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

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.

Conflict of interest statement

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

Figures

Figure 1. Expression of HCV RNA positive strand in serial plasma and PBMC samples and HCV RNA negative (replicative) strand in PBMC prior to, during and after completion of PegIFN/R treatment of 8/F patient with the initial diagnosis of CHC.

(A) HCV RNA positive strand detection using total RNA extracted from 250 µl or 750 µl of plasma. (B) HCV RNA positive strand identification using 2 µg of total RNA extracted from either mitogen-treated (*) or native (untreated) PBMC. (C) Detection of HCV RNA negative strand in HCV RNA positive strand reactive PBMC samples shown in B. Plasma and PBMC were collected in parallel (except 4A PBMC sample) at the time points (weeks) indicated under panels A and B. Minuses before week numbers indicate sample collections prior to initiation of PegIFN/R therapy, while pluses indicate collections after completion of the treatment. As positive controls for HCV RNA positive strand detection, RNA extracted from equivalent of 10 µl of HCV RNA-positive plasma (panel A) or 1 µg RNA from PBMC (panel B) of a patient with active CHC, and serial 10-fold dilutions of recombinant HCV 5′-UTR-E2 (rHCV UTR-E2) fragment carrying indicated copy numbers/reaction were used. For HCV RNA negative strand detection, 2 µg of total RNA from PBMC of the same control CHC patient as in panels A and B, and synthetic HCV RNA positive strand (sHCV RNA pos) and HCV synthetic RNA negative strand (sHCV RNA neg) at 104 copies/reaction were used as positive and specificity controls. Water amplified in direct (DW) and nested (NW) reactions and a mock (M) extraction served as contamination controls. Positive signals showed the expected 244-bp oligonucleotide fragments. Numbers under the panels represent relative densitometric units (DU) given by hybridization signals.

Figure 2. Phylogenetic analysis of the HCV 5′-UTR variants found in plasma and PBMC from patients 5/M and 8/F obtained prior to, during and after PegINF/R therapy.

The numbers 5 and 8 identify patient 5/M and 8/F, respectively. M indicates the variant and wild wild-type sequence found in the majority of the clones derived from a given patient prior to, during and after therapy. B stands for before treatment, D during treatment, and A after treatment with PegIFN/R. The variants from plasma are marked with S, while those from PBMC with C. The numbers 1–20 indicate individual clones. The genotype 2a and 2b nucleotide sequences serving as references are marked as Ref.