Introduction
Because hepatitis C virus (HCV) and HIV share parenteral modes of transmission, the prevalence of chronic HCV infection in HIV-infected patients is higher than 50% in intravenous drug users (IDU) [1–3].
Since HIV-infected patients have been treated by antiretroviral combinations, their life expectancy has been prolonged [4] and at present, they are more frequently exposed to the complications of chronic hepatitis C [2]. A number of cross-sectional studies have shown that HIV infection is associated with more severe liver disease in patients with chronic hepatitis C [5–9]. However, the actual impact of HIV infection on the natural course of HCV infection in IDU remains uncertain because of numerous co-factors, such as chronic alcohol consumption [5,10], hepatitis B and D virus-associated infections [1,2], and hepatotoxic medications, including antiretroviral therapy, which is suspected of aggravating chronic hepatitis C [11,12].
The aim of the present study was to determine the impact of HIV infection on the progression of liver disease in HCV-infected patients, disregarding the effects of advanced immunosuppression and antiretroviral therapy. In order to discriminate between different factors, we undertook a prospective case–control study and compared chronic hepatitis C associated or not with HIV infection in untreated patients with a CD4 cell count greater than 200/μl and no AIDS, who had become infected through intravenous drug use. In order to limit selection biases, we prospectively included consecutive HIV-positive infected patients.
Patients and methods
Cases
All consecutive HIV-infected patients attending for a medical examination between July 1995 and July 1996, in three departments of our medical centre, were prospectively and systematically screened for the following inclusion selection criteria: a history of intravenous drug use with no other risk factor, a CD4 cell count greater than 200/μl, no AIDS and no previous antiretroviral treatment, a positive test for anti-HCV antibody (enzyme-linked immunosorbent assay; ELISA 3), a negative test for hepatitis B surface antigen (HBsAg), and abnormal alanine aminotransferase (ALT) activity. Because patients with a low CD4 cell count received antiretroviral treatment, only those with a CD4 cell count greater than 200/μl were selected. Thirty-eight patients (cases) were eligible and underwent liver biopsy before the end of December 1996. The study was approved by the local ethics committee and all patients gave written informed consent.
Controls
Among 626 consecutive patients with positive anti-HCV antibody (ELISA 3) and abnormal ALT activity without HIV infection referred to our department during the same period, 38 (controls) with a history of intravenous drug use and no other risk factor were selected and matched with cases for age, sex and estimated duration of HCV infection. The indication for liver biopsy in cases and controls was a positive anti-HCV ELISA 3 test and elevated ALT activity. The duration of HCV infection was estimated as the time elapsed from the date of first intravenous drug use and that of liver biopsy [13]. Cases and controls were negative for HBsAg, and none of them had previously undergone interferon therapy.
Laboratory tests
All subjects were reactive for anti-HCV antibodies both by third-generation enzyme immunoassay (EIA; Ortho HCV 3.0 ELISA, Ortho Diagnostic Systems, Raritan, NJ, USA or Monolisa anti-HCV, Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) and third-generation recombinant immunoblot assay RIBA HCV 3.0 (Ortho Diagnostic Systems). Reactivity for anti-HIV by EIA (Genelavia mixte Pasteur, then Genscreen HIV 1 and 2, Sanofi Diagnostics Pasteur, or Abbott HIV-1 HIV-2 EIA third generation, Abbott laboratories, Chicago, IL, USA, or Ortho HIV-1 HIV-2 antibody capture ELISA, Ortho Diagnostic Systems) was confirmed by Western blot New Lav-Blot 1 (Sanofi Diagnostics Pasteur).
HCV RNA was tested in the sera using the reverse transcriptase–polymerase chain reaction (RT–PCR) Amplicor HCV technique (Roche Diagnostic Systems, Branchburg, NJ, USA). When the result of RT–PCR was positive, HCV RNA was quantified by the Quantiplex HCV RNA 1.0 Assay, or since 1996, by the Quantiplex HCV RNA 2.0 Assay (Chiron Diagnostics, Emeryville, CA, USA). Samples were frozen at −80°C within 4 h of collection and were not thawed until tested.
The determination of HCV genotypes was performed by Inno-Lipa HCV test, or since 1996, by Inno-Lipa II HCV (Innogenetics, Gent, Belgium). Whenever the result of RT–PCR was negative, the HCV type was determined using the serotyping test (Murex HCV serotyping 1–6 Assay, Murex Diagnostics, UK).
Histological evaluation
Paraffin-embedded biopsies were analysed by a single pathologist unaware of clinical and biological data except for the presence of chronic hepatitis C. This analysis was performed, following haematoxylin–phloxin–saffron, Perls, and picrosirius red staining using the METAVIR group items [14]. The presence of bile duct lesions and of haemosiderin was also assessed. Steatosis was graded according to the percentage of hepatocytes containing cytoplasmic vacuoles. Steatosis was considered as mild, moderate or severe, when less than 10, 10–30 or more than 30% of the hepatocytes were affected, respectively. Hepatitis was finally graded and staged using the METAVIR scoring system [15].
Study parameters
The following clinical, biochemical, virological and histological data were recorded at the time of liver biopsy: age, sex, estimated duration of HCV infection (i.e. between the first time of intravenous drug use and the time of liver biopsy), age at the presumed time of contamination, daily alcohol intake assessed by a senior hepatologist using a standard set of questions, ALT activity, CD4 cell count in HIV-infected patients, anti-HBc antibody, level of HCV viraemia, HCV genotype, METAVIR (fibrosis and activity), bile duct lesions and ductular proliferation, liver iron overload and steatosis. As one patient’s daily alcohol intake could be highly variable, the mean daily intake from the presumed date of contamination until the date of diagnosis of HCV infection was calculated, and the value of 40 g per day was considered as the threshold for excessive intake. Median values were chosen as cut-off points for age at liver biopsy, age at contamination and CD4 cell count. The progression rate of fibrosis per year was calculated as the ratio between the METAVIR fibrosis index and the estimated duration of HCV infection in years [16].
Statistical analysis
Qualitative and quantitative variables were expressed as percentages and as mean ± SD, respectively. Cases were compared with the corresponding controls by using the MacNemar’s test or the Wilcoxon test for paired data. Independent parameters significantly associated with the METAVIR score were assessed by using logistic regression. Progression rates of fibrosis according to demographic, biological and histological parameters in HIV–HCV-infected patients were compared by using the Mann–Whitney test. Differences were considered significant if the P value was 0.05 or less.
Results
Demographic characteristics of cases and controls
Thirty-eight IDU infected with HIV and HCV (cases) were prospectively recruited according to the selection criteria (i.e. CD4 cell count greater than 200/μl, no AIDS and no antiretroviral treatment). All patients with a CD4 cell count greater than 200/μl had compensated liver disease. Thirty-eight IDU infected with HCV (controls) were matched to cases for age at liver biopsy (36 ± 6 years), sex ratios (M/F 3.2) and estimated duration of HCV infection (14 ± 4 years). The general characteristics of cases and controls are shown in Table 1. Age, sex ratio, duration of HCV infection and age at contamination were similar in both groups. The mean alcohol intake was similar in cases and in controls (65 ± 15 versus 55 ± 10 g/day, P > 0.05). In addition, the proportion of patients whose mean alcohol consumption exceeded 40 g per day was not significantly different between both groups. In cases, the mean CD4 cell count was 500 ± 300/μl (Table 2). No patient had hepatotoxic medication at the time of liver biopsy.
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Table 1: Demographic characteristics of anti-HIV-positive (cases) and matched anti-HIV-negative (controls) patients with chronic hepatitis C.
Table 2: Comparison of biochemical, virological and histological characteristics according to HIV status (cases and controls).
Severity of liver disease
Table 2 shows the virological and histopathological findings in cases and controls. RT–PCR was positive in all patients. The level of HCV viraemia was significantly higher in cases than in controls (15 ± 13 versus 9.2 ± 10.4 × 106 Eq/ml, P = 0.05). They were infected predominantly by genotypes 1 (44 and 57%, respectively) and 3 (37% in both groups). Cases with CD4 cell counts less than 450/μl had significantly higher levels of HCV viraemia than cases with CD4 cell counts greater than 450/μl (17.3 ± 12.5 versus 12.8 ± 13.4, P = 0.05). The METAVIR fibrosis index was higher, without reaching statistical difference, and the METAVIR activity index was significantly higher in cases. The overall progression rate of fibrosis was significantly higher in cases than in controls (0.14 ± 0.1 versus 0.11 ± 0.08, P = 0.02). Cirrhosis appeared more frequent in cases than in controls (13 versus 3%), without reaching statistical difference (P = 0.1). In multivariate analysis, HIV infection was the only independent factor significantly associated with METAVIR score (odds ratio 2.9).
Factors associated with the progression rate of fibrosis in HIV–HCV-infected patients
The only parameters significantly associated with the progression rate of fibrosis in cases were the age at contamination and the METAVIR activity score (Table 3). There was no significant relationship between the CD4 cell count or the level of HCV viraemia and the METAVIR activity score.
Table 3: Risk factors for the progression rate of fibrosis in cases (HIV–HCV).
Discussion
This case–control study shows that, in IDU with chronic hepatitis C, HIV infection even in the early stages, is associated with more severe liver injury and with an apparent higher progression rate of fibrosis, regardless of age, sex and duration of HCV infection. To our knowledge, this is the only histological study on chronic hepatitis C in a population of HIV-infected patients before the onset of antiretroviral treatment.
A majority of patients with HIV infection in developed countries are currently undergoing antiretroviral therapy, usually including protease inhibitors (PI), which may influence the progression of chronic hepatitis C, although this issue is still debated [11,12,16,17]. Some authors have identified a deleterious effect of PI on hepatitis C [11,12], whereas others have not [16], or have even found a beneficial effect of PI on liver fibrogenesis [17]. None of our patients had received antiretroviral treatment at the time of liver biopsy, either because the HIV and HCV infections had both recently been discovered or because the patient had no current indication for antiretroviral treatment. In all cases, the CD4 cell count was higher than 200/μl, and besides this selection criteria, the study population was restricted to a subset of patients who were infected exclusively through intravenous drug use, who underwent a medical examination, who had elevated ALT activity, and no concomitant circulating HBsAg. Even if this selection was to introduce a bias, it should be emphasized that the same criteria were applied to the selection of controls. Cases and controls were paired according to strict parameters. In addition, all the patients were recruited prospectively, even though parameters such as alcohol consumption and the duration of HCV infection were determined retrospectively.
Many cross-sectional studies have documented a significant relationship between HIV infection and the severity of liver injury in IDU with chronic viral hepatitis [5–8,18]. However, the study populations in these reports were generally heterogeneous with regard to the stage of HIV infection, including AIDS patients [6,8]. In a recent retrospective case–control study, Benhamou et al.[9] documented a higher progression rate of fibrosis in HIV–HCV-infected patients. However, most of the patients had received antiretroviral therapy and some had advanced HIV infection, with CD4 cell counts of less than 200/μl. The present study provides further evidence that patients with HIV infection, even in the early stages, causes more severe liver inflammation and a higher progression rate of fibrosis during chronic hepatitis C. Although HCV disease is likely to progress even more rapidly in patients with advanced immunosuppression, the finding is important for the design of future anti-HCV strategies in HIV-infected patients.
The mechanisms that may account for the fact that HIV infection leads to increased fibrogenesis in patients with chronic hepatitis C are unknown. In our study, the level of HCV viraemia in HIV-infected patients was significantly higher than in controls, as previously reported [19,20], and was inversely related to the CD4 cell count [19]. However, there was no significant relationship between the level of HCV viraemia or the CD4 cell count and the progression rate of fibrosis. In the study by Benhamou et al.[9], a CD4 cell count of less than 200/μl was associated with a higher progression rate of fibrosis, whereas all the patients in the present study had a CD4 cell count higher than 200/μl. The distribution of genotypes was identical in cases and controls, and the progression rate of fibrosis was independent of genotype in cases, as demonstrated before in anti-HIV-negative patients [10,21]. It has previously been shown that isolated anti-HBc antibody in patients with chronic hepatitis C was frequently associated with occult hepatitis B virus infection and with more severe liver injury [22]. Although there was a trend towards a higher prevalence of anti-HBc positivity in cases, we did not find a significant relationship between the presence of anti-HBc antibodies and the progression rate of fibrosis. Alcohol consumption and the degree of steatosis, which may increase fibrogenesis through lipid peroxidation [23], were similar in cases and controls. Whereas Benhamou et al.[9] reported that alcohol consumption was associated with a higher progression rate of liver fibrosis, we and others [5] found no significant association between alcohol consumption and the severity of liver disease in HIV–HCV-infected patients. Methodological limitations, including the small sample size or the retrospective assessment of alcohol consumption, could explain this result. However, a significant association between alcohol intake and fibrosis progression was found among controls (data not shown). As previously hypothesized [5], the influence of HIV infection could mask that of alcohol use, at least temporarily in non-cirrhotic patients.
The METAVIR score activity was significantly higher in cases than controls, and was associated with a significant increase in the progression rate of fibrosis. Both the higher viral load and the greater degree of liver inflammation could result from abnormal immune functions in HIV-infected patients, even though their CD4 cell count was maintained above 200/μl. HIV infection of parenchymal and non-parenchymal cells in the liver [24], and subsequent local cytokine production [25], could also contribute to liver injury. Ductular proliferation tended to be more frequent in cases than in controls supporting this hypothesis, because cholangiolitis has been considered to be typical of HIV infection in patients with chronic hepatitis C [8]. More severe liver injury has also been attributed to iron overload in HIV-infected individuals [26], although we found no significant difference in the liver iron content between cases and controls.
Conclusion
HIV infection even in the early stages is associated with higher HCV viraemia and more severe liver injury, in untreated IDU with chronic hepatitis C. These results provide a solid rationale for therapeutic trials of hepatitis C in HIV-infected patients.
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