ENROLMENT OF PATIENTS AND COMPLIANCE

The details of the overall design of the Dionysos study have already been published.18-20 Briefly, all the 10 151inhabitants between 12 and 65 years of age of two Northern Italian towns received a written invitation to participate in the study. Of these, 6917 were enrolled during the two years of the study. This number represents 69% of the eligible population—that is, a fraction adequate to validate this type of study.20

INITIAL EVALUATION

As described more completely elsewhere,18 the following were performed.

(1)

A medical history was taken including family history of hepatitis in cohabiting people, presence of gallstone disease, history of past surgical operations, blood transfusions before 1990, drug abuse, dental procedures, dog or other domestic animal bites, homosexuality, acupuncture. The use of drugs was also registered.

(2)

A semiquantitative colour illustrated food questionnaire21-23 was administered which included detailed questions on the use of alcoholic beverages (wine, beer, alcoholic aperitifs, hard liquor) and the dose, duration of use and the time of drinking. Daily alcohol intake (in g) was computed by multiplying the frequency of consumption of each unit of beverage by the alcohol content of the specified portions, according to standard procedures.24,25

(3)

A detailed physical examination was carried out aimed at detecting physical signs related to chronic liver disease.

(4)

A blood sample was taken for determination of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), mean cell volume, platelet, erythrocyte and leucocyte count, hepatitis B surface antigen, hepatitis B surface antibody (Abbott Diagnostic Kits, North Chicago, Illinois, USA) and hepatitis C virus antibody (anti-HCV) (enzyme linked immunosorbent assay (ELISA), third generation; Ortho Diagnostic Systems, Raritan, New Jersey, USA).

The diagnosis of cirrhosis was clinically suspected when at least two of the following features were present: (a) spider nevi, scleral icterus, palmar erythema, ascites, flapping tremor, hepatic or splenic enlargement; (b) platelet count less than 140 000/mm³; (c) portal vein diameter greater than 12 mm or irregular margins of the liver parenchyma at ultrasonography.

VIROLOGICAL STUDIES

In all patients the presence of anti-HCV antibodies was assayed by a second generation (four antigen) immunoenzymatic screening test (Ortho-HCV; Ortho Diagnostic Systems). This assay detects specific reactivity to four HCV antigens, including three non-structural (C100-3, 5-1-1, C-33c) and one structural (C22). In positive samples the presence of anti-HCV antibodies was confirmed by a third generation immunoenzymatic test (Ortho Diagnostic Systems) and by a confirmatory test (RIBA; Chiron Corp, Emeryville, California, USA). Sera showing two or more positive bands were considered “positive”, whereas those with only one band (usually C22) were defined as “indeterminate” and those without HCV antigen bands as “negative”.

HCV RNA DETECTION

The presence of HCV RNA in the serum was assessed by polymerase chain reaction (PCR) amplification of the 5’ untranslated region of HCV, which is considered to be the most conserved region of the virus. The test was performed in all subjects testing positive for the presence of anti-HCV antibodies (screening test). The amplification was performed in two steps, so called nested PCR.

After extraction, cDNA synthesis was performed with an antisense external primer according to standard methods.26

HCV RNA detection was performed in the serum samples collected at presentation. If viral RNA was found, no additional determination was carried out; if HCV RNA was undetectable, the test was repeated on fresh serum samples collected during follow up.

HCV GENOTYPING

The HCV genotype was determined by PCR amplification of the core region as described by Okamoto,26 ,27 and classified according to the international Simmonds nomenclature.28Each HCV type was characterised by a different nucleotide length: 49 for type 1a, 144 for type 1b, 174 for type 2a, and 123 for type 2b. When a double infection was suspected because of the presence of several bands, a second PCR was repeated separately with each of four type-specific antisense primers. Primers were also added to enable detection of type 3a HCV.27 ,29

FOLLOW UP OF ANTI-HCV POSITIVE SUBJECTS

Each participant initially positive for anti-HCV atibody (and confirmed to be positive by third generation ELISA and the RIBA test) and each subject for whom the diagnosis of cirrhosis was suspected clinically (see above) underwent the following additional procedures every six months for at least three years: (a) repetition of baseline blood tests plus, as well as serum alkaline phosphatase, bilirubin, albumin, γ-globulin, prothrombin time, and α-fetoprotein, blood assay of glucose, cholesterol, and triglycerides; (b) ultrasonography of the liver, biliary system, pancreas, and spleen with the measurement of portal vein and retropancreatic splenic vein diameters (all sonograms were performed by one operator).

All of the 162 HCV RNA positive subjects with either ALT more than 1.5 times the upper limit of normal in at least two after one month interval check (n = 55) or with clinically suspected cirrhosis according to the criteria reported above (n = 25) were asked to undergo percutaneous liver biopsy. Of these, 79 met these criteria, and 77 had biopsies. Two patients with ALT more than 1.5 times the upper limit of normal, but without clinically suspected cirrhosis, declined to have a biopsy.

STATISTICAL ANALYSIS

Statistical analysis was performed with an SPSS/PC statistical package (SPSS Inc, Chicago, Illinois, USA). All p values reported are two tailed. Statistical comparisons between means were made by one way analysis of variance, and, when the variances were not homogeneous, by the Kruskal-Wallis one way analysis of variance.30

At the univariate analysis within the general Dionysos population, the following covariates were considered as possibly related to HCV infection: age, sex, daily alcohol intake, history of hepatitis among the cohabiting people, past surgical procedure, past dental procedures, intravenous drug use, acupuncture, blood transfusion before 1990, animal bites, and homosexuality. The association between the presence of cirrhosis/HCC and different HCV genotypes, after correction for age, sex, and daily alcohol intake, was also considered both within the general Dionysos population and the HCV infected population.

The factors selected were judged eligible to enter the multivariate model on the basis of the following tests31: (a) significance test on Pearson correlation coefficient under the null hypothesis of ρ = 0; (b) two-sample ttest (analysis of variance) on mean values with correction for unequal variances when detected; (c) likelihood ratio χ² statistic for testing the significance of explanatory variables, one at a time, from a linear logistic regression model. A significance level lower than 0.05 was considered the criterion for entry into the initial multivariate model.

The multivariate analysis was carried out using a linear logistic regression model,32 with a stepwise variable selection procedure (level for entry = 0.1, level for stay = 0.5). The models explain either the probability of HCV infection according to the different exposure risk factors or the probability of having cirrhosis/HCC according to the different HCV genotypes among both the total Dionysos cohort and the HCV infected population followed up for three years. As coinfection with two or three HCV genotypes was always associated with genotype 1b, results for patients infected with multiple genotypes were pooled with those for patients infected with HCV genotype 1b in the multivariate analysis. Independently of the used selection criteria, age and sex factors were always considered in the multivariate analysis in order to improve model fitting. During analysis for the association between the presence of cirrhosis/HCC and the presence of different HCV genotypes, daily alcohol intake was also always forced into the model. Odds ratios (OR) and 95% confidence intervals were also calculated.32

PREVALENCE OF HCV GENOTYPE INFECTION ACCORDING TO AGE AND SEX

The anti-HCV screening test was positive in 226 cases (3.2%). RIBA gave indeterminate results in 20 cases (9%), negative in 27 (12%), and positive in the remaining 179 cases (79%). Viral RNA was found in 154 of 179 RIBA positive cases (86%) and in six RIBA “indeterminate” cases (30%). Interestingly, two of the 27 RIBA negative cases (7%) were found to be HCV RNA positive. HCV RNA was detected in the serum in 162 cases (2.3%). The prevalence (P) of anti-HCV and HCV RNA positivity increased progressively with age. As shown in table 1, the prevalence was low in the age range 12–25 years, and more than two thirds of the positive subjects were older than 45.

Of the 162 HCV RNA positive subjects (table 1 and fig 1), seven (4.3%) were type 1a (P = 0.1%), 68 (42%) type 1b (P = 1.0%), 39 (24%) type 2a (P = 0.6%), one (0.6%) type 2b (P = 0.01%), four (2.5%) type 2c (P = 0.06%), three (1.8%) type 3a (P = 0.04%), three (1.8%) were coinfected with two genotypes (P = 0.04%), and 11 (6.8%) with three genotypes (P = 0.16%). Coinfection was always associated with the presence of genotype 1b. In 26 cases the core amplification was positive, but multiple or unexpected bands were found in the agarose and polyacrylamide gels; as PCR amplification was performed three times with the same results, these cases were considered as “untypable” (P = 0.38%).

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Figure 1

Distribution of different hepatitis C virus (HCV) genotypes in the HCV RNA positive subjects of the Dionysos cohort (n = 162).

The HCV genotype varied according to age (table 1). The percentage of subjects infected with all the HCV genotypes, except for genotype 3a, was significantly higher over 45 years (p<0.002) than under. The percentage of “untypable” genotype was significantly (p<0.05) higher in the 56–65 age range than in the others (table 1). The mean age of patients infected by the various HCV genotypes does not differ significantly. The prevalence of both anti-HCV and HCV RNA positivity was greater in women than men (ratio of men to women = 0.7) in all the age ranges considered.

RISK FACTORS FOR HCV INFECTION IN THE DIONYSOS COHORT

Tables 2 and 3 give the risk factors for exposure to HCV that were significantly (p<0.05) associated with positivity for HCV RNA and for different HCV genotypes both in the univariate and multivariate analysis. The univariate analysis (table 2) identified the presence of chronic hepatitis among the cohabiting, both surgical and dental procedures, intravenous drug use, blood transfusions before 1990, and animal (mainly dog) bites as possibly related risk factors for the presence of HCV infection. When these factors were entered into the logistic multiple regression analysis (table 3), after correction for sex and age, only history of intravenous drug use, blood transfusions, previous hepatitis among the cohabiting, and animal bites were significantly (p<0.05) associated with the presence of HCV infection.

RISK FACTORS FOR CIRRHOSIS/HCC IN THE DIONYSOS COHORT

The diagnosis of liver cirrhosis and/or HCC was confirmed by liver biopsy in all 78 cases in which the diagnosis was suspected clinically. As reported elsewhere,18 ,19 the main causes of cirrhosis/HCC were HCV and alcohol abuse. Of the 162 HCV RNA positive subjects, 20 (12%) had cirrhosis and five (3%) had HCC (four with cirrhosis as well). Diagnosis of cirrhosis was made at the initial evaluation. Fourteen of the 20 patients with cirrhosis (70%) were infected with genotype 1b, three (15%) with genotype 2a, and the remaining three with genotype 1a, “untypable” genotype, and multiple genotypes (types 1b, 2a, and 2b). Two of the five patients with HCC, infected with genotype 1b, had that diagnosis made at the initial evaluation, while the other three (one without cirrhosis, infected with genotype 1b and 2a, the other two with cirrhosis, infected with an untypable genotype) developed HCC during the three years of follow up, with an incidence rate of two per 22 patients with HCV induced cirrhosis per three years, or 3% per year. All five patients with HCC were alcohol abusers (mean (SD) daily alcohol intake was 122 (46) g; p<0.01 v other patients with cirrhosis).

Of the remaining 52 HCV RNA positive patients who underwent liver biopsy during the three years of follow up, 18 had minimal inflammation (11%) and 34 (21%) had chronic active hepatitis.

Fifty six HCV RNA positive patients (34%) showed no abnormality of ALT, AST, or GGT, and 29 (18%) had only a slight increase in either ALT or GGT.

Finally, of the 64 subjects positive for anti-HCV but negative for HCV RNA, 53 (83%) showed no clinical, biochemical, or ultrasonographic alterations during the three years follow up. In the remaining 10 cases (15.5%) a transient mild alteration in either GGT or ALT occurred. Of these 10 patients, one was obese, two had gallstones, and seven were alcohol abusers (60 g ethanol a day). Only one of these HCV RNA negative patients met the criteria for biopsy. He was found to have cirrhosis, but he was an alcohol abuser ( >120 g ethanol a day).

In order to discriminate which is the most important factor correlated with the presence of cirrhosis and/or HCC between different HCV genotypes, age, sex, and daily alcohol intake in the Dionysos cohort, we performed a multiple regression logistic analysis. As reported in table 4, after correction for age, sex, and daily alcohol intake, the odds for a patient infected by both HCV genotype 1b and multiple genotypes of having cirrhosis and/or HCC was 31 times higher (range 16–59) than for those patients not infected by HCV (p<0.0001). The risk for a subject infected by all the other HCV genotypes of having cirrhosis and/or HCC was lower (OR = 11.2, range 4.4–28.6, p<0.0001) but still highly significant compared with HCV RNA negative subjects.

When the multivariate analysis was performed considering only the HCV RNA positive population—that is, 162 subjects—after correction for sex, age, and daily alcohol intake, the only HCV genotype significantly associated (p = 0.002) with the risk of having cirrhosis and/or HCC was genotype 1b, either alone or combined with other genotypes (OR = 4.0, range 1.6–9.8).

Similarly, the prevalence of cirrhosis and/or HCC in HCV RNA positive patients was significantly higher (p<0.01) in patients infected by genotype 1 than in patients infected by all the other genotypes. In detail, 72% (18/25) of the liver cirrhosis with or without HCC was associated with genotype 1b. Conversely, only 25% of patients infected with genotype 1b showed consistently normal levels of ALT or GGT during the three years of follow up.

Finally, by taking 30 g daily alcohol intake as the risk threshold for alcohol induced liver damage, as we previously showed in the same Dionysos cohort (21), 36 of the HCV RNA positive subjects (22%) could be considered as drinkers at risk, while the remaining 126 were not. Thirteen of the 126 HCV RNA positive subjects not at risk (10%) had cirrhosis or HCC, while 12 of the 37 drinkers at risk (32%) had cirrhosis (p<0.05). The risk of developing cirrhosis for HCV RNA positive drinkers was three times higher than that for drinkers not at risk (95% confidence interval = 1.2–7.4, p<0.01). Two of the 12 HCV RNA positive cirrhotic patients who drank more than 30 g alcohol a day (17%) were younger than 45 compared with only one of 13 cirrhotic non-drinkers (7.7%, p<0.05).