This study detected HCV RNA in the plasma of a small proportion of treatment recovered patients (15/98, 15%) by an in-house nested RT-PCR even though they had been classified as sustained treatment responders based on undetectable plasma HCV RNA by Cobas Amplicor HCV Test. Because plasma samples from 14 of 15 HCV RNA positive patients tested positive only in the nested round of the PCR, the HCV RNA concentration was below the detection limit of the Cobas Amplicor HCV Test. Furthermore, HCV RNA positive bleed dates were interspersed with HCV RNA negative bleed dates for each patient, which suggests that the frequency of HCV RNA detection may be underestimated if only a single bleed date per patient is studied as typical for a cross-sectional study. The true prevalence of HCV persistence is likely higher than reported in this study because (1) patients were not tested at close intervals, (2) the liver was not studied, and (3) the sensitivity of the assay was limited to 40 copies/mL. Detection of HCV RNA was most frequent in the early years after cessation of treatment, and one of the patients in this group experienced high level HCV relapse in this time period similar to case reports of HCV relapse in 3 immune suppressed patients 0.5 years, 2 years,14
and 8 years13
after cessation of therapy. Whereas 15 of 77 plasma samples from patients within 8.5 years after cessation of therapy tested positive for HCV RNA, all 21 samples obtained at later time points tested negative (P
= .035, Fisher exact test), which supports the notion that HCV is eventually completely cleared. This is consistent with results by Morishima et al, who reported that HCV RNA level declined from transcription mediated assay (TMA) positive to TMA discordant to TMA undetectable.24
It is further supported by the observation that plasma samples from all spontaneously recovered patients, the majority of whom were studied more than 8 years after HCV infection, tested HCV RNA negative as well.
HCV RNA positive patients did not differ from HCV RNA negative patients in terms of age, gender, or HCV genotype. Also, treatment duration and regimen did not explain the persistence of low levels of HCV RNA. In fact, the majority of HCV RNA positive patients had received a 48-week course of PEG-IFN/RBV therapy, the optimal therapy according to the current state of the art. The post-treatment PCR results were unlikely to represent false positives because of the stringent precautions to prevent intersample contamination. In addition, the heterogeneity among the viral sequences obtained from patients precludes the presence of a common, contaminating laboratory strain. Finally, the HCV genotype after interferon-based treatment matched the pretreatment genotype in 11 of 13 (85%) of all patients, indicating persistence of autologous HCV RNA.
The topic of HCV RNA persistence has been controversially discussed in the literature. Several studies suggested that HCV is completely eradicated from serum and PBMC of spontaneously recovered or successfully treated patients.25,26
At the other extreme, 2 laboratories reported HCV RNA in more than 90% of serum and PBMC samples from spontaneously recovered2,17
and treatment-recovered patients.2,3
Whereas we employed RNA extraction, RT, and PCR techniques identical to the ones used by Pham et al,2
we detected HCV RNA only in a minority of samples. This discrepancy may be explained by the fact that some of the previous studies included a high proportion of injection drug users7
and subjects with unknown risk factors17
in whom the risk of reinfection/re-exposure is increased. In contrast, the majority of HCV RNA positive patients of this cohort acquired the initial HCV infection via transfusion and, thus, was less likely to have been exposed a second time. Also, in contrast to our study, neither Pham et al7
nor Radkowski et al17
confirmed that the detected HCV sequence represented persisting HCV RNA, ie, that it matched the sequence in the initial HCV infection. The percentage of HCV RNA positive test results in our study is more consistent with the results obtained by Morishima et al,24
Hoare et al,27
and George et al,28
who detected HCV RNA in a smaller proportion of treatment responders by nested RT-PCR or TMA, respectively, in approximately 6% of the studied patients.
What can be the source of the detected HCV RNA? We propose that PBMCs are not the source for the HCV RNA detected in plasma. First, HCV RNA was less frequently detected in the PBMC compartment than in plasma samples in our samples. Second, whereas HCV may bind to and be phagocytized by B cells and dendritic cells (reviewed in Timpe and McKeating29
), these cells do not support HCV replication in vitro30
and do not express claudin-1, an obligatory receptor for HCV infection that is solely expressed in the liver.31
Instead, we favor localized foci of HCV replication in the liver as the most likely source for the detected HCV RNA. This theory derives support from reports that HCV sequences were found in the liver even when they were not detectable in the blood.3,26,32
Studies to determine the infectious nature of the residual RNA are currently in progress in our laboratory.
Several interesting aspects should be noted about the T-cell response. First, the vigor of HCV-specific, T-cell responses was greater at HCV RNA positive time points than at HCV RNA negative ones. This result is reminiscent of a correlation between increased antiviral T-cell responses and low-level persisting virus in patients who had spontaneously recovered from acute hepatitis B.15
Second, the strongest increase in immune responsiveness in our study was observed against nonstructural proteins, which are not part of the virus particle and expressed solely when the virus infects cells and viral RNA is translated into proteins. This observation suggests that the increase in HCV-specific, T-cell responsiveness was because of an antigenic boost, derived from newly translated HCV RNA. Third, the preserved immune hierarchy within the HCV-specific, T-cell responses and the preserved pretreatment viral sequences support the notion that pretreatment HCV RNA persists. This interpretation is consistent with findings in other models that show persistent virus rarely induces new T-cell responses,33
whereas infection with a new virus of different sequence induces new T-cell responses and changes the hierarchy in the existing memory T-cell repertoire by stimulating and expanding some memory T-cell populations and contracting others.34
The observation that the sole patient who exhibited a change in immune hierarchy in our study also exhibited a change in viral genotype therefore suggests that this patient experienced a new infection.
In conclusion, HCV RNA may persist for a limited but not indefinite time after successful therapy and may sporadically reappear in the circulation, triggering T-cell responses. This may be missed in standard clinical evaluations, which typically assess the presence of HCV RNA at a single time point 6 months after cessation of therapy.