HCV Core Quasispecies Change Is Associated with End Stage Liver Disease in a Longitudinal Cohort of HCV and HCV/HIV Infected Patients with Inherited Bleeding Disorders

Hongxing Qin, Norah J. Shire, Susan D. Rouster, M. E. Eyster, James J. Goedert, Margaret J. Koziel, Kenneth E. Sherman



Patients with inherited bleeding disorders who received plasma clotting factor concentrates prior to 1987 have among the highest rates of HCV infection often associated with HIV coinfection. We evaluated the HCV core protein quasispecies evolution in longitudinally collected specimens comparing those with progression to decompensated, end-stage liver disease (ESLD) to those with compensated chronic hepatitis.



Serially collected serum samples reflecting long-term clinical outcomes were obtained from the NCI Multicenter Hemophilia Cohort Study. Index cases were derived from patients who progressed to ESLD. Nonprogressor controls were matched for age, gender, duration of infection, presence or absence of HIV coinfection, and HCV genotype. Samples from early infection (E) were compared to those obtained after onset of ESLD (L) in the index cases. The core protein coding region was amplified, subcloned and sequenced. Predicted amino acid sequences were compared for clonal variability and evolution. Complexity and diversity were determined.



       Over 425 subclones derived from 10 pairs of patients followed over a mean duration of 9.3 years (S.E.= 0.69 years) were sequenced and evaluated for genetic complexity and diversity

       80% (16/20) patients were HIV+

       At baseline, there were no statistically significant differences in complexity or diversity between progressors and nonprogressors



       Progressors demonstrated a slight increase in complexity over time compared to nonprogressors, although this change did not reach statistical significance

       Both groups of patients had dN/dS ratiors consistently <1, indicating a lack of selective pressure for this region

       Genetic distances in the core region between early and late clonal sets were likely to be greater for progressors (p=0.02), indicating greater divergence

       Mutation in the RKT motif (positions 9-11) that have been associated with NFkB activation were seen solely in progressors



       Differences in genetic diversity are apparent in the core region among interferon-nave progressors and nonprogressors with inherited bleeding disorders

       None of these differences were associated with HCV or HIV viral loads, demographic factors, or CD4+/CD 8+ counts

       In progressors, the changes that occur in the core region appear to be associated more frequently with hydrophotic elements that may have important functional significance

       Further understanding of these mechanisms may help identify factors related to rate of progression in HCV-infected patients with inherited bleeding disorders


Why Do Patients with Hepatitis C Not Attend Clinic Appointments? Results of a Focus Group Study

Eric W. Dieperink, Holly Groom, Ramsey C. Cheung, Margaret Celebreze, Barry Cohen, Samuel Ho, Mark L. Willenbring



The efficacy of antiviral treatment for chronic hepatitis C (HCV) has not been translated to clinical populations as most patients with HCV fail to access care. The purpose of this study was to determine patient's reasons for not seeking HCV specialty care.



We conducted seven focus groups with HCV patients. Two groups consisted of patients actively in HCV care and five groups who, despite extensive outreach, had failed to attend any appointments in a specialty care hepatitis clinic.


No significant differences in patient characteristics were found between groups, average age, years living with the HCV diagnosis and histories of substance use and mental illness disorders were similar in each group.


The following themes were identified as important barriers to seeking care:

       lack of perceived need for care, incorrect beliefs about progression of the illness and about treatment for HCV

       having a substance use disorder and stigma. HCV was frequently equated with HIV



This study identifies several potential barriers to care that may be common among veterans with HCV who do not attend appointments. Lack of knowledge regarding HCV and stigma appear to be the most important factors that influence help seeking behavior. Implications: Possible interventions to improve access include addressing logistical barriers and improving outreach by using alternative notification methods. Improved education and addressing stigma associated with HCV may improve patients help seeking and adherence with care. Efforts to improve access to care should increase the effectiveness of antiviral treatments for HCV.


Faciliators of hepatitis C care:

       Flexibility of scheduling

       Support Groups

       Interest and knowledge of VA Staff

Possible interventions

       Peer resources to enhance knowledge

       Improved resources specifically designed for friends or family to address concerns about stigma

       Improved education regarding asymptomatic disease vs. actual disease

       Addressing logistic barriers, in particular, improve, shorten or supplement notification of hepatitis C testing in order to decrease delays and more closely connect patient education with test results

The Effect of Hepatitis C Coinfection on the Risk of Fulminant Hepatic Failure in Patients with HIV in the HAART and Pre-HAART Eras

Jennifer R. Kramer, Thomas P. Giordano, Julianne Souchek, Hashem B. El-Serag



It is uncertain if patients coinfected with hepatitis C virus (HCV) and HIV are more likely to suffer fulminant hepatic failure (FHF) when compared to patients with HIV-only. It is also unclear if the risk of FHF changes when coinfected patients are on highly active antiretroviral therapy (HAART).



We conducted a retrospective cohort study using national administrative databases from the Department of Veterans Affairs (172 hospitals) in patients hospitalized for the first time with HIV and/or HCV between 10/1991 and 9/2000. FHF was defined by ICD-9 code 570 (acute liver failure) occurring after the index hospitalization through 9/2001 in the absence of pre-existing liver disease. We calculated incidence rates, Kaplan Meier (KM) cumulative incidence curves, and adjusted hazard ratios (HR) in Cox proportional hazards (PH) models while adjusting for age, race, and prior diagnoses of toxic hepatitis, diabetes, and alcoholism.



After excluding patients with pre-existing liver disease, we identified 11,678 patients with HIV-only and 4,761 patients with HCV-HIV coinfection.


There were 92 cases of FHF, which had a 6-week mortality rate of approximately 50%. There were 42 cases of FHF in the HIV-only group, for an incidence rate of 1.1/1000 person-years, and 50 cases in the coinfected group, for an incidence rate of 2.5/1000 person-years. In the KM analysis, the cumulative incidence of FHF in the coinfected group was higher than in the HIV-only group (p<0.0001). In a Cox PH model restricted to patients entering the cohort in the HAART era, coinfected patients had an adjusted HR for FHF of 5.86 compared to HIV-only patients (2.36-14.50). Patients with coinfection in the pre-HAART era did not have an increased risk of FHF (HR=0.99; 0.43-2.31).


Multivariate Results

The risk of FHF with HCV Coinfection:

Hazard ratios (HR) for HCV coinfection vs. HIV-only:

       Total follow-up:

       HR=2.66 (95% CI: 1.7.-4.1), p<0.0001

o      Pre-HAART era:

       HR=0.99, (95% CI: 0.4-2.3), p=0.98

o      HAART era:

       HR=5.86, (95% CI: 2.4-14.5), p=0.0001

       HR of interaction term (HAART era HCV coinfection status) from total follow-up:

       HR=3.96, (95% CI: 1.5-10.5

       Interpretation: The risk of FHF in patients with HCV confection compared to HIV-only during the HAART era was almost four fold higher than that calculated during the pre-HAART era.



       The risk of FHF in patients with HCV coinfection compared to HIV-only during the HAART era was several folds higher than that during the pre-HAART era.

       There is no question that patients with HCV-HIV coinfection should be receiving HAART therapy, but since FHF is associated with high mortality these patients should be monitored more closely.

       This is the first study of HIV-infected patients to examine the risk of FHF in relation to coinfection with HCV in the HAART era.

       The overall risk of FHF is low in HIV and HCV-HIV coinfected patients.


Comparison Study of Prometheus FIBROSpect II to Knodell and Ishak Fibrosis Scores in Alaska Native Hepatitis C Patients

Carol Christensen, Steve Livingston, Dana Bruden, Heike Dubner, Katie M. Smith, Jason Do, Esther Oh, Brian J McMahon



Methods less invasive to distinguish between mild and moderate/severe fibrosis in patients with chronic hepatitis C are desirable. We previously reported on 75 interpretable specimens tested with a panel of serologic markers for fibrosis (FIBROSpectSM: Prometheus Laboratories, San Diego, CA) as having a sensitivity of 89% (95% CI: 74-95%) and specificity of 85% (95% CI: 71-93%) in detecting moderate to severe fibrosis (Knodell 3-4). Sensitivity for detecting Ishak 4-6 was 97% (95% CI: 82-99%) and specificity was 78% (95% CI: 65-88%). 21% had an indeterminate result (Hepatology 2003;38:587A).



To evaluate the performance of FIBROSpectSM II in detecting severe fibrosis (Knodell and Ishak systems in stored sera of Alaska Natives participating in a Hepatitis C outcome study.



142 specimens were tested for serum hyaluronic acid, tissue inhibitor of metalloproteinase and alpha-2 macroglobulin, proteins involved in extracellular matrix remodeling. FIBROSpect II is a logistic regression model established for this panel of fibrosis markers to detect significant fibrosis (Metavir F2-4) in chronic HCV. Using the algorithm-derived index, all specimens were classified either as consistent with no/mild (Knodell 0-1) or severe fibrosis. Blinded liver biopsies were read and scored for fibrosis by a study pathologist. Clinical performance of the two FIBROSpec algorithms was compared.



Using FIBROSpect II, sensitivity for detecting Knodell fibrosis 3-4 increased to 92% (95% CI: 82-98%) with a specificity of 64% (95% CI: 54-74%), PPV of 60% and NPV of 94%. Sensitivity for Ishak 4-6 was 100% (95% CI: 89-100%) and specificity was 56% (95% CI: 46-65%), PPV of 41%, and NPV of 100%.



FIBROSpect II was clinically useful in ruling out severe fibrosis (Ishak 4-6). Comparing these results to our previous study with FIBROSpectSM, sensitivity improved but specificity decreased. The authors also noted that a longer biopsy length was associated with higher concordance between the FRIBOSpect II test and the Knodell biopsy score, indicating that biopsy sample size is important for an accurate assessment.



       Relating FIBROSpect II to the Knodell scoring system present problems because a Knodell score of 1 includes persons with mild and moderate fibrosis, which could account for the decrease in specificity.


Future Directions

       Evaluate the FIBROSpect II index score (range 0-100) in predicting the individual fibrosis, which could account for the decrease in specificity in this retrospective study cohort.

       Prespective studies evaluating FIBROSpect II test results with liver biopsy scores on patients with hepatitis B and C.


The Los Alamos Hepatitis C Sequence Database

Carla Kuiken, Russell Richardson, Karina Yusim



Providing web-based tools to retrieve and analyze annotated publicly available HCV sequence data.



The Los Alamos Hepatitis C virus (HCV) sequence database became publicly accessible in September 2003 (hcv.lanl.gov or hcv-db.org). It is based on the HIV sequence database (www.hiv.lanl.gov), which is used by thousands of researchers weekly.


Scientists who are familiar with that database will find it easy to navigate the HCV site. The database offers access to sequences using a flexible, user-friendly search interface that can retrieve aligned sequences based on the genomic region and a number of epidemiological and biological criteria like genotype, sampling country, risk group, and study. Alignments of all available complete-gene sequences are provided along with a large number of tools to process and analyze sequences. Using the website, researchers can obtain synonymous/non-synonymous calculations, multidimensional scaling of sequence data, primer and epitope alignments, BLAST searches of the HCV database, simple tree construction, and analysis of glycosylation sites. The sequences are annotated based on the literature and on phylogenetic analyses by the database staff.



Currently the database contains 22,000 sequences, of which 19,000 are annotated with genotype and 14,800 with country information. Annotation of sequences in the database currently includes host data (host species, health status at time of sampling, therapy history and response, sampling date and location, likely infection route, date and location), virus data (genotype and subtype, sequenced region), linkage data (transmission clusters, other sequences obtained from a host), and publication data (including publication links not annotated in GenBank). Sequences from patent applications, non-human hosts, and short sequences can easily be excluded from the results. Links to retrieve all sequences from a study are provided, as are links to GenBank and to external analysis tools.



The HCV sequence database has the potential to become a very powerful tool in HCV genetic research and vaccine design, just like the HIV database is now indispensable for HIV scientists. Future plans include building tools for automatic tree building, optimal tree selection, distance matrix analysis, and automatic alignment of user sequences to database sequences. A database of HCV immunological epitopes will be ready for release in the spring or summer of 2004.


Cost-Effectiveness of Peginterferon α-2b plus Ribavirin Treatment of Chronic Hepatitis C with F1 Fibrosis

John B. Wong, Thierry Poynard, Michael P. Manns, John G. McHutchison, Joann Harvey, Jamice K. Albrecht



Although not all patients with histologically mild chronic hepatitis C progress, prior studies suggest that antiviral treatment should be cost-effective, but those studies were based on mild inflammation and involved interferon alone or with ribavirin.



To determine the cost-effectiveness of peginterferon α-2b+ribavirin for histologically mild F1 fibrosis.



Using data from Manns Lancet 2001, we compared no antiviral therapy to peginterferon α-2b+>10.6 mg/kg ribavirin. Lifelong clinical and economic outcomes were based on Cox proportional hazard models estimating the likelihood of developing Metavir fibrosis stages F1-F4 over time (using 2313 liver biopsies), and on recent UNOS, SEER and NIH data (Wong, AASLD 2003). Drug costs applied the 12-week stopping rule. Cost-effectiveness results are presented as incremental cost per discounted (3%) quality-adjusted life year gained. Cost-effectiveness ratios below $50,000 per discounted quality-adjusted life year gained were considered to be cost-effective.



Observed sustained viral response rates for peginterferon α-2b+ribavirin treatment of F1 were 63% overall, 52% for genotype 1 and 91% for genotype 2/3.


Antiviral treatment reduced the 20-year incidence of cirrhosis from 20% to 7.6% overall, to 9.7% for genotype 1 and to 1.8% for genotype 2/3. Antiviral treatment extended life expectancy by 2.3 years overall, 1.9 years for genotype 1 and 3.4 years for genotype 2/3. Quality-adjusted life expectancy benefits of treatment were 4.7 years overall, 3.9 years for genotype 1 and 6.8 years for genotype 2/3.


Antiviral treatment reduced the future cost of hepatitis C complications by $27,500 overall, $22,900 for genotype 1 and $40,100 for genotype 2/3. Compared to no antiviral treatment, cost-effectiveness ratios for treatment were $5100 per discounted quality-adjusted life year gained overall, $9000 per discounted quality-adjusted life year gained for genotype 1 and $400 per discounted quality-adjusted life year gained for genotype 2/3.



Weight-based peginterferon α-2b+ribavirin should be cost-effective for patients with F1 fibrosis. Cost effectiveness improved in the presence of risk factors associated with more rapid fibrosis progression: advanced age, men, IVDA, A23 inflammation.

Does Non-Alcoholic Fatty Liver Disease (NAFLD) and Its Risk Factors Affect the Progression of Fibrosis in HCV Patients?

Joel Kertznus, Arie Regev, Enrique Molina, Jonatan Konfino, Patricia Mendez, Adriana Arcila, Eugene R. Schiff



Previous cross sectional studies suggest that patients with chronic hepatitis C (HCV) and superimposed NAFLD, or its risk factors, may be at increased risk of developing advanced fibrosis. Risk factors associated with NAFLD include: obesity, type II diabetes mellitus (DM), hyperlipidemia and hypertension (HTN). Previous studies were limited, as they did not assess the progression of fibrosis over time. Furthermore, none of the studies included hypertension as an independent risk factor for progression of fibrosis.



To asses the impact of body mass index (BMI), type II DM, hyperlipidemia, HTN and hepatic steatosis on the progression of HCV related fibrosis.



We retrospectively reviewed the medical records of 60 patients diagnosed with HCV, who underwent liver biopsies on two different occasions. Obtained data included demographics, BMI and medical history. All the pathology specimens were reviewed by a liver pathologist using the Scheuer fibrosis score. Extent of steatosis was graded based on the grading system proposed by Brunt et al. The rate of progression was assessed using the calculated difference between stages divided by the period of time elapsed between biopsies. Analysis of variance for repeated measures was used to study the variables.



Sixty patients (41 males, 19 females) were reviewed. The mean ages were 57 and 53 respectively, and the mean BMI was 27.8 4.6 (Kg/m2). The mean time between biopsies was 3.72 2.53 (years). 16 pts (27%) had HTN, 7 (11.7%) type II DM and 17 (28%) hyperlipidemia. None of the patients were active alcohol drinkers, but 13 (22%) reported a past history of alcohol >50g/day and 14 (23%) <50 g/day. Steatosis was documented in 38% of the cases. HCV genotype was 1 in 88%, 2 in 5%, 3 in 5% and 4 in 2% of the patients. Patients with HTN showed a mean stage of fibrosis progression of 1.1, compared to a mean stage of progression of 0.18 in the normotensive patients (p<.0125), determining a significant interaction between HTN and progression of fibrosis. There was no significant association between the presence of type II DM, hyperlipidemia, high BMI and the progression of liver fibrosis.



Hypertension was an independent predictor for the progression of HCV related fibrosis . However, none of the other NAFLD risk factors independently predicted a progression of fibrosis. In addition, steatosis was not identified as an independent risk factor for progression of fibrosis in HCV patients. Larger prospective studies are needed to clarify the association between NAFLD risk factors and the progression of HCV related fibrosis.