November 30, 2013

PLoS One. 2013 Nov 21;8(11):e80078. doi: 10.1371/journal.pone.0080078.

RESEARCH ARTICLE

Annie Y. Chen, Marija Zeremski, Ranjit Chauhan, Ira M. Jacobson, Andrew H. Talal, Tomasz I. Michalak

Abstract

Resolution of chronic hepatitis C is considered when serum HCV RNA becomes repeatedly undetectable and liver enzymes normalize. However, long-term persistence of HCV following therapy with pegylated interferon-α/ribavirin (PegIFN/R) was reported when more sensitive assays and testing of serial plasma, lymphoid cells (PBMC) and/or liver biopsies was applied. Our aim was to reassess plasma and PBMCs collected during and after standard PegIFN/R therapy from individuals who became HCV RNA nonreactive by clinical testing. Of particular interest was to determine if HCV genome and its replication remain detectable during ongoing treatment with PegIFN/R when evaluated by more sensitive detection approaches. Plasma acquired before (n = 11), during (n = 25) and up to 12–88 weeks post-treatment (n = 20) from 9 patients and PBMC (n = 23) from 3 of them were reanalyzed for HCV RNA with sensitivity <2 IU/mL. Clone sequencing of the HCV 5′-untranslated region from plasma and PBMCs was done in 2 patients. HCV RNA was detected in 17/25 (68%) plasma and 8/10 (80%) PBMC samples collected from 8 of 9 patients during therapy, although only 5.4% plasma samples were positive by clinical assays. Among post-treatment HCV RNA-negative plasma samples, 9 of 20 (45.3%) were HCV reactive for up to 59 weeks post-treatment. Molecularly evident replication was found in 6/12 (50%) among PBMC reactive for virus RNA positive strand collected during or after treatment. Pre-treatment point mutations persisted in plasma and/or PBMC throughout therapy and follow-up. Therefore, HCV is not completely cleared during ongoing administration of PegIFN/R otherwise capable of ceasing progression of CHC and virus commonly persists at levels not detectable by the current clinical testing. The findings suggest the need for continued evaluation even after patients achieve undetectable HCV RNA post-treatment.


Citation: Chen AY, Zeremski M, Chauhan R, Jacobson IM, Talal AH, et al. (2013) Persistence of Hepatitis C Virus during and after Otherwise Clinically Successful Treatment of Chronic Hepatitis C with Standard Pegylated Interferon α-2b and Ribavirin Therapy. PLoS ONE 8(11): e80078. doi:10.1371/journal.pone.0080078

Editor: Stephen J. Polyak, University of Washington, United States of America

Received: July 29, 2013; Accepted: October 2, 2013; Published: November 21, 2013

Copyright: © 2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This study was supported by an operating grant MOP-126056 from the Canadian Institutes of Health Research (CIHR) awarded to TIM. AYC is supported by the Canada Research Chair allowance provided by Memorial University. TIM holds the Senior Canada Research Chair in Viral Hepatitis/Immunology sponsored by the Canada Research Chair Program and funds from the CIHR and the Canada Foundation for Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Hepatitis C virus (HCV) is a single-stranded RNA virus that is the cause of clinically diagnosable chronic infection in approximately 170 million people worldwide. Of those acutely afflicted, 15% spontaneously resolve hepatitis, while the remaining develop chronic hepatitis C (CHC) [1]. Up to15% of the patients with CHC progress to fibrosis and cirrhosis, and they are at a greater risk of developing hepatocellular carcinoma (HCC) [2]. HCV is infectious even in trace amounts, with approximately 10 virions or 20 copies of viral RNA capable of transmitting infection in chimpanzees [3], [4] and with 20 to 50 virions able to establish productive infection in human T cells in vitro [5]. The introduction of nucleic acid amplification assays detecting HCV genomes with high sensitivity, i.e., <10 virus genome equivalents (vge) or copies/ml or <2.5 vge/µg RNA (<2 IU/ml), revealed that HCV persists at low levels (usually below 100 vge/ml) for years after clinical resolution of hepatitis either spontaneously or due to treatment with interferon-α (IFN) alone or pegylated IFN/ribavirin (PegIFN/R) [6], [7]. The long-term consequences of this essentially asymptomatic infection, termed as occult HCV infection (OCI), remains uncertain; however, OCI coincides with histologically evident protracted low grade liver inflammation and fibrosis in some patients for at least 10 years after completion of antiviral treatment [8][11]. Also, clinically diagnosed sustained virological response (SVR) achieved due to IFN or PegIFN/R does not universally prevent progression to HCC, which develops in up to 3.9% of these individuals [12][17]. Contrary to prevailing opinion based on the currently available clinical testing for HCV RNA, clinical diagnosis of SVR does not reflect molecular eradication of HCV, as evidenced by assays of enhanced sensitivity supplemented with examining of serial samples of plasma, peripheral blood mononuclear cells (PBMC) and, when available, liver biopsies, and by procedures enriching HCV in test material by amplifying viral RNA recovered from larger amounts of serum, liver biopsy material and/or from mitogen-stimulated PBMC [6][9], [18][20]. Further, the detection of HCV RNA replicative (negative) strand is not uncommon in OCI, particularly when ex vivo stimulated PBMC and liver biopsy material are analysed [6], [8], [11], [20]. Since discovery of OCI in 2004, persistence of HCV after SVR was the subject of studies by different groups which delineated virological and some unique immunological properties of this infection [6][9], [21][23]. Among others, OCI displays a distinct profile of antiviral cytokine expression in PBMC when compared to either CHC or healthy individuals, shows an antagonistic relation between HCV and IFN-α expression in PBMC, and that HCV replication in this compartment can be completely eliminated by activation of endogenous IFN-α [22], [23]. Nonetheless, OCI is rarely investigated and knowledge on this subject remains incomplete. To broaden characterization of this infection entity, in particular to learn about the fate of HCV during and shortly after completion of otherwise clinically successful treatment with PegIFN/R, we re-examined, using highly sensitive HCV genome detection methods, serial plasma and, in some cases, PBMC samples collected prior to, during and after completion of PegIFN/R therapy from patients with CHC who finally achieved clinical SVR.

Materials and Methods

Ethics Statement

The study was approved by the Weill Cornell Medical College institutional review board and was performed in accordance with the Declaration of Helsinki. The samples were collected after signing written informed consent.

Patients and samples

Serial plasma samples (n = 56) from 9 patients (3 men and 6 women; ages 38 to 62), who clinically resolved CHC in response to treatment with PegIFN/R, and sequential PBMC samples (n = 23) from 3 of them were investigated (Table 1). The patients were infected with HCV genotype 1 or 2 (Table 1). The origin and the route of HCV infection were undetermined; however none of the patients was an active drug user during treatment or follow-up. None of them also was co-infected with hepatitis B virus (HBV) or human immunodeficiency virus or was receiving immunosuppressive or anti-cancerous therapy. All patients received PegIFN/R treatment for 24 or 48 weeks (wks) with the exception of 6/F, 7/F and 2/F who were treated for 25, 44 or 68 wks, respectively (median treatment time for all 9 patients was 43.3 wks) (Table 1). The therapy resulted in the decline of plasma HCV RNA to undetectable levels, as measured by clinical laboratory tests (see below), and in normalization of liver enzymes, i.e., alanine aminotransferase (ALT) and aspartate aminotransferase (AST), starting within 3 to 4 wks after initiation of PegIFN/R. In regard to plasma samples, 11 samples from a total of 19 collected prior to initiation of the therapy (pre-treatment samples) were available for re-examination. These 11 samples were obtained between week 21 and one before the start of treatment (median time of collection 8.1 wks). Among 55 plasma samples collected during the treatment period (on-treatment samples), 25 were available for re-evaluation (Table 1). Also, from 34 plasma samples collected during follow-up lasting for up to 88 wks after completion of PegIFN/R therapy (post-treatment samples), 20 were available for reanalysis. The time of the last sample collection from individual patients ranged between 12 and 88 wks post-treatment (median 33.1 wks). Plasma was stored in 1-mL aliquots at −80°C until re-tested. One 1-mL aliquot per sample was available for investigation.

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