Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities

Differential Distribution of Hepatitis C Virus Genotypes in Patients With and Without Liver Function Abnormalities ENRICO SILINI,1 FULVIA BONO,1 AGOSTINO CIVIDINI,2 ANTONELLACERINO,2 SAVINO BRUNO,3 SONIA ROSSI,3 GIOVANNI BELLONI,4 BRUNO BRUGNETTI,5 EMILIO CIVARDI,5 LAURA SALVANESCHI,6 AND MARIO U. MONDELLI2

Hepatitis C virus (HCV) infection persists for an indefinite length of time in a major proportion of patients, i n d u c i n g chronic liver lesions that evolve to cirrhosis and hepatocellular c a r c i n o m a (HCC) in approximately 20% of cases. We studied HCV viremia and g e n o t y p e s by reverse t r a n s c r i p t i o n - p o l y m e r a s e chain reaction (RTPCR) in 341 c o n s e c u t i v e anti-HCV-positive patients. Of these, 167 patients had persistently normal or near normal alanine aminotransferase (ALT) levels (fluctuations -<5 IU above the upper limit of normal); the r e m a i n i n g 174 patients p r e s e n t e d w i t h elevated ALT and histological e v i d e n c e of chronic liver disease. S e v e n t y percent of patients w i t h normal ALT values h a d circulating HCV RNA despite the absence of biochemical indicators of liver d a m a g e and mild histological forms of chronic hepatitis w e r e detected in most patients w h o u n d e r w e n t liver biopsy. Isolated g e n o t y p e III infection w a s significantly more prevalent in this patient group with respect to control patients w i t h abnormal ALT values (70% vs. 39%; P < .001). Conversely, isolated g e n o t y p e II w a s more frequently f o u n d in patients w i t h elevated ALT values and e v i d e n c e of chronic liver disease (45% vs. 23%; P < .01) and it was progressively more represented in adv a n c e d liver disease, such as cirrhosis and HCC. Virological features of HCV infection might be associated with different clinical manifestations, suggesting a potential prognostic significance on disease outcome. (HEPATOLOGY 1995;21:285-290.) Abbreviations: HCV, hepatitis C virus; HCC, hepatocellular carcinoma; ALT, alanine aminotransferase; RT-PCR, reverse transcription-polymerase chain reaction; CLH, chronic lobular hepatitis; CPH, chronic persistent hepatitis; CAH, chronic active hepatitis; HBsAg, hepatitis B surface antigen; EIA II, second-generation enzyme immunoassay; RIBA II, second-generation immunoblot assay. From the Departments of 1Pathology, 2Infectious Diseases, 4Internal Medicine, and 6Transfusion Medicine, Instituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo and University of Pavia, Italy; the 3Third Department of Internal Medicine, Ospedale San Paolo, Milano, Italy; and the ~Laboratory Medicine, Policlinico Ponte San Pietro, Ponte San Pietro (Bergamo), Italy. Received March 23, 1994; accepted August 15, 1994. Supported in part by grants no. 030RFM93/01 and 240RCR91/10 from the Italian Ministry of Health to IRCCS Policlinico San Matteo and by grant no. 669 from Regione Lombardia to the University of Pavia. F.B. was supported by a fellowship of the Cividini-Rotary Club Bergamo Ovest. Presented in part at the 44th Annual Meeting of the American Association for the Study of Liver Diseases, Chicago, IL, November 4-7, 1993. Address reprint requests to: Mario U. Monde]li, MD, Istituto di Clinica delle Malattie Infettive, IRCCS Policlinico S. Matteo, via Taramelli 5, 27100 Pavia, Italy. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2102-000353.00/0

Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality worldwide and is highly heterogeneous with respect to sources of infection and clinico-pathological features. Its most remarkable characteristic is the high frequency with which chronic liver disease develops after acute infection, ranging from 50% to 62%, depending on patient category, criteria used to define chronicity, and length of follow-up. 1'2 It has been demonstrated t h a t chronic hepatitis C, despite its usually asymptomatic clinical course, will evolve to cirrhosis in approximately 20% of patients after 10 years 3'~ and to hepatocellular carcinoma (HCC) in a subset of them with a yearly incidence of 3%. ~ Demographic and clinical features of acute infection, as well as routes of transmission, are not predictive of chronicity or progression of disease and alanine aminotransferase (ALT) levels or antibody patterns are poor indicators of the severity of histological lesions. 2'3 Moreover, the natural history of the disease is considerably long, indolent, and influenced by host factors, and histological lesions are usually mild and unable to predict the long-term outcome of the disease. Previous studies suggested t h a t virological parameters of infection, such as qualitative and quantitative determinations of serum HCV RNA, are significantly associated with underlying liver pathology, irrespective of biochemical indicators of liver cell injury. 6-1° It has also been suggested t h a t part of the observed disease variability might be caused by the heterogeneous nature of HCV t h a t comprises several genetic variants (genotypes), 11'12 which have been associated with response to interferon treatment, viral titers, and antibody patterns. 13-17 To understand whether virological parameters of HCV infection might correlate with clinical manifestations and histological severity of liver disease, we analyzed the prevalence of HCV viremia and genotypes by reverse transcription-polymerase chain reaction (RTPCR) in anti-HCV-positive patients, of whom 167 patients had persistently normal or near normal ALT levels, whereas 174 patients had biochemical and histological evidence of chronic liver disease. PATIENTS AND METHODS P a t i e n t s . Three hundred forty-one anti-HCV-positive patients were studied and were divided into three groups. Group 1 consisted of 167 consecutive asymptomatic subjects (82 males, 85 females; median age, 52 years; range, 18 to 73) with persistently normal or near normal ALT levels.

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Occasional ALT fluctuations within 5 IU above the upper limit of normal (40 IU/mL) were tolerated for inclusion in this group. One hundred nine patients were volunteer blood donors and 16 of them (14.6%) had identifiable risk factors of acquiring HCV infection: 9 received blood transfusions and 7 were exposed to sexual or intrafamiliar contacts. Fifty-eight patients were tested for anti-HCV as part of an occasional laboratory check-up for no apparent specific reason or because of the presence of potential risk factors for HCV infection (33 of 58 patients; 57%): 9 patients received blood transfusions, 5 were health care workers, 2 were former intravenous drug abusers, and 17 were exposed to sexual or intrafamiliar contacts. Patients not donating blood were followed-up for a median of 9 months (6 to 28 months) with monthly ALT determinations, whereas blood donors were studied for a median of 26 months (10 to 46 months) with ALT determinations every 3 months. Fifty-six patients from group 1 underwent diagnostic liver biopsy, which showed chronic lobular hepatitis (CLH) in 5 patients, chronic persistent hepatitis (CPH) in 17 patients, mild chronic active hepatitis (CAH) in 12 patients, minimal histological changes in 14 patients, and normal liver in 8 patients. Group 2 consisted of 95 consecutive anti-HCV-positive patients (68 males, 27 females; median age, 51 years; range, 26-69) who presented with elevated ALT. A subsequent liver biopsy showed CPH in 8 patients, mild CAH in 11 patients, moderate or severe CAH in 64 patients, and cirrhosis in 12 patients. To investigate the distribution of HCV genotypes according to the histological severity of liver disease, 79 additional patients (group 3), showing liver lesions scarcely represented in group 1, (CPH in 8 patients, mild CAH in 8 patients, cirrhosis in 24 patients, and hepatocellular carcinoma [HCC] in 39 patients) were also included in the study. They were randomly selected from a wider population of more than 1,000 patients with chronic HCV infection. Risk factors of acquiring HCV infection were identified in 57 (33%) patients from groups 2 and 3:29 patients received blood transfusions, 11 patients were health care workers, 7 patients were former intravenous drug abusers, and 10 patients had sexual or intrafamiliar contacts. All patients from the three groups were residents of three neighboring provinces of Northern Italy, none were hepatitis B surface antigen (HBsAg) positive. Informed consent to participate in the study was obtained from all patients and the study protocol was approved by the hospital's ethical committee in conformity to the 1975 Declaration of Helsinki. Serology and Histology. All patients were screened for the presence of anti-HCV by a second-generation enzyme immunoassay (EIA II) (Ortho HCV, 2nd generation, Ortho Diagnostic Systems, Raritan, NJ), according to the manufacturer's instructions. Tests were performed in duplicate on aliquots stored frozen at -20°C and used only once. Anti-HCV immunoreactivity by EIA II was confirmed for selected patients from group 1 by second-generation immunoblot assay (RIBA II, Chiron Corporation, Emeryville, CA, and Ortho Diagnostic Systems). Histological diagnosis was performed according to standard diagnostic criteria. Minimal histological changes included the following patterns: steatosis, portal fibrosis, mild portal inflammation limited to a few portal tracts, occasional spotty necrosis, and mild cholestasis. HCV RNA Detection and Genotyping. HCV RNA was detected by nested RT-PCR using conserved primers localized in the 5' noncoding region of the viral genome ls'19 using RNA extracted from 100 #L of serum. HCV RNA detection was performed on the same serum sample used for serology. HCV genotyping was performed according to Okamoto et al 2°'21 using RT-PCR with type-specific primers of the core

HEPATOLOGYFebruary 1995 gene. Because the method used according to the published primer sequences resulted in several indeterminate samples with a remarkable absence of HCV type III infection, we cloned and sequenced core gene sequences of 18 indeterminate serum samples. Nucleotide analysis showed a marked uniformity of sequence among clones and the presence of motifs described as specific of HCV type III (Table 1). Several nucleotide substitutions were found in the region of the HCV type III specific primer in comparison with the Japanese sequences used for the development of the genotyping assay. Thus, the HCV type III specific primer was redesigned as follows: 5'-GCCCCATGAAGGGCGAGAAC-3'. Genotyping with the newly designed type III-specific primer was very efficient with an overall number of nontypable samples reproducib]y less than 3%. Genotyping fidelity was confirmed by cloning and sequencing of core region sequences from representative clones classified according to the modified procedure. HCV genotypes I, II, III, IV, and V according to Okamoto correspond to genotypes la, lb, 2a, 2b, and 3a, respectively, as classified by Simmonds 22 on the basis of seq u e n c e diversities within the 5' noncoding region of the genome. Statistical Analysis. Only viremic patients in whom HCV genotype could be successfully determined were considered for statistical analysis. Wilcoxon's two-sample test, X2 test, or Fisher's exact test were used as appropriate. Patients showing infection with genotypes I, V, and other, as indicated in our tables, were considered under the same category in contingency table analysis of genotype distribution. Other categories are as shown in the tables. RESULTS

H C V V i r e m i a a n d Genotype in P a t i e n t s With Persistently N o r m a l or N e a r N o r m a l A L T Levels (Group 1). D e t e r m i n a t i o n o f s e r u m H C V R N A s h o w e d t h a t 118 (71%) of 167 p a t i e n t s f r o m g r o u p 1 w e r e v i r e m i c d e s p i t e p e r s i s t e n t l y n o r m a l or n e a r n o r m a l A L T levels, w h e r e a s t h e r e m a i n d e r a p p a r e n t l y did not h a r b o r circ u l a t i n g virus. T h e a b s e n c e of H C V v i r e m i a w a s associa t e d w i t h a n i n d e t e r m i n a t e or n e g a t i v e R I B A I I a s s a y in t h e 126 p a t i e n t s f r o m g r o u p 1 for w h o m b o t h t e s t s w e r e a v a i l a b l e (Table 2). I n d e e d , 39 (86.6%) of 45 pat i e n t s w i t h no d e t e c t a b l e s e r u m H C V R N A w e r e R I B A I I - n e g a t i v e or i n d e t e r m i n a t e . H o w e v e r , R I B A I I indet e r m i n a t e r e s u l t s did n o t n e c e s s a r i l y i m p l y a b s e n c e of d e t e c t a b l e v i r e m i a , b e c a u s e also 23 (40.4%) of 57 R I B A i n d e t e r m i n a t e p a t i e n t s w e r e v i r e m i c (Table 2). Histological evidence of chronic h e p a t i t i s w a s p r e s e n t in 31 (62%) of 50 v i r e m i c p a t i e n t s f r o m g r o u p 1 in w h o m a liver b i o p s y h a d b e e n p e r f o r m e d . T w e n t y p a t i e n t s h a d e i t h e r C L H or C P H a n d 11 p a t i e n t s h a d m i l d CAH, w h e r e a s 19 h a d m i n i m a l histological c h a n g e s or n o r m a l liver. Six n o n v i r e m i c p a t i e n t s also h a d a liver biopsy, w h i c h s h o w e d m i l d C A H in 1 patient, C L H a n d C P H in 2 p a t i e n t s , a n d n o r m a l liver in 3 patients. H C V g e n o t y p e d e t e r m i n a t i o n in g r o u p 1 p a t i e n t s s h o w e d t h a t t h e m a j o r i t y (70%; 78 of 112) w a s infected b y H C V t y p e I I I , w h e r e a s only 23% (26 of 112) w e r e infected b y H C V t y p e I I a n d a m i n o r i t y b y H C V t y p e I or V (Table 3). H C V g e n o t y p e c o r r e l a t e d w i t h R I B A I I r e s u l t s in g r o u p 1 p a t i e n t s , b e c a u s e 20 of 21 (95%) RIBA II indeterminate, HCV RNA-positive sera that

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TABLE 1. C o m p a r i s o n o f H C V C o r e G e n e S e q u e n c e s A m o n g 14 I s o l a t e s

Type

Clone

Nucleotide 527

I I II III

HCV-1 Clone 1 Clone 2 HCV-J6 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 Clone 11 Clone 12

562

637

669

T--C--T-

-G- -TAGC

.........

.....

T--C- -T- -G- -TAGC

.........

.....

C-

T C A G C C T A T C C C C A A G G C T C G T C G G C C C G A G G C A G

......

- ......

. . . . . . . . . . . . . . . . . .

. .....

-T

. .....

-A

......

C C C C C C G A G G T T C C C G T C C C T C T T G G G G C C C C

......

- ......

A-

-A-G

G ................

...............

C-A

C .....

C .....

T ....

C .....

G .....

T--A-A-

C ......

C .....

T-

C .....

C .....

T-

C .....

C .....

...........

A - - - G - - C T - - A C T - - - A - -G--C---ACT-

-A-A-A-A-

- -A

--G--CT--ACT- -G-

-CA-

-ACT-

- -A

......

T .....

- ......

- -A

......

- - - -T-

-C

-T-

.....

T-

-C

T--A-A-

- -G-

-CA--ACT-

- -A

......

- ......

T-

-A-A-

- -G-

-CA-

-ACT-

- -A

......

- - - -T-

-C

.....

C .....

T-

-A-A-

- -G-

-CA-

-ACT-

- -A

......

- - - -T-

-C

.....

- -G-

-CA-

-ACT-

- -A

......

- ......

T-

-CG--ACT-

- -A

......

- ......

C .....

A-

C .....

C ...........

T ....

-C-

C .....

C .....

T--A-AC--G-

C .....

G .....

T-

-A-A-

- -G-

-CA-

-ACT-

- -A

......

- ......

C-

C . . . .

C .....

T-

-A-A-

- -G-

-CA-

-ACT-

- -A

......

- ......

C .....

-C-

G--TAG

T--C--T-

C--C-

C ........... C .....

.....

T-T-

-C-

-G-

-C-

-T-

T-

.........

-A

.........

.........

-A

.........

-A

.........

-C-

-T-

-A

.........

-C-

-T-

-A

.........

T--C--T-T-

-G

-C--T--A

T--C--TT-

..........

-A

.........

-C-

-T-

-A

.........

-C-

-T-

-G

.........

NOTE. HCV-1 and HCV-J6 are prototype strains of HCV type I and III, respectively. The region between nucleotides 527 and 562 is shown to demonstrate nucleotide motifs specific for each type. 1° The region encompassing nucleotides 637 to 669 includes the type III specific primer used for genotyping (bold face), several nucleotide substitutions are evident in Italian isolates with respect to prototype strain J6.

were genotyped showed infection with HCV type III, representing 38% (20 of 56) of all type III infections. An overall lack of reactivity with NS4 antigens (5.1.1 and C100-3) was observed in patients with HCV type III infection. Thus, 8 (16%) of 56 sera from patients with HCV type III infection were reactive with NS4 antigens, compared with 9 (53.3%) of 16 sera from patients infected with other HCV genotypes (P < .01). Similar results were previously reported by us 19 and others 17'22 and are probably related to the considerable sequence diversity of the NS4 region among genotypes} * Indeed, anti-NS4 antibodies in sera from HCV type III-infected patients are unlikely to recognize the NS4 protein fragments used for the RIBA II assay that are produced according to the sequence of the HCV prototype, HCV-1 (genotype I). HCV Genotype Distribution in Patients With Abnormal ALT Levels (Group 2). To investigate whether the HCV genotype distribution observed in group 1 patients was characteristic of this patient category or sireply reflected the overall prevalence of HCV genotypes in our geographic area, we determined the HCV genotype in 95 consecutive control patients who came to our observation with a positive anti-HCV test and elevated ALT values and who had a histological diagnosis of chronic hepatitis (group 2). All group 2 patients were TABLE 2. C o m p a r i s o n o f H C V R N A S t a t u s W i t h R I B A II R e a c t i v i t y i n 126 A n t i - H C V - P o s i t i v e P a t i e n t s W i t h P e r s i s t e n t l y N o r m a l or N e a r N o r m a l ALT V a l u e s HCV RNA RIBA II

Positive

Negative

Total

Positive Indeterminate Negative Total

57 23 1 81

6 34 5 45

63 57 6 126

viremic according to HCV RNA detection in serum by RT-PCR with primers of the 5'-noncoding region. HCV genotype distribution in patients from groups 1 and 2 is shown in Table 3. A statistically significant difference in the overall genotype distribution among the two groups was obtained by contingency table analysis (P < .001). Isolated HCV type III infection was significantly more prevalent in group 1 patients (P < .001) compared with group 2 patients. Conversely, isolated genotype II was more frequently found in group 2 patients (P < .01) compared with group 1 patients. Relationship Between HCV Genotype and Severity of L i v e r Disease. Because patients from group 2 showed more severe histological lesions compared with group 1 (see Patients and Methods), we asked whether differences in HCV genotype distribution among the two groups could be related to the higher ALT values found in group 2 or to the severity of the underlying histological lesions. To test this hypothesis we determined the HCV genotype in 79 randomly selected additional patients (group 3) with chronic liver lesions that were scarcely represented in group 2 patients. All group 3 patients had abnormal liver function tests. HCV genotype distributions according to histology of liver lesions in group 1 and in combined groups 2 and 3 are shown in Table 4. A significantly different distribution of HCV genotypes among histological categories was observed in patients from combined groups 2 and 3 (P < .05). This was mainly caused by the progressively higher frequency of HCV type II infections in severe liver disease, such as cirrhosis and HCC (P < .02), whereas this was not observed for other HCV types. To further clarify the relationship between HCV genotypes and biochemical and histological features of liver disease, we compared HCV genotype distribution in group 1 patients with CLH/CPH or mild CAH and patients from combined groups 2 and 3 showing liver disease of comparable severity. We found that the prev-

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TABLE 3. P r e v a l e n c e o f HCV G e n o t y p e s A c c o r d i n g t o A L T L e v e l s i n 207 A n t i - H C V - P o s i t i v e

Patients

HCV Genotype ALT Level

I

II

III

V

Normal (group 1) Elevated (group 2) Total

3 (3) 6 (6) 9

26 (23) 42 (45) 68

78 (70) 37 (39) 115

1 (1) 3 (3) 4

Other* 4 (4) 7 (7) 11

Total 112 95 207

NOTE. Differences in genotypes frequencies among the two groups calculated by )/e analysis were: HCV type II, P < .01; HCV type III, P < .001; other types, not significant. * Mixed infections and nontypable samples are grouped under this category. Group 1: type I-II (1), II-III (2), I-III (1). Group 2: type I-II (1), III-IV (2), II-III (2), nontypable (2). Percentages are reported in brackets.

alence of genotype III was significantly higher in subjects with normal or near normal ALT (P < .001), whereas genotype II was more frequently found in patients with biochemical features of chronic liver disease (P = .055). Thus, the distribution of HCV genotypes was influenced by ALT levels in patients with comparable histological lesions. Molecular Epidemiology of HCV Infection in Different Patient Categories. Known HCV genotypes (I, II, III, IV, and V) accounted, alone or in combination, for the majority of circulating viruses in our geographic area. This finding is in agreement with data obtained by other investigators in Italy 15'16'24 and Europe, 15'25 either by HCV serotyping or genotyping. We observed t h a t patients with HCV genotype I and V infections were younger than the remainder, their median age being 36 years (range, 27 to 63) and 29 years (range, 21 to 34), respectively, versus an overall median age of 53 years (range, 18 to 83). Seven of 9 patients with a history of intravenous drug abuse had infection with HCV type I or V. Drug addiction accounted for approximately 50% of infections with HCV type I (4 of 10 patients) or V (3 of 6 patients). The small number of patients infected with genotypes I and V in this series precluded statistical analysis of age differences among genotypes. No relationship with epidemiological or demographic features of HCV infection was observed for other genotypes.

DISCUSSION

HCV persists indefinitely in most infected patients inducing asymptomatic chronic liver disease, which is usually shown only by abnormal serum enzymes and histology. Current diagnostic approaches to HCV infection are effective in screening and in identifying patients who might benefit from antiviral treatment; however, they are unsatisfactory in the clinical management of patients with persistent anti-HCV positivity and no biochemical evidence of liver cell injury. To understand the features of HCV infection in this peculiar patient category, we integrated current diagnostic procedures with the study of virological parameters, such as HCV viremia and genotype. We observed t h a t a substantial proportion of patients with persistently normal or near normal ALT values had circulating HCV RNA. Most of these patients harbored HCV type III and had evidence of chronic liver disease, although of mild entity. Esteban et al, 26 studying anti-HCV-positive blood donors, observed milder histological lesions in subjects with normal ALT and indeterminate supplemental assay compared with donors with elevated ALT; however, HCV viremia was not assessed in their study. Alberti et al 6 found viremia to reliably predict liver lesions in the absence of biochemical indices of disease and in agreement with such findings most of our asymptomatic viremic patients

TABLE 4. P r e v a l e n c e o f H C V G e n o t y p e s A c c o r d i n g to L i v e r H i s t o l o g y i n 222 A n t i - H C V - P o s i t i v e

Patients

HCV Genotype ALT Level Normal (group 1)

Abnormal (Groups 2 and 3)

Liver Histology

I

II

Normal liver Minimal changes CLH/CPH Mild CAH CLH/CPH CAH Cirrhosis HCC Total

-1 (8) -1 (9) 2 (12) 5 (6) 1 (3) -10

-4 (33) 4 (20) 2 (18) 6 (35) 40 (49) 21 (58) 29 (74) 106

III 4 5 15 8 6 29 12 9

(80) (42) (75) (73) (35) (35) (33) (23) 88

V

Other*

Total

1 (20) ---2 (12) 3 (4) --6

-2 (17) 1 (5) -1 (6) 5 (6) 2 (6) 1 (3) 12

5 12 20 11 17 82 36 39 222

NOTE. Differences in genotype frequencies among histological categories in patients with abnormal liver function tests (groups 2 and 3), calculated by X2 analysis, were: HCV type II, P < .02; other types, not significant. * Mixed infections and nontypable samples are grouped under this category. Group 1: type I-II (1), II-III (1), I-III (1). Groups 2 and 3: type I-II (1), III-IV (2), II-III (3), nontypable (3). Percentages are reported in brackets.

HEPATOLOGYVol. 21, No. 2, 1995 showed some form of liver damage. However, in our study, HCV viremia did not invariably correlate with the presence of histological lesions in group 1 patients because 3 of 6 subjects without detectable viremia had chronic hepatitis and we found normal or slightly abnormal liver histology in 19 of 50 HCV RNA-positive patients. Moreover, in contrast with the findings of Alberti et al, ~ we observed uniformly mild liver lesions. The different criteria used for the selection of group 1 patients with respect to ALT values may explain these discrepancies. It must be also emphasized that the techniques used in this and other studies have intrinsic limitations that might in part explain the observed discrepancies between histology and HCV viremia. Indeed, circulating HCV RNA titers may vary considerably and occasionally fluctuate below the detection limit of the PCR a s s a y F On the other hand, liver biopsy m a y show intrinsic sampling variability. The possible coexistence of chronic viremia with normal liver histology has, nonetheless, been reported previously. 2s Our results suggest that, at least as far as HCV infection is concerned, the current clinical definition of chronic hepatitis that is presently based on abnormal liver function tests is probably inadequate because chronic liver disease is frequently found in patients with persistently normal ALT levels. Furthermore, anti-HCV patients with normal ALT levels should not be considered as "healthy carriers" because they are often viremic and, therefore, they should be studied with repeated ALT determinations and liver biopsies. Indeed, such patients might represent a large, overlooked reservoir of infections, which m a y have a significant role in HCV diffusion with important implications on long-term morbidity and mortality, considering the possibility of late reactivation of the disease. The frequency of HCV type III infection in biochemically silent patients, the highest ever reported thus far, prompted us to test HCV genotype distribution in a random sample of anti-HCV-positive patients with abnormal liver function tests and a histological diagnosis of chronic hepatitis. The objective was to exclude the possibility that the predominance of HCV type III infection observed in group I patients could be related to the geographic area from which our patients were recruited rather than to differences in clinico-pathological features of the disease. Indeed, subjects with overt clinical manifestations of disease resident in the same region of group 1 patients (group 2) showed a different distribution of HCV genotypes, with HCV genotype III being significantly more prevalent in patients with normal or near normal ALT levels and mild liver lesions, whereas HCV type II was more frequently found in patients with chronic hepatitis and elevated ALT. Patients from groups I and 2 were selected according to biochemical indices of liver cell injury, but liver biopsy showed a substantially different spectrum of underlying liver disease, which was significantly milder in group 1 patients. However, a higher frequency of genotype III infection in this group was still present when only patients with comparable histological lesions were considered in both groups, whereas the

SILINI ET AL 289 prevalence of genotype II was higher in patients with abnormal liver enzymes. It is possible that specific genotypes might be associated with different degrees of hepatocellular necrosis, the severity of which, at least as far as mild liver lesions are concerned, might be more accurately measured by repeated ALT determinations rather than by a single liver biopsy, which may be affected by sampling error. Further analysis of HCV genotype distribution as a function of histology in a larger cohort of patients with various forms of chronic liver disease, including a representative number of patients with HCV-related liver cirrhosis and HCC (groups 2 and 3) showed a possible association of specific genotypes with disease progression. In fact, an increased frequency of HCV type II infections was significantly associated with the severity of histological lesions, accounting for approximately 75% of all HCV infections in patients with HCC. Because most of the present concern raised by chronic HCV infection is about the long-term susceptibility to liver failure and cancer, this observation appears to be of major prognostic importance and should be confirmed in prospective studies. Age distribution of patients with HCV type II and III infections in our series was similar, excluding the possibility that a cohort effect might account for the higher frequency of HCV type II observed in older patients with advanced lesions. However, given the unexpectedly high frequency of HCV types I and V among young patients and their association with specific routes of transmission (intravenous drug abuse), larger surveys should be performed to investigate possible relationships between HCV genotypes and age or infection modalities. To this end, preliminary findings from a larger cohort of patients indicate that in our geographic area HCV genotypes I and V are almost exclusively detected in young patients, particularly in those with a history of intravenous drug a b u s e Y In conclusion, the results of the present study suggest that specific HCV genotypes might be associated with different clinical manifestations and severity of liver disease, reflecting intrinsic biological properties of the virus subtypes and suggesting a potential prognostic significance on disease outcome. These findings are in agreement with previous studies in which higher virus titers and a poorer response to interferon treatment were associated with different HCV genotypes. 13'14'16 Recent evidence suggests that also HCV RNA titers may independently correlate with the severity of inflammatory lesionsS-l°; further studies should help to establish the relative contributions of HCV genotype and titer in the management of HCV-infected patients. Nucleotide diversities have been found among genotypes in putative regulatory regions at the 5' and 3' ends of the genome 3°'3~ that m a y have pathogenetic implications as described for other RNA viruses. 32 Because the degree of nucleotide variability in hypervariable regions of envelope genes has been associated with response to interferon treatment, 33 it would be interest-

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HEPATOLOGYFebruary 1995

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ing to evaluate whether HCV type II and III differ under this profile.