Introduction
Hepatitis C virus (HCV) is a major etiologic agent of chronic hepatitis in the Western world. The prevalence of serum markers of hepatitis C is 1.8% in the general population in the United States [1]. The great majority of subjects infected by HCV go on to develop chronic hepatitis, which, in some instances, can evolve into cirrhosis and hepatocellular carcinoma. HCV is currently considered to be the main cause of end-stage liver disease requiring liver transplantation in the US and Europe. Although this RNA virus mainly affects the liver, there is ample evidence that supports the existence of extrahepatic replication [2–5]. One of the most studied sites is the lymphatic system. More than a decade ago, Willems et al. were able to demonstrate the ability of HCV to replicate in the peripheral blood mononuclear cells (PBMCs) of infected hemophiliac patients [6]. Later reports form other groups have also substantiated the replication of HCV in PBMCs [7, 8].

The current antiviral strategy is based on the use of interferon alfa (IFN) in combination with ribavirin. Through the years, the efficacy of treatment has consistently improved to the current response rate of over 50% when using the combination of pegylated-IFN and ribavirin [9–11]. It has been accepted that the clearance of hepatitis C from serum 6 months after the end of treatment is a marker for sustained viral response and this is generally thought to represent the ‘‘cure’’ of the infection, as the vast majority of subjects that reach this therapeutic goal go on to have a clinical, biochemical, and sometimes histological remission of their chronic hepatitis [12–16].

The concept of ‘‘viral eradication’’ has been recently challenged by different groups who have demonstrated the persistence of viral RNA in serum, liver, and PBMCs when sensitive assays are used to examine patients with spontaneous or treatment-induced infection resolution as conventionally defined [17, 18]. However, these reports have only considered patients treated with standard IFN, which has been proven to be less effective than peginterferon for the treatment of chronic hepatitis C [9, 10]. For this reason, we set out to study the frequency of HCV RNA J. F. Gallegos-Orozco Department of Internal Medicine, Mayo Clinic Arizona, Phoenix, AZ, USA

J. Rakela M. J. Rosati H. E. Vargas (&) V. Balan
Division of Transplantation Medicine, Mayo Clinic Arizona,
5777 Mayo Clinic Blvd., Phoenix, AZ 85054, USA
e-mail: vargas.hugo@mayo.edu 

HBV
DOI 10.1007/s10620-008-0323-x
persistence in serum and PBMCs of sustained viral responders (SVRs) to combination antiviral therapy with peginterferon alfa-2a and ribavirin.

Patients
Twenty-five SVRs to a complete course of peginterferon alfa-2a (Pegasys, Roche Pharmaceuticals, Nutley, NJ) and ribavirin therapy were included in this study. None of the patients had received previous anti-HCV therapy before combination with peginterferon alfa-2a and ribavirin. SVR was defined as the absence of detectable serum HCV RNA by commercial qualitative sensitive assays with a lower limit of detection of 50 IU/ml (Cobas Amplicar ver 2.0, Roche Diagnostics, Indianapolis, IN) 6 months after the end of therapy (EOT). The length of treatment was based on the infecting genotype, so that patients infected with genotype 1 were treated for 48 weeks, while patients with other genotypes (namely, genotypes 2 and 3) received only 24 weeks of combined therapy. The protocol was approved by the Mayo Clinic Institutional Review Board and all patients provided written informed consent.

Material and Methods
Blood samples were collected 6–56 months (mean, 22 months) after the end of treatment in all patients. Additionally, 13 of the 25 patients underwent a second collection 13–49 months after EOT (mean, 28 months). PBMCs from 10 ml of blood were isolated following centrifugation over a density gradient (Ficoll-Hypaque, Pharmacia, Kalamazoo, MI) and were immediately subjected to culture under mitogen and cytokine stimulation using variations of recently described methods [19]. In brief, PBMCs were placed in 2 ml of RPMI 1640 medium (Gibco BRL, Grand Island, NY) and 10% fetal bovine serum in six-well plates with the following combinations of mitogens and cytokines: (a) phitohemaglutinin (PHA, EY Laboratories Inc., San Mateo, CA) 5 lg/ml and interleukin- 2 (IL-2, Roche Diagnostics, Indianapolis, IN) 20 U/ml; (b) PHA 5 lg/ml, pokeweed (PWD, EY Laboratories Inc., San Mateo, CA) 5 lg/ml, IL-2 20 U/ml, and IL-4 (Roche Diagnostics, Indianapolis, IN) 1 ng/ml. PBMCs were incubated at 37 C for 48 h. After culture, the PBMCs were centrifuged at 400g, re-suspended in 750 ll of TRIzol LS (Invitrogen, Carlsbad, CA) and stored at -80 C until analysis. Two 500-ll aliquots of plasma obtained at the time of PBMC isolation were stored at -80 C until analysis.

RNA was extracted from plasma and cells following a modified guanidium thiocyanate–phenol/chloroform technique using commercially available kit (TRIzol LS). The RNA extracted from 500 ll of plasma was used in a single nested reverse-transcriptase polymerase chain reaction (PCR) using a previously described procedure for the 50UTR region [20]. RNA extracted from cultured PBMCs was diluted in 10 ll of DNase and RNase-free water, and stored at -20 C until analysis by PCR.

Real-Time PCR
An initial reverse transcriptase step was performed with Moloney murine leukemia virus (MMLV) using 5 ll of RNA template as previously described [20]. This was followed by a first round of PCR amplification using specific external primers for the 50UTR region (Table 1). Afterwards, samples were subjected to nested PCR using the internal primer-pair referred to in Table 1. The final 251- nucleotide-long amplicon was run on a 2% agarose gel and stained with ethidium bromide. Serial dilutions of synthetic RNA strand were used as positive controls and pertinent negative controls were also run in parallel. Extensive measures were carried out to avoid contamination. This inhouse assay has a detection limit of 10 copies/ml and was performed on both serum and PBMC samples obtained at all time points in all subjects included.

All PBMC samples were subjected to a qualitative realtime PCR using a LightCycler FastStart DNA Master SYBR Green I (Roche, Indianapolis, IN) and nested primer set as described previously [21]. Briefly, 2 ll of first-round product were added to an 18-ll mix of water, MgCl2 25 mM, 50 pmol of each internal primer (Table 1 ), and 2 ll of SYBR green. Real-time PCR was performed in a Light-Cycler apparatus (Roche) under the following conditions:

Table 1


10 min at 95 C for initial denaturation and enzyme activation, followed by 35 cycles of 95 C for 30 s, 55 C for 5 s, and 72 C for 30 s. A melting curve analysis was performed after each amplification to ensure that the appropriate product was amplified.
Statistical analysis was performed with SPSS version 11, using Student’s t-test to compare means between groups and the Chi-square or Fisher’s exact tests for the comparison of categorical data. A P-value \0.05 was considered to be statistically significant.

Results
There were 25 SVRs included in this study (14 female, 11 male), with a mean age of 52 years (range, 30–73 years). Fourteen patients (56%) were infected with HCV genotype 1 (eight with 1a, five with 1b, and one with 1a/1b), seven with genotype 2, and four with genotype 3. All patients completed a full course of peginterferon alfa-2a and RBV therapy (according to infecting genotype) and fulfilled the currently accepted definition of SVR. None of the 25 patients had detectable serum HCV RNA by commercial qualitative assay or with an in-house similarly sensitive RT-PCR at any time point analyzed.

The mean alanine aminotransferase (ALT) level before treatment was 103.4 ± 70.3, 33.3 ± 25.6 U/l at the EOT, and 22 ± 6.7 U/l 6 months after finishing antiviral treatment. Twenty-three patients had available pre-treatment liver biopsies, but only seven patients had post-treatment biopsy, as this was not a requirement after therapy. The mean pre-treatment hepatitis activity index (HAI) was 4.21 ± 1.96, with a mean fibrosis score of 1.45 ± 1.54, while the mean HAI score was 3.5 ± 2.43 and the fibrosis
score was 2 ± 2.52 after therapy.

Persistent HCV infection, defined as the presence of HCV RNA in PBMC cultures, was present in five of the 25 patients (20%), 3/25 from the first PBMC collection and 2/ 13 in the second PBMC collection. None of these five patients were positive on both collections. Among the five patients, there were three males and two females, with a mean age of 53 years. With regards to genotype distribution, three patients were infected by HCV genotype 1 and two patients by genotype 2. The mean re-treatment, end of treatment, and 6 months post-treatment serum ALT levels were 74.2 ± 47.4, 18.8 ± 5.5, and 23 ± 7.5 U/l, compared to 110.7 ± 74.1, 36.9 ± 27.5, and 21.8 ± 6.7 U/l in the 20 patients in whom PBMC HCV RNA was not detected (P[0.05 for all comparisons, Table 2). No conclusion could be made regarding the histological impact of persistent infection, as only one of the five patients underwent a post-treatment biopsy and the biopsy did not demonstrate any change in fibrosis score.

Discussion
In the current study, we were able to detect positive-strand HCV RNA in mitogen-stimulated PBMC from five of 20 individuals with chronic hepatitis C who fulfilled the current criteria for sustained viral response to a complete course of peginterferon alfa-2a and ribavirin combination antiviral therapy. Pham et al. [17] were the first to describe the occurrence of persistent HCV infection in patients that had cleared serum HCV RNA spontaneously or after treatment with standard interferon. Using a very sensitive nucleic acid detection method, they studied 16 patients that had cleared serum HCV RNA after infection (five with spontaneous clearance and 11 with treatment-induced clearance). They were able to establish the presence of serum HCV RNA in 88% of the samples, while HCV RNA was detected in 81% of the mitogen-stimulated PBMC cultures. The overall HCV RNA positivity in this group of patients was 100%; that is, HCV RNA was detected in at least one compartment (serum, PBMC, or tissue) in all of the studied subjects.

Table 2


In a more recent report, Radkowski et al. [18], using a similar approach, were able to detect HCV RNA in at least one compartment in 15 of 17 subjects (88%) considered to be SVRs after a complete trial of interferon and ribavirin combination therapy. In that study, patients were followed up for up to 9 years, with repeated sample collections at predefined time points. Low-levels of serum HCV RNA were detected in four of the 17 patients (24%), while nine of 17 (53%) were HCV RNA-positive in unfractioned PBMCs after 2–3 collections. These authors also analyzed the presence of HCV RNA in mitogen-stimulated lymphocytes and in cultured macrophages. Viral RNA was detected in 14 patients (82%) after two or three cell collections over time [18].

In the present report, 20% of SVRs had detectable HCV RNA in mitogen-stimulated PBMC cultures. This frequency of persistent HCV infection is lower than that previously reported [17, 18]. This could, in part, be due to the fact that the subjects described in the present report had received antiviral treatment with the most current recommended regimen, that of combined peginterferon with ribavirin, which has been previously demonstrated to be the most effective treatment to date. Another possibility would be the fact that the patients described had a shorter followup after the end of treatment than that reported by others.

It is unclear why we were unable to detect HCV RNA in both PBMC collections in the five patients identified, but it
is possible that the amount of viral RNA within the cultured PBMCs is so low that the assay employed in the current study was not sensitive enough to uniformly detect this low-level HCV RNA content.

Currently, sustained viral response has been equated to the ‘‘cure’’ of the HCV infection, as it undoubtedly produces long-term clinical, biochemical, and histological benefits. But it is also clear that, after long-term follow-up of subjects with sustained viral response, up to 8% of cases will relapse [22]. The relevance of persistent HCV RNA in tissue, serum, or blood cells is not yet apparent, but it is plausible that it could be the source of HCV recurrence under special circumstances, such as immunosuppression.

Support for this view has been recently provided by a well documented case in which a patient with IgG deficiency developed acute hepatitis C that responded to interferon therapy [23]. The patient was followed for several years, having persistently normal aminotransferase levels and the absence of serum HCV RNA by PCR assays repeated at least four times during her follow-up. Nine years later, the patient developed acute hepatitis. Six months before this episode of hepatitis, the patient had been receiving intravenous methylprednisolone with each IVIG infusion, and had also undergone several courses of prednisone for asthmatic episodes. Her aminotransferase levels were elevated greater than 15-fold and serum HCV RNA was detected.

Corticosteroids were discontinued and, after 2 months, the patient’s liver function tests normalized and HCV RNA became undetectable once more. The testing of frozen serum from previous episodes of hepatitis confirmed that the recurrence of infection was from the same virus. This clearly suggests that HCV persistence, after therapy or natural resolution, might have clinical implications, especially in patients subjected to immunosuppression. This could certainly be of concern in liver transplant candidates who have received successful antiviral therapy in the pretransplant period.

In conclusion, our results suggest that, in some patients with chronic hepatitis C, HCV RNA may persist in the PBMCs for up to 35 months after fulfilling SVR criteria following combined antiviral therapy with peginterferon alfa-2a and ribavirin. RNA persistence in PBMCs and sera was less frequent among our patients than that reported among those treated with standard interferon and ribavirin.

At present, the clinical relevance of this finding is unclear. It is possible that viral persistence and, specifically, the presence of PBMC HCV RNA may lead to HCV reactivation. Conversely, it may also provide a low level of antigen exposure to keep the immune status that would prevent the reactivation phenomenon.

Acknowledgments This work was supported in part by a grant from the Palumbo Foundation, the Edson Foundation and Roche Laboratories. The study sponsors did not participate in the design of the study, collection, analysis, or interpretation of the data, and were not involved in the writing of the manuscript.

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