Persistence of Hepatitis C Virus during and after Otherwise Clinically Successful Treatment of Chronic Hepatitis C with Standard Pegylated Interferon a-2b and Ribavirin Therapy Annie Y. Chen 1 , Marija Zeremski 2 , Ranjit Chauhan 1 , Ira M. Jacobson 2 , Andrew H. Talal 2,3 , Tomasz I. Michalak 1 * 1 Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John’s, Newfoundland and Labrador, Canada, 2 Center for the Study of Hepatitis C and Division of Gastroenterology and Hepatology, Weill Cornell Medical College, New York City, New York State, United States of America, 3 Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, State University of New York, Buffalo, New York State, United States of America 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-a/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 59- 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 a-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: ß 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. * E-mail: timich@mun.ca Introduction Hepatitis C virus (HCV) is a single-stranded RNA virus that is the cause of clinically diagnosable chronic infection in approxi- mately 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/mg 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-a (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 PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e80078