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Frequent Hepatitis C Virus Superinfection in Injection Drug Users
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The Journal of Infectious Diseases, October 15, 2004;190:1396-1403
Belinda L. Herring,1,2 Kimberly Page-Shafer,2,3 Leslie H. Tobler,1 and Eric L. Delwart1,2
1Blood Systems Research Institute, and 2Department of Medicine and 3Center for AIDS Prevention Studies, University of California, San Francisco
ABSTRACT
The frequency of hepatitis C virus (HCV) superinfection with a divergent viral strain was determined in a cohort of recently infected young injection drug users (IDUs) with an HCV incidence rate of 25%.
HCV was amplified, by use of polymerase chain reaction (PCR), from plasma samples collected from 25 HCV-infected individuals over an average period of 12 months, and their viral sequences were compared.
Phylogenetic analysis identified 5 IDUs with superinfection (20%) occurring after seroconversion: 2 IDUs were superinfected with different HCV genotypes, and 3 were superinfected with divergent strains of the same genotype. The superinfecting strains were not detected as minority variants (<0.5%) in the initial plasma HCV quasi species. Extensive measures were taken to exclude PCR contamination and mix-up of samples, and superinfection results were concordant at 2 HCV genetic loci.
HCV superinfection in IDUs, both intra- and intergenotype, is therefore a frequent event, with an incidence rate similar to that of de novo infections. These results suggest that no cross-protecting immunity develops during the first year of chronic infection with HCV.
INTRODUCTION
Hepatitis C virus (HCV) is estimated to infect 170 million people worldwide and can cause chronic hepatitis, cirrhosis, liver failure, and hepatocellular carcinoma. Spontaneous resolution of HCV viremia occurs in 〜15%–45% of infected individuals, and, in both humans and chimpanzees, improved control of viremia is associated with a cytotoxic T lymphocyte (CTL) response directed to numerous epitopes. Maintenance of a strong memory CD4+ response is also associated with improved control of HCV viremia.
The ability of a previously cleared or ongoing HCV infection to protect against subsequent infection with a divergent strain of HCV (i.e., superinfection) may reflect the ability of adaptive immune responses to provide effective cross-protection. Partial protection against superinfection was reported among HCV-seropositive subjects who had cleared viremia.
HCV superinfection has been seen in persons who recently received blood-product transfusions or organ transplants [14–20]. In the chimpanzee model system, previously infected chimpanzees injected with very high viral load inoculums showed transient or chronic viremia with the rechallenge strain. In contrast, when chimpanzees that had spontaneously cleared their initial viremia were rechallenged with HCV of different genotypes, they experienced reduced and only transient plasma viremia. In the present study, we measured the frequency of superinfection among highly exposed IDUs, currently the population most at risk for HCV infection.
Samples.
Twenty-five subjects were selected from a cohort of longitudinally followed young (<30 years of age) injection drug users (IDUs) who had recently undergone HCV seroconversion and from whom frozen plasma samples were available. Informed consent was obtained from all subjects. The demographic and behavioral characteristics of this cohort have been described elsewhere. Subjects with viremia lasting at least 4 months were selected. HCV RNA was detected by use of the HCV discriminatory version of the Procleix HIV-1/HCV assay (Chiron). This assay is based on transcription-mediated amplification technology and has a detection rate of 95% for 35 HCV RNA copies/mL. Viral loads were determined by use of the Roche COBAS amplicor HCV monitor 2.0 assay (limit of detection, 600 HCV RNA copies/mL). Seroconversion was determined by use of the Ortho HCV 3.0 ELISA system (Ortho-Clinical Diagnostics). The earliest collected plasma samples used for viral genetic analyses were labeled baseline samples, and the latest collected samples were labeled exit samples. Samples collected between these 2 time points were labeled intermediate samples.
IDU population.
HCV strains from 25 recently infected, HIV-negative subjects from a cohort of young urban IDUs were genetically characterized at the E1/E2 HVR1 locus by use of plasma samples collected at an average interval of 316 days (range, 134–715 days). All longitudinally collected samples from these subjects were found to be HCV RNA positive, at a limit of detection of 35 HCV RNA copies/mL. The baseline samples from 18 of 25 IDUs were found to be HCV seropositive.
AUTHOR DISCUSSION
Partial protection from recurring HCV viremia and reduced viral loads have been reported in seropositive subjects and chimpanzees who cleared viremia. In the present study, we sought to determine whether such protection from superinfection could be detected in viremic subjects with a high incidence rate of HCV infection. We found that the frequency of infection with highly divergent strains of HCV was similar to the incidence rate of new infection, indicating that little or no protection was afforded by seroconversion and persistent viremia.
The 5 cases of superinfection identified here were not the result of coinfection with multiple HCV strains, since the superinfecting strains were not detected in the baseline samples (limit of detection, ⩾0.5%). The absence of the superinfecting strains from the intermediate samples allowed a more precise timing of superinfection. IDU 32 was superinfected at least 62 days, IDU 59 at least 89 days, and IDU 54 at least 185 days after the initial seropositive sample was obtained. Therefore, although superinfections occurred in HCV-seropositive subjects, it is still possible that the relatively immature immune responses in these individuals may have been responsible for the absence of protection against superinfection detected here.
Mixed-genotype infections have been reported by use of nonsequencing methods; however, the methods used may have resulted in an overestimation of the frequency of mixed-genotype infections, since other studies found cases of mixed-genotype infections to be rare. A case report of cross-genotype superinfection after clearance of initial infection/viremia in an IDU was described; however, the data were not supported by detailed phylogenetic analysis or stringent controls for PCR contamination and mix-up of samples.
The high frequency of HCV superinfections that we detected among young IDUs indicates the ease with which a new viral strain can surmount immune responses directed at the resident strain and rapidly become the dominant quasi species. HCV superinfection was observed between viral strains belonging to the same, as well as different, genotypes. This result may reflect the difficulty for adaptive immune responses to recognize divergent strains, even those belonging to the same HCV genotype or subtype. The partial protection from reinfection reported in subjects who had apparently cleared their initial viremia may reflect a stronger immune response in this subset of individuals. Chimpanzees with lower viremia have shown stronger CTL responses targeting more epitopes.
Although examples of HIV superinfection have recently been reported, longitudinal sequence analyses have reported a low frequency of superinfection with strains of the same HIV subtypes. The higher probability of HCV infection after parenteral exposure, compared with that for HIV as well as the greater level of genetic variability of HCV, may, in part, account for the large difference between frequencies of HIV and HCV superinfection.
The present report has described the frequent occurrence of HCV superinfection in a cohort of highly exposed subjects. Superinfection in the 5 IDUs presented here was confirmed by sequencing at 2 HCV genetic loci, combined with detailed phylogenetic analysis, careful quality controls for PCR contamination and mix-up of samples, and exclusion of initial coinfection. The high frequency (20%) of superinfection in the cohort in the present study is similar to the underlying HCV incidence rate and, therefore, indicates that little or no protection is provided by seroconversion and ongoing viremia. The result of this natural-history experiment in HCV challenge indicates that successful vaccination against this highly diverse virus may prove to be difficult, except for the minority of individuals likely to otherwise spontaneously clear HCV. Although the clinical consequences of HCV superinfection remain unknown and no apparent effect on plasma viral load was detected here, such consequences might include a decreased probability of spontaneous HCV clearance and a greater resistance to therapy. At the very least, our data suggest that already-infected subjects and those whose viremia was cleared by antiviral therapy should protect themselves against subsequent HCV exposure.
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