HCV persistence: Cure is still a four letter word

Hepatitis C virus (HCV) infects between 170 and 350 million people worldwide and is currently the leading indication for orthotopic liver transplantation in the United States. While up to 50% of individuals clear HCV viremia following acute infection, most people develop persistent infection with chronic hepatitis that may progress to cirrhosis and, possibly, hepatocellular carcinoma.1 HCV, hepatitis C virus; SVR, sustained virological response; PBMC, peripheral blood mononuclear cell; HBV, hepatitis B virus The last decade has seen major improvements in antiviral therapy for chronic HCV infection. The initial aims were to normalize liver enzyme levels and to clear HCV viremia at the end of treatment (ETR). However, it soon became apparent that many patients quickly relapsed following a complete course of therapy. This led to the development of the concept of sustained virological response (SVR), defined as the absence of HCV viremia 6 months after the end of treatment. With current combination therapy with pegylated interferon and ribavirin, 56% of patients may achieve an SVR.2 Numerous long-term follow-up studies have shown that SVR appears to be a durable clinical endpoint.3, 4 Less than 4% of patients will relapse by 6 years following SVR, and in addition to viral clearance, liver biochemistry remains persistently normal and liver histology has been shown to improve in many patients.5 These observations have led some to cautiously consider the possibility that SVR may actually represent cure. Pham et al. recently reminded us that the word CURE must not be used prematurely.6 Using highly sensitive reverse transcription–polymerase chain reaction (RT-PCR)–nucleic acid hybridization assays, they looked for the presence of residual HCV RNA in individuals up to 5 years after apparent spontaneous or treatment-induced viral clearance. In addition to sera, peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells were examined because of known HCV lymphotropism. In all 16 patients studied, residual HCV RNA was detected. Importantly, after mitogen stimulation they were able to document the presence of negative-strand HCV RNA in 9 of (75%) 12 PBMC samples. Because HCV negative-strand RNA is a replicative intermediate in the viral life cycle, its presence is generally accepted as evidence of ongoing HCV replication. In this issue of HEPATOLOGY, Radkowski et al.7 confirm and expand upon the important finding of HCV persistence. Using similar methods to those employed by Pham's group, they looked for the presence of HCV RNA in serum and PBMCs from patients who were persistently HCV RNA negative by conventional assays after spontaneous or treatment-induced recovery. In addition, they examined liver tissue from biopsies performed 3 to 8 years after antiviral therapy. Of 17 patients studied, HCV RNA was found in at least one compartment in all but two patients. Negative-strand HCV RNA, suggestive of ongoing viral replication, was detected in lymphocytes from 2 patients and in macrophages from 4 other patients but not in any of the liver tissue examined. Sequencing of detected RNA allowed for genotype determination, which correlated with pretreatment genotype in all but one patient. This suggests that the HCV RNA detected was residual virus rather than the result of re-infection. The findings of these two studies are certainly intriguing and will potentially have important implications for our future understanding and management of HCV infection. Both studies confirm the presence of HCV RNA long after apparent resolution of infection; however, it is unclear what the significance may be of such low levels of virus. We have been here before. These observations are reminiscent of the data regarding occult hepatitis B infection. Numerous studies have documented low levels of hepatitis B virus (HBV) DNA in both liver and extra-hepatic compartments in patients negative for all serologic markers of HBV infection as well as in patients with isolated anti–hepatitis B core antibody. Occult HBV has been found in patients with apparently resolved infection (clearance of hepatitis B surface antigen and presence of anti-HBs),8, 9 patients with no known history of HBV,10, 11 patients with hepatocellular carcinoma,12 and patients with HCV infection. The prevalence of occult HBV varies greatly but is very high in certain populations; however, the clinical significance remains uncertain. Although the initial report documented more advanced liver disease in patients with HCV and evidence of occult HBV than in those with HCV alone,13 subsequent studies have not confirmed this finding.14, 15 Similarly, with isolated occult HBV the jury is still out. While Chan et al.16 found that one third of patients with cryptogenic cirrhosis had occult HBV in Hong Kong, Komori et al.17 showed that patients with persistent low-level HBV viremia after clearance of HBsAg actually had improved histology. Clearly, the significance of occult HBV still needs some clarification. What about HCV? Although the data from Radkowski et al. confirm the presence of HCV many years after apparent disease resolution, the clinical significance of this finding remains unclear. The observation of negative-strand HCV RNA in hepatocytes strongly suggests that they have not simply identified HCV remnants from past infection. This appears to be replicating virus and therefore has potentially important clinical and public health implications. Patients with persistent hepatic HCV RNA had no histological improvement, whereas all of the other patients had reduced fibrosis and inflammatory scores. While intriguing, more data are clearly needed before conclusions on clinical outcome can be made. In 2004, Castillo et al.18 described “occult hepatitis C infection” in 57 of 100 patients with persistently abnormal liver enzymes but no markers of HCV infection by commercial assays. They documented HCV RNA in hepatocytes as well as in PBMCs using highly sensitive RT-PCR and in situ hybridization. Negative-strand HCV RNA was also found in 48 patients (84.2%). Histologic analysis showed that patients with occult HCV infection were more likely to have both necroinflammatory activity and fibrosis on liver biopsy; however, the majority (65%) had only mild nonspecific changes or isolated steatosis. These provocative findings await confirmation. The clinical significance of low levels of HCV RNA in patients with apparently resolved infection is even less clear. The largest study looking at long-term duration of SVR showed that in 80 patients with up to 7.5 years of follow-up, 96% remained HCV RNA negative, 93% had persistently normal alanine aminotransferase, and 94% had clear histological improvement.5 McHutchison et al. found that only 7 of 170 sustained responders of the large treatment trials of interferon and ribavirin had detectable intrahepatic HCV RNA 24 weeks after treatment. Of those, only 2 (1.2% of all sustained responders) had a serological relapse up to 3.5 years later.19 It is likely that using the sensitive techniques of Radkowski et al.,7 HCV RNA would have been detected in a significant proportion of these cohorts; the implication being that despite low-level viral persistence, clinical improvement is still the most common outcome. Whether the presence, quantity, and location of replicating virus has any impact on clinical outcome remains to be seen. Other clinical issues of HCV persistence will also need clarification. HCV significantly increases the risk of the development of hepatocellular carcinoma, and to date, although it is commonly felt that successful HCV treatment reduces the risk of hepatocellular carcinoma, there are few data to support this contention. Even very low levels of circulating HCV may have an important effect on hepatocarcinogenesis in patients with cirrhosis. This certainly appears to be the case with occult HBV infection.12 Low levels of circulating virus may also account for the finding that many patients have persistent HCV-specific CD4+ and CD8+ T-cell populations many years after SVR or natural viral clearance. Low levels of antigen may be the necessary stimulus to maintain these cell populations.20 Unlike HBV, HCV reactivation after immunosuppression has not been reported, but it is possible that such a scenario will only become evident as more and more patients successfully treated for HCV are followed longer. Occult HCV persistence may account for the report of recurrent HCV infection after liver transplantation in a small number of patients treated successfully with antiviral therapy prior to the transplantation.21 Understanding the mechanisms by which the virus persists at low levels for extended periods of time may also be important. Although the complete mechanisms of occult HBV (HBsAg negative) are not yet entirely clear, the presence of covalently closed circular HBV DNA in an episomal form in the nuclei of hepatocytes accounts partially for HBV's ability to persist in the face of immunological surveillance and viral suppression. HCV is not known to have a similar latent stage in its replication cycle. However, HCV has been found in numerous nonhepatic compartments, some of which are immunologically privileged (e.g., central nervous system),22 and this may account for the low-level persistence after apparently successful antiviral therapy. It has been proposed that HCV compartmentalization may occur, in which HCV confined to a given “compartment” may not be capable of “infecting” other compartments.23 Such a theory could explain the finding of HCV virions in PBMCs but not in liver in many of the patients studied. Perhaps even more important than the clinical consequences of persistent HCV are the public health implications. If patients with no markers of HCV infection still carry infectious virions, they are a potential source of HCV spread in the community. This may be significant for blood donation and organ transplantation programs, as patients may well be missed by current screening strategies. However patients carrying such a low level of virus may not be infectious at all, as we have learned that the risk of transmission of low-level or occult HBV is minimal. To help clarify this issue, examination of high-risk cohorts with no standard markers of HCV infection will be very important, as will the demonstration that this is in fact infectious virus. Radkowski et al.7 have certainly opened our eyes with their provocative findings. The significance of occult HCV persistence remains to be seen, and future work in this area is anxiously anticipated. For now, we will have to watch our tongues before using the word cure.