- Email: [email protected]
Hepatitis C virus serotypes and viremia in patients with chronic infection
S62
Posters 2
I P2Cl/149]
P2Cl/147 ] DETECTION OF HEPATITIS C VIRAL RNA : COMPARISON OF AMPLICOR ONE STEP PCR AND NESTED SET PCR WITH HCV SEROLOGY ON I-IF._2vlODIALYZEDPATIENTS. **lk
*
M. Bolzard2-. C. S a v a d a - - . M.C. Demachv-,.M. Louvel~*, Kamalodme-:-2,,..C_=C.' ** Barbanel ** - *Molecular biology unit, ** Hemodialysis unit, Centre Hospitalier, Meaux, France - *** Roche Molecular Systems, Neuilly, France. Several recent studies have demonsta-ated utility of RNA PCR (RT-PCR) in the diagnosis and txeatment of hepatitis C infections. The two-step nested set PCR technique is widely used to enhance the sensitivity of detection of HCV genomie sequences. The limitation of this technique is that second round PCR reactions are vulnerable to potential contaminations by first round amplification products. The Amplicor@ HCV RNA PCR assay avoids two-step PCR by using the rTth polymerase and AmpErase enzymes. We have compared the one step with nested set PCR : all patients in a dialysis unit at Meaux hospital were tested for the presence of HCV RNA. Sera obtained from 77 hemodialyzed patients prepared within 2 hours of collection and stored at 80"C were analyzed. Anti-HCV status was determined by ELISA 2 (Abbott) and all samples reactive by ELISA were tested by RIBA 2 or RIBA 3 assay. The results are expressed below : ELISA PCR Amplicor + +
PCR +[11/12 nested/Amplicor -
0 65
I
nested + l i t PCR - l
0 I 65
Anti-HCV antibodies were associated with HCV RNA in all cases. One patient who had anti-HCV antibodies detectable in second generation tests was repeatedly HCV RNA negative with the nested polymerase chain reaction and positive with one step PCR. The sera of three patients were RIBA-3 indeterminate (two had isolate NS3 band and one an isolate Core band) but HCV RNA positive. Conclusion : these results suggest that the one step RT PCR is at least as much sensible as nested RT PCR. Carryover contamination risks are minimized by the AmpErase® system included in the Amplieor assay and by avoiding two-step PCR. The Amplicor® HCV RNA PCR assay is therefore a useful tool for the HCV infection diagnosis of hemodialyzed patients ; in our hands, although the presence of anti-HCV is closely associated with viremia, even 3rd generation RIBA gave indeterminate status for some hemodialyzed patients.
P2 Cl/148 ] LONG TERM ADMINISTRATION OF INTERFERON-o~ LIMITS PROGRESSION OF LIVER FIBROSIS IN NON-RESPONDER PATIENTS WITH CHRONIC HEPATITIS C
P. Chossegros", M. Chevallier" S. Guerret', C. Tr6po", J.A. Grimaud" "CNRS URA 1459 Institut Pasteur de Lyon, France "*Unit~ d'H~patologie, HOtel Dieu, Lyon, France A 6 months course of IFN-e treatment has been shown to reduce collagen deposit in hepatic lobule (Hepatology 1993,18:1344-1349). The long term evolution (59 +/- 3,8 mths) of fibrosis was investigated in liver biopsies from 25 non responders patients with CAH C treated with successive courses of IFN-ot (total: 533 +/- 72 MU). Liver fibrosis was assessed from repeated liver biopsies using a scoring system (SS: Hepatology 1994, In press) and the activity graded according to Knodell score (KS). Respectively 14 and II patients have been treated for less (L:7,5 mths, 313 MU) and more (M:21,8 mths, 791 MU) than 12 months. No difference was observed between the 2 groups on the initial biopsy for necrosis inflammation and total KS and SS as well as for transaminase response rate and duration of follow up (61/57 mths). At the end of follow up all the patients had elevated transaminases and HCV viremia. The mean inflammation and necrosis of KS was increased compared to pretreatment levels. The difference between initial and terminal SS of L (+0,2) and M (-6,1) was significant (p < 0,05). 2 progressions to cirrhosis were observed in the L group. Conclusion : Despite persistent viremia and elevation of transaminases, aggravation of inflammation and necrosis of the KS, long term treatment with IFN-ot2b is associated with a regression of liver fibrosis. These promising results have to be confirmed on a larger series of patients.
HEPATITIS C VIRUS SEROTYPES PATIENTS WITH CHRONIC INFECTION
AND
VIREMIA
IN
R Romeo, MG Rumi, R Soffredini~ MF Donato, E Del Ninno, ML Parravicini, S Pacchetti, M Colombo. Inst. Intern. Med., University of Milan, Italy. Recent data from Japan and USA suggest that severity of hepatitis C could be predicted by such virus factors as serum types and HCV-RNA levels. Aim of our study was to assess whether this was also true in our geographical area. ~atlents: 197 consecutive patients (129 M, x age 50 yr, 21-77) anti-HCV positive (EIA2) with biopsy ~roven chronic liver disease: 36 c h r o n i c p e r s l s t e n t hepatitis (CPH), 29 mild chronic active hepatitis (CAH), 73 CAH, 32 active cirrhosis and 27 HCC. Methods: HCV-RNA was detected by nested PCR (5'UTR primers) and quantitated by branched DNA assay (bDNA, Chiton Co). HCV serotypes were detected using an ELISA assay based on branched pestides corresponding to t h e h y p e r v a r i a b l e antigenic regions of t h e N S - 4 p r o t e i n of H C V {Dr. P. Simmonds). Results: 134/197 patients (68%) tested b-DNA ~ositive. HCV-RNA levels were not statistlcally different among liver disease groups. RNA ~evels expressed as genome equivalent/mlxl0 were: 724 in CPH, 686 in mild CAH, 1022 in CAH, 890 in cirrhosis and 916 in HCC. The 63 patients (32%) who tested b-DNA negative were PCR positive. 107 patients (54%) had serotype 1, 22 (11%) type 2, 9 (5%) type 3, 17 (9%) mixed types (1+2, 1+3, 1+2+3) and 42 (21%) had no specified serotype. Serotype 2 a l o n e or p l u s serotype 1 was associated with significantly lower median l e v e l s of v i r e m i a as c o m p a r e d to t h e remaining serotypes (type 2 v s 1 P= .0002; type 1+2 v s 1 P=.0008; type 2 v_ss non specified P=.0153; t y p e 1+2 vs 1+3 P=. 0602; type 1+2 v_ss n o n specified P=--.0129). All serotypes were equally distributed in t h e various disease groups. Conclusions: patients with chronic hepatitis C show a wide range of HCV viremia and are p r e d o m i n a n t l y infected by serotype 1. There was no c l e a r cut c o r r e l a t i o n between disease stage, viremia and serotype.
I P2C1/150 I COMPARISON OF DIFFERENT TYPES AND DOSES OF ALPHA IFN FOR TREATMENT OF CHRONIC HEPATITIS C. C.Casarin, L.Chemello, P.Bonctti, LBenvegnfi, LDe Molincr, P.Pontisso, F.Tremolada, A.Albcrti and TVVH group. Institute of Clinical Medicine, University of Padua (Italy). We have conducted a randomized trial to assess efficacy, as to ALT normalization and HCV-RNA clearance, of two different types and dosagesof IFN. 59 patients were treated with 10 MU tiw for 4 weeks followed by 6 MU tiw for 20 weeks using either alpha2b (29 cases group A l) or l.vmphoblastoid (30 casesgroup A2) IFN. 64 patients received 3 MU daily and then tiw of alpha2b (35 cases group B l) or lymph. (29 casesgroup B2)IFN. Rates of biochemical
and virological response at different check-points are shown in the table:
Duriug IFN normal ALT End of IFN normal ALT HCV-RNA neg HCV-RNA pos 6 months offlFN normal ALT HCV-RNA ncg HCV-RNA pos
A1
A2
BI
B2
77% 55% 100% 0 30% gl~% 12%
75% 68% 100% 0 32% 88% 12%
70% 50% 78% 22% 32% 67% 33%
60% 54% 80% 20% 14% 75% 25%
Breakthroughs dnring IFN were more frequent with alpha2b IFN (29%) than with lymph. IFN 10%, while relapsesafter IFN did the opposit, Icadiqg to similar rates of sustained rcspo,sc. The efficacy of the two types of lFN was similar, although the kinetic of reactivation after primary responsewas different. The high dose regimen induced more efficient eradication of HCV-RNA in patients with sustained bichcmical response.
Posters 2
I P2Cl/149]
P2Cl/147 ] DETECTION OF HEPATITIS C VIRAL RNA : COMPARISON OF AMPLICOR ONE STEP PCR AND NESTED SET PCR WITH HCV SEROLOGY ON I-IF._2vlODIALYZEDPATIENTS. **lk
*
M. Bolzard2-. C. S a v a d a - - . M.C. Demachv-,.M. Louvel~*, Kamalodme-:-2,,..C_=C.' ** Barbanel ** - *Molecular biology unit, ** Hemodialysis unit, Centre Hospitalier, Meaux, France - *** Roche Molecular Systems, Neuilly, France. Several recent studies have demonsta-ated utility of RNA PCR (RT-PCR) in the diagnosis and txeatment of hepatitis C infections. The two-step nested set PCR technique is widely used to enhance the sensitivity of detection of HCV genomie sequences. The limitation of this technique is that second round PCR reactions are vulnerable to potential contaminations by first round amplification products. The Amplicor@ HCV RNA PCR assay avoids two-step PCR by using the rTth polymerase and AmpErase enzymes. We have compared the one step with nested set PCR : all patients in a dialysis unit at Meaux hospital were tested for the presence of HCV RNA. Sera obtained from 77 hemodialyzed patients prepared within 2 hours of collection and stored at 80"C were analyzed. Anti-HCV status was determined by ELISA 2 (Abbott) and all samples reactive by ELISA were tested by RIBA 2 or RIBA 3 assay. The results are expressed below : ELISA PCR Amplicor + +
PCR +[11/12 nested/Amplicor -
0 65
I
nested + l i t PCR - l
0 I 65
Anti-HCV antibodies were associated with HCV RNA in all cases. One patient who had anti-HCV antibodies detectable in second generation tests was repeatedly HCV RNA negative with the nested polymerase chain reaction and positive with one step PCR. The sera of three patients were RIBA-3 indeterminate (two had isolate NS3 band and one an isolate Core band) but HCV RNA positive. Conclusion : these results suggest that the one step RT PCR is at least as much sensible as nested RT PCR. Carryover contamination risks are minimized by the AmpErase® system included in the Amplieor assay and by avoiding two-step PCR. The Amplicor® HCV RNA PCR assay is therefore a useful tool for the HCV infection diagnosis of hemodialyzed patients ; in our hands, although the presence of anti-HCV is closely associated with viremia, even 3rd generation RIBA gave indeterminate status for some hemodialyzed patients.
P2 Cl/148 ] LONG TERM ADMINISTRATION OF INTERFERON-o~ LIMITS PROGRESSION OF LIVER FIBROSIS IN NON-RESPONDER PATIENTS WITH CHRONIC HEPATITIS C
P. Chossegros", M. Chevallier" S. Guerret', C. Tr6po", J.A. Grimaud" "CNRS URA 1459 Institut Pasteur de Lyon, France "*Unit~ d'H~patologie, HOtel Dieu, Lyon, France A 6 months course of IFN-e treatment has been shown to reduce collagen deposit in hepatic lobule (Hepatology 1993,18:1344-1349). The long term evolution (59 +/- 3,8 mths) of fibrosis was investigated in liver biopsies from 25 non responders patients with CAH C treated with successive courses of IFN-ot (total: 533 +/- 72 MU). Liver fibrosis was assessed from repeated liver biopsies using a scoring system (SS: Hepatology 1994, In press) and the activity graded according to Knodell score (KS). Respectively 14 and II patients have been treated for less (L:7,5 mths, 313 MU) and more (M:21,8 mths, 791 MU) than 12 months. No difference was observed between the 2 groups on the initial biopsy for necrosis inflammation and total KS and SS as well as for transaminase response rate and duration of follow up (61/57 mths). At the end of follow up all the patients had elevated transaminases and HCV viremia. The mean inflammation and necrosis of KS was increased compared to pretreatment levels. The difference between initial and terminal SS of L (+0,2) and M (-6,1) was significant (p < 0,05). 2 progressions to cirrhosis were observed in the L group. Conclusion : Despite persistent viremia and elevation of transaminases, aggravation of inflammation and necrosis of the KS, long term treatment with IFN-ot2b is associated with a regression of liver fibrosis. These promising results have to be confirmed on a larger series of patients.
HEPATITIS C VIRUS SEROTYPES PATIENTS WITH CHRONIC INFECTION
AND
VIREMIA
IN
R Romeo, MG Rumi, R Soffredini~ MF Donato, E Del Ninno, ML Parravicini, S Pacchetti, M Colombo. Inst. Intern. Med., University of Milan, Italy. Recent data from Japan and USA suggest that severity of hepatitis C could be predicted by such virus factors as serum types and HCV-RNA levels. Aim of our study was to assess whether this was also true in our geographical area. ~atlents: 197 consecutive patients (129 M, x age 50 yr, 21-77) anti-HCV positive (EIA2) with biopsy ~roven chronic liver disease: 36 c h r o n i c p e r s l s t e n t hepatitis (CPH), 29 mild chronic active hepatitis (CAH), 73 CAH, 32 active cirrhosis and 27 HCC. Methods: HCV-RNA was detected by nested PCR (5'UTR primers) and quantitated by branched DNA assay (bDNA, Chiton Co). HCV serotypes were detected using an ELISA assay based on branched pestides corresponding to t h e h y p e r v a r i a b l e antigenic regions of t h e N S - 4 p r o t e i n of H C V {Dr. P. Simmonds). Results: 134/197 patients (68%) tested b-DNA ~ositive. HCV-RNA levels were not statistlcally different among liver disease groups. RNA ~evels expressed as genome equivalent/mlxl0 were: 724 in CPH, 686 in mild CAH, 1022 in CAH, 890 in cirrhosis and 916 in HCC. The 63 patients (32%) who tested b-DNA negative were PCR positive. 107 patients (54%) had serotype 1, 22 (11%) type 2, 9 (5%) type 3, 17 (9%) mixed types (1+2, 1+3, 1+2+3) and 42 (21%) had no specified serotype. Serotype 2 a l o n e or p l u s serotype 1 was associated with significantly lower median l e v e l s of v i r e m i a as c o m p a r e d to t h e remaining serotypes (type 2 v s 1 P= .0002; type 1+2 v s 1 P=.0008; type 2 v_ss non specified P=.0153; t y p e 1+2 vs 1+3 P=. 0602; type 1+2 v_ss n o n specified P=--.0129). All serotypes were equally distributed in t h e various disease groups. Conclusions: patients with chronic hepatitis C show a wide range of HCV viremia and are p r e d o m i n a n t l y infected by serotype 1. There was no c l e a r cut c o r r e l a t i o n between disease stage, viremia and serotype.
I P2C1/150 I COMPARISON OF DIFFERENT TYPES AND DOSES OF ALPHA IFN FOR TREATMENT OF CHRONIC HEPATITIS C. C.Casarin, L.Chemello, P.Bonctti, LBenvegnfi, LDe Molincr, P.Pontisso, F.Tremolada, A.Albcrti and TVVH group. Institute of Clinical Medicine, University of Padua (Italy). We have conducted a randomized trial to assess efficacy, as to ALT normalization and HCV-RNA clearance, of two different types and dosagesof IFN. 59 patients were treated with 10 MU tiw for 4 weeks followed by 6 MU tiw for 20 weeks using either alpha2b (29 cases group A l) or l.vmphoblastoid (30 casesgroup A2) IFN. 64 patients received 3 MU daily and then tiw of alpha2b (35 cases group B l) or lymph. (29 casesgroup B2)IFN. Rates of biochemical
and virological response at different check-points are shown in the table:
Duriug IFN normal ALT End of IFN normal ALT HCV-RNA neg HCV-RNA pos 6 months offlFN normal ALT HCV-RNA ncg HCV-RNA pos
A1
A2
BI
B2
77% 55% 100% 0 30% gl~% 12%
75% 68% 100% 0 32% 88% 12%
70% 50% 78% 22% 32% 67% 33%
60% 54% 80% 20% 14% 75% 25%
Breakthroughs dnring IFN were more frequent with alpha2b IFN (29%) than with lymph. IFN 10%, while relapsesafter IFN did the opposit, Icadiqg to similar rates of sustained rcspo,sc. The efficacy of the two types of lFN was similar, although the kinetic of reactivation after primary responsewas different. The high dose regimen induced more efficient eradication of HCV-RNA in patients with sustained bichcmical response.