- Email: [email protected]
HBV genotyping analysis from the surface antigen region using the trugene™ HBV genotyping kit and a PCR-based homebrew method
Category 5: Viral hepatitis: basic aspects ~]
HEPATIC IRON DISTRIBUTION IN 153 PATIENTS WITH CHRONIC VIRAL HEPATITIS (CH): RELATION WITH GRADING, STAGING ANO HFE MUTATIONS
Chiara Corengia ] , Giorgio Bovo 2, Cristina Arosio 3, Angelica Chie sara 4, Alessandra Salvionil, Viviana Mauril, Massimo Pozzil, Cristina Cestari 4, Anna Vergani l , Alberto Piperno I . t Clinica Medica
Universitgz Milano Bicocca, 2Dipartimento di Patologia, Ospedale S Gerardo Monza; 31stituto Auxologico Italiano, Ospedale S Luca Milano ; 4Divisione di Medicina 2, Ospedale S Gerardo Monza, Italy
Background: Hepatic iron overload is common in patients with CH. The relationship between iron, grading and staging and the role of HFE mutations in the developement of iron overload and fibrosis are controversial. Aim: To evaluate the relationship between a) hepatoceUular iron distribution, grading and staging; b) HFE genotypes, hepatocellular iron distribution and fibrosis. Methods: 253 CH patients that underwent liver biopsy from 1995 to 1999 were studied. Hepatic iron was assessed by Deugnier's score modified and grading and staging by Ishak's score. HFE mutations were assesed in 147 patients and in 139 healthy controls. Results: Hepatic iron overload was present in 124 patients (49%) (group A) and absent in 129 (51%) (group B). Two groups did not differ for age, sex and body mass index (BMI). Hepatic iron store did not correlate with BMI. Fibrosis and steatosis grade were significantly higher in group A than B (p < 0.0001 and p = 0.05). Cirrhosis was more frequent in group A than B (27% vs 13%, p = 0.007). In group A the staging score was significantly correlated with portal iron (p < 0.001), particulary with connective and endothelial deposits (p < 0.001 and p < 0.01). C282Y and H63D were more frequent in group A than in B (p = 0.033) and in controls (p = 0.0002). A significant correlation between HFE genotypes and hepatocytic iron, that increases with the increasing HFE genotype severity, was found (p = 0.0014). Conclusion: The correlation between fibrosis and portal iron support the fibrogenetic role of macrophagic iron. HFE mutations contribute to the pathogenesis of hepatocytic iron accumulation in CH.
~-]
LAMIVUDINE-RESISTANT HBV IS CROSS-RESISTANT TO L-dT AND L-dC IN VITRO
William Delaney, Huiling Yang, Michael Miller, Craig Gibbs, Shelly Xiong. Gilead Sciences, Foster Oty, CA, USA Objective: Clinical resistance of HBV to lamivudine is hallmarked by the development of rtM204I or rtL180M + rtM204V mutations in the viral polymerase. These mutations confer in vitro resistance to lamivudine (beta-L-2'-3'-dideoxy-3'-thiacytidine) and structurally-related L-nucleoside analogs such as b'TC and L-FMAU. Novel L-nucleosides with anti-HBV activity have recently been reported; these include betaL-thymidine (L-dT) and beta-L-deoxycytosine (L-dC), which are each in phase I/II clinical trials. The aim of this study, was to determine whether lamivudine-resistant HBV was sensitive to L-dC and L-dT in vitro. Methods: HepG2 cells were transfected with wild-type, rtL180M, rtM204I, or ~L180M + rtM204V mutant HBV and were treated with L-dC or L-dT. Following drug treatment, intraceUular replicative intermediates were quantified by Southern blotting and the IC50 of each drug was calculated for each strain of HBV. Results: L-dC and L-dT inhibited the replication of wild-type HBV with IC50 values of 0.26 and 0.28 microMolar, respectively. The ~L180M mutation conferred approximately 10-fold resistance to both compounds, similar to the level of resistance conferred to 3TC. The rtM204I and rtL180M + rtM204V mutations conferred high levels resistance to both L-dC and L-dT (IC50 values increased > 300-fold). Conclusions: These in vitro studies indicate that HBV mutants selected during lamivudine therapy are cross-resistant to L-dC and L-dT and provide further evidence that YMDD mutations confer broad cross-resistance to Lnucleosides.
~0-]
89
ULTRASTRUCTURAL HEPATOCYTE MODIFICATIONS IN HCV INFECTED HUMAN LIVER
Laura Falasca l, Fabiola Ciccosantit, Roberta Nardacci t, Oreste Lo Iacono 2, Manro Piacentini L3. t Lab. Electron Microscopy, INMI-1RCCS
'L. SpaUanzani' Rome; 2University of Study of Palermo, Palermo; SUniversity of Rome 'Tor Vergata', Rome, Italy Despite many efforts, the search for the morphological features of HCV inside the hepatocytes has been hampered by the low level of viral particles present in the liver tissue of infected individuals. Little information is avalaible on intracellular localization of the virus and on prerequisites for its assembly, as a consequence poorly understood is the pathogenic mechanism of liver disease. In this work we analysed, by transmission electron microscopy, 30 hepatic biopsies from HCV infected patients. Liver sampies were obtained from individuals infected by hepatitis C virus with different genotypes, and with a different degree of liver damage in order to find possible common ultrastructural modification useful to understand the progression of the disease. A variety of alterations were frequentely seen in hepatocytes, mostly concerning alteration of mitochondria, dilatation of endoplasmic reticulum, and presence of lipid inclusions in the cytoplasm. In addition, in a limited number of hepatocytes we have found peculiar changes of nuclear morphology (indentation and chromatin condensation). The most relevant result is that we found, in association with nucleus modification, the presence of vires-like particles. The morphology and the size of the particles were consistent with the predicted HCV virions based on other studies.
-] DIFFERENT MODULATION OF PKR GENE EXPRESSION BY HCV GENOTYPES Martina Gerotto, Francesca Dal Pero, Silvia Boccato, Gladis Bortoletto, Alessia Ferrafi, Stefano Realdon, Afredo Alberti. University of Padova,
Padova, Italy In hepatitis C, the cellular protein kinase PKR may play a role in the pathogenesis and response to interferon. Some HCV proteins, including NS5A, were shown in vitro to bind PKR and to upregulate or downregulate its expression and activation. Little is know on the in vivo expression of PRK in patients with hepatitis C. Our aim was to measure PKR expression in HCV positive patients. PKR-mRNA was measured by competitive RTPCR in PBMCs from 24 patients (18 with HCV-1 and 6 with HCV-2) and from control healthy subjects. PBMCs were studied before and after incubation with IFN. Mean baseline PKR mRNA levels were significantly lower in HCV1 patients (0.40 + 0.24 fg) compared to HCV2 cases (2.45 + 2.9 fg p -- 0.006) and controls (1.37 + 1.0 p = 0.005). No correlation was found between mutations in the NS5A-PKR binding domain and PKRmRNA levels, that were also independent of viraemia, ALT and histologic activity. After in vitro incubation with IFN, PKR mRNA was enhanced to similar levels in HCV1 (3.3 + 2.3) and HCV2 (3.2 + 2.4) patients but remained significantly lower compared to control (8.0 + 5.6 p = 0.003). These results indicate that PKR gene expression is downregnlated in HCV infection, particularly in patients with HCV 1, and this is independent of the NS5A PKR-binding domain sequence. Preliminary evidence suggests that modulation of PKR may affect response to IFN therapy, currently ongoing in these patients.
-'] HBV GENOTYPING ANALYSIS FROM THE SURAFCE ANTIGEN REGION USING THE TRUGENE TM HBV GENOTYPING KIT AND A PCR-BASED HOMEBREW METHOD Joe Huong, Kristy Reece, Aileen Grunwald, Cynthia Sturchio, Abel De La Rosa, Mimi Healy. Visible Genetics Corp., Suwanee, GA, USA Background: Hepatitis B virus (HBV) infects approximately 400 million people worldwide, of those 5-10% become chronically infected. Recent reports suggest some HBV genotypes may influence the outcome of chronic
Poster Sessions
90
infections. Currently, therapy may consist of immune modulators and/or antivirals. Similar to HIV, HBV develops resistance to antivirals through mutations in the pol gene. This study compares the results obtained by direct sequencing using the TRUGENE HBV Genotyping Kit (RUO v 1.0) and the HBV GeneLibrarianTM module (Visible Genetics Inc., Toronto, Canada) to a rapid Surface Anitgen (SA) PCR-based homebrew genotyping method. Methods: One Hundred (100) HBV antibody-positive plasma samples were used to evaluate the performance of the TRUGENE HBV Genotyping Kit and the OpenGeneTM DNA Sequencing System. The samples were genotyped with the HBV GeneLibrarian module and by BLAST (NCBI). Twenty-five of the 100 samples were evaluated with a PCR-based genotyping method amplifying from lares 1 to SA. Results: There was 100% concordance between the genotyping results of the TRUGENE assay and the BLAST searches. Eighteen of the 25 samples amplified using the PCR-based method were concordant with the sequencing method. The remaining seven samples, including a genotype G, were untypeable by fragment analysis. Conclusions: The influence of viral genotype on disease progression and therapy efficacy may or may not be similar for HBV as it is for HCV. For this reason, researchers should continue to acquire HBV genotype data for correlation with disease outcome. The TRUGENE HBV Genotyping Kit provides both genotype and pol mutations in a single assay.
~ 1 - ~ QUANTITATIVE DETERMINATION OF HEPATITIS C VIRUS CORE ANTIGEN (HCVAg) AND PREDICTION OF RESPONSE TO THERAPY IN CHRONIC HCV INFECTION Ruth Jacobs, David Brown, John Diment, Josephine Whitehe, Geoffrey Dusheiko. Centre for Hepatolgy, Royal Free and University College Medical School, London NW3 2PF; Ortho-Clinical Diagnostics, Buckinghamshire, HP7 OHJ, UK The recent development of a highly sensitive and specific enzyme linked immunosorbent assay for HCVAg (Total HCV Core Antigen Microwell Plate Assay, Ortho-Clinical Diagnostics, NJ) has allowed us the opportunity to evaluate the clinical usefulness of this test. 124 serum samples from 78 patients were tested. Eight patients were undergoing alpha interferon and ribavirin therapy, for which serial samples were assayed. Viral load was known for 52 samples and qualitative HCV RNA for 102. Regression analysis and Fishers exact test were used to evaluate significance. A significant correlation was seen between HCVAg level and viral load (p < 0.0001). Fifty one samples were positive for both HCV RNA and HCVAg, 34 negative for both, 14 HCV RNA positive but HCVAg negative and 3 HCV RNA negative and HCVAg positive. This again, represented a significant association (p < 0.0001) between HCV RNA and HCV core antigenaemia. Of eight patients undergoing treatment, 5 were genotype 1, and 3 were genotype 3a. Three had a viraemic and biochemical response while the rest remained HCV RNA positive. Seven patients became HCVAg negative within one month of starting treatment and six of these remained so throughout. The patient who remained antigenaemic showed reduced HCVAg from 29 pg/ml to 3-5 pg/ml but remained viraemic and did not exhibit any reduction in ALT. In conclusion, measurement of HCVAg can be used to monitor HCV infection although it is not as sensitive as HCV RNA. Reduction of HCVAg load to negative values cannot as yet be used to indicate recovery or response to treatment.
~-]
ORTHO TOTAL HCV CORE ANTIGEN ASSAY CAN AID EARLY PREDICTION OF RESPONSE IN PATIENTS TREATED WITH INTERFERON/RIBAVIRIN
Francoise Lunel, Pascal Veillon, Christopher Payan. Laboratoire De Bacterio- Virologie, CHU Angers, Angers, France Aim: Evaluate the predictive value of Total HCV Core Antigen Assay and viral kinetics in patients with chronic HCV.
Methods: 122 patients infected by genotype 1, 4, 5 or pretreatment viral load (bDNA 2.0, Chiron) >3 Meq/ml with no previous treatment, received 6 MU IFN during 12 months (M). Ribavirin was given with IFN after 3 months therapy, for 9 months in patients with detectable RNA. Viral load was expressed as Log (IU/mL) and HCV Ag as Log (pg/mL x 10 000). Results: Pre-treatment Ag values were correlated with viral load (r2 = 0.779). We observed a rapid decrease of Ag (5.2 Log pg/mL) and viral load (5.1 Log IU/mL) after M1 in sustained responders (SR). In patients who relapsed (RR) after IFN alone, the fall was less important (2.6 Log pg/mL, 3.6 Log IU/mL) during M1. In SR and RR to combination therapy, the decrease of Ag and viral load at M1 were, respectively, (Ag: 1.2 and 1.4 Log pg/mL; RNA: 2.4 and 1.5 Log IU/mL). We did not observed significant variation of Ag and viral load in non responders. The negative predictive value of HCV RNA and Ag after M1 of treatment were 100%, and positive predictive values were 81% and 82%. After one month of IFN alone, the HCV Ag decrease was highly predictive of SR, correlated with RNA negativation and early reduction of HCV RNA (>2 log). Conclusion: Early measurements of Total HCV Core Antigen are useful to predict long term response to treatment.
[-~
EVALUATION OF ORTHO TOTAL HCV CORE ANTIGEN ASSAY IN ASSESSMENT AND FOLLOW UP OF PATIENTS TREATED FOR CHRONIC HCV
Francoise Lunel, Pascal Veillon, Christopher Payan. Laboratoire de Bacterio- Virologie, Angers, France An assay to quantitate "Total" HCV core antigen in serum or plasma, in the presence of anti-HCV antibodies, has been developed by OCD. Total Core Ag assay is theoretically capable of measuring viremia, and may reflect viral load. We evaluated HCV Ag with 2 quantitative assays for HCV RNA: bDNA 2.0 or 3.0 (Bayer) and Monitor 2.0 (Roche). Methods: We studied 191 samples before treatment and 237 during treatment from 144 patients with chronic HCV treated with IFN or IFN/Ribavirin during one year. Results: Correlation of Ag and quantitative assays was high (r = 0.85 and 0.83, respectively). We did not find any difference between the levels of RNA and Ag among HCV genotypes (r = 0.82-0.96). In untreated RNA positive patient's samples (n = 144), HCV Ag was under the cut-off in 8 pts. bDNA 2.0 and Monitor 2.0 failed to detect 5 and 1 samples respectively. Ag values, before treatment, were significantly lower in sustained responders than in other groups (5.4 versus 6 pg/mL, p < 0.001). In treated patients, we found very good correlation between decrease or negativation of Ag and viral load: 2 Log IU/ml decline of HCV RNA after M1 was significantly correlated with the decrease or negativation of HCV Ag and response. 38/41 of sustained responders had a RNA load decrease >2 Log IU/ml and 39/41 had a negativation of HCV Ag after M1. Conclusion: Total HCV Core Ag appears to be a new tool for monitoring patients with HCV infection.
~']
APOPTOSIS OF PERIPHERAL BLOOD LYMPHOCYTES IN CHRONIC VIRAL HEPATITIS C
Alexey Boueverov, Ioulia Choulpekova, Marina Maevskaya, Vladimir Vashkin, Suleiman Mammaev. Department of Internal Diseases Propedeutics, Moscow Sechenov Medical Academy, Moscow, Russia Aim: to study the mechanisms of peripheral blood lymphocytes' apoptosis in chronic HCV-infection. Materials and Methods: 25 pts with chronic viral hepatits C (CVH C), HCV-RNA positive, were enrolled into the study. 20 healthy donors served as controls. The antigens' expression on peripheral blood lymphocytes was assessed using monoclonal antibodies CD3-, CD4-, CD25- Fits and CD8 PRE (Catlag, USA), by the flowing cytoflnrometria (Partec, USA). The proportion of lymphocytes in apoptosis immediately after selection and
HEPATIC IRON DISTRIBUTION IN 153 PATIENTS WITH CHRONIC VIRAL HEPATITIS (CH): RELATION WITH GRADING, STAGING ANO HFE MUTATIONS
Chiara Corengia ] , Giorgio Bovo 2, Cristina Arosio 3, Angelica Chie sara 4, Alessandra Salvionil, Viviana Mauril, Massimo Pozzil, Cristina Cestari 4, Anna Vergani l , Alberto Piperno I . t Clinica Medica
Universitgz Milano Bicocca, 2Dipartimento di Patologia, Ospedale S Gerardo Monza; 31stituto Auxologico Italiano, Ospedale S Luca Milano ; 4Divisione di Medicina 2, Ospedale S Gerardo Monza, Italy
Background: Hepatic iron overload is common in patients with CH. The relationship between iron, grading and staging and the role of HFE mutations in the developement of iron overload and fibrosis are controversial. Aim: To evaluate the relationship between a) hepatoceUular iron distribution, grading and staging; b) HFE genotypes, hepatocellular iron distribution and fibrosis. Methods: 253 CH patients that underwent liver biopsy from 1995 to 1999 were studied. Hepatic iron was assessed by Deugnier's score modified and grading and staging by Ishak's score. HFE mutations were assesed in 147 patients and in 139 healthy controls. Results: Hepatic iron overload was present in 124 patients (49%) (group A) and absent in 129 (51%) (group B). Two groups did not differ for age, sex and body mass index (BMI). Hepatic iron store did not correlate with BMI. Fibrosis and steatosis grade were significantly higher in group A than B (p < 0.0001 and p = 0.05). Cirrhosis was more frequent in group A than B (27% vs 13%, p = 0.007). In group A the staging score was significantly correlated with portal iron (p < 0.001), particulary with connective and endothelial deposits (p < 0.001 and p < 0.01). C282Y and H63D were more frequent in group A than in B (p = 0.033) and in controls (p = 0.0002). A significant correlation between HFE genotypes and hepatocytic iron, that increases with the increasing HFE genotype severity, was found (p = 0.0014). Conclusion: The correlation between fibrosis and portal iron support the fibrogenetic role of macrophagic iron. HFE mutations contribute to the pathogenesis of hepatocytic iron accumulation in CH.
~-]
LAMIVUDINE-RESISTANT HBV IS CROSS-RESISTANT TO L-dT AND L-dC IN VITRO
William Delaney, Huiling Yang, Michael Miller, Craig Gibbs, Shelly Xiong. Gilead Sciences, Foster Oty, CA, USA Objective: Clinical resistance of HBV to lamivudine is hallmarked by the development of rtM204I or rtL180M + rtM204V mutations in the viral polymerase. These mutations confer in vitro resistance to lamivudine (beta-L-2'-3'-dideoxy-3'-thiacytidine) and structurally-related L-nucleoside analogs such as b'TC and L-FMAU. Novel L-nucleosides with anti-HBV activity have recently been reported; these include betaL-thymidine (L-dT) and beta-L-deoxycytosine (L-dC), which are each in phase I/II clinical trials. The aim of this study, was to determine whether lamivudine-resistant HBV was sensitive to L-dC and L-dT in vitro. Methods: HepG2 cells were transfected with wild-type, rtL180M, rtM204I, or ~L180M + rtM204V mutant HBV and were treated with L-dC or L-dT. Following drug treatment, intraceUular replicative intermediates were quantified by Southern blotting and the IC50 of each drug was calculated for each strain of HBV. Results: L-dC and L-dT inhibited the replication of wild-type HBV with IC50 values of 0.26 and 0.28 microMolar, respectively. The ~L180M mutation conferred approximately 10-fold resistance to both compounds, similar to the level of resistance conferred to 3TC. The rtM204I and rtL180M + rtM204V mutations conferred high levels resistance to both L-dC and L-dT (IC50 values increased > 300-fold). Conclusions: These in vitro studies indicate that HBV mutants selected during lamivudine therapy are cross-resistant to L-dC and L-dT and provide further evidence that YMDD mutations confer broad cross-resistance to Lnucleosides.
~0-]
89
ULTRASTRUCTURAL HEPATOCYTE MODIFICATIONS IN HCV INFECTED HUMAN LIVER
Laura Falasca l, Fabiola Ciccosantit, Roberta Nardacci t, Oreste Lo Iacono 2, Manro Piacentini L3. t Lab. Electron Microscopy, INMI-1RCCS
'L. SpaUanzani' Rome; 2University of Study of Palermo, Palermo; SUniversity of Rome 'Tor Vergata', Rome, Italy Despite many efforts, the search for the morphological features of HCV inside the hepatocytes has been hampered by the low level of viral particles present in the liver tissue of infected individuals. Little information is avalaible on intracellular localization of the virus and on prerequisites for its assembly, as a consequence poorly understood is the pathogenic mechanism of liver disease. In this work we analysed, by transmission electron microscopy, 30 hepatic biopsies from HCV infected patients. Liver sampies were obtained from individuals infected by hepatitis C virus with different genotypes, and with a different degree of liver damage in order to find possible common ultrastructural modification useful to understand the progression of the disease. A variety of alterations were frequentely seen in hepatocytes, mostly concerning alteration of mitochondria, dilatation of endoplasmic reticulum, and presence of lipid inclusions in the cytoplasm. In addition, in a limited number of hepatocytes we have found peculiar changes of nuclear morphology (indentation and chromatin condensation). The most relevant result is that we found, in association with nucleus modification, the presence of vires-like particles. The morphology and the size of the particles were consistent with the predicted HCV virions based on other studies.
-] DIFFERENT MODULATION OF PKR GENE EXPRESSION BY HCV GENOTYPES Martina Gerotto, Francesca Dal Pero, Silvia Boccato, Gladis Bortoletto, Alessia Ferrafi, Stefano Realdon, Afredo Alberti. University of Padova,
Padova, Italy In hepatitis C, the cellular protein kinase PKR may play a role in the pathogenesis and response to interferon. Some HCV proteins, including NS5A, were shown in vitro to bind PKR and to upregulate or downregulate its expression and activation. Little is know on the in vivo expression of PRK in patients with hepatitis C. Our aim was to measure PKR expression in HCV positive patients. PKR-mRNA was measured by competitive RTPCR in PBMCs from 24 patients (18 with HCV-1 and 6 with HCV-2) and from control healthy subjects. PBMCs were studied before and after incubation with IFN. Mean baseline PKR mRNA levels were significantly lower in HCV1 patients (0.40 + 0.24 fg) compared to HCV2 cases (2.45 + 2.9 fg p -- 0.006) and controls (1.37 + 1.0 p = 0.005). No correlation was found between mutations in the NS5A-PKR binding domain and PKRmRNA levels, that were also independent of viraemia, ALT and histologic activity. After in vitro incubation with IFN, PKR mRNA was enhanced to similar levels in HCV1 (3.3 + 2.3) and HCV2 (3.2 + 2.4) patients but remained significantly lower compared to control (8.0 + 5.6 p = 0.003). These results indicate that PKR gene expression is downregnlated in HCV infection, particularly in patients with HCV 1, and this is independent of the NS5A PKR-binding domain sequence. Preliminary evidence suggests that modulation of PKR may affect response to IFN therapy, currently ongoing in these patients.
-'] HBV GENOTYPING ANALYSIS FROM THE SURAFCE ANTIGEN REGION USING THE TRUGENE TM HBV GENOTYPING KIT AND A PCR-BASED HOMEBREW METHOD Joe Huong, Kristy Reece, Aileen Grunwald, Cynthia Sturchio, Abel De La Rosa, Mimi Healy. Visible Genetics Corp., Suwanee, GA, USA Background: Hepatitis B virus (HBV) infects approximately 400 million people worldwide, of those 5-10% become chronically infected. Recent reports suggest some HBV genotypes may influence the outcome of chronic
Poster Sessions
90
infections. Currently, therapy may consist of immune modulators and/or antivirals. Similar to HIV, HBV develops resistance to antivirals through mutations in the pol gene. This study compares the results obtained by direct sequencing using the TRUGENE HBV Genotyping Kit (RUO v 1.0) and the HBV GeneLibrarianTM module (Visible Genetics Inc., Toronto, Canada) to a rapid Surface Anitgen (SA) PCR-based homebrew genotyping method. Methods: One Hundred (100) HBV antibody-positive plasma samples were used to evaluate the performance of the TRUGENE HBV Genotyping Kit and the OpenGeneTM DNA Sequencing System. The samples were genotyped with the HBV GeneLibrarian module and by BLAST (NCBI). Twenty-five of the 100 samples were evaluated with a PCR-based genotyping method amplifying from lares 1 to SA. Results: There was 100% concordance between the genotyping results of the TRUGENE assay and the BLAST searches. Eighteen of the 25 samples amplified using the PCR-based method were concordant with the sequencing method. The remaining seven samples, including a genotype G, were untypeable by fragment analysis. Conclusions: The influence of viral genotype on disease progression and therapy efficacy may or may not be similar for HBV as it is for HCV. For this reason, researchers should continue to acquire HBV genotype data for correlation with disease outcome. The TRUGENE HBV Genotyping Kit provides both genotype and pol mutations in a single assay.
~ 1 - ~ QUANTITATIVE DETERMINATION OF HEPATITIS C VIRUS CORE ANTIGEN (HCVAg) AND PREDICTION OF RESPONSE TO THERAPY IN CHRONIC HCV INFECTION Ruth Jacobs, David Brown, John Diment, Josephine Whitehe, Geoffrey Dusheiko. Centre for Hepatolgy, Royal Free and University College Medical School, London NW3 2PF; Ortho-Clinical Diagnostics, Buckinghamshire, HP7 OHJ, UK The recent development of a highly sensitive and specific enzyme linked immunosorbent assay for HCVAg (Total HCV Core Antigen Microwell Plate Assay, Ortho-Clinical Diagnostics, NJ) has allowed us the opportunity to evaluate the clinical usefulness of this test. 124 serum samples from 78 patients were tested. Eight patients were undergoing alpha interferon and ribavirin therapy, for which serial samples were assayed. Viral load was known for 52 samples and qualitative HCV RNA for 102. Regression analysis and Fishers exact test were used to evaluate significance. A significant correlation was seen between HCVAg level and viral load (p < 0.0001). Fifty one samples were positive for both HCV RNA and HCVAg, 34 negative for both, 14 HCV RNA positive but HCVAg negative and 3 HCV RNA negative and HCVAg positive. This again, represented a significant association (p < 0.0001) between HCV RNA and HCV core antigenaemia. Of eight patients undergoing treatment, 5 were genotype 1, and 3 were genotype 3a. Three had a viraemic and biochemical response while the rest remained HCV RNA positive. Seven patients became HCVAg negative within one month of starting treatment and six of these remained so throughout. The patient who remained antigenaemic showed reduced HCVAg from 29 pg/ml to 3-5 pg/ml but remained viraemic and did not exhibit any reduction in ALT. In conclusion, measurement of HCVAg can be used to monitor HCV infection although it is not as sensitive as HCV RNA. Reduction of HCVAg load to negative values cannot as yet be used to indicate recovery or response to treatment.
~-]
ORTHO TOTAL HCV CORE ANTIGEN ASSAY CAN AID EARLY PREDICTION OF RESPONSE IN PATIENTS TREATED WITH INTERFERON/RIBAVIRIN
Francoise Lunel, Pascal Veillon, Christopher Payan. Laboratoire De Bacterio- Virologie, CHU Angers, Angers, France Aim: Evaluate the predictive value of Total HCV Core Antigen Assay and viral kinetics in patients with chronic HCV.
Methods: 122 patients infected by genotype 1, 4, 5 or pretreatment viral load (bDNA 2.0, Chiron) >3 Meq/ml with no previous treatment, received 6 MU IFN during 12 months (M). Ribavirin was given with IFN after 3 months therapy, for 9 months in patients with detectable RNA. Viral load was expressed as Log (IU/mL) and HCV Ag as Log (pg/mL x 10 000). Results: Pre-treatment Ag values were correlated with viral load (r2 = 0.779). We observed a rapid decrease of Ag (5.2 Log pg/mL) and viral load (5.1 Log IU/mL) after M1 in sustained responders (SR). In patients who relapsed (RR) after IFN alone, the fall was less important (2.6 Log pg/mL, 3.6 Log IU/mL) during M1. In SR and RR to combination therapy, the decrease of Ag and viral load at M1 were, respectively, (Ag: 1.2 and 1.4 Log pg/mL; RNA: 2.4 and 1.5 Log IU/mL). We did not observed significant variation of Ag and viral load in non responders. The negative predictive value of HCV RNA and Ag after M1 of treatment were 100%, and positive predictive values were 81% and 82%. After one month of IFN alone, the HCV Ag decrease was highly predictive of SR, correlated with RNA negativation and early reduction of HCV RNA (>2 log). Conclusion: Early measurements of Total HCV Core Antigen are useful to predict long term response to treatment.
[-~
EVALUATION OF ORTHO TOTAL HCV CORE ANTIGEN ASSAY IN ASSESSMENT AND FOLLOW UP OF PATIENTS TREATED FOR CHRONIC HCV
Francoise Lunel, Pascal Veillon, Christopher Payan. Laboratoire de Bacterio- Virologie, Angers, France An assay to quantitate "Total" HCV core antigen in serum or plasma, in the presence of anti-HCV antibodies, has been developed by OCD. Total Core Ag assay is theoretically capable of measuring viremia, and may reflect viral load. We evaluated HCV Ag with 2 quantitative assays for HCV RNA: bDNA 2.0 or 3.0 (Bayer) and Monitor 2.0 (Roche). Methods: We studied 191 samples before treatment and 237 during treatment from 144 patients with chronic HCV treated with IFN or IFN/Ribavirin during one year. Results: Correlation of Ag and quantitative assays was high (r = 0.85 and 0.83, respectively). We did not find any difference between the levels of RNA and Ag among HCV genotypes (r = 0.82-0.96). In untreated RNA positive patient's samples (n = 144), HCV Ag was under the cut-off in 8 pts. bDNA 2.0 and Monitor 2.0 failed to detect 5 and 1 samples respectively. Ag values, before treatment, were significantly lower in sustained responders than in other groups (5.4 versus 6 pg/mL, p < 0.001). In treated patients, we found very good correlation between decrease or negativation of Ag and viral load: 2 Log IU/ml decline of HCV RNA after M1 was significantly correlated with the decrease or negativation of HCV Ag and response. 38/41 of sustained responders had a RNA load decrease >2 Log IU/ml and 39/41 had a negativation of HCV Ag after M1. Conclusion: Total HCV Core Ag appears to be a new tool for monitoring patients with HCV infection.
~']
APOPTOSIS OF PERIPHERAL BLOOD LYMPHOCYTES IN CHRONIC VIRAL HEPATITIS C
Alexey Boueverov, Ioulia Choulpekova, Marina Maevskaya, Vladimir Vashkin, Suleiman Mammaev. Department of Internal Diseases Propedeutics, Moscow Sechenov Medical Academy, Moscow, Russia Aim: to study the mechanisms of peripheral blood lymphocytes' apoptosis in chronic HCV-infection. Materials and Methods: 25 pts with chronic viral hepatits C (CVH C), HCV-RNA positive, were enrolled into the study. 20 healthy donors served as controls. The antigens' expression on peripheral blood lymphocytes was assessed using monoclonal antibodies CD3-, CD4-, CD25- Fits and CD8 PRE (Catlag, USA), by the flowing cytoflnrometria (Partec, USA). The proportion of lymphocytes in apoptosis immediately after selection and