Evaluation of a new assay for direct qualitative detection of serum HCV RNA

232A

AASLD ABSTRACTS

HEPATOLOGYO c t o b e r 2001

233

234

STANDARDIZED QUANTIFICATION OF HEPATITIS C VIRUS RNA W I T H THIRD-GENERATION "BRANCHED DNA" (BDNA) SIGNAL AMPLIFICATION ASSAY. Magali Bouvier-Alias, Jocelyne Remire, Francoise Darthuy, Daniel Dhumeaux, Jean-Michel Pawlotsky, Hopital Henri Mondor, Creteil France

SURVIVAL IN HCV/HIV CO-INFECTED PATIENTS. Tarek I Hassanein, Nina Aronson, Deanna L Oliver, Ed Barber, Robin C Hilsabeek, Eileen Chatfield, Marl Stewart, Christopher Mathews, University of California, San Diego, San Diego

Hepatitis C virus (HCV) RNA quantification is useful to tailor interferon (IFN) alfa-ribavirin treatment duration and to monitor HCV replication kinetics during treatment. The latter application will probably become more important in the next future when more sensitive assays and additional therapeutic agents are available. Standardization of HCV RNA quantification units recently represented a major achievement, opening the way to the wide clinical use of H CV RNA quantification. The 3rd-generation bDNA assay (Versant HCV RNA 3.0 Quantitative Assay, Bayer Diagnostics) is a new version of the signal amplification-based bDNA assay that can be semi-automated and quantifies HCV RNA in standardized international nnits/ml (IU/ml). We evaluated the performance of this new assay in the clinical setting. RESULTS: Quantification in IU/ml was accurate, based on a panel of samples containing increasing HCV RNA concentrations calibrated to the W H O HCV RNA standard (NAP-HCV, AcroMetrix Inc. ; r=0.997, p=0.0002). Quantification was linear over the entire stated dynamic range of quantification (615 to 7,700,000 IU/ml, ie 2.8 to 6.9 log IU/ml). The assay displayed excellent precision and reproducibility over the entire range of quantifiable values (intra-run variability : 0.9% to 14.2% ; inter-run variability : 11.0% to 19.0%). Specificity was 99.0% in a series of 107 HCV-negative samples. Equal and linear quantification of the major HCV genotypes was achieved. HCV RNA loads in 3rd-generation bDNA were compared with those in the SuperQuant RT-PCR assay (National Genetics Institute) in 409 clinical samples from patients infected with HCV genotype lb and included in a therapeutical 1FN-ribavirin trial with frequent sampling for the study of HCV replication kinetics. The two assays strongly correlated (r=0.968, p < 0 . 0 0 0 1 ) and HCV RNA kinetics during therapy were identical with both tests. CONCLUSIONS: The 3rd-generation bDNA-based Versant HCV RNA 3.0 Quantitative Assay provides semi-automated HCV RNA quantification in IU/ml. The performance of this assay makes it a good tool for HCV RNA quantification in clinical routine, both before and during treatment in order to optimize the results of antiviral therapy of chronic hepatitis C.

The impact of HCV infection on survival of HIV patients has been inconsistent. Prior results from our lab suggested that HCV status did not significantly impact survival in HIV patients, while others have shown that HCV status shortens patients' survival. The purpose of the present study was to test the hypothesis that co-infected patients who seek specialized treatment for HCV will have better survival rates than those who do not. Methods: 2969 HIV patients seen over a 5 1/2-year period were included in this study. Mean age of the sample was 40.9 years, and 85% were male. 603 patients were co-infected with HCV. Co-infected patients were divided into two groups, according to compliance with medical care. 62 co-infected patients were judged to be compliant, and 549 were not. A third group was comprised of 2366 HIV-infected patients. To examine group differences in survival, Kaplan Meier and Cox regression stratified by group were used to analyze both unadjusted and adjusted survival distributions. We determined independent risk factors for survival, and variables that were significant were entered as covariates in a multivariate Cox regression. Results: The unadjusted model showed that there was significant difference between the three groups survival functions by log rank test (X2 = 18.43, p = 0.0001). Analyses revealed that the compliant co-infected group had a significantly longer survival rate than both the HIV and noncompliant groups. In turn, the HIV group had a significantly longer survival rate than the non-compfiant co-infected group. The hazard ratios were adjusted for demographics (age, race, gender), baseline CD4 and HIV viral load, and time dependant CD4 and HIV viral load, in three separate models. The difference in hazard ratios between the three groups remained consistent in all three models. Thus, even when accounting for these covariates, there were significant group differences in survival rate. Summary: 1) There is a significant difference in survival between the three groups studied. 2) This difference is not a function of demographics, baseline CD4 and HIV viral load, nor time dependant CD4 and HIV viral load. In conclusion, co-infected patients who actively seek specialized care for their HCV infection have significantly better survival rates than patients who do not.

235

236

ASSOCIATION BETWEEN HEPATITIS C, DIABETES MELLITUS AND RACE: A CASE-CONTROL STUDY. Preeti R John, Paul J Thuluvath, The Johns Hopkins University School of Medicine, Baltimore, MD Ahigherprevalenceof type1l diabetesmellitus(typeII DM)has beenreportedin patientswithhepatitis C (HCV)infection.However,in mostpublishedstudies, the controlpopulationwasnot matchedfor severity of liverdisease,bodymassindex (BMI)or race, knownrisk factorsfor the developmentof type II DM. Objective:To determinethe prevalenceof typeI1DMin 97 patientswith HCVcirrhosis (cases)and 194 non-HCV cirrhosis (case-controls)matchedfor age, sex, body mass index (BMI)and severityof liver disease. Patientsand Methods: In this case-controlstudy, all patientshad OLT (to reduceascertainment ands referralbias). For eachpatientwith HCV,2 case-controls,matchedfor age,sex and severityof liver diseasewereselectedin a consecutivemannerfromthe livertransplantregistryafterconcealingprimary liver disease and diabeticstatus to reducebias. The etiologyof liver diseasein the case~controlgroup included58alcohoIiccirrhosis,38 PBC,23 PSC,15HBV,14cryptogeniccirrhosis,9 autoimmunecirrhosis, 9 NASH,9 hemochromatosisand 19 others.Allpatientshad clinicaland histologicalevidenceofcirrhosis, witha ChildPughscoregreaterthan7 (ChildBor C). Prevalenceofpre-andpost-transplanttypeII diabetes wasdeterminedin casesand case-controls.Results:Theage,sexand severityofliverdiseaseweresimilarin both groupsbut thereweremoreAfricanAmericans(AA)in theHCVgroup(23/97,24%)comparedto case controls (16/194,8%).32%of casesand 18%controlshad BMI>30. The meanageof the AAcases,White American(WA)cases,AAcontrolsandWAcontrolswassimilar.Theprevalenceofpre-transplantDMwas higherin theHCVgroup(19.6%)comparedto case-controls(7.7%)(P =0.003,O.R.2.9,95%CI 1.4 - 6.0). The prevalencewas similarin obesegroup (BMI>30),but significantlydifferentin the non-obesegroup (24.5% vs 12.4%,P =0.04, OR 2.3, CI 1.1-5.2. The prevalenceof pre-transplantDM in AAwith HCV (33.3%) was higherthan WAwith HCV(ll.8%)(P =0.02, O.R. =3.8, 95% CI =1.2 - 11.5)andAAcasecontro|s(6.3%)(P =0.04, O.R.7.5,95%CI = 0.8 - 67.4).AmongWA,the prevalenceof DMwassimilarin theHCVgroup(] 1.8%)and ease-controls(11.7%).Afteradjustingforage,BMIand case-controlstatus,AA were2.7 timesmorelikelyto developDMthan%VA(P = 0.0I3, 95%CI 1.2- 5.9).NewonsetDMwassimilar in HCVgroup(13.4%)and case-controls(7.2%,P ~0.09),but in the non-obesegroup,therewas a higher incidence of new onset DM in the HCV group (18.9%versus8.5%, P =.05, OR 2.5, CI 1.0-6.3). The incidenceof new onset DMwas similarin AAwith HCV (20.8%)and AAcase-controls(18.8%.Conclusions Our studyshowsthat theprevalenceof typeII DMis higherin patientswithHCVcirrhosiscompared to a matchedgroupof patients with other liverdiseasesof equalseverity.Thiswas mainlybecauseof a higherprevalenceof diabetesmellitusin AAw~thHCVcirrhosis.

EVALUATION OF A N E W ASSAY FOR DIRECT QUALITATIVE DETECTION OF SERUM HCV RNA. Michelle M Martinot-Peignoux, INSERM U481, Clichy France; Nathalie Boyer, Dominique Valla, Patrick Marcellin, Service d'H~patologie, Clichy France

Demographics of Cases (HCV group) and Case-Controls

HCV 9roup (N --97) Case-Controls (N =194) P-value Age (~SD) Race(white/black/other) Sex(M/F) Albumin Total Bilirubin Direct Bilirubin Prothrombin Time Body Mass Index (BMI) BMI ~30 (Obese)

47.7 ~8.g 68/23/6 22/75 2,86 ~0.66 5,95 ~8.62 2.82 ~5.08 15.2 ~ 1.84 29.4 ~6.0

31 (%)

48.2 [~9.4 171/16/7 441150 2.81 ~0.99 7.07 ~8,69 3,61 ~5.52 15.34 ~3.21 27.4 ~5,4 35 (%)

NS NS NS 0.7 0.4 0.3 0.4 0.03 0,008

The aim of our study was to evaluate the performances of the new sensitive qualitative HCV RNA assay based on transcription-mediated amplification (TMA) (VERSANTTM HCV RNA QUALITATIVE ASSAY, GEN-PROBE, BAYER) allowing the detection of serum HCV RNA in a single tube. Specificity, sensitivity and reproducibility were tested. Specificity. For the specificity study 514 anti-HCV negative samples from blood donation were tested. 8/514 (1,55%) were tested positive, 2/8 were tested positive after a second confirmation r u n and were PCR positive (HCV COBAS AMPLICOR, ROCHE). The overall specificity of the assay was 98.8%. Sensitivity. For the sensitivity study serial dilutions of the W H O standard were used and tested 10 times; 100% of the dilutions calibrated at 20 IU/mL, 10 IU/mL, 5 IU/mL and 2.5 IU/mL were detected, and 60% of the dilution calibrated at 1 IU/mL was detected. Reproducibility. For the reproducibility study two dilutions calibrated at 50 HCV copies/mL (10 IU/mL) and 25 HCV copies/mL (5 IU/mL) (VERSANT HCV RNA 3.0 ASSAY (bDNA,), BAYER) were tested 10 times for HCV genotypes la, lb, 2, 3, 4, 5, and 6. 100% of the dilution calibrated at 50 HCV copies/mL were detected, for each HCV genotype. 100% of the dilutions calibrated at 25 HCV copies/mL were detected for genotypes la, 3, 4; and 6, and 80% of the dilutions calibrated at 25 HCV copies/mL were detected for genotypes lb, 2, and 5. Conclusion. The VERSANTTM HCV RNA QUALITATIVE ASSAY (TMA) assay provides a specificity of 98.8%, a high sensitivity (below 2,5 IU/ml) and an excellent reproducibility for each HCV genotype. This assay appears to be a good tool for monitoring patients during and after therapy.