Sequential Serum Hepatitis C Viral RNA Levels Longitudinally Assessed by Branched DNA Signal Amplification

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1 Sequential Serum Hepatitis C Viral RNA Levels Longitudinally Assessed by Branched DNA Signal Amplification STUART C. GORDON, 1,2 PETER J. DAILEY, 3 ANN L. SILVERMAN, 1,2 BILAL A. KHAN, 1 VALLISTRAM P. K ODALI, 1 AND JUDITH C. WILBER 3 The aim of this study was to determine the stability of viral load over an extended period in patients with chronic hepatitis C virus (HCV). Sequential serum specimens collected from fourteen non-alcoholic adult patients with chronic HCV between 1990 and 1997 were tested retrospectively for HCV RNA levels by branched DNA assay (Quantiplex HCV RNA 2.0 [Chiron Diagnostics, Emeryville, CA]). A minimum of three serum samples was obtained at various intervals from each patient. None of the patients received antiviral therapy. Liver biopsies, available for 10 of 14 patients, showed mild or moderate hepatitis in seven and cirrhosis in three (one developed cirrhosis during followup). RIBA strip immunoassay showed that 7, 3, and 4 patients had viral genotypes 1, 2, and 3, respectively. The follow-up time averaged 5.3 years (range, 3.7 to 6.6 years). Eight patients (57.2%) showed increased viral levels from baseline to follow-up, the remaining six patients (42.8%) showed decreased viral levels. The three cirrhotic patients had the highest viral levels over time. The mean change was a 0.29-fold decrease (median, 5.08; range, to 7.32). A less than twofold change in either direction was demonstrated for six patients (42.8%), and a less than threefold change was demonstrated for 10 patients (71.4%). Variation from baseline to last follow-up as calculated by log determination showed that the viremic load varied less than one log 10 in all but one individual. These results show that viral load remains relatively stable over prolonged periods in most untreated patients with chronic hepatitis C. (HEPA- TOLOGY 1998;28: ) Abbreviations: HCV, hepatitis C virus; ALT, serum alanine aminotransferase; bdna, branched DNA; HIV, human immunodeficiency virus; CV, coefficient of variance. From the 1 Division of Gastroenterology-Hepatology, William Beaumont Hospital, Royal Oak, MI; the 2 Wayne State University School of Medicine, Detroit, MI; and the 3 Chiron Corporation, Emeryville, CA. Received March 13, 1998; accepted July 31, Address reprint requests to: Stuart C. Gordon, M.D., Division of Gastroenterology- Hepatology, William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, Michigan Fax: (248) ; sgordon@beaumont.edu. Copyright 1998 by the American Association for the Study of Liver Diseases /98/ $3.00/ Before the advent of the molecular technology to measure hepatitis C virus (HCV) RNA, it was recognized that serum alanine aminotransferase (ALT) levels fluctuated in a sawtooth pattern throughout the course of this chronic infection. 1 Subsequent studies reported no relationship between serum ALT and HCV RNA levels, 2 and it is therefore unclear whether viral load fluctuates in a manner analogous to that of liver biochemistries. The relationship between HCV RNA levels and liver histology and clinical outcome is controversial. 3,4 However, successful viral eradication by alpha interferon therapy clearly depends on pretreatment viral load. 5,6 As such, the viral level is clinically significant. It is assumed, but not confirmed, that viral load remains relatively stable within a given patient. Serum HCV RNA may be quantified by several methods, including target amplification (polymerase chain reaction assays), 7 signal amplification, 8 and isothermal transcriptionbased nucleic acid amplification. 9 Of these, branched DNA (bdna) signal amplification technology is standardized and has high precision and reproducibility, 10 and may therefore detect potentially small fold changes in HCV RNA levels. The bdna assay uses a microtiter well format to directly measure HCV RNA by linear signal amplification and chemiluminescent detection. The first generation bdna assay showed reduced sensitivity for HCV genotypes 2 and 3, 11,12 and therefore previous reports that used this assay should be interpreted with caution. 13 The second-generation bdna assay equally measures RNA from HCV genotypes 1, 2, and 3 and can detect HCV RNA levels as low as 200,000 copies/ ml. 13 The natural fluctuation of viral load during the course of chronic hepatitis C infection is not well defined. Knowledge of the spontaneous variation of HCV RNA levels may shed light on the pathogenesis of viral persistence of this disease. In this study, we sought to determine the stability of viral load over an extended period in untreated patients with chronic HCV. PATIENTS AND METHODS Study Population. From a well defined cohort of patients with chronic HCV, 14 we studied 14 untreated individuals who were seen on a regular basis at our outpatient Hepatology Clinic and for whom frozen serum samples were available for analysis. These patients (nine women, five men; age range at follow-up, 34 to 80 years old) were followed at varying time intervals between August, 1990 and April, All patients were initially referred for evaluation of a positive anti-hcv blood test. HCV viremia was confirmed by a qualitative polymerase chain reaction assay in all patients, and each was HBsAg- and anti-hiv-negative. No other cause for chronic liver disease existed in any patient. No patient was treated with antiviral or other immunosuppressive agent (each declined therapy). Aliquots of serum samples were stored at 80 C within 1 hour of collection at the time of the patient s clinic visit. Each patient had a minimum of three serum samples obtained at various intervals throughout the study period (maximum, six samples). Epidemiological data from the 14 individuals are given in Table 1. Seven patients reported prior injection drug use, and the presumed disease duration for these patients was calculated according to the first year of drug use. Five persons were previously transfused, each on one occasion, and the estimated disease duration for these patients was calculated according to the year of transfusion. Two patients, including a 70-year-old Italian-born man, reported un-

2 HEPATOLOGY Vol. 28, No. 6, 1998 GORDON ET AL Patient ID Age at Follow-Up (yrs) TABLE 1. Clinical Features of Patient Population Sex HCV Genotype Mode of Transmission Presumed Disease Duration (yrs) Biopsy Result A 39 M 1 IDU 15 mild B 38 M 3 IDU 18 C 41 F 3 IDU 17 D 40 M 3 IDU 16 moderate* E 41 F 3 IDU 23 F 74 M 2 BT 25 G 34 F 1 IDU 9 moderate H 41 M 1 Unknown mild I 69 F 1 BT 19 moderate J 70 M 1 Unknown cirrhosis K 37 F 1 IDU 14 mild L 75 F 2 BT 18 mild M 80 F 1 BT 28 mild N 74 F 2 BT 20 cirrhosis Abbreviations: F, female; M, male; IDU, injection drug use; BT, blood transfusion. *Developed cirrhosis during follow-up. known risk factors. Diagnostic liver biopsies were done on 10 of the 14 patients. All had varying degrees of histologic chronic hepatitis, including two patients who had cirrhosis and one who initially had moderate chronic hepatitis and then developed cirrhosis during the follow-up period. Four patients declined liver biopsy. Hepatitis C Virus RNA Quantification. Serum HCV RNA determinations were done retrospectively on frozen serum samples using the Quantiplex HCV RNA 2.0 assay (Chiron Diagnostics, Emeryville, CA), which is based on bdna technology. The bdna assay uses a set of synthetic oligonucleotide probes to bind HCV RNA molecules to a solid phase, followed by linear signal amplification to generate a chemiluminescent signal that is proportional to the amount of HCV RNA in the sample. 15 Included in each assay run were high and low control samples. The high control sample was a normal human serum with a known concentration of recombinant single-stranded DNA phage containing the 5 untranslated sequence of HCV. The low control was a normal human serum sample containing propiolactone-treated HCV. The concentration of HCV RNA in each sample was determined from a standard curve, and results were expressed in copies per ml. A quantification limit of copies per ml and a high level of reproducibility (mean coefficient of variance, 14%) have been reported for this assay. 13 All samples and controls were tested in duplicate according to the instructions in the package insert. As described in a previous report, 3 serum samples collected early in this study from patients infected with HCV genotype 1 had been tested with the first generation bdna assay (Quantiplex HCV RNA 1.0, [Chiron Diagnostics]). A subset of these specimens was stored at 60 C or lower for 4 years and then retested with the Quantiplex HCV RNA 2.0 assay (Chiron Diagnostics) in this study. Accurate quantification of HCV RNA from patients infected with HCV genotype 1 has been documented for the first and second generation bdna assays. 11,13 Other Methods. The HCV genotype for each patient was determined by using a strip immunoblot assay system. 16 Histological analysis of liver biopsy specimens was based on standard nomenclature classifications of chronic hepatitis. 17 Statistical Analysis. SAS version 6.12 (SAS Institute, Inc., Cary, NC) was used for all statistical calculations. All HCV RNA levels were analyzed after log 10 transformation. RESULTS Retrospective analysis of HCV RNA levels in sequential serum specimens was performed using the Quantiplex HCV RNA 2.0 assay (Chiron Diagnostics). In addition to assay standards, both high and low control samples were included in each assay run. The high control sample gave a mean quantification equivalent to 7.8 log 10 HCV RNA copies/ml and a coefficient of variance (CV) of 17%. The low control gave a mean quantification equivalent to 6.15 log 10 HCV RNA copies/ml and a CV of 12%. The effect of long-term storage on HCV RNA level in serum specimens collected in this study was assessed by comparing quantification values measured before and after 4 years of storage at 60 C or lower. Six serum samples collected at early time points had been previously tested by using the first generation bdna assay (Quantiplex HCV RNA 1.0 [Chiron Diagnostics]). These samples were stored for 4 years at 60 C or lower and then retested with the Quantiplex HCV RNA 2.0 assay (Chiron Diagnostics). The analysis was limited to samples from patients infected with HCV genotype 1 because this viral genotype would be expected to be quantified accurately by both the first and second generation bdna assays. 11,13 There was no significant change in HCV RNA levels in these samples during storage (P.67). The HCV RNA levels in sequential serum samples from all 14 patients with chronic HCV are shown in Fig. 1. The follow-up time for this patient population averaged 5.3 years (range, 3.7 to 6.6 years). Eight patients showed an overall increase in HCV RNA levels from baseline to last follow-up, and the remaining 6 patients showed an overall decrease in viral load. The three cirrhotic patients (patients D, J, and N) showed the highest HCV RNA levels over time. In addition to HCV RNA measurements, serum ALT determinations were made at several time points during follow-up. Figure 2 shows the comparison of HCV RNA and ALT values on a per-patient basis over time. Immunoblot analysis showed that seven individuals were infected with HCV genotype 1, three with HCV genotype 2, and four with HCV genotype 3 (Table 1). No difference in variability of HCV RNA levels over time between those infected with different HCV genotypes was found. The overall fold-change and log 10 variation in viral load as determined by comparing HCV RNA levels at baseline and last follow-up is given in Table 2. The mean fold change in HCV RNA levels for all 14 patients was a 0.29-fold decrease (median, 5.08; range, to 7.32). Six patients (42.8%) showed a less than twofold change in viral load in either direction, and 10 patients (71.4%) showed a less than threefold change. Variation from baseline to last follow-up as calculated by log determination showed that the HCV RNA levels varied by less than one log 10 in all but one individual (patient L). A t-test was used to test whether there was a significant mean change in log 10 scale between the first and last HCV RNA level for this cohort and whether the rate of change was significantly different from zero. The rate of change was defined as the change between the first and last log 10 HCV RNA levels divided by the follow-up time from baseline to last visit. Because the lengths of follow up for patients was not equal, both the weighted and unweighted mean log 10 differences and rate of change were evaluated. No significant change was seen in the mean level of HCV RNA (unweighted means, P.92; weighted means, P.88) or in the mean rate of change of HCV RNA over time (unweighted means, P.89; weighted means, P.73). A mixed model approach was used to evaluate all of the

3 1704 GORDON ET AL. HEPATOLOGY December 1998 FIG. 1. HCV RNA levels in sequential serum samples from 14 chronic HCV patients as measured with the bdna assay. cohort data. Because of the hierarchical structure of the viral load data (i.e., observations were nested within persons) and irregular follow-up intervals, the mixed model used in the analysis was a linear regression model with random coefficients, which can be considered as a two-level model. In the first level, a regression model was created for each individual using time since baseline as an independent variable, and HCV RNA (in log 10 scale) as a dependent variable. In the second level, the model assumed that the random slopes from each individual were normally distributed around a global mean slope across the whole cohort with a variance component that could be estimated from the data. Fitting this model to the data, the global slope was estimated as , which is not significantly different from zero (P.9286). DISCUSSION The correlation of degree of viremia with other parameters of infection assumes that viral load is a relatively stable parameter in a given patient. 10,18 Initial reports suggested that viremia itself periodically disappears during the course of acute HCV, 19,20 wherein sharp fluctuations in viral levels may represent the opposing forces of viral replication and immunity. Titers of hepatitis C viral levels generally decline in the months following acute infection. 20 Early reports among chronically infected individuals suggested the possibility of intermittent viremia in hemophiliacs with chronic HCV 21 and in a study of community-acquired chronic HCV. 22 Using quantitative viral assays, recent reports suggest that level of viremia is a relatively stable phenomenon. In the study by Nguyen et al., 23 serum HCV RNA levels were assayed daily, weekly, and monthly for 3 months. These investigators showed minuscule fluctuations throughout the study period and concluded that serum viral levels remained relatively stable over this brief interval. Hollingsworth et al. 18 followed the viral loads of 11 individuals with chronic HCV over a mean of 11 months and found that HCV RNA levels varied little over this period of time. Yoshimura et al. 24 likewise found no significant change in viral load over a 3-year period among a group of patients with chronic HCV that included hemodialysis patients. The present report essentially extends and validates these shorter-term follow-up studies. These observations confirm the relative stability of viremic load over time and suggest that the concept of intermittent viremia without intervention is an unlikely phenomenon in chronic HCV. The observations of the present study differ from those of an earlier study by Eyster et al. 25 in which viral levels over time in both HIV coinfected and noncoinfected hemophiliac individuals were compared. These investigators describe an increase of HCV RNA levels of nearly threefold over several years, and a much more dramatic increase in viral levels among HIV coinfected patients. However, the majority of the HCV /HIV patients had nondetectable HCV RNA levels at baseline, usually from samples dating to the early 1980s. One recent report 26 suggested that freezer-stored serum samples from greater than 5 years may yield significantly lower values as a result of RNA degradation. We found no evidence of RNA degradation in samples stored for 4 years in the present study. In addition, it is now realized that the first-generation bdna assay may have underestimated the viral levels of HCV genotype 3, 11 which comprise the majority of these hemophiliac patients. 27 Previous reports 28,29 have shown a correlation between HCV RNA levels and degree of liver damage. Studies of the

4 HEPATOLOGY Vol. 28, No. 6, 1998 GORDON ET AL FIG. 2. Comparison of serum ALT ( ) and HCV RNA ( ) levels in individual patients over time. relationship between serum HCV RNA levels and liver viral quantification show conflicting data. 30,31 Specific host factors regulating viral replication during chronic infection are not well defined, and it is not known whether a continuum of immune competence may in part determine degree of viremia among apparently immunocompetent patients with chronic HCV. Because of the small sample size, the present study did not Patient ID TABLE 2. Comparison of HCV RNA Levels at Baseline and Last Visit Baseline HCV RNA (log 10 copies/ml) HCV RNA at Last Visit (log 10 copies/ml) Fold Change Log 10 Change A B C D E F G H I J K L M N attempt to establish a relationship between viral levels and other clinical parameters, such as age, gender, liver histology or mode of transmission. We have previously reported such associations 3 among a larger cohort of patients, and found that patients with more aggressive histological activity (including cirrhosis) had higher levels of viremia. The present study sample size was too small to make such a correlation, but it was noted that one patient (patient D) evolved to cirrhosis over the 5-year follow-up of this study, and the two other cirrhotic patients (patients J and N) had the highest viral levels over time. Patient J has now developed hepatic decompensation and hepatocellular carcinoma. As increasing numbers of HCV patients undergo antiviral therapy, cohorts of untreated individuals such as those described herein will become increasingly scarce. This is unfortunate, because it remains to be established whether a subset of patients with chronic hepatitis C are nonprogressive. Among HIV type-1 infected persons, for example, HIV RNA plasma levels are considerably lower in patients with nonprogressive disease. 32,33 Such data suggest the need to evaluate prospectively chronic HCV patients to determine whether viral levels correlate with disease progression and long-term prognosis. Note added in proof: Patient L, the one patient in this study in whom viral levels dropped by greater than one log 10 over time, was HCV RNA negative by bdna assay (although

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