Journal of Infection (1995) 30, 235-239

Relationship of Genotype to Level of Hepatitis C Viraemia Determined by Competitive Polymerase Chain Reaction

Jun Hayashi 1, Yasuhiro Kishihara 1, Eriko Yoshimura ~, Yoshiki TanP, Kouzaburo YamajP, Hideyuki Ikematsu 1, Hiroaki Ishiko 2, Seizaburo KashiwagP

~1"he Department of General Medicine, Kyushu University Hospital, 2Mitsubishi Bio-Clinical Laboratories Incorporation

Accepted for publication 16 September 1994

To compare the levels of hepatitis C virus (HCV) viraemia in carriers of the same genotype in various stages of chronic HCV infection, we quantified the amount of HCV RNA by competitive polymerase chain reaction and determined HCV genotype using type-specific primers. The study population included 255 patients with chronic HCV infection (asymptomatic 33, chronic hepatitis 141, liver cirrhosis 50, hepatocellular carcinoma 31). Of these 255, the prevalence of HCV RNA genotype II was 67.8%, genotype Ill, 17.3% and genotpye IV, 14.9%; no genotype I was found. The level of HCV RNA (logarithmic transformed copy numbers per 50 #I of serum) was significantly higher in subjects of genotype II than in those of genotypes III or IV (mean titre 5.8+ 1.0 vs. 5.1 + 1.2 and 4.8+ 1.1, P<O.05, respectively). There was no significant difference in the level of HCV RNA between genotypes III and IV. Of 173 patients of genotype II, there were no significant differences between the level of HCV RNA and the stage of liver disease or in the level of HCV RNA by age. Of the 129 with genotype II with a history of blood transfusion, there was no significant difference between the level of HCV RNA of patients with and without a history of transfusion or between that of patients with a history of blood transfusion and the time elapsed since blood transfusion. The level of HCV viraemia depended on the genotype of HCV RNA and did not correlate to age or to the stage of liver disease. We conclude that the genotype of HCV RNA should be given attention in order to provide more effective treatment.

Introduction

Since the development of diagnostic tests for hepatitis C virus (HCV) infection, the assay for antibody to HCV (anti-HCV) has shown that HCV is the aetiological agent for most cases of non-A, non-B hepatitis 1'2 and is the most frequent cause of chronic liver diseases in J apan) Several investigators have recently used two-stage poly- merase chain reaction (PCR) for the detection of HCV RNA. ~-7 Circulating HVC RNA is a marker of viraemia, and can be useful in the detection and diagnosis of HCV carriers. 6'7 The level of HCV viraemia by competitive PCR is useful in evaluating the state of viral replication in chronic HCV infection 8-11 and is one of the predictive markers of effective interferon therapy, ix HCV has four genomic determinants, I, II, III and IV, assayed by PCR with type-specific primers. These genotypes were shown to be identical in mother and baby in each of two examples of perinatal transmission, and were also identical in donor and recipient in a case of accidental needle ex- posure. 12 The genotypes of HCV have different responses

Address correspondence to: Jun Hayashi, MI), Department of General Medicine, Kyushu University Hospital, Higashi-Ku, Fukuoka 812, Japan.

to interferon, 13'14 because they may also differ in viral replication.

We quantified the amount of HCV RNA by competitive PCR and determined HCV genotype in asymptomatic HCV carriers and patients with chronic liver disease by HCV infection. To elucidate the relationship between the levels of HCV viraemia and progression of liver disease, we compared the levels of HCV viraemia in carriers of the same genotype in various stages of chronic HCV infection.

Materials and Methods

Study population

The 255 patients studied, all living in Fukuoka Prefecture, Japan, were positive for both anti-HCV and HCV RNA for more than 6 months; 33 were blood donors (18 men and 15 women) without abnormal serum alanine aminotransferase (ALT) elevation and 222 were chronic liver disease patients who had been admitted to Kyushu University Hospital in Fukuoka between 1988 and 1993. Of the 222 patients, liver biopsy was performed on 191. Histological examinations revealed chronic hepatitis in

0163-4453/95/030235 +05 $08.00/0 © 1995 The British Society for the Study of Infection

236 Relationship of Genotype to Hepatitis C

141 (83 males and 58 females) and liver cirrhosis in 50 (33 males and 17 females). The remaining 31 patients (23 males and 8 females) were diagnosed as cases of hepatocellular carcinoma (HCC) by ultrasonography plus histological examination, arteriography and elevated concentration of serum alphafetoprotein. Background information concerning episodes of blood transfusion was obtained from 191 of the 222 patients.

Hepatitis B virus infection was ruled out using com- mercial serological tests (absence of detectable hepatitis B surface antigen and high titre of antibody to hepatitis B core antigen).

Table 1. Distribution by genotype of hepatitis C virus RNA in chronic liver disease patients with hepatitis C virus infection

Genotype Stage of No. liver disease patients II (%) III (%) IV (%)

Chronic 141 hepatitis Cirrhosis 50 HCC 31 Total 222

8 6 ( 5 7 . 3 ) 36 85.7)

38 ( 2 5 . 3 ) 4: 9.5) 26 ( 1 7 . 3 ) 2 4.8)

150 (100.0) 42 (100.0)

19 63.3)

8 26.7) 3 lO.O)

30 (lOO.O)

HCC: hepatocellular carcinoma

Assay methods

All serum samples were separated and stored at - 2 0 ° C until testing for anti-HCV, HCV RNA, genotypes of HCV RNA and level of HCV viraemia. Anti-HCV was measured by enzyme linked immunosorbent assay (ELISA) (HCV EtA II, Abbott Laboratories, North Chicago, [L, U.S.A.p HCV RNA was detected by two-stage PCR, using primers from the 5'-noncoding region of HCV genome: ~ 5'-CTGTGAGGAACTACTGTCTT-3' (sense; nt 28-47) and 5'-AACACTACTCGGCTAGCAGT-3' (antisense; nt 2 2 9 - 248) in the first stage, and 5'-TTCACGCA- GAAAGCGTCTAG-3' (sense; nt 46-65) and 5'- GTTGATCCAAGAAAGGACCC-3' (antisense; nt 171- 190) in the second stage.

The HCV genotype was determined by two-stage PCR, using universal and type-specific primers from the pu- tative C gene of the HCV genome, according to the methods by Okamoto et al. 12 The first stage of PCR was performed with primers consisting of 5'-TGCGCGCGAC- (TA)AGGAAGACTTC-3' (nt 137 to 158 from the HC-]4 isolate, 15 sense) and 5'-ATGTACCCCATGAGGTCGGCGA- 3' (nt 391 to 412 from the HC-]I isolate, 15 antisense). The second stage of PCR was performed with a sense primer consisting of 5"-AGGAAGACTTCCGAGCGGTC-3" (nt 148 to 167 from the HC-]I) and a mixture of four type-specific antisense primers. Antisense primers specific for the four HCV types were 5'-TGCCTTGGGGA- TAGGCTGAC-3' (nt 185 to 204, type I), 5'- GAGCCATCCTGCCCACCCCA-3' (nt 272 to 291, type II), 5"-CCAAGAGGGACGGGAACCTC-3' (nt 302 to 321, type [II) and 5'-ACCCTCGTTTCCGTACAGAG-3' (nt 251 to 270, type IV). They were deduced from regions of the putative C gene where the sequences were preserved only by a group of isolates with minor variations. The expected sizes of the products by the second stage of PCR were 57 bp (type I), 144 bp (type II), 174 bp (type III) and 123 bp (type IV), respectively.

To quantify HCV RNA by competitive PCR, mutant

HCV RNA restricted by EcoRI in the 5"-non-coding region was obtained. HCV RNA from patients was mixed in each tube with the diluted mutant HCV RNA (102-7 copies/50 ~tl), and competitive PCR with 5'-non-coding region was carried out. The amplified products were then restricted by EcoRI and were analysed by electrophoresis, with mutant HCV RNA demonstrated in 105 and 113 base pairs (bp) and HCV RNA from patients in 218 bp. The size of the PCR product from patients was compared with that of the diluted mutant HCV RNA} °

Statistical analysis

Chi-square test and Kruskal Wallis one-way analysis of variance were used to determine the statistical sig- nificance of the data. Variables in the text are expressed as mean ± S.D.

Results

In 255 patients with chronic HCV infection the pre- valence of HCV RNA genotype II was 67.8%, genotype III, 17.3% and genotype IV, 14.9%; no genotype I was found. The distribution by genotype of HCV RNA for 222 patients with chronic liver disease is shown in Table 1. The prevalences of cirrhosis and HCC were relatively higher in patients with genotypes II and IV than in those of genotype III, with no significance.

The relationship between HCV RNA genotype and the level of HCV RNA (logarithmic transformed copy numbers per 50 gl of serum) among subjects in this study is shown Table 2. The level of HCV RNA was significantly higher in subjects of genotype II than in those of genotypes III or IV (5.8___1.0 vs. 5.1___1.2 and 4 .8+1 .1 , P<0.05, respectively). There was no significant difference in the level of HCV RNA between genotypes [II and IV. There was also no difference between the sexes.

The levels of HCV RNA by the stage of liver disease are compared among patients with genotypes II, III and

Jun Hayashi e t al,

Table 2. Relation between the genotype and level of hepatitis C virus RNA in patients with chronic hepatitis C virus infection

237

Genotype Males Level of Females Level of Total HCV RNA HCV RNA

Level of HCV RNA

H 103 5.7-+1,1 70 5.8_+0.9 173 III 27 5.0_+1.1 17 5.2_+1.3 44 IV 27 4 . 8 ± 1 . 2 11 4.8_+0.6 38 ~ t a l 157 5.4_+1,2 98 5.6_+1.0 255

5 .8 -+1 .0 ' 5 .1 -+1 .2 ' 4 . 8 ± 1 . 1 " 5.5-+1.1

HCV RNA; hepatitis C virus RNA I-ICV RNA level of defined as log 10 (copies per 50 gl of serum) Data expressed as mean + S.D, * P<0.05

Table 3. Relation between the level of hepatitis C virus RNA and the stage of liver disease by hepatitis C virus RNA genotype

Stage of Genotype II Genotype III Genotype IV liver disease

No, Level of No, Level of No, Level of patients HCV RNA patients HCV RNA patients HCV RNA

Asymptomatic 23 6.0___ 1.1" 2 6.5 _+0.7 8 5.3 _+0.5* carriers Chronic 86 5.7 ± 1.1" 36 5.1 -+ 1.0" 19 4.6 -+ 1 .4 ' hepatitis Cirrhosis 38 5.8 -+ 0.8* 4 5.3 -t- 1.5 8 4.9 ± 0.8* HCC 26 5.8-+0.9 2 4.0-+2.8 3 4.7-+0.6 Total 173 5.8 -+ 1.0" 44 5.1 -t- 1.2" 38 4.8 -t- 1.1"

HCV RNA; hepatitis C virus RNA HCV RNA level of defines as log 10 (copies per 50 gl of serum) Data expressed as mean-t-S.D, HCV; hepatocellular carcinoma * P<0.05

IV in Table 3. Among 173 subjects with genotype II, the mean level of HCV RNA was 6.0-t-1.1 in asymptomatic HCV carriers, 5.7 + 1.1 in chronic hepatitis, 5.7 ___ 0.8 in cirrhosis and 5.8 i -0 .9 in HCC. In asymptomatic carriers, the mean level of HCV RNA of carriers with genotype II was significantly higher than that in carriers with gen- otype IV (5.34-0.5, P<0.05). In chronic hepatitis, the mean level of HCV RNA of patients with genotype II was significantly higher than that found in those with genotypes III (5.1___1.0, P<0.05) and IV (4 .6_ 1.4, P<0.05). In cirrhosis, the mean level of HCV RNA of patients with genotype II was significantly higher than that of patients with genotype IV (4.9 ± 0.8, P<0.05). In HCC, the mean level of HCV RNA of patients with genotype II was relatively higher than in those with other genotypes, however, there were no significant differences.

The levels of HCV RNA by age are compared among

patients with genotypes II, III and IV (Table 4). Among patients with genotype II the mean level of HCV RNA was 5.6 + 1.5 in the under 39 years age group, 5.8 _+ 1.0 in 40 -49 year age group, 5.7__0.8 in 50-59 year age group and 5.8 + 1.0 in those over 60. The mean level of HCV RNA of patients with genotype II was significantly higher than that of patients with genotype IV in the 50-59 (4 .4+1 .4 , P<0.05) and over 60 (4 .6+0 .5 , P<0.05) age groups, and that of patients with genotype III (5.7_+ 1.0) was also significantly higher than that of patients with genotype IV in the over 60 year age group (P<0.05). However, there were no significant differences in the HCV RNA titre by age when compared with patients of the same genotype.

Of 191 patients in whom a history of blood transfusion was obtained, 129 were of genotype II. The level of HCV RNA and time elapsed since blood transfusion among

238 Relationship of Genotype to Hepatitis C

Table 4. Relation between the level of hepatitis C virus RNA and age by hepatitis C virus RNA genotype

Age (years) Genotype II Genotype lII Genotype 1V

No. Level of No. Level of No, Level of patients HCV RNA patients HCV RNA patients HCV RNA

- 3 9 25 5.6 _+ 1.5 15 4.9 _+ 1.2 6 5.8 _+ 1.0 40 -49 30 5.8 _+ 1.0 11 4.9 _+ 1.3 6 4.8 _+ 1.6 50-59 63 5.7_+0.8* 11 5.1 + 1.0 8 4.4_+ 1.4" 6 0 - 55 5.8 _+ 1.0" 7 5.7 ± 1 .0 ' 18 4,6 _+ 0.5* Total 173 5.8 _+ 1.0" 44 5.1 ± 1.2" 38 4.8 ± 1.1"

HCV RNA; hepatitis C virus RNA HCV RNA level of defined as log 10 (copies per 50 ~tl of serum) Data expressed as mean + S.D. * P<0.05

Table 5. Time elapsed since blood transfusion in 129 patients of genotype II

Time since transfusion Patients Level of (years) HCV RNA

(-) 72 5.6 + 1.2 O- 9 17 5 . 9 + 0 . 6

10-19 9 5.9_+1.0 20 -29 19 6.1 _ 1.0 3 0 - 12 6 . 0 + 0 . 7

HCV RNA; hepatitis C virus RNA HCV RNA level of defined as log 10 (copies per 50 gl of serum) Data expressed as m e a n _ S,D. (-) indicated no history of blood transfusion

those of genotype II are shown in Table 5 to clarify the relation between the level of HCV RNA and the duration of HCV infection. The mean level of HCV RNA was 5 . 6 _ 1.2 in patients without a history of blood trans- fusion, 5.9_+0.6 in patients who had received blood transfusion within the previous 9 years, 5 . 9 + 1 . 0 in those with transfusion 10-19 years before, 6.1 + 1.0 in those 20 -29 years before and 6.0___ O. 7 in those over 30 years before. There was no relationship between the level of HCV RNA of patients with and without a history of blood transfusion and the time elapsed since the blood transfusion.

Discussion

The effectiveness of interferon in the treatment of patients with chronic hepatitis C depends on the level of HCV viraemia, which was found to be significantly lower prior to t reatment in complete responders to interferon than in non-responders} 1'16'17 However, the relationship between levels of viraemia and the stage of liver disease has remained controversial. Methods used to calculate the

level of HCV viraemia include serial dilution PeR, com- petitive PeR, 8-11 and branched DNA signal am- plification} 6 Since methods based on PeR are useful for quantifying small amounts of RNA, we used a competitive PeR assay, with synthetic mutan t HCV RNA as a com- petitor, according to the descriptions given by Kato et

a l5 ° We quantified the amount of HCV RNA in the serum and found the range to be from 102 to 108 genomes/ 50 gl serum.

This study clearly shows that levels of HCV viraemia depend on the genotype of HCV RNA. Because the nuc- leotide sequence of genotype II differs from those of genotypes III and IV and because genotype III is similar to that of genotype IV, 12 the level of HCV viraemia in subjects with genotype II was compared to that for genotypes III and IV and was found to be significantly higher. There was no difference in HCV viraemia level between patients with genotypes III and IV. This difference was seen in each age group and at each stage of liver disease. The genotype of HCV RNA is a factor related to the response to interferon, with genotype II being unfavorable for the effective treatment of chronic HCV infection and hepatitis. 18 We reported elsewhere that the subtype of hepatitis B surface antigen was one factor of seroconversion of hepatitis B e antigen (HBeAg) to anti- body to HBeAg. 19 It seems probable that the different subtypes or genotypes of hepatitis viruses may have differentiated viral activity.

Hagiwara et a l } ° found that the H C V viraemia level was higher in patients with advanced liver disease. However, when we compared the titre by specific genotype, there was no difference in HCV viraemia level at the various stages of liver disease. Recently, a new branched DNA signal amplification technique was developed, and Lau et al., 16 using this method, found that high levels of HCV viraemia were not associated with the histological diagnosis. Since the results of these studies show that a

Jun Hayashi ot al. 239

high level of viral activity may not play an important role in the progression of liver disease, the genotype of HCV RNA might be a pertinent factor, as indicated by our finding that the prevalence of severe liver disease was high in patients of genotypes II and IV. Unfortunately, the number of patients examined in our study was too small to make a definite conclusion. Because the method of study used (a cross-sectional survey) may have biased the results, a cohort study needs to be done to answer the questions. The correlation between the level of HCV viraemia and the duration of HCV infection is also con- troversial. 16'2° Because our results included asymptomatic carriers and were obtained from patients with the same genotype, it is clear that there is no correlation between the HCV viraemia level and the estimated duration of HCV infection.

In hepatitis B virus (HBV) infection, the replicative level of HBV usually declines with age. 2i In contrast, quantitation of circulating HCV RNA revealed that the serum concentration of HCV did not decline with age, remaining unchanged throughout the course of the dis- ease. These results plus data in our previous reports of numerous HCV carriers without elevated ALT, 22 suggest that the etiology of liver injury is not only the result of a direct cytopathic effect of HCV. 23

in conclusion, the level of HCV viraemia depended on the genotype of HCV RNA and did not correlate to age or stage of liver disease. Thus, in examining subjects with chronic HCV infection, it may be more beneficial to assay for the genotype of HCV RNA rather than for the level of HCV viraemia.

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