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

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.

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.

The effect of adherence to therapy on sustained response in daily or three times a week interferon alpha-2b plus ribavirin treatment of naive and nonresponder chronic hepatitis C patients.

The aim was to demonstrate adherence to treatment has been suggested to enhance rates of sustained response in patients with hepatitis C. In this study, we evaluated the effect of drug dosage reduction or the duration of the expected therapy in patients treated with interferon (IFN)-alpha2b plus ribavirin. Virologic response rates were re-analysed according to compliance to therapy in (i) 301 naive and (ii) 142 nonresponders to previous IFN therapy treated with either IFN 5 MU TIW for 8 weeks followed by IFN 3 MU TIW for 40 weeks plus ribavirin or IFN 3 MU QD for 16 weeks followed by IFN 3 MU TIW for 24 weeks plus ribavirin. Patients were separated into those who adhered to > or =80% of their intended treatment schedule (dose of both drugs and duration) and those who did not. Compliance to treatment resulted in significantly higher response rates in both groups of patients: 43.93% compared with 6.90% of noncompliant naive patients and 30.77% compared with 10.53% of nonresponder patients. Compliance to treatment was found to have a similar effect when the results were analysed according to HCV genotype. Our findings suggest that compliance to treatment for > or =80% of the intended treatment schedule results in significantly higher sustained response rates in both naive and nonresponder patients. Consequently, every effort should be made to improve patient adherence to therapy.

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.

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.

Multiple viral/self immunological cross-reactivity in liver kidney microsomal antibody positive hepatitis C virus infected patients is associated with the possession of HLA B51.

Liver Kidney Microsomal autoantibody type 1(LKM1) directed to cytochrome P4502D6 (CYP2D6) characterises autoimmune hepatitis type-2 (AIH-2), but is also found in a proportion of chronic hepatitis C virus (HCV) infected patients, CYP2D6252-271 being a major B- cell autoepitope. Molecular mimicry and immunological cross-reactivity between CYP2D6252-271, HCV polyprotein and the infected cell protein 4 (ICP4) of herpes simplex virus type 1 (HSV-1) have been suggested as triggers for the induction of LKM1, but reactivity and cross-reactivity to the relevant sequences have not been investigated experimentally. CYP2D6252-271 and its viral homologues were constructed and tested by ELISA in the sera of 46 chronically infected HCV patients, 23 of whom were LKM1 positive. Reactivity to the E1 HCV and ICP4 HSV1 mimics was frequently found in HCV infected patients irrespectively of their LKM1 status; viral/self cross-reactivity (as indicated by inhibition studies), however, was present in the only 2 of the 23 LKM1 seropositive HCV patients, who possessed the HLA allotype B51. Our results indicate that in HCV infected patients virus/self cross-reactivity is dependent on a specific immunogenetic background, a finding awaiting confirmation by studies in larger series of patients.

A randomized trial to assess the efficacy of interferon-alpha daily in combination with ribavirin in the treatment of naïve patients with chronic hepatitis C.

A randomized trial was conducted to assess the efficacy of interferon-alpha (IFN) daily in combination with ribavirin in 301 naïve patients with chronic hepatitis C (CHC). Patients were randomized to receive ribavirin 1.2 g daily (QD) for 48 weeks with either IFN 5 MU (thrice weekly) TIW for 8 weeks followed by IFN 3 MU TIW for 40 weeks (IFN TIW, n = 154) or IFN 5 MU QD for 8 weeks followed by IFN 3 MU QD for 16 weeks followed by IFN 3 MU TIW for 24 weeks (IFN QD, n = 147). Treatment discontinuation rates, because of adverse events, were similar in the two arms (14.9% in IFN TIW and 14.3% in IFN QD, P = 0.87). The proportion of patients with sustained virological response (SVR) was 27.9% for patients treated TIW and 38.8% for those treated QD (P = 0.046). According to logistic regression analysis, patients in the IFN QD arm had 1.7 times higher probability of achieving SVR, than those receiving IFN TIW (P = 0.038). Low baseline viral load (P = 0.017) and genotype non-1 (P = 0.036) were associated with higher SVR rates. Combination of IFN/ribavirin for 48 weeks is more effective when IFN is administered daily for the first 24 weeks in naïve patients with CHC.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in health care workers (HCWs): guidelines for prevention of transmission of HBV and HCV from HCW to patients.

The transmission of viral hepatitis from health care workers (HCW) to patients is of worldwide concern. Since the introduction of serologic testing in the 1970s there have been over 45 reports of hepatitis B virus (HBV) transmission from HCW to patients, which have resulted in more than 400 infected patients. In addition there are six published reports of transmissions of hepatitis C virus (HCV) from HCW to patients resulting in the infection of 14 patients. Additional HCV cases are known of in the US and UK, but unpublished. At present the guidelines for preventing HCW to patient transmission of viral hepatitis vary greatly between countries. It was our aim to reach a Europe-wide consensus on this issue. In order to do this, experts in blood-borne infection, from 16 countries, were questioned on their national protocols. The replies given by participating countries formed the basis of a discussion document. This paper was then discussed at a meeting with each of the participating countries in order to reach a Europe-wide consensus on the identification of infected HCWs, protection of susceptible HCWs, management and treatment options for the infected HCW. The results of that process are discussed and recommendations formed. The guidelines produced aim to reduce the risk of transmission from infected HCWs to patients. The document is designed to complement existing guidelines or form the basis for the development of new guidelines. This guidance is applicable to all HCWs who perform EPP, whether newly appointed or already in post.

A randomized trial to assess the efficacy of interferon alpha in combination with ribavirin in the treatment of interferon alpha nonresponders with chronic hepatitis C: superior efficacy of high daily dosage of interferon alpha in genotype 1.

A randomized trial was conducted to assess the efficacy of daily (QD) or thrice weekly (TIW) administration of interferon-alpha (IFN) in high doses in combination with ribavirin (1.0-1.2 g/day) in patients with chronic hepatitis C (CHC) who were nonresponders to previous IFN monotherapy. Interferon was administered as 10 MU IFN (QD or TIW) for 4 weeks, followed by 5 MU IFN (QD or TIW) for 20 weeks, and then by 3 MU IFN (QD or TIW) for 24 weeks. Sustained virological response (SVR) was evaluated in 142 patients who received at least one dose of medication. One-fourth of the patients achieved SVR, 26% of those treated with IFN QD and 25% of those treated with IFN TIW (P = 0.85). For genotype 1 patients, SVR rates were 32.4 and 15.8% for IFN QD and IFN TIW, respectively, whereas for genotype non-1 patients the corresponding SVR rates were 20.6 and 36.4%, respectively (test of homogeneity: P = 0.031). This finding was further confirmed by multivariate logistic regression analysis where a statistically significant interaction (P = 0.012) was found between treatment and HCV genotype indicating that the IFN QD regimen was superior to IFN TIW among genotype 1 patients whereas, among genotype non-1 patients, the two treatments were similar (odds ratio of SVR in IFN QD vs IFN TIW: 3.33 among genotype 1 patients, 95% CI: 1.00-11.14). In conclusion, re-treatment of patients not responding to previous IFN monotherapy with a combination of high daily dose of IFN with ribavirin may be beneficial for genotype 1 infected patients.

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.

Objective assessment of the contribution of each diagnostic test and of the ordering sequence in jaundice caused by pancreatobiliary carcinoma.

BACKGROUND: Computer-assisted diagnostic systems are not substantially more accurate than the clinician in the differential diagnosis of jaundice but may help in optimal selection and sequencing of tests. The present study aimed to assess with an electronic diagnostic tool the pattern of ordering tests and the diagnostic contribution and related financial cost of each test in jaundiced patients with pancreatobiliary carcinoma, in an effort to make the clinician's diagnostic behaviour more efficient and economical. METHODS: Clinical and diagnostic test data were prospectively gathered from 356 jaundiced patients with pancreatobiliary carcinoma and entered in a Bayesian diagnostic programme. The test results were added to the existing diagnostic evidence, and the programme calculated the diagnostic contribution of each test. RESULTS: A total of 1804 diagnostic tests were ordered. Quantitative assessment of the diagnostic contribution of each test showed that percutaneous transhepatic cholangiography and computed tomography were associated with the highest provision of information. The most cost-effective tests were ultrasonography and liver function tests. CONCLUSIONS: It is possible to determine objectively the diagnostic contribution of each test in establishing the diagnosis of pancreatobiliary carcinoma. The observed physician behaviour in ordering the various diagnostic tests might be improved with regard to its efficacy and its cost-effectiveness profile.

The contribution of telemedicine to cardiology.

The Telemedicine Centre at Sismanoglion Hospital of Athens is connected to seven primary-care units (HCCs) on the mainland and six HCCs on Aegean islands. Telemedicine activity from 1992 to 1995 was reviewed. During the study period, the data relating to 1947 cardiac patients were transmitted through the telemedicine network: 681 (35%) of the patients presented with cardiological problems and 333 (17%) of them had an urgent cardiac event. The telemedicine network brought a number of benefits, including better access to cardiology specialists, improved decisions about patient transportation, a reduction in isolation and continuing professional education.

Autoimmunity to a liver membrane lipoprotein and liver damage in alcoholic liver disease.

Antibodies reacting with a liver membrane lipoprotein (LSP) have been detected by radioimmunoassay in the sera of 15 (27%) of 55 patients with alcohol-related liver lesions. There was a close association between the presence of the anti-LSP antibody and the findings on liver biopsy of a lymphocytic infiltrate in the portal tracts together with piecemeal necrosis of periportal hepatocytes. These histological features are characteristically found in the autoimmune disorder of chronic active hepatitis, in which anti-LSP antibodies are almost invariably present. It is suggested that in these cases of alcoholic liver disease there is loss of tolerance, and continued production of anti-LSP could promote periportal inflammation and accelerate the progression to cirrhosis. In the cases of acute alcoholic hepatitis without periportal inflammation studied, anti-LSP was not detected demonstrating that production of this autoantibody is not simply secondary to liver damage.

Failure to obtain an autoimmune response following cryosurgery to the normal rat liver.

Smooth muscle antibody (SMA) and anti-liver-specific lipoprotein (anti-LSP) responses were investigated following five different freeze thaw regimes to the normal rat liver. The livers were examined histologically for evidence of autoimmune liver disease. No SMA or anti-LSP was found in any animal and on histological examination the unfrozen part of all livers was normal. It is concluded that cryosurgical damage to the liver is unlikely to provoke an autoimmune response.

Antibodies to a human liver membrane lipoprotein (LSP) in primary biliary cirrhosis.

Antibodies reacting with a human liver-specific membrane lipoprotein (LSP) have been detected using a sensitive and specific radioimmunoassay in 19 (51%) of 37 patients with primary biliary cirrhosis. The anti-LSP antibodies were found only in the later stages of the disease as judged by histological criteria, being present in 73% of those in stage IV, 44% of those in stage III, and none of those in stage I or II. Although there was no relationship between percentage binding and standard liver function tests, there was a close correlation between percentage binding of 125I-LSP by serum and the extent of piecemeal necrosis of periportal hepatocytes on liver biopsy. The timing of the anti-LSP response makes it very unlikely that it is involved in the pathogenesis of the early bile duct damage but the results of this and other studies suggest that antibodies to this hepatocyte membrane lipoprotein may be an important cause of periportal liver cell necrosis in both primary biliary cirrhosis and chronic active hepatitis and could be one of the factors determining progression to cirrhosis in both these conditions.

Leukocyte migration inhibition in response to biliary antigens in primary biliary cirrhosis, sclerosing cholangitis, and other chronic liver diseases.

The leukocyte migration inhibition test has been used to investigate cellular immune responses to antigens in a protein fraction (BPF) of normal human gallbladder bile in patients with a variety of intra- and extrahepatic diseases. Inhibition of leukocyte migration in the presence of BPF was observed in 30 (81%) of 37 patients with PBC, in 8 (80%) of 10 patients with sclerosing cholangitis, and in 7 (26%) of 27 patients with chronic active hepatitis. Only 1 of 31 patients with other liver diseases or with uncomplicated ulcerative colitis showed a similar response to BPF. The BPF was found to contain three antigens which were distinct from plasma proteins. Immunofluorescence studies revealed that one of these antigens appears to be derived from that part of the hepatocellular membrane which forms the bile canaliculus and that a second appears to be associated with the epithelial cell membranes of interlobular and septal bile ducts. The site of origin of the third antigen could not be established. It is suggested that cellular immune responses to biliary antigens could be involved in the progressive bile duct destruction of chronic biliary disease.

The Budd-Chiari syndrome in pregnancy.

A case of Budd-Chiari syndrome in a young woman, which started probably in the last trimester of pregnancy, is described. The diagnosis was made clinically and was confirmed by inferior venacavography and on exploratory laparotomy. The possible connection of the syndrome with the pregnancy is discussed.

Sensitisation to halothane-altered liver components in severe hepatic necrosis after halothane anaesthesia.

In-vitro sensitisation (inhibiton or stimulation of leucocyte migration) in response to a liver homogenate obtained from rabbits pretreated with halothane was found in eight of twelve patients with halothane-associated hepatitis. Sensitisation was not observed when the homogenates were obtained from animals pretreated with ether. Furthermore, leucocyte migration in response to "halothane homogenate" was normal in eleven patients who had shown no abnormality in liver function after halothane anaesthesia and in thirty patients with other liver diseases. These studies provide direct evidence that sensitisation to halothane-altered liver-cell components is present in those occasional patients in whom severe liver damage develops after halothane anaesthesia.

The significance of stimulation in the leucocyte migration test as an indicator of cellular hypersensitivity--studies using Tamm-Horsfall glycoprotein as antigen.

To determine whether stimulation of leucocyte migration in the presence of an antigen is a reliable indicator of sensitization, the results of leucocyte migration using a kidney antigen (Tamm-Horsfall glycoprotein) were compared with those of lymphocyte transformation using the same antigen and with the lymphocyte cytotoxicity for cells known to synthesize this antigen. There was a close correlation between the results of all three tests and these findings strongly suggest that the stimulation of leucocyte migration as an immunological phenomenon was as valid as inhibition in demonstrating sensitization.