Hepatitis C virus (HCV) causes deaths

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1 Original Article / Liver Hepatobiliary & Pancreatic Diseases International Diagnostic accuracy of enhanced liver test to assess liver in patients with chronic hepatitis C Roberto Catanzaro, Michele Milazzo, Silvia Arona, Chiara Sapienza, Dario Vasta, Domenico Arcoria and Francesco Marotta Catania, Italy BACKGROUND: The prognosis and clinical management of patients with chronic liver diseases are closely related to the severity of liver. Liver biopsy is considered the gold standard for the staging of liver. However, it is an invasive test sometimes related to complications. This study aimed to assess the diagnostic value of enhanced liver (ELF) test to predict liver in patients with chronic hepatitis C. METHODS: This study included 162 patients with liver disease and 67 healthy controls. Hyaluronic acid, tissue inhibitor of matrix metalloproteinase type 1, and amino-terminal propeptide type III procollagen were measured by enzymelinked immunosorbent assay with the ELF test ADVIA Centaur (Siemens Healthcare Diagnostics Inc.). Fibrosis stage was determined using the Metavir scoring system. RESULTS: In our study, for the diagnosis of significant (Metavir F 2) a cut-off value >7.72 provides a sensitivity of 93.0% and a specificity of 83.0%. The areas under the receiver operator characteristic curve, sensitivity, specificity, and positive and negative predictive values were 0.94, 93.3%, 81.0%, 93.3%, and 81.0%, respectively (P<0.001). For the diagnosis of cirrhosis (Metavir F=4) a cut-off value >9.3 provides a Author Affiliations: Department of Medical and Pediatric Sciences, Institute of Internal Medicine "A. Francaviglia", Section of Gastroenterology, University of Catania, "G. Rodolico" Hospital, Via S. Sofia, Catania, Italy (Catanzaro R, Milazzo M, Arona S and Sapienza C); Laboratory analysis "MedLab-Analisi Cliniche", Catania, Italy (Vasta D); Specialist in diabetes and metabolic diseases, Catania, Italy (Arcoria D); and ReGenera Research Group for Aging Intervention, Milano, Italy (Marotta F) Corresponding Author: Professor Roberto Catanzaro, Department of Medical and Pediatric Sciences, Institute of Internal Medicine "A. Francaviglia", Section of Gastroenterology, University of Catania, "G. Rodolico" Hospital, Pad. 4, I Piano, Stanza 17, Via S. Sofia, Catania, Italy (Tel: ; Fax: ; rcatanza@ unict.it) 2013, Hepatobiliary Pancreat Dis Int. All rights reserved. doi: /S (13)60079-X sensitivity of 93.0% and a specificity of 86.0%. The areas under the receiver operator characteristic curve, sensitivity, specificity, and positive and negative predictive values were 0.94, 79.1%, 90.8%, 75.6%, and 92.3%, respectively (P<0.001). CONCLUSIONS: The ELF test is a promising non-invasive method for assessing liver in patients with chronic hepatitis C. It is effective in the diagnosis of both and cirrhosis. (Hepatobiliary Pancreat Dis Int 2013;12: ) KEY WORDS: enhanced liver test; non-invasive diagnosis; liver ; blood marker Introduction Hepatitis C virus (HCV) causes deaths worldwide each year. Viral hepatitis is the leading cause of liver cirrhosis and liver cancer, which in turn ranks as the third cause of cancer in the world. In the WHO European region, approximately nine million people are chronically infected with HCV. [1] Liver is characterized by the course of chronic liver disease. It can progress to cirrhosis, resulting in complications such as portal hypertension, liver failure and hepatocellular carcinoma. The degree of liver is decisive for the assessment and appropriate management of the disease and strongly is indicative of its prognosis. The severity of is important to determine the need of treatment and follow-up. It can also predict the response to a treatment. The true gold standard for assessing the degree of liver would be a histological analysis of the liver as a whole. Since this is not possible in living patients, liver biopsy has been adopted as the reference standard. However, this 500 Hepatobiliary Pancreat Dis Int,Vol 12,No 5 October 15,2013

2 Liver and ELF test diagnostic method has some pitfalls that have led to several questions about its actual value. [2, 3] The procedure involves certain risks such as pain, bleeding, perforation of other organs, and needs a histological interpretation from a special operator. Thus it affects the cost of medical care and causes strong anxiety of the patient. [4-6] Recent studies focused on alternative diagnostic methods for the assessment of the degree of [7, 8] involving either elastic waves or serum markers. The algorithms for the interpretation of these serum biomarkers have developed in the last decade. Among them, enhanced liver (ELF) test, which combines hyaluronic acid (HA), the tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1), and the aminoterminal propeptide of type III procollagen (PIIINP), has been accurate in detecting in a large cohort of patients with chronic liver disease. [9] [10, 11] Further studies have evaluated the diagnostic algorithm in patients with various liver diseases. The ELF test has been capable in predicting the progression of the disease in different clinical settings. [12, 13] It is recognized that the heterogeneous etiology of the cohorts in these studies has been a major limitation. The present study aimed to evaluate prospectively the ELF test in the assessment of its effectiveness in the diagnosis of liver in patients with chronic hepatitis C with reference to biopsy. It also aimed to examine the development, accuracy, clinical utility and limitations of biomarkers as diagnostic tools for the assessment of liver. The ELF test is useful to assess the stage and rate of progression of liver. It comprises three serum biomarkers: HA, TIMP-1 and PIIINP. [13, 14] Biomarkers are direct indicators or the metabolism and degradation of the extracellular matrix, which indicate liver. A higher concentration of individual markers leads to a higher ELF score and indicates a greater likelihood of more severe. The ELF test has received the Conformité Européenne mark in May [15] The test for the staging of liver has been validated in patients with chronic hepatitis C, alcoholic liver disease and non-alcoholic fatty liver disease. [16] This method can predict the presence of with a sensitivity of 90% and the absence of with a negative value of 92%. The test does not show any precautions and contraindications. It uses direct markers of fibrogenesis (HA and TIMP-1) and therefore unreliable results will be seen in patients with chronic diseases characterized by fibrogenesis in other organs rather than the liver. [17] Furthermore in this study, the ELF test was compared with the aspartate aminotransferase (AST)-to-platelet ratio index (APRI score), another diagnostic serologic test. APRI score=[(ast/uln)/platelet count] 100. Methods The present prospective study included 162 patients with chronic hepatitis C. They were consecutively admitted to our Complex Unit for a liver biopsy in 27 months from January 2011 to March Only biopsies longer than 15 mm with at least 6 portal tracts were accepted. Exclusion criteria included the previous history of antiviral therapy, the presence of ascites, chronic kidney failure or chronic coinfection HBV/HCV or HIV/HCV, chronic liver disease of other etiology (HBV, non-alcoholic steatohepatitis, hemochromatosis, Wilson's disease, autoimmune hepatitis and α-1 antitrypsin deficiency), liver failure, patients with alcohol abuse (taking more than 30 g/d of ethanol), heart failure or pregnancy, and patients with BMI >30 kg/m 2. The diagnosis of chronic hepatitis C was determined according to the positivity of anti-hcv and HCV- RNA for at least 6 months. The levels of HCV-RNA were determined by RNA extracted from serum, with reverse transcription and amplification of cdna in real time PCR with TaqMan probes, with a sensitivity of 10 IU/ ml. Sixty-seven healthy volunteers with no indication for liver biopsy were recruited and served as controls. These subjects were regarded as healthy on the basis of normal liver function tests, negative serology for HBV, HCV and autoimmune hepatitis, normal abdominal ultrasonography, and normal renal function test. These healthy subjects had never suffered from hepatitis and had neither history of alcohol abuse nor use of hepatotoxic drugs. Written informed consents were obtained from all patients and healthy volunteers, and the study was approved by the local ethics committee according to the Declaration of Helsinki and Good Clinical Practice guidelines. ELF test The ELF test was carried out in two weeks after liver biopsy. The patients were subjected to laboratory analysis of 0.3 ml of blood taken at MedLab of Catania. Alcohol affects many of the variables used in the ELF test. Abstinence from alcohol prior to sampling was respected. [18-21] Serum sample was processed through the ELF test ADVIA Centaur (Siemens Healthcare Diagnostics Inc.). As an in vitro diagnostic test for multivariate indices in the assessment of liver, it generates a single score (ELF score) combined with doses of HA, PIIINP and TIMP-1. This newer algorithm has been shown to maintain its diagnostic performance compared to the Original European Liver Fibrosis (OELF) panel including the variable "age". [22] This score measures the qualitative and quantitative variations of Hepatobiliary Pancreat Dis Int,Vol 12,No 5 October 15,

3 Hepatobiliary & Pancreatic Diseases International the extracellular matrix of the liver, allowing a dynamic assessment of the activity of fibrogenesis and fibrinolysis. The ELF score is a numerical value with no units of measurement. In calculating the ELF score, ADVIA Centaur analyzers use the ADVIA Centaur doses of HA, PIIINP and TIMP-1 in the following formulae: ELF score per ADVIA Centaur XP= ln[cha] ln[cpiiinp] ln[ctimp-1]; ELF score per ADVIA Centaur CP= ln[cha] ln[cpiiinp] ln[ctimp-1]. The test was based on the first formula. The ELF test is for exclusive use of the ADVIA Centaur. The interpretation of the severity of liver with the ELF score is shown as none/mild, ELF score <7.7; moderate, ELF score ; and severe, ELF score 9.8. Liver histology Percutaneous liver biopsies were performed under ultrasound guidance by a specialist, using an 18-G disposable needle. The biopsy specimens were fixed with formalin and stained with hematoxylin and eosin. All of the liver biopsies were evaluated by expert pathologists, who were blinded to the patients' clinical histories. The stage of was evaluated according to the histological staging of Metavir. Fibrosis was classified by the Metavir scoring system into five stages: 0 (no ), 1 (portal without septa), 2 (portal with rare septa), 3 (many septa without cirrhosis), and 4 (cirrhosis). [23] Statistical analysis Quantitative variables were expressed as median (range) or mean±standard deviation and qualitative variables in percentage. The diagnostic values of the ELF test in predicting significant and cirrhosis were assessed by calculating the areas under the receiver operator characteristic () curve. was also expressed with standardization according to stages, advanced and non-advanced, to prevent a spectrum bias. [24] Adjusted (Adj) was independent from stages and was calculated by observed (Ob) using the formula Adj=Ob+[ (2.5-ObDANA)], according to the difference in advanced [F2-F3-F4] and non-advanced [F0-F1] (DANA method). Best cut-off values were determined by optimization of the Younden index. Sensitivity (Se), specificity (Sp) and positive and negative predictive values (PPV and NPV) were calculated. Positive likelihood ratios were calculated based on the values of sensitivity and specificity. Statistical analysis was performed with SPSS 20.0 (SPSS Inc., Chicago, IL, USA), except for comparisons performed with MedCalc P values lower than 0.05 and 0.01 were considered significant and highly significant, respectively. Results One hundred and sixty-two patients were enrolled in this study, with a mean age of 55.19±9.53 years and a female/male ratio of 1.8. Sixty-seven healthy subjects served as controls, with a mean age of 51.43±10.24 years and a female/male ratio of 1.5. The results were grouped by two different cut-off values: significant (Metavir F 2) or non-significant (Metavir F<2) and presence (Metavir F=4) or absence of cirrhosis (Metavir F<4). [25] Following such classification, we divided the patients into two groups (Tables 1 and 2). The increased mean values of the ELF test were in parallel to the stages of (7.63±0.42 in F1, 8.70 ± 0.88 in F2, 9.07±0.68 in F3 and 10.10±0.54 in F4; all P<0.05 between adjacent stages) (Fig. 1). Moreover, there was no overlap between F=1 and F 2 or between F<4 and F=4 (Fig. 2). Significant The for the diagnosis of significant Table 1. Baseline characteristics of patients and healthy controls Characteristics Patients (n=162) Healthy controls (n=67) Age (yr) 55.19± ±10.24 Gender (male, n, %) 57/162 (35) 27/67 (40) BMI (kg/m 2 ) 26.2± ±5.1 ELF score 8.82± ±0.77 Male: ELF score 9.16±1.19 * 6.49±0.77 Female: ELF score 8.64±1.06 * 6.27±0.77 F0/ELF score 6.36±0.77 F1/ELF score 7.63±0.42 # F2/ELF score 8.70±0.88 # F3/ELF score 9.07±0.68 # F4/ELF score 10.10±0.54 # APRI score 0.99± ±0.07 F0/APRI score 0.25±0.01 F1/APRI score 0.59±0.32 # F2/APRI score 0.86±0.33 # F3/APRI score 1.07±0.41 # F4/APRI score 1.56±0.56 # Platelet count ( 10 9 /L) ± ±30.39 AST (U/L) 63.03± ±6.34 Data are expressed as mean±standard deviation. *: P=0.005, significant; #: P<0.001, highly significant. BMI: body mass index; ELF: enhanced liver ; F0-F1-F2-F3-F4, Metavir; APRI: AST-to-platelet ratio index; AST: aspartate aminotransferase. 502 Hepatobiliary Pancreat Dis Int,Vol 12,No 5 October 15,2013

4 Liver and ELF test Table 2. Staging and ELF test (n, %) Factors Data Histological staging of Metavir Healthy subject * 67/229 (29.3) Metavir F=1 42/229 (18.3) Metavir F=2 66/229 (28.8) Metavir F=3 11/229 (4.8) Metavir F=4 43/229 (18.8) ELF test Mild liver <7.7 42/162 (25.9) Moderate liver /162 (46.3) Severe liver >9.8 45/162 (27.8) Fibrosis (biopsy) Non-significant (Metavir F<2) 42/162 (25.9) Significant (Metavir F 2) 120/162 (74.1) Cirrhosis (biopsy) Absent cirrhosis (Metavir F<4) 119/162 (73.5) Present cirrhosis (Metavir F=4) 43/162(26.5) Fibrosis (ELF test) Non-significant (ELF test <7.7) 42/162 (25.9) Significant (ELF test 7.7) 120/162 (74.1) Cirrhosis (ELF test) Absent cirrhosis (ELF test <9.8) 117/162 (72.2) Present cirrhosis (ELF test 9.8) 45/162 (27.8) *: These subjects were regarded as healthy on the basis of normal liver function tests, negative serology for HBV, HCV and autoimmune hepatitis, normal abdominal ultrasonography, and normal renal function test. ELF: enhanced liver. Fig. 2. ELF score concerning healthy controls compared to patients with liver. Boxes and horizontal lines within boxes represent interquartile ranges (IQRs) and median values, respectively. The upper and lower whiskers indicate 75th percentile plus 1.5 IQR and 25th percentile minus 1.5 IQR, respectively. ELF: enhanced liver. Fig. 3. Receiver operator characteristic (ROC) curves for ELF test and APRI in the diagnosis of significant (F 2). APRI: ASTto-platelet ratio index; ELF: enhanced liver. Fig. 1. ELF score in the different histological staging of Metavir. Boxes and horizontal lines within boxes represent interquartile ranges (IQRs) and median values, respectively. The upper and lower whiskers indicate 75th percentile plus 1.5 IQR and 25th percentile minus 1.5 IQR, respectively. o, mild outlier: a value more than 75th percentile plus 1.5 IQR, but less than 75th percentile plus 3.0 IQR. ELF: enhanced liver. (Metavir F 2) was 0.94 (95% CI: ), with an optimal ELF test cut-off value for the diagnosis of significant equal to 7.72 that may provide a sensitivity of 93.0% and a specificity of 83.0% (Fig. 3). The sensitivity and specificity of the ELF test in the diagnosis of significant were 93.3% and 81.0%, respectively (Table 3). Cirrhosis The for the diagnosis of cirrhosis (Metavir F=4) was 0.94 (95% CI: ), with an optimal ELF test cut-off value for the diagnosis of cirrhosis equal to 9.30 that may provide a sensitivity of 93.0% Hepatobiliary Pancreat Dis Int,Vol 12,No 5 October 15,

5 Hepatobiliary & Pancreatic Diseases International Fig. 4. Receiver operator characteristic (ROC) curves for ELF test and APRI in the diagnosis of cirrhosis (F=4). APRI: AST-toplatelet ratio index; ELF: enhanced liver. Table 3. Diagnostic values of ELF test for predicting (Metavir F 2) and cirrhosis (Metavir F=4) 95% CI Se Sp PPV NPV (%) (%) (%) (%) LH+ Fibrosis 0.94 (P<0.001) Cirrhosis 0.94 (P<0.001) ELF: enhanced liver ; : areas under the receiver operator characteristic curve; CI: confidence interval; Se: sensitivity; Sp: specificity; PPV: positive predictive value; NPV: negative predictive value; LH + : positive likelihood ratio. Table 4. Diagnostic values of APRI score for predicting (Metavir F 2) and cirrhosis (Metavir F=4) 95% CI Se Sp PPV NPV (%) (%) (%) (%) LH+ Fibrosis 0.84 (P<0.001) Cirrhosis 0.89 (P<0.001) ELF: enhanced liver ; : areas under the receiver operator characteristic curve; CI: confidence interval; Se: sensitivity; Sp: specificity; PPV: positive predictive value; NPV: negative predictive value; LH + : positive likelihood ratio. Table 5. Sensitivity analysis of observed and standardized of ELF test and APRI score for the diagnosis of advanced, according to prevalence of stages Cutoff Cutoff Ob- for the diagnosis of advanced Mean stage in nonadvanced F0-F1 Mean stage in advanced F2-F3-F4 Difference in mean advanced minus Adjmean nonadvanced (DANA score) ELF test APRI score ELF: enhanced liver ; APRI: AST-to-platelet ratio index; : areas under the receiver operator characteristic curve; DANA: difference in advanced [F2-F3-F4] minus non-advanced [F0-F1]. Fig. 5. Receiver operator characteristic (ROC) curves for ELF test and APRI score to distinguish healthy subjects (F=0) from patients with liver (F 1). APRI: AST-to-platelet ratio index; ELF: enhanced liver. Table 6. Sensitivity analysis of observed and standardized of ELF test and APRI score for the diagnosis of cirrhosis according to prevalence of stages Ob- Mean for the stage in diagnosis absence of of cirrhosis cirrhosis F0-F1-F2-F3 Mean stage in cirrhosis F4 Difference in mean cirrhosis stage minus mean noncirrhosis stage (DANA score) ELF test APRI score Adj- ELF: enhanced liver ; APRI: AST-to-platelet ratio index; : areas under the receiver operator characteristic curve; DANA: Difference in advanced [F4] minus non-advanced [F0-F1-F2-F3]. and a specificity of 86.0% (Fig. 4). The sensitivity and specificity of the ELF test in the diagnosis of cirrhosis are 79.1% and 90.8%, respectively (Table 3). Furthermore, was calculated to distinguish the healthy controls from patients with liver. It was 0.96 (95% CI: ), with an optimal ELF test cut-off value for F 1 equal to 7.35 that provides a sensitivity of 93.8% and a specificity of 85.1% (Fig. 5). The same statistical analysis was performed for the APRI score (Figs. 3-5) and the results are shown in Table 4. For this dignostic serologic test, in the diagnosis of significant in patients with chronic hepatitis C, a cut-off value 0.63 may provide a sensitivity of 87.5% and a specificity of 76.2%, whereas in the diagnosis of cirrhosis a cut-off value 1.19 is related to a sensitivity of 74.4% and a specificity of 87.4%, respectively. The Adjs for the ELF test and APRI score obtained by the DANA method are shown in Tables 5 and Hepatobiliary Pancreat Dis Int,Vol 12,No 5 October 15,2013

6 Liver and ELF test Discussion To assess the diagnostic accuracy of the ELF test, we considered its correlation with liver in subjects who underwent liver biopsy. The multivariable logistic regression model develops a predictive algorithm whose accuracy was examined by the analysis of the ROC curve. Binary analysis was performed by dividing patients into two groups based on their stage. The degree of was dichotomized into significant (no /portal without septa versus portal with rare septa/many septa without cirrhosis/cirrhosis) and liver cirrhosis (present or absent). The validity of a biomarkers model should be verified through the examination of accuracy in different groups, including those with concomitant diseases, with different age groups and with liver disease of different etiology. Lichtinghagen et al [26] have recently demonstrated the importance of variables such as age and gender. In our study we found a significant difference (P<0.01) between men (9.16±1.19) and women (8.64±1.06). Moreover, liver disease progression in response to treatment and prognosis in terms of morbidity and mortality are both important parameters. The accuracy of diagnostic biomarkers as mentioned above has been reported in terms of with sensitivity, specificity and predictive values calculated at specific cut-off points. Typically, a range of values at one end of the test result spectrum will produce a high sensitivity and a low specificity, while a range of values at the opposite end of the spectrum will have a low sensitivity and a high specificity. The intermediate results of the tests often determine moderate values of sensitivity and specificity, which are not clinically significant and, therefore, comprise an "indeterminate range". In our study the ELF test showed a number of results ranging between 7.10 and For the diagnosis of significant in patients with chronic liver disease, a cut-off value 7.72 may provide a sensitivity of 93.0% and a specificity of 83.0%. A cut-off value 9.30 for the diagnosis of cirrhosis may provide a sensitivity of 93.0% and a specificity of 86.0%. For example, a comparison can be made with the APRI score, which typically provides a number of results ranging from 0.1 to 8.0. In the diagnosis of significant in patients with chronic hepatitis C, according to the literature, a cut-off value 0.5 provides a sensitivity of 81.0% and a specificity of 50.0%, whereas a cut-off value 1.5 is related to a sensitivity of 35.0% and a specificity 91.0% respectively. [27, 28] Thus, most of the biomarkers will produce inconclusive results for patients who fall in the indeterminate range for a specific end-point of. Parkes et al [28] examined the performance of 10 algorithms serum biomarkers and found that 65% of subjects had indeterminate results for the prediction of significant. However, the values in the indeterminate range for an end-point of (for example, clinically significant ) can still be useful for the diagnosis of with other end-points (for example, cirrhosis). The values of APRI between 0.5 and 1.5, for example, are not useful in determining a significant, but those higher than 1.0 are related to a sensitivity of 89.0% and a specificity of 75.0% in the diagnosis of HCV-related cirrhosis. [27] The APRI score, in our cohort, showed similar results. The outcomes were slightly better than those reported in the literature. [27] In the diagnosis of significant in patients with chronic hepatitis C, a cut-off value 0.63 may provide a sensitivity of 87.5% and a specificity of 76.2%, whereas in the diagnosis of cirrhosis a cutoff value 1.19 is related to a sensitivity of 74.4% and a specificity of 87.4%, respectively. In our study, the ELF test in the diagnosis of significant (Metavir F 2) showed a sensitivity of 93.3% and a specificity of 81.0% (P<0.001). In the diagnosis of liver cirrhosis (Metavir F=4), instead, the test showed a sensitivity of 79.1%, but a specificity of 90.8% (P<0.001). PPV and NPV represent a more useful interpretation of the test results performed on a small number of patients. Predictive values, sensitivity, and specificity depend on the prevalence of the underlying disease. Thus, a test can be highly specific for the diagnosis of cirrhosis, but it is related to a low PPV if the underlying prevalence is very low (Table 7). For example, an APRI cut-point of 2.0 has a specificity of 91.0% in the diagnosis of cirrhosis; however, if the prevalence of cirrhosis is only 15.0%, the PPV is 50.0%. Therefore, it is important to understand that the characteristics of the tests with biomarkers vary according to the setting. Selection bias may exist in studies which include patients who have undergone liver biopsy. In our study, the prevalence of significant and cirrhosis was 74.1% and 26.5%, respectively, which are higher than those (2.8% and 0.3%) in the general community. [27] Table 7. Predictive values of diagnostic biomarker with a sensitivity and specificity of 90%, depends on the underlying prevalence of disease Prevalence of (%) PPV (%) NPV (%) PPV: positive predictive value; NPV: negative predictive value. Hepatobiliary Pancreat Dis Int,Vol 12,No 5 October 15,

7 Hepatobiliary & Pancreatic Diseases International Therefore, for a given biomarker, the PPV will be significantly lower but the NPV significantly higher in the general community than in a clinical trial (Table 7). In our study, the PPV and NPV in the diagnosis of significant were 93.3% and 81.0%, respectively. In the diagnosis of liver cirrhosis, the PPV and NPV were 75.6% and 92.3%, respectively. Moreover, the varies because of the prevalence of at different stages. This represents a spectrum bias that has important implications of non-invasive methods, when analyzing outcomes of a diagnostic method used in different populations. If extreme stages of (F0 and F4) are overrepresented in a given population, the sensitivity and specificity of a diagnostic test will be higher than in another population of patients with middle stages of (F1 and F2). Several ways of preventing "spectrum bias" have been proposed including the Adj using the DANA method. Clinically, an accurate determination of liver is not as important as in other pathological scoring systems. For each diagnostic method for evaluating patients with significant, both sensitivity and specificity of above 85% can be considered sufficient since no relevant clinical consequences are found in case of false positives or false negatives. [29] The present study showed that the ELF test is a promising non-invasive method for assessing liver in patients with chronic hepatitis C. The test has proven to be useful in determining the intermediate stages of or the presence or absence of cirrhosis. The ELF test was proved more reliable than the APRI score in the diagnosis of significant and cirrhosis. However, it was not effective in discriminating healthy volunteers from patients with liver (Figs. 3-5). The assessment of the liver through the ELF test combined with other non-invasive diagnostic methods may provide high PPV or NPV, highlighting the stage of with a high level of confidence. Thus a number of patients can avoid liver biopsy. At present, liver biopsy is still considered as the essential method for the staging of, and the ELF test might be one of the best non-invasive diagnostic methods in the selection of patients eligible for biopsy and in the follow-up of patients who already received antiviral therapy and/or dietary supplement. Contributors: VD and AD proposed the study. MM wrote the first draft, analyzed the data and contributed to the design and interpretation of the study. All authors participated in the data monitoring. MF coordinated the study. CR is the guarantor. Funding: None. Ethical approval: the study was approved by the local ethics committee according to the Declaration of Helsinki and Good Clinical Practice guidelines. 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