Hepatitis C virus (HCV) infection and liver-related mortality: a population-based cohort study in southern Italy

Alberto R Osellaa, Giovanni Misciagnaa, Vito M Guerraa, Marisa Chiloirob, Renato Cupponec, Aldo Cavallinid, Alfredo Di Leod and the Association for the Study of Liver Diseases in Puglia,e

Laboratories of Epidemiology and Biostatistics,
a Experimental Pathophysiology
b and Biochemistry;
d Department of Medicine,
c Medical Research Institute IRCCS ‘Saverio De Bellis’ Castellana Grotte, Italy.

Reprint requests to: Alberto R Osella, Laboratory of Epidemiology and Biostatistics, Via F Valente 4, 70013 Castellana G (BA), Italy. E-mail: osellar{at}libero.it


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background Hepatitis C virus (HCV) is a common cause of chronic liver diseases but the degree to which these diseases contribute to liver-related mortality is not well established. The aim of this study was to estimate the absolute and relative effects of HCV infection on liver-related mortality.

Methods A population random sample of 2472 subjects aged >=30 years was enrolled and followed up from 1985 to 1996. At enrolment, a structured interview and a clinical evaluation were performed. Serum samples were tested using HCV ELISA and RIBA HCV. Outcomes were overall and liver-related mortality and tracing procedures included review of office and hospital records, death certificates, and interviews with general practitioners, attending hospital and next of kin. Statistical analysis was performed using Poisson and binomial prospective data regression.

Results Crude overall and liver-related mortality rates were 7.66 (95% CI : 6.68–8.79) and 0.9 (95% CI : 0.3–2.2) per 103 person-years, respectively. For HCV infection effect, incidence rate ratio and difference (per 103 person-year), risk ratio and difference were 27.5 (95% CI : 6.5–115.6), 4 (95% CI : 3–7), 33.1 (95% CI : 7.8– 139.3) and 0.06 (95% CI : 0.04–0.08), respectively; all measures were adjusted for age at death, sex and daily alcohol intake.

Conclusions The results show a strong relative but weak absolute effect of HCV infection on liver-related mortality in the 10-year period considered. Poisson and binomial models are virtually equivalent, but the choice of the summarizing measure of effect may have a different impact on health policy.

Keywords Binomial models, cohort study, HCV infection, liver-related mortality, Poisson models

Accepted 17 March 2000


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Hepatitis C virus (HCV) is a common cause of chronic liver disease.1 Chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) are well established sequellae of HCV infection, but the degree to which these diseases contribute to the liver-related mortality rate is highly variable worldwide.211

In different studies liver-related mortality rates ranging from <1% to 11.7% have been reported.8,9 Most of these studies were conducted in tertiary care centres and used different designs to assess the impact of HCV on mortality. Estimates of the HCV effect may therefore be biased as a result of a form of selection bias operating toward the less favourable prognostic segment of the HCV-related liver disease spectrum. Two carefully controlled studies by Seef et al.4,11 showed a small statistical significant difference in liver-related deaths between post-transfusional hepatitis C patients and controls after 18 years of follow-up and, that a proportion of them had mild disease. In these studies, most patients who died from liver-related disease were identified as heavy drinkers.

On the other hand, in a few population-based studies, albeit with strong geographical variability, a high prevalence of HCV infection has been shown in older subjects; the great majority being viraemic and without clinically obvious disease.12,13 As previously reported,14 HCV infection is highly prevalent in Castellana G (small town in southern Italy) with more than 40% of subjects aged >=50 years being anti-HCV positive. The longitudinal phase of this study showed a moderate incidence rate of HCV infection. However, the impact of HCV infection on mortality in this population remains unknown.

Antiviral therapy15 is now available and certain responders' characteristics have also been identified. However, as antiviral therapy has significant side effects, estimation of the magnitude of liver-related death associated with HCV exposure outside the clinical setting is an issue of public health concern, as is identification of groups of patients at risk who could benefit from therapy.

Analysis of epidemiological data is generally based on multiplicative models which are useful in aetiological research, but others such as additive ones are more useful from the point of view of public health.1618

To evaluate the potential impact of different models on health policy, we conducted a prospective, population-based study. The aim of this study was to estimate the absolute and relative effects of HCV infection on liver-related mortality rate and risk.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The cohort
Details of the overall design of this population-based cohort study have been published elsewhere.14,19 Briefly, in 1985 a systematic random sample of 3500 subjects (2000 men and 1500 women) aged >=30 years was drawn from the electoral register of Castellana G (17 334 residents at the 1981 Census); 30% worked in the agricultural sector. In 1985–1986, 2472 (1429 men and 1043 women) of 3500 subjects agreed to take part in the study (70.6% response rate). A total of 1028 people did not respond: 127 had changed their address or migrated, 36 had died, and 865 refused to take part. There was no difference in age, sex or job between responders and non-responders. A structured standard interview was conducted to collect information about socio-demographic characteristics, health status, personal history and selected behavioural variables and a validated questionnaire probing dietary habits was administered.20 A clinical evaluation of each participant was also performed. A blood sample was taken and deep-frozen at –90°C until testing. In 1992–1993, 2158 of 2472 enrolled subjects (87.2% response rate) were followed-up and the same procedures repeated for all subjects. The time of observation from date of enrolment to death, migration or end of study, whichever occurred first, was recorded for each subject.

Exposure assessment
Serum samples were tested using the HCV ELISA test system (Ortho Diagnostic System, Milan, Italy). The cutoff values for HCV ELISA test ranged from 0.632 to 0.672 nm and a serum sample was considered reactive if its optical density value was twice the cutoff value. All reactive serum were confirmed using RIBA HCV 2.0 SIA (Ortho Diagnostic System, Milan, Italy). Serum HCV RNA was tested by nested RT-PCR using conserved primers in the 5'- non-coding region.21 The HCV genotyping was performed by type-specific primers in the core region according to Okamoto's method and subsequent modifications.21,22

At enrolment, HCV antibody (anti-HCV) serological status was established only in 1969 of 2472 respondents because the serum samples of the 503 remaining subjects had been used in a previous study on the association between endogenous sex hormones and cholesterol gallstones.23 In all 417 of the 503 enrolled subjects whose serum samples were not available (not included in the HCV cohort) were followed-up from 1992 to 1993. To check possible selection bias we established the anti-HCV prevalence in these subjects. It was similar to that of the subjects included in the cohort. Furthermore, the anti-HCV test was repeated for 1699 available paired serum samples. We obtained an overall concordance of 99.5%.

Tracing procedures and outcome assessment
The follow-up of the enrolled subjects ended on 31 December 1996 and their vital status on that date was obtained from the town's Registry. Two outcomes were considered: overall and liver-related mortality (International Classification of Diseases, Ninth Revision, [ICD-9] codes 155.0 and 571). Outcome was assessed by the following means (per cent of dead subjects): (1) interviews with the general practitioner (100%) and attending hospital (11.7%); (2) review of office (22.0%) and hospital records (78%); (3) review of medical history at enrolment (100%); (4) death certificates (81% of anti-HCV positive subjects, 76% of anti-HCV negative and 80% of those with unknown status) and (5) interviews with the next of kin (10%).

Clinical data were evaluated for the presence of cirrhosis and HCC on the basis of established clinical and histological criteria.2426 Subjects with a history of liver disease who did not fulfil the criteria for cirrhosis or HCC were considered to have chronic liver disease on the basis of the presence of biopsy-proven chronic hepatitis27 or when, along with abnormal liver function tests, at least one of the following was present: (1) thrombocytopenia; (2) ultrasonographic or endoscopic signs of portal hypertension; or (3) ascites and either liver or spleen enlargement. Subjects who did not meet any of the above criteria were considered to have no evidence of chronic liver disease. The cause of death for each subject was established after careful evaluation of all data collected, performed always by a single clinician who specialised in hepatology and was unaware of the HCV status of the dead subjects.

Statistical analysis
Differences among continuous variables were evaluated by one-way analysis of variance (ANOVA); multiple comparisons were performed with Scheffeé's method. Categorical variables were analysed by {chi}2 test. Exploratory analysis did not reveal differences between subjects whose sera were not available and the other groups and so further analysis considered only anti-HCV positive and negative subjects.

Absolute and relative effects of HCV exposure on mortality were estimated for two types of measures: rates (incidence rates) and risks (incidence proportion) along with their 95% CI. The general framework of the Poisson regression model for grouped cohort data was used to estimate the incidence rate ratio (IRR) and difference (IRD);28,29 an algorithm was used to allocate person-time exactly.30 Risk ratio (RR) and difference (RD) were estimated as suggested for prospective binomial data.18 In these models, the data were categorized into strata of the covariates (confounding factors) for each of the risk groups. The strata correspond to age at death (<45, 45–54,55–64 and >=65 years), sex (male, female) and daily alcohol intake (<31, >=31 g/ethanol). Daily alcohol intake was dichotomized taking into account the regional and national drinking pattern.31,32 From previous analysis,14 it was known that HCV infection was homogeneously distributed in terms of socioeconomic characteristics. The main variability in anti-HCV prevalence was due to age and sex.

Agreement between observed and expected cases, Pearson {chi}2 and deviance statistic were used to evaluate the model's goodness-of-fit. In all analysis, any other cause of death was assumed to operate independently of the cause of death under study.

Computations for rates were performed using the AMFIT33 module in the statistical package Epicure 2.1 whereas the generalized linear interactive modelling (GLIM)34 was used for estimating risks.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Details of the study base at enrolment are shown in Table 1Go. Mean age was significantly higher in anti-HCV positive subjects whereas daily alcohol intake was more or less equally distributed among groups.


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Table 1 Characteristics of the study base at enrolment. Castellana G (Italy), 1985–1986
 
As of 31 December 1996, cumulative follow-up was 26 623.2 person-years, with a mean (±SD) of 10.8 (1.8). At the end of the study, vital status was assessed for 503 of 511 HCV exposed subjects (98.4 %), 1435 of 1458 unexposed (98.4%), and 490 of 503 (97.4%) subjects with unknown exposure status; the remaining 44 untraced subjects contributed 199 person-years of observation until they migrated. Crude mortality rates from all causes and liver disease-related were 7.66 (95% CI : 6.68–8.79) and 0.9 (95% CI : 0.3–2.2) per 103 person-years, respectively. Cohort mortality details are shown in Table 2Go. There were 75 deaths among HCV-positive subjects: 30 (40%) due to liver-related disease (14 HCC, 16 cirrhosis), 21 (28%) from cardiovascular diseases, 14 (18.7%) from non-hepatic neoplastic disease and 10 (13.3%) due to miscellaneous causes (4 pulmonary diseases, 2 renal diseases, 4 alia). No anti-HCV positive dead subject had ever received interferon therapy. Among HCV-negative subjects, two died of liver-related diseases (one HCC, one cirrhosis), whereas there were five liver-related deaths among subjects with unknown HCV status (all cirrhosis).


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Table 2 Cohort's mortality characteristic. Castellana G (Italy), 1985–1996
 
Hepatitis C virus RNA was detected in 56.0% (42/75) of HCV positive dead subjects. The results of genotyping were as follows: 1b 43% (18/42), 2a 50% (21/42), other groups 7% (3/43).

The summarizing measures of effects for Poisson models (IRR and IRD) and prospective binomial data (RR and RD), along with the expected and observed number of cases, the deviance associated with each model, and Pearson's {chi}2 are reported in Tables 3 and 4GoGo, respectively. The four models appeared to be roughly equivalent, especially for ratio measures. However, the performance of risk models was better, as reflected by a higher agreement between observed and fitted cases, a smaller deviance, and thus a better fit (Table 4Go). The relative measures of effect obtained were 27.5 (95% CI 6.5–115.6) for IRR and 33.1 (95% CI : 7.8–139.3) for RR. The IRD was 4 (95% CI : 3–7) per 103 person-years whereas the RD estimate, in the 10-year period considered, was 0.06 (95% CI : 0.04–0.08).


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Table 3 Incidence rates: relative and absolute effect of hepatitis C virus (HCV) infection exposure on liver disease-related mortality. Castellana G (Italy), 1985–1996
 

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Table 4 Incidence proportion: relative and absolute effect of hepatitis C virus (HCV) infection exposure on liver disease-related mortality. Castellana G (Italy), 1985–1996
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
These results show a strong relative effect (IRR = 27.5, 95% CI : 6.5–115.6; RR = 33.1, 95% CI : 7.8–139.3) and a weak absolute effect (IRD = 0.004, 95% CI : 0.003–0.007; RD = 0.06, 95% CI : 0.04–0.08) of HCV infection on liver-related mortality in the 10-year period considered. Two models (Poisson and binomial prospective) were fitted and two measures (relative and absolute) of the effect of HCV infection on liver disease-related mortality were estimated. The results suggest that the Poisson and binomial models are virtually equivalent, but the choice of the type of summarizing measure of effect may have a different impact on health policy.

Competing risks of death related to both HCV infection and other unmeasured exposures, such as Hepatitis B virus (HBV) infection, may have produced biased measures of effect in this study. This implies that subpopulations particularly susceptible to liver-related death due to HCV infection could have been selectively censored from our cohort by other causes of death.35 However, most of the liver-unrelated deaths were due to cardiovascular and non-hepatic neoplastic diseases; besides, none of the subjects who died from renal disease had been diagnosed as having membranoproliferative glomerolunephritis.36 In order to control for confounding due to alcohol consumption, a validated questionnaire was administered. In this geographical area the drinking pattern is characterized by a steady daily intake of wine and drinking is common at meals as it is considered part of the normal diet. Alcohol abuse is not a public health concern. Within Italy, the proportion of mortality from liver cirrhosis associated with alcohol consumption has been reported to be low in this area31 and chronic viral infections are responsible for most cases of cirrhosis.37 It has recently been suggested that, at the population level,32 the risk of developing alcohol induced liver damage is mediated not only by the amount of alcohol drunk, but also by drinking alcohol and multiple different alcoholic beverages outside mealtimes. Other unmeasured confounding factors may also have biased our results, but, since this was a large population random sample, it should have been well able to prevent confounding by unmeasured factors.38

Several studies reporting mortality rates among HCV infected patients have been published. Although these studies were remarkably different in design, characteristics, number of patients and length of follow-up, liver-related mortality rates ranged from 0.8% to 11.7%.210 Several parameters, such as age at infection, gender, genotype, virus load and route of infection have been identified as having a major influence on the natural history of HCV infection,39 with patients infected at an older age being more likely to develop progressive disease. Besides, it has been shown that patients with mild liver disease are the most likely to respond to antiviral therapy.15 However, care should be taken in generalizing results from clinical and epidemiological studies conducted at tertiary care centres.40 As previously reported, HCV infection is highly prevalent in this area of Southern Italy14 with a strong negative cohort effect and a moderate HCV infection incidence rate. HCV types 1b and 2 a/c are the most frequent and are distributed across the population with no particular pattern.41

Since HCV infection is widespread in this area, the comparison of different measures of HCV exposure effect on the mortality rate raises issues concerning public health and patient management. Our results show a strong relative effect of HCV exposure (IRR and RR) on liver-related mortality; this leads to consideration of HCV exposure as an important component cause in at least one complete causal mechanism of liver-related death.42 On the other hand, absolute measures of effect (IRD and RD) were virtually equivalent and showed a weak effect. Subjects in this population-based cohort probably became HCV infected early in life and, taking into account time elapsed from infection to liver disease, HCV exposure may reasonably be lagged for a longer period than 10 years. Consequently, absolute measures should reflect the actual impact of HCV exposure effect on liver-related mortality from the public health point of view in this population. Indeed, the IRD was only 4 x 103 person-years and RD 6% in 10 years and age at death was no different between HCV exposed and unexposed dead subjects. In this sense, a more precise knowledge of the biological mechanisms is needed to establish the effect of HCV exposure on the incidence time of liver-related death and, consequently, to obtain valid estimates of both excess and aetiological fractions attributable to exposure.43

The availability of interferon and antiviral therapies may have a great impact on the course of HCV infection. Results from clinical trials15 have shown that HCV infected young patients with mild disease become virological responders. These seem to be the characteristics of most subjects in this area where HCV infection is highly prevalent.12,13 Thus, from the public health viewpoint, it is important to identify patients at risk of developing progressive disease and the effectiveness, risks, benefits and cost of treatment should also be carefully evaluated. Besides, HCV infection seems to play an important role in decreasing other risk factors for chronic diseases.44,45

Liver disease due to HCV infection requires clinical and epidemiological approaches to find out the causes of cases and determinants of incidence at the population level.46 This means that different causal pathways should be assessed in order to implement prevention strategies for those at high risk and strategies aimed at the population should fight the underlying causes of incidence. Other impact measures such as reduction in life expectancy43 in the meantime may be useful as a means of estimating the impact of HCV infection on mortality, for public health purposes.


    Acknowledgments
 
The authors thank A Bolognini, A Beatrice, A Farina, R Pascale, GP Pinque, and MR Sabbatelli, M Bianco and P Longo (Medical Research Institute IRCCS ‘Saverio De Bellis’, Castellana Grotte, Italy), for their contribution to data collection and technical help. In addition, we thank the Municipality of Castellana Grotte, and G Silvestri, M Veneziani and M Colonna, Azienda Socio-Sanitaria BA/5 for their kind co-operation.


    Notes
 
e The following members of the Association for the Study of Liver Diseasesin Puglia were active participants: D Bonelli, C Campanella, A Carpinelli,A Cortese, G Di Munno, A Elia, M Guzzoni Iudice, S Lomuscio, A Mazzarisi, V Pagliarulo, N Perrone, E Piccigallo, S Simone, L Stagno, D Taccone,N Tauro and D Valentini. Back


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 Methods
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 Discussion
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