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Evaluation of ortho total HCV core antigen assay in assessment and follow up of patients treated for chronic HCV
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.
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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.
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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
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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