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Persistence of hepatitis C virus

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  • Shshonee (Alley)
    (I highlighted some areas in bold.. Alley) It is unclear whether the current antiviral treatment for chronic hepatitis C virus (HCV) infection results in
    Message 1 of 1 , Jan 10, 2005
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      (I highlighted some areas in bold.. Alley)

      It is unclear whether the current antiviral treatment for chronic hepatitis C
      virus (HCV) infection results in complete elimination of the virus, or
      whether small quantities of virus persist. Our study group comprised 17 patients
      with chronic HCV who had sustained virological response (SVR) after
      interferon/ribavirin treatment

      http://natap.org/pipermail/doctors_natap.org/2005-January/000429.html
      ---------------------------------------------------------------------------------------------------

      NATAP] Persistence of HCV after SVR- 2 Editorialsdoctors at natap.org doctors at natap.org
      Mon Jan 3 13:02:57 EST 2005

      NATAP - www.natap.org

      Persistence of hepatitis C virus in patients successfully treated for chronic
      hepatitis C

      Hepatology
      Volume 41, Issue 1, January 2005

      This report contains original published study abstract, 2 Editorials, and
      followed by study article text. Despite the study finding low-level residual hep
      C virus in some compartments for patients who achieved SVR (sustained viral
      response), Harvey Alter in his Editorial below says: ��..This residual
      low-level replicative state may not have clinical relevance since there is coexistent
      histological improvement�..�

      Study authors reported these results:
      HCV RNA in Liver Tissue.
      Follow-up liver biopsies were performed 41 to 98 months (mean, 63.6 � 16.7
      months) after the end of treatment in 11 of 17 patients. Importantly, all
      patients in whom follow-up biopsies were done showed improvement in
      necroinflammatory changes (mean, 5.5 � 2.2 vs. 1.5 � 1.4; P = .001 by Wilcoxon matched pairs
      test), and 9 patients also had improvement in liver fibrosis. Mean fibrosis
      score in all 11 patients with follow-up biopsy was significantly lower after
      treatment when compared with pretreatment values (2.5 � 0.9 vs. 1.0 � 0.9; P =
      .039). HCV RNA sequences were detected in liver samples from three patients.
      Interestingly, the only 2 cases without improvement in fibrosis were also HCV RNA
      positive (cases 1 and 4). This result raises the possibility that the
      detection of viral sequences in the liver despite ostensible SVR could have prognostic
      value with relation to fibrosis.

      Study authors: Marek Radkowski 1 2, Juan F. Gallegos-Orozco 1, Joanna
      Jablonska 2, Thomas V. Colby 1, Bozena Walewska-Zielecka 3, Joanna Kubicka 2, Jeffrey
      Wilkinson 1, Debra Adair 1, Jorge Rakela 1, Tomasz Laskus 1 *�

      1Department of Medicine, Mayo Clinic Scottsdale, Scottsdale, AZ
      2Institute of Infectious Diseases, Medical Academy, Warsaw, Poland
      3National Institute of Hygiene, Warsaw, Poland

      ABSTRACT
      It is unclear whether the current antiviral treatment for chronic hepatitis C
      virus (HCV) infection results in complete elimination of the virus, or
      whether small quantities of virus persist. Our study group comprised 17 patients
      with chronic HCV who had sustained virological response (SVR) after
      interferon/ribavirin treatment. Serum and peripheral blood mononuclear cells were collected
      2 to 3 times at 3- to 6-month intervals starting 40 to 109 months (mean, 64.2
      � 18.5 months) after the end of therapy.

      In addition, lymphocyte and macrophage cultures were established at each
      point. In 11 patients, frozen liver tissue samples were available from follow-up
      biopsies performed 41 to 98 months (mean, 63.6 � 16.7 months) after therapy.

      Presence of HCV RNA was determined by sensitive reverse-transcriptase
      polymerase chain reaction, and concentration of positive and negative strands was
      determined by a novel quantitative real-time reverse transcriptase polymerase
      chain reaction.

      Only 2 of 17 patients remained consistently HCV RNA negative in all analyzed
      compartments. HCV RNA was detected in macrophages from 11 patients (65%) and
      in lymphocytes from 7 patients (41%). Viral sequences were also detected in 3
      of 11 livers and in sera from 4 patients. Viral replicative forms were found in
      lymphocytes from 2 and in macrophages from 4 patients. In conclusion, our
      results suggest that in patients with SVR after therapy, small quantities of HCV
      RNA may persist in liver or macrophages and lymphocytes for up to 9 years.

      This continuous viral presence could result in persistence of humoral and
      cellular immunity for many years after therapy and could present a potential risk
      for infection reactivation. BELOW FOLLOWING THE TWO EDITORIALS IS THE ARTICLE
      TEXT, RESULTS, & AUTHOR DISCUSSION FROM THIS STUDY.

      1st EDITORIAL

      An Unsustained Sustained Virological Response
      Harvey J. Alter

      Yesterday upon the stair

      I saw a man who wasn't there

      He wasn't there again today

      How I wish he'd go away?

      I was reminded of this old nursery rhyme as I read the study of Radkowski et
      al. describing the long-term persistence of hepatitis C virus (HCV) RNA in
      patients who met the treatment criteria for sustained virologic responders
      (SVRs). Although caution has always prevented equating SVR with cure, a decade-long
      experience with interferon mono or combination therapy has shown that in SVRs
      serum HCV RNA remains negative, liver histology improves, molecular relapse is
      rare, and clinical relapse is near nil. Thus, it is discouraging that these
      investigators found residual RNA in 15 (88%) of 17 patients followed for a mean
      of 64.2 � 18.5 months after a sustained virological response to interferon
      (IFN)/ribavirin. RNA detection was enhanced by stimulating lymphocytes and
      macrophages in culture before testing, and by testing serial samples over long
      intervals. HCV RNA was found in macrophages from 11 (65%), in lymphocytes from 7
      (41%), in serum from 4 (24%), and in liver from 3 (27%) of 11 tested. Viral
      replicative forms (negative strands) were found in lymphocytes from 2 and
      macrophages from 4. Of note, residual viral loads were very low; only 1 of 4 serum
      samples had quantifiable virus, and that level was only 208 gEq/mL. In cultured
      lymphocytes and macrophages, quantitation was possible in only the minority
      and here the levels were 166 to 560 gEq/106 cells. Importantly, 9 of 11 patients
      with follow-up biopsies showed significant improvement in necroinflammatory
      and fibrosis scores despite the presence of residual virus. Interestingly, the
      two patients who did not show histologic improvement were among the three with
      residual virus in the liver.

      Thus, it appears that small amounts of HCV RNA persist in the tissues of
      apparent sustained responders for up to 9 years. Such persistence has been
      suspected because of vigorous long-term cell-mediated immune responses in patients
      who clear viremia either spontaneously or following antiviral therapy.
      Nonetheless, it was hoped that viral clearance was permanent because HCV does not
      integrate into the host genome and because there is no recalcitrant replicative
      intermediate such as covalently closed circular DNA (cccDNA). Despite these
      hopes, it appears that apparent virologic recovery in HCV infection may be more
      akin to the persistent replicative state that has been shown following recovery
      in hepadnavirus and some non-HCV flavivirus infections. This residual low-level
      replicative state may not have clinical relevance since there is coexistent
      histological improvement and maintenance of a strong humoral and cell-mediated
      immune response that should hold the virus in check. The major concern is
      whether a virological and clinical exacerbation could occur if such patients were
      subsequently immunosuppressed. That issue cannot be answered at present and
      sends a signal that sustained virological responders need to be followed long
      term and perhaps advised of potential recurrence should they develop an immune
      deficient state through disease or medication.

      2nd EDITORIAL

      HCV persistence: Cure is still a four letter word

      Jordan J. Feld *�, T. Jake Liang

      Liver Disease Branch, National Institute of Diabetes & Digestive & Kidney
      Diseases, National Institutes of Health, Bethesda, MD

      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]

      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.

      References
      1 Alter MJ, Kruszon-Moran D, Nainan OV, McQuillan GM, Gao F, Moyer LA, et
      al. The prevalence of hepatitis C virus infection in the United states, 1988
      through 1994. N Engl J Med 1999; 341: 556-562.
      2 Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL Jr, et
      al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus
      infection. N Engl J Med 2002; 347: 975-982.
      3 McHutchison JG, Davis G, Esteban-Mur R, Poynard T, Ling MH, Garaud JJ,
      et al. Durability of sustained virologic response in patients with chronic
      hepatitis C after treatment with interferon alpha-2B alone or in combination with
      ribavirin [abstract]. HEPATOLOGY 2001; 34(Suppl): 244A.
      4 Swain M, Heathcote EJ, Bain V, Feinman SV, Sherman M, Kaita KD, et al.
      Long-lasting sustained virological response in chronic hepatitis C patients
      previously treated with 40 Kda peginterferon alpha-2A (pegasys) [abstract].
      HEPATOLOGY 2001; 34(Suppl): 33A.
      5 Marcellin P, Boyer N, Gervais A, Martinot M, Pouteau M, Castelnau C, et
      al. Long-term histologic improvement and loss of detectable intrahepatic HCV
      RNA in patients with chronic hepatitis C and sustained response to
      interferon-alpha therapy. Ann Intern Med 1997; 127: 875-881.
      6 Pham TN, MacParland SA, Mulrooney PM, Cooksley H, Naoumov NV, Michalak
      TI. Hepatitis C virus persistence after spontaneous or treatment-induced
      resolution of hepatitis C. J Virology 2004; 78: 5867-5874.
      7 Radkowski M, Gallegos-Orozco JF, Jablonska J, Colby TV,
      Walewska-Zielecka B, Kubicka, J, et al. Persistence of hepatitis C virus in patients
      successfully treated for chronic hepatitis C. HEPATOLOGY 2005; 41: 106-114.
      8 Liang TJ, Baruch Y, Ben-Porath E, Enat R, Bassan L, Brown NV, et al.
      Hepatitis B virus infection in patients with idiopathic liver disease.
      Hepatology 1991; 13: 1044-51.
      9 Liang TJ, Bodenheimer HC Jr, Yankee R, Brown NV, Chang K, Huang J, et
      al. Presence of hepatitis B and C viral genomes in US blood donors as detected
      by polymerase chain reaction amplification. J Med Virol 1994; 42: 151-157.
      10 Torbenson M, Kannangai R, Astemborski J, Strathdee SA, Vlahov D,
      Thomas DL. High prevalence of occult hepatitis B in Baltimore injection drug users.
      HEPATOLOGY 2004; 39: 51-57.
      11 Uchida T, Shimojima M, Gotoh K, Shikata T, Tanaka E, Kiyosawa K.
      Silent hepatitis B virus mutants are responsible for non-A, non-B, non-C, non-D,
      non-E hepatitis. Microbiol Immunol 1994; 38: 281-285.
      12 Paterlini P, Gerken G, Nakajima E, Terre S, D'Errico A, Grigioni W, et
      al. Polymerase chain reaction to detect hepatitis B virus DNA and RNA
      sequences in primary liver cancers from patients negative for hepatitis B surface
      antigen. N Engl J Med 1990; 323: 80-85.
      13 Cacciola I, Pollicino T, Squadrito G, Cerenzia G, Orlando ME, Raimondo
      G. Occult hepatitis B virus infection in patients with chronic hepatitis C
      liver disease. N Eng J Med 1999; 341: 22-26.
      14 Kao JH, Chen PJ, Lai MY, Chen DS. Occult hepatitis B virus infection
      and clinical outcomes of patients with chronic hepatitis C. J Clin Microbiol
      2002; 40: 4068-4071.
      15 Fabris P, Brown D, Tositti G, Bozzola L, Giordani MT, Bevilacqua P, et
      al. Occult hepatitis B virus infection does not affect liver histology or r
      esponse to therapy with interferon alpha and ribavirin in intravenous drug users
      with chronic hepatitis C. J Clin Virol 2004; 29: 160-166.
      16 Chan HL, Tsang SW, Leung NW, Tse CH, Hui Y, Tam JS, et al. Occult HBV
      infection in cryptogenic liver cirrhosis in an area with high prevalence of
      HBV infection. Am J Gastroenterol 2002; 97: 1211-1215.
      17 Komori M, Yuki N, Nagaoka T, Yamashiro M, Mochizuki K, Kaneko A, et
      al. Long-term clinical impact of occult hepatitis B virus infection in chronic
      hepatitis B patients. J Hepatol 2001; 35: 798-804.
      18 Castillo I, Pardo M, Bartolome J, Ortiz-Movilla N, Rodriguez-Inigo E,
      Lucas S, Salas C, Jimenez-Heffernan JA, Perez-Mota A, Graus J, Lopez-Alcorocho
      JM, Carreno V. Occult hepatitis C virus infection in patients in whom the
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      2004; 189: 7-14.
      19 McHutchison JG, Poynard T, Esteban-Mur R, Davis GL, Goodman ZD, Harvey
      J, et al. Hepatic HCV RNA before and after treatment with interferon alone or
      combined with ribavirin. HEPATOLOGY 2002; 35: 688-693.
      20 Takaki A, Wiese M, Maertens G, Depla E, Seifert U, Liebetrau A, et al.
      Cellular immune responses persist and humoral responses decrease two decades
      after recovery from a single-source outbreak of hepatitis C. Nat Med 2000; 6:
      578-582.
      21 Forns X, Garcia-Retortillo M, Serrano T, Feliu A, Suarez F, de la Mata
      M, et al. Antiviral therapy of patients with decompensated cirrhosis to
      prevent recurrence of hepatitis C after liver transplantation. J Hepatol 2003; 39:
      389-396.
      22 Radkowski M, Wilkinson J, Nowicki M, Adair D, Vargas H, Ingui C, et
      al. Search for hepatitis C virus negative-strand RNA sequences and analysis of
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      Persistence of hepatitis C virus in patients successfully treated for chronic
      hepatitis C

      Article Text

      Hepatitis C virus (HCV) is the major etiologic agent of parenterally
      transmitted non-A, non-B hepatitis.[1][2] In most infected patients, HCV persists
      indefinitely, leading to chronic hepatitis, cirrhosis, and hepatocellular
      carcinoma.[3-5] The overall prevalence of anti-HCV in the United States is 1.8%, and
      approximately 2.7 million Americans carry the virus.[6] Seeff et al.[7] have
      recently reported on the long-term outcome in HCV-infected patients in several
      different transfusion studies. Twenty-five year follow-up of the HCV cases
      showed viremia with chronic hepatitis in 38%, viremia without chronic hepatitis in
      39%, anti-HCV without viremia in 17%, and only 7% of patients had no residual
      markers of HCV infection. Thirty-five percent of HCV patients who underwent
      biopsies for biochemically defined chronic hepatitis displayed cirrhosis,
      representing 17% of all patients.[7]

      Modern antiviral therapy is successful in approximately 50% of infected
      patients, resulting in clearance of HCV RNA from serum, which is usually
      accompanied by normalization of liver biochemical tests and improvement of liver
      histology.[8][9] Currently accepted criteria for sustained virological response (SVR)
      require the patient to remain HCV RNA negative in serum for 6 months after
      termination of treatment when tested with an assay with a sensitivity of at
      least 100 viral copies/mL.[8] Long-term virological outcome in patients with SVR
      has been analyzed only recently. Recurrence of infection, defined as
      reappearance of HCV RNA in serum, was found to be below 2% at 1 to 4 years after
      induction of SVR,[10][11] although in one study in which patients were followed for
      3.5 to 8.8 years, the relapse rate was as high as 8%.[12] Obviously, because
      the length of follow-up of treated patients is still limited, durability of SVR
      may turn out to be lower over extended periods. It has also been reported that
      in 2% of SVR patients viral sequences could be detected in liver, and some of
      these patients ultimately experienced recurrent infection.[13] However, the
      presence of HCV in other compartments except liver has not been analyzed so far
      in SVR patients.

      HCV is not a strictly hepatotropic virus, and there is evidence that it can
      also replicate in peripheral blood mononuclear cells (PBMCs). The infected
      cells were reported to contain HCV RNA-negative strand, which is a viral
      replicative intermediate, and viral genomic sequences were often found to be distinct
      from those found in serum and liver.[14-17] Furthermore, it was also reported
      that human T- and B-cell lines are capable of supporting HCV infection in
      vitro,[18][19] and some viral strains were found to be lymphotropic both in vitro
      and in vivo in infected chimpanzees.[20] Within the population of PBMCs, the
      cells harboring replicating virus have been identified as belonging to T-cell
      and B-cell lineage and monocytes/macrophages.[21-23] Although the presence of
      extrahepatic replication of HCV is becoming well recognized, whether it is
      affected by current antiviral treatment is unclear. Persistence of extrahepatic
      sites of HCV replication could potentially play a role in late recurrence after
      treatment.

      In the current study, we provide evidence that in most patients with SVR,
      low-level HCV RNA can be detected in lymphocytes and monocytes/macrophages, and
      occasionally in liver and serum for up to 9 years after the end of therapy.

      Patients and Methods

      Biological Samples.
      The study group comprised 17 randomly chosen patients with chronic HCV who
      responded to antiviral therapy and fulfilled the criteria for SVR by being
      negative for HCV RNA in serum at the end of therapy and 6 months afterwards. The
      other inclusion criteria were willingness to participate in the study and at
      least 3 years' follow-up after the end of treatment. Moreover, patients had to be
      persistently HCV RNA negative in serum by routine testing performed at least
      once a year after the end of therapy. During the 4- to 9-year follow-up, HCV
      RNA status was checked in all patients first by in-house assay (sensitivity
      limit, 500 viral copies/mL) and from 1997 on by Amplicor HCV version 2.0
      (sensitivity limit, 135 viral copies/mL). Liver function tests, which were repeated
      every 6 months during the follow up, were consistently normal. Before treatment,
      the diagnosis of chronic hepatitis was based on the presence of HCV RNA and
      anti-HCV in serum, increased activity of serum aminotransferases, and on liver
      biopsy findings, which was performed in 15 patients. Five patients treated
      between 1993 and 1997 received interferon (IFN) a2b (Intron-A) monotherapy 3 to 5
      million units for 24 to 72 weeks 3 times per week, whereas 12 patients
      received combination treatment consisting of intron-A 5 million units 3 times per
      week and ribavirin 1,200 mg daily. The likely sources of infection were surgery
      with or without blood transfusions (10 cases), occupational exposure (3
      cases), and past intravenous drug abuse (2 cases). In 2 patients, no risk factors
      could be identified. The study protocol conformed to institutional review board
      guidelines at participating institutions.

      For the current study, serum and PBMC samples were collected 2 to 3 times at
      3- to 6-month intervals from each patient starting 40 to 109 months (mean,
      64.2 � 18.5 months) after the end of therapy. In addition, frozen tissue samples
      kept at -80�C were available from 11 control liver biopsies performed 41 to 98
      months (mean, 63.6 � 16.7 months) after therapy. Liver histopathology was
      read in a blinded fashion as to clinical data by a trained pathologist (T.V.C.),
      using Hepatitis Activity Index scoring system proposed by Knodell et al.[24]

      Control sera and PBMC samples were collected from 15 healthy
      anti-HCV-negative subjects. In 13 of 15 controls, sera and PBMCs were collected again after 1
      month.

      MORE RESULTS

      HCV RNA-positive livers were examined for viral RNA load by our real-time
      strain-specific assay. All three samples had low but quantifiable amounts of
      virus ranging from 180 genomic Eq/g RNA in patients 10 to 245 and 466 genomic Eq
      in patients 4 and 1, respectively. However, HCV RNA-negative strand was not
      detected in any of the 3 liver samples. This result is not unexpected, because
      the viral-negative strand is typically 1- to -2-log lower than the positive
      strand,[28][29] and thus it was likely to have been below the sensitivity of the
      assay. In all 3 cases, the HCV genotype found in the liver was identical to
      that present in serum before initiation of treatment.

      HCV RNA in Sera.
      Sera were collected starting 40 to 109 months after the end of antiviral
      treatment. All 17 patients were repeatedly nonreactive for HCV RNA by commercial
      tests (Amplicor 2.0, Roche). Whereas all initial sera were HCV RNA negative
      with our current in-house assay (sensitivity limit 10 genomic Eq/mL), 4 sera
      collected from 4 different patients at later times were positive (Table 2). All
      sera, which were positive by the RT-PCR/hybridization assay, were tested for RNA
      viral load by the positive-strand real-time PCR (sensitivity 102 genomic
      Eq/mL). Of 4 samples, only 1 contained quantifiable amounts of HCV RNA (208
      genomic Eq/mL). Because the remaining 3 sera were negative when tested by the
      real-time RT-PCR, the quantity of HCV RNA must have been below the sensitivity limit
      of this assay and was therefore likely to be between 10 and 100 viral
      copies/mL.

      HCV RNA in PBMCs and Lymphocyte and Macrophage Cultures.
      Two of 17 studied patients were HCV RNA positive in unfractioned PBMCs on
      first testing, and this number increased to 9 when additional samples collected
      at 3- to 6-month intervals were analyzed (Table 2). The presence of viral
      sequences was confirmed in 4 cases by positive-strand real-time RT-PCR, and in one
      of these samples HCV RNA-negative strand was detected as well. The quantity of
      viral load varied from 166 to 560 genomic Eq per 106 cells, and the
      proportion of positive to negative HCV RNA strand in the single case where both were
      detected was 6.3.

      We have previously found that mitogen stimulation of lymphocytes may enhance
      HCV replication,[21] and similar observations have been made by other authors
      with respect to woodchuck hepatitis virus.[30] We have also found that HCV
      positive and negative strands can be detected in cultured macrophages from
      infected patients[21] and that cultured native human macrophages are susceptible to
      HCV infection in vitro.[31] For these reasons, we analyzed the presence of
      viral RNA in phytohemagglutinin mitogen (PHA) stimulated lymphocytes and in
      cultured macrophages. As seen in Table 2, HCV RNA was detectable either in
      stimulated lymphocytes or cultured macrophages in 9 patients (53%) on the first
      testing, and 14 patients (82%) were positive in at least one culture when the
      analysis was repeated 1 to 2 times using cells collected at later times. In 16 of 29
      positive lymphocyte and macrophage samples, there was enough viral RNA to
      allow for quantification of HCV RNA-positive strand, which ranged from 1.43 � 102
      to 4.26 � 103 per 106 cells (mean, 9.51 � 102 genomic Eq/106 cells). HCV
      RNA-negative strand, which is a viral replicative form, was detected in 6 (21%) of
      29 HCV RNA-positive cultures (Table 3). The ratio of positive to negative
      strand in the samples ranged from 2.1 to 11.3 (mean, 6.6), and is lower than the
      ratio reported for liver but similar to the ratio determined previously in
      HCV-infected macrophage cell cultures.[28] Reactions detecting viral-negative
      strand were unlikely to represent false-positive results because nonspecific
      detection of the incorrect strand might be expected when the latter is present at
      a concentration of at least 107 to 108 genomic Eq/reaction. However, the
      concentration of HCV RNA in samples containing viral-negative strand was below 104
      genomic Eq/reaction.

      Overall, taking into account the results of all testing, including cell
      cultures as well as detection of HCV RNA in liver and serum, evidence of infection
      was found in 15 (88%) of 17 patients. In one patient, the only evidence of
      infection was HCV RNA presence in the liver tissue, whereas in the remaining
      patients viral sequences were detected in serum or cultured macrophages and
      lymphocytes. Thus, HCV RNA was detected in macrophages from 11 patients (65%) and in
      lymphocytes from 7 patients (41%); in 4 patients (24%), both cell types
      tested positive. Viral replicative forms were found in lymphocytes from 2 (12%) and
      in macrophages from 4 (24%) patients. Only 2 of 17 patients in the study
      remained consistently HCV RNA negative in all analyzed compartments; however,
      follow-up liver biopsy sample was unavailable for analysis in one of these cases.
      Because of limitations in clinical material available, repeated independent
      confirmation of positive results was limited to liver samples and 16 cell
      cultures. All 3 liver samples and 15 cultures (94%) were positive on repeated
      testing with our RT-PCR. However, 24 (53%) of 45 originally positive samples were
      confirmed by the less sensitive real-time quantitative RT-PCR.

      Comparison of 5UTR sequences amplified from different compartments was done
      in 14 patients. For this purpose, respective RT-PCR products were cloned (TA
      cloning system, Invitrogen) and 2 to 7 individual clones were sequenced
      directly. In all 3 liver-positive cases, viral sequences amplified after therapy from
      liver tissue were identical to those found in pretreatment serum. Altogether,
      in 6 patients, all analyzed sequences were identical in the same patient. In 7
      cases, there were small sequence differences, suggesting a possibility of
      viral evolution, and in one patient (patient 8), sequence differences were
      significant enough to qualify pretreatment and posttreatment viral variants as
      belonging to various genotypes. Figure 4 shows comparison of sequences amplified
      from different compartment in patients 2, 8, and 13. In case 2, the pretreatment
      serum-derived virus differed from all posttreatment sequences by two
      nucleotide substitutions (C to A at position 204 and G to A at position 243) and one
      nucleotide insertion (C between positions 126 and 127). Interestingly, all of
      these changes were previously identified by us in sequences amplified from
      extrahepatic sites,[16][32] and substitutions at positions 204 and 243 were also
      described by others.[18][33] The C to A change at position 204 and G to A
      change at position 243 were also found in patient 16 (not shown). In patient 13,
      pretreatment and posttreatment sequences differed by 3 to 4 substitutions.

      Importantly, the genotypes after treatment were compatible with the genotypes
      found in pretreatment serum, the only exception being patient 8. However,
      this patient had a history of intravenous drug use, and thus the genotype
      discordance may have been the result of fresh superinfection. Alternatively, there
      could have been different genotypes in PBMCs and serum compartments before
      treatment. However, no PBMCs from the pretreatment period were available for
      analysis.

      All sera and PBMC samples as well as all lymphocyte and macrophage cultures
      from 15 control subjects were HCV RNA negative.

      Author Discussion

      Currently available antiviral therapy for chronic hepatitis C leads to SVR in
      approximately 50% of patients.[8][9] However, whether successful treatment
      results in sterilization, or whether low viral replication persists and is
      perhaps kept in check by cellular and humoral immune responses, is unclear. Thus,
      the observation that specific antibodies and cellular immune response may
      persist for 2 decades after spontaneous resolution of acute hepatitis C could imply
      continuous antigen stimulation.[7][34] The critical role of virus-specific
      CD4+ T cells in long-term control of the virus is suggested by the observation
      that loss of this specific T-cell response is followed by HCV recurrence in
      patients recovering from acute hepatitis C.[35] Similarly, in the chimpanzee
      hepatitis C model, depletion of CD4+ T cells results in incomplete control of
      viremia associated with emergence of viral escape mutations.[36]

      The current study provides evidence that HCV RNA can indeed persist for years
      in patients fulfilling the accepted criteria for SVR after treatment of
      chronic hepatitis C.[8] Whereas HCV RNA was detectable in sera from only 4 of 17
      patients, 14 patients harbored viral sequences in circulating lymphocytes or
      macrophages. This situation may be analogous to infection with hepatitis B virus,
      which also demonstrates lymphotropism.[37][38] Thus, hepatitis B virus DNA
      sequences were found in PBMCs over 5 years after complete clinical and
      serological recovery from acute viral hepatitis.[39] Small quantities of hepatitis B
      virus DNA have been shown to persist in serum for decades after recovery from
      acute hepatitis, and these traces of virus maintain specific cytotoxic
      T-lymphocyte response.[40] Also, in woodchuck hepatitis virus infection model, a
      lifelong persistence of scanty amounts of replicating virus occurs in both the liver
      and the lymphatic system after spontaneous resolution of experimental
      hepatitis.[41]

      HCV RNA was detected in unfractioned PBMCs in 9 patients, and viral-negative
      strand was detected only in one. However, culture of lymphoid cells with PHA
      as well as culture of macrophages increased the number of patients harboring
      HCV sequences to 14, and in 6 of these patients, viral replicative forms were
      detected as well. This finding suggests that HCV replication may be more
      efficient in activated cells. PHA is nonspecific inducer of T-cell proliferation
      stimulating CD8+ and CD4+ cells via lectin binding to glycosylated T-cell receptor
      complex proteins, whereas culturing of macrophages on polystyrene delivers a
      powerful activation signal for the latter cells.[42] Interestingly, it was
      shown that unspecific mitogen stimulation of lymphocytes facilitates detection of
      woodchuck hepatitis virus, which is becoming recognized as being an
      inherently lymphotropic virus.[41] Another important factor aiding HCV RNA detection
      was repeated testing. Whereas 9 patients were positive in serum or lymphocyte
      and macrophage culture on testing of initial samples, this number increased to
      14 when samples collected at 2 to 3 different points were analyzed. Because
      viral load was typically low and close to the detection limit of used assays,
      this intermittent detection could be explained by sampling differences related to
      stochastic phenomena.

      Infection of monocytes/macrophages by HCV is not unexpected, because these
      cells are known to be permissive to a wide range of viruses, including some
      other flaviviruses,[43] and many RNA and DNA viruses are lymphotropic.[44] We have
      previously reported the presence of viral replicative forms in
      monocytes/macrophages from HCV-infected patients,[21] and in subsequent studies we showed
      that native human macrophages are susceptible to HCV infection in vitro and that
      this infection may be facilitated by concomitant human immunodeficiency virus
      (HIV) infection.[28] Whereas the mere presence of HCV RNA in phagocytic cells
      could come from virions entrapped inside these cells or adsorbed on their
      surface, presence of viral-negative strand in macrophage and lymphocyte cultures
      argues for the presence of genuine viral replication. HCV RNA-negative strand
      was detected only in a minority of lymphocyte and macrophage cultures, but it
      is likely that the strand-specific assays are not sensitive enough to detect
      low-level extrahepatic replication. Indeed, in several studies they were found
      to be at least 1 log less sensitive than standard RT-PCR.[27][29] Moreover, in
      cells supporting HCV replication, negative RNA strands are generally detected
      at levels lower than the levels of positive strands,[27][29] and the titer of
      positive strand HCV RNA in cultured cells was low to begin with.
      Interestingly, using a novel real-time quantitative PCR, we found the ratio of positive to
      negative strands in lymphocytes and macrophages may be lower than that found
      in the liver,[27][29] which suggests that replication dynamics in extrahepatic
      and hepatic sites may be different. However, it is also possible that the
      high ratio in the liver tissues is caused by contamination by circulating virions
      containing positive-strand HCV RNA.

      Our findings of the continuing presence of HCV RNA years after ostensible
      successful treatment are compatible with the very recent findings by Pham et
      al.,[45] who were able to amplify viral sequence from follow-up sera or PBMC in 11
      of 11 SVR patients for up to 5 years after therapy. Most monocyte-derived
      dendritic cell cultures and mitogen-stimulated PBMCs contained HCV RNA-negative
      strand as well. All of those patients were HCV RNA negative in serum by
      commercial assays. However, liver tissue was not analyzed in this report, and
      pretreatment samples were not available for genotype comparison.

      In summary, our results suggest that in patients with SVR after IFN or
      IFN/ribavirin therapy, small quantities of HCV RNA may persist in liver or PBMCs for
      up to 9 years. This continuous presence of HCV RNA could explain the
      phenomenon of relatively common persistence of humoral and cellular immunity for many
      years after supposed viral clearance and could present a potential risk for
      transmission or infection reactivation.

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