J Gastroenterol 1994; 29:147-151 Journal of- Gastroenterology �9 Springer-Verlag 1994

Detection of HCV RNA in subjects with antibody to hepatitis C virus among the general population of Fukuoka, Japan

JUN HAYASHI, 1 KOYA NAKASHIMA, 1 ERIKO YOSHIMURA, 1 MIKI HIRATA, 1 YOSHIAKI MAEDA, 2 and SEIZABURO KASHIWAGI 1

1 The Department of General Medicine, Kyushu University Hospital, Higashi-ku, Fukuoka, 812 Japan 2 Fukuoka Red Cross Blood Center, Kamikoga, Chikushino, 818 Japan

Abstract: Volunteer blood donors and aged people who came to hospitals for a thorough physical checkup were surveyed to evaluate the exact prevalence of hepatitis C virus (HCV) infection in the general popu- lation of Fukuoka, Japan. We tested for antibody to HCV (anti-HCV) by second-generation assay and, to distinguish active HCV infection from past resolved infection, we tested for HCV RNA in reactive serum samples by polymerase chain reaction. The prevalence of anti-HCV was 286 (2.0%) of 14341 subjects, in- creasing with advancing age, from 0.4% in the under- 29 age group to 12.0% in the over-70 age group. There were no differences between sexes. HCV RNA was detected in 170 of 286 (59.4%) anti-HCV-positive subjects. The ratio of HCV RNA-positive to anti- HCV-positive subjects was higher in males than in females (P < 0.05) and decreased with advancing age, from 72.2% to 46.5%. The prevalence of elevated alanine aminotransferase (ALT) was only 15.9% in subjects with HCV RNA, higher in males (21.4%) than in females (8.3%) (P < 0.05). This study revealed that the prevalence of anti-HCV was high in the aged population, but that the ratio of HCV RNA-positive to anti-HCV-positive subjects was low, and most of the HCV RNA-positive subjects had normal ALT levels.

Key words: HCV RNA, anti-HCV, general population

Offprint requests to: J. Hayashi (Received for publication on Mar. 29, 1993; accepted on July 15, 1993)

Introduction

The genome of hepatitis C virus (HCV), the major causative factor of non-A, non-B hepatitis, was iso- lated in 1989 a and radioimmune and enzyme-linked immunosorbent assays have been developed to detect the antibody, i.e., antibody to c100-3 protein (anti- c100), to the protein expressed in the cloning ex- periments. 2 Anti-c100 has been widely used for epidemiologic studies of HCV infection. The pre- valence of anti-cl00 in blood donors is 1.3% in Japan, 3 0.4%-0.8% in Europe, 4-6 and 0.9%-1.4% in the United States. 7 Since these investigations did not include members of the aged population; survey of HCV infection in the over-70 age group is therefore necessary.

On the other hand, the anti-c100 does not cover all HCV infections, 8 since the c100-3 protein con- tains expression products only from non-structural region 4 of the HCV genome. 1 A second genera- tion assay for antibody to HCV (anti-HCV) has been developed. Because the protein used in this assay contains expression products from non-structural regions 3 and 4 and from the nucleocapsid region of the HCV genome, this assay increases the sen- sitivity of antibody detection in infected patients. 9-It However, anti-HCV positivity does not reflect the state of HCV viremia. The direct detection of HCV RNA by polymerase chain reaction (PCR) is currently the only tool that can be employed for the detection of HCV viremia, and it remains the only way to differentiate active from past resolved infection. 12,13

We surveyed a wide range of age groups in the general population of Fukuoka, Japan for the pre- valence of anti-HCV, and we tested all reactive serum samples of HCV RNA by PCR to identify HCV viremia. We also noted the prevalence of liver dys- function in subjects with HCV viremia.

148 J. Hayashi et al.: HCV RNA in the general population of Japan

Materials and methods

Study population

A total of 14 341 serum samples, 13 944 from volunteer blood donors (7028 males, 6916 females; age 16-64 years) from the Fukuoka Red Cross Blood Center and 397 from people who came to Hara Doi Hospital in Fukuoka City for a thorough physical checkup were collected in March, 1992. Volunteer blood donors in Japan are screened for anemia and hepatitis.

Assay methods

Serum samples were separated and stored at -20~ until tested for anti-HCV by passive hemagglutination assay (HCV PHA, Dainabot Co. Ltd., Tokyo, Japan). 13 The test for anti-HCV was repeated and the neutralization tests were performed to confirm pos'itivity. The reactivity of the antibody was deter- mined by absorbing specimens with purified antigen, which contain c22, c33c, and c100-3 antigens, then testing the absorbed and unadsorbed sera for anti- bodies, using a commercial PHA procedure. Serum samples were judged positive for anti-HCV if re- peatedly proven positive by the same method and absorbed by purified antigen.

All the anti-HCV-positive serum samples were tested for HCV RNA by two-stage PCR. RNA was extracted with acid guanidinium thiocyanate-phenol- chloroform. Primers and a probe were constructed for the 5'-noncoding region (where the base sequence is highly conserved), based on sequence data for HCV.13 The outer primers used were 5'-CTGTGAGGAACT ACTGTCTT-3' (sense) and 5'-AACACTACTCGGC TAGCAGT-3' (antisense). The cDNA was amplified by 35 cycles of PCR. One portion of the PCR products was sampled, and another pair of primers inside the ones listed above were designed. Another 30

cycles of the PCR were run. The sense primer used was 5'-TTCACGCAGAAAGCGTCTAG-3', and the antisense primer was 5 '-GTTGATCCAAGAAAG GACCC-3'. Sizes of the PCR products estimated on gel electrophoresis were as predicted: 221 bp by the first stage of PCR and 145 by the second stage. After 20min, the PCR was repeated with the same serum samples; three negative and three positive controls were included in each PCR. This approach made feasible the evaluation of reproducibility and allowed for the checking of false-positive results arising from contamination and false-negative results caused by un- toward events related to the PCR.13

Conventional liver function tests were performed, using a multiple autoanalyzer. The presence of liver dysfunction was defined as an elevated serum alanine aminotransferase (ALT) level, above 36 IU/1.

Statistical analys&

The chi-square test was used for paired data to examine the significance of differences between the sexes, and the Cochran-Armitago test was used to evaluate the tendency of the prevalence to increase or decrease with advancing age, with P = 0.05 being as the minimum level of significance.

Results

The distribution of anti-HCV by sex and age in the general population of Fukuoka is shown in Table 1. Anti-HCV was detected in 286 (2.0%) of 14341 sub- jects. There were no significant differences between sexes. The prevalence of anti-HCV increased with ad- vancing age, in both sexes, P < 0.001, from 0.4% in the under-29 age group to 12.0% in the over-70 age group.

Table l. Distribution of antibody to hepatitis C virus among 14341 subjects in the general population of Fukuoka, Japan, 1992

Males Females Total

Age group No. Anti-HCV No. Anti-HCV No. Anti-HCV (years) tested + (%) tested + (%) tested + (%)

-29 2840 19 (0.7) 4072 12 (0.3) 6 912 31 (0.4) 30-39 2099 34 (1.6) 1209 20 (1.7) 3 308 54 (1.6) 40-49 1273 30 (2.4) 701 20 (2.9) 1 974 50 (2.5) 50-59 612 33 (5.4) 621 32 (5.2) 1233 65 (5.3) 60-69 226 20 (8.8) 331 23 (6.9) 557 43 (7.7) 70- 102 13 (12.7) 255 30 (11.8) 357 43 (12.0)

Total 7152 149 (2.1) 7189 137 (1.9) 14341 286 (2.0)

Anti-HCV, Antibody to hepatitis C virus detected by second-generation assay

J. Hayashi et al.: HCV RNA in the general population of Japan 149

Table 2. Prevalence of hepatitis C virus RNA by sex and age among subjects with antibody to hepatitis C virus in Fukuoka, Japan, 1992

Males Females Total

Age group No. of subjects HCV RNA No. of subjects HCV RNA No. of subjects HCV RNA (years) with anti-HCV + (%) with anti-HCV + (%) with anti-HCV + (%)

-29 19 15 (78.9) 12 4 (33.3) 31 19 (61.3) 30-39 34 26 (76.5) 20 13 (65.0) 54 39 (72.2) 40-49 30 21 (70.0) 20 12 (60.0) 50 33 (66.0) 50-59 33 23 (69.7) 32 16 (50.0) 65 39 (60.0) 60-69 20 7 (35.0) 23 13 (56.5) 43 20 (46.5) 70- 23 6 (46.2) 30 14 (46.7) 43 20 (46.5)

Total 149 98 (65.8)* 137 72 (52.6)* 286 170 (59.4)

* P < 0.05 (P value for the relative number of HCV RNA-positive sera among groups, determined by chi-square test) HCV RNA, Hepatitis C virus RNA; anti-HCV, antibody to hepatitis C virus detected by second-generation assay

Table 3. Distribution of elevated alanine aminotransferase by sex and age among subjects with hepatitis C virus RNA in Fukuoka, Japan, 1992

Males Females Total

Age group No. with No. with liver No. with No. with liver No. with No. with liver (years) HCV RNA + dysfunction (%) HCV RNA + dysfunction (%) HCV RNA + dysfunction (%)

-29 15 1 (6.7) 4 0 30-29 26 10 (38.5)* 13 0* 40-49 21 4 (19.0) 12 1 (8.3) 50-59 23 3 (13.0) 16 1 (6.3) 60-69 7 1 (14.3) 13 2 (15.4) 70- 6 2 (33.3) 14 2 (14.3)

Total 98 21 (21.4)* 72 6 (8.3)*

19 1 (5.3) 39 10 (25.6) 33 5 (15.2) 39 4 (10.3) 20 3 (15.0) 20 4 (20.0)

170 27 (15.9)

* P < 0.05 (All P values for the relative number of subjects with liver dysfunction among groups were determined by the chi-square test) HCV RNA, Hepatitis C virus RNA

The prevalence of HCV RNA by sex and age in anti-HCV-positive subjects is shown in Table 2. HCV RNA was detected in 170 (59.4%) of 286 anti-HCV- positive subjects and was significantly more prevalent in males (65.8%) than in females (52.6%) (P < 0.05). The ratio of HCV RNA-positive to anti-HCV-positive subjects decreased with advancing age (P < 0.01), from 72.2% in the 30-39 age group to 46.5% in the over-60 age group. This decrease was significant in males (P < 0.01), but not in females. In the over-60 age group, more than 50% of the anti-HCV-positive subjects were not currently infected with HCV.

Elevated ALT levels were detected in 34 (11.9%) of 286 anti-HCV-positive subjects. The prevalence of H C V RNA was significantly higher in the 27 (79.4%) of 34 subjects with liver dysfunction than in the 143 (56.7%) of 252 subjects without liver dysfunction (P < 0.05). Seven (20.6%) anti-HCV-positive subjects had liver dysfunction, but HCV RNA was not detected in their sera, and they were negative for hepatitis B surface antigen.

Distribution of elevated A LT levels by sex and age in subjects with H CV RN A is shown in Table 3. Of the 170 subjects with H CV RNA, 27 (15.9%) had an elevated ALT. The prevalence of liver dysfunction was significantly higher in males (21.4%) than in fe- males (8.3%) (P < 0,05), and was lowest (5.3%) in the under-29 age group and highest (25.6%) in the 30-39 age group. There was no significant difference by age group. There were no female subjects under 39 years of age with liver dysfunction.

Discussion

This study revealed that the prevalence of anti-HCV was 2.0% in the general population of Fukuoka, Japan, but 12.0% in those over 70 years of age. Be- cause previous reports did not include H CV infection rates for the aged population and used the original anti-cl00, 3-7 their prevalence rates were relatively lower than the results of the present study. Because

150 J. Hayashi et al.: HCV RNA in the general population of Japan

we were not able to obtain medical histories (e.g., histories of blood transfusion, etc.) of the subjects, the reasons for the high prevalence of anti-HCV-positive subjects in the aged population are unclear. The pre- valence of anti-HCV increased with advancing age and, therefore, we concluded that HCV was probably more endemic in this area in the past than at present. The anti-HCV (by second-generation assay) used in this study gave a more sensitive and specific appraisal of viral infection. 11 Such improved sensitivity also con- tributed to the high prevalence of HCV infection found in this study.

The present study showed that approximately 60% of anti-HCV-positive subjects in the general popu- lation of Fukuoka have HCV viremia. This result was lower than we expected and the question remains whether the PCR for HCV RNA was sensitive enough. Since HCV RNA was detected in 89.3% of anti'-cl00-positive and/or anti-GOR-positive serum samples 15 and in 91.0% of anti-HCV-positive serum samples 16 from patients with chronic non-A, non-B liver disease, in 86.4% of anti-HCV-positive serum samples from hemodialysis patients, and in 80.2% of anti-HCV-positive serum samples from the inhabitants of Iki Island in Nagasaki Prefecture (our unpublished data) by the same PCR method, it would seem that the results of the present study are reliable. Approxi- mately 60% of the aged population have no HCV RNA in their sera, suggesting either that the HCV genome is eliminated from the sera of infected sub- jects with advancing age or that they had a greater chance of HCV infection in the past.

Another striking finding of our study was that 85% of the subjects with HCV viremia had normal liver function. This suggests that HCV RNA positivity in the general population is little associated with elevated ALT levels. This is consistent with a previous report on HCV transmission by blood donors with normal ALT levels. 17 The lack of a consistent correlation between viral replication and elevated ALT has sug- gested the absence of a direct cytopathic effect of HCV 18 and, at least in some subjects with HCV viremia, the existence of extrahepatic sites of repli- cation, a9 The prevalence of ALT abnormality among subjects with HCV RNA was unexpectedly low, pos- sibly because ALT was measured only once for each subject. Alberti et al. 2~ reported that anti-HCV- positive donors with HCV viremia had biopsy- determined chronic hepatitis despite a normal ALT. Precise examinations, including liver biopsy and follow up of ALT, need to be done to determine the status of subjects with HCV viremia so that the indications for interferon therapy may be considered. 2a

The prevalence of anti-HCV-positive subjects with HCV viremia was significantly higher in males than in

females, in contrast to the prevalence of anti-HCV- positive subjects without HCV viremia, which was higher in females than in males. It appears that fe- males are more capable of eliminating HCV than males. Liver dysfunction was also more prevalent in males with HCV viremia. HCV appears to act dif- ferently in the two sexes, as is the case for hepatitis B virus. 22

The results of this study led us to conclude that, in Fukuoka, Japan the prevalence of HCV infection was high, especially in the elderly group in whom the ratio of subjects with HCV viremia to anti-HCV-positive subjects was low, and that most subjects with HCV viremia appeared to be healthy, as determined by con- ventional liver function tests.

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