Natural Fluctuations of Hepatitis C Viral Load in a Homogeneous Patient Population: A Prospective Study

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1 Natural Fluctuations of Hepatitis C Viral Load in a Homogeneous Patient Population: A Prospective Study LIAM FANNING, 1 ELIZABETH KENNY-WALSH, 1 JOHN LEVIS, 1 KINGSHUK ROY CHOUDHURY, 2 BRIDIN CANNON, 1 MARGARET SHEEHAN, 3 MICHAEL WHELTON, 1 AND FERGUS SHANAHAN 1 The aim of this study was to determine the variation in hepatitis C viral load over an extended period of patient follow up. Serum samples were collected from 49 female individuals who were identified as having been infected from the same source of hepatitis C contaminated anti-d immunoglobulin during the period from 1977 (May) to 1978 (November). All patients attended the hepatitis C clinic at Cork University Hospital, Cork, Ireland. The study group was homogeneous with respect to gender, hepatitis C virus (HCV) genotype (1b), and duration of infection. None of the patients had received antiviral therapy at the time of completion of study. Viral load quantifications were assessed using the Roche Monitor (F. Hoffmann-La Roche, Ltd., Basel, Switzerland) assay. The mean age of the study group at time of infection was 30.3 years (SD 6.1) with a range from 18.5 to 43 years. The mean time of follow-up was 4.1 years (SD 1.0) with a range from 1.2 to 5 years. The mean rate of change of viral load per year was 0.23 log 10 viral copies per ml serum for the study group (SD 0.19) with a range of 0.18 to 0.78 that was significantly different from zero, P F The rate of change of viral load per year was negatively correlated with viral load at first determination, r.35, P.01. Age at infection did not correlate with the slope of change of viral load, P.10. In conclusion, most women infected with HCV 1b will have an increase in viral load over time but a few patients who acquire infection early in adult life will show a decrease in viral load. (HEPATOLOGY 2000;31: ) Abbreviations: HCV, hepatitis C virus; RT-PCR, reverse transcription polymerase chain reaction. From the Hepatitis C Unit, Department of Medicine, 1 Department of Statistics, 2 and Department of Pathology, 3 National University of Ireland, Cork, University College Cork, Cork, Ireland. Received July 21, 1999; accepted October 25, Address reprint request to Liam Fanning, Ph.D., Hepatitis C Unit, Department of Medicine, Clinical Sciences Building, Cork University Hospital, University College Cork, Cork, Ireland. l.fanning@ucc.ie; fax: (21) Copyright 2000 by the American Association for the Study of Liver Diseases /00/ $3.00/0 225 Hepatitis C virus (HCV) infection is the main cause of parenteral non-a, non-b hepatitis. 1-5 HCV consists of a heterogeneous mix of isolates defined by genotype, each of which is further classified into subtypes. A number of factors have been identified as important in predicting the outcome of disease progression. These include age at infection, viral type/subtype, viral load, quasispecies, and mode of infection Clinical heterogeneity in disease progression may reflect either viral heterogeneity or variations in host response. However, the fluctuations in viral load during the natural history of hepatitis C infection are poorly understood. The purpose of the present study was to determine if viral load remains at a steady state or is in dynamic flux during the course of an HCV infection. To avoid heterogeneity of risk factors and confounding variables in viral type/subtype, we studied a unique cohort of individuals all infected by anti-d/ blood product from a single source of HCV 1b. The sole source of the infectious agent was identified as HCV 1b contaminated anti-d immunoglobulin. The contaminating HCV 1b was derived from a single donor The patients were of similar ethnogeographic background and had an absence of other risk factors for liver disease. Serum HCV RNA can be quantitated by a range of different technologies (reverse transcription-polymerase chain reaction [RT-PCR], RT-PCR coupled to different signal amplification systems such as colorimetric assays, and the branched chain technology) The level of sensitivity achieved by the more recent versions of these technologies has improved qualitative assessment of HCV serum status. Clinically relevant sensitivities of 100 viral genomes per ml of serum are now readily achievable. The work reported here used the Roche Monitor system for amplification of the HCV. The high degree of reproducible quantification of viral load and a uniform linear range of amplification between the different versions of the Roche HCV Monitor assays for HCV of genotype 1b provide a means in which quantifications over several generations of assays are suitable for longitudinal studies. In addition, amplification of HCV genotype 1b by the Monitor system does not suffer from the reported difficulties associated with other genotypes. 21 The linear range of amplification of the Roche Monitor assay is 2,000 to 500,000 viral copies per ml of serum (3.3 to 5.7 log 10 viral copies per ml of serum), however, with appropriate dilution this can be extended to viral copies per ml of serum (7.7 log 10 viral copies per ml of serum). Assessment of variations in the viral load during chronic hepatitis C infection may enable the development of models that allow for the better prediction of when patients are likely or not to enter a phase in which rapid progression of the hepatitis C viral infection occurs and in which antiviral therapy is less likely to effect clearance. In this study we sought to assess the natural flux of hepatitis C serum viral load over an extended period in patients with chronic disease who have not had antiviral therapy. PATIENTS AND METHODS The study group defined here is part of a previously wellcharacterized cohort of women infected with HCV 1b contaminated

2 226 FANNING ET AL. HEPATOLOGY January 2000 anti-d immunoglobulin in Ireland during the period from May 1977 to November ,20 All patients attended the hepatitis C clinic at Cork University Hospital, Cork, Ireland. All patients were evaluated sequentially at this single center. Demographic profiling of the patients in this cohort confirms the homogeneity of this group, i.e., the study population consisted of 49 women, all were Rhesus negative with chronic hepatitis C type 1b, and all were recipients of HCV-contaminated anti-d immunoglobulin by intravenous injection from 1977 to The study group were all of the same ethnic origin, all were healthy childbearing women at time of infection, and all had an absence of competing risk factors for infectious and other liver diseases. None of the patients had received antiviral treatment at the time of study. Each patient had a minimum of 3 separate serum viral load determinations. Investigations were performed with informed consent and complied with a standardized protocol in compliance with standards of care and in accordance with guidelines of the University Hospital ethics committee. This included a detailed history regarding competing risk factors for hepatitis C such as other blood or blood products, previous or continuing intravenous drug abuse, tattooing, occupational health exposure, sexual or household contact with a person who had hepatitis, homosexual contact, promiscuity, and organ transplant. Alcohol intake was assessed by direct questioning in a structured format as to the number of drinks per day and was measured in units, in which 1 unit of alcohol was defined as a half a pint of beer, or a glass of wine, with each unit taken as containing 10 g of alcohol. 22 The alcohol intake for the patient study group was between 0 to 4 units of alcohol per week. Sample Storage and Virological Assessment. Each serum specimen was aliquoted upon receipt and immediately transferred to a 80 o C storage facility. All of the samples used for this study were analyzed within 1 month of receipt. Each specimen used for HCV qualitative assessment and viral load measurement was exposed to a single freeze-thaw cycle. The virologist was blind to the clinical status of each patient. Qualitative RT-PCR was performed on patient serum using the Roche Amplicor HCV kit (F. Hoffmann-La Roche, Ltd., Basel, Switzerland). Serum quantitative RT-PCR was performed using the Roche HCV Monitor kit (F. Hoffmann-La Roche, Ltd.). All hepatitis C viral quantification from RNA extraction to end point colorimetric detection was performed in triplicate. On first determination those samples found to be above the linear range of the assay were diluted accordingly and the quantification repeated in triplicate. Secondary standards, of defined transcript number, were used in each assay to validate the viral-load quantification. Baseline viral-load quantification was paired with liver biopsy for each individual. Liver Biopsy. Liver biopsy was performed using a Menghini needle under ultrasound guidance on all individuals in the study. All specimens were examined by the same pathologist who was blind to the biochemical and serological data. Liver histology was scored as recommended by the International Association for the Study of the Liver and other Working Parties, using the Histological Activity Index HCV Genotype Analysis. HCV type/subtype analysis was performed on all individuals using the amplicons resulting from the qualitative HCV RT-PCR. Identification of HCV type/subtype was by line-probe assay (Inno-Lipa HCV II; INNOGENETICS, Zwijndrecht, Belgium). Description of Statistical Testing. All HCV RNA determinations were analyzed after log 10 transformation. Measurement of the change of viral load over time was done in 2 ways, similar to the measures used by Gordon et al. 26 The first measure was the rate of change, defined as the change between the first and last log 10 HCV levels divided by the follow-up time between baseline and last follow up, i.e., HCV(T last ) log 10 HCV(T first ) rate of change log 10 T last T first However, this measure does not use the information contained in visits between the first and the last. The alternative mixed model approach used by Gordon et al. 26 fits a separate linear regression for each individual, with time since baseline as the independent variable and log 10 HCV levels as the dependent variable. The change then is measured as the slope (b) of this regression, i.e., log 10 HCV(T) c bt. In our analysis, we examined both the rates of change and slope as measures of the change in viral load. RESULTS As previously reported all the women were confirmed positive for the HCV and infected with HCV of genotype 1b. 20 The mean age subsequent to infection, in 1979, was 30.3 years (SD 6.1) with a range from 18.5 to 43 years (Fig. 1). The duration of follow-up was 4.1 years (SD 1.0 years) range 1.2 to 5 years. The grade of inflammation ranged from 0 to 7 (mean 3, SD 1) (Fig. 1) and the stage of fibrosis ranged from 0 to 4 (mean 1, SD 1). The range of the fold difference between baseline and last viral load assessment was 0.22 ( 0.65 log 10 viral copies/ml) to (2.33 log 10 viral copies/ml) with a mean of (1.38 log 10 viral copies/ml) SD (1.64 log 10 viral copies/ml). Only 8.2% (n 4) of the individuals in the study group showed a decrease in viral load between initial base line assessment and last follow-up sample, range 0.65 to log 10 viral copies per ml serum, mean 0.42 (SD 0.27) log 10 viral copies per ml (Fig. 1). The range of the viral load increase for the remaining 91.2% (n 45) of individuals in the study group was to 2.33 log 10 viral copies per ml of serum, mean 0.83 (SD 0.77) (Fig. 1). None of the individuals greater than 30 years of age at infection showed a decrease in serum viral load by completion of the study. The rate of change in viral load during the study period was calculated as a log 10 determination per year. This showed that the mean rate of viral load change per year was 0.23 log 10 viral copies for the study group (SD 0.19) with a range of 0.18 to 0.78 log 10 viral copies per ml. The rate of change of viral load per year was negatively correlated with viral load at first determination, r.35, P.01 (correlation coefficient r) (Fig. 2). The age of the infected women in 1979 did not correlate with the baseline assessment of viral load, r.08, P.585. However, age in 1979 did correlate with end of FIG. 1. Change in viral load and degree of inflammation. For each of the 49 patients, the symbols above the individual patient identification numbers show the change in viral load during the study period (P for first and w for last determination) and the degree of inflammation for the same patient at baseline (s). The 4 women who showed a decrease in hepatitis C viral load during the study are identified by open symbols.

3 HEPATOLOGY Vol. 31, No. 1, 2000 FANNING ET AL. 227 per ml per year, which was significantly different from zero, P (Fig. 3). The mean slope of change of viral load was numerically almost identical to the rate of change, with a mean value of 0.24 log 10 viral copies per ml per year, which was also significantly different from zero, P FIG. 2. Scatter plot of the rate of change of viral load per year and baseline viral load. Rate of change was defined as the log 10 difference between the last and first viral load determinations divided by the length of follow up in years between the first and last viral load quantification. The negative correlation between rate of change of viral load and baseline viral load of HCV 1b was statistically significant, r.35, P.01. DISCUSSION As therapeutic options for the treatment of chronic hepatitis C infection become more efficacious, relatively large numbers of untreated individuals will be unavailable for study. Consequently, there will be diminishing opportunities for investigation of the natural variance in virological and clinical parameters currently used to assess disease progression and long-term prognosis. The observations of the present study accrue from a large-defined group of untreated individuals with the same gender, same defined source of infection, same viral type/subtype, and a known duration of infection. The results of our investigation show that viral load increases over time in women infected with HCV 1b and that intermittent or spontaneous clearance without antiviral therapy is an unlikely event. The results detail the significance of the age at infection and its associations with both increasing viral load and the rate of change of viral load per year during the study period. Specimen processing and storage conditions can influence the stability, and hence the detectability, of HCV RNA. Well controlled sample processing and storage conditions are critical to the quantitative analysis of HCV RNA. 27 Cryopreservation of specimens at 80 o C appears to offer the most stable conditions for long-term storage 26,28 in which possible prompt measurement of viral load without long-term cryopreservation is the preferred option. Long-term cryopreservation for several years with subsequent batch analysis may yield follow-up viral load assessment, r.346, P.015. The mean rate of change of viral load for the study group was 0.23 log 10 viral copies per ml per year. Exclusion of the 4 women who showed a decreased viral load over time did not significantly alter the mean rate of change of viral load, log 10 viral copies per ml per year, P.41. The mean rate of change of viral load for those women who were 30 and 30 years of age in 1979 was statistically different, P.032, at 0.15 and 0.31 log 10 viral copies per ml per year, respectively. The mean rate of change of viral load between the 2 age groups remained significantly different if the 4 women with a decrease in viral load were excluded from the analysis, P.029. The rate of change of viral load was found to be associated with age of acquisition of HCV r.42, P.003 (Fig. 3). Furthermore, if the 4 women who had a negative rate of change were excluded from the analysis, the association between age at acquisition and rate of change of viral load remains statistically significant, r.39, P.009. A correlation between the slope of change of viral load with age for the entire study group was identified, but was of borderline significance, r.28, P.053. The strength of this observation was weakened if those 4 women with a negative slope of change were excluded from the analysis, r.24, P.10. The mean rate of change of viral load for those women who showed an increase in viral load was 0.22 log 10 viral copies FIG. 3. Combination scatter and box plots of the rate of change of viral load per year and age at infection. Rate of change was defined as the log 10 difference between the last and first viral load determinations divided by the length of follow-up in years between the first and last viral load quantification. Median values are indicated as a solid white line between the 25th and 75th percentile markers. The rate of change of viral load was positively correlated with age of acquisition of HCV 1b, r.42, P.003. A closed circle identifies individuals who had a positive rate of change, n 45. An open circle highlights individuals who had a negative rate of change, n 4.

4 228 FANNING ET AL. HEPATOLOGY January 2000 artefactual changes in viral load and hence affect accurate quantification of the viral RNA. HCV RNA is not stable when stored at ambient or 4 o C for longer than a few days. 28 Multiple freeze-thaw cycles can decrease the integrity of the specimens, resulting in artefactual modifications of viral load. 21,29 All the specimens used for this study were exposed to a single freeze-thaw cycle before qualitative and quantitative evaluations. A single methodology was used to measure the serum viral load in this study group. This has the advantage of providing a quantification methodology with a constant uniformity of linear amplification (3.3 to 5.7 log 10 viral copies per ml) of the template over several generations of the assay, thereby avoiding artefactual variances in viral load due to methological differences. The homogeneity of the viral genotype in this study group also avoids any confounding genotype-associated amplification variances of viral load. 30 We have identified an inverse relationship between the rate of change of viral load and baseline viral load for this cohort of individuals infected with HCV 1b, r.35, P.01 (Fig. 2). This observation may reflect a window in the natural history of hepatitis C infection in which the equilibrium between viral clearance by the host and viral replication is in dynamic flux with a trend towards increased host-viral burden. It is probable that a steady state of viral production and clearance once achieved will exist as the status quo until hepatitis C progresses into a more advanced disease category. There is an apparent lack of consensus with regard to the natural variation in viral load in chronic hepatitis C infection in the published literature. 26,31-33 This diversity of opinion is undoubtedly influenced by the heterogeneity of patient populations with respect to age at infection, gender, duration of disease, viral type/subtype, probable mode of transmission of the virus, duration of study period, and means of viral load quantification. A notable feature of the study group presented here is that most patients have mild to moderate liver disease (Fig. 1), the study group is homogeneous with respect to gender and HCV type/subtype, a single mode of transmission of the HCV, a defined duration of infection, viral load quantification methodology, and competing etiologies such as alcoholic- or acetaminophen-induced liver disease are not present. 20 Disease progression in hepatitis C infection is positively associated with age at exposure. It is accepted that individuals infected after the age of 50 progress more rapidly to active liver disease than those infected before 50 years of age. 34 In this study, we observed a strong trend toward higher rates of increase in viral load and slope of change with more advanced age at time of acquisition of HCV. However, when the 4 women who had a negative slope of change in viral titers were excluded from the analysis, the statistical association between age and slope of change was no longer evident. Based on the assumption of constant viral load increase one can postulate a simple model for changing viral load over time: the mean fold increase in viral load per year for the study group was (on the absolute scale), consequently, the predicted viral load (VL 2 ) at time y is VL y (VL 1 at y ), in which y is any time at which the viral load has been measured and y is the time interval between the two viral load assessments in years. This model may provide a clinically useful tool for the estimation of the period of time in which patients are likely to have a serum viral load correlated with likely response to antiviral therapy However, a greater understanding of the natural history of chronic HCV infection and the correlations between viral load, histological activity indices, age, duration of disease, and gender may lead to more refined models for the prediction of viral load fluctuations over time. In summary, this opportunity to control for the variables that can influence the natural history of hepatitis C infection has helped illuminate some clinically relevant characteristics of this persistent viral infection such as the magnitude of viral load modulation over time and the rate of change of viral load in HCV 1b infection and its relationship to age at infection. There is a natural variance in viral load, and an increased viral load significantly different from zero is evident over time in this study group. 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