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Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C
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
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.
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.
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.
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. Numerous long-term follow-up studies have shown that SVR appears to be a durable clinical endpoint. 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. These observations have led some to cautiously consider the possibility that SVR may actually
Pham et al. recently reminded us that the word CURE must not be used prematurely. 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. 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), patients with no known history of HBV, patients with hepatocellular carcinoma, 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, subsequent studies have not confirmed this finding. Similarly, with isolated occult HBV the jury is still out. While Chan et al. found that one third of patients with cryptogenic cirrhosis had occult HBV in Hong Kong, Komori et al. 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. 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. 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. It is likely that using the sensitive techniques of Radkowski et al., 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. 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. 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. 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), 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. 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. 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.
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 response 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 etiology of persistently abnormal results of liver-function tests is unknown. JID 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 viral sequences in the central nervous system: evidence of replication. J Virol 2002; 76: 600-608.
23 Ducoulombier D, Roque-Afonso AM, Di Liberto G, Penin F, Kara R, Richard Y, et al. Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes. HEPATOLOGY 2004; 39: 817-825.
Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C
Hepatitis C virus (HCV) is the major etiologic agent of parenterally transmitted non-A, non-B hepatitis. 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. Seeff et al. 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.
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. 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. 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, although in one study in which patients were followed for 3.5 to 8.8 years, the relapse rate was as high as 8%. 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. 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, and some viral strains were found to be lymphotropic both in vitro and in vivo in infected chimpanzees. 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
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.
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.
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, 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, and similar observations have been made by other authors with respect to woodchuck hepatitis virus. We have also found that HCV positive and negative strands can be detected in cultured macrophages from infected patients and that cultured native human macrophages are susceptible to HCV infection in vitro. 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. 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, and substitutions at positions 204 and 243 were also described by others. 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.
Currently available antiviral therapy for chronic hepatitis C leads to SVR in approximately 50% of patients. 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. 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. 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.
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. 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. Thus, hepatitis B virus DNA sequences were found in PBMCs over 5 years after complete clinical and serological recovery from acute viral hepatitis. 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. 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
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. 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. 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, and many RNA and DNA viruses are lymphotropic. We have previously reported the presence of viral replicative forms in monocytes/macrophages from HCV-infected patients, 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. 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. Moreover, in cells supporting HCV replication, negative RNA strands are generally detected at levels lower than the levels of positive strands, 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, 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., 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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