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13C-methacetin-breath test compared to also noninvasive biochemical blood tests in predicting hepatic fibrosis and cirrhosis in chronic hepatitis C
Available online at www.sciencedirect.com
Digestive and Liver Disease 40 (2008) 743–748
Liver, Pancreas and Biliary Tract 13
C-methacetin-breath test compared to also noninvasive biochemical blood tests in predicting hepatic fibrosis and cirrhosis in chronic hepatitis C
L. Dinesen b , W.F. Caspary a , R.W. Chapman b , C.F. Dietrich c , C. Sarrazin a , B. Braden a,b,∗ a
Medical Department I, Johann Wolfgang Goethe University of Frankfurt/Main, Germany b John Radcliffe Hospital, Oxford, United Kingdom c Medical Department II, Caritas Hospital, Bad Mergentheim, Germany Received 6 August 2007; accepted 29 January 2008 Available online 12 March 2008
Abstract Background. The 13 C-methacetin-breath test and also several noninvasive blood tests comprising routine laboratory parameters have been proposed to predict fibrosis and cirrhosis in chronic hepatitis C. The aim of the study was to compare the diagnostic accuracy between these tests referring to hepatic histology as gold standard. Methods. 96 patients with chronic hepatitis C virus infection underwent percutaneous liver biopsy and the 13 C-methacetin-breath test. The Fibroindex, the aspartate aminotransferase to platelet ratio index , and the aspartate aminotransferase to alanine aminotransferase ratio were used as parameters for the staging of fibrosis. The main endpoint was the area under the characteristic curves for the diagnosis of advanced fibrosis (F3-F4) and cirrhosis (F4) according to the Batts Ludwig criteria. Results. ROC analysis revealed a cut-off <14.6‰ best with 92.6% sensitivity and 84.1% specificity for the 13 C-methacetin-breath test, for the Fibroindex >1.82 70.4% sensitivity and 91.3% specificity, for the aspartate aminotransferase to platelet ratio >1.0 a 66.7% sensitivity and 75.4% specificity, and for the aspartate aminotransferase to alanine aminotransferase ratio >1.0 63.0% sensitivity and 59.4% specificity in predicting liver cirrhosis. The areas under the curve for the breath test, the Fibroindex, aspartate aminotransferase to platelet ratio and the aspartate aminotransferase to alanine aminotransferase ratio were 0.958, 0.845, 0.799, and 0.688, respectively, when predicting cirrhosis. For identifying patients with advanced fibrosis, the areas under the curve were 0.827, 0.804, 0.779, and 0.561, respectively. Discordances between Fibroindex (21%), aspartate aminotransferase to platelet ratio (29%) or aspartate aminotransferase to alanine aminotransferase ratio (37.6%) and liver biopsy were significantly more frequent than between 13 C-breath test (11.6%) and liver biopsy (P < 0.05). Conclusion. The 13 C-methacetin-breath test is more reliable in predicting advanced fibrosis and cirrhosis than simple biochemical parameters (aspartate aminotransferase to platelet ratio; aspartate aminotransferase to alanine aminotransferase ratio). © 2008 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: Cirrhosis; Cytochrome P450; Liver function; Stable isotope
1. Introduction The knowledge of the stage of liver fibrosis is important for the prognosis and for decisions on antiviral treatment in patients with chronic hepatitis C infection. Treatment is ∗ Corresponding author at: Gastroenterology Department, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, United Kingdom. Tel.: +44 18 6522 1466; fax: +44 18 6575 1100. E-mail address: [email protected] (B. Braden).
recommended for patients with significant fibrosis (guidelines), which is according to current practice assessed by liver biopsy. Percutaneous liver biopsy allows histological examination of the liver tissue, but is invasive, costly, and includes a risk of complications of about 1% including death [1,2]. Furthermore, inter- and intra-observer variability and sampling error limit the diagnostic accuracy. Because of these problems associated with liver biopsies a noninvasive marker of fibrosis and cirrhosis is desirable. Ideally, such a test should be simple, readily available,
1590-8658/$30 © 2008 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. doi:10.1016/j.dld.2008.01.013
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inexpensive, and reliable and accurate in predicting liver fibrosis. Several markers or indices of readily available laboratory tests have been proposed as predictors of hepatic fibrosis. According to Wai et al. [3] the aspartate aminotransferase (AST) to platelet ratio (APRI) identifies patients with significant fibrosis and cirrhosis. The aspartate aminotransferase to alanine aminotransferase ratio (AAR) has also been used as predictor of fibrosis [4–6]. Recently, Koda et al. [7] presented a Fibroindex derived from the platelet count, AST, and gamma globulin measurements for predicting significant fibrosis. Quantitative liver function tests describe accurately the functional hepatic mass, but are cumbersome in clinical diagnostic routine as they are more complex to perform and more expensive than conventional biochemical tests. The 13 Cmethacetin-breath test (C-MBT) noninvasively also assesses the hepatic functional mass. The metabolism of the substrate 13 C-methacetin to 13 C-labelled carbon dioxide that is exhaled quantitatively evaluates the hepatic microsomal enzyme system (cytochrome P450 1A2) [7–11]. Previous studies have demonstrated that the 13 C-methacetin-breath test is reliable and accurate in predicting the severity of liver cirrhosis [8–14]. The aim of this study was to compare the diagnostic power of the 13 C-methacetin-breath test as a functional test with the simple biochemical parameters APRI, AAR, and platelets in predicting advanced stages of liver fibrosis and cirrhosis.
2. Materials and methods 2.1. Patients Ninety-six patients with chronic hepatitis C virus (HCV) infection were included in the study. The diagnosis was established by the presence of hepatitis C virus RNA using polymerase chain reaction assays. We have excluded patients with clinical evidence of liver cirrhosis, alcohol intake of more than 30 g alcohol per day, positivity for hepatitis B surface antigen or human immunodeficiency virus antibodies. Patients with autoimmune liver disease were also excluded. All patients underwent biochemical evaluation of blood samples that included full blood count, aspartate aminotransferase, alanine aminotransferase, ␥-glutamyltranspeptidase, alkaline phosphatase, total bilirubin, albumin, gamma globulin, and prothrombin activity. Routine biochemical tests were carried out using commercially available test kits. Ninety-four patients were classified grade A in the Child Pugh classification, two were graded B owing to markedly elevated bilirubin. Ultrasonographic evaluation of the abdomen including Doppler ultrasonographic measurements of the portal vein and liver veins were performed in all patients. Patients with
Table 1 Demographic, biochemical and clinical characteristics of patients with chronic hepatitis C virus infection Characteristics
Patients
No (male/female) Mean age (yr)a
96 (57/39) 48 ± 11 (23–75)
Biochemistrya,b Alanine aminotransferase (U/L) Aspartate aminotransferase (U/L) Prothrombin time Albumin (g/L) Bilirubin (mol/L)
38 ± 34 (7–230) 26 ± 16 (7–87) 92 ± 15 (50–115) 4.1 ± 0.5 (2.7–5.6) 14.7 ± 9.8 (5.1–75.2)
Mean ± standard deviation (range). Normal reference ranges: 4–22 U/L for alanine aminotransferase, 6–18 U/L for aspartate aminotransferase, >70% for prothrombin time, 32–55 g/L for albumin and 3.4–20.5 mol/L for bilirubin. a
b
a thrombosis of the portal vein, hepatic veins or an occlusion of the hepatic artery were excluded from the study. The demographic and biochemical characteristics of the patients are given in Table 1. The study was conducted according to the Declaration of Helsinki and approved by the research committee of our institute. All patients were at least 18 years old, and their informed consent was obtained. 2.2.
13 C-methacetin-breath
test
After an overnight fast, each subject ingested 75 mg 13 Cmethacetin (99% APE, Wagner-Analysentechnik, Bremen, Germany) dissolved in 200 mL water. Breath samples were collected at baseline and 15 min after ingestion of the substrate. During the test period, the patients were at rest and fasted to avoid influences due to variations in CO2 production and food intake. The 13 CO2 /12 CO2 isotope ratio in the breath samples was analysed by nondispersive isotope-selective infrared spectrometry (Wagner Analysentechnik, Worpswede, Germany). The delta (δ) values obtained after 15 min were related to the baseline δ values as delta over baseline value (DOB) 15 min [15]. In a previous study including 104 patients with various liver diseases the DOB 15 min proved to be a reliable parameter for the 13 C-methacetin-breath test in receiver operator curve analysis when compared with liver histology (92.6% sensitivity and 94.1% specificity) [16]. The technicians performing the breath tests were blinded to all clinical findings and liver biopsy results. The clinicians and pathologists were blinded to the breath test results. 2.3. Liver biopsy histology Percutaneous liver biopsy was carried out using the Menghini technique. The liver histology was classified according to the Batts Ludwig criteria on a fibrosis scale from F0 to F4: F0, no fibrosis; F1, enlarged fibrous portal tracks; F2, periportal septa; F3, fibrous septa with architectural distortion, but no cirrhosis; F4, cirrhosis [17]. The pathologist
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had no other clinical information than the patients’ diagnosis of chronic hepatitis C virus infection. 2.4. Statistics Data are shown as mean and standard deviation or median and range as appropriate. P < 0.05 was considered statistically significant. The APRI was calculated as AST/(upper limit of normal)/platelet counts (109 /L) × 100. The AAR was calculated as AST/(upper limit of normal)/ALT/(upper limit of normal). The Fibroindex was deducted according to [7] as Fibroindex = 1.738–0.064 × (platelets [×104 /L]) + 0.005 × AST [IU/L] + 0.463 gamma globulin [g/dL]. Receiver operating characteristic curves were used to define the cut-off levels with the best sensitivities and specificities for identifying the presence of cirrhosis or advanced fibrosis. The diagnostic power of the tests was assessed using the area under the receiver operating characteristic (ROC) area curve. The ROC area is a reliable measure to quantify the discriminative power of a diagnostic model [18]. A test that correctly classified all subjects would have an area of 1.0 (perfect discrimination) and a test without sufficient discriminatory value would have an area of 0.5 or less. A value >0.8 was considered to represent good discrimination. 95% confidence intervals are given for sensitivities and specificities. 2 -Test was used to compare the frequencies of discordances to liver histology between the different fibrosis tests. Statistic analyses were performed using the programme BIAS.
3. Results According to the Batts Ludwig criteria 9 patients (9.3%) had early fibrosis with enlarged fibrous portal tracks (F1), 30 patients (31.2%) showed periportal septa (F2), and 57 patients (59.4%) had advanced fibrosis (F3 and F4) with fibrous septa and architectural distortion (F3, n = 30; 31.2%) or complete cirrhosis (F4, n = 27; 28.1%). The demographic and biochemical characteristics of the patients are given in Table 1.
Fig. 1. Receiver operating characteristic curve analysis of the 13 Cmethacetin-breath test with delta over baseline values at 15 min in predicting the histological presence of liver cirrhosis (F4) (black line) and advanced fibrosis (F3 and F4) (grey line).
The area under the curve was 0.688. The receiver operating characteristic analysis of AAR is depicted in Fig. 4. Table 2 lists test qualities of all noninvasive tests in predicting the histological presence of liver cirrhosis (F4). 3.2. Predicting advanced fibrosis Receiver operator curve analysis of the 13 C-methacetinbreath test results (Fig. 1 and Fig. 5) revealed a cut-off <21‰ best with 75.4% sensitivity and 79.5% specificity for predicting advanced fibrosis. The area under the receiver operator curve was 0.827. The Fibroindex >1.35 obtained 66.7% sensitivity and 84.6% specificity as predictor of advanced fibrotic alterations. The area under the curve was 0.804 (Fig. 2).
3.1. Predicting cirrhosis Receiver operating characteristic analysis of the 13 Cmethacetin-breath test results (Fig. 1) revealed a cut-off <14.6‰ best with 92.6% sensitivity and 84.1% specificity in predicting cirrhosis. The area under the receiver operator curve was 0.957. The Fibroindex >1.82 obtained 70.4% sensitivity and 91.3% specificity as predictor of cirrhotic alterations. The area under the curve was 0.845 (Fig. 2). APRI >1.0 could predict cirrhosis with 66.7% sensitivity and 75.4% specificity. The area under the curve was 0.799. The receiver operating characteristics are presented in Fig. 3. Patients with cirrhosis could be identified with 63.0% sensitivity and 59.4% specificity using an AST to APT ratio >1.0.
Fig. 2. Receiver operating characteristic curve analysis of the Fibroindex in predicting the histological presence of liver cirrhosis (F4) (black line) and advanced fibrosis (F3 and F4) (grey line).
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Table 2 Test qualities of the noninvasive tests in predicting the histological presence of liver cirrhosis (F4) or advanced fibrosis (F3 and F4) 13 C-methacetin-breath
Fibroindex
APRI
AAR
92.6 (75.7–99.1) 84.1 (73.3–91.8) 69.4 (51.9–83.7) 96.7 (88.5–99.6) 86.5 (78.0–92.3)
70.4 (49.8–86.3) 91.3 (82.0–96.7) 76.0 (54.9–90.6) 88.7 (79.0–95.0) 85.4 (76.7–91.8)
66.7 (46.0–83.5) 75.4 (63.5–85.0) 51.4 (34.0–68.6) 85.3 (73.8–93.0) 72.9 (62.9–81.5)
63.0 (42.4–80.6) 59.4 (46.9–71.1) 37.8 (23.8–53.5) 80.4 (66.9–90.2) 60.4 (49.9–70.3)
Predicting advanced fibrosis (F3 and F4) Sensitivity [%] 75.4 (62.2–85.9) Specificity [%] 79.5 (63.5–90.7) Positive predictive value 84.3 (71.4–93.0) Negative predictive value 68.9 (53.4–81.8) Accuracy 77.1 (67.4–85.1)
66.7 (52.9–78.6) 84.6 (69.5–94.1) 86.4 (72.6–94.8) 63.5 (49.0–76.4) 74.0 (64.0–82.4)
64.9 (51.1–77.1) 84.6 (69.5–94.1) 86.1 (72.1–94.7) 62.3 (47.9–75.2) 72.9 (62.9–81.5)
70.2 (56.6–81.6) 48.7 (32.4–65.2) 66.7 (53.3–78.3) 52.8 (35.5–69.6) 61.5 (51.0–71.2)
Predicting cirrhosis (F4) Sensitivity [%] Specificity [%] Positive predictive value Negative predictive value Accuracy
test
95% confidence intervals are given in brackets.
Fig. 5. 13 C-methacetin-breath test results as delta over baseline values at 15 min in patients with chronic hepatitis C virus infection in different stages of fibrosis. The horizontal lines present the median delta over baseline at 15 min in each fibrosis group. Fig. 3. Receiver operating characteristic curve analysis of the AST to platelets ratio in predicting the histological presence of liver cirrhosis (F4) (black line) and advanced fibrosis (F3 and F4) (grey line).
APRI >0.75 could predict advanced fibrosis with 64.9% sensitivity and 84.6% specificity. The area under the curve was 0.779. The receiver operating characteristics are presented in Fig. 3. Patients with advanced fibrosis could be identified with 70.2% sensitivity and 48.7% specificity using an AST to APT ratio >0.85. The area under the curve was 0.561. The receiver operating characteristic analysis of AAR is depicted in Fig. 4. The test performances of the noninvasive tests in predicting the histological presence of advanced fibrosis (F3 and F4) are given in Table 2. In total, discordances between Fibroindex (21.0%), APRI (29%) or AAR (37.6%) and liver biopsy were significantly more frequent than between 13 C-breath test (11.6%) and liver biopsy (P < 0.05).
4. Discussion
Fig. 4. Receiver operating characteristic curve analysis of the AAR in predicting the histological presence of liver cirrhosis (F4) (black line) and advanced fibrosis (F3 and F4) (grey line).
Liver biopsy is recommended before starting antiviral treatment, particularly for patients with hepatitis C virus genotype 1 infection. However, it implies a risk of complication, and it is limited by sampling errors. Therefore, several noninvasive tests comprising routine laboratory parameters
L. Dinesen et al. / Digestive and Liver Disease 40 (2008) 743–748
have been proposed to predict fibrosis in chronic HCV in order to avoid liver biopsy. These simple biochemical blood tests base on liver enzymes, platelets, and gamma globulins. In chronic hepatitis C virus infection the APRI, the AAR and the Fibroindex identify patients with advanced stages of fibrosis or cirrhosis [3,7,19–22]. We have previously demonstrated that breath testing with 13 C-labelled methacetin provide a safe, noninvasive means for evaluating hepatic metabolism that is correlated with liver histology [13]. However, all these noninvasive tests base on different pathogenetic mechanisms. 13 C-methacetin-breath tests measure the functional metabolic capacity of the residual hepatic cells and indirectly reflect how many hepatic cells are already lost. Increased AST and ALT, on the other hand, indicate the acute damage of hepatic cells. Decreased platelets might mainly result from hypersplenism due to portal hypertension. Elevated gamma globulins might be explained by shunting of bacterial antigens away from the liver to lymphoid tissue. This means that different surrogates are used, which indirectly reflect the difficult pathophenomenon of fibrosis. Each test can assess only one aspect of the complex process of fibrosis. In fact, no single biochemical test can be considered as a sensitive index of the overall hepatic function and is able alone to predict the severity and prognosis of hepatic diseases, whether acute or chronic. Therefore, combining different tests in indices appears a promising approach. We observed significantly more frequent discordances between APRI or AAR (29%, 37.6%) and liver biopsies than between 13 C-breath test (11.6%) and liver biopsy (P < 0.005). The Fibroindex also showed more discordances compared with histology (21.0%) than the 13 C-methacetin-breath test (P < 0.05). The 13 C-methacetin-breath test is more expensive than the biochemical markers (about 16 Euro substrate costs), but it is also widely available. The breath samples can be sent by mail to specialized centres or can be directly analysed as bedside test by nondispersive isotope-selective infrared spectrometers. The 13 C-methacetin-breath test is more practicable than other 13 C-based liver function tests because it can be performed as a simple two-point measurement as known from the 13 C-urea breath test [16]. The rapid metabolism of methacetin allows a short sampling interval for the breath test. On the other hand, methacetin undergoes a high hepatic extraction rate, which makes the methacetin breath test susceptible to variations in hepatic blood flow [10]. Special fibrotic markers, such as serum hyaluronic acid, type IV collagen, type IV collagen 7s domain, and P III P, have also been reported to predict liver fibrosis and cirrhosis [19,23]. However, their measurement is less standardized and expensive. Serum hyaluronic acid seems to be the best of these markers and reliably identifies cirrhosis, but its diagnostic value for less advanced fibrosis is not significantly
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higher than that of the Fibroindex [7]. Another disadvantage in the hyaluronic acid measurement is that diet and a history of gastrectomy influence the results. Fibrosis scores, which include the measurement of hyaluronic acid (i.e. FibroSpect [24]) have the same restrictions. The 13 C-methacetin-breath test reliably detected cirrhosis. Therefore, it might be a helpful diagnostic tool to identify patients that are candidates for regular screening for hepatocellular cancer and the development of varices. In conclusion, the 13 C-methacetin-breath test is more reliable in predicting advanced fibrosis and cirrhosis than the simple biochemical parameters (Fibroindex, APRI, AAR). The 13 C-methacetin-breath test is a safe and precise quantitative liver function test. The Fibroindex and the 13 Cmethacetin-breath test might serve as surrogate markers for advanced fibrosis and cirrhosis and can help to avoid liver biopsies.
Practice points • The Fibroindex and the 13 C-methacetinbreath test indicate the grade of hepatic fibrosis in patients with chronic hepatitis C virus infection. • The 13 C-methacetin-breath test is superior to simple biochemical indices in predicting advanced fibrosis and cirrhosis. • The modification of the 13 C-methacetin as a simple two-point measurement at baseline and after 15 min reliably reflects the hepatic metabolic functional capacity.
Research agenda • As the Fibroindex and the 13 C-methacetin reflect the hepatic grade of fibrosis they might serve as surrogate markers and might help to avoid liver biopsies.
Conflict of interest statement None declared.
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[3] Wai CT, Greenson JK, Fontana RJ, Kalbfleisch JD, Marrero JA, Conjeevaram HS, et al. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology 2003;38:518–26. [4] Sheth SG, Flamm SL, Gordon FD, Chopra S. AST/ALT ratio predicts cirrhosis in patients with chronic hepatitis C virus infection. Am J Gastroenterol 1998;93:44–8. [5] Imperiale TF, Born LJ. Clinical utility of the AST/ALT ratio in chronic hepatitis C. Am J Gastroenterol 2001;96:919–20. [6] Park G, Jones DB, Katelaris P. Value of AST/ALT ratio as fibrotic predictor in chronic hepatitis C. Am J Gastroenterol 2005;100:1623–4 [author reply 1624]. [7] Koda M, Matunaga Y, Kawakami M, Kishimoto Y, Suou T, Murawaki Y. Fibroindex, a practical index for predicting significant fibrosis in patients with chronic hepatitis C. Hepatology 2007;45: 297–306. [8] Adamek RJ, Goetze O, Boedeker C, Pfaffenbach B, Luypaerts A, Geypens B. 13 C-methacetin breath test: isotope-selective nondispersive infrared spectrometry in comparison to isotope ratio mass spectrometry in volunteers and patients with liver cirrhosis. Z Gastroenterol 1999;37:1139–43. [9] Pfaffenbach B, Gotze O, Szymanski C, Hagemann D, Adamek RJ. The 13 C-methacetin breath test for quantitative noninvasive liver function analysis with an isotope-specific nondispersive infrared spectrometer in liver cirrhosis. Dtsch Med Wochenschr 1998;123:1467–71. [10] Klatt S, Taut C, Mayer D, Adler G, Beckh K. Evaluation of the 13 C-methacetin breath test for quantitative liver function testing. Z Gastroenterol 1997;35:609–14. [11] Matsumoto K, Suehiro M, Iio M, Kawabe T, Shiratori Y, Okano K, et al. [13 C]methacetin breath test for evaluation of liver damage. Dig Dis Sci 1987;32:344–8. [12] Holtmeier J, Leuschner M, Schneider A, Leuschner U, Caspary WF, Braden B. 13 C-methacetin and 13 C-galactose breath tests can assess restricted liver function even in early stages of primary biliary cirrhosis. Scand J Gastroenterol 2006;41:1336–41. [13] Braden B, Faust D, Sarrazin U, Zeuzem S, Dietrich CF, Caspary WF, et al. 13 C-methacetin breath test as liver function test in patients with chronic hepatitis C virus infection. Aliment Pharmacol Ther 2005;21:179–85. [14] Festi D, Capodicasa S, Sandri L, Colaiocco-Ferrante L, Staniscia T, Vitacolonna E, et al. Measurement of hepatic functional mass by means
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Liver, Pancreas and Biliary Tract 13
C-methacetin-breath test compared to also noninvasive biochemical blood tests in predicting hepatic fibrosis and cirrhosis in chronic hepatitis C
L. Dinesen b , W.F. Caspary a , R.W. Chapman b , C.F. Dietrich c , C. Sarrazin a , B. Braden a,b,∗ a
Medical Department I, Johann Wolfgang Goethe University of Frankfurt/Main, Germany b John Radcliffe Hospital, Oxford, United Kingdom c Medical Department II, Caritas Hospital, Bad Mergentheim, Germany Received 6 August 2007; accepted 29 January 2008 Available online 12 March 2008
Abstract Background. The 13 C-methacetin-breath test and also several noninvasive blood tests comprising routine laboratory parameters have been proposed to predict fibrosis and cirrhosis in chronic hepatitis C. The aim of the study was to compare the diagnostic accuracy between these tests referring to hepatic histology as gold standard. Methods. 96 patients with chronic hepatitis C virus infection underwent percutaneous liver biopsy and the 13 C-methacetin-breath test. The Fibroindex, the aspartate aminotransferase to platelet ratio index , and the aspartate aminotransferase to alanine aminotransferase ratio were used as parameters for the staging of fibrosis. The main endpoint was the area under the characteristic curves for the diagnosis of advanced fibrosis (F3-F4) and cirrhosis (F4) according to the Batts Ludwig criteria. Results. ROC analysis revealed a cut-off <14.6‰ best with 92.6% sensitivity and 84.1% specificity for the 13 C-methacetin-breath test, for the Fibroindex >1.82 70.4% sensitivity and 91.3% specificity, for the aspartate aminotransferase to platelet ratio >1.0 a 66.7% sensitivity and 75.4% specificity, and for the aspartate aminotransferase to alanine aminotransferase ratio >1.0 63.0% sensitivity and 59.4% specificity in predicting liver cirrhosis. The areas under the curve for the breath test, the Fibroindex, aspartate aminotransferase to platelet ratio and the aspartate aminotransferase to alanine aminotransferase ratio were 0.958, 0.845, 0.799, and 0.688, respectively, when predicting cirrhosis. For identifying patients with advanced fibrosis, the areas under the curve were 0.827, 0.804, 0.779, and 0.561, respectively. Discordances between Fibroindex (21%), aspartate aminotransferase to platelet ratio (29%) or aspartate aminotransferase to alanine aminotransferase ratio (37.6%) and liver biopsy were significantly more frequent than between 13 C-breath test (11.6%) and liver biopsy (P < 0.05). Conclusion. The 13 C-methacetin-breath test is more reliable in predicting advanced fibrosis and cirrhosis than simple biochemical parameters (aspartate aminotransferase to platelet ratio; aspartate aminotransferase to alanine aminotransferase ratio). © 2008 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. Keywords: Cirrhosis; Cytochrome P450; Liver function; Stable isotope
1. Introduction The knowledge of the stage of liver fibrosis is important for the prognosis and for decisions on antiviral treatment in patients with chronic hepatitis C infection. Treatment is ∗ Corresponding author at: Gastroenterology Department, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, United Kingdom. Tel.: +44 18 6522 1466; fax: +44 18 6575 1100. E-mail address: [email protected] (B. Braden).
recommended for patients with significant fibrosis (guidelines), which is according to current practice assessed by liver biopsy. Percutaneous liver biopsy allows histological examination of the liver tissue, but is invasive, costly, and includes a risk of complications of about 1% including death [1,2]. Furthermore, inter- and intra-observer variability and sampling error limit the diagnostic accuracy. Because of these problems associated with liver biopsies a noninvasive marker of fibrosis and cirrhosis is desirable. Ideally, such a test should be simple, readily available,
1590-8658/$30 © 2008 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l. doi:10.1016/j.dld.2008.01.013
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inexpensive, and reliable and accurate in predicting liver fibrosis. Several markers or indices of readily available laboratory tests have been proposed as predictors of hepatic fibrosis. According to Wai et al. [3] the aspartate aminotransferase (AST) to platelet ratio (APRI) identifies patients with significant fibrosis and cirrhosis. The aspartate aminotransferase to alanine aminotransferase ratio (AAR) has also been used as predictor of fibrosis [4–6]. Recently, Koda et al. [7] presented a Fibroindex derived from the platelet count, AST, and gamma globulin measurements for predicting significant fibrosis. Quantitative liver function tests describe accurately the functional hepatic mass, but are cumbersome in clinical diagnostic routine as they are more complex to perform and more expensive than conventional biochemical tests. The 13 Cmethacetin-breath test (C-MBT) noninvasively also assesses the hepatic functional mass. The metabolism of the substrate 13 C-methacetin to 13 C-labelled carbon dioxide that is exhaled quantitatively evaluates the hepatic microsomal enzyme system (cytochrome P450 1A2) [7–11]. Previous studies have demonstrated that the 13 C-methacetin-breath test is reliable and accurate in predicting the severity of liver cirrhosis [8–14]. The aim of this study was to compare the diagnostic power of the 13 C-methacetin-breath test as a functional test with the simple biochemical parameters APRI, AAR, and platelets in predicting advanced stages of liver fibrosis and cirrhosis.
2. Materials and methods 2.1. Patients Ninety-six patients with chronic hepatitis C virus (HCV) infection were included in the study. The diagnosis was established by the presence of hepatitis C virus RNA using polymerase chain reaction assays. We have excluded patients with clinical evidence of liver cirrhosis, alcohol intake of more than 30 g alcohol per day, positivity for hepatitis B surface antigen or human immunodeficiency virus antibodies. Patients with autoimmune liver disease were also excluded. All patients underwent biochemical evaluation of blood samples that included full blood count, aspartate aminotransferase, alanine aminotransferase, ␥-glutamyltranspeptidase, alkaline phosphatase, total bilirubin, albumin, gamma globulin, and prothrombin activity. Routine biochemical tests were carried out using commercially available test kits. Ninety-four patients were classified grade A in the Child Pugh classification, two were graded B owing to markedly elevated bilirubin. Ultrasonographic evaluation of the abdomen including Doppler ultrasonographic measurements of the portal vein and liver veins were performed in all patients. Patients with
Table 1 Demographic, biochemical and clinical characteristics of patients with chronic hepatitis C virus infection Characteristics
Patients
No (male/female) Mean age (yr)a
96 (57/39) 48 ± 11 (23–75)
Biochemistrya,b Alanine aminotransferase (U/L) Aspartate aminotransferase (U/L) Prothrombin time Albumin (g/L) Bilirubin (mol/L)
38 ± 34 (7–230) 26 ± 16 (7–87) 92 ± 15 (50–115) 4.1 ± 0.5 (2.7–5.6) 14.7 ± 9.8 (5.1–75.2)
Mean ± standard deviation (range). Normal reference ranges: 4–22 U/L for alanine aminotransferase, 6–18 U/L for aspartate aminotransferase, >70% for prothrombin time, 32–55 g/L for albumin and 3.4–20.5 mol/L for bilirubin. a
b
a thrombosis of the portal vein, hepatic veins or an occlusion of the hepatic artery were excluded from the study. The demographic and biochemical characteristics of the patients are given in Table 1. The study was conducted according to the Declaration of Helsinki and approved by the research committee of our institute. All patients were at least 18 years old, and their informed consent was obtained. 2.2.
13 C-methacetin-breath
test
After an overnight fast, each subject ingested 75 mg 13 Cmethacetin (99% APE, Wagner-Analysentechnik, Bremen, Germany) dissolved in 200 mL water. Breath samples were collected at baseline and 15 min after ingestion of the substrate. During the test period, the patients were at rest and fasted to avoid influences due to variations in CO2 production and food intake. The 13 CO2 /12 CO2 isotope ratio in the breath samples was analysed by nondispersive isotope-selective infrared spectrometry (Wagner Analysentechnik, Worpswede, Germany). The delta (δ) values obtained after 15 min were related to the baseline δ values as delta over baseline value (DOB) 15 min [15]. In a previous study including 104 patients with various liver diseases the DOB 15 min proved to be a reliable parameter for the 13 C-methacetin-breath test in receiver operator curve analysis when compared with liver histology (92.6% sensitivity and 94.1% specificity) [16]. The technicians performing the breath tests were blinded to all clinical findings and liver biopsy results. The clinicians and pathologists were blinded to the breath test results. 2.3. Liver biopsy histology Percutaneous liver biopsy was carried out using the Menghini technique. The liver histology was classified according to the Batts Ludwig criteria on a fibrosis scale from F0 to F4: F0, no fibrosis; F1, enlarged fibrous portal tracks; F2, periportal septa; F3, fibrous septa with architectural distortion, but no cirrhosis; F4, cirrhosis [17]. The pathologist
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had no other clinical information than the patients’ diagnosis of chronic hepatitis C virus infection. 2.4. Statistics Data are shown as mean and standard deviation or median and range as appropriate. P < 0.05 was considered statistically significant. The APRI was calculated as AST/(upper limit of normal)/platelet counts (109 /L) × 100. The AAR was calculated as AST/(upper limit of normal)/ALT/(upper limit of normal). The Fibroindex was deducted according to [7] as Fibroindex = 1.738–0.064 × (platelets [×104 /L]) + 0.005 × AST [IU/L] + 0.463 gamma globulin [g/dL]. Receiver operating characteristic curves were used to define the cut-off levels with the best sensitivities and specificities for identifying the presence of cirrhosis or advanced fibrosis. The diagnostic power of the tests was assessed using the area under the receiver operating characteristic (ROC) area curve. The ROC area is a reliable measure to quantify the discriminative power of a diagnostic model [18]. A test that correctly classified all subjects would have an area of 1.0 (perfect discrimination) and a test without sufficient discriminatory value would have an area of 0.5 or less. A value >0.8 was considered to represent good discrimination. 95% confidence intervals are given for sensitivities and specificities. 2 -Test was used to compare the frequencies of discordances to liver histology between the different fibrosis tests. Statistic analyses were performed using the programme BIAS.
3. Results According to the Batts Ludwig criteria 9 patients (9.3%) had early fibrosis with enlarged fibrous portal tracks (F1), 30 patients (31.2%) showed periportal septa (F2), and 57 patients (59.4%) had advanced fibrosis (F3 and F4) with fibrous septa and architectural distortion (F3, n = 30; 31.2%) or complete cirrhosis (F4, n = 27; 28.1%). The demographic and biochemical characteristics of the patients are given in Table 1.
Fig. 1. Receiver operating characteristic curve analysis of the 13 Cmethacetin-breath test with delta over baseline values at 15 min in predicting the histological presence of liver cirrhosis (F4) (black line) and advanced fibrosis (F3 and F4) (grey line).
The area under the curve was 0.688. The receiver operating characteristic analysis of AAR is depicted in Fig. 4. Table 2 lists test qualities of all noninvasive tests in predicting the histological presence of liver cirrhosis (F4). 3.2. Predicting advanced fibrosis Receiver operator curve analysis of the 13 C-methacetinbreath test results (Fig. 1 and Fig. 5) revealed a cut-off <21‰ best with 75.4% sensitivity and 79.5% specificity for predicting advanced fibrosis. The area under the receiver operator curve was 0.827. The Fibroindex >1.35 obtained 66.7% sensitivity and 84.6% specificity as predictor of advanced fibrotic alterations. The area under the curve was 0.804 (Fig. 2).
3.1. Predicting cirrhosis Receiver operating characteristic analysis of the 13 Cmethacetin-breath test results (Fig. 1) revealed a cut-off <14.6‰ best with 92.6% sensitivity and 84.1% specificity in predicting cirrhosis. The area under the receiver operator curve was 0.957. The Fibroindex >1.82 obtained 70.4% sensitivity and 91.3% specificity as predictor of cirrhotic alterations. The area under the curve was 0.845 (Fig. 2). APRI >1.0 could predict cirrhosis with 66.7% sensitivity and 75.4% specificity. The area under the curve was 0.799. The receiver operating characteristics are presented in Fig. 3. Patients with cirrhosis could be identified with 63.0% sensitivity and 59.4% specificity using an AST to APT ratio >1.0.
Fig. 2. Receiver operating characteristic curve analysis of the Fibroindex in predicting the histological presence of liver cirrhosis (F4) (black line) and advanced fibrosis (F3 and F4) (grey line).
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Table 2 Test qualities of the noninvasive tests in predicting the histological presence of liver cirrhosis (F4) or advanced fibrosis (F3 and F4) 13 C-methacetin-breath
Fibroindex
APRI
AAR
92.6 (75.7–99.1) 84.1 (73.3–91.8) 69.4 (51.9–83.7) 96.7 (88.5–99.6) 86.5 (78.0–92.3)
70.4 (49.8–86.3) 91.3 (82.0–96.7) 76.0 (54.9–90.6) 88.7 (79.0–95.0) 85.4 (76.7–91.8)
66.7 (46.0–83.5) 75.4 (63.5–85.0) 51.4 (34.0–68.6) 85.3 (73.8–93.0) 72.9 (62.9–81.5)
63.0 (42.4–80.6) 59.4 (46.9–71.1) 37.8 (23.8–53.5) 80.4 (66.9–90.2) 60.4 (49.9–70.3)
Predicting advanced fibrosis (F3 and F4) Sensitivity [%] 75.4 (62.2–85.9) Specificity [%] 79.5 (63.5–90.7) Positive predictive value 84.3 (71.4–93.0) Negative predictive value 68.9 (53.4–81.8) Accuracy 77.1 (67.4–85.1)
66.7 (52.9–78.6) 84.6 (69.5–94.1) 86.4 (72.6–94.8) 63.5 (49.0–76.4) 74.0 (64.0–82.4)
64.9 (51.1–77.1) 84.6 (69.5–94.1) 86.1 (72.1–94.7) 62.3 (47.9–75.2) 72.9 (62.9–81.5)
70.2 (56.6–81.6) 48.7 (32.4–65.2) 66.7 (53.3–78.3) 52.8 (35.5–69.6) 61.5 (51.0–71.2)
Predicting cirrhosis (F4) Sensitivity [%] Specificity [%] Positive predictive value Negative predictive value Accuracy
test
95% confidence intervals are given in brackets.
Fig. 5. 13 C-methacetin-breath test results as delta over baseline values at 15 min in patients with chronic hepatitis C virus infection in different stages of fibrosis. The horizontal lines present the median delta over baseline at 15 min in each fibrosis group. Fig. 3. Receiver operating characteristic curve analysis of the AST to platelets ratio in predicting the histological presence of liver cirrhosis (F4) (black line) and advanced fibrosis (F3 and F4) (grey line).
APRI >0.75 could predict advanced fibrosis with 64.9% sensitivity and 84.6% specificity. The area under the curve was 0.779. The receiver operating characteristics are presented in Fig. 3. Patients with advanced fibrosis could be identified with 70.2% sensitivity and 48.7% specificity using an AST to APT ratio >0.85. The area under the curve was 0.561. The receiver operating characteristic analysis of AAR is depicted in Fig. 4. The test performances of the noninvasive tests in predicting the histological presence of advanced fibrosis (F3 and F4) are given in Table 2. In total, discordances between Fibroindex (21.0%), APRI (29%) or AAR (37.6%) and liver biopsy were significantly more frequent than between 13 C-breath test (11.6%) and liver biopsy (P < 0.05).
4. Discussion
Fig. 4. Receiver operating characteristic curve analysis of the AAR in predicting the histological presence of liver cirrhosis (F4) (black line) and advanced fibrosis (F3 and F4) (grey line).
Liver biopsy is recommended before starting antiviral treatment, particularly for patients with hepatitis C virus genotype 1 infection. However, it implies a risk of complication, and it is limited by sampling errors. Therefore, several noninvasive tests comprising routine laboratory parameters
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have been proposed to predict fibrosis in chronic HCV in order to avoid liver biopsy. These simple biochemical blood tests base on liver enzymes, platelets, and gamma globulins. In chronic hepatitis C virus infection the APRI, the AAR and the Fibroindex identify patients with advanced stages of fibrosis or cirrhosis [3,7,19–22]. We have previously demonstrated that breath testing with 13 C-labelled methacetin provide a safe, noninvasive means for evaluating hepatic metabolism that is correlated with liver histology [13]. However, all these noninvasive tests base on different pathogenetic mechanisms. 13 C-methacetin-breath tests measure the functional metabolic capacity of the residual hepatic cells and indirectly reflect how many hepatic cells are already lost. Increased AST and ALT, on the other hand, indicate the acute damage of hepatic cells. Decreased platelets might mainly result from hypersplenism due to portal hypertension. Elevated gamma globulins might be explained by shunting of bacterial antigens away from the liver to lymphoid tissue. This means that different surrogates are used, which indirectly reflect the difficult pathophenomenon of fibrosis. Each test can assess only one aspect of the complex process of fibrosis. In fact, no single biochemical test can be considered as a sensitive index of the overall hepatic function and is able alone to predict the severity and prognosis of hepatic diseases, whether acute or chronic. Therefore, combining different tests in indices appears a promising approach. We observed significantly more frequent discordances between APRI or AAR (29%, 37.6%) and liver biopsies than between 13 C-breath test (11.6%) and liver biopsy (P < 0.005). The Fibroindex also showed more discordances compared with histology (21.0%) than the 13 C-methacetin-breath test (P < 0.05). The 13 C-methacetin-breath test is more expensive than the biochemical markers (about 16 Euro substrate costs), but it is also widely available. The breath samples can be sent by mail to specialized centres or can be directly analysed as bedside test by nondispersive isotope-selective infrared spectrometers. The 13 C-methacetin-breath test is more practicable than other 13 C-based liver function tests because it can be performed as a simple two-point measurement as known from the 13 C-urea breath test [16]. The rapid metabolism of methacetin allows a short sampling interval for the breath test. On the other hand, methacetin undergoes a high hepatic extraction rate, which makes the methacetin breath test susceptible to variations in hepatic blood flow [10]. Special fibrotic markers, such as serum hyaluronic acid, type IV collagen, type IV collagen 7s domain, and P III P, have also been reported to predict liver fibrosis and cirrhosis [19,23]. However, their measurement is less standardized and expensive. Serum hyaluronic acid seems to be the best of these markers and reliably identifies cirrhosis, but its diagnostic value for less advanced fibrosis is not significantly
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higher than that of the Fibroindex [7]. Another disadvantage in the hyaluronic acid measurement is that diet and a history of gastrectomy influence the results. Fibrosis scores, which include the measurement of hyaluronic acid (i.e. FibroSpect [24]) have the same restrictions. The 13 C-methacetin-breath test reliably detected cirrhosis. Therefore, it might be a helpful diagnostic tool to identify patients that are candidates for regular screening for hepatocellular cancer and the development of varices. In conclusion, the 13 C-methacetin-breath test is more reliable in predicting advanced fibrosis and cirrhosis than the simple biochemical parameters (Fibroindex, APRI, AAR). The 13 C-methacetin-breath test is a safe and precise quantitative liver function test. The Fibroindex and the 13 Cmethacetin-breath test might serve as surrogate markers for advanced fibrosis and cirrhosis and can help to avoid liver biopsies.
Practice points • The Fibroindex and the 13 C-methacetinbreath test indicate the grade of hepatic fibrosis in patients with chronic hepatitis C virus infection. • The 13 C-methacetin-breath test is superior to simple biochemical indices in predicting advanced fibrosis and cirrhosis. • The modification of the 13 C-methacetin as a simple two-point measurement at baseline and after 15 min reliably reflects the hepatic metabolic functional capacity.
Research agenda • As the Fibroindex and the 13 C-methacetin reflect the hepatic grade of fibrosis they might serve as surrogate markers and might help to avoid liver biopsies.
Conflict of interest statement None declared.
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