Journal of Infection (1992) 25, 47-53
H e p a t i t i s C virus R N A and hepatitis C virus a n t i b o d y in the s e r u m o f patients with a b n o r m a l liver f u n c t i o n Hiroshi Ichimura,*~ Ikuo Tamura,* Osamu Yamada,~ Ei-ichi Takezaki,t Tetsuzo Koda,* Osamu Kurimura* and Takashi Kurimura~ * Institute of Clinical Research, and t Department of Internal Medicine, Kure National Hospital, Kure and ~.Department of Pathology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan Accepted for publication 28 November I99I Summary In order to elucidate the relation between hepatitis C virus (HCV) RNA and antibody to HCV (anti-HCV) in serum, we examined samples of serum collected from 228 HBsAg-negative patients, with abnormal alanine aminotransferase (ALT) values, for H C V - R N A by nested polymerase chain reaction (PCR) assay and for anti-HCV using ClOO protein as the antigen. H C V - R N A was detected in 99 (92'5 %) of lO7 anti-HCVIgG-positive samples, regardless of E L I S A optical density cut-off value (ELISA ratio), and in 34 (28.1%) of 121 anti-HCV-IgG-negative samples in which the frequency of the presence of H C V - R N A became higher in proportion to the E L I S A ratio. Among 42 discordant cases (34 anti-HCV-IgG-negative, RNA-postive cases and eight anti-HCV-IgG-positive, RNA-negative cases), IO were positive for antiH C V - I g M (8/34 and 2/8, respectively) irrespective of clinical status. These findings suggest that in patients with abnormal A L T values, even if they are anti-HCV-IgG negative, HCV infection cannot be excluded. Furthermore, PCR assay for detecting H C V - R N A may be more suitable for identifying patients with infectious virus than is detection of anti-HCV-IgG. Detection of anti-HCV-IgM may also be useful.
Introduction A n R N A virus, designated hepatitis C virus ( H C V ) , has been p r o p o s e d as one of the major aetiological agents for n o n - A , n o n - B hepatitis. 1 I g G antibody to C l o o ( a n t i - H C V - I g G ) , a r e c o m b i n a n t non-structural protein from H C V , is detectable b y an enzyme-linked i m m u n o s o r b e n t assay ( E L I S A ) . 2,3 E v e n so, the current m e t h o d of detecting a n t i - H C V - I g G cannot identify all s e r u m samples containing H C V and the presence of the antibody does not imply viral replication. 4-6 I g M antibody to C l o o ( a n t i - H C V - I g M ) has also been reported not necessarily to reflect replication of H C V . 7 A test for viraemia and antigenaemia of H C V is not yet available. D e t e c t i o n of R N A sequences of H C V b y a polymerase chain reaction (PCR), therefore, m a y indicate a good marker for the presence of H C V . In this study we examined samples of serum from outpatients, with abnormal serum alanine aminotransferase ( A L T ) values, for a n t i - H C V - I g G and - I g M using C l o o protein as the antigen as well as for H C V - R N A b y a § Address correspondence to: Dr H. Ichimura, Institute of Clinical Research, Kure National Hospital, Aoyama 3-1, Kure 737, Japan.
oi63-4453/92/o4oo47+o7 $03.00/0
© I99Z The British Society for the Study of Infection
48
H. ICHIMURA
ET AL.
nested P C R using primers derived from the putative core region of the H C V genome. ~ Materials and methods Serum samples
D u r i n g October and N o v e m b e r I99O, a total of 264 samples of serum were obtained from 264 outpatients attending Kure National Hospital, Hiroshima, Japan, and who had abnormal serum A L T values ( > 38 U/I). Of the 264 patients, 36 (I3"6 %) were positive for hepatitis B surface antigen (HBsAg). T h e y were therefore excluded from this study. T h e remaining 228 serum samples from 228 patients (mean age + S.D. : 57"2 _+ z4"7 years; male to female: 15o to 78) were stored at - 3 o °C until used. A n t i - H C V assay
Serum samples were tested for the presence of I g G antibody to C~oo (antiH C V - I g G ) with the Ortho Diagnostic E L I S A kit. For the detection of a n t i - H C V - I g M in serum, horseradish peroxidase(HRP-) conjugated anti-human # chain antibody (Eiken Immunochemical, Ohtawara, Japan), diluted I in Iooo in 5o % fetal calf serum, was used together with I m m o l E D T A as a second antibody instead of H R P - c o n j u g a t e d antih u m a n I g G in the Ortho E L I S A kit. T h e cut-off value was calculated as follows: mean E L I S A optical density of 3o serum samples from healthy antiH C V - I g G - n e g a t i v e persons plus 6 S.D.8 (Fig. I). P C R for H C V R N A s e q u e n c e s
For the detection of hepatitis C viral sequences, serum R N A was extracted, reverse-transcribed and amplified. T h e reverse-transcription/PCR primers and internal probe sequences (Table I) were derived from the known sequence of H C - J I . ~ A search of a D N A / R N A database (Gen Bank) was made in order to determine the specificity of the selected primers and internal sequences. R N A was extracted from serum samples by means of glass powder and c D N A synthesis according to the m e t h o d described previously. 9 c D N A was then amplified by a first-stage P C R in a D N A thermal cycler (Perkin-Elmer Cetus, C T , U.S.A.) for 32 cycles with a Gene A m p D N A amplification reagent kit (Perkin-Elmer Cetus) after the m e t h o d originally described by Saiki e t a l . 1° Each reaction cycle involved denaturation at 94 °C for ~ min, primer annealling at 45 °C for I min and primer extension at 72 °C for I min. Re-amplification was then attempted on presumptive P C R products from the first stage of PCR. T h e second stage of P C R employed the pair of primers, no. 2, designed to be internal, in sequence to primers no. I, which were used for the first stage of P C R (Table I). T h e reaction conditions were the same as in the first. An aliquot of the reaction mixture was subjected to electrophoresis on a composite gel of 3 % NuSieve G T G ( F M C Bioproducts, M O , U.S.A.) and z % agarose gel in I x T B E buffer, p H 8.o (90 mM Trisborate, 2 m m o l E D T A ) , and D N A species on the gel were stained with ethidium bromide for observation under ultraviolet light. Specificity of the D N A band was confirmed by Southern blot hybridisation analysis in which [~2p] -end-labelled oligonucleotide was used as a probe (Table I).
Hepatitis C virus and abnormal liver function I-I
49
1-083
0.5
0.4
,% c~ o
5: ~m
0.3
ee
> o -r
I c 0-2
Cut-off ( 0 . 1 9 8 ) M e o n + 6 S.D.
•
: O.I
i
• Meon ( O . O 4 2 )
Anti-HCV IgG (-) HCV-RNA(*)
n--54
(+) Anti-
Anli-HCV IgG
Heolthy* HCV
HCV-RNA(-)
IgG (-)
n-'8
n:50
F i g . I. Presence of anti-HCV-IgM in the serum of persons with abnormal alanine aminotransferase values. * Healthy: healthy controls (normal alanine aminotransferase values).
Throughout this study, false-positive PCR results were avoided by strict application of the measures of Kwok and Higuchi for preventing contamination. 1t Results T h e relation between the presence of H C V - R N A and anti-HCV-IgG in the serum of patients with abnormal A L T values is shown in Table II. H C V R N A was detected in I33 (58"3 %) of the 228 serum samples; that is, in 99 (92"5 %) of zo7 anti-HCV-IgG-positive samples and in 34 (28.I %) of i 2 i anti-HCV-IgG-negative samples, while I4I (6I'8%) of the 228 serum samples were positive for either H C V - R N A or anti-HCV-IgG. We further determined the frequency of H C V - R N A in these samples according to E L I S A optical density/cut-off value ( E L I S A ratio) (Table III). H C V - R N A was detected in more than 9o % anti-HCV-IgG-positive samples, even in cases where the E L I S A ratio was less than i. In anti-HCV-IgGnegative samples, H C V - R N A was detected in nine (I2"7 %) of 7I samples in
ET AL.
H. I C H I M U R A
5°
T a b l e I Sequences of oligonucleotide primer pairs and probe used for P C R on
c D N A of H C V genome*
Primer No. i Sense Anti-sense No. 2 Sense Anti-sense Internal probe
Nucleotide positions
Sequence (5' ~ 3')
PCR products (bp)
343-363 699-679
CCT CAA AGA AAA ACC AAA CGT G G T ATC GAT GAC C T T ACC CAA
357
475-495 595-575 555-588
AAG ACT TCC GAG CGG TCG CAA AGC CCT CAT TGC CAT AGA GGG TCA GCC CGG GTA CCC T T G GCC CCT CTA T G G CAA T
I2I
* Copied from and numbered according to the recorded sequence of HC-J1 (Okamoto et
al.*2). T a b l e I I Relation between the presence of H C V - R N A
and anti-HCV-IgG in the serum of patients with abnormal alanine aminotransferase values HCV-RNA
Anti-HCV-IgG
+ -
Total
+
-
Total
99 34 I33
8 87 95
io 7 12i 228
T a b l e I I I Frequency of H C V - R N A
in sera of patients with abnormal alanine aminotransferase with reference to anti-HCV-IgG E L I S A ratio Anti-HCV-IgG ELISA ratio*
n
Number of HCV-RNA positive cases (%)
Positive
> 5 2-5
63 32
59 (93'7) 29 (90"6)
< 2
I2
I I (91'7)
Negative
> 0.6
20
I i (55"0)
0'2-0'6 < 0"2
30 71
14 (46'7) 9 (I2"7)
* ELISA ratio: optical density/cut-off value. w h i c h t h e E L I S A ratio was less t h a n 0"2, a n d in 25 (5o'0 % ) o f 5o s a m p l e s in w h i c h t h e E L I S A ratio was m o r e t h a n 0-2. T h e r e is a significant d i f f e r e n c e in t h e f r e q u e n c y o f H C V - R N A b e t w e e n t h e s e r u m s a m p l e s in w h i c h t h e E L I S A ratio was m o r e a n d t h o s e in w h i c h it was less t h a n 0"2 (X~ t e s t : P < o ' o o I ) . I n Fig. I t h e results o f t h e a n t i - H C V - I g M assay are s u m m a r i s e d . I g M
Hepatitis C virus and abnormal liver function
5I
Table IV Relation between the presence of H C V - R N A , anti-HCV-IgG and -IgM in the serum of patients with abnormal alanine aminotransferase values Anti-HCV Sample I 2 3 4 5 6 7 8 9
(ELISA
IgG -
IgM
(0'06) (0.06) (0"07) (o.o8) (0'09)
-
(o"15) (o'15) (o'I9)
-
(o'2I) (o.21)
-
(0"21)
-
-
-- ( O ' 2 I )
I 0
HCV-RNA
- (0"32) - (0"35) - (0'50) - (0'53) - (0"23)
(O'I4)
-
-
ratio*)
--
+ + + + +
(O'18)
+
(o'46) (o'6o) (o'o8) (0'49) (0'69) (1-2o) (I"33) (0-39) (0"24) (0'94) (o"31) (0"29) (0"38) (5'47)
+ + + + + + + + + + + + + +
II i2 13 I4 15 I6 17 18 19 20
-- (0'22) (0'28) (0"28) - (0"29) (0"32) - (0"34) -- (o'4o)
21 22
-- (0"52) -(0"56)
--
(0"21) (0"18)
+ +
23 24 25 26 27 28 29 30
- (0"59) -- (o'64) -- (0'67) (0-68) -- ( o ' 7 I ) -- (0"76) -- (0"84) - (0"85)
+ (1"76) + (1'43) - (0'32) (0-49) + (I'O4) -- (0"22) -- (0'62) - (0'33)
+ + + + + + + +
31
-- (0"88)
-- (0"78)
+
32 33 34
-
(o'91) (0'95) (0"99)
+ (1"47) + (r'o7) -- (0"47)
+ + +
35 36 37 38 39 40 41 42
+ + + + + + + +
(1"65) (2'63) (4'73) (4"77)
--+
(0"75) (0-24) (0-86) (i.o6)
---
( > 5)
-- ( 0 ' I 0 )
--
( > 5) ( > 5) ( > 5)
(0"29) -- (0"43) + (2"21)
--
-
-
+ +
--
-
-
-
-
-
-
-
-
-
+ --
-
-
-
-
-
-
-
-
* ELISA ratio: optical density/cut-off value. J- P a t i e n t s w i t h a h i s t o r y o f b l o o d t r a n s f u s i o n .
Disease
-
Hepatoma Liver cirrhosis
Liver cirrhosis
Acute hepatitist Hepatoma
Liver cirrhosis
Liver cirrhosis Acute hepatitist
Malignant lymphoma t
-
Liver cirrhosis
52
H. I C H I M U R A
ET AL.
antibody was sought in 42 discordant cases [34 a n t i - H C V - I g G ( + ) and H C V R N A ( - ) ; 8 a n t i - H C V - I g G ( - ) and H C V - R N A ( + ) ] together with 30 healthy controls who had normal A L T values. I g M antibody against C l o o was detected in IO of the discordant cases (Table IV). Discussion
We found that in patients with abnormal A L T values, even if they were antiHCV-IgG-negative, the possibility of H C V infection could not be excluded. H C V - R N A was detected in 34 (28.I %) of the I2I anti-HCV-IgG-negative samples. T h e frequency of H C V - R N A detection was related to the E L I S A ratio. In 50% (25/50) of anti-HCV-IgG-negative samples, in which the E L I S A ratio was more than o.2, H C V - R N A was detected. T h u s , the E L I S A for a n t i - H C V - I g G appears to underestimate the true incidence of H C V infection if the current cut-off value is used. In this study, in four patients who had high concentrations of anti-HCVI g G in their serum ( E L I S A ratio > 5), H C V - R N A was not detected (Table III). Preliminary experiments, in which 5'-non-coding sequences of H C V - R N A were used as PCR-primers, and which were thought to be the most sensitive for detecting H C V - R N A , 1~ gave the same result (data not shown). These findings suggest that a n t i - H C V - I g G positivity in serum may not always indicate viraemia, even if the serum shows an abnormal A L T value. A further study of these patients, using the four-antigen recombinant i m m u n o b l o t assay (reported to be a better indication of viraemia than E L I S A la) would be needed. In these four patients, high values for anti-HCVI g G may reflect the presence in their serum of neutralising antibody to H C V which may have cleared H C V from the bloodstream. For the present study, 264 serum samples showing abnormal A L T values were collected randomly from outpatients of K u r e National Hospital, which is a general hospital. Of these, 36 (13"6 %) were positive for HBsAg and 133 (58"3 %) of the remaining 228 were positive for H C V - R N A . These findings indicate that H C V infection was the major cause of abnormal A L T values in our patients. In conclusion, our results suggest that in patients with abnormal A L T values, H C V infection cannot be excluded even if they are anti-HCVnegative; that P C R assay for detecting H C V - R N A may be more suitable for identifying patients with infectious virus than a n t i - H C V ; and that H C V infection is the major cause of people having abnormal A L T values in Japan. It is also noted that, in some patients, a n t i - H C V - I g M could be detected irrespective of a n t i - H C V - I g G or H C V - R N A . Furthermore, the presence of I g M antibody did not correlate with clinical findings. T h e mechanism of antiH C V - I g M production during the clinical course of H C V infections remains to be elucidated. (This work was supported by a grant-in-aid from the Ministry of Health and Welfare, Japan. We thank Dr Masao Itoh for technical advice.)
H e p a t i t i s C virus a n d a b n o r m a l liver f u n c t i o n
53
References 1. Choo Q - L , Kuo G, Weiner AJ, Overby LR, Bradley D W , Houghton M. Isolation of a c D N A clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244: 359-362. 2. Kuo G, Choo Q-L, Alter HJ et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989; 244: 362-364. 3. Alter HJ, Purcell RH, Shih J W et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N E n g l J Med 1989; 32x: 1494-I5OO. 4. Kaneko S, Unoura M, Kobayashi K, Kuno K, Murakami S, Hattori N. Detection of serum hepatitis C virus RNA. Lancet I99o; 335: 9765. Okamoto H, Okada S, Sugiyama Y et al. T h e 5'-terminal sequence of the hepatitis C virus genome. 07pn 07 Exp Med 199o; 6o: 167-177. 6. Weiner A~, Kuo G, Bradley D W et al. Detection of hepatitis C virus sequences in non-A, non-B hepatitis. Lancet 199o; 335: 1-3. 7. Quiroga JA, Campillo NiL, CatiUo I, Bartolome J, Porres JC, Carreno V, I g M antibody to hepatitis C virus in acute and chronic hepatitis C. Hepatology 199o; x4: 38-43. 8. Watanabe J, Minegishi K, Mitsumori T et al. Prevalence of anti-HCV antibody in blood donors in the Tokyo area. Vox Sang I99I ; 59: 86-88. 9. Yamada O, Matsumoto T , Nakashima M et al. A new method for extracting D N A or R N A for polymerase chain reaction. 07 Viro! Methods I99o ; 27 : 2o3-21o. zo. Saiki RK, Gelfand D H , Stoffel S e t al. Primer-directed enzymatic amplification of D N A with a thermostable D N A polymerase. Science I988 ; 2399 : 487-491. I i . Kwok S, Higuchi R. Avoiding false positives with PCR. Nature I989; 339: 237-238. i2. Okamoto H, Okada S, Sugiyama Y e t al. Detection of hepatitis C virus R N A by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region. 07pn 07 Exp Med 199o; 6o: 215-222. 13. Van der Poel CL, Cuypers H T M , Reesink H W et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991 ; 337 : 317-319.
H e p a t i t i s C virus R N A and hepatitis C virus a n t i b o d y in the s e r u m o f patients with a b n o r m a l liver f u n c t i o n Hiroshi Ichimura,*~ Ikuo Tamura,* Osamu Yamada,~ Ei-ichi Takezaki,t Tetsuzo Koda,* Osamu Kurimura* and Takashi Kurimura~ * Institute of Clinical Research, and t Department of Internal Medicine, Kure National Hospital, Kure and ~.Department of Pathology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan Accepted for publication 28 November I99I Summary In order to elucidate the relation between hepatitis C virus (HCV) RNA and antibody to HCV (anti-HCV) in serum, we examined samples of serum collected from 228 HBsAg-negative patients, with abnormal alanine aminotransferase (ALT) values, for H C V - R N A by nested polymerase chain reaction (PCR) assay and for anti-HCV using ClOO protein as the antigen. H C V - R N A was detected in 99 (92'5 %) of lO7 anti-HCVIgG-positive samples, regardless of E L I S A optical density cut-off value (ELISA ratio), and in 34 (28.1%) of 121 anti-HCV-IgG-negative samples in which the frequency of the presence of H C V - R N A became higher in proportion to the E L I S A ratio. Among 42 discordant cases (34 anti-HCV-IgG-negative, RNA-postive cases and eight anti-HCV-IgG-positive, RNA-negative cases), IO were positive for antiH C V - I g M (8/34 and 2/8, respectively) irrespective of clinical status. These findings suggest that in patients with abnormal A L T values, even if they are anti-HCV-IgG negative, HCV infection cannot be excluded. Furthermore, PCR assay for detecting H C V - R N A may be more suitable for identifying patients with infectious virus than is detection of anti-HCV-IgG. Detection of anti-HCV-IgM may also be useful.
Introduction A n R N A virus, designated hepatitis C virus ( H C V ) , has been p r o p o s e d as one of the major aetiological agents for n o n - A , n o n - B hepatitis. 1 I g G antibody to C l o o ( a n t i - H C V - I g G ) , a r e c o m b i n a n t non-structural protein from H C V , is detectable b y an enzyme-linked i m m u n o s o r b e n t assay ( E L I S A ) . 2,3 E v e n so, the current m e t h o d of detecting a n t i - H C V - I g G cannot identify all s e r u m samples containing H C V and the presence of the antibody does not imply viral replication. 4-6 I g M antibody to C l o o ( a n t i - H C V - I g M ) has also been reported not necessarily to reflect replication of H C V . 7 A test for viraemia and antigenaemia of H C V is not yet available. D e t e c t i o n of R N A sequences of H C V b y a polymerase chain reaction (PCR), therefore, m a y indicate a good marker for the presence of H C V . In this study we examined samples of serum from outpatients, with abnormal serum alanine aminotransferase ( A L T ) values, for a n t i - H C V - I g G and - I g M using C l o o protein as the antigen as well as for H C V - R N A b y a § Address correspondence to: Dr H. Ichimura, Institute of Clinical Research, Kure National Hospital, Aoyama 3-1, Kure 737, Japan.
oi63-4453/92/o4oo47+o7 $03.00/0
© I99Z The British Society for the Study of Infection
48
H. ICHIMURA
ET AL.
nested P C R using primers derived from the putative core region of the H C V genome. ~ Materials and methods Serum samples
D u r i n g October and N o v e m b e r I99O, a total of 264 samples of serum were obtained from 264 outpatients attending Kure National Hospital, Hiroshima, Japan, and who had abnormal serum A L T values ( > 38 U/I). Of the 264 patients, 36 (I3"6 %) were positive for hepatitis B surface antigen (HBsAg). T h e y were therefore excluded from this study. T h e remaining 228 serum samples from 228 patients (mean age + S.D. : 57"2 _+ z4"7 years; male to female: 15o to 78) were stored at - 3 o °C until used. A n t i - H C V assay
Serum samples were tested for the presence of I g G antibody to C~oo (antiH C V - I g G ) with the Ortho Diagnostic E L I S A kit. For the detection of a n t i - H C V - I g M in serum, horseradish peroxidase(HRP-) conjugated anti-human # chain antibody (Eiken Immunochemical, Ohtawara, Japan), diluted I in Iooo in 5o % fetal calf serum, was used together with I m m o l E D T A as a second antibody instead of H R P - c o n j u g a t e d antih u m a n I g G in the Ortho E L I S A kit. T h e cut-off value was calculated as follows: mean E L I S A optical density of 3o serum samples from healthy antiH C V - I g G - n e g a t i v e persons plus 6 S.D.8 (Fig. I). P C R for H C V R N A s e q u e n c e s
For the detection of hepatitis C viral sequences, serum R N A was extracted, reverse-transcribed and amplified. T h e reverse-transcription/PCR primers and internal probe sequences (Table I) were derived from the known sequence of H C - J I . ~ A search of a D N A / R N A database (Gen Bank) was made in order to determine the specificity of the selected primers and internal sequences. R N A was extracted from serum samples by means of glass powder and c D N A synthesis according to the m e t h o d described previously. 9 c D N A was then amplified by a first-stage P C R in a D N A thermal cycler (Perkin-Elmer Cetus, C T , U.S.A.) for 32 cycles with a Gene A m p D N A amplification reagent kit (Perkin-Elmer Cetus) after the m e t h o d originally described by Saiki e t a l . 1° Each reaction cycle involved denaturation at 94 °C for ~ min, primer annealling at 45 °C for I min and primer extension at 72 °C for I min. Re-amplification was then attempted on presumptive P C R products from the first stage of PCR. T h e second stage of P C R employed the pair of primers, no. 2, designed to be internal, in sequence to primers no. I, which were used for the first stage of P C R (Table I). T h e reaction conditions were the same as in the first. An aliquot of the reaction mixture was subjected to electrophoresis on a composite gel of 3 % NuSieve G T G ( F M C Bioproducts, M O , U.S.A.) and z % agarose gel in I x T B E buffer, p H 8.o (90 mM Trisborate, 2 m m o l E D T A ) , and D N A species on the gel were stained with ethidium bromide for observation under ultraviolet light. Specificity of the D N A band was confirmed by Southern blot hybridisation analysis in which [~2p] -end-labelled oligonucleotide was used as a probe (Table I).
Hepatitis C virus and abnormal liver function I-I
49
1-083
0.5
0.4
,% c~ o
5: ~m
0.3
ee
> o -r
I c 0-2
Cut-off ( 0 . 1 9 8 ) M e o n + 6 S.D.
•
: O.I
i
• Meon ( O . O 4 2 )
Anti-HCV IgG (-) HCV-RNA(*)
n--54
(+) Anti-
Anli-HCV IgG
Heolthy* HCV
HCV-RNA(-)
IgG (-)
n-'8
n:50
F i g . I. Presence of anti-HCV-IgM in the serum of persons with abnormal alanine aminotransferase values. * Healthy: healthy controls (normal alanine aminotransferase values).
Throughout this study, false-positive PCR results were avoided by strict application of the measures of Kwok and Higuchi for preventing contamination. 1t Results T h e relation between the presence of H C V - R N A and anti-HCV-IgG in the serum of patients with abnormal A L T values is shown in Table II. H C V R N A was detected in I33 (58"3 %) of the 228 serum samples; that is, in 99 (92"5 %) of zo7 anti-HCV-IgG-positive samples and in 34 (28.I %) of i 2 i anti-HCV-IgG-negative samples, while I4I (6I'8%) of the 228 serum samples were positive for either H C V - R N A or anti-HCV-IgG. We further determined the frequency of H C V - R N A in these samples according to E L I S A optical density/cut-off value ( E L I S A ratio) (Table III). H C V - R N A was detected in more than 9o % anti-HCV-IgG-positive samples, even in cases where the E L I S A ratio was less than i. In anti-HCV-IgGnegative samples, H C V - R N A was detected in nine (I2"7 %) of 7I samples in
ET AL.
H. I C H I M U R A
5°
T a b l e I Sequences of oligonucleotide primer pairs and probe used for P C R on
c D N A of H C V genome*
Primer No. i Sense Anti-sense No. 2 Sense Anti-sense Internal probe
Nucleotide positions
Sequence (5' ~ 3')
PCR products (bp)
343-363 699-679
CCT CAA AGA AAA ACC AAA CGT G G T ATC GAT GAC C T T ACC CAA
357
475-495 595-575 555-588
AAG ACT TCC GAG CGG TCG CAA AGC CCT CAT TGC CAT AGA GGG TCA GCC CGG GTA CCC T T G GCC CCT CTA T G G CAA T
I2I
* Copied from and numbered according to the recorded sequence of HC-J1 (Okamoto et
al.*2). T a b l e I I Relation between the presence of H C V - R N A
and anti-HCV-IgG in the serum of patients with abnormal alanine aminotransferase values HCV-RNA
Anti-HCV-IgG
+ -
Total
+
-
Total
99 34 I33
8 87 95
io 7 12i 228
T a b l e I I I Frequency of H C V - R N A
in sera of patients with abnormal alanine aminotransferase with reference to anti-HCV-IgG E L I S A ratio Anti-HCV-IgG ELISA ratio*
n
Number of HCV-RNA positive cases (%)
Positive
> 5 2-5
63 32
59 (93'7) 29 (90"6)
< 2
I2
I I (91'7)
Negative
> 0.6
20
I i (55"0)
0'2-0'6 < 0"2
30 71
14 (46'7) 9 (I2"7)
* ELISA ratio: optical density/cut-off value. w h i c h t h e E L I S A ratio was less t h a n 0"2, a n d in 25 (5o'0 % ) o f 5o s a m p l e s in w h i c h t h e E L I S A ratio was m o r e t h a n 0-2. T h e r e is a significant d i f f e r e n c e in t h e f r e q u e n c y o f H C V - R N A b e t w e e n t h e s e r u m s a m p l e s in w h i c h t h e E L I S A ratio was m o r e a n d t h o s e in w h i c h it was less t h a n 0"2 (X~ t e s t : P < o ' o o I ) . I n Fig. I t h e results o f t h e a n t i - H C V - I g M assay are s u m m a r i s e d . I g M
Hepatitis C virus and abnormal liver function
5I
Table IV Relation between the presence of H C V - R N A , anti-HCV-IgG and -IgM in the serum of patients with abnormal alanine aminotransferase values Anti-HCV Sample I 2 3 4 5 6 7 8 9
(ELISA
IgG -
IgM
(0'06) (0.06) (0"07) (o.o8) (0'09)
-
(o"15) (o'15) (o'I9)
-
(o'2I) (o.21)
-
(0"21)
-
-
-- ( O ' 2 I )
I 0
HCV-RNA
- (0"32) - (0"35) - (0'50) - (0'53) - (0"23)
(O'I4)
-
-
ratio*)
--
+ + + + +
(O'18)
+
(o'46) (o'6o) (o'o8) (0'49) (0'69) (1-2o) (I"33) (0-39) (0"24) (0'94) (o"31) (0"29) (0"38) (5'47)
+ + + + + + + + + + + + + +
II i2 13 I4 15 I6 17 18 19 20
-- (0'22) (0'28) (0"28) - (0"29) (0"32) - (0"34) -- (o'4o)
21 22
-- (0"52) -(0"56)
--
(0"21) (0"18)
+ +
23 24 25 26 27 28 29 30
- (0"59) -- (o'64) -- (0'67) (0-68) -- ( o ' 7 I ) -- (0"76) -- (0"84) - (0"85)
+ (1"76) + (1'43) - (0'32) (0-49) + (I'O4) -- (0"22) -- (0'62) - (0'33)
+ + + + + + + +
31
-- (0"88)
-- (0"78)
+
32 33 34
-
(o'91) (0'95) (0"99)
+ (1"47) + (r'o7) -- (0"47)
+ + +
35 36 37 38 39 40 41 42
+ + + + + + + +
(1"65) (2'63) (4'73) (4"77)
--+
(0"75) (0-24) (0-86) (i.o6)
---
( > 5)
-- ( 0 ' I 0 )
--
( > 5) ( > 5) ( > 5)
(0"29) -- (0"43) + (2"21)
--
-
-
+ +
--
-
-
-
-
-
-
-
-
-
+ --
-
-
-
-
-
-
-
-
* ELISA ratio: optical density/cut-off value. J- P a t i e n t s w i t h a h i s t o r y o f b l o o d t r a n s f u s i o n .
Disease
-
Hepatoma Liver cirrhosis
Liver cirrhosis
Acute hepatitist Hepatoma
Liver cirrhosis
Liver cirrhosis Acute hepatitist
Malignant lymphoma t
-
Liver cirrhosis
52
H. I C H I M U R A
ET AL.
antibody was sought in 42 discordant cases [34 a n t i - H C V - I g G ( + ) and H C V R N A ( - ) ; 8 a n t i - H C V - I g G ( - ) and H C V - R N A ( + ) ] together with 30 healthy controls who had normal A L T values. I g M antibody against C l o o was detected in IO of the discordant cases (Table IV). Discussion
We found that in patients with abnormal A L T values, even if they were antiHCV-IgG-negative, the possibility of H C V infection could not be excluded. H C V - R N A was detected in 34 (28.I %) of the I2I anti-HCV-IgG-negative samples. T h e frequency of H C V - R N A detection was related to the E L I S A ratio. In 50% (25/50) of anti-HCV-IgG-negative samples, in which the E L I S A ratio was more than o.2, H C V - R N A was detected. T h u s , the E L I S A for a n t i - H C V - I g G appears to underestimate the true incidence of H C V infection if the current cut-off value is used. In this study, in four patients who had high concentrations of anti-HCVI g G in their serum ( E L I S A ratio > 5), H C V - R N A was not detected (Table III). Preliminary experiments, in which 5'-non-coding sequences of H C V - R N A were used as PCR-primers, and which were thought to be the most sensitive for detecting H C V - R N A , 1~ gave the same result (data not shown). These findings suggest that a n t i - H C V - I g G positivity in serum may not always indicate viraemia, even if the serum shows an abnormal A L T value. A further study of these patients, using the four-antigen recombinant i m m u n o b l o t assay (reported to be a better indication of viraemia than E L I S A la) would be needed. In these four patients, high values for anti-HCVI g G may reflect the presence in their serum of neutralising antibody to H C V which may have cleared H C V from the bloodstream. For the present study, 264 serum samples showing abnormal A L T values were collected randomly from outpatients of K u r e National Hospital, which is a general hospital. Of these, 36 (13"6 %) were positive for HBsAg and 133 (58"3 %) of the remaining 228 were positive for H C V - R N A . These findings indicate that H C V infection was the major cause of abnormal A L T values in our patients. In conclusion, our results suggest that in patients with abnormal A L T values, H C V infection cannot be excluded even if they are anti-HCVnegative; that P C R assay for detecting H C V - R N A may be more suitable for identifying patients with infectious virus than a n t i - H C V ; and that H C V infection is the major cause of people having abnormal A L T values in Japan. It is also noted that, in some patients, a n t i - H C V - I g M could be detected irrespective of a n t i - H C V - I g G or H C V - R N A . Furthermore, the presence of I g M antibody did not correlate with clinical findings. T h e mechanism of antiH C V - I g M production during the clinical course of H C V infections remains to be elucidated. (This work was supported by a grant-in-aid from the Ministry of Health and Welfare, Japan. We thank Dr Masao Itoh for technical advice.)
H e p a t i t i s C virus a n d a b n o r m a l liver f u n c t i o n
53
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