Journal of Medical Virology 37:87-92 (1992)

Prevalence of Hepatitis C Virus Antibodies and Hepatitis C Virus-RNA in an Urban Population M. Rapicetta, A.F. Attili, A. Mele, A. De Santis, P. Chionne, K. Cristiano, E. Spada, E. Giuliani, L. Carli, F. Goffredo, and L. Capocaccia Departments of Virology (M.R., P.C., K.C., E.S.) and Epidemiology (A.M.), Istituto Superiore di Sanita, the Department of Gastroenterology (A.F.A., A.D.S., E.G., L.C., F.G., L.C.), University ‘ Z a Sapienza,” Rome, Italy Several studies had been carried out on anti-hepatitis C virus (HCV) prevalence in populations with blood exposure risks and in blood donors. New tests are now available which allow the investigation t o extend to other parameters such as antibody type and HCV-RNA. In this study the prevalence of anti-HCV c100-3 and the associated epidemiological, clinical, and virological markers were evaluated in subjects from an urban population located in central Italy. In positive cases the time persistence of HCVRNA and anti-HCV antibody pattern was studied. For this purpose, sera from 1,484 randomly sampled individuals, aged 30-69 years, collected in 1985 and stored at -80°C were retrospectively tested. The prevalence was 0.87% (i.e., 13 antiHCV c100-3 positive cases). A significant association was observed with raised alanine transaminase (ALT) levels ( P < 0.001). Paired serum samples from 11 out of the 13 subjects collected in 1985 and 1991 were tested by nested polymerase chain reaction (PCR) using primers from the 5’ non-coding region and by 4-RIBA. Concordant RIBA patterns between 1985 and 1991 were observed in the majority of positive paired sera (7/9) as well as for HCV-RNA (619). HCV-RNA was present in sera simultaneously positive t o both types of antibody or t o anti-cl00-3 or anti-c22 alone. A wide spectrum of viral and antibody patterns in anti-HCV c100-3 positive sera was observed in this urban population and persisted for at least 6 years. o 1992 Witey-Liss, Inc.

KEY WORDS: hepatitis C antibodies, HCVRNA, nested PCR, blood donors

INTRODUCTION Hepatitis C virus (HCV) has been identified as a n etiological agent of non-A, non-B post-transfusion hepatitis (NANBH) [Choo et al., 1990; Kuo et al., 1989; Alter et al., 19891. Anti-HCV antibodies, as detected by immunoassay using the non-structural c100-3 protein a s antigen LKuo et al., 19891, have been shown to be 0 1992 WILEY-LISS, INC.

present at a high rate (60-80%) in many communities a t increased risk of infection with blood born viruses such as intravenous drug users [Widell et al., 1991; Esteban et al., 1989; Roggerdorf et al., 1989; Mortimer et al., 19891, patients with haemophilia, and patients who have received multiple transfusions [Widell et al., 1991; Makris et al., 1990; Simmonds et al., 19901. Its role in community-acquired NANBH infection has also been determined [Bortolotti et al., 19911. Studies carried out in blood donors showed different prevalence in different geographical areas [Choo et al., 1990; Kuo et a]., 1989; Janot et al., 1989; Kuhnl et al., 1989; Brind et al., 1990; Sirchia et al., 19901. The prevalence ranges from 0.4-1.7% in Italy [Sirchia e t al., 19901. It was suggested that the 4 antigen immunoblot assays (4RIBA) (Ortho Diagnostics) be used as a confirmatory assay [Van der Poel et al., 19911. This test includes as antigens 3 non-structural proteins coded from the NS3 and the NS4 regions of the viral genome (c33, 5.1.1, c100-3) and a core-associated antigen (c22). Specific and sensitive polymerase chain reactions (PCR) methods for HCV-RNA detection have been developed [Cristiano et al., 19911. Currently no data are available on anti-c100-3 prevalence in the general population, its confirmation and specific pattern of antibody reactivity by 4-RIBA test, and its correlation with HCV-RNA presence. The purpose of this study was to evaluate the prevalence of anti-HCV c100-3 in a n urban population and to evaluate its association with epidemiological, clinical, and virological markers. To this end, we tested retrospectively the sera from one of the 18 populations of randomly selected individuals, aged 30-69, previously involved in a n epidemiological survey on gallstone disease [The M.I.Co1. group, 19911. In addition, subjects with anti-HCV c100-3 positive sera in 1985 were reexamined in January 1991to determine the persistence of HCV-RNA and HCV antibody.

Accepted for publication October 10,1991. Address reprint requests to Dr. Maria Rapicetta, Laboratory of Virology, Istituto Superiore di Sanita, Viale Regina Elena, 29900161 Rome, Italy.

Rapicetta et al.

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MATERIALS AND METHODS Study Population The study population was a randomly selected subgroup (N = 1,484; 848 maled636 females) of the subjects (N = 2,622; age range 30-69 years) participating in a n epidemiological study on gallstone disease carried out in the general population of Tivoli (Rome, Italy) between June 1985 and July 1986. Field activities included collection of information by means of a n administered questionnaire, ultrasonographic examination of the upper abdomen, and blood sampling. This was part of a multicentre study on cholelithiasis (M.I.Col.), details of the protocol having been reported elsewhere [The M.I.Co1. group, 19911. Sera were stored a t -80°C until tested in September 1990. There were no differences between subjects who were and were not tested for anti-HCV antibodies a s regards sex, age distribution, weight, height, education, type of work, and marital status. Anti-HCV c100-3 positive individuals were serologically and clinically re-examined in January 1991. Serological Assays Anti-HCV antibodies in the 1,484 serum samples were tested using the c100-3 Ortho enzyme-linked immunosorbent assay (ELISA) test. Positive samples were tested further by a second generation ELISA which uses a s antigen the c22 protein in addition to NS3 and NS4 genome regions coded proteins and a second generation RIBA test (4-RIBA, Ortho Diagnostics) which uses c22, 5.1.1, c100-3, and c33. Sera were considered confirmed ( a t least two bands present), indeterminate (only one band present), or not confirmed, according to the manufacturer’s instructions. Four hundred seventy-three randomly selected sera were also tested for HBsAg and anti-HBc by ELISA (Abbott, IL, USA). Serum alanine transaminase (ALT) activity was measured by spectrophotometry. Serum ALT levels twice the upper normal value (24 IU/l) were considered “altered.” A percutaneous liver biopsy was carried out using a modified Menghini needle (Surecut 16-G). Six millimicron paraffin embedded sections were stained with a) haematoxylin-eosine and b) Masson’s stain.

PCR Assay HCV-RNA was carried out by nested PCR with primers from the 5’ non-coding region, using as the template RNA extracted from serum samples and copied into cDNA by reverse transcription. Nucleotide sequences and positions selected according to Okamoto et al. 119901 were for the outer primers: 5’ GGC GAC ACT CCA CCA TAG ATC (nucleotides 1-21], 3’ GGT GCA CGG TCT ACG AGA CCT (nucleotides 324-304) (PCR product size 324 bp) and for the inner primers: 5’ CTG TGA GGA ACT ACT GTC TTC (nucleotides 2 8 4 8 ) , 3 ’ CCC TAT CAG GCA GTA CCA CAA (nucleotides 284264) (PCR product size 256 bp).

TABLE I. Age Prevalence of Anti-HCV (ELISA c100-3) in Urban PoDulation Age group 30-39 40-49 50-59 60-69 Total

No.

Anti-HCV+

%

361 382 445 296 1.484

2

0.55 0.78 1.35 0.67 0.87

3 6 2 13

The procedure included the 2 following steps: a) RNA extraction: 50 pl of serum samples were extracted by mixing each with 150 pl of extraction buffer consisting of 4.2 M guanidinium isothiocyanate, 0.5% sodium lauryl sarkosate, and 25 mM Tris HCl pH 8.0, followed by one extraction with a n equal volume of parts of phenol and chloroform and one with chloroform alone. The aqueous phase was precipitated with a n equal volume of isopropyl alcohol and the resulting pellet washed with 70% ethanol. The total nucleic acid extracted by this procedure from 50 p1 of serum was resuspended in 50 pl of RNAse-free water and 0.5 p1 of RNasin (100 U/pl), stored at -80°C, and used a s the template in the PCR reaction. b) cDNA synthesis and amplification: The first PCR reaction was combined with the reverse transcription step in the same tube containing a 100 p1 reaction made up a s follows: 25 pl of serum nucleic acid extract, 0.5 pM of each outer primer, 200 pM of each of the 4 deoxynucleotides, 2.5 units of recombinant Taq DNA polymerase, 10 units of avian myeloblastosis virus reverse transcriptase and 1 x Taq buffer consisting of 10 mM Tris HC1 pH 8.3,50 mM KC1, 1.5 mM MgCl,, and 0.01% gelatin. The thermocycler was programmed to first incubate the samples for 20 minutes a t 43°C for the initial reverse transcription step and then to carry out 35 cycles consisting each of 94°C for 1minute, 45°C for 1.5 minutes, and 72°C for 1.5 minutes. For the second reaction, 10 pl were removed from the first reaction and added to a tube containing the second set of primers and all the other PCR reagents as in the first reaction. PCR was then carried out for another 35 cycles a s described above. The PCR products were analysed by electrophoresis on 2% Nusieve and 0.5% regular agarose gels containing 0.5 pgiml ethidium bromide. The gels were viewed and photographed on a n ultraviolet (UV) light box. As positive control, a sample from patient with chronic hepatitis C with a n unusually high level of HCV-RNA was used with a titre of 105/25 pl by this PCR assay. As negative controls, 4 HBV DNA-positive sera and 3 normal sera were tested under the same conditions: none of these reacted by this test.

Statistics Chi square test was performed to assess the differences between proportions.

RESULTS Table I shows anti-HCV c100-3 prevalence in the

Anti-HCV and HCV-RNA in Urban Population TABLE 11. Association Between Anti-HCV ELISA c100-3 and ALT Levels or Anamnestic Data of Previous Henatitis Anti-HCVS Variables no./total % P value* ALT levels Normal 911,460 0.7

Prevalence of hepatitis C virus antibodies and hepatitis C virus-RNA in an urban population.

Several studies had been carried out on anti-hepatitis C virus (HCV) prevalence in populations with blood exposure risks and in blood donors. New test...
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