GASTROENTEROLOGY 2000;118:760–764

LIVER, PANCREAS, AND BILIARY TRACT

Natural History of Hepatitis C Virus Carriers With Persistently

Normal Aminotransferase Levels

MARCELLO PERSICO, ELIANA PERSICO, ROSALBA SUOZZO, SALVATORE CONTE,

MASSIMILIANO DE SETA, LEONARDO COPPOLA, BRUNO PALMENTIERI,
FERDINANDO CARLO SASSO, and ROBERTO TORELLA
Internal Medicine and Hepatology Unit, II University of Naples, Naples, Italy

Background & Aims: Some patients with serum hepati- Several studies suggest that these patients do not
tis C virus (HCV) have persistently normal aminotrans- benefit from interferon treatment.11–14 However, no data
ferase (ALT) levels and are affected by cirrhosis. This are available on the natural history of chronic hepatitis C
study prospectively evaluated progression of the dis- associated with persistently normal ALT levels that
ease in a group of anti-HCV–positive patients with
support this observation. In particular, whether there is
persistently normal ALT levels. Methods: Thirty-seven
subjects were studied. Each subject underwent liver
any progress in grading and staging of chronic hepatitis
biopsy at baseline and after 5 years of follow-up. At in these patients is not known.
baseline, serum samples were tested for genotypes This study was therefore designed to evaluate the
and HCV RNA load. ALT levels and serum HCV RNA were natural history of a group of anti-HCV–positive patients
tested every other month and every 6 months, respec- with persistently normal ALT levels during 7 years of
tively. Patients with increased ALT were discharged prospective follow-up to provide further insights into the
from the study and treated with IFN. Five years after concept of disease progression. We found that patients
the end of IFN therapy, a liver biopsy was performed. with persistently normal ALT levels have chronic hepati-
Results: Liver biopsy at baseline showed chronic hepa- tis whose grade of activity does not increase over 5 years
titis in 34 patients and normal histology in 3 patients, 2
of observation. This lends support to the concept that
of whom were negative for HCV RNA and 1 positive.
HCV genotypes were distributed as follows: 2a, 56%;
these patients may not benefit from interferon treatment.
1b, 41%; and 1a, 3%. At the end of 7-year follow-up,
73% of the patients still had normal ALT values. Liver Materials and Methods
histology after 5 years was comparable to that ob-
Inclusion Criteria
served at entry to study. Conclusions: Most patients
with persistently normal ALT serum levels have very Between January 1990 and 1992, all anti-HCV–
mild chronic hepatitis. However, healthy anti-HCV– positive patients routinely attending the Internal Medicine and
positive subjects exist. In patients with HCV-related Liver Unit at our institution were prospectively enrolled in the
chronic hepatitis associated with persistently normal study if they met the following criteria: (1) persistently (at least
ALT levels, the grade of disease activity does not 3 times during the last year) normal ALT levels, (2) no
increase over years and progression to cirrhosis is slow symptoms and/or other signs of chronic liver disease, and (3)
or absent. willingness to undergo physical and blood examinations at
scheduled intervals and consent to invasive diagnostic proce-
dures if needed. The prevalence of this group of patients was
any subjects with hepatitis C virus (HCV) infec-
M tion have persistently normal aminotransferase
(ALT) levels and are considered asymptomatic anti-HCV
10.7% (21 of 195 patients) at the first year and 9.2% (6 of 175
patients) at the second year of enrollment. Intravenous drug
abusers were excluded from the study. Some of the enrolled
carriers.1–4 Most of these patients have been documented patients have been part of a previous investigation.5
to have some degree of histologically proven chronic liver
damage ranging from mild chronic hepatitis to liver
cirrhosis.5,6 HCV genotype distribution7,8 and viral load9,10 Abbreviation used in this paper: PCR, polymerase chain reaction.

have also been explored, and available data in literature r 2000 by the American Gastroenterological Association
0016-5085/00/$10.00
are still conflicting. doi:10.1053/gg.2000.5966
April 2000 NATURAL HISTORY OF ASYMPTOMATIC C VIRUS CARRIERS 761

Routine Investigations Follow-up

Routine evaluation of the patients included medical Enrollment of the patients started on January 1990 and
history, physical examination, complete blood count, and closed on January 1992. Patients were then prospectively seen
measurement of ALT and markers for viral hepatitis. Markers as follows.
for hepatitis B infection were tested by radioimmunoassays Genotype and viral load were determined and liver biopsy
(Abbott Laboratories, Chicago, IL). Antibody to hepatitis C was performed at the time of entry in the study. ALT levels
virus was assayed by an enzyme-linked immunoassay test of were evaluated every other month. Every 6 months, patients
second generation (ELISA; Ortho Diagnostic Systems, Raritan, underwent physical examination and serum HCV RNA deter-
NJ). Specific investigations included a reverse-polymerase mination. Patients who had increase in ALT levels were
chain reaction (PCR) in a 9600 thermal cycler (Perkin Elmer, discharged from the follow-up and treated with interferon.
Emeryville, CA) to detect HCV RNA, HCV RNA load, and Patients who finished 5 years of follow-up were asked to repeat
HCV genotype. liver biopsy.
In patients with increased ALT levels, a liver biopsy was
RNA Preparation and HCV performed 5 years after the end of interferon therapy. Grading
RNA Quantification and staging were retrospectively compared with those of
All manipulations were performed under ribonuclease- patients with constantly normal ALT levels.
free conditions.
Total RNA was isolated from serum according to Chomczyn- Histological Analysis
ski and Sacchi15 and reverse transcribed using random hexamers.
All serum samples were treated in parallel with negative The Kaplan–Meier method20 was used to calculate the
controls. percentage of patients with normal ALT levels during and at
A competitive PCR to quantify HCV RNA was used the end of follow-up.
successfully. Other investigators have attempted the use of a
similar procedure.16 The most conserved region (58NC) of Results
HCV RNA was retrotranscribed and amplified with outer
primers and cloned. In the middle, 70 base pairs of exogenous Patients
DNA were added, the longer product was quantified, and Between January 1990 and January 1992, 37
different dilutions were amplified to obtain a standard curve asymptomatic anti-HCV–positive patients were enrolled and
with 4 increasing points, the lowest of which was comparable followed up. Table 1 shows their demographic and clinical
to 10 copies of genoma per microliter. Coamplification of the characteristics. No sex difference was found, 25 patients were
complementary DNA samples with known amounts of the
blood donors, and all other patients underwent anti-HCV
DNA competitor was performed, and resolution of the 2 PCR
products was obtained by gel electrophoresis.
antibody blood testing because at least 1 member in their
Carryover PCR contamination was avoided by applying the family had tested positive for anti-HCV antibodies. Apparent
measures suggested by Kwok and Higuchi.17 risk factors of HCV infection were collected only in 10
subjects who reported previous surgical procedures in their
Procedure for HCV Genotyping clinical history. The apparent duration of the disease could be
Serum PCR products were hybridized to type- and determined in only 12 subjects, with a mean value of
subtype-specific probes 1a, 1b, 2a, 2b, and 3a to classify HCV 10.8 years (range, 5–20 years). All patients had normal
genotypes. The probes used fulfill 2 main criteria: no more than ALT levels, and no clinical symptom was present.
2 mismatches to the corresponding published sequences of the
same subtype, and they differ by 3 or more mismatches
Table 1. Demographic and Clinical Features of Asymptomatic
compared with published sequences of other types and sub-
Patients With HCV
types. The only exception is probe 2b showing only 2
mismatches to the corresponding sequence of type 3a.18 No. of patients 37
Sex (M/F) 20/17
Age, yr (range) 40.8 ⫾ 14.5 (22–65)
Liver Histology ALT, U/L (range) a 23 (12–37)
Ultrasound-assisted liver biopsy was performed using a Blood donors 15
Surgical procedures 10
modified Menghini needle of 1.6-mm diameter (Hepafix B;
HCV RNA (⫹/⫺) 35/2
Braun, Melsungen AG, Germany). Liver specimens, previously Genotype: 1a, 1b, 2a 2/14/19
fixed in formalin and embedded in paraffin, were stained and Grading 8 (2–16)
reviewed at 2 different times by the same observer who was Staging 2 (0–4)
blinded to biochemical and clinical data. Histological features Total HAI 10 (3–20)
were scored according to Ishak et al.,19 and grading, staging, HAI, histological activity index.
and total score were reported. aNormal values (12–40).
762 PERSICO ET AL. GASTROENTEROLOGY Vol. 118, No. 4

Table 2. Liver Histology of the 7 Patients Who Left the Study histological progression over 5 years with that observed
Because of Increase of ALT Level at Baseline and over the same period in the patients with persistently
After 5 Years of Suspension of Interferon Therapy
normal ALT levels (Table 2). The 2 patients with normal
Score at baseline Score after 5 yr
liver histology and negative HCV RNA were excluded
Genotype Grading Staging Total Grading Staging Total from the analysis.
1b 2 1 3 3 1 4 Including the dropout subjects, in the seventh year of
1b 3 0 3 3 1 4 follow-up, according to the Kaplan–Meier method, 22%
1b 3 1 4 4 1 5 of patients enrolled left the study and 78% still had
2a 13 4 17 14 6a 20 a
2a 9 2 11 9 3 12 normal ALT levels (Figure 1).
2a 9 2 11 10 3 13
2a 3 1 4 2 0b 2b
Serum HCV RNA, Liver Histology,
aLivercirrhosis.
bMinimal changes.
and Genotype
HCV RNA at time 0 was positive in 35 patients
Follow-up and negative in 2 patients who also had a normal pattern
The overall follow-up was 7 years, and to date each at liver histology. The mean ⫾ SD findings of grading,
patient has completed at least 5 years of follow-up. Three staging, and total histological score of liver biopsy
patients missed a follow-up visit, 1 in the first year and 2 specimens at baseline, evaluated according to Ishak’s
in the third year, and were considered dropouts. At their classification, are shown in Table 3. Table 3 also shows
last visit, they still had normal ALT values. median and range of grading, staging, and total score of
Because of the increase in ALT level, 2 patients left the liver biopsy specimens of the 24 patients still in the study
study in the second year, 4 in the third year, and 1 in the after 5 years of follow-up. No difference was found. Of
fourth year. The severity of the histological score in these the 7 patients who left the follow-up for ALT increase and
patients was not related to the increase in ALT. Risk who underwent interferon treatment, 1 responded to the
factors other than HCV were excluded as being respon- therapy and the others were nonresponders. In these
sible for the increase in ALT level (i.e., control of alcohol patients, histological data 5 years after suspension of
consumption as well as control of markers of major and interferon therapy showed the following findings. The
minor infectious agents). The patients started interferon single ‘‘sustained responder’’ had histological improve-
therapy, and no further histological examinations were ment from mild chronic hepatitis to minimal liver
programmed before treatment. For these patients, we change. Among the other 6 patients, 5 did not have any
retrospectively collected data from liver biopsies per- significant difference between liver histology at entry to
formed 5 years after suspension of treatment to study the study and after 5 years, although 2 patients showed a
outcome to interferon therapy and to compare the higher staging score, likely documenting a progression

Figure 1. Kaplan–Meier curve

of asymptomatic patients. After
7 years of follow-up, 73% of
patients had normal and 23%
had increased ALT values. No
patients had increases in ALT
levels after the fourth year.
April 2000 NATURAL HISTORY OF ASYMPTOMATIC C VIRUS CARRIERS 763

Table 3. Median and Range of Histological Score of Patients infected subjects with persistently normal ALT levels
With Persistently Normal ALT Levels at Baseline have any progression of liver damage after 5 years of
and After 5 Years of Follow-up According to
HCV Genotype
follow-up. This might be helpful in deciding whether
these patients need to be treated with interferon. The
Genotype Ishak score Grading Staging Total
prospective follow-up supports the concept that all
1b–1a a Basal 10.5 2 12.5 HCV-positive patients with persistently normal ALT
(3–16) (1–4) (4–20)
After 5 yr 11.5 2 13.5 levels have chronic hepatitis with slow progression, or no
(3–16) (1–4) (4–20) progression at all, to more severe liver disease (i.e.,
2ab Basal 5 1.5 6.5 cirrhosis). The first consequence of this might be the
(2–13) (0–4) (3–17)
After 5 yr 5 1.5 6.5
inefficacy of interferon therapy in asymptomatic patients
(2–13) (0–4) (3–17) in favor of a wait-and-see approach. This is similar to data
a1b ⫽ 9; 1a ⫽ 1.
from a recent study of interferon treatment in asymptom-
b2a ⫽ 14. atic patients14 concluding that such treatment is not
effective in these patients. Nevertheless, interferon is
suggested as a therapeutic choice for patients with mild
of fibrosis, and 1 had progression to liver cirrhosis (Table disease and flaring ALT. The question is whether and
2). Patients with normal liver histology were not asked to when interferon should be a therapeutic option. In our
repeat liver biopsy, and their serum HCV RNA was study, no patients had increases in ALT values after the
persistently negative. Serum HCV RNA titer at baseline fourth year of follow-up. Possibly because of the accurate
was widely distributed with a mean copy number of follow-up of such patients, we selected a very strict group
8.06 ⫻ 104 (range, 750 ⫻ 105 to 1.00 ⫻ 105) copies/µL. of subjects who really represent ‘‘asymptomatic’’ carriers
No significant variation of HCV RNA titer was reported of HCV. Interestingly, patients with increases in ALT
during the follow-up, neither was a significant difference levels did not have the worst histological score, suggest-
found for HCV viremia after 5 years (mean copy number, ing that ALT levels are not a valuable predictor of
9.04 ⫻ 104; range, 750 ⫻ 105 to 1150 ⫻ 105). Genotype progression of disease. Most of these subjects6,7 treated
distribution is shown in Table 1 and the HCV genotype with interferon did not benefit from the therapy, and the
prevalence according to histology in Tables 2 and 3. No histological score 5 years after the end of interferon
relation was found between HCV genotype and liver treatment showed progression to cirrhosis in 1 patient
histology. In particular, HCV genotype and HCV serum and mild progression of fibrosis in 2 patients. Only 1
titers were not different in patients discharged by the patient responded to therapy; his histology improved
follow-up because of the increase of ALT compared with from mild chronic hepatitis to minimal liver changes. A
those found in patients with persistently normal ALT recent demonstration of a lower degree of fibrosis in
levels. These results support data reported by other patients with persistently normal ALT activity21 supports
investigators on similar cohort of patients of larger size.21 the concept of very slow progression, if any, of liver
disease in these subjects. However, whereas Mathurin et
Discussion al.21 evaluated progression of liver fibrosis over a virtual
Most patients infected with HCV have different period of time of assumed years before biopsy, our data
degrees of liver damage that can worsen and lead to liver instead strongly demonstrate a lack of progression of
cirrhosis in 20% of the cases, although subjects with disease activity in 2 liver biopsies performed several years
normal liver histology despite ongoing HCV viremia apart in a prospective designed study.
have been documented.2–5 However, the long-term natu- The diversion of the 2 groups (asymptomatic and
ral history of the so-called asymptomatic carriers of HCV symptomatic), at the beginning of the study under the
is not clear. In general, although ALT levels do not relate same name, may suggest that the pathological potential
to the entity of the liver damage,22 patients with of HCV is expressed only in a particular genetic back-
persistently normal ALT levels have less severe disease.8 ground. The immune system of ‘‘asymptomatic HCV
Supporting these data, Mathurin et al.21 recently showed carriers’’ might be able to satisfactorily react to the
in a large cohort of patients that those with normal ALT turnover of the quasi species, avoiding the escape mecha-
had less severe fibrosis than those with increased ALT. nism.
Nevertheless, some reports show the presence of cirrhosis The mean value of the apparent duration of the disease
in such patients.6 Whether these patients should be (determined only in a small sample and still under
treated with interferon is still subject to debate.11,13,14 observation at the time of second biopsy) was about 10
This study was designed to evaluate whether HCV- years. This, added to the 5 years of follow-up, represents a
764 PERSICO ET AL. GASTROENTEROLOGY Vol. 118, No. 4

long period of observation to draw conclusions on the of hepatitis C virus carriers with persistently normal or abnormal
alanine aminotransferase levels. Hepatology 1997;26:1393–
natural history of the disease in these patients. Neverthe- 1398.
less, a longer follow-up might be needed. 7. Silini E, Bono F, Cividini A, Cerino A, Bruno S, Belloni G, et al.
The indifferent HCV genotype distribution together Differential distribution of hepatitis C virus genotypes in patients
with and without liver function abnormalities. Hepatology 1995;21:
with the lack of association of any particular HCV
285–290.
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Through initial screening of anti-HCV–positive pa- normal liver biochemical values. Hepatology 1995;22:418–425.
10. Naito M, Hayashi N, Mita E, Natto M, Kasahara A, Fusamoto H,
tients, we recognized 2 patients who were HCV RNA Kamada T. Serum hepatitis C virus quantity and histological
negative and who had normal liver histology. These features of hepatitis C virus carriers with persistently normal ALT
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11. Van Thiel DH, Caraceni P, Molloy PJ, Hassanein T, Kania RJ,
might have recovered from the acute disease showing a
Gurakar A, Friedlander L. Chronic hepatitis C in patients with
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