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Hepatology, November 1999, p. 1307-1311, Vol. 30, No. 5
Clinical Features of Hepatitis C-Infected Patients With Persistently Normal Alanine Transaminase Levels in the Southwestern United States
M. Mazen Jamal1,
From the Divisions of 1Gastroenterology and 2Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM.
ABSTRACT
Approximately one third of patients with chronic hepatitis C virus (HCV) infection have normal alanine transaminase (ALT) levels. We studied the clinical, biochemical, virological, and histological features in patients with persistently normal ALT. A case-control study was conducted on 275 patients with chronic HCV infection, including 75 patients with persistently normal ALT and 200 patients with abnormal ALT. Persistently normal ALT was defined as 4 consecutive ALT values in each patient within a period of 12 months. The average age of the patients was 44 years (range 18 to 69 years). More non-Hispanic whites had persistently normal ALT. The mean serum ferritin level was significantly lower in patients with persistently normal ALT as compared with abnormal ALT (128 ± 92 ng/mL and 224 ± 128 ng/mL), respectively (P = .017). The mean HCV-RNA level was significantly lower in patients with persistently normal ALT as compared with abnormal ALT (12 × 105 ± 2.8 × 10 6 copies/mL and 33 × 105 ± 8.0 × 10 6), respectively (P = .02). Histologically, patients with persistently normal ALT had less severe portal inflammation (P < .05), lobular inflammation (P = .003), piecemeal necrosis (P = .002), fibrosis (P < .05), lower prevalence of cirrhosis (P = .007), as well as a slower fibrosis progression rate (P < .001). Chronic hepatitis C patients with persistently normal ALT have low-activity grade and stage on liver biopsy. In these patients the hepatitis C RNA level was lower compared with abnormal ALT patients, which may explain the slower fibrosis progression rate. (HEPATOLOGY 1999;30:1307-1311.)
INTRODUCTION
Hepatitis C viral infection often causes chronic liver disease and leads to serious consequences including cirrhosis and hepatocellular carcinoma.1-4 The development of serological and virological tests to detect hepatitis C infection has contributed greatly to our understanding of the natural history and clinical features of this infection.5,6 Evaluation of hepatitis C infection involves standard biochemical tests. Elevation of alanine transaminase (ALT) is indicative of hepatocellular inflammation and necrosis and considered to be a hallmark of chronic hepatitis C infection. However, up to a third of patients infected with hepatitis C have normal ALT levels.7,8 Therefore, ALT level may not always predict histological evidence of chronic hepatitis C.9 The aim of this study is to evaluate the clinical, biochemical, virological, and histological features of chronic hepatitis C patients with persistently normal ALT and to compare the results in patients with abnormal ALT.
PATIENTS AND METHODS
A case-control study was conducted at the University of New Mexico Health Sciences Center from 1995 to 1997. During this time, 275 consecutive patients were eligible for the study. Case and control patients participated in a structured interview conducted by a Research Coordinator who was blinded to the study. Data collected included: age, race, sex, risk factors, symptoms (depression, fatigue, weakness, and abdominal pain), comorbid illnesses (rheumatoid arthritis, diabetes mellitus, coronary artery disease, chronic renal failure, and congestive heart failure), and alcohol consumption history. Information about alcohol consumption included both frequency of drinking and amount consumed. Laboratory data included complete blood count, serum biochemistry, prothrombin time, iron, total iron binding capacity, and ferritin. The ALT measurements were performed on a Johnson & Johnson Vitros 950 Chemistry system (Rochester, NY) using Vitros chemistry calibrator kit 3 and Vitros reagents. Reference interval for women was 9 to 52 (SI U/L) and for men was 21 to 72 (SI U/L). The reference intervals are the central 95% of results from a study of 2,445 healthy adults after excluding 5% of the extreme values. The hepatitis C virus (HCV)-RNA viral load measurement was performed using a second generation reverse-transcription polymerase chain reaction (RT-PCR) assay (Roche Amplicor Monitor Kit, Branchburg, NJ). The assay involves measurements made on patients’ serum samples diluted serially: 1:1, 1:5, 1:25, 1:125, 1:625, and compared against serially diluted control serum. This assay is reported to have approximately a 3-fold variability (0.311 log).10 HCV genotyping was performed at the Tricore Specialty Labs (Albuquerque, NM). The assay for genotyping uses extraction of HCV RNA, RT coupled with PCR to specifically amplify a 450-nucleotide portion of the NS5b region of the HCV-RNA genome, which is then directly sequenced.11 An ABI 377 automatic sequencer (PE Biosystems, Perkin-Elmer, Foster City, CA) was used for cycle sequencing.
Eligibility and Definitions.
All patients were positive for hepatitis C antibody by enzyme-linked immunosorbent assay (ELISA II; Abbott Laboratory, Evanston, IL); had positive hepatitis C RNA by quantitative PCR (HCV Monitor; Roche Diagnostic Systems, Branchburg, NJ); and had negative autoimmune markers (antinuclear and antismooth muscle antibodies). All the patients had an HCV- genotype test. Patients younger than 18 years, those with previous treatment with interferon alfa, those with fluctuating ALT level (patients with even one abnormal value with others being within the normal range on serial ALT testing), liver transplant patients, those with undetectable HCV RNA, those with hepatitis B surface antigen and patients with hepatocellular carcinoma, and hemochromatosis were all excluded. The patients were divided into two groups based on ALT level. Group I consisted of 75 patients with persistently normal serum ALT level, which was defined as at least 4 normal ALT values within 12 months and at least 3 normal levels within 6 months. Group II had 200 patients with abnormal serum ALT level. Duration of infection was defined as the duration since the first exposure to a blood-borne risk factor (time since a blood transfusion or first exposure to intravenous drug use [IDU]). This could be determined in 67 out of 75 patients (89.3%) in the persistently normal ALT group and 180 out of 200 (90%) in the abnormal ALT group. All patients signed a consent form to participate in the study and the study was approved by the institutional review committee of the University of New Mexico.
Liver Biopsy Specimen Preparation and Evaluation.
All liver biopsy specimens were fixed in 10% neutral-buffered formalin. Sections were cut at 3- to 4-µm thickness and stained with hematoxylin-eosin, prussian blue (for Iron), and Masson’s trichrome stain and reviewed by a staff pathologist who was blinded with respect to the clinical data. Biopsy specimens were graded with respect to the degree of piecemeal necrosis, portal and lobular inflammation, and fibrosis according to a revised scoring system.12,13 Fibrosis progression rate was defined as the ratio of the fibrosis score over the estimated duration of the infection.
Statistical Analysis.
The statistical analysis was performed using SAS/STAT Software (SAS Institute, Carey, NC). Any P value less than .05 was taken to be indicative of statistical significance. Continuous variables were analyzed by unpaired t tests and nonparametric tests. Binary variables were analyzed using 
2 tests and Fisher’s exact test. Quantitative variables were expressed as means ± SD; the median and interquartile were used for alcohol consumption because the distribution was not normal. The multivariate statistical analysis using logistic regression included biologically relevant variables and those that showed a P value of less than .20 in the univariate analysis. Odds ratio and their 95% confidence interval were used to indicate the strength of influence. In the multivariate model, fibrosis progression rate was coded as dichotomous variable using the code 0 or 1 to indicate a rate of 
the mean value or a rate greater than the mean.
RESULTS
Four hundred forty-three patients were diagnosed with hepatitis C in the Gastroenterology clinic between June 1995 and June 1997. Two hundred seventy-five patients were eligible for the study and 168 patients were excluded for the following reasons: 89 patients with previous treatment with interferon; 44 patients with negative HCV RNA; 15 patients had fluctuating ALT level; 8 patients with a history of liver transplantation; 4 patients younger than 18 years; 3 patients with hepatitis B; 2 patients with acquired immune deficiency syndrome; 2 patients with autoimmune hepatitis; and 1 patient with hemochromatosis.
Demographics.
Demographic features of the two groups are shown in table 1. There was no significant difference between the two groups based on age and sex. Average age of the patients in the two groups was 44 years. There were more non-Hispanic whites in the persistently normal ALT group (P = .03). Subanalysis of the ALT level in Hispanics and non-Hispanic whites in the two groups did not show any significant difference. The mean level of ALT in the persistently normal ALT group in Hispanics and non-Hispanic whites was 38.3 ± 12.9 and 37.8 ± 12.0, respectively. There was no significant difference between the patients of the two groups in terms of duration of infection, or the risk factors for hepatitis C. Average alcohol consumption was the same in both groups even when we took into consideration heavy alcohol consumption (>50 g/d).
| table 1. Demographics of Patients With Persistently Normal and Abnormal ALT |
Biochemical Data.
The biochemical data of the two groups are shown in table 2. There was no significant difference in both groups in regards to serum iron level, total iron-binding capacity, iron saturation, albumin, bilirubin, and prothrombin time. However, the serum ferritin and the HCV-RNA levels in the two groups were significantly different. Mean serum ferritin levels for persistently normal ALT and abnormal ALT patients were 128 ± 92 ng/dL and 224 ± 128 ng/dL, respectively (P = .017). HCV-RNA level was 12 × 105 ± 2.8 × 10 6 and 33 × 105 ± 8.0 × 10 6, respectively (P = .02). To examine carefully whether the difference in mean values is more than the intrinsic variability of the RT-PCR assay we applied logarithm to the base 10 and found a 1.4-order difference in means of the log-transformed values and this is more than the reported intrinsic variability for this assay. The transformed-log values for persistently normal ALT and abnormal ALT groups were 4.06 ± 2.0 and 5.67 ± 1.6, respectively, withP < .001.
| table 2. Biochemistry of Patients With Persistently Normal and Abnormal ALT |
Symptoms and Comorbid Illnesses.
There was no significant difference in the presenting symptoms or the presence of comorbid illnesses in the patients of the two groups (table 3). Although there was no statistically significant difference in terms of depression as a symptom, there was a trend towards depression being more common in the subjects with abnormal ALT (P = .08).
| table 3. Symptoms and Comorbid Diseases in Both Groups |
Genotype.
There was no significant difference in HCV genotype between the two groups (Fig. 1). Genotype 1b was most commonly encountered in our patients followed up by Genotype 3. Four patients in each group had mixed viral infection.
 Fig. 1. Comparison of the percentage of the different genotype distribution in the two groups. Persistently normal ALT group ( ) and abnormal ALT group ( ). |
Histological Features.
Patients with persistently normal ALT had histologically less severe liver disease as compared with those with abnormal ALT (Fig. 2). The mean scores for portal inflammation in patients with persistently normal and abnormal ALT were (1.4 ± 0.73) and (1.8 ± 0.8), respectively, (P < .05), for piecemeal necrosis (0.87 ± 0.78) and (1.44 ± 0.87) (P = .002), for lobular inflammation (0.97 ± 0.41) and (1.40 ± 0.73) (P = .003), and for fibrosis (1.4 ± 1.66) and (2.05 ± 1.63), respectively, (P < .05). Six percent of patients with persistently normal ALT had cirrhosis compared with 19% in the abnormal ALT group (P= .007). Fibrosis progression rate per year was evaluated in 67 patients from group I and 180 patients from group II (in whom duration of infection was known). The fibrosis progression rate in both groups was (0.08 ± 0.07) and (0.15 ± 0.1), respectively, (P < .001). We analyzed the fibrosis progression rate per year after excluding the patients with alcohol consumption of more than 50 g/d and it remained significantly lower in persistently normal ALT group (0.05 ± 0.06) compared with the abnormal ALT group (0.11 ± 0.10) (P < .001). A subanalysis of the fibrosis progression rate per year in patients with alcohol consumption of more than 50 g/d again was significantly lower in the persistently normal ALT group (0.13 ± 0.11) compared with the abnormal ALT group (0.23 ± 0.22) (P < .001). Multivariate analysis was performed using logistic regression with fibrosis progression rate as a binary outcome variable. We developed our model by repetitive elimination and inclusion of predictive variables. In our model age, ALT and alcohol were predictive factors of fibrosis progression. Alcohol, race, sex, ferritin, or RNA levels were not found to be confounding variables. The odds ratio (OR) and 95% CI for ALT level and alcohol were 3.7 (1.4, 9.7) P = .008, and 1.8 (1.2, 2.9) P < .05, respectively. For age, the OR and 95% CI per decade were 1.9 (1.2, 3.0) P = .006. The histological grading was analyzed in the two groups and further subanalysis was performed to compare Hispanics and non-Hispanic whites with regard to the mean grading score (table 4). The mean grading score for these two groups was 4.15 ± 2.1 and 6.6 ± 2.9, respectively, (P = .003). The mean grading score in Hispanics in the two groups was 4.12 ± 2.05 and 7.2 ± 2.8, respectively, (P < .001), and for the non-Hispanic whites was 4.2 ± 2.15 and 6.1 ± 2.9, respectively, (P < .001). There was no statistical difference between Hispanics and non-Hispanic whites in the persistently normal ALT group with regard to the mean grading score. There was no difference in the iron-stain grade on liver biopsy between the two groups.
).
DISCUSSION
The clinical characteristics of chronic HCV-infected patients with persistently normal ALT are not completely defined. Controversies still exist regarding the severity of disease on liver biopsy in these patients.14-16 A clear understanding of these features is important in making rational therapeutic decisions regarding the care of these patients. A crucial aspect of this study is our definition of persistently normal ALT, which is at least 4 consecutive, normal ALT values within a period of 12 months and at least 3 values within 6 months. This decreases the chance of including patients with fluctuating ALT level, which would have inflated our case population and confounded the results.
Our study highlights several important features of HCV-infected patients with persistently normal ALT level. It is important to note that there were no healthy hepatitis C carriers in our study (i.e., no normal biopsy specimens) in contrast to a previous study.15 We found histologically significant liver disease even in patients with persistently normal ALT, although it was less severe compared with subjects with persistently abnormal ALT. Using the revised scoring system we have shown significantly less severe portal inflammation, piecemeal necrosis, and lobular inflammation as well as degree of fibrosis, and incidence of cirrhosis in patients with persistently normal ALT compared with patients with abnormal ALT. Similar results were reported by other investigators,14,15 which are in contrast to the findings of at least 1 study16 showing moderate to severe disease in persistently normal ALT patients.
One of the main results in our study was the estimate of fibrosis progression rate suggesting a slower progression of liver fibrosis in the normal ALT group, even after excluding patients with heavy alcohol consumption (>50 g/d). However, our results showed that alcohol hastened fibrosis progression equally in persistently normal and abnormal ALT groups, but did not contribute to the difference between the two groups. Therefore, we suggest that the natural history of HCV infection in patients with persistently normal ALT is associated with delay in the development of severe liver disease.
Genotyping has become an important tool in the armamentarium of a hepatologist. It is important both as a prognostic tool and also in determining the duration of treatment compared with a more favorable genotype. There have been studies on genotype distribution in patients with persistently normal ALT with varied results.17,18 We did not find any difference in genotype distribution among patients of the two groups. These differences maybe attributed to geographical variation and differences in mode of transmission of infection as have been pointed out in the past. To determine the replicative level of HCV in patients infected with the virus, quantification of the HCV RNA in the serum by RT-PCR assay is routinely done. There is evidence that HCV cytopathogenicity may contribute to hepatocellular damage in liver cells with elevated level of HCV RNA, thereby suggesting a correlation between HCV-RNA level and severity of liver disease.19-21 Higher HCV-RNA levels have also been reported in patients with chronic active hepatitis compared with those with chronic persistent hepatitis C.22 In our study we found that patients with persistently normal ALT had significantly lower HCV-RNA levels thereby signifying less severe disease. It is interesting, although not surprising, that subjects with persistently normal ALT have a significantly lower serum ferritin level because it has been reported that a high ferritin level is associated with more severe hepatitis and advanced liver disease.23 Many HCV-positive patients with elevated aminotransferase activity have a serum ferritin level above the normal range, but only a minority of these patients have iron overload.24 Two studies found a close relationship between serum ALT activity and ferritin level.25,26 Therefore, we feel that a high ferritin level in a chronic HCV-infected patient maybe indicative of more severe disease.
Contrary to previous reports,16,27 our study shows no sex difference between the persistently normal and abnormal ALT groups, which might be a limitation of our study. However, based on multivariate analysis, sex was not found to be a confounding variable for fibrosis progression. It is not clear why more Hispanic patients have an abnormal ALT level. It has been postulated that race might play a role in progression of HCV-related chronic liver disease.28 A vast majority of chronic HCV patients either have asymptomatic disease or report nonspecific symptoms. Fatigue is a common complaint and other symptoms include depression, weakness, nausea, anorexia, abdominal discomfort, and difficulty with concentration. We analyzed some of these symptoms and found no association between any individual symptom and type of chronic HCV infection. However, there was a trend towards depression being more common in abnormal ALT patients compared to their persistently normal ALT counterparts. The analysis also included comorbid chronic illnesses that might have a role in expression of some of these nonspecific symptoms. Patients in both groups were matched in terms of the chronic comorbid illnesses studied. Therefore, depression, though nonspecific, might be an important clinical symptom marker of more severe disease.
It is difficult to estimate the duration of infection in a majority of subjects owning to multiple risk factors. However, it is easier to predict the duration of disease in subjects who have had a single blood transfusion or used intravenous drugs for a short duration of time. There has been a suggestion that the mode of transmission of infection influences the severity of liver disease. Patients who acquired the infection through a blood transfusion had more severe disease compared with the other risk factors. 29-31 However, because there was no difference between the two groups, as far as mode of transmission is concerned, this variable would not influence the disease course and confound our results.
In conclusion, our report showed that patients with persistently normal ALT have less severe hepatitis and less advanced disease histologically, which supports the concept that the lower level of viremia may play a role in slowing down the progression of disease.
Abbreviations
Abbreviations: ALT, alanine transaminase; HCV, hepatitis C virus; RT-PCR, reverse-transcription polymerase chain reaction.
FOOTNOTES
Received May 13, 1999; accepted August 10, 1999.
Address reprint requests to: M. Mazen Jamal, M.D., Division of Gastroenterology and Hepatology, University of New Mexico Health Sciences Center, 2211 Lomas Blvd. NE, ACC-5, Albuquerque, NM 87131-5271. E-mail: mmjamal@salud.unm.edu; fax (505) 272-6839.
REFERENCES
Copyright © 1999 by the American Association for the Study of Liver Diseases.
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