 cirrhosis, 20 received three injections of hepatitis B vaccine at monthly intervals and 12 had a 4th injection one month later. Effectiveness was evaluated on the anti-HBs titer at the 6th month; it did not differ between the 3- and 4-injection groups. Two patients were good responders (anti-HBs greater than 300 mU/ml), 14 had a low response (m-30 mU/ml at the peak) and 16 (50%) had no detectable anti-HBs. All alcoholics without cirrhosis were given 4 injections; all had detectable anti-HBs but their mean antibody response was 97.7 +/- 4 SD. No obvious statistical difference in the response to the vaccine was noted on the basis of sex or age, or on the discontinuation or not of alcohol intake. Deficient antibody response to vaccines has not previously been demonstrated in patients with cirrhosis or in alcoholics. It remains to be determined whether this response is specific of HBV and how it could be related to the role of HBV in the occurrence of liver alterations in alcoholics.

Monoclonal anti‐HBs antibodies radioimmunoassay and serum HBV‐DNA hybridization as diagnostic tools of HBV infection: relative prevalence among HBsAgnegative alcoholics, patients with chronic hepatitis or hepatocellular carcinomas and blood donors

Abstract. Anti‐HBs monoclonal antibodies radioimmunoassay (m‐RIA) and HBV‐DNA hybridization techniques were used to detect HBs antigen (HBsAg)—associated determinants (evidence of HBV on‐going infection) and HBV‐DNA sequences (evidence of viral multiplication) in the serum samples of 479 patients who were HBsAg negative by standard solid‐phase radioimmunoassay. They included 128 alcoholics, 104 patients with chronic hepatitis, fifty‐four with an hepatocellular carcinoma, 100 with coagulation disorders and ninety‐three blood donors. The aim of this study was the comparison in these populations of the prevalence of the various HBV markers. m‐RIA detected HBsAg‐associated determinants in 1% of blood donors, 3% of coagulation disorders, 3·1% of the alcoholics, 21·1% of chronic hepatitis and 16·6% of hepatocellular carcinoma; hybridization identified HBV‐DNA sequences in 0·9%, 2·2%, 10·9%, 9·6% and 5·5% of these cases, respectively. The combined prevalence of both markers of an on‐going HBV infection (with or without viral multiplication) was 14·16%, 26·9% and 22·2% in the latter groups, respectively, as compared with only 3% in patients with coagulation disorders and 2·1% of blood donors. These results confirm the frequency of HBV or HBV‐related virus infection in alcoholics, in chronic hepatitis and hepatocellular carcinomas, despite the absence of HBsAg by standard RIA (or even of any other usual marker); this gives further evidence for variations in the expression of HBV infection. A high and quite similar prevalence of usual serum markers and hybridization results was observed in the alcoholics and in the patients with chronic hepatitis. By contrast, in the former there was an about seven‐fold lower positivity of m‐RIA, in which alcohol consumption could play a role by impairing HBsAg secretion.

Non-radioactive hepatitis B virus DNA probe for detection of HBV-DNA in serum

A diagnostic test has been developed to detect hepatitis B virus (HBV) DNA in human sera. This test involves a dot-blot technique in which non-radioactive nucleic acid labelled with 2-acetylaminofluorene (AAF) is used as probe. Two series of human sera from 228 blood donors and 113 HBsAg chronic carriers were tested by hybridization with the same DNA probe labelled either with AAF or with 32P. A correlation between the techniques was observed for 328 sera (96%), and using the non-radioactive test it was possible to detect 56 (86%) of the 65 HBV-DNA-positive patients. A comparative study of the HBeAg/anti-HBe status and the presence of HBV-DNA was carried out on the sera from 113 HBsAg chronic carriers, 65 of which were positive for HBeAg and 29 of which were positive for anti-HBe antibodies. With the AAF test, 44 of the HBeAg-positive sera were positive, while 5 of the anti-HBe-positive sera were positive. This study shows that, although this non-radioactive test is slightly less sensitive than the radioactive hybridization assay (RHA), it can be used for a survey of HBV carriers. Dissociation of the viral multiplication and the HBe/anti-HBe status was identified with the AAF test as well as with the RHA. It would therefore appear that the AAF test described here may be used for the extensive survey of HBV multiplication.

Influence of human immunodeficiency virus infection on hepatitis δ virus superinfection in chronic HBsAg carriers

SUMMARY. It is generally agreed that hepatitis B virus (HBV) replication is reduced by hepatitis δ virus infection (HDV) and augmented by human immunodeficiency virus (HIV) infection. However, the precise nature of the interactions between HBV. HDV and HIV is controversial. The aim of this study was to evaluate the impact of HIV infection on HBV and HDV replication, and on histological scores during δ virus super‐infection in HDV‐positive. chronic carriers of hepatitis B surface antigen (HBsAg). We studied 38 men and six women, 15 of whom were HIV‐positive and all of whom had at least one marker of HDV infection. Serum hepatitis B e antigen (HBeAg), HBV DNA, HDV RNA, anti‐δ antigen antibodies (anti‐HD) IgM, anti‐HD IgG and hepatitis δ antigen (HDAg) were tested for in the serum and liver, respectively; anti‐hepatitis C virus (HCV) antibodies were detected using a second‐generation recombinant immunoblot assay. Histological specimens were scored blindly according to Knodells classification for periportal and intralobular necrosis, portal inflammation and fibrosis. HBV DNA was detected more frequently in the HIV‐positive patients than in those who were HIV‐negative (25 vs 0% P = 0.01), while markers of HDV replication (serum anti‐HD IgM. serum HDV RNA and liver HDAg) were as frequent in the HIV‐positive patients (69%, 40% and 50% respectively) as in those who were HIV‐negative (75%, 52% and 30%. respectively; P>0.05). By contrast, 31% of the HIV‐positive patients were serum HDAg‐positive compared to only 6% of the HIV‐negative patients (P = 0.001). HDV antigenaemia and anti‐HD antibodies usually fluctuated in the HIV‐positive patients during follow‐up. The mean Knodell score was similar in the HIV‐positive (11.5 ± 3.2) and HIV‐negative (10.7 ± 2) subgroups, as was the mean semi‐quantitative index of hepatic necrosis, inflammation and fibrosis. Our results provide evidence that in HDV‐positive patients: (1) HIV infection counters the inhibitory effect of HDV superinfection on HBV replication; (2) serum anti‐HD IgM, HDV RNA and liver HDAg are not more frequent in HIV‐positive than in HIV‐negative patients, suggesting that HIV infection has no effect on HDV replication (although the significance of the increased frequency of HD antigenaemia remains unclear): (3) the histological severity of liver disease is not influenced by HIV status.

Hepatitis B defective virus with rearrangements in the preS gene during HBV chronic infection

We have found a defective form of hepatitis B in an anti-HBe positive chronic HBsAg carrier with liver cancer (HCC). Viral deletions were identified in the preS coding region using PCR and sequencing. The presence of deleted HBV forms was observed in serum, PBMC, and liver tissue. To conclude, rearrangements and deletions in the preS gene would potentially lead to an impairment in viral clearance without affecting viral penetration in liver cells, possibly accounting for chronic HBV infection.

Hepatitis G virus infection in hepatitis C virus‐positive patients co‐infected or not with hepatitis B virus and/or human immunodeficiency virus

This was a retrospective study to evaluate the prevalence and impact of hepatitis G virus (HGV) infection in hepatitis C virus (HCV)‐positive drug addicts, according to the serological status of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) co‐infection. Two hundred and thirty‐five randomly selected intravenous drug addicted patients (147 French, 88 Italian) were studied. All patients were positive for antibodies to HCV (anti‐HCV). HGV RNA positivity was measured by reverse transcriptase–polymerase chain reaction (RT–PCR). Comparisons of HCV RNA positivity rate, and biological and histopathological variables, were made between HGV RNA‐positive and negative patients, according to their HBV and HIV status. HGV prevalence was around 30% in both French and Italian groups. No clear association between HGV infection and a particular HCV genotype was observed. The rate of HCV RNA positivity did not differ between HGV‐positive and HGV‐negative patients after stratification for hepatitis B surface antigen (HBsAg) and HIV positivity. Histological severity of the underlying chronic hepatitis did not differ according to the HGV status; however, in HIV‐positive HBsAg‐negative patients, the hepatitis activity was moderately increased in HGV‐positive patients. A striking negative influence of HBsAg positivity on HCV replication was observed in HIV‐negative patients: an HCV RNA‐positive rate of 25% was found in HBsAg‐positive patients vs 86% in HBsAg‐negative patients; similar significant results were observed in HIV‐positive patients, although to a lesser extent. The underlying chronic hepatitis was significantly more severe in HBsAg‐positive than in HBsAg‐negative HIV‐negative patients. Hence, HGV infection is highly prevalent in anti‐HCV positive drug addicts but the co‐infection with HCV does not seem to influence HCV replication nor to worsen the underlying chronic hepatitis, in HIV‐negative patients at least. Reciprocal influence between HBV, HCV and HIV appears rather complex, HBsAg carriage seeming to exert per se a negative effect on HCV replication, particularly in HIV‐negative patients, suggesting that interactions between hepatitis viruses should always be analysed in the light of HIV status.

Clinical significance of the polymerase chain reaction (PCR) assay in chronic HBV carriers.

PCR was evaluated as a clinical tool for use in accurate identification of the specific etiologic agent in chronic HBV carriers. The method was found to be valuable in diagnosis and for monitoring therapy, as well as for elucidation of genotypic variants of HBV in chronic HBV cases. By this means an HBV defective variant with alterations in the preS1/preS2 sequence was detected and is consequently described here.