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Hepatitis C virus genotype distribution in Egyptian diabetic patients: A preliminary study
Arab Journal of Gastroenterology 14 (2013) 14–19
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Original Article
Hepatitis C virus genotype distribution in Egyptian diabetic patients: A preliminary study Omayma Saleh a,⇑, Azza A. Baiomy b, Ayman El-desouky a, Hosam Zaghloul b, Mohammad El-Arman b, Gamal M. Dahab c, Mohamed S. Abdel-Rahman d a
Internal Medicine Department, Faculty of Medicine, Mansoura University, Mansoura City, Egypt Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura City, Egypt Clinical Pharmacology Department, Faculty of Medicine, Mansoura University, Mansoura City, Egypt d Pharmacology/Physiology Department, New Jersey Medical School, UMDNJ, Newark, NJ, USA b c
a r t i c l e
i n f o
Article history: Received 12 November 2011 Accepted 29 December 2012
Keywords: Hepatitis C virus HCV genotypes Insulin resistance Egypt Diabetes
a b s t r a c t Background and study aims: There is controversy regarding whether a specific hepatitis C virus (HCV) genotype is associated with diabetes mellitus. This study aimed to investigate HCV genotype distribution in diabetics and its relation to some clinical and laboratory variables in HCV-positive diabetic versus nondiabetic Egyptians in East Delta. Patients and methods: The study included 100 HCV-positive patients of which 66 were diabetic in addition to 35 healthy adults as a control group. Clinical assessment, laboratory measurements of plasma glucose, insulin, C-peptide, C-reactive protein (CRP), tumour necrosis factor-a (TNF-a) and liver functions (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT)) as well as HCV genotype determination were done, and AST/platelet ratio index (APRI) and Homoeostasis Model of Assessment-Insulin Resistance (HOMA-IR) were calculated. Results: The main results were the presence of HCV genotype 3, in 31.8% of the diabetic group and in 26.5% of the non-diabetic group, while the remainder of cases had genotype 4, the predominant genotype in Egypt. This is the first report of the presence of HCV genotype 3 in about 30% of an Egyptian cohort. However, there was no significant difference in genotype distribution between both groups. Further, there were significantly higher values of HOMA-IR, insulin and C-peptide in HCV-positive groups in comparison to the control group, while TNF-a was significantly higher in the HCV-positive diabetic group. However, there were no significant differences between both genotypes regarding these parameters. Conclusion: Although this study reveals for the first time the presence of HCV genotype 3 in a significant percentage of a group of Egyptian patients, where the majority were diabetic, the association between diabetes and certain HCV genotypes could not be confirmed on the basis of our findings. Hence, taking into consideration the impact of such a finding on the treatment decisions of those patients, further studies are warranted to explore these findings to a greater extent. Ó 2013 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.
Introduction Hepatitis C virus (HCV) infection is a disease with a significant global impact and an important cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. According to a World Health Organization (WHO) report, more than 3% of the world population are infected. There are considerable regional differences, where the prevalence is as high as 20% in some countries. In Africa and western Pacific, the prevalence is higher than North America and Europe [1–3]. ⇑ Corresponding author. Address: Internal Medicine Department, Endocrinology and Diabetes Unit, Specialized Medical Hospital, Mansoura University, Mansoura City, Egypt. Tel.: +20 1119951003. E-mail address: [email protected] (O. Saleh).
In Egypt, HCV is the most common aetiology of chronic liver disease, where the prevalence of antibodies to HCV is 10-fold higher than in the United States and Europe [4,5]. Among the six major HCV genotypes found worldwide, genotype 4 is the most predominant genotype in Egypt, which was thought to have more than 90% preponderance [6–9]. The geographical distribution of HCV genotypes may be useful for epidemiological purposes; however, besides its epidemiological use, HCV genotype determination is relevant both as a predictor of response to therapy as well as to decide the length of time required for antiviral treatment, being less in the case of genotypes 2 and 3 [10]. There are recent reports about a change in HCV genotype distribution among certain areas in Europe and Africa, which were suggested to be attributed to changes in the mode of transmission
1687-1979/$ - see front matter Ó 2013 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ajg.2013.01.005
O. Saleh et al. / Arab Journal of Gastroenterology 14 (2013) 14–19
and immigration status in these areas [19]. Furthermore, several studies have shown that HCV infection is associated with modest but significantly increased risk of developing type 2 diabetes mellitus (DM) [10–17]. Therefore, in the light of the international epidemic of type 2 DM and the increasing incidence of HCV infection, the association of HCV and DM is potentially a major health problem [20]. The controversy whether a specific HCV genotype is diabetogenic is still going on. Further, studies of the relation of HCV genotype and other variables are limited and there is a paucity of recent data [21]. Hence, given the importance of HCV genotype determination regarding its prognostic significance and its impact on the treatment decisions and cost of therapy, the aim of this study was to determine the HCV genotype status and its association with selected clinical and laboratory variables related to either diabetes or HCV in a group of HCV-positive diabetic versus non-diabetic Egyptians. Further, whether diabetes might have an association with a certain HCV genotype would be another objective. Patients and methods Patients Inclusion criteria The patients included in the study were recruited from the outpatient diabetes and gastroenterology clinics at the Specialized Medical Hospital of Mansoura University, in Egypt. This cross-sectional comparative study included 100 HCV-positive patients (66 diabetic and 34 non-diabetic patients), in addition to 35 healthy subjects of matched age and gender selected as a control group. Exclusion criteria Patients were excluded from this study if they had the following: advanced cirrhosis showing manifestations of liver cell failure; a history of other liver disease including hepatitis B virus infection; previous anti-HCV therapy; having hepatocellular carcinoma; associated kidney or cardiac disease; pregnancy; infections; smoking; and corticosteroid treatment. These factors can affect DM status or may precipitate or aggravate insulin resistance and might affect some laboratory measurements particularly tumour necrosis factor (TNF-a). Methods 1. The included patients and controls were subjected to complete clinical assessment and laboratory and radiological investigations. Informed consent was taken from each patient before enrolment in the study, according to the ethics committee guidelines. 2. Anthropometric measurements including weight, height and waist circumference were taken and body mass index (BMI) was calculated. BMI more than 25 kg m 2 is considered overweight while BMI P 30 kg m–2 is considered obese. 3. High-resolution ultrasonographic analysis of the liver surface was performed. 4. The following laboratory measurements were taken: Routine laboratory tests including fasting glucose and liver function tests including serum albumin, bilirubin, aspartic transaminase (AST), alanine transaminase (ALT) and gamma glutaryl transpeptidase (SGT) were assayed by commercially available kits supplied by Human, Wiesbaden, Germany. Fasting serum insulin assay was performed by enzyme-amplified sensitivity immunoassay supplied by Biosource Technologies, Fleunes, Belgium.
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Homoeostasis Model of Assessment-Insulin Resistance (HOMAIR) was calculated according to the formula of Mathews et al. [22] as follows: HOMA-IR = Fasting serum insulin (lIU ml 1) Fasting plasma glucose (mmol l–1)/22.5. Fasting C-peptide was determined by solid-phase competitive chemiluminescent enzyme immunoassay using Immulite automated analyser supplied by DPC, Los Angeles, CA, USA [23]. Serum TNF-a was assayed by AviBion Human TNF-a enzymelinked immunosorbent assay (ELISA) kits supplied by Orgenium Laboratories, Vantaa, Finland [24]. 5. The method of genotype determination is the following: HCV genotype determination was performed by polymerase chain reaction (PCR) with a type-specific primer (Promega 2800 Woods Hollow Road, Madison, WI, USA) depending on the amplification of a C gene sequence by PCR using a universal primer (sense) and a mixture of four type-specific primers (anti-sense). HCV types were determined by the size of the products specific to each of them. Sequences of the C gene of different HCV types were amplified by PCR with type-specific primers subjected to electrophoresis and stained with ethidium bromide. The different HCV types (I–IV) were recognized by distinct sizes of the PCR products, 57, 144, 174 and 123 bp, respectively. Statistical analysis Data were analysed using the Statistical Package of Social Sciences (SPSS) computer software for Windows, version 11 (SPSS Inc., Chicago, IL, USA). Qualitative variables were presented as numbers and percentages. The chi-squared (X2) or Fisher exact test was used for comparison between groups, as appropriate. Quantitative variables were presented as mean ± standard deviation (SD). The Student’s t-test was used for comparison of two groups. Analysis of variance (ANOVA) with Bonferroni multiple comparisons was used for more than two groups. The Kruskal–Wallis was used for non-parametric data comparison. Pearson’s correlation coefficient was used to calculate the correlation between variables within the same group. P < 0.05 was considered statistically significant. Results The study groups were matched regarding gender distribution. However, there was a significantly higher mean age in the HCV-positive diabetic group versus the non-diabetic groups. BMI showed significantly higher mean values among the diabetic HCV-positive group versus the non-diabetic and the control groups. However, a non-significant difference in the percentage of overweight and obese subjects was found among the three groups (Table 1). HCV genotypes Genotype 4 was found to be the predominant genotype found in 45 out of the 66 diabetic patients (68.2%) and in 25 out of 34 nondiabetic patients (73.5%). Genotype 3 was found among the remainder of the patients including 21 diabetic and 9 non-diabetic patients (31.8% and 26.5%, respectively). To the best of our knowledge, this is the first report of the presence of HCV genotype 3 in about 30% of an Egyptian group. There was statistically no significant difference between the diabetic and non-diabetic groups regarding the genotype distribution (Table 1). Liver function tests HCV-positive patients (diabetics and non-diabetics) showed significantly higher ALT and AST values in comparison to control
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O. Saleh et al. / Arab Journal of Gastroenterology 14 (2013) 14–19
Table 1 Clinical characteristics of the studied groups. Group variable
HCV + DM (Number = 66)
HCV + Non-diabetic (Number = 34)
Control (Number = 35)
p value
Gender N (%) Men Age mean ± SD (years) BMI mean ± SD (kg m–2)
41 (62.1) 51.6 ± 7.9a,c 29.3 ± 4a,c
25 (73.5) 43.6 ± 9.6a 27.2 ± 3.6a
18 (51.4) 46.4 ± 10c 27.2 ± 2.8c
NS <0.05 <0.05
Overweight/obesity Present N (%) Absent N (%)
58 (75.9) 8 (12.1)
25 (73.5) 9 (26.5)
29 (82.9) 6 (17.1)
NS NS
NS: non-significant difference. a Significant difference between HCV-positive diabetic and non-diabetic groups. c Significant difference between HCV-positive diabetic and control groups.
Table 2 HCV genotypes and liver function parameters in the studied groups.
a b c
Group variable
HCV + DM (66)
HCV non-diabetic (34)
Genotype 4 N (%) Genotype 3 N (%) AST (u ml–1) mean ± SD ALT (u ml–1) mean ± SD GGT (u l–1) mean ± SD APRI mean ± SD
45 (68.2) 21 (31.8) 65.1 ± 34.8c 61.2 ± 34.9a,c 101.8 ± 71.5a,c 0.75 (0.5)c
25 (73.5) 9 (26.5) 76.1 ± 35b 84.5 ± 49a,b 50.6 ± 31a,b 0.8 (0.5)b
Control (35)
p value NS NS <0.05 <0.05 <0.05 <0.05
c,b
29.9 ± 4.6 30.9 ± 4.6b,c 24.9 ± 8.1b,c 0.13 (0.03)b,c
Significant difference between HCV-positive diabetic and non-diabetic groups. Significant difference between HCV-positive non-diabetic and control groups. Significant difference between HCV-positive diabetic and control groups.
but there was a non-significant difference between the diabetic and non-diabetic group regarding these parameters (Table 2). On the other hand, levels of GGT were significantly higher among the HCV-positive diabetic group versus the control and the HCV-positive non-diabetic groups (Table 2). AST–platelet ratio index
Tumour necrosis factor-a TNF-a showed higher values among HCV-positive diabetic versus non-diabetic and control groups (Table 3).
Table 4 Comparison between subjects having HCV genotype 3 and genotype 4.
This is considered a surrogate measure of liver fibrosis [25]. Higher values were found among HCV-positive diabetic and nondiabetic groups versus the control group but no significant difference between the HCV-positive diabetic and non-diabetic groups was found (Table 2). Metabolic parameters Regarding serum insulin, C-peptide and HOMA-IR, there were significantly higher insulin levels and HOMA-IR values among HCV-positive diabetics in comparison to controls, suggesting a degree of insulin resistance in those patients (Table 3). C-reactive protein There were no significant differences between groups regarding C-reactive protein (CRP) positivity (Table 3).
Gender M(%) F(%) Over-weight/obese Number(%) BMI mean ± SD CRP Negative N (%) Positive N (%) Age mean ± SD C-peptide mean ± SD TNF-a mean ± SD AST mean ± SD ALT mean ± SD GGT mean ± SD APRI mean ± SD
Genotype 3 (30)
Genotype 4 (70)
Sig.
17(56.7) 13(43.3)
49(70) 21(30)
NS
24(80) 28.8 ± 4.4
59(84) 28.4 ± 3.8
NS NS
19(633) 11(36.7) 46.8 ± 9.8 2.2 ± 1.3 30.5 ± 44.9 64.6 ± 36.9 62.1 ± 42.6 97.1 ± 81.5 0.7 ± 0.4
51(72.9) 19(27.1) 49.8 ± 8.9 2.3 ± 1.4 26.9 ± 11.8 70.6 ± 41 72.1 ± 41 78.96 ± 50 0.8 ± 0.5
NS NS NS NS NS NS NS
NS = non-significant (p > 0.05).
Table 3 Metabolic and inflammatory markers among the studied groups. Group variable
a b c
HCV + DM (66)
HCV non-diabetic (34)
Control (35)
p value
Serum insulin (uIU ml–1) mean ± SD Serum C peptide (ng ml–1) mean ± SD HOMA-IR mean ± SD
2.8 ± 1.3a,c –
14.3 ± 6.8b 1.2 ± 0.8a 3.6 ± 2b
9.6 ± 2.1b 0.9 ± 0.3c 21 ± 0.6b
<0.05 <0.05 <0.05
CRP Negative N (%) Positive N (%) TNF-a (pg ml 1) mean ± SD
43(64.2) 23 (34.8) 38.11 ± 8.6a,c
27 (79.4) 7 (20.6) 9.7 ± 4.6a
– – 8.2 ± 4.3c
<0.05 <0.05 <0.05
Significant difference between HCV-positive diabetic and non-diabetic groups. Significant difference between HCV-positive non-diabetic and control groups. Significant difference between HCV-positive diabetic and control groups.
O. Saleh et al. / Arab Journal of Gastroenterology 14 (2013) 14–19 Table 5 Correlation between HOMA-IR and TNF-a and liver function indices in both HCV positive diabetic and non-diabetic groups. Non-diabetic Group (34) TNF-a AST ALT GGT APRI *
r = 0.601 r = 0.082 r = 0.062 r = 0.247 r = 0.178
Diabetic Group (66) p < 0.001* p = 0.62 p = 0.72 p = 0.159 p = 0.313
r = 0.248 r = 0.183 r = 0.082 r = 0.222 r = 0.093
p = 0.054 p = 0.141 p = 0.512 p = 0.073 p = 0.457
Significant correlation (p < 0.001).
Comparison between genotype 4 and genotype 3 HCV-positive cases Comparison of HCV-positive genotype 4 versus genotype 3 revealed no significant difference regarding either the clinical or the biochemical parameters (Table 4). A significant correlation between HOMA-IR and TNF-a was found in the non-diabetic group only (Table 5). Discussion The aim of the present work was to study HCV genotypes in relation to other clinical and laboratory variables in a group of diabetic Egyptians in East Delta. It is known that the predominant HCV genotype in Egypt is genotype 4, which was found to exceed 90% prevalence in previous studies, while no recent data studying the relation between genotype and diabetes were found [6–9]. However, it is worth noting, that the present study shows for the first time (to the best of our knowledge) the presence of HCV genotype 3 in a significant proportion of this mainly diabetic group (about 30%, Table 2), while the remainder of the patients (about 70%) were genotype 4. Although this is a relatively small number of patients, this result might be considered an important finding due to its potential impact on clinical and therapeutic decisions [10,26]. Either this finding could denote an epidemiologic shift [19], as reported in other countries such as France [8] and Italy [27], or could be a coincidence, which may warrant further studies with greater numbers of patients. This finding might be explained on the basis that the majority of cases in this cohort were diabetic and HCV genotype 3 was previously suggested as one of the genotypes commonly associated with diabetes [20]. However, we did not find a statistically significant difference between diabetic and non-diabetic HCV-positive cases regarding the percentage of genotype 3 cases in both groups (Table 2). It is well known that Egypt has a high prevalence of HCV infection, which was previously attributed in some studies and metaanalyses to the mass treatment of schistosomiasis with parenteral antimony compounds decades ago [28,5]. However, this could only explain the HCV prevalence among patients in their 50s, but cannot explain the newly diagnosed subjects in their early adulthood. Regarding the previously mentioned epidemiologic change of HCV genotype distribution in Europe, it was partly explained by changes in mode of transmission where there was a decline of cases transmitted through infected blood after adoption of blood transfusion safety measures since 1992, all over the world including Egypt. Blood supplies since then are safe in Europe and no case of HCV transmission due to administration of plasma-derived products has been reported since 1994 [29]. This could suggest other modes of transmission of HCV, such as intravenous drug use and nosocomial infection, in newly diagnosed cases. In this context, in a cohort of Greek patients with chronic hepatitis C, HCV genotypes 3 and 4 were detected in a significant proportion of patients with nosocomial or community acquired infection
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[30]. However, in the present study, apart from this, the mode of transmission remains unknown with no other risk factor found in a significant number of cases (data not shown), as was reported in other countries as well [19]. Hence, this newly reported genotype distribution could be partly explained by the other unidentified modes of transmission of the virus. Therefore, further studies on a nationwide scale would be suggested to explore this finding and its role in preventive and treatment measures for such cases. It is now widely recognized that chronic HCV infection is associated with insulin resistance and type 2 DM [11–18,31] and it is now considered by some authors as a metabolic disease [31]. In this study, significantly higher values of serum insulin, C-peptide and HOMA-IR were found among HCV-positive non-diabetic patients versus HCV-negative patients (Table 3), suggesting that HCV status is associated with insulin resistance as previously reported in other studies [17,32–35]. Further, hyperinsulinaemia rather than insulin deficiency in association with chronic HCV infection has been also reported [36]. The association of hyperinsulinaemia with liver cirrhosis has been suggested to, most probably, be due to decreased hepatic extraction of insulin rather than pancreatic hyper-secretion. Hence, measurement of serum C-peptide in addition to insulin is important when assessing the relative contribution of pancreatic secretion and hepatic extraction of diseased liver [37,38]. In the present study, measurement of serum insulin and accordingly, the HOMA score in HCV-positive diabetics, would be confounded by exogenous insulin treatment in significant number of cases, so, we presented only the results of insulin and HOMA-IR in HCV-positive non-diabetic patients versus controls (Table 3), since the HOMA score has been considered a non-reliable index in the presence of diabetes especially if insulin treated [39]. In this context, C-peptide determination was more reliable and showed significantly higher values among diabetic HCV-positive patients in comparison with HCV-negative controls and non-diabetics as well (Table 3). In agreement with these findings, Hui et al. [40], investigating 121 HCV patients with early stages of fibrosis, reported higher levels of insulin, C-peptide and HOMA-IR compared to healthy controls. Regarding the relation between HCV genotypes and insulin resistance, studies have reported that it was more common in patients with predominant genotype 1 [32,40]. Subsequent studies confirmed this association in genotype 4 [32], genotype 2 [41] and more recently genotype 3 [42]. However, in our study, there were no significant differences between both genotypes (Table 4). Since liver cirrhosis, irrespective of the aetiology, can itself cause insulin resistance and hyperinsulinaemia [43–45], it is important to exclude patients with advanced cirrhosis during the assessment of the relationship between HCV and insulin resistance, which is the case in this study. Regarding liver function parameters, the present study showed significantly higher levels of AST, ALT and GGT in HCV-positive patients in comparison to controls. However, no difference was found between diabetic and non-diabetic HCV-positive patients apart from a significantly higher GGT level among the diabetic group versus the non-diabetic group (Table 2). It was found that the presence of high GGT levels in HCV-positive diabetics is correlated with lower sustained virologic response rate in such patients. A close relationship between serum GGT levels and hepatic steatosis, advanced fibrosis and insulin resistance has been described [26]. In our study, there was no significant direct correlation between HOMA-IR and liver function parameters. It was previously reported that not all patients with HCV infection develop insulin resistance, suggesting complex interactions between the virus and the host factors that are only partially understood [31]. Regarding inflammatory parameters, this study showed markedly increased levels of TNF-a in the HCV-positive diabetic group
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versus non-diabetic and control groups (Table 3), but it showed no significant difference between genotypes. It is worth noting that TNF-a has been previously suggested to mediate the effect of HCV infection on glucose metabolism [46]. This finding is in agreement with previous studies showing the pro-inflammatory TNF-a induced insulin resistance in patients with HCV infection irrespective of the degree of fibrosis [18]. However, in this study, TNF-a showed a significant correlation with the HOMA score in the non-diabetic group only (Table 5) and showed no significant difference among both genotypes, which was the rationale behind the assay of TNF-a here (Table 4). It is also worth mentioning that subjects with clinical cirrhosis were excluded from the present study and APRI, which is a surrogate marker of liver fibrosis [25], showed no significant difference between the diabetic and the non-diabetic HCV-positive group (Table 2). In conclusion, the results of this study report for the first time the presence of HCV genotype 3 in a significant percentage of cases in an Egyptian group (where the majority were diabetic). This finding could have an impact on the prognosis and plan of therapy for those patients, particularly because HCV genotype 3 has a more favourable response to interferon/ribavirin anti-HCV therapy, with shorter duration of treatment than either genotype 4 or 1 [10]. In this regard, it has been recently reported by investigators from Turkey, where the major HCV genotype is genotype 1 and the sum of genotypes 2 and 3 was found to be about 6% [47], that determination of HCV genotypes and tailoring the treatment thereafter is cost-effective even in countries where the prevalence of genotype 2 or 3 is low. Hence, based on our results, genotype-based studies are warranted to verify these findings on larger groups of patients in Egypt before considering treatment options. Further, studies are required to follow up such patients and their response to therapy in comparison to the other predominant genotype 4 HCV-positive patients. Conflicts of interest The authors declared that there was no conflict of interest. Acknowledgement This work was supported, in part, by a grant from the Department of Pharmacology/Physiology, New Jersey Medical School, UMDNJ, Newark, NJ, USA. We appreciate the contribution of Professor Dr. Abdel Hady ElGilany in the performance of the statistical analysis of the results of this study. References [1] Proceedings of the European Association for the study of the liver international consensus conference on hepatitis C, 26–27 February 1999, Paris, France; 1993. J Hepatol;31(Suppl. 1):1–268. [2] Intitative for vaccine research: hepatitis C [homepage on the Internet], 2008. [3] Wong T, Lee SS. Hepatitis C: a review for primary care physicians. CMAJ 2006;174(5):59–649. [4] Strickland GT, Elhefni H, Salman T, et al. Role of hepatitis C infection in chronic liver disease in Egypt. Am J Trop Med Hyg 2002;67(4):42–436. [5] Marzouk D, Sass J, Bakr I, et al. Metabolic and cardiovascular risk profiles and hepatitis C virus infection in rural Egypt. Gut 2007;56(8):10–1105. [6] El-Zayadi A, Simmonds P, Dabbous H, et al. Hepatitis C virus genotypes among HCV-chronic liver disease patients in Egypt: a leading trial. J Egypt Public Health Assoc 1994;69(5–6):327–34. [7] Ray SC, Arthur RR, Carella A, et al. Genetic epidemiology of hepatitis C virus throughout Egypt. J Infect Dis 2000;182(3):698–707. [8] Payan C, Roudot-Thoraval F, Marcellin P, et al. Changing of hepatitis C virus genotype patterns in France at the beginning of the third millenium: the GEMHEP GenoCII study. J Viral Hepat 2005;12(4):405–13. [9] Kamal SM, Nasser IA. Hepatitis C genotype 4: what we know and what we don’t yet know. Hepatology 2008;47(4):1371–83.
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[45] Picardi A, D’Avola D, Gentilucci UV, et al. Diabetes in chronic liver disease: from old concepts to new evidence. Diabetes Metab Res Rev 2006;22(4):274–83. [46] Valenti L, Pulixi E, Fracanzani AL, et al. TNFa genotype affects TNFa release, insulin sensitivity and the severity of liver disease in HCV chronic hepatitis. J Hepatol 2005;43(6):944–50. [47] Tahan V, Ozaras R, Karaca C, et al. Is HCV genotyping cost-effective even when the prevalences of genotypes 2 and 3 are low? Hepatogastroenterology 2009; 56(94–95):1425–8.
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Original Article
Hepatitis C virus genotype distribution in Egyptian diabetic patients: A preliminary study Omayma Saleh a,⇑, Azza A. Baiomy b, Ayman El-desouky a, Hosam Zaghloul b, Mohammad El-Arman b, Gamal M. Dahab c, Mohamed S. Abdel-Rahman d a
Internal Medicine Department, Faculty of Medicine, Mansoura University, Mansoura City, Egypt Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura City, Egypt Clinical Pharmacology Department, Faculty of Medicine, Mansoura University, Mansoura City, Egypt d Pharmacology/Physiology Department, New Jersey Medical School, UMDNJ, Newark, NJ, USA b c
a r t i c l e
i n f o
Article history: Received 12 November 2011 Accepted 29 December 2012
Keywords: Hepatitis C virus HCV genotypes Insulin resistance Egypt Diabetes
a b s t r a c t Background and study aims: There is controversy regarding whether a specific hepatitis C virus (HCV) genotype is associated with diabetes mellitus. This study aimed to investigate HCV genotype distribution in diabetics and its relation to some clinical and laboratory variables in HCV-positive diabetic versus nondiabetic Egyptians in East Delta. Patients and methods: The study included 100 HCV-positive patients of which 66 were diabetic in addition to 35 healthy adults as a control group. Clinical assessment, laboratory measurements of plasma glucose, insulin, C-peptide, C-reactive protein (CRP), tumour necrosis factor-a (TNF-a) and liver functions (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT)) as well as HCV genotype determination were done, and AST/platelet ratio index (APRI) and Homoeostasis Model of Assessment-Insulin Resistance (HOMA-IR) were calculated. Results: The main results were the presence of HCV genotype 3, in 31.8% of the diabetic group and in 26.5% of the non-diabetic group, while the remainder of cases had genotype 4, the predominant genotype in Egypt. This is the first report of the presence of HCV genotype 3 in about 30% of an Egyptian cohort. However, there was no significant difference in genotype distribution between both groups. Further, there were significantly higher values of HOMA-IR, insulin and C-peptide in HCV-positive groups in comparison to the control group, while TNF-a was significantly higher in the HCV-positive diabetic group. However, there were no significant differences between both genotypes regarding these parameters. Conclusion: Although this study reveals for the first time the presence of HCV genotype 3 in a significant percentage of a group of Egyptian patients, where the majority were diabetic, the association between diabetes and certain HCV genotypes could not be confirmed on the basis of our findings. Hence, taking into consideration the impact of such a finding on the treatment decisions of those patients, further studies are warranted to explore these findings to a greater extent. Ó 2013 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.
Introduction Hepatitis C virus (HCV) infection is a disease with a significant global impact and an important cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. According to a World Health Organization (WHO) report, more than 3% of the world population are infected. There are considerable regional differences, where the prevalence is as high as 20% in some countries. In Africa and western Pacific, the prevalence is higher than North America and Europe [1–3]. ⇑ Corresponding author. Address: Internal Medicine Department, Endocrinology and Diabetes Unit, Specialized Medical Hospital, Mansoura University, Mansoura City, Egypt. Tel.: +20 1119951003. E-mail address: [email protected] (O. Saleh).
In Egypt, HCV is the most common aetiology of chronic liver disease, where the prevalence of antibodies to HCV is 10-fold higher than in the United States and Europe [4,5]. Among the six major HCV genotypes found worldwide, genotype 4 is the most predominant genotype in Egypt, which was thought to have more than 90% preponderance [6–9]. The geographical distribution of HCV genotypes may be useful for epidemiological purposes; however, besides its epidemiological use, HCV genotype determination is relevant both as a predictor of response to therapy as well as to decide the length of time required for antiviral treatment, being less in the case of genotypes 2 and 3 [10]. There are recent reports about a change in HCV genotype distribution among certain areas in Europe and Africa, which were suggested to be attributed to changes in the mode of transmission
1687-1979/$ - see front matter Ó 2013 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ajg.2013.01.005
O. Saleh et al. / Arab Journal of Gastroenterology 14 (2013) 14–19
and immigration status in these areas [19]. Furthermore, several studies have shown that HCV infection is associated with modest but significantly increased risk of developing type 2 diabetes mellitus (DM) [10–17]. Therefore, in the light of the international epidemic of type 2 DM and the increasing incidence of HCV infection, the association of HCV and DM is potentially a major health problem [20]. The controversy whether a specific HCV genotype is diabetogenic is still going on. Further, studies of the relation of HCV genotype and other variables are limited and there is a paucity of recent data [21]. Hence, given the importance of HCV genotype determination regarding its prognostic significance and its impact on the treatment decisions and cost of therapy, the aim of this study was to determine the HCV genotype status and its association with selected clinical and laboratory variables related to either diabetes or HCV in a group of HCV-positive diabetic versus non-diabetic Egyptians. Further, whether diabetes might have an association with a certain HCV genotype would be another objective. Patients and methods Patients Inclusion criteria The patients included in the study were recruited from the outpatient diabetes and gastroenterology clinics at the Specialized Medical Hospital of Mansoura University, in Egypt. This cross-sectional comparative study included 100 HCV-positive patients (66 diabetic and 34 non-diabetic patients), in addition to 35 healthy subjects of matched age and gender selected as a control group. Exclusion criteria Patients were excluded from this study if they had the following: advanced cirrhosis showing manifestations of liver cell failure; a history of other liver disease including hepatitis B virus infection; previous anti-HCV therapy; having hepatocellular carcinoma; associated kidney or cardiac disease; pregnancy; infections; smoking; and corticosteroid treatment. These factors can affect DM status or may precipitate or aggravate insulin resistance and might affect some laboratory measurements particularly tumour necrosis factor (TNF-a). Methods 1. The included patients and controls were subjected to complete clinical assessment and laboratory and radiological investigations. Informed consent was taken from each patient before enrolment in the study, according to the ethics committee guidelines. 2. Anthropometric measurements including weight, height and waist circumference were taken and body mass index (BMI) was calculated. BMI more than 25 kg m 2 is considered overweight while BMI P 30 kg m–2 is considered obese. 3. High-resolution ultrasonographic analysis of the liver surface was performed. 4. The following laboratory measurements were taken: Routine laboratory tests including fasting glucose and liver function tests including serum albumin, bilirubin, aspartic transaminase (AST), alanine transaminase (ALT) and gamma glutaryl transpeptidase (SGT) were assayed by commercially available kits supplied by Human, Wiesbaden, Germany. Fasting serum insulin assay was performed by enzyme-amplified sensitivity immunoassay supplied by Biosource Technologies, Fleunes, Belgium.
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Homoeostasis Model of Assessment-Insulin Resistance (HOMAIR) was calculated according to the formula of Mathews et al. [22] as follows: HOMA-IR = Fasting serum insulin (lIU ml 1) Fasting plasma glucose (mmol l–1)/22.5. Fasting C-peptide was determined by solid-phase competitive chemiluminescent enzyme immunoassay using Immulite automated analyser supplied by DPC, Los Angeles, CA, USA [23]. Serum TNF-a was assayed by AviBion Human TNF-a enzymelinked immunosorbent assay (ELISA) kits supplied by Orgenium Laboratories, Vantaa, Finland [24]. 5. The method of genotype determination is the following: HCV genotype determination was performed by polymerase chain reaction (PCR) with a type-specific primer (Promega 2800 Woods Hollow Road, Madison, WI, USA) depending on the amplification of a C gene sequence by PCR using a universal primer (sense) and a mixture of four type-specific primers (anti-sense). HCV types were determined by the size of the products specific to each of them. Sequences of the C gene of different HCV types were amplified by PCR with type-specific primers subjected to electrophoresis and stained with ethidium bromide. The different HCV types (I–IV) were recognized by distinct sizes of the PCR products, 57, 144, 174 and 123 bp, respectively. Statistical analysis Data were analysed using the Statistical Package of Social Sciences (SPSS) computer software for Windows, version 11 (SPSS Inc., Chicago, IL, USA). Qualitative variables were presented as numbers and percentages. The chi-squared (X2) or Fisher exact test was used for comparison between groups, as appropriate. Quantitative variables were presented as mean ± standard deviation (SD). The Student’s t-test was used for comparison of two groups. Analysis of variance (ANOVA) with Bonferroni multiple comparisons was used for more than two groups. The Kruskal–Wallis was used for non-parametric data comparison. Pearson’s correlation coefficient was used to calculate the correlation between variables within the same group. P < 0.05 was considered statistically significant. Results The study groups were matched regarding gender distribution. However, there was a significantly higher mean age in the HCV-positive diabetic group versus the non-diabetic groups. BMI showed significantly higher mean values among the diabetic HCV-positive group versus the non-diabetic and the control groups. However, a non-significant difference in the percentage of overweight and obese subjects was found among the three groups (Table 1). HCV genotypes Genotype 4 was found to be the predominant genotype found in 45 out of the 66 diabetic patients (68.2%) and in 25 out of 34 nondiabetic patients (73.5%). Genotype 3 was found among the remainder of the patients including 21 diabetic and 9 non-diabetic patients (31.8% and 26.5%, respectively). To the best of our knowledge, this is the first report of the presence of HCV genotype 3 in about 30% of an Egyptian group. There was statistically no significant difference between the diabetic and non-diabetic groups regarding the genotype distribution (Table 1). Liver function tests HCV-positive patients (diabetics and non-diabetics) showed significantly higher ALT and AST values in comparison to control
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O. Saleh et al. / Arab Journal of Gastroenterology 14 (2013) 14–19
Table 1 Clinical characteristics of the studied groups. Group variable
HCV + DM (Number = 66)
HCV + Non-diabetic (Number = 34)
Control (Number = 35)
p value
Gender N (%) Men Age mean ± SD (years) BMI mean ± SD (kg m–2)
41 (62.1) 51.6 ± 7.9a,c 29.3 ± 4a,c
25 (73.5) 43.6 ± 9.6a 27.2 ± 3.6a
18 (51.4) 46.4 ± 10c 27.2 ± 2.8c
NS <0.05 <0.05
Overweight/obesity Present N (%) Absent N (%)
58 (75.9) 8 (12.1)
25 (73.5) 9 (26.5)
29 (82.9) 6 (17.1)
NS NS
NS: non-significant difference. a Significant difference between HCV-positive diabetic and non-diabetic groups. c Significant difference between HCV-positive diabetic and control groups.
Table 2 HCV genotypes and liver function parameters in the studied groups.
a b c
Group variable
HCV + DM (66)
HCV non-diabetic (34)
Genotype 4 N (%) Genotype 3 N (%) AST (u ml–1) mean ± SD ALT (u ml–1) mean ± SD GGT (u l–1) mean ± SD APRI mean ± SD
45 (68.2) 21 (31.8) 65.1 ± 34.8c 61.2 ± 34.9a,c 101.8 ± 71.5a,c 0.75 (0.5)c
25 (73.5) 9 (26.5) 76.1 ± 35b 84.5 ± 49a,b 50.6 ± 31a,b 0.8 (0.5)b
Control (35)
p value NS NS <0.05 <0.05 <0.05 <0.05
c,b
29.9 ± 4.6 30.9 ± 4.6b,c 24.9 ± 8.1b,c 0.13 (0.03)b,c
Significant difference between HCV-positive diabetic and non-diabetic groups. Significant difference between HCV-positive non-diabetic and control groups. Significant difference between HCV-positive diabetic and control groups.
but there was a non-significant difference between the diabetic and non-diabetic group regarding these parameters (Table 2). On the other hand, levels of GGT were significantly higher among the HCV-positive diabetic group versus the control and the HCV-positive non-diabetic groups (Table 2). AST–platelet ratio index
Tumour necrosis factor-a TNF-a showed higher values among HCV-positive diabetic versus non-diabetic and control groups (Table 3).
Table 4 Comparison between subjects having HCV genotype 3 and genotype 4.
This is considered a surrogate measure of liver fibrosis [25]. Higher values were found among HCV-positive diabetic and nondiabetic groups versus the control group but no significant difference between the HCV-positive diabetic and non-diabetic groups was found (Table 2). Metabolic parameters Regarding serum insulin, C-peptide and HOMA-IR, there were significantly higher insulin levels and HOMA-IR values among HCV-positive diabetics in comparison to controls, suggesting a degree of insulin resistance in those patients (Table 3). C-reactive protein There were no significant differences between groups regarding C-reactive protein (CRP) positivity (Table 3).
Gender M(%) F(%) Over-weight/obese Number(%) BMI mean ± SD CRP Negative N (%) Positive N (%) Age mean ± SD C-peptide mean ± SD TNF-a mean ± SD AST mean ± SD ALT mean ± SD GGT mean ± SD APRI mean ± SD
Genotype 3 (30)
Genotype 4 (70)
Sig.
17(56.7) 13(43.3)
49(70) 21(30)
NS
24(80) 28.8 ± 4.4
59(84) 28.4 ± 3.8
NS NS
19(633) 11(36.7) 46.8 ± 9.8 2.2 ± 1.3 30.5 ± 44.9 64.6 ± 36.9 62.1 ± 42.6 97.1 ± 81.5 0.7 ± 0.4
51(72.9) 19(27.1) 49.8 ± 8.9 2.3 ± 1.4 26.9 ± 11.8 70.6 ± 41 72.1 ± 41 78.96 ± 50 0.8 ± 0.5
NS NS NS NS NS NS NS
NS = non-significant (p > 0.05).
Table 3 Metabolic and inflammatory markers among the studied groups. Group variable
a b c
HCV + DM (66)
HCV non-diabetic (34)
Control (35)
p value
Serum insulin (uIU ml–1) mean ± SD Serum C peptide (ng ml–1) mean ± SD HOMA-IR mean ± SD
2.8 ± 1.3a,c –
14.3 ± 6.8b 1.2 ± 0.8a 3.6 ± 2b
9.6 ± 2.1b 0.9 ± 0.3c 21 ± 0.6b
<0.05 <0.05 <0.05
CRP Negative N (%) Positive N (%) TNF-a (pg ml 1) mean ± SD
43(64.2) 23 (34.8) 38.11 ± 8.6a,c
27 (79.4) 7 (20.6) 9.7 ± 4.6a
– – 8.2 ± 4.3c
<0.05 <0.05 <0.05
Significant difference between HCV-positive diabetic and non-diabetic groups. Significant difference between HCV-positive non-diabetic and control groups. Significant difference between HCV-positive diabetic and control groups.
O. Saleh et al. / Arab Journal of Gastroenterology 14 (2013) 14–19 Table 5 Correlation between HOMA-IR and TNF-a and liver function indices in both HCV positive diabetic and non-diabetic groups. Non-diabetic Group (34) TNF-a AST ALT GGT APRI *
r = 0.601 r = 0.082 r = 0.062 r = 0.247 r = 0.178
Diabetic Group (66) p < 0.001* p = 0.62 p = 0.72 p = 0.159 p = 0.313
r = 0.248 r = 0.183 r = 0.082 r = 0.222 r = 0.093
p = 0.054 p = 0.141 p = 0.512 p = 0.073 p = 0.457
Significant correlation (p < 0.001).
Comparison between genotype 4 and genotype 3 HCV-positive cases Comparison of HCV-positive genotype 4 versus genotype 3 revealed no significant difference regarding either the clinical or the biochemical parameters (Table 4). A significant correlation between HOMA-IR and TNF-a was found in the non-diabetic group only (Table 5). Discussion The aim of the present work was to study HCV genotypes in relation to other clinical and laboratory variables in a group of diabetic Egyptians in East Delta. It is known that the predominant HCV genotype in Egypt is genotype 4, which was found to exceed 90% prevalence in previous studies, while no recent data studying the relation between genotype and diabetes were found [6–9]. However, it is worth noting, that the present study shows for the first time (to the best of our knowledge) the presence of HCV genotype 3 in a significant proportion of this mainly diabetic group (about 30%, Table 2), while the remainder of the patients (about 70%) were genotype 4. Although this is a relatively small number of patients, this result might be considered an important finding due to its potential impact on clinical and therapeutic decisions [10,26]. Either this finding could denote an epidemiologic shift [19], as reported in other countries such as France [8] and Italy [27], or could be a coincidence, which may warrant further studies with greater numbers of patients. This finding might be explained on the basis that the majority of cases in this cohort were diabetic and HCV genotype 3 was previously suggested as one of the genotypes commonly associated with diabetes [20]. However, we did not find a statistically significant difference between diabetic and non-diabetic HCV-positive cases regarding the percentage of genotype 3 cases in both groups (Table 2). It is well known that Egypt has a high prevalence of HCV infection, which was previously attributed in some studies and metaanalyses to the mass treatment of schistosomiasis with parenteral antimony compounds decades ago [28,5]. However, this could only explain the HCV prevalence among patients in their 50s, but cannot explain the newly diagnosed subjects in their early adulthood. Regarding the previously mentioned epidemiologic change of HCV genotype distribution in Europe, it was partly explained by changes in mode of transmission where there was a decline of cases transmitted through infected blood after adoption of blood transfusion safety measures since 1992, all over the world including Egypt. Blood supplies since then are safe in Europe and no case of HCV transmission due to administration of plasma-derived products has been reported since 1994 [29]. This could suggest other modes of transmission of HCV, such as intravenous drug use and nosocomial infection, in newly diagnosed cases. In this context, in a cohort of Greek patients with chronic hepatitis C, HCV genotypes 3 and 4 were detected in a significant proportion of patients with nosocomial or community acquired infection
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[30]. However, in the present study, apart from this, the mode of transmission remains unknown with no other risk factor found in a significant number of cases (data not shown), as was reported in other countries as well [19]. Hence, this newly reported genotype distribution could be partly explained by the other unidentified modes of transmission of the virus. Therefore, further studies on a nationwide scale would be suggested to explore this finding and its role in preventive and treatment measures for such cases. It is now widely recognized that chronic HCV infection is associated with insulin resistance and type 2 DM [11–18,31] and it is now considered by some authors as a metabolic disease [31]. In this study, significantly higher values of serum insulin, C-peptide and HOMA-IR were found among HCV-positive non-diabetic patients versus HCV-negative patients (Table 3), suggesting that HCV status is associated with insulin resistance as previously reported in other studies [17,32–35]. Further, hyperinsulinaemia rather than insulin deficiency in association with chronic HCV infection has been also reported [36]. The association of hyperinsulinaemia with liver cirrhosis has been suggested to, most probably, be due to decreased hepatic extraction of insulin rather than pancreatic hyper-secretion. Hence, measurement of serum C-peptide in addition to insulin is important when assessing the relative contribution of pancreatic secretion and hepatic extraction of diseased liver [37,38]. In the present study, measurement of serum insulin and accordingly, the HOMA score in HCV-positive diabetics, would be confounded by exogenous insulin treatment in significant number of cases, so, we presented only the results of insulin and HOMA-IR in HCV-positive non-diabetic patients versus controls (Table 3), since the HOMA score has been considered a non-reliable index in the presence of diabetes especially if insulin treated [39]. In this context, C-peptide determination was more reliable and showed significantly higher values among diabetic HCV-positive patients in comparison with HCV-negative controls and non-diabetics as well (Table 3). In agreement with these findings, Hui et al. [40], investigating 121 HCV patients with early stages of fibrosis, reported higher levels of insulin, C-peptide and HOMA-IR compared to healthy controls. Regarding the relation between HCV genotypes and insulin resistance, studies have reported that it was more common in patients with predominant genotype 1 [32,40]. Subsequent studies confirmed this association in genotype 4 [32], genotype 2 [41] and more recently genotype 3 [42]. However, in our study, there were no significant differences between both genotypes (Table 4). Since liver cirrhosis, irrespective of the aetiology, can itself cause insulin resistance and hyperinsulinaemia [43–45], it is important to exclude patients with advanced cirrhosis during the assessment of the relationship between HCV and insulin resistance, which is the case in this study. Regarding liver function parameters, the present study showed significantly higher levels of AST, ALT and GGT in HCV-positive patients in comparison to controls. However, no difference was found between diabetic and non-diabetic HCV-positive patients apart from a significantly higher GGT level among the diabetic group versus the non-diabetic group (Table 2). It was found that the presence of high GGT levels in HCV-positive diabetics is correlated with lower sustained virologic response rate in such patients. A close relationship between serum GGT levels and hepatic steatosis, advanced fibrosis and insulin resistance has been described [26]. In our study, there was no significant direct correlation between HOMA-IR and liver function parameters. It was previously reported that not all patients with HCV infection develop insulin resistance, suggesting complex interactions between the virus and the host factors that are only partially understood [31]. Regarding inflammatory parameters, this study showed markedly increased levels of TNF-a in the HCV-positive diabetic group
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O. Saleh et al. / Arab Journal of Gastroenterology 14 (2013) 14–19
versus non-diabetic and control groups (Table 3), but it showed no significant difference between genotypes. It is worth noting that TNF-a has been previously suggested to mediate the effect of HCV infection on glucose metabolism [46]. This finding is in agreement with previous studies showing the pro-inflammatory TNF-a induced insulin resistance in patients with HCV infection irrespective of the degree of fibrosis [18]. However, in this study, TNF-a showed a significant correlation with the HOMA score in the non-diabetic group only (Table 5) and showed no significant difference among both genotypes, which was the rationale behind the assay of TNF-a here (Table 4). It is also worth mentioning that subjects with clinical cirrhosis were excluded from the present study and APRI, which is a surrogate marker of liver fibrosis [25], showed no significant difference between the diabetic and the non-diabetic HCV-positive group (Table 2). In conclusion, the results of this study report for the first time the presence of HCV genotype 3 in a significant percentage of cases in an Egyptian group (where the majority were diabetic). This finding could have an impact on the prognosis and plan of therapy for those patients, particularly because HCV genotype 3 has a more favourable response to interferon/ribavirin anti-HCV therapy, with shorter duration of treatment than either genotype 4 or 1 [10]. In this regard, it has been recently reported by investigators from Turkey, where the major HCV genotype is genotype 1 and the sum of genotypes 2 and 3 was found to be about 6% [47], that determination of HCV genotypes and tailoring the treatment thereafter is cost-effective even in countries where the prevalence of genotype 2 or 3 is low. Hence, based on our results, genotype-based studies are warranted to verify these findings on larger groups of patients in Egypt before considering treatment options. Further, studies are required to follow up such patients and their response to therapy in comparison to the other predominant genotype 4 HCV-positive patients. Conflicts of interest The authors declared that there was no conflict of interest. Acknowledgement This work was supported, in part, by a grant from the Department of Pharmacology/Physiology, New Jersey Medical School, UMDNJ, Newark, NJ, USA. We appreciate the contribution of Professor Dr. Abdel Hady ElGilany in the performance of the statistical analysis of the results of this study. References [1] Proceedings of the European Association for the study of the liver international consensus conference on hepatitis C, 26–27 February 1999, Paris, France; 1993. J Hepatol;31(Suppl. 1):1–268. [2] Intitative for vaccine research: hepatitis C [homepage on the Internet], 2008. [3] Wong T, Lee SS. Hepatitis C: a review for primary care physicians. CMAJ 2006;174(5):59–649. [4] Strickland GT, Elhefni H, Salman T, et al. Role of hepatitis C infection in chronic liver disease in Egypt. Am J Trop Med Hyg 2002;67(4):42–436. [5] Marzouk D, Sass J, Bakr I, et al. Metabolic and cardiovascular risk profiles and hepatitis C virus infection in rural Egypt. Gut 2007;56(8):10–1105. [6] El-Zayadi A, Simmonds P, Dabbous H, et al. Hepatitis C virus genotypes among HCV-chronic liver disease patients in Egypt: a leading trial. J Egypt Public Health Assoc 1994;69(5–6):327–34. [7] Ray SC, Arthur RR, Carella A, et al. Genetic epidemiology of hepatitis C virus throughout Egypt. J Infect Dis 2000;182(3):698–707. [8] Payan C, Roudot-Thoraval F, Marcellin P, et al. Changing of hepatitis C virus genotype patterns in France at the beginning of the third millenium: the GEMHEP GenoCII study. J Viral Hepat 2005;12(4):405–13. [9] Kamal SM, Nasser IA. Hepatitis C genotype 4: what we know and what we don’t yet know. Hepatology 2008;47(4):1371–83.
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