Hepatitis C Virus Persistence After Sustained Virological Response to Antiviral Therapy in Patients With or Without Past Exposure to Hepatitis B Virus

T. N. Q. Pham; C. S. Coffin; N. D. Churchill; S. J. Urbanski; S. S. Lee; T. I. Michalak

Disclosures

J Viral Hepat. 2012;19(2):103-111. 

In This Article

Patients, Clinical Samples and Methods

Patients and Clinical Samples

Twenty-four patients determined to have resolved CHC according to clinical and laboratory assay criteria were randomly selected for the study. They were followed at the University of Calgary Liver Unit, Alberta, Canada. These individuals had achieved a sustained virological response (SVR) for duration of up to 6 years following treatment with interferon alpha (IFN) or pegylated IFN S1). None of them was subjected toand ribavirin (p-IFN/R) (Supplementary Table immunosuppressive or anticancerous therapy during follow-up. Liver function tests were repeatedly normal and serum HCV RNA negative by Roche Amplicor HCV v2.0 assay (sensitivity 500 IU/mL or 1000 virus genome equivalent (vge)/mL; Roche Molecular Diagnostics, Pleasanton, CA, USA). All patients were reactive for antibodies to HCV (anti-HCV) by immunoassays (Abbott Diagnostics, Mississauga, Canada) at the time of the first blood sample collection. Clinical charts revealed that eight of them were also positive for antibodies to HBV core S1), indicative of a past(HBV-C) antigen (anti-HBc) (Supplementary Table exposure to HBV, by the Abbot Corzyme assay (Abbott Laboratories, North Chicago, IL, USA). One individual was negative for anti-HBc while positive for antibodies to HBsAg (anti-HBs); however, plasma and PBMC from this person were HBV DNA reactive when tested by research polymerase chain reaction (PCR) assays employed in this study.

Plasma and corresponding PBMC were available from all 24 subjects (Supplementary Table S1). Up to three plasma and PBMC pairs were collected from 21 individuals every 6–12 months, while a single sample pair from the remaining three was obtained at 18 months post-SVR. Liver biopsies were available before and after therapy (up to 60 months post-SVR) for eight individuals and only after SVR for an additional patient. The study protocol was approved by local Human Investigation Committees. All patients provided written informed consent to participate in the study.

Preparation of Peripheral Blood Mononuclear Cells and Cell Cultures

Peripheral blood mononuclear cells were isolated by Ficoll-HyPaque (Pharmacia Biotech, Quebec, Canada) gradient fractionation and cultured, if required, for 72 h in the presence of phytohemagglutinin (PHA; 5 μg/mL; Sigma, Mississauga, Canada) and interleukin-2 (IL-2; 20 U/mL; Roche) prior to analysis for HCV RNA or HBV DNA.[14]

Nucleic Acid Extraction

RNA was usually extracted from 250 μL of plasma using Trizol LS (Invitrogen Life Technologies, Burlington, Ontario, Canada) or 1 × 107 naive (uncultured) PBMC using Trizol (Invitrogen).[14] DNA was extracted[34] from the same volume of plasma or number of PBMC. When necessary, extraction of 1-mL sample of test plasma was carried out and the nucleic acid analysed. If the sample remained virus nonreactive, 4 mL of plasma was ultracentrifuged at 40 000 rpm for 20 h at 4 °C and nucleic acid extracted from the resulting pellet was tested for HCV RNA or HBV DNA.

Hepatitis C Virus RNA-positive and Hepatitis C Virus RNA-negative Strand Detection

RNA from PBMC (1–2 μg RNA; equivalent of 1–2 × 106 cells), plasma (equivalent of 250 μL, 1 mL or 4 mL) or liver tissue (2 μg RNA) was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) for HCV RNA-positive strand using HCV 5'-untranslated region (5'-UTR)-specific primers, cycling conditions and quantification standards reported earlier.[14] In all instances, specificity of amplicons was confirmed by nucleic acid hybridization (NAH) using 32P-labelled recombinant HCV 5'-UTR-E2 fragment as a probe.[14] Sensitivity of this assay is <10 vge/mL or <2.5 vge/reaction. HCV RNA-negative strand was detected by strand-specific RT-PCR/NAH using rTth DNA polymerase.[14] This assay detects ~100 copies of the correct (negative) strand, while nonspecifically identifying ≥106 vge of the positive strand.[14] Specificity of PCR amplicons and validity of the controls were confirmed by NAH. In every analysis, we included a number of negative and contamination controls, as reported.[14] Briefly, for each group of samples subjected to nucleic acid extraction, a mock sample containing no patient material was extracted and analysed with test samples. In the PCR step, a water sample and a mock (no test cDNA) were amplified as controls.

Hepatitis B Virus DNA Detection

Hepatitis B virus DNA in plasma, PBMC or liver was detected by PCR/NAH using primers specific for HBV-C and X (HBV-X) genes (GenBank accession number X72702). For first-round amplification, the primers were as follows: 1847-TGTTCATGTCCCACTGTTCAAGC (HBV-C outer sense), 2274-AAGATAGGGGCATTTGGTGG (HBV-C outer antisense), 1266-CCATACTGCGGAACTCCTAGC (HBV-X outer sense) and 1779-ACAGACCAATTTATGCCTACAGCC (HBV-X outer antisense). For second-round amplification, the primers were 1893-TTTGGGGCATGGACATTGACC (HBV-C inner sense), 2301-ATAAGCTGGAGGAGTGCGAATCC (HBV-C inner antisense), 1310-CTGGAGCAAACATTATCGGG (HBV-X inner sense) and 1748-CAAAGACCTTTAACCTGATCTCC (HBV-X inner antisense). In all cases, amplifications were carried out for 40 cycles under the following conditions: 95 °C for 60 s (denaturation), 52 °C for 60 s (annealing) and 72 °C for 60 s (extension). Specificity of amplicons was confirmed by NAH using 32P-labelled recombinant full-length HBV DNA as a probe.[11] This assay detects ≤10 vge/mL or ≤2.5 vge/reaction. Negative, positive and contamination controls were routinely included, as reported.[11,34]

Clonal Sequencing

To assess possible sequence variations and compartmentalization of HCV, 5'-UTR amplicons were cloned and at least 10 clones from each PCR product sequenced bidirectionally.[8] Polymorphisms were analysed by Sequencher software version 4.7 (Gene Codes Corp., Ann Arbor, MI, USA).

Immunoelectron Microscopy

To examine the presence of HCV virions, 500 μL of plasma was incubated with anti-HCV E2 monoclonal antibody (mAb; AP33; provided by Dr. Arvind Patel, University of Glasgow, UK) or mAb isotype control, as reported.[35,36] In the case of 16–45/M plasma, HCV RNA-positive fractions recovered after ultracentrifugation (4 mL plasma) over a 30% sucrose cushion[36] were similarly incubated with anti-E2 mAb. HCV particles were detected with anti-mouse IgG conjugated with 12-nm gold particles (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and, after counterstaining with 1% phosphotungstic acid, examined under a JEM 120 EX microscope (JEOL Ltd., Tokyo, Japan).

Liver Histology

After routine processing, liver biopsies were blindly evaluated by a hepatopathologist according to the METAVIR scoring system.[37]

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