Viral Load and Clinicopathological Features of Chronic Hepatitis C (1b) in a Homogeneous Patient Population LIAM FANNING,1 ELIZABETH KENNY,1 MARGARET SHEEHAN,2 BRIDIN CANNON,1 MICHAEL WHELTON,1 JOE O’CONNELL1 J. KEVIN COLLINS,1 AND FERGUS SHANAHAN1
Monitoring the progression of hepatitis C virus (HCV) includes clinical, biochemical, and histological parameters. Quantitation of viral load by reverse-transcription polymerase chain reaction (RT-PCR) may offer a more reliable marker of disease status. Conflicting reports on viral titers may reflect heterogeneity of patient population, mode of infection, and viral type/subtype. The aim of this study was to correlate quantitative serum viral load with alanine transaminase (ALT) and histological status in a homogenous population. The study population consisted of 77 Rhesus-negative women with chronic hepatitis C type 1b. Homogenous features of this study population included: same defined source of infection (contaminated anti-D immunoglobulin); same duration of disease (17 years at the time of study); same viral type/subtype; same ethnic origin; all healthy child-bearing females at the time of infection; and 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. Liver biopsy was performed on all patients. All biopsies were scored by a single histopathologist who was blinded to the clinical and viral status of each patient. A weak, but statistically significant, correlation (rs ⴝ .26; P F .05) between serum viral load and the degree of inflammation (mean value: 3.87 ⴞ 2.17 [SD]) was found. There was no significant correlation between serum viral load and the degree of fibrosis (mean value: 0.84 ⴞ 0.8 [SD]; P ⴝ .06). There was no significant correlation between serum viral load and ALT, although there was a correlation between ALT and the degree of inflammation (rs ⴝ .241; P ⴝ .035). (HEPATOLOGY 1999;29:904-907.) Hepatitis C virus (HCV) infection is the main cause of parenteral non-A, non-B hepatitis.1-5 The prevalence rate varies in the general population from 0.15% (Scandinavia) to as high as 44% (northwest Egypt and southern Cameroon).6
Abbreviations: HCV, hepatitis C virus; PCR, polymerase chain reaction; ALT, alanine transaminase; RT, reverse transcription; RUQP, right upper quadrant pain; ELISA, enzyme-linked immunosorbent assay. From the Departments of 1Medicine and 2Pathology, National University of Ireland, Cork, University College Cork, Ireland. Received June 5, 1998; accepted October 27, 1998. Supported by research grant HC07/96 (to L.F.) from the Health Research Board of Ireland. Address reprint requests to: Liam Fanning, Department of Medicine, Clinical Sciences Building, Cork University Hospital, University College Cork, Cork, Ireland. E-mail:
[email protected]; fax: 21-345300. Copyright r 1999 by the American Association for the Study of Liver Diseases. 0270-9139/99/2903-0039$3.00/0
An estimated 80% of infected individuals develop chronic disease, with progression to cirrhosis in 20% and hepatocellular carcinoma in 4%.7 It is also the most common indication for liver transplantation in the United States.7 The rate of disease progression is variable. A number of factors have been identified as important in predicting the outcome of progression. These include age at infection, gender, viral type/ subtype, viral load, and mode of infection.8-12 Monitoring the rate of progression from chronic hepatitis to cirrhosis and hepatocellular cancer is suboptimal with standard clinical and biochemical techniques. Histology is the main criterion for assessing severity and disease progression.13 Advances in polymerase chain reaction (PCR) technology allow identification and quantitation of serum HCV RNA, but its precise clinical role remains unclear.13-17 A number of studies have suggested a relationship between HCV type, subtype, and serum HCV levels.18-21 However, there are conflicting reports on the relationship between the biochemical markers of inflammation (alanine transaminase [ALT]), histological degree of inflammation, and serum HCV-RNA levels by reverse-transcription (RT)-PCR.22-25 These conflicting results may relate, in large part, to the heterogeneity of the patient groups studied. Patient groupings are often of mixed gender, ethnic origin, ill-defined duration of disease, and mixed HCV genotype/ subtype. The identification of a group of women who were all infected with HCV type 1b through intravenous anti-D immunoglobulin administration in 1977 provides a uniquely homogenous cohort to study the progression of HCV infection.26 The homogenous features of this study population included: gender, ethnic origin, similar duration of disease (17 years at the time of obtaining specimens), single defined source of infection and likely similar inoculum size (contaminated anti-D immunoglobulin), same viral type/subtype, and an absence of competing risk factors and other possible causes of concomitant liver disease. None of the patients had received antiviral treatment at the time of study. Patients were initially identified as having been exposed to HCV through a national screening program for all anti-D recipients since the introduction of anti-D therapy in Ireland in the late 1960s. This screening program was set up in response to a link identified between HCV infection in a female patient population whose only linking risk factor for hepatitis C was the administration of intravenous anti-D immunoglobulin in their postpartum period in 1977.26 The purpose of this study was to investigate the relationship between viral load, serum transaminase levels, and liver histology 17 years’ post-HCV infection in a homogenous patient population.
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PATIENTS AND METHODS Study Population. In total, 82 viremic patients (HCV type 1b) were identified as being in receipt of HCV-infected anti-D immunoglobulin. Archival samples of the batches of anti-D administered in 1977 have been tested and found positive for HCV type 1b. This was given to them postpartum in 1977. Of the 82 patients assessed, 5 were excluded from the study, because the serum viral load was unavailable within 6 weeks of liver biopsy. After exclusion of these patients, a total of 77 patients were assessed. Demographic profiling of the patients in this cohort confirms the homogeneity of this group, i.e., the study population consisted of 77 women (with a mean age of 44.6 ⫾ 4.5 years at the time of diagnosis and all were Rhesus-negative) with chronic hepatitis C type 1b; all were recipients of HCV-contaminated anti-D immunoglobulin by intravenous injection in 1977; same duration of disease (17 years at the time of study); same ethnic origin; all healthy child-bearing females at the time of infection; and 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. A detailed analysis of the spectrum of symptoms exhibited by the patients in the study group gives the following breakdown: arthralgia/myalgia, n ⫽ 10; arthralgia/myalgia and right upper quadrant pain (RUQP), n ⫽ 2; depression/anxiety, n ⫽ 3; depression/anxiety and RUQP, n ⫽ 1; epigastric discomfort, n ⫽ 1; fatigue, n ⫽ 20; fatigue and arthralgia/myalgia, n ⫽ 1; fatigue, arthralgia/myalgia, and right upper quadrant pain, n ⫽ 2; fatigue and depression/anxiety, n ⫽ 2; fatigue and skin rash, n ⫽ 1; RUQP, n ⫽ 5; and asymptomatic, n ⫽ 17. Investigations were performed with informed consent and complied with a standardized protocol in compliance with the standard of care, and therefore in accordance with guidelines of the University Hospital ethic’s 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 transplantation. Alcohol intake was assessed by direct questioning in a structured format as to the number of drinks per day and was measured in units, where 1 unit of alcohol was defined as a half-pint of beer or a glass of wine, with each unit taken as containing 10 g of alcohol.27 The alcohol intake for the patient study group was between 0 to 4 units of alcohol per week. Biochemical investigations included serum transaminases, ALT, aspartate transaminase, and ␥-glutamyl transferase. 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.). Histological Assessment. Liver biopsy was performed using a Menghini needle under ultrasound guidance in all 77 viremic patients. The biopsy specimens were fixed in formalin for histological examination. All specimens were examined by a pathologist who was blinded 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; Histological Activity Index.28-30 Inflammation was scored on an 18-point scale: interface change/piecemeal necrosis 0-4, confluent necrosis 0-6, lobular inflammation 0-4, and portal inflammation 0-4. Fibrosis was staged as 0 ⫽ no fibrosis; 1 ⫽ fibrous expansion of some portal areas with or without short fibrous septa; 2 ⫽ fibrous expansion of most of the portal area with or without short fibrous septa; 3 ⫽ fibrous expansion of most of the portal areas with occasional portal-portal bridging; 4 ⫽ fibrous expansion of portal areas with marked bridging; 5 ⫽ marked bridging with occasional nodules (incomplete cirrhosis); 6 ⫽ cirrhosis, probable or definite. Qualitative Detection of Serum HCV. At the commencement of the study, all 77 patients were confirmed positive for serum HCV RNA. Qualitative HCV RT-PCR was performed using the Roche Amplicor Hepatitis C virus test (F. Hoffmann-La Roche Ltd.) in accordance
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with the manufacturer’s specifications. HCV RNA was isolated from 100 µL of serum using a guanidine thiocyanate lysis buffer. After heat treatment at 60°C for 10 minutes, an equal volume of isopropanol was added. Following incubation at room temperature for 2 minutes, the RNA was precipitated by centrifugation. The pellet was washed once in 70% ethanol and resuspended in a bicine buffer containing manganese. Briefly, RT and PCR of the 58 untranslated region was performed on isolated RNA in a single reaction using the Tth DNA polymerase. Analysis of the amplicons was by enzyme-linked immunosorbent assay (ELISA). Three HCV-positive control reactions (consisting of five transcripts per reaction) and one negative control reaction were included in each qualitative HCV RT-PCR. Quantitative Measurement of Serum HCV Load. Quantitative HCV RT-PCR was performed using the Roche Monitor Hepatitis C virus test (F. Hoffmann-La Roche Ltd.). Each specimen was analyzed in triplicate and the mean value reported as the viremic level in the serum. Briefly, HCV RNA was isolated from 100 µL of serum using a guanidine thiocyanate lysis buffer. After heat treatment at 60°C for 10 minutes, an equal volume of isopropanol was added. Following incubation at room temperature for 2 minutes, the RNA was precipitated by centrifugation. The pellet was washed once in 70% ethanol and resuspended in a bicine buffer containing manganese. A known quantity of internal standard was included in each preparation of HCV RNA. RT-PCR of a region in the 58 untranslated region was performed as suggested by the manufacturer, i.e., 50°C for 2 minutes, 60°C for 30 minutes, 1 minute at 95°C, followed by two cycles of 95°C for 20 seconds and 20 seconds at 60°C. This was followed by a further 33 cycles at 90°C for 20 seconds and 20 seconds at 60°C. Quantitation of the amount of amplicon in each RT-PCR was by ELISA. HCV Genotype Analysis. HCV type/subtype analysis was performed using the amplicons resulting from the qualitative HCV RT-PCR. Identification of HCV type/subtype was by line-probe assay (InnoLipa HCV II, INNOGENETICS, Zwijndrecht, Belgium). Briefly, this was performed by using nitrocellulose with type- and subtypespecific DNA probes attached, hybridizing denatured RT-PCR products at 50°C, removing nonhybridizing DNA, and visualizing the banding pattern; thus, identification of HCV type/subtype was achieved by ELISA. Statistical Analysis. Spearman Rank correlation (rs) coefficients were used to assess the correlation among serum ALT, serum quantitative HCV RNA, and the degree of histological damage. Hepatitis A and Hepatitis B Virus Status. Testing for hepatitis B surface antigen, antibody to core antigen, and hepatitis A was performed using commercially available ELISAs (Abbott Laboratories, North Chicago, IL). RESULTS
A statistically significant relationship was found between the serum quantitative HCV-RNA titers (n ⫽ 77) and the degree of inflammation, with rs ⫽ .26 and P ⬍ .05 (Fig. 1). However, no significant relationship was found to exist between the HCV-RNA quantitative level and the degree of fibrosis of disease (Fig. 1, inset), with rs ⫽ .22 and P ⬎ .05. While there was a statistically significant relationship identified between the serum ALT and grade of inflammation (rs ⫽ .241; P ⫽ .035) (Fig. 2), no statistically significant relationship was identified between serum ALT and stage of fibrosis (rs ⫽ .17; P ⬎ .05) (Fig. 2, inset), and there was no statistically significant relationship identified between the degree of serum ALT elevation and serum viral load. DISCUSSION
Our results indicate a relationship between serum viral load and the degree of inflammation in this relatively homogenous group of patients with chronic HCV infection. There is also a statistically significant relationship between
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FIG. 1. Correlation between serum HCV titer and the histological degree of inflammation yields a correlation value of rs ⫽ .26, P ⬍ .05. Inset: Statistical analysis of serum HCV load and the histological degree of fibrosis gives a correlation value of rs ⫽ .22, P ⬎ .05.
the level of serum transaminase and the degree of inflammation. Analysis of disease progression in the cohort of HCVinfected patients presented in this study is undoubtedly influenced by the homogenous nature, the narrow histological spread of disease severity to date (with the majority being within mild to mild-moderate categories on the histological activity index), and perhaps the specific immunological time frame at infection, i.e., within 48 hours’ postpartum. The apparent conflict in previously reported studies may in part reflect: 1) the heterogeneity of patient populations with respect to age, gender, duration of disease, viral type/subtype, and method of contamination; 2) the inherent risk of sampling error in liver biopsy specimens; and 3) the use of different quantitative methods to assess serum HCV-RNA titers at unspecified time spans between serology and liver biopsy. A notable feature of the study group presented here is that most patients have mild to moderate disease, and few have severe disease at this stage. Our study cohort is representative and reflects the relative mild course of this particular form of hepatitis C 1b in Ireland (i.e., HCV
FIG. 2. Statistical analysis of serum ALT levels and the degree of inflammation yields a correlation value of rs ⫽ .241, P ⫽ .035. Inset: Statistical analysis of serum ALT levels and the degree of fibrosis gives a correlation value of rs ⫽ .17, P ⬎ .05.
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1b-contaminated Anti-D, from a single source), and clinical details from a larger patient population infected with HCV from the same source in Ireland support this.31 The influences of competing etiologies such as alcoholicor acetaminophen-induced liver disease are not present in this cohort. Other modifiers, which may impact on disease activity, are HLA genotype and quasispecies diversity. Immunogenetic factors may influence susceptibility not only to chronicity, but also to severity of inflammation in hepatitis C.32,33 The degree of serum ALT abnormality has been used and interpreted clinically as an indicator of histological damage. It has also served as a criterion for requesting repeat liver biopsy, and starting and monitoring antiviral response to treatment. The statistically significant relationship between serum ALT and the degree of inflammation found in the group of HCV type 1b-infected patients described here supports this. However, this observation has not been seen uniformly in all other investigations. In some other patient groups with normal or near-normal ALT, significant histological abnormalities exits; moreover, in other patients groups, the degree of ALT correlated poorly with histological abnormalities.34-36 Lau et al., Nousbaum et al., and McCormick et al., in patient groups of 34, 220, and 44 patients, respectively, found no correlation between viral load, histological damage, and elevated ALT.37-39 In summary, we have identified a statistically significant relationship between: 1) serum viral load and the degree of inflammation; and 2) serum transaminase levels and the degree of inflammation. Correlations similar to those presented here have previously been published by others using more heterogeneous cohorts.15,40-42 Notwithstanding these findings, the strength of the patient population presented here is the ability to control for variables that impact on the natural history of HCV infection. It is now evident that additional long-term longitudinal studies using serial viral load estimations and histopathology are needed to better define the role of HCV RNA in monitoring disease progression in hepatitis C. REFERENCES 1. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood borne non-A, non-B hepatitis genome. Science 1989;44:359-632. 2. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;44:362-364. 3. Kato N, Hijikata M, Ootsuyama Y, Nakigawa M, Ohkoshi S, Sugimura T, Shimotohno K. Molecular cloning of the human hepatitis C genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A 1990;7:9524-9528. 4. Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo QL, Kuo G. Detection of antibody to hepatitis virus in prospectively followed transfusion recipients with acute chronic non-A, non-B hepatitis. N Engl J Med 1989;21:1494-1500. 5. Esteban JI, Esteban R, Viladomiu L, Lopez-Talavera JC, Gonzalez A, Hernandez JM, Roget M et al. Hepatitis C virus antibodies among risk groups in Spain. Lancet 1989;2(8658):294-297. 6. Harrison TJ, Zuckernam AJ. The Molecular Medicine of Viral Hepatitis. West Sussex, UK: Wiley, 1997. 7. Alter MJ, The epidemiology of hepatitis C in the west. Semin Liver Dis 1995;15:5-14. 8. Alter MJ. Transmission of hepatitis C virus—route, dose, titer. N Engl J Med 1994;30:784-786. 9. Tong MJ, El-Farra NS, Reikes AR, Co RL. Clinical outcomes after transfusion-associated hepatitis C. N Engl J Med 1995;32:1463-1466. 10. Van der Poel CL, Cuypers HT, Reesink HW. Hepatitis C virus six years on. Lancet 1994;344:1475-1479.
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