Liver 1997: 17: 133-138 Printed in Denmark , All rights reserved

Copyr ighr 0 Munksgaurd 1997

LNER ISSN 0106-9543

Virological characterization and liver histology in HCV positive subjects with

elevated ALT levels normal and Rossini A, Ravaggi A, Agostinelli E, Bercich L, Gazzola GB, Radaeli E, Callea F, Cariani E. Virological characterization and liver histology in HCV positive subjects with normal and elevated ALT levels. Liver 1997: 17: 133-138. 0 Munksgaard, 1997

Abstract: We analyzed HCV genotype and RNA titer in 36 chronically infected subjects, 20 with persistently normal or near-normal alanine aminotransferase (ALT) activity and 16 with raised ALT activity. All sub- jects underwent liver biopsy and evaluation of the histological activity index (HAI) by both Knodell's and Ishak's scoring systems. Genotype 2 was detected in most subjects with normal ALT activity, whereas genotype 1 was more frequent among subjects with raised ALT activity. HCV-RNA titer was higher in subjects with increased ALT. Histological evidence of chronic hepatitis was documented in all cases, but higher scores for grad- ing and for staging were associated with increased ALT activity. HCV genotype had no statistical relationship with RNA titer or with liver his- tology. In logistic regression analysis, viral genotype, RNA titer and his- tological scores for grading and staging were correlated independently with the ALT profile. The evidence of chronic hepatitis in all subjects with persistently normal ALT activity suggests that healthy HCV carriage is a rare event.

Infection with the hepatitis C virus (HCV) may lead to different clinical profiles, ranging from acute resolving hepatitis to chronic infection with variable patterns of alanine aminotransferase (ALT) activity (1). A subset of anti-HCV positive subjects displays persistently normal ALT values despite evidence of viral replication, detected by amplification of serum HCV-RNA (2). This clin- ical profile has suggested the existence of healthy HCV carriers (3), but this concept is challenged by the histological evidence of chronic hepatitis in anti-HCV and HCV-RNA positive subjects with normal ALT values (4, 5).

The aim of the present study was the analysis of virological and histological features of HCV infec- tion associated with different ALT profiles.

Patients and methods Patients

The study population consisted of 36 consecutive subjects positive for anti-HCV and HCV-RNA, re-

Angelo Rossinil, Antonella Ravaggi2, Enrica Agostinelli3, Luisa Bercich4, Gian Biattista Gazzola3, Enrico Radaelil, Francesco Callea4 and Elisabetta CarianiZ 'Third Department of Internal Medicine, 'Third Laboratory of Clinical Chemistry, 4First Department of Pathology, Hospital of Brescia and 3Blood Transfusion Service, Hospital of Chiari, Italy

Key words: competitive polymerase chain reaction (PCR) - histological activity index - HCV genotypes - HCV-RNA - HCV-RNA titer

Elisabetta Cariani, 1 1 1 Laboratorio Analisi, A. 0. Spedali Civili, P.le Spedali Civili, 1, 25123 Brescia, Italy

Received 3 July 1996, accepted for publication 28 January 1997

ferred from the Blood Transfusion Centre of the Hospital of Chiari to our department (Internal Medicine) for clinical and laboratory evaluation. Twenty-one subjects had normal ALT levels and 12 of them had been regular volunteer blood donors for 2 to 28 years before detection of anti-HCV. The remaining 15 subjects had been excluded from do- nations because of abnormal ALT values before detection of anti-HCV: However, five of them had previously donated blood for 2 to 17 years with normal ALT at controls performed 3 to 4 times a year.

After detection of anti-HCV, all subjects were monitored by determination of ALT levels at 1-3 month intervals for 18 to 50 months. All subjects with normal ALT values underwent at least 12 consecutive monthly determinations. Most of them maintained normal enzyme levels during fol- low-up, whereas five showed sporadic fluctuations at monthly monitoring. In only one case, after 6 months ALT levels became persistently elevated for a further 12 months of follow-up. The 15 sub-

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jects with elevated ALT levels showed increased values during follow-up.

According to the enzymatic profile, the study population was divided into two groups. Group 1 consisted of 20 subjects with persistently normal ALT or with occasional fluctuations within 5 IU above the upper limit of normal (40 IU/l) (1 1 men and 9 women, mean age 49.6k10.9 years). Group 2 consisted of 16 subjects with ALT levels exceed- ing 1.5-fold normal values (1 1 men and 5 women, mean age 49.1 -+ 11.1 years).

None of the subjects included in this study re- ported alcohol or drug abuse, possible sources of parenteral infections or sexual activity with multiple partners. Seven subjects had previously received blood transfusion(s). Markers of active HBV infection (HBsAg or high-titer anti-HBcAg) were negative in all cases. Markers of resolved HBV infection (anti-HBcAg and anti-HBsAg) were detected in nine cases (Group 1: n=6, Group 2: n=3). Six subjects (Group 1: n=1, Group 2: n=5) had anti-HBcAg alone. All sub- jects with normal ALT and/or with anti-HBcAg alone were negative for serum HBV-DNA tested by polymerase chain reaction. Since the relation- ship between hepatitis G virus (HGV) infection and liver disease is still debated (6), HGV-RNA was not investigated. Other noninfective causes of hepatitis (metabolic diseases or hepatotoxic drugs) were excluded. All subjects were negative for antibodies against human immunodeficiency virus and for non-organ specific autoantibodies. None of them was treated with interferon before entering this study.

Serological tests

HBV markers were tested by commercially avail- able kits (Abbott Laboratories, North Chicago, IL, USA). Anti-HCV antibodies were assayed in duplicate by two ELISA assays (Ortho Diagnostic Systems, Raritan, NJ, USA, and Abbott Labora- tories) and by the second and third generation Re- combinant Immunoblot Assay (RIBA, Ortho Di- agnostic Systems).

Liver histology

All subjects gave their informed consent to liver biopsy. Histological examination was carried out by one pathologist according -to conventional cri- teria. Histological scores were determined accord- ing to the Knodell’s histological activity index (HAI) scoring system (7) and to modifications pro- posed by Ishak et al. (8).

HCV genotype determination

Total RNA was isolated from 100 pl of serum with RNAzol B (Cinna-Biotecx, Houston, TX, USA), following the manufacturer’s instructions. After denaturation, RNA samples were reverse tran- scribed and amplified by nested PCR with primers located in the El region. PCR products were then used for the determination of HCV types la and 1 b by differential hybridization on microtiter plates (9). The amplified fragments that did not hybridize with probes specific for subtypes l a and 1 b were cloned and sequenced.

Samples that failed to be amplified with El primers were subjected to nucleotide sequence analysis of the 5’ untranslated region (UTR), ob- tained by nested PCR as previously described (10).

Cloning and nucleotide sequence analysis

The PCR products were cloned in the Bluescript plasmid vector (Stratagene, La Jolla, CA, USA) and at least three independent clones were ana- lyzed for each isolate to deduce a consensus se- quence.

Nucleotide sequence analysis was performed on denatured double stranded DNA using the dide- oxy-chain termination method (1 1). Both strands were entirely sequenced for each clone using the Sequenase version 2.0 kit (United States Biochem- ical Corp., Cleveland, OH, USA). Sequence data were analyzed with the computer programme PC/ GENE (Intelligenetics, Geel, Belgium).

Quantification of HCV-RNA

The determination of HCV-RNA copy number was carried out on serum samples collected at the time of liver biopsy with a competitive PCR-differ- ential hybridization assay (12). Briefly, a competi- tive RNA template containing 2 point mutations, compared with the 5’ UTR sequence, was added in known amount to each sample. After reverse transcription and nested PCR, each sample was subjected to differential hybridization with the DNA enzyme immunoassay (GEN-ETI-K DEIA, Sorin Biomedica, Saluggia, Italy), using probes specific for wild-type and competitor. The ratio be- tween the optical density values obtained on each probe, plotted on a standard curve, allowed the es- timation of RNA copy number present in serum samples.

Statistical analysis

Student’s t test and chi squared test were used to compare the characteristics of patients. Serum

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ficient. A p value of 0.05 or less was considered statistically significant. A logistic regression model (Egret statistical package) was used to identify characteristics independently associated with the ALT profile.

Table 1. Virological characteristics of subjects with different ALT profiles

Group 1 Group 2 P

HCV genotype (number of subjects)

1 2

8 12

13 3 10.05

HCV-RNA titer (log copies/rnl) median (range) 7.4 (5-8.6) 7.9 (6-8.9) (0.05

HCV-RNA levels were compared by the Kruskal- Wallis test. The Mann-Whitney U test was applied to the analysis of the histological activity index and sub-indices. The relationship between HCV- RNA titers and histological scores was analyzed by the Spearman rank-order correlation coef-

Results Virological characteristics

For the determination of HCV genotype, the HCV El region was amplified by nested PCR. Amplifi- cation was successful in 30 cases, 15 in Group 1 (normal or near-normal ALT level) and 15 in Group 2 (raised ALT level). Differential hybridiza- tion detected subtype l b in 20 subjects, 7 from Group 1 and 13 from Group 2. Subtype la was detected in only one case (Group 1). The determi- nation of HCV genotypes in the remaining subjects

la*

A3 3

1 bo

B7

A1 4

A3 2

2aA

2cA

A2 7

B15

B16

A1 1

B3 0

A1 0

A2 4

A3

A2 2

A2 3

2b*

Fig. I. Alignment of deduced amino acid sequences of the El region (residues 298-383) derived from subjects in Groups 1 (A) or 2 (B) infected with HCV genotype 1 (top) or 2 (bottom). *: reference sequences for genotype l a (14); O: reference sequence for genotype 1 b (1 5); ": reference sequences for genotypes 2a, 2b and 2c (1 3, consensus sequences of several isolates).

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lb 87 I 1

pI+, c, A1 1 t A22 -1 I A23 2b 2b

Fig. 2. Dendrogram of the genetic relatedness of HCV isolates derived from subjects in Group 1 (A) or 2 (B) based on the percent homology of partial E l amino acid sequence (residues 298-383). Reference sequences for genotypes l a (14), l b (15) and 2a-c (1 3) are indicated in bold letters.

was performed by partial sequence analysis of the El and/or 5' UTR and detected genotype 2 in all cases. The prevalence of genotypes 1 and 2 in the two groups was significantly different ( jKO.05) (Table 1).

Deduced amino acid sequence alignment of the terminal part of the El region, which allows the discrimination of 12 different viral genotypes/sub- types (13), showed that sequences belonging to genotype 2 were more similar to type 2c than to 2a and 2b (Fig. 1). The analyzed sequences were de- rived from subjects in Group 1 (n=8) and in Group 2 (n=2). Homology to genotype 2c ranged from 85 to 93%, to genotype 2a from 79 to 85%, and to genotype 2b from 73 to 80%. Sequences classified as genotypes l a and lb by differential hybridization showed high homology to references (14,15) (91.9 to 96.5%) (Fig. 2).

Since the clinical profile of HCV infection might be linked to different rates of viral replication, serum HCV-RNA titer was analyzed by means of competitive PCR in the two groups of subjects. Our results indicated significantly different values of logarithmic transformed HCV-RNA copies/ml (p<0.05) (Table 1). The HCV-RNA titer, however, was not statistically different between subjects in- fected with genotype 1 or 2 (p=0.36).

Liver histology

Histological examination revealed the presence of inflammatory changes in all cases. Chronic inter- face hepatitis (formerly CAH) was observed in 33 cases, chronic portal hepatitis (formerly CPH) in 2 and liver cirrhosis in 1.

The severity of liver lesions was estimated both

Table 2. Histological features of subjects with different ALT profiles

Group 1 Group 2 P

Liver histology (n) NS Chronic interface hepatitis 19 14 Chronic portal hepatitis 1 1 Cirrhosis 0 1

Necro-inflammation (Grading) median (range) Knodell's score 4 (2-7) 47 (1-11) 10.05

Piecemeal necrosis 1 (0-3) 3 (0-5) 10.05 Lobular necrosis 1 (1-3) 1 (0-3) NS Portal inflammation* 2 (1-3) 3 (1-3) NS

Ishak's score 5 (3-9) 8 (1-14) 10.05 Piecemeal necrosis 1.5 (0-3) 3 (0-3) 10.05 Confluent necrosis 0 (0-1) 0 (0-5) NS Lobular necrosis 1 (1-3) 2 (0-3) NS Portal inflammation 2 (1-3) 3 (1-3) NS

Fibrosis - Cirrhosis (Staging) median (range)

Knodell's score 1 (0-3) 2 (1-4) 10.01 Ishak's score 1 (0-3) 2.5 (1-5) 10.01

NS: not significant; * : correlated with HCV-RNA titer (~10 .05 )

Table 3. Independent variables associated with ALT profile on logistic re- gression analysis

Variable P

HCV genotype (n) 0.013 HCV-RNA titer (log copies/ml) 0.021 ' Histological grading

Knodeli's score Ishak's score

Histological staging Knodell's score Ishak's score

0.01 1 0.012

0.001 0.001

by the Knodell's histological activity index (HAI) (7) and by the criteria proposed by Ishak et al. (8) (Table 2). The scores for necro-inflammation (grading) and for fibrosis (staging) were both higher in Group 2 (p<0.05 and p<O.Ol, respec- tively). Among single scores in grading, only peri- portal necrosis was significantly different between Groups 1 and 2 (pC0.05).

The histological diagnosis and single HA1 indexes had no statistical relationship with the in- fecting genotype (data not shown). Independent of ALT levels, a significant correlation was observed between HCV-RNA titer and the degree of portal inflammation (p<0.05) in the Knodell's, but not in the Ishak's, scoring system.

Multivariate analysis of characteristics associated with the ALT profile

A logistic regression model was used to investigate which virological and histological characteristics

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levels (24). The different methods used for the quantification of the viral RNA might partly ac- count for this discrepancy. Furthermore, the deter- mination of enzyme levels made on a single serum sample (24) might not be related to the ALT profile over time. In our study subjects with increased ALT had significantly higher serum HCV-RNA tit- er than those with normal or near-normal enzyme levels on long-term follow-up. However, in some cases with normal ALT the RNA titer was higher than 8 log copies/ml, suggesting that a low rate of HCV replication cannot be considered a distinctive characteristic of this group of subjects.

Little is known about the role played by host or viral factors in the pathogenesis of liver damage due to HCV. Our results suggest that the features of HCV infection can be influenced by the viral genotype, consistent with previous reports of dif- ferent pathogenic potential of HCV strains. Geno- type 1 was found in a higher percentage of chronic active hepatitis and cirrhosis with respect to other viral types (22, 25), and the rate of response to interferon, determined by ALT normalization, was higher in patients infected with genotypes 2 and 3 (20, 25, 26). In the present study, the histological scores for both grading and staging were signifi- cantly higher in subjects with raised ALT levels, which were more frequently infected with type 1, compared with donors with normal ALT levels. On the other hand, consistent with another report (27), no statistical relationship was found between liver histology (whatever grading and staging sys- tem) and HCV genotype. Logistic regression analysis confirmed this view, showing that HCV genotype, RNA titer and histological scores for grading and staging were independently associated with the biochemical expression of HCV infection.

Several reports suggested that the viral titer may influence the severity of liver damage (18, 23, 27, 28). However, the timing of quantitative HCV- RNA determination might be critical for the detec- tion of a possible relationship. Indeed, the time course of HCV-RNA fluctuations and of liver his- tological changes in natural HCV infection has still to be determined. Independent of ALT levels, we found a correlation between HCV-RNA titer determined at the time of liver biopsy and the score for portal inflammation in the Knodell’s, but not in the Ishak’s, scoring system. This observation might suggest a relationship between the rate of HCV replication and the degree of inflammation in the portal tracts. The discrepancy between the two scoring systems might be explained by the fact that the Knodell’s score does not allow intermedi- ate values (e.g., 2).

The results of this study show that although sub- jects with raised ALT levels had higher scores for

were independently associated with the ALT pro- file. The analysis of data showed that HCV geno- type, HCV-RNA titer, histological grading and staging (whatever scoring system) were indepen- dently associated with the profile of ALT (Table 3).

Discussion

The development of serological tests for the detec- tion of antibodies to HCV has allowed the identi- fication of anti-HCV positive subjects without symptoms or biochemical signs of liver damage. However, the existence of healthy HCV carriers is a matter of controversy. In a significant percentage of cases, anti-HCV reactivity is associated with the presence of HCV-RNA in serum (16, 17) and with histological signs of liver damage (9, irrespective of ALT levels.

The absence of ALT elevation despite evidence of chronic hepatitis might be related to infection with a specific HCV genotype and/or to a lower degree of viral replication. In this study, we ana- lyzed these issues by comparing two groups of sub- jects with HCV-RNA and different ALT profiles.

Genotype 2 was mainly associated with persist- ently normal or near-normal ALT levels, whereas genotype l(b) was prevalent among subjects with elevated ALT. This observation is in contrast with previous reports detecting genotype I1 (lb) in the majority of Japanese patients with normal ALT levels (18, 19). The discrepancy could be due to a different geographical distribution of viral geno- types, since several studies indicate a large preva- lence of HCV genotype 1 over genotype 2 in Japan (20, 21). In line with our results, a recent study on Italian patients reported increased prevalence of HCV genotype I11 (2a) in patients with normal or near-normal ALT levels with respect to patients with abnormal ALT levels (22). The characteriza- tion of samples with type 2 had required the syn- thesis of specific PCR primers, suggesting the existence of a different subtype of genotype 2 in the Italian population. Consistent with these find- ings, in the present study nucleotide sequence analysis of the El region indicated that all geno- type 2 sequences shared higher homology with subtype 2c than with subtypes 2a and 2b. Our re- sults suggest that subtype 2c might be widely dis- tributed in Italy and responsible for most of bio- chemically silent HCV infections.

The possible relationship between the profile of ALT and the titer of HCV viremia is a matter of controversy. A previous report indicated a positive correlation between serum HCV-RNA titer and mean ALT levels (23), whereas another study failed to detect any difference in the viral titer between blood donors with normal or with elevated ALT

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grading and staging, liver damage of some degree was detected in all those with normal ALT levels. These observations raise questions about the exist- ence of the HCV healthy carrier state, or at least suggest the need for a reassessment of this defi- nition. Moreover, our data further support the role of histological examination for the clinical evalu- ation of subjects with active HCV replication, in- dependent of ALT levels. Obviously much remains to be known about the meaning and the final out- come of the inflammatory process almost in- variably present in the liver of individuals with normal ALT levels chronically infected with HCV.

Acknowledgements

This work was partially supported by a grant from Regione Lombardia (Progetto Ricerche Finalizzate n. 25).

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