Research Article
Prevalence of occult hepatitis C infection in chronic hemodialysis and kidney transplant patients Seema Baid-Agrawal1,⇑, Ralf Schindler1, Petra Reinke1, Adrienne Staedtler2, Sunda Rimpler3, Barbara Malik4, Ulrich Frei1, Thomas Berg5 1
Dept of Nephrology and Medical Intensive Care, Campus Virchow Klinikum, Charite Universitaetsmedizin Berlin, Germany; 2Dept of Internal Medicine, Sana Klinikum Lichtenberg, Berlin, Germany; 3Dept of Internal Medicine, Vivantes Wenckebach Klinikum, Berlin, Germany; 4Dept of Gastroenterology and Hepatology, Campus Virchow Klinikum, Charite Universitaetsmedizin Berlin, Germany; 5 Dept of Hepatology, Universitaetsklinikum Leipzig, Leipzig, Germany
Background & Aims: Detection of hepatitis C virus (HCV) RNA in peripheral blood mononuclear cells (PBMC) and/or hepatocytes in absence of HCV RNA in serum, designated as ‘occult HCV infection’, has been a matter of controversy in recent years. We investigated for the first time the prevalence of occult HCV infection in large cohorts of chronic hemodialysis (CHD) and kidney transplant (KTx) patients. Methods: We enrolled 417 CHD patients, 417 KTx recipients and 2 control groups – 25 anti-HCV (antibody against HCV)-positive and HCV RNA-positive patients with chronic hepatitis C, and 40 antiHCV-, HCV RNA-, and HBsAg-negative healthy subjects. HCV RNA was tested in serum and PBMC using a sensitive commercial assay. Results: In CHD patients, the prevalence of anti-HCV was 3.6% (15/417) and of positive serum HCV RNA 2.4% (10/417). HCV RNA was detected in PBMC in 1/407 (0.25%) HCV serum RNA-negative patients (‘‘occult HCV infection’’). In KTx recipients, prevalence of anti-HCV was 4.8% (20/417) and of positive serum HCV RNA 4.6% (19/417). Occult HCV infection was found in 2/398 (0.5%) serum HCV RNA-negative patients. On a mean longitudinal follow-up of 30 months of the 3 patients with occult HCV infection, there was no clinical or virological evidence of HCV infection. Conclusions: The prevalence of occult HCV infection was very low in our CHD and KTx patients, and it did not appear to be clinically relevant. Further studies in geographic populations with high HCV endemicity are required to clarify the significance of occult HCV infection in these patient groups. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Occult hepatitis C; HCV; PBMC; Peripheral blood mononuclear cells; Prevalence; Kidney transplant; Hemodialysis; HCV RNA; Anti-HCV; TMA. Received 27 September 2013; received in revised form 13 January 2014; accepted 13 January 2014; available online 18 January 2014 ⇑ Corresponding author. Address: Dept of Nephrology and Medical Intensive Care, Campus Virchow-Klinikum, Charite Universitatsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: +49 30 450 553 373; fax: +49 30 450 553 916. E-mail address: [email protected] (S. Baid-Agrawal). Abbreviations: HCV, Hepatitis C; Anti-HCV, antibody against HCV; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; CHD, chronic hemodialysis; KTx, kidney transplant; TMA, transcription-mediated amplification.
Introduction Chronic infection with hepatitis C virus (HCV) is a serious public health problem associated with increased morbidity and mortality, affecting approximately 2.8% of the world’s population [1,2]. The diagnosis of HCV is made currently by detection of antiHCV (antibody against HCV) and HCV RNA in serum [2,3]. Interestingly, in recent years, HCV RNA has been detected in nonserum reservoirs such as peripheral blood mononuclear cells (PBMC) and/or hepatocytes even in the absence of HCV RNA in serum, which has raised the controversial issue of an intriguing but not unequivocally accepted entity designated as ‘‘occult HCV infection’’ [4–10]. As the presence of viral replicating ability in PBMC could possibly lead to HCV spread, patients with occult HCV infection may be potentially infectious. Clinically significant occult HCV infection was first described in 2004 in patients with persistently elevated liver function tests and who had negative anti-HCV and HCV RNA in serum, but positive HCV RNA in PBMC [4]. The existence of this entity was supported by subsequent detection of low concentrations of HCV RNA in PBMC in immunocompetent patients who cleared of HCV either spontaneously or after treatment [5–10]. Occult HCV infection in liver tissue/PBMC has been described in two clinical settings: (1) In patients with liver disease of unknown origin (anti-HCV-negative, serum HCV RNA negative, persistently elevated liver transaminases) [4,11,12], and (2) In patients with spontaneous/treatment-induced HCV RNA clearance from serum (anti-HCV-positive, serum HCV RNA-negative, normal liver transaminases) [13,14]. Recently, occult HCV infection was also reported in an apparently disease-free state in absence of liver disease, anti-HCV and HCV RNA in serum [15,16]. However, several other investigators have failed to detect HCV RNA in PBMC in various clinical settings such as cryptogenic liver disease, HCVassociated systemic vasculitis, or connective tissue disease [17], and in patients with resolved HCV infection, mixed cryoglobulinemia, onco-hematological disease or HIV infection [13,18–21]. Therefore, the jury is still out on the existence and significance of occult HCV infection. We hypothesized that if HCV RNA does persist at low levels in PBMC, then it should be more easily detectable in patients with
Journal of Hepatology 2014 vol. 60 j 928–933
JOURNAL OF HEPATOLOGY impaired immune function such as those on chronic hemodialysis (CHD) [22] and kidney transplant (KTx) recipients than in immunocompetent patients. Therefore, these patients would constitute an ideal model to explore the existence of occult HCV infection, all the more so as the presence of occult HCV infection may have deleterious consequences in these high-risk groups of patients. In this study, we aimed to investigate for the first time the prevalence of occult HCV infection in PBMC in large cohorts of CHD and KTx patients. As serum transaminases may be normal in these patients even in the presence of liver disease [23,24], we included all consecutive CHD and KTx patients irrespective of their liver function.
HCV RNA detection by Transcription-Mediated Amplification (TMA) The thawed samples of both serum and PBMC were tested for HCV RNA in batches using Versant HCV RNA qualitative TMA assay (Siemens Healthcare Diagnostics, Eschborn, Germany), according to the manufacturer’s instructions and with all due precautions for prevention of contamination. The test was performed using 500 ll of serum and a minimum of 2.5 million PBMC. The Versant TMA assay is a highly sensitive test with a detection limit of 9.6 IU/ml for HCV RNA. Negative and positive controls were simultaneously tested in each run. A repeat confirmatory testing was performed in all samples that were positive for occult HCV in different runs to exclude contamination occurrence.
Materials and methods
Statistical analyses
Study design
To determine the prevalence with an accuracy of 5% (95% confidence interval), we calculated the required sample size before the initiation of the study to be 400 patients for each group. Results of continuous measured data are presented as means (standard deviation, SD) and categorical variables are expressed as proportion, if not stated otherwise.
Both cross-sectional and longitudinal.
Anti-HCV detection Anti-HCV was tested using either the chemiluminescence microparticle immunoassay ARCHITECT anti-HCV (Abbott Diagnostics, Wiesbaden, Germany) or first generation Elecys chemiluminescence anti-HCV assay (Roche Diagnostics, Mannheim, Germany).
Setting and participants We enrolled two cohorts of patients on CHD and KTx recipients and two control groups as follows: CHD patients 417 consecutive clinically stable adult patients undergoing chronic hemodialysis at six dialysis centers in Berlin between August 2009 and May 2010. KTx recipients 417 clinically stable adult KTx recipients followed in our outpatient transplant clinic between May 2010 and March 2011.
Results Patient characteristics The demographic and baseline characteristics in the two study cohorts and two control groups are shown in Table 1 and their virological results in Table 2. Prevalence of HCV infection in CHD and KTx Patients
Positive controls 25 contemporaneous anti-HCV-positive, HCV serum RNA-positive non-transplant patients with chronic HCV infection without cirrhosis, and who had not yet been initiated on antiviral therapy. Negative controls 40 healthy asymptomatic volunteers taking no medications with normal liver function tests and no known history of HCV or hepatitis B infection, and who were anti-HCV-, HCV serum RNA-, and HBsAg-negative. The study protocol was in accordance with the ethical guidelines of the Declaration of Helsinki and was approved by our institution’s Ethics Committee. Fully informed written consent was obtained from all participants before enrolment. Demographic and clinical data were collected from the electronic medical records. Definition Occult HCV infection was defined as presence of HCV RNA in PBMC in the absence of HCV RNA in serum, irrespective of the anti-HCV status. Laboratory methods Serum and PBMC preparation and preservation Samples of 20 ml citrate blood and 10 ml serum were taken from each patient on the study entry day (before the initiation of dialysis in CHD patients) and transported on the same day to our laboratory. The serum was centrifuged and stored in several aliquots at 20 °C until further analysis. PBMC were immediately prepared from the citrate blood by the standard density gradient centrifugation on Ficoll-Paque using Leucosep tubes (Greiner Bio One GmbH, Germany), isolated and washed as per the manufacturer’s instructions. The cells were then counted using a hemocytometer (Neubauer chamber). Aliquots of approximately 2.5 million cells were stored at 80 °C until further analysis.
CHD patients (N = 417) The prevalence of anti-HCV was 3.6% (15 of 417); 10 had detectable HCV RNA in serum and 5 were HCV RNA-negative. Prevalence of positive serum HCV RNA was 2.4% (10 of 417) and all 10 were anti-HCV positive. Of five CHD patients who had received antiviral therapy in the past, three had positive antiHCV but negative HCV RNA in both serum and PBMC suggestive of a sustained virologic response, and the remaining two were positive for anti-HCV and HCV RNA both in serum and PBMC suggestive of an ongoing viral infection. Occult HCV infection defined as detection of HCV RNA in PBMC in absence of HCV RNA in serum was found in one single patient of the 407 (0.25%) HCV RNA-negative patients. The test was positive with 6 million PBMC, and weak positive, i.e., the relative light units (RLU) of the analysed sample were lower compared to those of the positive calibrator, when repeated with the remaining 2.5 million cells from the same sample. KTx recipients (N = 417) The prevalence of anti-HCV was 4.8% (20 of 417); 16 had detectable HCV RNA in serum and four were HCV RNA-negative. Prevalence of positive HCV RNA was 4.6% (19/417), of which three were anti-HCV-negative. All three recipients who had received antiviral therapy in the past had positive anti-HCV and positive HCV RNA in both serum and PBMC suggestive of an ongoing HCV infection. There was a very low prevalence of occult HCV infection in PBMC (as defined above) in two of 398 (0.5%) HCV RNA-negative
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Research Article Table 1. Baseline and clinical characteristics of all patients and controls.
Age in years (Mean ± SD) Sex: Males (%) Race: Caucasians (%) Anti-HCV-positive (%) HCV RNA-positive (%) Prevalence of HCV by positive anti-HCV and/or HCV RNA False-negative serology (%) Duration of CHD or time since last KTx in years (Mean ± SD) Serum AST values (Mean ± SD) Serum ALT values (Mean ± SD) Serum GGT values (Mean ± SD) Patients with elevated serum ALT and/or AST values (%)* Previous antiviral therapy (%) HBsAg-positive patients (%) Previous history of blood transfusion
CHD patients (n = 417) 66.1 ± 14.9 225 (54%) 414 (99.3%) 15 (3.6%) 10 (2.4%) 15 (3.6%) 0 4.9 ± 5.2
KTx recipients (n = 417) 53.0 ± 12.8 250 (60%) 413 (99.0%) 20 (4.8%) 19 (4.6%) 23 (5.5%) 3/19 (15.8%) 6.5 ± 8.6
Positive controls (n = 25) 60.2 ± 12.5 8 (32%) 24 (96%) 25 (100%)§ 25 (100%)§ 25 (100%) 0 -
Negative controls (n = 40) 32.6 ± 8.3 16 (40%) 40 (100%) 0§ 0§ 0 0 -
25 ± 17 26 ± 26 61 ± 82 61 (15.3%) 5 (1.2%) 2 (0.5%) 57 (13.7%)
27 ± 24 29 ± 62 44 ± 87 50 (12.5%) 3 (0.7%) 14 (3.4%) 87 (20.8%)
60 ± 36 64 ± 35 79 ± 63 25 (100%) 0 0 n.a.
25 ± 13 7±7 n.a. 0 0 0 n.a.
⁄
Normal values of liver enzymes are as follows: ALT:
Prevalence of occult hepatitis C infection in chronic hemodialysis and kidney transplant patients Seema Baid-Agrawal1,⇑, Ralf Schindler1, Petra Reinke1, Adrienne Staedtler2, Sunda Rimpler3, Barbara Malik4, Ulrich Frei1, Thomas Berg5 1
Dept of Nephrology and Medical Intensive Care, Campus Virchow Klinikum, Charite Universitaetsmedizin Berlin, Germany; 2Dept of Internal Medicine, Sana Klinikum Lichtenberg, Berlin, Germany; 3Dept of Internal Medicine, Vivantes Wenckebach Klinikum, Berlin, Germany; 4Dept of Gastroenterology and Hepatology, Campus Virchow Klinikum, Charite Universitaetsmedizin Berlin, Germany; 5 Dept of Hepatology, Universitaetsklinikum Leipzig, Leipzig, Germany
Background & Aims: Detection of hepatitis C virus (HCV) RNA in peripheral blood mononuclear cells (PBMC) and/or hepatocytes in absence of HCV RNA in serum, designated as ‘occult HCV infection’, has been a matter of controversy in recent years. We investigated for the first time the prevalence of occult HCV infection in large cohorts of chronic hemodialysis (CHD) and kidney transplant (KTx) patients. Methods: We enrolled 417 CHD patients, 417 KTx recipients and 2 control groups – 25 anti-HCV (antibody against HCV)-positive and HCV RNA-positive patients with chronic hepatitis C, and 40 antiHCV-, HCV RNA-, and HBsAg-negative healthy subjects. HCV RNA was tested in serum and PBMC using a sensitive commercial assay. Results: In CHD patients, the prevalence of anti-HCV was 3.6% (15/417) and of positive serum HCV RNA 2.4% (10/417). HCV RNA was detected in PBMC in 1/407 (0.25%) HCV serum RNA-negative patients (‘‘occult HCV infection’’). In KTx recipients, prevalence of anti-HCV was 4.8% (20/417) and of positive serum HCV RNA 4.6% (19/417). Occult HCV infection was found in 2/398 (0.5%) serum HCV RNA-negative patients. On a mean longitudinal follow-up of 30 months of the 3 patients with occult HCV infection, there was no clinical or virological evidence of HCV infection. Conclusions: The prevalence of occult HCV infection was very low in our CHD and KTx patients, and it did not appear to be clinically relevant. Further studies in geographic populations with high HCV endemicity are required to clarify the significance of occult HCV infection in these patient groups. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Occult hepatitis C; HCV; PBMC; Peripheral blood mononuclear cells; Prevalence; Kidney transplant; Hemodialysis; HCV RNA; Anti-HCV; TMA. Received 27 September 2013; received in revised form 13 January 2014; accepted 13 January 2014; available online 18 January 2014 ⇑ Corresponding author. Address: Dept of Nephrology and Medical Intensive Care, Campus Virchow-Klinikum, Charite Universitatsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: +49 30 450 553 373; fax: +49 30 450 553 916. E-mail address: [email protected] (S. Baid-Agrawal). Abbreviations: HCV, Hepatitis C; Anti-HCV, antibody against HCV; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; CHD, chronic hemodialysis; KTx, kidney transplant; TMA, transcription-mediated amplification.
Introduction Chronic infection with hepatitis C virus (HCV) is a serious public health problem associated with increased morbidity and mortality, affecting approximately 2.8% of the world’s population [1,2]. The diagnosis of HCV is made currently by detection of antiHCV (antibody against HCV) and HCV RNA in serum [2,3]. Interestingly, in recent years, HCV RNA has been detected in nonserum reservoirs such as peripheral blood mononuclear cells (PBMC) and/or hepatocytes even in the absence of HCV RNA in serum, which has raised the controversial issue of an intriguing but not unequivocally accepted entity designated as ‘‘occult HCV infection’’ [4–10]. As the presence of viral replicating ability in PBMC could possibly lead to HCV spread, patients with occult HCV infection may be potentially infectious. Clinically significant occult HCV infection was first described in 2004 in patients with persistently elevated liver function tests and who had negative anti-HCV and HCV RNA in serum, but positive HCV RNA in PBMC [4]. The existence of this entity was supported by subsequent detection of low concentrations of HCV RNA in PBMC in immunocompetent patients who cleared of HCV either spontaneously or after treatment [5–10]. Occult HCV infection in liver tissue/PBMC has been described in two clinical settings: (1) In patients with liver disease of unknown origin (anti-HCV-negative, serum HCV RNA negative, persistently elevated liver transaminases) [4,11,12], and (2) In patients with spontaneous/treatment-induced HCV RNA clearance from serum (anti-HCV-positive, serum HCV RNA-negative, normal liver transaminases) [13,14]. Recently, occult HCV infection was also reported in an apparently disease-free state in absence of liver disease, anti-HCV and HCV RNA in serum [15,16]. However, several other investigators have failed to detect HCV RNA in PBMC in various clinical settings such as cryptogenic liver disease, HCVassociated systemic vasculitis, or connective tissue disease [17], and in patients with resolved HCV infection, mixed cryoglobulinemia, onco-hematological disease or HIV infection [13,18–21]. Therefore, the jury is still out on the existence and significance of occult HCV infection. We hypothesized that if HCV RNA does persist at low levels in PBMC, then it should be more easily detectable in patients with
Journal of Hepatology 2014 vol. 60 j 928–933
JOURNAL OF HEPATOLOGY impaired immune function such as those on chronic hemodialysis (CHD) [22] and kidney transplant (KTx) recipients than in immunocompetent patients. Therefore, these patients would constitute an ideal model to explore the existence of occult HCV infection, all the more so as the presence of occult HCV infection may have deleterious consequences in these high-risk groups of patients. In this study, we aimed to investigate for the first time the prevalence of occult HCV infection in PBMC in large cohorts of CHD and KTx patients. As serum transaminases may be normal in these patients even in the presence of liver disease [23,24], we included all consecutive CHD and KTx patients irrespective of their liver function.
HCV RNA detection by Transcription-Mediated Amplification (TMA) The thawed samples of both serum and PBMC were tested for HCV RNA in batches using Versant HCV RNA qualitative TMA assay (Siemens Healthcare Diagnostics, Eschborn, Germany), according to the manufacturer’s instructions and with all due precautions for prevention of contamination. The test was performed using 500 ll of serum and a minimum of 2.5 million PBMC. The Versant TMA assay is a highly sensitive test with a detection limit of 9.6 IU/ml for HCV RNA. Negative and positive controls were simultaneously tested in each run. A repeat confirmatory testing was performed in all samples that were positive for occult HCV in different runs to exclude contamination occurrence.
Materials and methods
Statistical analyses
Study design
To determine the prevalence with an accuracy of 5% (95% confidence interval), we calculated the required sample size before the initiation of the study to be 400 patients for each group. Results of continuous measured data are presented as means (standard deviation, SD) and categorical variables are expressed as proportion, if not stated otherwise.
Both cross-sectional and longitudinal.
Anti-HCV detection Anti-HCV was tested using either the chemiluminescence microparticle immunoassay ARCHITECT anti-HCV (Abbott Diagnostics, Wiesbaden, Germany) or first generation Elecys chemiluminescence anti-HCV assay (Roche Diagnostics, Mannheim, Germany).
Setting and participants We enrolled two cohorts of patients on CHD and KTx recipients and two control groups as follows: CHD patients 417 consecutive clinically stable adult patients undergoing chronic hemodialysis at six dialysis centers in Berlin between August 2009 and May 2010. KTx recipients 417 clinically stable adult KTx recipients followed in our outpatient transplant clinic between May 2010 and March 2011.
Results Patient characteristics The demographic and baseline characteristics in the two study cohorts and two control groups are shown in Table 1 and their virological results in Table 2. Prevalence of HCV infection in CHD and KTx Patients
Positive controls 25 contemporaneous anti-HCV-positive, HCV serum RNA-positive non-transplant patients with chronic HCV infection without cirrhosis, and who had not yet been initiated on antiviral therapy. Negative controls 40 healthy asymptomatic volunteers taking no medications with normal liver function tests and no known history of HCV or hepatitis B infection, and who were anti-HCV-, HCV serum RNA-, and HBsAg-negative. The study protocol was in accordance with the ethical guidelines of the Declaration of Helsinki and was approved by our institution’s Ethics Committee. Fully informed written consent was obtained from all participants before enrolment. Demographic and clinical data were collected from the electronic medical records. Definition Occult HCV infection was defined as presence of HCV RNA in PBMC in the absence of HCV RNA in serum, irrespective of the anti-HCV status. Laboratory methods Serum and PBMC preparation and preservation Samples of 20 ml citrate blood and 10 ml serum were taken from each patient on the study entry day (before the initiation of dialysis in CHD patients) and transported on the same day to our laboratory. The serum was centrifuged and stored in several aliquots at 20 °C until further analysis. PBMC were immediately prepared from the citrate blood by the standard density gradient centrifugation on Ficoll-Paque using Leucosep tubes (Greiner Bio One GmbH, Germany), isolated and washed as per the manufacturer’s instructions. The cells were then counted using a hemocytometer (Neubauer chamber). Aliquots of approximately 2.5 million cells were stored at 80 °C until further analysis.
CHD patients (N = 417) The prevalence of anti-HCV was 3.6% (15 of 417); 10 had detectable HCV RNA in serum and 5 were HCV RNA-negative. Prevalence of positive serum HCV RNA was 2.4% (10 of 417) and all 10 were anti-HCV positive. Of five CHD patients who had received antiviral therapy in the past, three had positive antiHCV but negative HCV RNA in both serum and PBMC suggestive of a sustained virologic response, and the remaining two were positive for anti-HCV and HCV RNA both in serum and PBMC suggestive of an ongoing viral infection. Occult HCV infection defined as detection of HCV RNA in PBMC in absence of HCV RNA in serum was found in one single patient of the 407 (0.25%) HCV RNA-negative patients. The test was positive with 6 million PBMC, and weak positive, i.e., the relative light units (RLU) of the analysed sample were lower compared to those of the positive calibrator, when repeated with the remaining 2.5 million cells from the same sample. KTx recipients (N = 417) The prevalence of anti-HCV was 4.8% (20 of 417); 16 had detectable HCV RNA in serum and four were HCV RNA-negative. Prevalence of positive HCV RNA was 4.6% (19/417), of which three were anti-HCV-negative. All three recipients who had received antiviral therapy in the past had positive anti-HCV and positive HCV RNA in both serum and PBMC suggestive of an ongoing HCV infection. There was a very low prevalence of occult HCV infection in PBMC (as defined above) in two of 398 (0.5%) HCV RNA-negative
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Research Article Table 1. Baseline and clinical characteristics of all patients and controls.
Age in years (Mean ± SD) Sex: Males (%) Race: Caucasians (%) Anti-HCV-positive (%) HCV RNA-positive (%) Prevalence of HCV by positive anti-HCV and/or HCV RNA False-negative serology (%) Duration of CHD or time since last KTx in years (Mean ± SD) Serum AST values (Mean ± SD) Serum ALT values (Mean ± SD) Serum GGT values (Mean ± SD) Patients with elevated serum ALT and/or AST values (%)* Previous antiviral therapy (%) HBsAg-positive patients (%) Previous history of blood transfusion
CHD patients (n = 417) 66.1 ± 14.9 225 (54%) 414 (99.3%) 15 (3.6%) 10 (2.4%) 15 (3.6%) 0 4.9 ± 5.2
KTx recipients (n = 417) 53.0 ± 12.8 250 (60%) 413 (99.0%) 20 (4.8%) 19 (4.6%) 23 (5.5%) 3/19 (15.8%) 6.5 ± 8.6
Positive controls (n = 25) 60.2 ± 12.5 8 (32%) 24 (96%) 25 (100%)§ 25 (100%)§ 25 (100%) 0 -
Negative controls (n = 40) 32.6 ± 8.3 16 (40%) 40 (100%) 0§ 0§ 0 0 -
25 ± 17 26 ± 26 61 ± 82 61 (15.3%) 5 (1.2%) 2 (0.5%) 57 (13.7%)
27 ± 24 29 ± 62 44 ± 87 50 (12.5%) 3 (0.7%) 14 (3.4%) 87 (20.8%)
60 ± 36 64 ± 35 79 ± 63 25 (100%) 0 0 n.a.
25 ± 13 7±7 n.a. 0 0 0 n.a.
⁄
Normal values of liver enzymes are as follows: ALT:
