Nicolaos C. Tassopoulos

Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV.

Hepatitis E infection is associated with areas in which hepatitis E virus (HEV) infection is endemic. Acute infections in industrialized nations are usually linked to travel to endemic areas. Recently, an acute hepatitis infection in a patient from the United States (US), with no recent foreign travel history, was linked to a novel strain of HEV. Although a few additional cases have been reported from patients who have not traveled to endemic areas, the source of these infections has not been determined. The objective of this study was to identify additional HEV isolates from patients with acute infection who had no recent history of travel to areas where HEV is considered endemic, and to determine the genetic relationship between these and other HEV isolates. Viral RNA was isolated from serum and polymerase chain reaction (PCR) was performed using consensus primers based on a number of HEV isolates. HEV sequence in open reading frame (ORF) 1 and ORF2 was identified in three patients from nonendemic areas, one from Italy and two from Greece. Comparative and phylogenetic analyses were performed. The Greek and Italian isolates were significantly divergent from two isolates from the US and isolates identified previously from HEV‐endemic regions. The Italian isolate was distinct from the two Greek isolates. In addition, the two Greek isolates were significantly divergent from each other. Phylogenetic analysis indicated that the Italian and two Greek isolates represent three new genotypes of HEV, distinct from the Burmese, Mexican, and US genotypes. J. Med. Virol. 57:243–251, 1999.

Natural history of acute hepatitis B surface antigen-positive hepatitis in Greek adults

We prospectively followed up 821 adults with acute viral hepatitis hospitalized at the Athens Hospital for Infectious Diseases between May 1981 and May 1983. Radioimmunoassays for the detection of serologic markers of hepatitis A virus, hepatitis B virus, and hepatitis delta virus, and molecular hybridization techniques for the detection of serum hepatitis B virus deoxyribonucleic acid and hepatitis delta virus ribonucleic acid were used. Based on the results of an enzyme immunoassay for the detection of immunoglobulin M antibody to hepatitis B core antigen (Corzyme-M), 563 cases were diagnosed as acute hepatitis B and 45 as acute hepatitis superimposed on hepatitis B surface antigen carriage. Development of the hepatitis B surface antigen carrier state was observed in only 1 (0.2%) of the 507 cases with acute hepatitis B that were followed. In contrast, hepatitis B surface antigen persisted in all the latter cases. Acute hepatitis superimposed on hepatitis B surface antigen carriage was attributed to hepatitis A virus superinfection in 2 (4.4%), hepatitis delta virus superinfection in 22 (48.9%), reactivation of chronic type B hepatitis in 12 (26.7%), seroconversion from hepatitis B e antigen-positive to anti-hepatitis B e antibody-positive in 2 (4.4%), presumed superinfection by non-A, non-B agent(s) in 6 (13.4%), and the first clinical manifestation of chronic active hepatitis in 1 (2.2%) case. These data show that acute clinical hepatitis B in adults seems to be a self-limited disease and rarely leads to the development of the carrier state in this epidemiologic setting and hepatitis delta virus superinfection and spontaneous reactivation of chronic hepatitis B are the principal causes of acute hepatitis superimposed in hepatitis B surface antigen carriers in an area with a moderately high prevalence of hepatitis B virus infections.

Molecular epidemiology of hepatitis C virus (HCV) in Greece: temporal trends in HCV genotype-specific incidence and molecular characterization of genotype 4 isolates

Summary.  This study aimed to estimate the overall HCV genotype distribution and to reconstruct the HCV genotype‐specific incidence in Greece during the recent decades. It also focused at the identification of genotype 4 subtype variability in Greek isolates. A total of 1686 chronically infected HCV patients with detectable serum HCV RNA by RT‐PCR, belonging to different risk groups were studied. Amplified products from the 5′‐noncoding region were typed using a commercially available assay based on the reverse hybridization principle. The HCV genotype‐specific incidence was estimated using a previously described back calculation method. HCV genotype 1 was the most prevalent (46.9%) followed by genotype 3 (28.1%), 4 (13.2%), 2 (6.9%) and 5 (0.4%). A high prevalence of genotype 1 (66.3%) in haemophilia patients was recorded whereas HCV genotype 3 was found mainly among patients infected by I.V. drug use (58.2%). Data on the temporal patterns of HCV genotype‐specific incidence in Greece revealed a moderate increase (1.3–1.6 times) for genotypes 1 and 4, and a decrease (1.5 times) for genotype 2 from 1970 to 1990, whereas there was a sharp (13‐fold) increase for genotype 3. The molecular characterization of 41 genotype 4 HCV isolates belonging to various risk groups revealed that, subtype 4a was the most frequently detected (78%). Phylogenetic comparison of the Greek 4a isolates with all HCV‐4a isolates reported worldwide so far revealed a topology which does not discriminate Greek isolates from the others. HCV‐4 does not represent a recent introduction in Greece.

Development and assessment of a novel real-time PCR assay for quantitation of HBV DNA

HBV DNA quantitation is used extensively for the monitoring of treatment of hepatitis B virus (HBV) infection. The aim of this study was to develop a highly sensitive and reproducible real-time PCR (RTD-PCR) assay for the quantitation of HBV DNA using the LightCycler system. The performance of this assay was assessed by analyzing serial dilutions of HBV genomic DNA of known concentration and the lower limit of detection was found to be 1 DNA copy/reaction. By using serial dilutions of plasmid standard, RTD-PCR was determined to quantify HBV DNA in a 10-log10 dynamic range. RTD-PCR was found to be more sensitive than the commercially available tests such as the Quantiplex HBV DNA and the AMPLICOR HBV MONITOR assays. The median coefficient of variation of interexperimental variability was 3.2%. The HBV DNA values obtained with RTD-PCR were highly correlated with assays available commercially. These findings suggest that our RTD-PCR assay combines high sensitivity and reproducibility for HBV DNA quantitation in an incomparable high dynamic range of quantitation.

Investigation of SEN Virus Infection in Patients with Cryptogenic Acute Liver Failure, Hepatitis-Associated Aplastic Anemia, or Acute and Chronic Non–A–E Hepatitis

SEN virus (SENV) has been tentatively linked to transfusion-associated non-A-E hepatitis. We investigated SENVs role in unexplained hepatitis in other settings. Polymerase chain reaction amplification was used to detect 2 SENV variants (SENV-D and SENV-H) in 1706 patients and control subjects. SENV was detected in 54 (22%) of 248 patients with acute or chronic non-A-E hepatitis, 9 (35%) of 26 patients with hepatitis-associated aplastic anemia, and 0 of 17 patients with cryptogenic acute liver failure, compared with 150 (24%) of 621 control subjects with liver disease and 76 (10%) of 794 healthy control subjects. When controlling for geographic region, the prevalence of SENV among case and control subjects was not significantly different. The severity of acute or chronic hepatitis A, B, or C was not influenced by coexisting SENV infection. No etiological role for SENV in the cause of cryptogenic hepatitis could be demonstrated.

Hepatitis C virus 1b is the dominant genotype in HCV‐related carcinogenesis: A case‐control study

In an ongoing case‐control study in Athens on the etiology of hepatocellular carcinoma (HCC), an analysis was made in order to assess whether HCV genotype 1b is associated with hepatocellular carcinoma (HCC). The HCV genotype was determined in 17 HCC patients, 87 patients with chronic hepatitis C (CHC) without cirrhosis (NC‐CHC) and 23 patients with CHC and cirrhosis (C‐CHC). HCV genotype 1b was detected in 14/17, 16/23 and 23/87 of HCC, C‐CHC and NC‐CHC respectively, The age‐ and gender‐adjusted odds ratios contrasting HCC with NC‐CHC and C‐CHC with NC‐CHC were 8.3 and 3.8 respectively. These data strongly support the hypothesis that HCV 1b is a stronger liver carcinogen than other HCV genotypes, probably through increased HCV replication and enhanced liver cytopathicity.

Reconstructing and predicting the hepatitis C virus epidemic in Greece: increasing trends of cirrhosis and hepatocellular carcinoma despite the decline in incidence of HCV infection

Summary.  In this study, a comprehensive methodology for modelling the hepatitis C virus (HCV) epidemic is proposed to predict the future disease burden and assess whether the recent decline in the incidence of HCV may affect the future occurrence of cirrhosis and hepatocellular carcinoma (HCC) cases. Using the prevalence of HCV, the distribution of chronic hepatitis C (CHC) patients within the various transmission groups and their infection‐onset times, it was possible to reconstruct the incident infections per year in the past that progressed to CHC in Greece. The natural history of the disease was simulated in subcohorts of newly infected subjects using transition probabilities derived either empirically between fibrosis stages 0–4 or from literature review. Annual estimates of the incidence and prevalence of CHC by fibrosis stage, HCC and mortality in Greece were obtained up to 2030. HCV incidence peaked in the late 1980s at five new infections/10 000 person‐years. Under the assumption of 20–100% decline in HCV incidence after 1990, the cumulative number of incident cirrhosis and HCC cases from 2002–2030 was projected to be lower by 9.6–48.2% and 5.9–29.5%, respectively, than that estimated under the assumption of no decline. However, the prevalent cirrhotic/HCC cases and HCV‐related deaths are predicted to decline in the next 30 years only under the assumption of complete elimination of new HCV infections after 1990. Despite the progress in the reduction of HCV transmission, primary prevention does not seem adequate to reverse the rise in the incidence of cirrhosis and HCC.

Fulminant hepatitis due to Epstein-Barr virus infection

Epstein-Barr virus infection is a benign disease, which may occasionally be fatal, particularly in children. Epstein-Barr virus infection is rare in elderly subjects and appears to have a self-limited course. An unusual case of fulminant hepatitis due to primary Epstein-Barr virus infection in a 62-year-old male 18 days after a cardiosurgical operation and blood transfusions is described in the present paper. Post-mortem examination of the liver showed massive hepatic necrosis. The etiology was established by increase in IgM antibodies to Epstein-Barr virus (titer 1:3.120) in serum and by cellular expression of Epstein-Barr virus DNA in liver tissue.

Future trends of HCV-related cirrhosis and hepatocellular carcinoma under the currently available treatments

Summary.  The epidemic of hepatitis C virus (HCV) infection is a major public health issue. We conducted a comprehensive analysis to estimate future HCV‐related morbidity and mortality, using a model which is the first to take into account currently available treatments. We reconstructed the incident infections per year in the past that progressed to chronic hepatitis C (CHC) in Greece. Then, the natural history of the disease was simulated in subcohorts of newly infected subjects in the presence or absence of treatment using yearly estimates of the number of treated patients obtained from national databases. Annual estimates of the incidence and prevalence of CHC by fibrosis stage, hepatocellular carcinoma (HCC) and mortality were obtained up to 2030. The current proportion of naïve CHC patients receiving treatment in Greece is 1.2% per year. Treatment of 1.2–10% of naïve CHC patients per year would reduce the cumulative number of incident cirrhosis and HCC cases from 2002 to 2030 by 10.8–39.4% and 12.8–39.8%, respectively and decrease the number of prevalent cirrhosis and HCC cases in 2030 by approximately 17–48% compared with the number estimated under the assumption of no treatment. Approximately 17 cirrhosis cases or six HCC cases or 10 premature deaths would be prevented for every 100 treated patients. However, the prevalent cirrhotic/HCC cases because of HCV and HCV‐related deaths would not plateau until 2030. Despite the introduction of effective treatment, HCV‐related morbidity and mortality will likely increase during the next 20–30 years in Greece. Intensive primary prevention efforts coupled with increased access to the currently available treatments are necessary to control the chronic consequences of HCV epidemic.

Recombinant interferon-alpha therapy for acute hepatitis B: a randomized, double-blind, placebo-controlled trial.

Summary. In spite of the availability of hepatitis B vaccine, acute hepatitis B continues to be a worldwide problem for which no specific therapy is available. We investigated the safety and the effectiveness of recombinant interferon‐α2b (rIFN‐α2b) in the treatment of acute hepatitis B by determining overall severity and duration of symptoms, time required to clear viral antigens and hepatitis B virus (HBV) DNA, and titre of antibodies to hepatitis B surface antigen (HBsAb), 24 weeks after the onset of therapy. One hundred patients were randomly assigned to treatment with either 3 million units (MU) (n= 34) or 10 MU (n= 33) rIFN‐α2b or to placebo (n= 33), three times weekly for 3 weeks. Follow‐up was for 24 weeks. A significantly shorter duration of the symptoms and signs of acute hepatitis was observed in patients who received 3 MU rIFN‐α2b compared with those who received 10 MU rIFN‐α2b or placebo. Twenty‐one weeks post‐therapy, patients treated with 10MU rIFN‐α2b showed a significantly higher geometric mean HBsAb titre than those treated with placebo (85.1 vs 35.5 IU1‐1, P < 0.05), rIFN‐α2b administration was well tolerated even in jaundiced patients. No serious side‐effects were observed necessitating reduction in dose or discontinuation of the drug. The effect of rIFN‐α2b on transition of HBV infection to chronicity could not be evaluated in this trial because such an unfavourable course was not seen in any of the treated or the control patients. In conclusion, rIFN‐α2b was safe in acute hepatitis B, and at low dose was found to ameliorate symptoms and to shorten significantly the duration of illness.