The Impact of Diet on Liver Fibrosis and on Response to Interferon Therapy in Patients With HCV-Related Chronic Hepatitis

Carmela Loguercio, MD; Alessandro Federico, MD; Mario Masarone, MD; Roberto Torella, MD; Marcello Persico, MD


Am J Gastroenterol. 2008;103(12):3159-3166. 

In This Article

Patients and Methods


Informed written consent was obtained from each subject and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee. We prospectively enrolled patients affected by HCV from Southern Italy referred to our Department between 2002 and 2005. Excluded from the study were patients with chronic HBV and HIV infection, decompensated cirrhotics, and patients with such other clinically relevant associated diseases as decompensated diabetes, kidney diseases, pulmonary diseases, collagen diseases, tumors. A total of 1,084 patients with HCV-related chronic hepatitis were enrolled in the study. Of these, 408 patients had well compensated Child A cirrhosis and 432 were naive patients (including chronic hepatitis and Child A cirrhosis) who had undergone antiviral therapy. HCV genotypes were collected before starting antiviral treatment. As a control group, we studied 2,326 apparently healthy blood donors who were negative for HCV antibody and hepatitis B surface antigen (HBsAg), and had normal liver tests.

The 1,084 patients with chronic liver disease underwent a liver biopsy, which was scored according to Ishak et al..[22] Steatosis was graded as absent (0), mild (<33%, 1), moderate (>33% and ≤66%, 2), or severe (>66%, 3).[23] The subgroup of patients, in whom interferon (IFN) therapy was indicated, received pegylated-IFN + ribavirin and were treated for 48 (in genotype 1-4 patients) or 24 wk (in genotype 2-3 patients) according to current guidelines.[24] The virological response to therapy was assessed by HCV-RNA measurements at baseline, at 12 and 48 wk of treatment, and 24 wk after treatment ended. Based on qualitative HCV-RNA results, the patients were defined either sustained virological responders (SVRs) (no detectable HCV-RNA after 24/48 wk of treatment and 6 months thereafter) or non-responders (NRs) (viral breakthrough, and virological nonresponse, with persistence of HCV-RNA at the end of treatment). Treatment was discontinued in virological nonresponders at week 12 of therapy, based on a positive quantitative HCV-RNA test.[25]


Patients and controls with a BMI between 25 kg/m2 (for men) and 24 kg/m2 (for women) and 29.9 kg/m2 were considered overweight (determined as kg/m2, and those with a BMI ≥30 kg/m2 were considered obese. Plasma levels of blood glucose, nitrogen, creatinine, cholesterol, triglycerides after 12 h of fasting (routine kits) were measured, and patients and controls underwent routine liver tests. Dietary habits were recorded for all enrolled subjects. For this purpose, we used the Winfood Software 2.0 package (Medimatica s. r. l., Martinsicuro, Italy) program, which has previously been used to assess alimentary history.[26,27] On the basis of the quantities and qualities of foods consumed, the program elaborates the energy intake and the percentage of macronutrients and micronutrients, and calculates the elements in each food. Proteins are reported as animal and vegetal proteins. Carbohydrates are divided in soluble and amide, lipids in saturated, polyunsaturated, monounsaturated fatty acids, and cholesterol. The complete elaboration of intakes shows the list of diet components, the ratio among components and calories, and the subdivision in breakfast, lunch, and dinner. We recorded the food intake of a complete week, including working days and the weekend. The dietary examination was randomly repeated in a large number of subjects (202 patients and 334 controls, equally distributed in men and women) to evaluate variability. Data collected were similar in all interviews (Concordance: k = 0, 8). The data were compared with the tables of food consumption and recommended dietary intakes of the Italian National Institute of Nutrition and Food Composition Database in Italy.[28,29]

Alcohol use was evaluated with a standardized precodified questionnaire (complete AUDIT test).[30] We considered as constant a continuous daily alcohol intake in the last 3 yr at least. The quantity of daily alcohol intake was calculated based on a drink that corresponds to about 12 g of pure ethanol.[31]

RNA Preparation and HCV RNA Determination

All steps were carried out under RNase-free conditions. The polymerase chain reaction (PCR) procedure was used to determine HCV RNA. Sera were rapidly (within 30 min of blood drawing) frozen at -20ºC. RNA was extracted according to Chomczynsky and Sacchi,[32] and c-DNA was derived. To identify HCV-RNA, a nested PCR was performed using primers that expanded the highly conserved 5' noncoding genomic region. Carryover PCR contamination was avoided by applying the measures suggested by Kwok and Higuchi.[33]

Hcv Genotyping

To classify the HCV genotypes, serum PCR products were hybridized to type- and subtype-specific probes 1a, 1b, 2a, 2b, and 3a. The probes had to fulfill two main criteria: no more than two mismatches compared with the corresponding published sequences of the same subtype, and they had to differ by three or more mismatches compared with published sequences of other types and subtypes. The only exception is probe 2b, which had only two mismatches compared with the corresponding sequence of type 3a.[34]


Data were considered significant at P < 0.051. Continuous normally distributed variables were summarized as mean ± standard deviation (SD) and categorical variables as frequency and percentage. Variance analysis (ANOVA) was used to evaluate the differences among means and the c2 test for the differences of percentages among groups. The degree of association between continuous, normally distributed variables was assessed by Pearson's correlation test. Spearman rank correlations were also used to assess the significance of associations between ordinal or continuous predictor variables and liver histology (inflammation, steatosis, fibrosis). Univariate logistic regression was used to quantify the association among all clinical and laboratory variables and the histological findings or the response to therapy. Using the univariate predictors as input variables, multiple logistic regression, performed with forward stepwise selection of variables, identified the independent predictor of liver steatosis, grading, and staging as well as response to therapy. The adjusted odds ratios (OR) for the association of liver histology and response to therapy with each category of alimentary history were calculated by multiple logistic regression analysis.[35] In the model, liver histology (inflammation, steatosis or fibrosis) and the response to treatment were outcome variables, whereas the amount of daily food intake, expressed both as total calories and as single components, as well as alcohol intake, age, BMI, HCV genotypes and viremia, and metabolic parameters were the independent variables. For OR estimates, the condition of absence or of normality of each parameter was the reference category (OR = 1). Data handling and analyses were performed with the Statistical Package for Social Sciences (SPSS 13.0; SPSS Inc., Chicago, IL).


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