Hepatitis C virus genotypes in northern Italy:
clinical and virological features.
A Ravaggi, A Rossini, C Mazza, M Puoti, M G Marin and E Cariani
J. Clin. Microbiol. 1996, 34(11):2822.
These include:
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JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1996, p. 2822–2825
0095-1137/96/$04.0010
Copyright q 1996, American Society for Microbiology
Vol. 34, No. 11
Hepatitis C Virus Genotypes in Northern Italy:
Clinical and Virological Features
ANTONELLA RAVAGGI,1 ANGELO ROSSINI,2 CINZIA MAZZA,1 MASSIMO PUOTI,3
MARIA GRAZIA MARIN,1 AND ELISABETTA CARIANI1*
III Laboratory of Clinical Chemistry1 and III Department of Internal Medicine,2 Hospital of Brescia,
and Institute of Infectious Diseases, University of Brescia,3 Brescia, Italy
We analyzed the characteristics of subjects from the same area who were infected with hepatitis C virus
genotypes 1 through 4 and subtypes 1a and 1b. Our data are consistent with a rapid evolution in the
epidemiology of HCV genotypes and argue against different pathogenic potentials for genotypes 1b and 2.
The analysis of risk factors revealed a similar prevalence of
blood transfusions among subjects with genotypes 1, 2, and 4.
The absence of transfusions among individuals with type 3
might be linked to the lower mean age. Intravenous (i.v.)-drug
users were infected with genotypes 1, 3, and 4, with a significantly increased frequency of genotype 3 (P , 0.01). Among
subjects infected with subtype 1a or with mixed 1a-1b infections, i.v.-drug use was more frequent than among subjects
infected with subtype 1b only (P , 0.01). Subjects without
known parenteral risk factors were more often infected with
genotype 2 or subtype 1b (P , 0.01).
Samples from 176 cases were tested for anti-HCV antibodies
by the recombinant immunoblot assay (Ortho Diagnostic Systems, Raritan, N.J.); of these cases, 119 were with genotype 1,
43 were with genotype 2, 9 were with genotype 3, and 5 were
with genotype 4. Seventy-seven samples were tested by secondgeneration assay, and 99 were tested by third-generation assay.
Anti-C100/5.1.1 was significantly less frequent in genotype 2
samples (n 5 21; 48.8%) than in genotype 1 samples (n 5 99;
83.2%) (P , 0.01), consistent with previous data (1, 13). These
results had no relationship to the generation of the assay used
(data not shown).
The presence of anti-human immunodeficiency virus (HIV)
antibodies, for which we tested samples from 178 subjects, was
associated with HCV genotypes 1 and 3 (P , 0.01) and with
subtype 1a or mixed 1a-1b infections (P , 0.01) (Table 2).
Alanine aminotransferase (ALT) values were available for
176 subjects (120 with genotype 1, 39 with genotype 2, 10 with
genotype 3, and 7 with genotype 4). Lower mean levels (P ,
0.05) and a higher percentage of normal values (,50 IU/liter)
(P , 0.01) were detected in subjects with genotype 2 than in
subjects with genotype 1.
During the last year before sampling, liver biopsies were
The sequence analysis of hepatitis C virus (HCV) isolates
allows the identification of at least six genotypes with distinctive patterns of geographic distribution (11). Previous studies
with Italian patients have identified age- and risk-related
prevalences of viral genotypes (4, 5, 7, 9, 10). We analyzed the
characteristics of HCV genotypes 1 through 4 and of subtypes
1a and 1b in our geographic area of northern Italy.
The study population consisted of 237 subjects (age [mean 6
standard deviation], 45.4 6 15.9 years), including 160 patients
with previous diagnoses of chronic HCV infection and 77 subjects occasionally found to be positive for anti-HCV. The latter
group included 52 women undergoing pregnancy screening
and 25 subjects screened for blood donations.
HCV genotypes 1 through 4 were determined by differential
hybridization on microtiter plates (7) of the amplified 59 untranslated region (6) with genotype-specific probes (Table 1).
The viral genotypes could be determined for all samples, indicating that additional genotypes are infrequent in Italy. Genotype 1 was detected in 158 cases (66.7%), genotype 2 was
detected in 57 cases (24.1%), genotype 3 was detected in 14
cases (5.9%), and genotype 4 was detected in 8 cases (3.3%)
(Table 2). Subtypes 1a and 1b were determined by hybridization of PCR products of the E1 gene (7). The subtyping procedure was successful in 98 of 110 samples with genotype 1
available for analysis, detecting subtype 1a in 25 cases (25.5%),
subtype 1b in 67 cases (68.4%), and mixed 1a-1b infections in
6 cases (6.1%). Typing results were consistent with the sequence analysis of the 59 untranslated region and/or of genes C
and E1, performed on 35 samples (data not shown).
The characteristics of subjects infected with different genotypes and/or subtypes were compared by parametric or nonparametric tests as appropriate (EPI.INFO 5.0 statistical package; Centers for Disease Control and Prevention, Atlanta,
Ga.). The numbers of males and females were not statistically
different among patients infected with each genotype. Subjects
infected with genotype 2 were older than those infected with
genotypes 1, 3, and 4 (P , 0.01). Furthermore, infections with
genotypes 3 (P , 0.01) and 4 (P , 0.05) were associated with
lower mean ages than infection with genotype 1, although
these results may have been affected by the small sample sizes.
Subtype 1a was detected in a younger age group than subtype
1b (P , 0.01).
TABLE 1. Oligonucleotide probes used for the determination of
HCV genotypesa
Probe
Genotype
Sequence (59339)b
G1
G2
G3
G4
1
2
3
4
GAATTGCCAGGACGACC
GAATTGCCGGGAAGACT
GAATCGCTGGGGTGACC
GAATCGCCGGGATGACC
Nucleotide positions 2171 through 2155.
Derived from the alignment of published 59 untranslated region sequences
(12) with those obtained from 25 isolates from our geographic area. The typespecific mutations are underlined.
a
* Corresponding author. Mailing address: III Laboratory of Clinical
Chemistry, Hospital of Brescia, P.le Spedali Civili, 1, 25123 Brescia,
Italy. Phone: 39 30 3995555. Fax: 39 30 307251.
b
2822
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Received 11 April 1996/Returned for modification 21 June 1996/Accepted 2 August 1996
VOL. 34, 1996
NOTES
2823
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FIG. 1. Distribution of HCV RNA titers according to HCV genotype and subtype, ALT level, and the presence or absence of anti-HIV. The horizontal lines
represent median HCV RNA levels within the population samples.
2824
J. CLIN. MICROBIOL.
NOTES
TABLE 2. Epidemiological and serological features of subjects infected with genotypes 1 through 4 and subtypes 1a and 1b
HCV genotypes
HCV subtypes
Characteristic
Total no. of
subjects
Age (mean 6
SD) (yr)
Male/female
ratio
No. positive for
anti-HIV/no.
tested (%)
2
3
4
1a
158
57
14
8
25
1b
67
1a 1 1b
6
45.5 6 15.1
51.4 6 15.3
26.4 6 6.2
33.5 6 18.6
30.9 6 8
47 6 14.2
37.8 6 15.4
77/81
34/23
3/11
3/5
7/18
36/31
4/2
20.9
20.9
3.2
24.6
0
3.5
0
64.3
14.3
25
12.5
12.5
8
76
12
19.4
11.9
1.5
0
67.7
0
0.6
0
0
0
0
0
0
0.6
53.8
0
71.9
0
21.4
12.5
37.5
0
4
0
67.2
16.7
16.7
32/120 (26.7)
2/37 (5.4)
7/13 (53.8)
2/8 (25)
19/25 (76)
7/56 (12.5)
4/6 (66.7)
performed on 149 subjects, 107 with genotype 1, 36 with genotype 2, 2 with genotype 3, and 4 with genotype 4. Histological analysis detected chronic persistent hepatitis in 14 cases
(12 with genotype 1 and 2 with genotype 2), chronic active
hepatitis in 89 (63 with genotype 1, 20 with genotype 2, 2 with
genotype 3, and 4 with genotype 4), liver cirrhosis in 17 (15
with genotype 1 and 2 with genotype 2), and hepatocellular
carcinoma in 29 (17 with genotype 1 and 12 with genotype 2).
The severity of histological lesions did not differ significantly
between subjects with genotype 1 and those with genotype 2.
Histological activity scores (2) were analyzed for patients
with subtype 1b (n 5 33) and genotype 2 (n 5 21); they show
similar epidemiological features but different biochemical and
serological features. Statistical analysis failed to show significant differences (data not shown).
The titers of HCV RNA in 110 serum samples adequately
stored for quantitative analysis were determined by competitive PCR (8). The viral titers were not related to epidemiological or histological characteristics (data not shown) but were
lower in samples with genotype 3 than in samples with genotype 1 (P , 0.01) (Fig. 1). A differential sensitivity of the assay
according to the viral type is unlikely, since primers and probe
were based on a sequence highly conserved among different
genotypes (8). Further studies are needed to assess whether
the lower viral load is an intrinsic characteristic of genotype 3
infection or whether it is linked to specific features of the
population infected with this genotype.
The presence of anti-HIV was associated with higher median titers of HCV RNA, even though the difference did not
reach statistical significance. Subjects with increased ALT levels had higher viral loads than those with normal ALT levels
(P , 0.05), suggesting a relationship between the rate of viral
replication and the degree of liver cell necrosis.
Altogether, HCV types 1b and 2 were both detected in older
subjects and were associated with community-acquired infections. By contrast, genotype 3 and subtype 1a were associated
with lower age, i.v.-drug use, and HIV infection. These observations are consistent with a rapid evolution in the prevalence
of HCV genotypes in Italy. Our results argue against different
pathogenic potentials for genotypes 1b and 2, despite the biochemically silent clinical course associated with genotype 2 in
this and other reports (1, 9). However, this issue is still controversial (3, 9) and needs further confirmation by long-term
prospective studies. The changing incidence of viral types in
younger age groups underlines the need for the reevaluation of
the prevalences of HCV genotypes at different times.
This work was supported by a grant from Regione Lombardia (Progetto Ricerche Finalizzate n. 25).
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VOL. 34, 1996
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NOTES
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