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Detection of hepatitis C virus by polym erase chain reaction in haemodialysed patients in relationship to anti-HCV status
Q INSTITUT PASrEuR/EI.SEVlL-R Paris 1993
Res. Virol. 1993, 144, 233-242
Detection of hepatitis C virus by polym erase chain reaction in haemodialysed patients in relationship to anti-HCV status F. Bouchardeau (~), P. Chauveau (2), N. Le Marrec (i), A. Girault (]1, B. Zins (2) and A . M . Courouc6 (I)(*) m lnstitut national de Transfusion sanguine, 6, rue Alexandre Cabanel, 75015 Paris, and ¢2) Centre m~dical Edouard Rist, 14, rue Boileau, 75016 Paris
SUMMARY HCV RNA was determined by the polymerase chain reaction (PCR) in 41 haemodialysed patients with a known anti-HCV status (ELISA and RIBA-2) and a monthly alanine aminotransferase (ALT) level determination. No histological examination of the liver tissue was available. Four samples from each patient were collected at 6 month intervals for 18 months. Seven patients negative for anti-HCV during the entire follow-up gave negative PCR results on the four samples. Two patients who were anti-HCV-negative upon entry into the study seroconverted to HCV during follow-up. HCV RNA was detected during the acute phase of hepatitis. HCV RNA was no longer detectable after antiviral therapy was administered to one patient. Out of 27 anti-HCV-positive patients, 24 had persistent viraemia, 2 had transient viraemia (1 sample PCR-negative and 3 samples PCR-positive) and 1 was PCR-negative on the 4 samples. Thirteen of the 26 viraemic patients had a normal ALT level during the preceding 3 years. Three patients with a C22-3 band alone by RIBA-2 were negative by PCR, whereas two patients with a C33-c band alone were PCR-positive on the four samples. These results suggest that HCV viraemia was strongly associated with anti-HCV in haemodialysed patients with or without biological hepatitis.
Key-words: Viraemia, Hepatitis, HCV, Haemodialysis, Alanine aminotransferase, RNA, PCR; RIBA-2, ELISA, Anti-HCV status.
INTRODUCTION
The cloning of part of the hepatitis C virus (HCV) genome was reported in 1989 (Choo et al., 1989). The HCV genome was shown to contain a positive-stranded RNA genome of about 9,400 nucleotides (Choo et al., 1989) that encoded a polyprotein precursor of 3,011 amino acids
Submitted December 24, 1992, accepted April 29, 1993. (*) Corresponding author.
(Choo et al., 1991). In mid-1990, first generation assays to detect antibody to a non-structural antigen of HCV were available (Kuo et ai., 1989). They were adopted for the routine screening of blood donations and for the diagnosis of hepatitis. Second generation assays which include polypeptides that are additional gene products (core and protease epitopes) were de-
F. B O U C H A R D E A U E T AL.
234
veloped a n d used in early 1991 (Van der Po~l et aL, 1991 ; W a n g et al., 1992). T h e d e t e c t i o n o f H C V R N A o f f e r s great advantages in the diagnosis o f H C V infection. T h e a p p l i c a t i o n o f the p o l y m e r a s e chain r e a c t i o n ( P C R ) t e c h n i q u e to a m p l i f y reverse t r a n s c r i b e d c D N A permits a very sensitive assay f o r viral R N A circulating in b l o o d .
41 patients who had been tested by anti-HCV ELISA 2nd generation (Ortho Diagnostics Systems, Raritan, NY, USA). All samples reactive by ELISA were tested by RIBA-2 immunoblot assay (Chiron Corporation, Emeryville, CA, USA). This assay qualitatively detects antibodies to four HCV-encoded antigens, two from a non-structural NS4 region (C5-1-1, CI00-3), one from a non-structural NS3 region (C33-c) and one structural core antigen (C22-3). A positive result requires reactivity to two HCV antigens. Reactivity to only one HCV antigen results in an indeterminate interpretation.
In this study, we a n a l y s e d H C V i n f e c t i o n in a p o p u l a t i o n o f h a e m o d i a l y s e d patients by detecting b o t h viral R N A by P C R and a n t i b o d i e s to H C V ( a n t i - H C V ) by second generation assays. F o r m o r e a c c u r a t e results a n d a b e t t e r c o m p a r ison o f v i r a e m i a a n d a n t i b o d i e s , a f o l l o w - u p s t u d y was p e r f o r m e d o v e r an 1 8 - m o n t h p e r i o d .
- - g r o u p 1 = 9 patients with normal ALT level and anti-HCV-negative ;
MATERIALS AND METHODS
- - g r o u p 2 = 1 3 patients with ALT level > o r = 2 × n o r m a l value and anti-HCV-positive by RIBA-2;
Population
In a haemodialysis unit, 41 patients known to be negative or positive for anti-HCV were selected for the PCR study. Follow-up of 18 mont-hs appeared to be possible due to different parameters, and in particular no forecasted renal transplant during that period. A total of 26 men and 15 women entered the study. The mean age was 44.1_+ 17.8 years and 46.6_+21 years respectively. The duration o f haemodialysis was 10.2_+years for men and 12_+4.7 years for women (table I). Subjects were not infected by human immunodeficiency virus (HIV). Two were hepatitis B virus (HBV) chronic carriers with circulating HBV DNA, while all others were immunized against HBV, 20 o f them by vaccination. Alanine aminotransferase (ALT) levels were determined at least twice a month from the first dialysis up to the end of follow-up. No recent liver biopsy had been performed on these patients, for 3 reasons : no treatment was available until 1991, no renal transplantation was anticipated for them, and liver biopsy p r e s e n t e d some risk for these c h r o n i c haemodialysed patients. It would therefore not have been ethical to propose liver biopsy to these patients simply for the purpose of this study. Upon entry into the study, anti-HCV status was known for all the
ALT cDNA ELISA HBsAg HBV
= = = =
alanine aminotransferase. complementary DNA. enzyme-linked immunosorbent assay. hepatitis B surface antigen. = hepatitis B virus.
For the PCR stu.dy, patients were divided into four groups according to the mean ALT levels during the three years preceding the study and the antiHCV status upon entry into the study (table 1):
- - group 3 = 14 patients with normal ALT level and anti-HCV-positive by RIBA-2; - - group 4 = 3 patients with C22-3 band alone and normal ALT, and 2 with C33-c band alone and elevated A L T ; the latter two are chronic HBV carriers. Patients from groups 2, 3 and 4 were known to be anti-HCV-positive since at least 1989, except for 2 who seroconverted in January 1990 (1 in group I and I in group 2). All samples were tested by anti-HCV ELISA and RIBA-2 during follow-up.
Single P C R and " n e s t e d " P C R
Samples Blood samples were collected specifically for PCR from January 1991 to June 1992, twice a year at 6-month interval (4 samples). For patients who had seroconverted during the study, blood samples were collected more frequently during acute hepatitis. The best conditions for specimen collection and storage were used (Busch et al., 1992). Sera were
HCV HIV NS PCR RIBA
= hepatitis C virus. = human immunodeficiency virus. -- " non-slructural region o f the H C V genome, = polymerase chain reaction. = recombinant i m m u n o b l o t assay.
D E T E C T I O N O F H E P A T I T I S C VIRUS B Y P C R I N H A E M O D I A L Y S E D P A T I E N T S
235
Table I. Patients' distribution in terms of anti-HCV status, age, time in dialysis and A L T level at the entry in the study. Men
Group Group Group Group
1 2 3 4
Women
Anti-HCV
No.
Mean age (years)
Time in dialysis (years)
ALT U/I
No.
Mean age (years)
Time in dialysis (years)
ALT U/I
Total
Neg Pos Pos C22-3 ~°) C33-c ~.~
8 (") 7 8 2 1 ¢"'~ 26 ( " )
54.9 38.6 41.7 57.5 53 44.9
6.4 14.4 10.4 6.7 14.3 10.2
12.1 28.9 12.8 7.5 20 --
1 6 6 1 1 ¢'°') 15
65.5 41.2 50.2 59 26.9 46.6
11.2 12 13 9.3 10.5 12
18 30 12.6 8.4 24.7 --
9 I'°) 13 14 3 2 ¢'") 41 ( ' ' )
Total
(*) Indeterminate profile by RIBA-2 (only one band). (**) Two patients seroconverted to HCV during the study. (***) HBs Ag carriers. ALT level was the mean level observed during the three years preceding the study.
separated within 2-3 h of venipuncture. They were quickly frozen and kept out at - 80°C until RNA extraction, cDNA and PCR were performed.
S R 2 = inner antisense primer (21nt): 5'-GGGCACTCGCAAGCACCCTAT-3' ; SFI = outer sense primer (20nt): 5 ' - G C C A T G G C G T T A G T A T G A G T - 3 ' , and
R N A extraction RNA was extracted by a method using acid guanidinium t h i o c y a n a t e / p h e n o l / c h l o r o f o r m as previously described (Chomczynski and Sacchi, 1987); 200 ~! of serum (thawed at 4°C only once), 600 ~.1 denaturing solution containing 4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7, 0.5 % sarcosyl, 0.1 M 2-mercaptoethanol and 600~zl Tris/EDTA-saturated phenol were mixed, 100 ~1 chloroform were added and the tube was incubated on ice for 15 rain. After centrifugation, the aqueous phase was twice re-extracted with an equal volume of chloroform. RNA was precipited with an equal volume of isopropanol and l ~ g of glycogen (Boehringer) overnight at - 20°C. After centrifugation for 15 rain at 4°C, RNA was washed with 1 ml of 70 % ethanol and air-dried for 30 min. The pellet was redissoived in 20 ~tl of sterile distilled water.
SF2 = inner sense primer (20nt): 5'-GTGCAGCCTCCAGGACCCCC-3'. The PCR-amplified products a~'e respectively 258 bp and 211 bp.
cDNA synthesis Extracted RNA was reverse-transcribed in 20 i~1 cDNA reaction mixture containing 50 pmol of the SRI antisense primer, 0.5 m M / l of dNTP, 10 m M / l Hepes, 0.2 m M / l EDTA, 200 units of M-MLV reverse transcriptase (Gibco BRL) and 20 units o f RNase inhibitor (Boehringer). cDNA synthesis was performed at 37°C for 1 h. The reaction was stopped for 10 min at 72°C (Sellner et al., 1992).
Amplification Primers Two pairs of primers, located in the highly conserved 5' non-coding region of the HCV genome, were used (Okamoto et al., 1990a, b): SRI = outer antisense primer (20nt) : 5'-TGCACGGTCTACGAGACCTC-3',
The PCR reaction mixture contained 0.2 m M / l d N T P , 1.5 mM/1 Mg CI 2, 50 mM/I KCI, 10 m M / l Tris-HCl pH 8.3 and 1.2 units of "Ampli Taq DNA Polymerase" (Perkin Elmer Cetus, Norwalk, CT), and 50 pmol o f primers. For single PCR, 5 I.d o f cDNA were added to 50 g.l o f PCR reaction mixture containing SR2 and SF2 primers. Thirty-five cycles
236
F. B O U C H A R D E A U E T A L .
were performed: 1 min at 92°C for denaturation, 1 min at 55°C for annealing and 1 min at 72°C for extension in a programmable DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). For "nested" PCR, the first amplification step was clone with 5 ~tl ofcDNA added to 50 ~l of PCRreaction mixture with SR1 and SFI primers; 25 cycles were performed in the same conditions as those used for single PCR. After the first amplification step, 50 ~.l of PCR reaction mixture containing the corresponding inner primer pair (SR2 and SF2) were added through the oil layer in the same tube. For the second PCR reaction, the same conditions described for single PCR were used. Each serum was analysed by single PCR. For each patient with negative or transient positive results, the four samples were reanalysed by "nested" PCR. The PCR products were analysed by electrophoresis on 1 070agarose gel and visualized by ultraviolet fluorescence after staining with ethidium bromide. To reduce the risk of contamination during PCR, a number of precautions were taken as recommended by Kwok and Higuchi (1989). All reagents were prepared and all PCR steps were carried out in one room. The PCR products were analysed in a separate room. Specific pipettes were used for sample and reagent preparations. To control the risk of contamination, several negative controls including water were used at each step of the PCR. To validate the procedure, 20 samples from negative blood donors and 30 samples from individuals with chronic hepatitis C were analysed. In addition, we participated in different HCV RNA standardization studies, including the "Eurohep" HCV RNA test panel containing 22 samples from human and chimpanzee (Zaaijer et al., 1993) and we obtained correct results. Reference materials for quantitation will be provided in the next phase of HVC RNA Eurohep standardization.
RESULTS All 20 negative controls were negative by " n e s t e d " PCR and the 30 positive controls were found positive by single PCR. Seven patients from group l, negative for anti-HCV and with normal ALT, were negative in single PCR and " n e s t e d " PCR in four consecutive samples. In this group, two patients seroconverted during the study, one upon entry and the second a year later. The first patient received packed red cells in June and August 1990 and the onset o f hepati-
tis occurred in October 1990. The last known anti-HCV-negative sample was collected in June 1990 and the first known anti-HCV positive sample (C22-3 and C33-c bands by RIBA-2) in January 1991. HCV RNA was positive by single PCR and " n e s t e d " PCR in the first available sample (January 1991) corresponding to the first A L T peak (300 U/l) and in 3 successive samples collected from February to October 1991. For ten months, HCV RNA remained positive until the patient received interferon treatment. In the first sample following 'the beginning of therapy (3 months later), HCV RNA was negative in single PCR and in " n e s t e d " PCR and remained negative 6 months later. At that time, the A L T level was normal and the RIBA-2 profile was unchanged (4 bands). These results are illustrated in figure I. The second patient seroconverted after 12 months of follow-up (anti-HCV-negative on J a n u a r y 1992 and -positive on February 1992). He was never transfused. PCR was negative on the two 1991 specimens and positive during the rise in the A L T level (January 1992) in two successive samples. In the first PCR-positive sample (January 1992), anti-HCV were negative by EL1SA and RIBA-2. In the second positive sample (February 1992), one m o n t h later, RIBA-2 exhibited 3 bands on C5-1-1, C l 0 0 and C33-c. In March 1992, when the A L T level was still elevated, HCV RNA was negative by single PCR and " n e s t e d " PCR, and in June 1992, weak positivity was detected by single PCR and clearly confirmed by " n e s t e d " l~CR. No C22-3 band was detectable by RIBA-2 during the entire follow-up. No other specimens remained available. Results are presented i n figure 2. In group 2, 13 patients exhibited either 3 (5 patients) or 4 (8 patients) bands by RIBA-2, with no change during follow-up. All 13 patients had antibodies to C33-c; only one patient had no antibodies to C22-3. Eleven o f the patients were positive by single PCR in the four samples and the two others gave transient positive results in the four samples. For one of them, the first and the last sample were weakly positive by single PCR and clearly positive by " n e s t e d " PCR,
D E T E C T I O N OF H E P A T I T I S C VIRUS B Y PCR I N H A E M O D I A L Y S E D P A T I E N T S
35O
+
.'.~:
+
+++
++
C100-3
++÷.(. ++++
+,0-4.+ .... ....
C33-c
+++
+÷
++
+++
II
::::
237
5.1.1
{:22-3
3OO
250
200
150
100
50 Interferon/~'
1990
1991
1992
Fig. I. ALT level, RIBA-2 profile and HCV-RNA results in the follow-upof a patient who seroconverted to HCV upon entry into the study and who was treated early by interferon. RIBA-2 profile: band intensityexpressed from + to + + + + on the four HCV antigens,C5-1-1, CI00-3, C33-c and C22-3.
the second sample was negative by single PCR but positive by "nested" PCR and the third sample remained negative by "nested" PCR. For the other patient, the first three samples were positive by single and "nested" PCR and the last one was negative by single PCR and positive by " n e s t e d " PCR. From the beginning to the end of follow-up, these two patients had ALT levels which were not significantly different from those observed in other patients from group 2 and exhibited four bands by RIBA-2. In group 3, the 14 patients had antibodies to C22-3 and C33-c along with antibodies to C5-1-1 in 3 patients, antibodies to C100-3 in 1 patient and to antibodies to both in 8 patients. HCV RNA was positive in 12 patients by single PCR in the four samples. In one patient, only three samples were found to be positive by single PCR and " n e s t e d " PCR, with the second sample
negative by both single and nested PCR. This patient seroconverted in January 1990 and never acquired antibodies to C100-3. For the final patient, who had the lowest ALT level and was known to be anti-HCV-positive with 3 bands by RIBA-2 during the entire follow-up (negative for C100-3), HCV RNA was negative by " n e s t e d " PCR in the four samples. In group 4, the 3 patients who had only a C22-3 band by RIBA-2 were negative by single PCR and " n e s t e d " PCR during the 18-month follow-up. They had normal ALT level. The 2 patients with a C33-c band alone were strongly positive by single PCR on all samples. They had an elevated ALT level but were also chronic HBV carriers. These results are summarized in table II. Positive results by " n e s t e d " PCR in samples negative by single PCR were observed in 2 of the 64 tested samples (table III).
238
F. B O U C H A R D E A U E T A L .
450
+
÷÷
5-1-1
4-+
+-('+
CI00-3
+++
+++
C33-c
"
C22-3
400
350
3OO
250
20O
150
|
++4-
~
HCV-RNA
100
50
0
1991
1992
Fig. 2. ALT level, RIBA-2 profile and HCV-RNA results in the follow-up of a patient who seroconverted to HCV after one year of follow-up. RIBA-2 profile: band intensity expressed from + to + + + + on the four HCV antigens, C5-1-1, C100-3, C33-c and C22-3.
Table I1. RNA-HCV detection by PCR on the 4 successive samples from 39 patients.
Group 4 PCR
Group 1
Group 2
Negative on the 4 samples
7 (100 %)
0
Positive on the 4 samples
0
12 (92.3 °7,0)
Transient results (3 positive, 1 negative)
0
1
Group 3 1 12 (85.7 %) 1
C22-3
C33-c
3
0
0
2
0
0
In group 1, two patients who seroconverted at the beginning or during follow-up were excluded from this table.
DISCUSSION
In this s t u d y , the results o b t a i n e d by a n t i , HCV antibody determination and HCV RNA
detection by P C R could be c o m p a r e d . In addition, this analysis was p e r f o r m e d o n 4 consecutive samples d u r i n g an 18-month f o l l o w - u p to a p p r e c i a t e the variations in b o t h a n t i - H C V an-
D E T E C T I O N OF H E P A T I T I S C VIRUS B Y PCR I N H A E M O D I A L Y S E D
239
PATIENTS
Table !II. Comparative results between single and "nested" PCR in the four groups of patients. Negative No. patients No. samples
Single PCR results Tested by "nested" PCR Positive by "nested" PCR
Group 1 Seroconverted
7 28
Group 2
Group 3
Group 4
13 52
14 56
5 20
2 12
Pos
Neg
Pos
Neg
Pos
Neg
Pos
Neg
Pos
Neg
0
28
7
5
49
3
51
5
8
12
0
28
7
5
5
3
3
5
0
12
0
0
7
0
5
2
3
0
0
0
tibodies and viraemia assessed by PCR. Adequate care was taken to avoid erroneous diagnosis (Kwok and Higuchi, 1989) and to obtain specific and reproducible results as the choice of the method for RNA extraction (Chomczynski and Sacchi, 1987) and of the primers in the 5' non-coding region (Okamoto et al., 1990a, b; Garson et al., 1990) since this region is highly conserved among all HCV isolates studied to date (Han et al., 1991 ; Garson et al., 1990). In this population and in our technical conditions, " n e s t e d " PCR offered a minor advantage compared to single PCR : of the 117 samples positive by PCR, l l5 were positive by single PCR and 2 by " n e s t e d " PCR only. A total of 51 samples were negative by both single and " n e s t e d " PCR. The 28 samples from the 7 patients who remained anti-HCV-negative during the entire follow-up were HCV-RNA-negative, including by " n e s t e d " PCR. Negative PCR results in an anti-HCV-negative population have been generally reported (Evans et aL, 1992), except when anti-HCV was detected by firstgeneration assays (Kato et aL, 1990) which have been shown to be less sensitive than secondgeneration assays (Chauveau et al., 1992; Van der Po~l et al., 1991). HCV RNA was detected in the majority of anti-HCV-positive sera from patients with (group 2) or without (group 3) biological hepa-
titis. As previously said, no correlation with histology could be made. This study revealed that the presence of antiHCV antibody was associated with transient or persistent viraemia in 26 of the 27 patients (96 °7o)known to be anti-HCV-positive upon entry into the study. This viraemia was regularly observed on the 4 samples during the entire follow-up for 24 patients (89 o70).For 2 patients, one of the 4 samples collected durh)g the followup gave negative "nested" PCR results. This observation indicated the necessity to perform successive HCV RNA detection before concluding as to the absence of viraemia as already observed in humans (Shimizu et al., 1990) and in chimpanzees (Farci et al., 1991). It is probable that these two patients had too weak a level of circulating virus to be detectable, since successive samples were HCV-RNA-positive. In this study, HCV RNA was highly concordant with anti-HCV, as observed in a Japanese study conducted in haemodialysed patients (Tokunaga et al., 1991) where 86 o70 of the 21 patients who were anti-HCV-positive by RIBA-2 were HCV-RNA-positive. This good correlation between PCR and serology was also reported in 94 °70 of blood donors (Follet et al., 1991) and in patients with post-transfusion hepatitis C (Wang et al., 1992; Van der Po~l et al., 1991). Only one anti-HCV positive patient remained P C R negative on the 4 samples and ex-
240
F. B O U C H A R D E A U E T AL.
hibited 3 bands by RIBA-2. These negative PCR results associated with the absence of anti-C 100-3 shown to be a marker of infectivity (Weiner et al., 1990) might suggest a resolved infection in this patient. However, four other patients with no anti-C100-3 were HCV-RNA-positive. Consequently, it seems that the profile of anti-HCV antibodies cannot be used to estimate the evolution of the infection. No correlation between HCV RNA and ALT results could be made in these chronic infected patients, since similar HCV RNA results were observed in patients from group 2 with elevated A L T and patients from group 3 with normal ALT. This observation was also reported in blood donors (Van der Po~l et al., 1991). However, the patient with PCR-negative results had the lowest ALT level. Several hypotheses can be put forth to explain these negative results: absence of viraemia, too weak a level of circulating virus or a different HCV strain not recognized by the used primers (Irwing et al., 1992; Xu et al., 1992). Different primers should be used in this PCR-negative patient before making a definite conclusion as to potential recovery from HCV infection. In the two patients with acute hepatitis C, PCR results were clearly correlated with A L T levels. For one patient, no sample was available immediately before or after the first rise in A L T to determine the first appearance of HCV viraemia and the time of antibody seroconversion. Both antibody and RNA were detected on the first available specimen following the rise in ALT. For the second patient who seroconverted, HCV RNA was detected before anti-HCV, as previously observed (Zonaro et aL, 1991 ; Farci et aL, 1992). Viraemia was present in both patients during the acute phase, showing that HCV RNA detection represents an available means of identifying primary HCV infection. This detection is also useful for monitoring antiviral therapy. In the patient who received interferon treatement very soon after the infection, HCV RNA was no more detectable after this therapy. A longer follow-up will be necessary to assess the therapy efficacy. Kanai et aL (1990) had observed a decrease HCV RNA in serum after interferon treatement in 5 patients but no
HVC-negative results. For Hagiwara et al. (1992a), (1992b), HCV RNA became undetectable in 100 °7o of patients treated by interferon when ALT level fell into a normal range during therapy. For the other patient, who presented a transient HCV RNA after the acute hepatitis, the follow-up was too short for a long-term prognosis. In the 5 patients with indeterminate results by RIBA-2, the 3 with a C22-3 band alone were HCV-RNA-negative and the 2 with a C33-c band alone were HCV-RNA-positive. However, the number of patients was too low to extend there PCR results to all patients with this profile. Other studies have demonstrated that patients reactive only for C22-3 antigen could be infectious: 6/13 (46 °/0) for Halfon et al. (1992) and 8/26 ( 3 1 % ) for Follett et al. (1991). Our results on patients reactive to C33-c alone and PCR-positive correlate with other reports : one case described by Follett et al. (1992) and two cases described by Halfon et al. (1992). This RIBA-2 profile was not systematically associated with viraemia (Follett et al., 1991 ; Allain et al., 1992). However, it is clear that an indeterminate profile by RIBA-2 can be found in infectious subjects. Consequently, it is important that screening tests for anti-HCV contain antigen from the NS3 region (Follett et al., 1991). This study revealed that the presence of antiHCV antibody in haemodialysed patients was associated with viraemia in almost all cases. These results obtained on four consecutive samples suggest that PCR is a reliable and reproducible technique for HCV RNA determination, particularly when primers from the highly conserved 5' noncoding region are used. Such PCR assays provide information concerning the viraemic status when anti-HCV is present but liver function is normal, or when anti-HCV has an indeterminate profile. Such PCR assays demonstrate the necessity of follow-up before confirming that an HCV-RNA-negative, anti-HCV-positive patient had potentially eradicated the virus. The detection of HCV RNA represents the only available test to identify primary HCV infection early in its course. Moreover, PCR provides a valuable tool for monitoring the efficacy of antiviral therapy.
D E T E C T I O N O F H E P A T I T I S C VIRUS B Y P C R I N H A E M O D I A L Y S E D Acknowledgements We thank Ortho Diagnostic System (France) for their support, and the staff of the Edouard Rist Medical Center for collecting serum samples from patients.
D6tection du virus de I'h~patite C par la PCR chez des sujels h~modialys~s et stalut en anticorps anti-HCV L ' A R N du virus de I'h6patite C (ARN VHC) a 6t6 recherch6 par <>(PCR) chez 41 h6modialys6s dont le statut en anticorps antiV H C 6tait connu par ELISA et RIBA-2 ainsi que le taux des A L T (alanine aminotransf6rase) d6termin6 chaque mois. De r6cents examens histologiques du foie n'6taient pas disponibles. Pour chaque sujet, 4 6chantillons ont 6t6 pr61ev6s tousles 6 mois pendant 18 tools. Sept cas anti-VHC n6gatifs durant tout le suivi 6taient n6gatifs par PCR, sur les 4 6chantillons. Deux sujets anti-VHC n6gatifs Iors de leur entr6e dans 1'6tude ont s6roconverti au cours du suivi. L ' A R N VHC 6tait pr6sent au cours de la phase aigui5 de I'h6patite. Chez un malade trait6 par interf6ron, la vir6mie n'6tait plus d6tectable apr~s ce traitement. Parmi les 27 sujets anti-VHC positifs, 24 pr6sentaient une vir6mie persistante, 2 une vir6mie transitoire (1 6chantillon n6gatif par PCR et 3 6chantillons positifs par PCR) et un sujet 6tait n6gatif par PCR sur les 4 pr616vements. Treize des 26 sujets vir6miques avaient des transaminases normales au cours des trois ann6es pr6c6dant l'6tude. Trois sujets poss6dant une bande isol6e C22-3 en RIBA-2 6taient n6gatifs par PCR alors que deux, ayant une bande isol6e en C33-c, 6taient positifs pour les 4 6chantillons. Ces r6sultats suggSrent que la vir6mie VHC est largement associ6e ~ I'anticorps anti-HCV chez les patients h6modialys6s, avec ou sans h6patite biologique. Mots-clds: Vir6mie, H6patite, HCV, H6modialyse, Alanine-aminotransf~rase, ARN, P C R ; RIBA-2, ELISA, Statut en anticorps anti-HCV.
References Allain, J.P., Rankin, A., Kuhns, M.C. & McNamara, A. (1992), Clinical importance of HCV confirmatory testing in blood donors. Lancet, 339, 1171-1172. Busch, M.P., Wilber, J.C., Johnson, P., Tobler, L. & Evans, C.S. (1992), Impact of specimen handling and storage on detection of hepatitis C virus RNA. Transfusion, 32, 420-425. Chauveau, P., Courouc~, A.M., Lemarrec, N., Naret, C., Poignet, J.L., Girault, A., Ramdame, M. & Delons,
PATIENTS
241
S. (1992), Antibodies to hepatitis C virus by second generation test in hemodialyzed patients. Kidney Int., 43 (suppl. 41), I-3. Chomczynski, P. & Sacchi, N. (1987), Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Analyt. Biochem., 162, 156-159. Choo, Q.L., Kuo, G., Weiner, A.J., Overby, L.R., Bradley, D.W. & Houghton, M. (1989), Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science, 244, 359-362. Choo, Q.L., Richman, K.H., Han, J.H., Berger, K., Lee, C., Dong, C., Gallegos, C., Colt, D., Medina-Selby, A., Barr, P.J., Weiner, A.J., Bradley, D.W., Kuo, G. & Houghton, M. (1991), Genetic organisation and diversity of the hepatitis C virus. Proc. nat. Acad. Sci. (Wash.), 88, 2451-2455. Evans, C.S., Tobler, L., Polito, A., Stewart, J., Chien, D., Wilber, J., Quan, S., Delaney, S., Kuo, G. & Busch, M.P. (1992), Comparative evaluation of supplemental hepatitis C virus antibody test systems. Transfusion, 32, 408-414. Farci, P., Alter, H.J., Wong, D., Miller, R.H., Shih, J.W., Jett, B. & Purcell, R.H. (1991), A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. New Engl. J. Med., 325, 98-104. Farci, P., l.ondon, W.T., Wong, D.C., Dawson, G.J., Vallari, D.S., Engle, R. & Purcell, R.H. (1992), The natural history of infection with hepatitis C virus (HCV) in chimpanzees: comparison of serologic responses measured with first- and second-generation assays and relationship to HCV viremia. J. Infect. Dis., 165, 1006-1011. Follett, E.A.C., Dow, B.C., McOmish, F., Lee Yap, P., Hughes, W., Mitchell, R. & Simmonds, P. (1991), HCV confirmatory testing of blood donors. Lancet, 338, 1024. Follett, E.A.C., Dow, B.C., McOmish, F., Lee Yap, P., Crawford, R.J., Mitchell, R. & Simmonds, P. (1992), HCV confirmatory testing of blood donors. Lancet, 339, 928. Garson, J.A., Ring, C., Tuke, P. & Tedder, R.S. (1990), Enhanced detection by PCR of hepatitis C virus RNA. Lancet, 336, 878-879. Hagiwara, H., Hayashi, N., Mita, L., Ueda, K., Fakehara, T., Kasahara, A., Fusamoto, H. & Kamada, T. (1992), Detection of hepatitis C virus RNA in serum of patients with chronic hepatitis C treated with interferon-alpha. Hepatology, 13, 37-41. Hagiwara, H., Hayashi, N., Kasahara, A., Mita, L., Fakehara, T., Fusamoto, H. & Kamada, T. (1992), Three cases of posttransfusion hepatitis C treated with interferon-alpha. Confirmation of a carrier state by detection of hepatitis C virus RNA after interferon therapy. Dig. Dis. Sci., 37, 631-634. Halfon, P., Rousseau, S., Tamalet, C., Antoni, M., Gerolami, V., Levy, M., Bourli~re, M., Planells, R. & Cartouzou, G. (1992), Indeterminate secondgeneration hepatitis C recombinant immunoblot test : detection of hepatitis C virus infection by polymerase chain reaction. J. Infect. Dis., 166, 449. Han, J.H., Shyamala, V., Richman, K.H., Brauer, M.J., lrvine, B., Urdea, M.S., Tekamp-Olson, P., Kuo, G., Choo, Q.-L. & Houghton, M. (1991), Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' un-
242
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translated region and poly(A) tails at the 3' end. Proc. nat. Acad. Sci. (Wash.), 88, 1711-1715. Irwing, W.L., Day, S., Eglin, R.P., Bennett, D.P., Jones, D.A., Nuttall, P. & James, V. (1992), HCV and PCR negativity. Lancet, 339, 1425. Kanai, K., lwata, K., Nakao, K., Kako, M. & Okamoto, H. (1990), Suppression of hepatitis C virus RNA by interferon-alpha. Lancet, 336, 245. Kato, N., Yokosuka, O., Omata, M., Hosoda, K. &Ohto, M. (1990), Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction. J. Clin. Invest., 86, 1764-1767. Kuo, G., Choo, Q.-L., Alter, H.J., Gitnick, G.L., Redeker, A.G., Purcell, R.H., Miyamura, T., Dienstag, J.L., Alter, M.J., Stevens, C.E., Tegtmeier, G.E., Bonino, F., Colombo, M., Lee, W.-S., Kuo, C., Berger, K., Shuster, J.R., Overby, L.R., Bradley, D.W. & Houghton, M. (1989), An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science, 244, 362-364. Kwok, S. & Higuchi, R. (1989), Avoiding false positives with PCR. Nature (Lond.), 339, 237-238. Martinot-Peignoux, M., Marcellin, P., Xu, L.-Z., Bernuau, J., Erlinger, S., Benhamou, J.P. & Larzul, D. (1992), Reactivity to c33c antigen as a marker of hepatitis C virus multiplication. 3". Infect. Dis., 165, 595-596. Okamoto, H., Okada, S., Sugiyama, Y., Tanaka, T., Sugai, Y., Akahane, Y., Machida, A., Mishiro, S., Yoshizawa, H., Miyakawa, Y. & Mayumi, M. (1990), Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region. Jap. J. Exp. Med., 60, 215-222. Okamoto, H., Okada, S., Sugiyama, Y., Yotsumoto, S., Tanaka, T., Yoshizawa, H., Tsuda, F., Miyakawa, Y. & Mayumi, M. (1990), The 5'-terminal sequence of the hepatitis C virus genome. Jap. J. Exp. Med., 60, 167-177. Seliner, L.N., Coelen, R.J. & Mackenzie, J.S. (1992), Reverse transcriptase inhibits Taq polymerase activity. Nucl. Acids Res., 20, 1487-1490. Shimizu, Y.K., Weiner, A.J., Rosenblatt, J., Wong, D.C.,
Shapiro, M., Popkin, T., Houghton, M., Alter, H.J. & Purcell, R.H. (1990), Early events in hepatitis C virus infection of chimpanzees, in "Viral hepatitis and liver disease" (F.B. Hollinger, S.M. Lemon, H.S. Margolis) (pp. 393-396). Williams and Wilkins, Baltimore. Tokunaga, S., Oguchi, H., Higuchi, M., Kasahara, H., Matsumoto, M., Tanaka, E., Kiyosawa, K. & Furuta, S. (1991), Hepatitis C virus (HCV)-RNA titers in hemodialysis (HD) patients. American Society of Nephrology. 24th annual meeting. Abstract 92 P. Tong, C.Y.W. & Codd, A.A. (1992), RIBA-2 band intensity and PCR in HCV infection. Lancet, 340, 117. Van der Po~l, C.L., Cuypers, H.T.M., Reesink, H.W., Weiner, A.J., Quan, S., Di Nello, R., Van Boven, J.J.P., Winkel, 1., Mulder-Folkerts, D., ExeI-Oehlers, P.J., Schaasberg, W., Leentvaar-Kuypers, A., Pollto, A., Houghton, M. & Lelie, P.N. (1991), Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet, 337, 317-319. Wang, J.T., Wang, T.H., Lin, J.T., Sheu, J.C., Lee, C.Z. & Chert, D.S. (1992), Improved serodiagnosis of posttransfusion hepatitis C virus infection by a secondgeneration immunoassay based on multiple recombinant antigens. Vox Sang., 62, 21-24. Weiner, A.J., Kuo, G., Bradley, D.W., Bonino, F., Saracco, G., Lee, C., Rosenblatt, J., Choo, Q.-L. & Houghton, M. (1990), Detection of hepatitis C viral sequence in non-A, non-B hepatitis. Lancet, 335, 1-3. Xu, L.Z., Martinot-Peignoux, M., Marcellin, P., Benhamou, J.P. & Larzul, D. (1992), Lack of polymerase chain reaction amplification in the 5' region of a hepatitis C virus isolate. J. Infect. Dis., 165, 1164-1165.
Zaaijer, H.L., Cuypers, H.T.M., Reesink, H.W., Winkel, I.N., Gerken, G. & Lelie, P.N. (1993), Reliability of polymerase chain reaction for detection of hepatitis C virus. Lancet, 341, 722-724. Zonaro, A., Puoti, M., Fiordalisi, G., Mantero, G., Castelnuovo, F., Primi, D. & Cariani, E. (1991), Detection of serum hepatitis C virus RNA in acute non-A, non-B hepatitis. J. Infect. Dis., 163, 923-924.
Res. Virol. 1993, 144, 233-242
Detection of hepatitis C virus by polym erase chain reaction in haemodialysed patients in relationship to anti-HCV status F. Bouchardeau (~), P. Chauveau (2), N. Le Marrec (i), A. Girault (]1, B. Zins (2) and A . M . Courouc6 (I)(*) m lnstitut national de Transfusion sanguine, 6, rue Alexandre Cabanel, 75015 Paris, and ¢2) Centre m~dical Edouard Rist, 14, rue Boileau, 75016 Paris
SUMMARY HCV RNA was determined by the polymerase chain reaction (PCR) in 41 haemodialysed patients with a known anti-HCV status (ELISA and RIBA-2) and a monthly alanine aminotransferase (ALT) level determination. No histological examination of the liver tissue was available. Four samples from each patient were collected at 6 month intervals for 18 months. Seven patients negative for anti-HCV during the entire follow-up gave negative PCR results on the four samples. Two patients who were anti-HCV-negative upon entry into the study seroconverted to HCV during follow-up. HCV RNA was detected during the acute phase of hepatitis. HCV RNA was no longer detectable after antiviral therapy was administered to one patient. Out of 27 anti-HCV-positive patients, 24 had persistent viraemia, 2 had transient viraemia (1 sample PCR-negative and 3 samples PCR-positive) and 1 was PCR-negative on the 4 samples. Thirteen of the 26 viraemic patients had a normal ALT level during the preceding 3 years. Three patients with a C22-3 band alone by RIBA-2 were negative by PCR, whereas two patients with a C33-c band alone were PCR-positive on the four samples. These results suggest that HCV viraemia was strongly associated with anti-HCV in haemodialysed patients with or without biological hepatitis.
Key-words: Viraemia, Hepatitis, HCV, Haemodialysis, Alanine aminotransferase, RNA, PCR; RIBA-2, ELISA, Anti-HCV status.
INTRODUCTION
The cloning of part of the hepatitis C virus (HCV) genome was reported in 1989 (Choo et al., 1989). The HCV genome was shown to contain a positive-stranded RNA genome of about 9,400 nucleotides (Choo et al., 1989) that encoded a polyprotein precursor of 3,011 amino acids
Submitted December 24, 1992, accepted April 29, 1993. (*) Corresponding author.
(Choo et al., 1991). In mid-1990, first generation assays to detect antibody to a non-structural antigen of HCV were available (Kuo et ai., 1989). They were adopted for the routine screening of blood donations and for the diagnosis of hepatitis. Second generation assays which include polypeptides that are additional gene products (core and protease epitopes) were de-
F. B O U C H A R D E A U E T AL.
234
veloped a n d used in early 1991 (Van der Po~l et aL, 1991 ; W a n g et al., 1992). T h e d e t e c t i o n o f H C V R N A o f f e r s great advantages in the diagnosis o f H C V infection. T h e a p p l i c a t i o n o f the p o l y m e r a s e chain r e a c t i o n ( P C R ) t e c h n i q u e to a m p l i f y reverse t r a n s c r i b e d c D N A permits a very sensitive assay f o r viral R N A circulating in b l o o d .
41 patients who had been tested by anti-HCV ELISA 2nd generation (Ortho Diagnostics Systems, Raritan, NY, USA). All samples reactive by ELISA were tested by RIBA-2 immunoblot assay (Chiron Corporation, Emeryville, CA, USA). This assay qualitatively detects antibodies to four HCV-encoded antigens, two from a non-structural NS4 region (C5-1-1, CI00-3), one from a non-structural NS3 region (C33-c) and one structural core antigen (C22-3). A positive result requires reactivity to two HCV antigens. Reactivity to only one HCV antigen results in an indeterminate interpretation.
In this study, we a n a l y s e d H C V i n f e c t i o n in a p o p u l a t i o n o f h a e m o d i a l y s e d patients by detecting b o t h viral R N A by P C R and a n t i b o d i e s to H C V ( a n t i - H C V ) by second generation assays. F o r m o r e a c c u r a t e results a n d a b e t t e r c o m p a r ison o f v i r a e m i a a n d a n t i b o d i e s , a f o l l o w - u p s t u d y was p e r f o r m e d o v e r an 1 8 - m o n t h p e r i o d .
- - g r o u p 1 = 9 patients with normal ALT level and anti-HCV-negative ;
MATERIALS AND METHODS
- - g r o u p 2 = 1 3 patients with ALT level > o r = 2 × n o r m a l value and anti-HCV-positive by RIBA-2;
Population
In a haemodialysis unit, 41 patients known to be negative or positive for anti-HCV were selected for the PCR study. Follow-up of 18 mont-hs appeared to be possible due to different parameters, and in particular no forecasted renal transplant during that period. A total of 26 men and 15 women entered the study. The mean age was 44.1_+ 17.8 years and 46.6_+21 years respectively. The duration o f haemodialysis was 10.2_+years for men and 12_+4.7 years for women (table I). Subjects were not infected by human immunodeficiency virus (HIV). Two were hepatitis B virus (HBV) chronic carriers with circulating HBV DNA, while all others were immunized against HBV, 20 o f them by vaccination. Alanine aminotransferase (ALT) levels were determined at least twice a month from the first dialysis up to the end of follow-up. No recent liver biopsy had been performed on these patients, for 3 reasons : no treatment was available until 1991, no renal transplantation was anticipated for them, and liver biopsy p r e s e n t e d some risk for these c h r o n i c haemodialysed patients. It would therefore not have been ethical to propose liver biopsy to these patients simply for the purpose of this study. Upon entry into the study, anti-HCV status was known for all the
ALT cDNA ELISA HBsAg HBV
= = = =
alanine aminotransferase. complementary DNA. enzyme-linked immunosorbent assay. hepatitis B surface antigen. = hepatitis B virus.
For the PCR stu.dy, patients were divided into four groups according to the mean ALT levels during the three years preceding the study and the antiHCV status upon entry into the study (table 1):
- - group 3 = 14 patients with normal ALT level and anti-HCV-positive by RIBA-2; - - group 4 = 3 patients with C22-3 band alone and normal ALT, and 2 with C33-c band alone and elevated A L T ; the latter two are chronic HBV carriers. Patients from groups 2, 3 and 4 were known to be anti-HCV-positive since at least 1989, except for 2 who seroconverted in January 1990 (1 in group I and I in group 2). All samples were tested by anti-HCV ELISA and RIBA-2 during follow-up.
Single P C R and " n e s t e d " P C R
Samples Blood samples were collected specifically for PCR from January 1991 to June 1992, twice a year at 6-month interval (4 samples). For patients who had seroconverted during the study, blood samples were collected more frequently during acute hepatitis. The best conditions for specimen collection and storage were used (Busch et al., 1992). Sera were
HCV HIV NS PCR RIBA
= hepatitis C virus. = human immunodeficiency virus. -- " non-slructural region o f the H C V genome, = polymerase chain reaction. = recombinant i m m u n o b l o t assay.
D E T E C T I O N O F H E P A T I T I S C VIRUS B Y P C R I N H A E M O D I A L Y S E D P A T I E N T S
235
Table I. Patients' distribution in terms of anti-HCV status, age, time in dialysis and A L T level at the entry in the study. Men
Group Group Group Group
1 2 3 4
Women
Anti-HCV
No.
Mean age (years)
Time in dialysis (years)
ALT U/I
No.
Mean age (years)
Time in dialysis (years)
ALT U/I
Total
Neg Pos Pos C22-3 ~°) C33-c ~.~
8 (") 7 8 2 1 ¢"'~ 26 ( " )
54.9 38.6 41.7 57.5 53 44.9
6.4 14.4 10.4 6.7 14.3 10.2
12.1 28.9 12.8 7.5 20 --
1 6 6 1 1 ¢'°') 15
65.5 41.2 50.2 59 26.9 46.6
11.2 12 13 9.3 10.5 12
18 30 12.6 8.4 24.7 --
9 I'°) 13 14 3 2 ¢'") 41 ( ' ' )
Total
(*) Indeterminate profile by RIBA-2 (only one band). (**) Two patients seroconverted to HCV during the study. (***) HBs Ag carriers. ALT level was the mean level observed during the three years preceding the study.
separated within 2-3 h of venipuncture. They were quickly frozen and kept out at - 80°C until RNA extraction, cDNA and PCR were performed.
S R 2 = inner antisense primer (21nt): 5'-GGGCACTCGCAAGCACCCTAT-3' ; SFI = outer sense primer (20nt): 5 ' - G C C A T G G C G T T A G T A T G A G T - 3 ' , and
R N A extraction RNA was extracted by a method using acid guanidinium t h i o c y a n a t e / p h e n o l / c h l o r o f o r m as previously described (Chomczynski and Sacchi, 1987); 200 ~! of serum (thawed at 4°C only once), 600 ~.1 denaturing solution containing 4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7, 0.5 % sarcosyl, 0.1 M 2-mercaptoethanol and 600~zl Tris/EDTA-saturated phenol were mixed, 100 ~1 chloroform were added and the tube was incubated on ice for 15 rain. After centrifugation, the aqueous phase was twice re-extracted with an equal volume of chloroform. RNA was precipited with an equal volume of isopropanol and l ~ g of glycogen (Boehringer) overnight at - 20°C. After centrifugation for 15 rain at 4°C, RNA was washed with 1 ml of 70 % ethanol and air-dried for 30 min. The pellet was redissoived in 20 ~tl of sterile distilled water.
SF2 = inner sense primer (20nt): 5'-GTGCAGCCTCCAGGACCCCC-3'. The PCR-amplified products a~'e respectively 258 bp and 211 bp.
cDNA synthesis Extracted RNA was reverse-transcribed in 20 i~1 cDNA reaction mixture containing 50 pmol of the SRI antisense primer, 0.5 m M / l of dNTP, 10 m M / l Hepes, 0.2 m M / l EDTA, 200 units of M-MLV reverse transcriptase (Gibco BRL) and 20 units o f RNase inhibitor (Boehringer). cDNA synthesis was performed at 37°C for 1 h. The reaction was stopped for 10 min at 72°C (Sellner et al., 1992).
Amplification Primers Two pairs of primers, located in the highly conserved 5' non-coding region of the HCV genome, were used (Okamoto et al., 1990a, b): SRI = outer antisense primer (20nt) : 5'-TGCACGGTCTACGAGACCTC-3',
The PCR reaction mixture contained 0.2 m M / l d N T P , 1.5 mM/1 Mg CI 2, 50 mM/I KCI, 10 m M / l Tris-HCl pH 8.3 and 1.2 units of "Ampli Taq DNA Polymerase" (Perkin Elmer Cetus, Norwalk, CT), and 50 pmol o f primers. For single PCR, 5 I.d o f cDNA were added to 50 g.l o f PCR reaction mixture containing SR2 and SF2 primers. Thirty-five cycles
236
F. B O U C H A R D E A U E T A L .
were performed: 1 min at 92°C for denaturation, 1 min at 55°C for annealing and 1 min at 72°C for extension in a programmable DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). For "nested" PCR, the first amplification step was clone with 5 ~tl ofcDNA added to 50 ~l of PCRreaction mixture with SR1 and SFI primers; 25 cycles were performed in the same conditions as those used for single PCR. After the first amplification step, 50 ~.l of PCR reaction mixture containing the corresponding inner primer pair (SR2 and SF2) were added through the oil layer in the same tube. For the second PCR reaction, the same conditions described for single PCR were used. Each serum was analysed by single PCR. For each patient with negative or transient positive results, the four samples were reanalysed by "nested" PCR. The PCR products were analysed by electrophoresis on 1 070agarose gel and visualized by ultraviolet fluorescence after staining with ethidium bromide. To reduce the risk of contamination during PCR, a number of precautions were taken as recommended by Kwok and Higuchi (1989). All reagents were prepared and all PCR steps were carried out in one room. The PCR products were analysed in a separate room. Specific pipettes were used for sample and reagent preparations. To control the risk of contamination, several negative controls including water were used at each step of the PCR. To validate the procedure, 20 samples from negative blood donors and 30 samples from individuals with chronic hepatitis C were analysed. In addition, we participated in different HCV RNA standardization studies, including the "Eurohep" HCV RNA test panel containing 22 samples from human and chimpanzee (Zaaijer et al., 1993) and we obtained correct results. Reference materials for quantitation will be provided in the next phase of HVC RNA Eurohep standardization.
RESULTS All 20 negative controls were negative by " n e s t e d " PCR and the 30 positive controls were found positive by single PCR. Seven patients from group l, negative for anti-HCV and with normal ALT, were negative in single PCR and " n e s t e d " PCR in four consecutive samples. In this group, two patients seroconverted during the study, one upon entry and the second a year later. The first patient received packed red cells in June and August 1990 and the onset o f hepati-
tis occurred in October 1990. The last known anti-HCV-negative sample was collected in June 1990 and the first known anti-HCV positive sample (C22-3 and C33-c bands by RIBA-2) in January 1991. HCV RNA was positive by single PCR and " n e s t e d " PCR in the first available sample (January 1991) corresponding to the first A L T peak (300 U/l) and in 3 successive samples collected from February to October 1991. For ten months, HCV RNA remained positive until the patient received interferon treatment. In the first sample following 'the beginning of therapy (3 months later), HCV RNA was negative in single PCR and in " n e s t e d " PCR and remained negative 6 months later. At that time, the A L T level was normal and the RIBA-2 profile was unchanged (4 bands). These results are illustrated in figure I. The second patient seroconverted after 12 months of follow-up (anti-HCV-negative on J a n u a r y 1992 and -positive on February 1992). He was never transfused. PCR was negative on the two 1991 specimens and positive during the rise in the A L T level (January 1992) in two successive samples. In the first PCR-positive sample (January 1992), anti-HCV were negative by EL1SA and RIBA-2. In the second positive sample (February 1992), one m o n t h later, RIBA-2 exhibited 3 bands on C5-1-1, C l 0 0 and C33-c. In March 1992, when the A L T level was still elevated, HCV RNA was negative by single PCR and " n e s t e d " PCR, and in June 1992, weak positivity was detected by single PCR and clearly confirmed by " n e s t e d " l~CR. No C22-3 band was detectable by RIBA-2 during the entire follow-up. No other specimens remained available. Results are presented i n figure 2. In group 2, 13 patients exhibited either 3 (5 patients) or 4 (8 patients) bands by RIBA-2, with no change during follow-up. All 13 patients had antibodies to C33-c; only one patient had no antibodies to C22-3. Eleven o f the patients were positive by single PCR in the four samples and the two others gave transient positive results in the four samples. For one of them, the first and the last sample were weakly positive by single PCR and clearly positive by " n e s t e d " PCR,
D E T E C T I O N OF H E P A T I T I S C VIRUS B Y PCR I N H A E M O D I A L Y S E D P A T I E N T S
35O
+
.'.~:
+
+++
++
C100-3
++÷.(. ++++
+,0-4.+ .... ....
C33-c
+++
+÷
++
+++
II
::::
237
5.1.1
{:22-3
3OO
250
200
150
100
50 Interferon/~'
1990
1991
1992
Fig. I. ALT level, RIBA-2 profile and HCV-RNA results in the follow-upof a patient who seroconverted to HCV upon entry into the study and who was treated early by interferon. RIBA-2 profile: band intensityexpressed from + to + + + + on the four HCV antigens,C5-1-1, CI00-3, C33-c and C22-3.
the second sample was negative by single PCR but positive by "nested" PCR and the third sample remained negative by "nested" PCR. For the other patient, the first three samples were positive by single and "nested" PCR and the last one was negative by single PCR and positive by " n e s t e d " PCR. From the beginning to the end of follow-up, these two patients had ALT levels which were not significantly different from those observed in other patients from group 2 and exhibited four bands by RIBA-2. In group 3, the 14 patients had antibodies to C22-3 and C33-c along with antibodies to C5-1-1 in 3 patients, antibodies to C100-3 in 1 patient and to antibodies to both in 8 patients. HCV RNA was positive in 12 patients by single PCR in the four samples. In one patient, only three samples were found to be positive by single PCR and " n e s t e d " PCR, with the second sample
negative by both single and nested PCR. This patient seroconverted in January 1990 and never acquired antibodies to C100-3. For the final patient, who had the lowest ALT level and was known to be anti-HCV-positive with 3 bands by RIBA-2 during the entire follow-up (negative for C100-3), HCV RNA was negative by " n e s t e d " PCR in the four samples. In group 4, the 3 patients who had only a C22-3 band by RIBA-2 were negative by single PCR and " n e s t e d " PCR during the 18-month follow-up. They had normal ALT level. The 2 patients with a C33-c band alone were strongly positive by single PCR on all samples. They had an elevated ALT level but were also chronic HBV carriers. These results are summarized in table II. Positive results by " n e s t e d " PCR in samples negative by single PCR were observed in 2 of the 64 tested samples (table III).
238
F. B O U C H A R D E A U E T A L .
450
+
÷÷
5-1-1
4-+
+-('+
CI00-3
+++
+++
C33-c
"
C22-3
400
350
3OO
250
20O
150
|
++4-
~
HCV-RNA
100
50
0
1991
1992
Fig. 2. ALT level, RIBA-2 profile and HCV-RNA results in the follow-up of a patient who seroconverted to HCV after one year of follow-up. RIBA-2 profile: band intensity expressed from + to + + + + on the four HCV antigens, C5-1-1, C100-3, C33-c and C22-3.
Table I1. RNA-HCV detection by PCR on the 4 successive samples from 39 patients.
Group 4 PCR
Group 1
Group 2
Negative on the 4 samples
7 (100 %)
0
Positive on the 4 samples
0
12 (92.3 °7,0)
Transient results (3 positive, 1 negative)
0
1
Group 3 1 12 (85.7 %) 1
C22-3
C33-c
3
0
0
2
0
0
In group 1, two patients who seroconverted at the beginning or during follow-up were excluded from this table.
DISCUSSION
In this s t u d y , the results o b t a i n e d by a n t i , HCV antibody determination and HCV RNA
detection by P C R could be c o m p a r e d . In addition, this analysis was p e r f o r m e d o n 4 consecutive samples d u r i n g an 18-month f o l l o w - u p to a p p r e c i a t e the variations in b o t h a n t i - H C V an-
D E T E C T I O N OF H E P A T I T I S C VIRUS B Y PCR I N H A E M O D I A L Y S E D
239
PATIENTS
Table !II. Comparative results between single and "nested" PCR in the four groups of patients. Negative No. patients No. samples
Single PCR results Tested by "nested" PCR Positive by "nested" PCR
Group 1 Seroconverted
7 28
Group 2
Group 3
Group 4
13 52
14 56
5 20
2 12
Pos
Neg
Pos
Neg
Pos
Neg
Pos
Neg
Pos
Neg
0
28
7
5
49
3
51
5
8
12
0
28
7
5
5
3
3
5
0
12
0
0
7
0
5
2
3
0
0
0
tibodies and viraemia assessed by PCR. Adequate care was taken to avoid erroneous diagnosis (Kwok and Higuchi, 1989) and to obtain specific and reproducible results as the choice of the method for RNA extraction (Chomczynski and Sacchi, 1987) and of the primers in the 5' non-coding region (Okamoto et al., 1990a, b; Garson et al., 1990) since this region is highly conserved among all HCV isolates studied to date (Han et al., 1991 ; Garson et al., 1990). In this population and in our technical conditions, " n e s t e d " PCR offered a minor advantage compared to single PCR : of the 117 samples positive by PCR, l l5 were positive by single PCR and 2 by " n e s t e d " PCR only. A total of 51 samples were negative by both single and " n e s t e d " PCR. The 28 samples from the 7 patients who remained anti-HCV-negative during the entire follow-up were HCV-RNA-negative, including by " n e s t e d " PCR. Negative PCR results in an anti-HCV-negative population have been generally reported (Evans et aL, 1992), except when anti-HCV was detected by firstgeneration assays (Kato et aL, 1990) which have been shown to be less sensitive than secondgeneration assays (Chauveau et al., 1992; Van der Po~l et al., 1991). HCV RNA was detected in the majority of anti-HCV-positive sera from patients with (group 2) or without (group 3) biological hepa-
titis. As previously said, no correlation with histology could be made. This study revealed that the presence of antiHCV antibody was associated with transient or persistent viraemia in 26 of the 27 patients (96 °7o)known to be anti-HCV-positive upon entry into the study. This viraemia was regularly observed on the 4 samples during the entire follow-up for 24 patients (89 o70).For 2 patients, one of the 4 samples collected durh)g the followup gave negative "nested" PCR results. This observation indicated the necessity to perform successive HCV RNA detection before concluding as to the absence of viraemia as already observed in humans (Shimizu et al., 1990) and in chimpanzees (Farci et al., 1991). It is probable that these two patients had too weak a level of circulating virus to be detectable, since successive samples were HCV-RNA-positive. In this study, HCV RNA was highly concordant with anti-HCV, as observed in a Japanese study conducted in haemodialysed patients (Tokunaga et al., 1991) where 86 o70 of the 21 patients who were anti-HCV-positive by RIBA-2 were HCV-RNA-positive. This good correlation between PCR and serology was also reported in 94 °70 of blood donors (Follet et al., 1991) and in patients with post-transfusion hepatitis C (Wang et al., 1992; Van der Po~l et al., 1991). Only one anti-HCV positive patient remained P C R negative on the 4 samples and ex-
240
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hibited 3 bands by RIBA-2. These negative PCR results associated with the absence of anti-C 100-3 shown to be a marker of infectivity (Weiner et al., 1990) might suggest a resolved infection in this patient. However, four other patients with no anti-C100-3 were HCV-RNA-positive. Consequently, it seems that the profile of anti-HCV antibodies cannot be used to estimate the evolution of the infection. No correlation between HCV RNA and ALT results could be made in these chronic infected patients, since similar HCV RNA results were observed in patients from group 2 with elevated A L T and patients from group 3 with normal ALT. This observation was also reported in blood donors (Van der Po~l et al., 1991). However, the patient with PCR-negative results had the lowest ALT level. Several hypotheses can be put forth to explain these negative results: absence of viraemia, too weak a level of circulating virus or a different HCV strain not recognized by the used primers (Irwing et al., 1992; Xu et al., 1992). Different primers should be used in this PCR-negative patient before making a definite conclusion as to potential recovery from HCV infection. In the two patients with acute hepatitis C, PCR results were clearly correlated with A L T levels. For one patient, no sample was available immediately before or after the first rise in A L T to determine the first appearance of HCV viraemia and the time of antibody seroconversion. Both antibody and RNA were detected on the first available specimen following the rise in ALT. For the second patient who seroconverted, HCV RNA was detected before anti-HCV, as previously observed (Zonaro et aL, 1991 ; Farci et aL, 1992). Viraemia was present in both patients during the acute phase, showing that HCV RNA detection represents an available means of identifying primary HCV infection. This detection is also useful for monitoring antiviral therapy. In the patient who received interferon treatement very soon after the infection, HCV RNA was no more detectable after this therapy. A longer follow-up will be necessary to assess the therapy efficacy. Kanai et aL (1990) had observed a decrease HCV RNA in serum after interferon treatement in 5 patients but no
HVC-negative results. For Hagiwara et al. (1992a), (1992b), HCV RNA became undetectable in 100 °7o of patients treated by interferon when ALT level fell into a normal range during therapy. For the other patient, who presented a transient HCV RNA after the acute hepatitis, the follow-up was too short for a long-term prognosis. In the 5 patients with indeterminate results by RIBA-2, the 3 with a C22-3 band alone were HCV-RNA-negative and the 2 with a C33-c band alone were HCV-RNA-positive. However, the number of patients was too low to extend there PCR results to all patients with this profile. Other studies have demonstrated that patients reactive only for C22-3 antigen could be infectious: 6/13 (46 °/0) for Halfon et al. (1992) and 8/26 ( 3 1 % ) for Follett et al. (1991). Our results on patients reactive to C33-c alone and PCR-positive correlate with other reports : one case described by Follett et al. (1992) and two cases described by Halfon et al. (1992). This RIBA-2 profile was not systematically associated with viraemia (Follett et al., 1991 ; Allain et al., 1992). However, it is clear that an indeterminate profile by RIBA-2 can be found in infectious subjects. Consequently, it is important that screening tests for anti-HCV contain antigen from the NS3 region (Follett et al., 1991). This study revealed that the presence of antiHCV antibody in haemodialysed patients was associated with viraemia in almost all cases. These results obtained on four consecutive samples suggest that PCR is a reliable and reproducible technique for HCV RNA determination, particularly when primers from the highly conserved 5' noncoding region are used. Such PCR assays provide information concerning the viraemic status when anti-HCV is present but liver function is normal, or when anti-HCV has an indeterminate profile. Such PCR assays demonstrate the necessity of follow-up before confirming that an HCV-RNA-negative, anti-HCV-positive patient had potentially eradicated the virus. The detection of HCV RNA represents the only available test to identify primary HCV infection early in its course. Moreover, PCR provides a valuable tool for monitoring the efficacy of antiviral therapy.
D E T E C T I O N O F H E P A T I T I S C VIRUS B Y P C R I N H A E M O D I A L Y S E D Acknowledgements We thank Ortho Diagnostic System (France) for their support, and the staff of the Edouard Rist Medical Center for collecting serum samples from patients.
D6tection du virus de I'h~patite C par la PCR chez des sujels h~modialys~s et stalut en anticorps anti-HCV L ' A R N du virus de I'h6patite C (ARN VHC) a 6t6 recherch6 par <
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