Abstract

In hepatitis C virus (HCV) infection, virus load and the risk for HCV-related end-stage liver disease (ESLD) are increased among persons with human immunodeficiency virus (HIV) coinfection. To clarify these relationships, 42 hemophilic patients who developed ESLD and random samples from 164 hemophilic patients with HCV infection alone and 146 with HCV-HIV coinfection were tested for HCV load and genotype. HCV genotype was unrelated to HIV and age. In contrast, HCV load was higher with older age (Ptrend=.0001) and with HIV coinfection (6.2 vs. 5.9 log10 genome equivalents/mL, P=.0001). During 16 years of follow-up of dually infected patients, ESLD risk was unrelated to HCV load overall (Ptrend=.64) or separately to HCV genotype 1 and genotypes 2 or 3 (Ptrend⩾.70). Irrespective of virus load, incidence of ESLD was marginally increased 2-fold (95% confidence interval, 0.8–5.6) with HCV genotype 1. Understanding the discordance between HCV load and ESLD, despite HIV’s link to each of these, may help clarify the pathogenesis of HCV-related disease

By the mid-1980s, about half of persons with hemophilia were infected with both hepatitis C virus (HCV) and human immunodeficiency virus (HIV) and an additional one-quarter to one-third were infected with HCV but not HIV [1]. About 80% of such patients develop persistent HCV infection, manifested as detectable viremia with or without chronic hepatic aminotransferase elevations [2]. End-stage liver disease (ESLD) is the major outcome of chronic HCV infection. Risk of ESLD, particularly for older hemophilic patients, appears to be increased with HIV coinfection [1, 3, 4] and may relate to an aberrant immune response to HCV infection [5]. Here, we investigated whether HCV genotype or virus load was predictive of ESLD in a prospectively followed cohort of HCV-HIV coinfected patients with hemophilia

Methods

Participants and specimensThe distributions of HCV load and genotypes were examined in stratified random samples of HCV-HIV coinfected and HCV-positive, HIV-negative participants of the Multicenter Hemophilia Cohort Study (MHCS): 20 participants each were from 8 birth cohorts (1 cohort consisting of those born before 1950, six 5-year cohorts of those born between 1950 and 1979, and 1 cohort of those born after 1979) [6]. Only MHCS centers in the United States were included. ESLD was defined as persistent ascites, bleeding esophageal varices, hepatic encephalopathy, or death, excluding nonhepatic causes [1]. The random samples did not differ from the MHCS US population in severe hemophilia A prevalence (P=.80), cumulative 16-year survival rate (P=.44), or ESLD incidence rate (P=.51; data not shown). Of the participants in the random samples, 310 (97%) had sufficient reposited specimens for determination of HCV load and genotype. In addition to the 12 ESLD case patients in the coinfected random sample, the 30 other coinfected US ESLD case patients with a prediagnostic specimen were included for case-cohort analysis of ESLD risk [7]. Differences over time in HCV load by HIV and ESLD status were evaluated by testing a second specimen of the same type (serum or plasma) that was obtained ∼5 years earlier

Virus load and genotype testingHCV load was determined with branched DNA (bDNA) technology (Quantiplex HCV RNA 2.0 assay, Chiron) with a lower limit of sensitivity of 200,000 (5.3 log10) genome equivalents (GEq)/mL. HCV load was transformed to log10 for all analyses, and, for continuous analyses, it was input as 100,000 (5.0 log10) GEq/mL for bDNA-negative specimens. For genotyping, HCV RNA was reverse transcribed and amplified by the polymerase chain reaction (PCR), using nested biotinylated primers to the highly conserved 5′ noncoding region developed in our laboratory (A.H.) and/or using a commercially available HCV RNA assay (Amplicor HCV, Roche Diagnostic Systems). Genotyping of the PCR products was done on the labeled amplicons, using a line-probe assay (LiPA, version 1.0; Immunogenetics) [8]. Specimens with no PCR product for the LiPA were considered to be PCR negative. Specimens with atypical band patterns were referred to as genotype unspecified. The rest were grouped into major genotypes/subtypes (HCV-1a, -1b, -2, -3, or -4)

Statistical analysisThe coinfected cohort was followed, on average, for 16 years from individual input HIV seroconversion dates [6] to the date of the onset of ESLD, death, or last follow-up (typically in late 1998). Use of alternate starting dates, such as individual birth dates or the median HIV seroconversion date for the MHCS as a whole, had no substantive effect on the results (data not presented). Proportional hazards modeling (Epicure, HiroSoft International) for a case-cohort study [7] was used to determine the relative hazard and 95% confidence interval of ESLD by HCV genotype and virus load, adjusted for age. The relationship of HCV load to HIV status, genotype, hemophilia type and severity, and age was examined by analysis of variance. A Student’s t test was used to compare change in HCV load by HIV or ESLD status. The distribution of genotypes by birth cohort and HIV status was tested by χ2 and Fisher’s exact tests

Results

Of the 310 randomly selected US hemophilic participants, 173 (56%) had HCV genotype 1, including 92 (30%) with subtype 1a and 81 (26%) with subtype 1b. Thirty-two participants had genotype 2, 30 had genotype 3, and 3 had genotype 4. Seven participants (1% of HIV-negative and 3% of HIV-positive participants) had multiple genotypes detected, 17 (5%) had genotypes that could not be specified, and 48 (15%) were HIV negative, as determined by PCR. Genotypes did not differ by birth cohort or HIV status (table 1), but significantly more HIV-negative than HIV-positive participants had PCR-negative results (23% vs. 8%, P=.0001)

Table 1

Hepatitis C virus (HCV) genotypes and virus load, by human immunodeficiency virus (HIV) status in relation to prevalence and risk of end-stage liver disease (ESLD)

Table 1

Hepatitis C virus (HCV) genotypes and virus load, by human immunodeficiency virus (HIV) status in relation to prevalence and risk of end-stage liver disease (ESLD)

HCV load was higher among HIV-positive, compared with HIV-negative, participants (6.2 vs. 5.9 log10 GEq/mL, P=.0001), and, over a mean of 4.9 years, it increased even more (0.42 vs. 0.16 log10 GEq/mL, P=.0002). HCV load also was higher with older age, for both the HIV-negative participants (0.017 log10 GEq/mL per year, P=.0001) and the HIV-positive participants (0.018 log10 GEq/mL per year, P=.0001) (figure 1)

Figure 1

Hepatitis C virus (HCV) load among participants of the Multicenter Hemophilia Cohort Study, by age (at time of sampling) and human immunodeficiency virus (HIV) infection status. Virus load was higher in HIV-positive than in HIV-negative subjects and increased with age in both groups. HCV RNA was measured by branched DNA (bDNA) technology; specimens with no detectable viremia, by polymerase chain reaction, were excluded. For reference, 6.0 log10 is 106 viral genome equivalents (GEq)/mL. The lower limit of detection is 5.3 log10 (200,000 GEq/mL); specimens with levels not detected by bDNA were set to 5.0 log10 (100,000 GEq/mL)

Figure 1

Hepatitis C virus (HCV) load among participants of the Multicenter Hemophilia Cohort Study, by age (at time of sampling) and human immunodeficiency virus (HIV) infection status. Virus load was higher in HIV-positive than in HIV-negative subjects and increased with age in both groups. HCV RNA was measured by branched DNA (bDNA) technology; specimens with no detectable viremia, by polymerase chain reaction, were excluded. For reference, 6.0 log10 is 106 viral genome equivalents (GEq)/mL. The lower limit of detection is 5.3 log10 (200,000 GEq/mL); specimens with levels not detected by bDNA were set to 5.0 log10 (100,000 GEq/mL)

Irrespective of HIV status, mean HCV load was 6.3 log10 GEq/mL with HCV types 1a, 1b, and 2, slightly lower (6.1 log10 GEq/mL) with genotype 3, and nonsignificantly higher (6.6 log10 GEq/mL) among the 7 participants with multiple genotypes. Likewise, mean virus loads were 6.1 log10 GEq/mL in participants with mild, moderate, or severe hemophilia A or with factor VIII inhibitors. HCV load was lower (5.8 log10 GEq/mL) among participants with hemophilia B, but this difference was not significant with adjustment for HIV status and age (data not shown)

Risk of ESLD with HCV-HIV coinfection appeared to be 2-fold higher with genotype 1 than with genotype 2 or 3 (P=.09; table 1). ESLD risk was unrelated to HCV load overall (Ptrend=.64; table 1) and in subgroups with HCV genotype 1 or genotypes 2 or 3 (Ptrend=.70 and .81, respectively). Over a mean interval of 4.9 years between paired samples, HCV load increased 0.48 log10 GEq/mL in coinfected participants who developed ESLD, compared with 0.41 log10 GEq/mL in coinfected participants who did not develop ESLD (P=.61)

Discussion

ESLD in an HCV-HIV coinfected patient with hemophilia is a major clinical event that often leads rapidly to death [1, 4]. HCV-related liver fibrosis and, presumably, ESLD are associated with duration of infection, older age, male sex, high alcohol consumption, HIV infection, and low CD4+ lymphocyte count [2, 9]. We found recently that ESLD risk was increased with an atypical HCV antibody pattern (high anti-c100 and low anti-c22) [5]. We studied 310 patients who are highly representative of the US hemophilia population, to clarify the interaction of HIV with HCV genotypes, virus load, and ESLD risk

Overall, with or without HIV, we found no relationship between HCV load and genotype, as noted usually but not always [3, 10, 11]. The associations noted between HCV load and HIV, including a significantly higher prevalence of HCV viremia and a nonsignificantly higher prevalence of multiple genotypes, deserve further study. HIV-related immune dysfunction presumably impairs clearance of HCV, but the components involved are unknown. Detection of multiple genotypes, as well as switches in genotypes over time [3, 8], in this repeatedly exposed population were not limited to those with HIV and may be a function of HCV load, which was 0.3 log10 GEq/mL higher than in those with a single HCV genotype. The distribution of genotypes that we found was similar to that found in a previous study [12]. In addition, our representative sampling enabled us to show that HCV genotypes were independent of birth cohort, suggesting that there were no large genotype shifts in the plasma donor population before the mid-1980s

As in the study by Thomas et al. [13], HCV load in our study was higher with older age. In addition, we and others found that HCV load was much higher and increased more steeply over time with HIV coinfection [3, 13, 14]. Despite this association, age-adjusted ESLD risk was unrelated to HCV load in the coinfected participants overall and in the coinfected subgroups with HCV genotype 1 or with genotypes 2 or 3. As in other studies [3, 10, 11], our power to investigate subgroups was limited except with genotype 1. Irrespective of virus load, the risk of ESLD was nonsignificantly increased 2-fold for coinfected participants with HCV genotype 1. In contrast, in a recent multivariate analysis of 125 HCV-HIV coinfected and 185 HCV singly infected hemophilic patients, genotype 1 was associated with a 7.8-fold (95% confidence interval, 2.0–30.8) increased risk of liver death [4]. Additional studies will be needed to reconcile these findings. We found no difference in ESLD risk between HCV subtypes 1a and 1b

In summary, HIV infection is associated with increased HCV load, with an increased rate of liver fibrosis [9], and with ESLD [1, 3, 4]. Despite these links, we found that HCV load was not associated with ESLD, even in subgroups defined by HCV genotype. This paradox is quite unlike the straightforward association between HIV load and AIDS, perhaps reflecting the relationship between liver fibrosis and intrahepatic T lymphocyte cytotoxicity, which appears to be unrelated to HCV load [15]. These and other such studies are likely to lead to improved understanding of the pathogenesis of HCV-related diseases and their treatment and prevention

Acknowledgements

We thank Brad Goebel, Mike Walters, and Andrea Stossel (Scientific Applications International Corporation), for performing the assays; Susan Wilson (Research Triangle Institute) and Frances Yellin (Computer Sciences Corporation), for computer programming; and Virginia Lamprecht and Barbara Kroner (Research Triangle Institute), for study management. We are especially grateful to the study participants, hemophilia center staff members, and collaborators of the Multicenter Hemophilia Cohort Study for their tireless contributions

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Informed consent was obtained from the patients or their parents or guardians, and human experimentation guidelines of the US Department of Health and Human Services and those of the authors’ institutions were followed in the conduct of clinical research
Financial support: National Cancer Institute (CP-33002) in association with Research Triangle Institute