In this large multi-racial cohort of IDUs with chronic hepatitis C infection, HCV RNA levels were independently associated with six factors: age, gender, racial ancestry, HIV-1 infection, HCV genotype and host IL28B rs12979860 genotype.
HCV RNA levels tended to be higher with older age and longer duration of drug injection, variables that are highly correlated in this study. The average time since initiation of drug use in these IDUs is 19 years and, at least until recently, most IDUs who enrolled in UHS became infected with HCV relatively soon after initiating drug injection(9
). We believe, therefore, that reported years of injection drug practices is a reasonable proxy for the time since initial infection with HCV. Our data suggest that HCV RNA levels may increase over time. Consistent findings were previously reported in another cross-sectional study of IDUs, (6
) but results from longitudinal studies of HCV RNA are mixed. The study with the longest follow-up period (median, 9.2 years) found that HCV RNA levels increased over time (13
), but studies based on shorter follow-up periods (average 1-5 years), which may have lacked the statistical power to exclude modest increases, did not(14
), .We speculate that HCV may propagate more efficiently over time, perhaps due to selection of HCV variants with high replicative efficiency or host loss of immunological control of HCV.
In the absence of HIV-1 infection, HCV-RNA levels tended to be lower for women compared to men and this difference remained after potential confounding variables were considered. Among the 237 HIV-infected UHS participants, however, median HCV RNA levels were similar in women and men. In the ALIVE study of IDUs, lower HCV RNA levels were observed in women compared to men among HIV-uninfected subjects, although that association was not statically significant in a multivariate analysis (6
). As in our study, HCV RNA levels did not differ by gender among the HIV-infected ALIVE participants. Among HCV-infected Alaska Natives, women had much lower levels of HCV RNA than men (17
Comparing HCV RNA by racial ancestry, African American UHS participants tended to have higher levels than participants of European or Asian ancestry, even after we considered other factors, including IL28B
genotype. Few previous studies have been able to make such comparisons. In ALIVE, no difference in HCV RNA levels was seen between African American subjects and those of other races, however, only 40 non-African American subjects were included in that analysis.(6
) Among patients enrolled in treatment trials for chronic hepatitis C, pre-treatment HCV RNA levels did not different between African American and European American subjects in either the VirahepC (18
) or IDEAL (19
HCV-RNA levels were considerably higher among UHS participants who were infected with HIV-1 compared to those who were not (6.73 versus 6.40 log10
copies/ml), which is consistent with the results from a number of previous studies. (6
) In our study, we were able to control for a fuller range of potential confounding, but this association remained strong even when these factors were considered.
Among the subjects for whom we could determine viral genotype, almost 80% were infected with HCV genotype 1A or 1B; the median HCV RNA level in this group was 6.51 log10
copies/ml. Nonetheless, consistent with other studies among injection drug users (6
), we found a diversity of HCV genotypes in this population - 321 UHS participants had HCV genotypes 2, 3, or 4. Those who were infected with HCV genotype 2 had higher HCV RNA levels (median, 6.69 log10
) than those infected with genotype 1, although this difference reached statistical significance only in the subsample with IL28B
genotype data available. We observed lower viral levels in participants who were infected with genotype 3 (median, 6.34 log10
). Those findings remained significant in the multivariable analysis of the whole sample, but lost significance when the analysis was restricted to the subsample with IL28B
genotype data, perhaps due to insufficient statistical power. Among the 17 subjects with HCV genotype 4 infection, the median HCV RNA level was 6.12 log10
. Consistent with our findings, an earlier report of Swiss blood transfusion recipients co-infected with HIV-1 showed the highest HCV RNA levels in patients with genotype 2, and the lowest levels in patients with genotype 4 (24
). In a multi-national study (predominantly IDUs), HCV RNA levels were lowest among subjects infected with genotypes 3 or 4, and similar among those with genotypes 1 and 2, although relatively few subjects with genotype 2 were included in this analysis(7
). Among Alaska Natives, the lowest HCV RNA levels were found in persons infected with HCV genotypes 3a and the highest in those infected with genotype 2b. In that population, no patients were found to be infected with genotype 4. (17
Several variables that we found to be associated with higher HCV RNA among UHS participants (older age, male gender, African ancestry and HIV infection) were previously associated with failure to spontaneously clear HCV infection in this cohort, (8
) as well as in other studies.(2
) The IL28B
genotype is an exception to this pattern. This genotype is associated with a higher frequency of spontaneous HCV clearance in UHS (25
)and other studies (26
) and a higher likelihood of a successful response to peginterferon-ribavirin combination therapy, but paradoxically, it was also associated with higher HCV RNA (among the European American participants and UHS subjects overall). A number of prior reports also found the otherwise favorable IL28B
genotype to be associated with higher baseline HCV RNA, (4
) (although some other studies did not (26
)). The association of IL28B
-CC genotype with both better response to therapy and higher serum HCV-RNA in the absence of treatment seems counterintuitive, but, prior to therapy, patients with the IL28B
-CC genotype have lower expression of interferon stimulated genes induced by the JAK-STAT pathway.(33
) Thus, patients with the favorable genotype appear to have less endogenous interferon activity, but greater responsiveness to exogenous interferon-α.
Comparing participants by racial ancestry, African American UHS participants had the highest HCV RNA levels despite having the lowest frequency of the IL28B-CC genotype. Thus, not only does the lower prevalence of the IL28B-CC genotype among African Americans not explain their higher viral loads, but controlling for IL28B genotype actually increases the disparity in viral loads between African Americans and both whites and Asian/Amerindian participants. Furthermore, we did not see the association between higher HCV RNA and IL28B-CC among the African American participants. It is possible, therefore, that additional genetic factors lead to poorer viral control among persons of African ancestry.
Our study has a number of strengths. UHS is a cohort of street-recruited IDUs, therefore, we could compare HCV RNA across ancestral groups or individual infected with different viral genotypes without the potential biases caused by markedly differing sources of HCV infection or socioeconomic status. Few, if any, of the UHS participants had been treated for HCV infection, therefore, the HCV RNA values among these subjects were not subject to selection by previous HCV treatment. The relatively large size of the cohort provided good statistical power for many comparisons, although our power was low for certain variable categories, including Hispanic or Asian ancestry and viral genotypes 3 or 4. The limitations of our study should be considered as well. The cross-sectional design did not allow us to determine the timing of HCV, HBV and HIV infections among the participants and we also could not differentiate the effect of duration of infection (as estimated by number of years of drug injection) from the effect of age because these factors are highly correlated. As mentioned above, we could not determine whether the relationship between duration of infection might represent super-infection, immune senescence or some other factor that varies with time or age. CD4+ lymphocytes counts were not measured for UHS subjects, therefore, we could not consider the extent of immunodeficiency present among the 13.9% of participants in this analysis who were co-infected with HIV-1. Successful antiretroviral therapy in HIV-HCV co-infected individuals may increase HCV RNA levels, at least temporarily, especially among individuals with lower CD4+ counts,(35
) but our data were too limited to allow us to examine this effect.
We performed viral genotyping by direct sequencing, the ‘gold standard’ technique for discriminating HCV types and subtypes. (36
) This genotyping was based on the NS5B region, which tends to produce more accurate results than the 5′NC region, (37
) but this method allowed us to detect only the dominant circulating strain of HCV. An important concern in this analysis is whether methodological differences may account for the discrepancies in HCV RNA levels between different genotypes. We used a third generation bDNA) assay with an analytic sensitivity of 2.5×103
copies/ml to measure viral levels. This method amplifies signal rather than target, which is the basis for classical reverse transcription polymerase chain reaction (PCR) and transcription-mediated amplification assays. First-generation bDNA assays underestimated levels of HCV genotype 2 and 3 (40
), but third-generation bDNA tests are accurate, reproducible andwell calibrated to the World Health Organization HCV RNA standard. (41
) In support of our findings, a previous report of an association between HCV genotype 4 infection and lower HCV RNA levels was based on measurement by PCR and determined that the results were not influenced by viral genotype-specific amplification bias (24
In conclusion, the level of HCV viremia, an important predictor of response to HCV treatment, is itself influenced by a wide range of demographic, viral and host genetic factors. A better understanding of the determinants of HCV viremia might lead to improved treatment of patients with chronic hepatitis C.