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

Results

Occult Hepatitis C Virus Infection in Individuals With Clinical Sustained Virological Response Not Exposed to Hepatitis B Virus

Using the highly sensitive RT-PCR/NAH assays established earlier,[14] 15 of 28 plasma samples from 13 of 15 individuals were found positive for HCV RNA when 250-μL samples were tested (Table 1). Of those samples that were virus nonreactive at 250 μL, further testing with one or 4 mL of plasma increased HCV RNA detection[38] in an additional four and two samples, respectively. Overall, 21 of 28 (75%) of plasma samples were HCV RNA reactive (Table 1). HCV RNA levels varied considerably (2.5–400 vge/mL) at different time points within the same patient and also between different patients. For example, plasma HCV levels fluctuated from 400 vge/mL in the first sample to undetectable 6 months later (e.g. 5–45/F and 18–54/F), only to be detected again at 100 vge/mL 1 year thereafter (e.g. 18–54/F). In PBMC, 8 of 28 unstimulated samples were positive for HCV RNA, with ex vivo stimulation[39,40] increasing virus identification in another nine (Table 1). As in plasma, viral genome levels in untreated or mitogen-treated PBMC varied between individuals, ranging between 5 and 500 vge/106 cells, but not substantially in the same patient.

Overall, of the 11 cases with sequential samples, HCV RNA was detectable at least once in plasma in all cases and in unstimulated PBMC in six. Ex vivo stimulation of PBMC led to HCV RNA detection in the other five cases, making all cases ultimately positive for HCV RNA in PBMC. Hepatic HCV RNA was identified in one of three cases from this study group (Fig. 1).

Figure 1.

Detection of hepatitis C virus (HCV) in liver biopsies from individuals years after clinical diagnosis of sustained virological response following treatment of chronic hepatitis C. Total liver RNA (4 μg) was amplified by nested reverse transcriptase-polymerase chain reaction (RT-PCR) with 5'-UTR-specific primers and amplicon specificity verified by nucleic acid hybridization (NAH). (a) Identification of HCV RNA-positive strand. (b) Detection of HCV RNA-negative strand using strand-specific RT-PCR/NAH. Tenfold serial dilutions of HCV sRNA-positive or HCV sRNA-negative strands were used as quantitative standards, respectively. Dilutions of HCV sRNA-positive strands were included in the negative strand assay as a specificity control. Contamination controls included water added instead of cDNA and amplified by direct (DW) and nested (NW) reactions and mock (M) treated as test RNA. Positive signals showed the expected 244-bp 5'-UTR sequence fragments.

The detection of HCV RNA-positive strand was unlikely due to viral adsorption to the cells because of the concurrent detection of the viral negative strand in liver and PBMC (Table 1 and Fig. 1), of viral sequence polymorphisms in PBMC relative to plasma or liver (2–53/M; Supplementary Table S2) and of circulating HCV-like particles (3–46/F; Fig. 2). The particles usually occurred singly and, occasionally, in small aggregates with estimated diameters between 50 and 75 nm. Collectively, these findings clearly indicated the persistence of authentic HCV replication at low level in persons with clinical resolution of CHC.

Figure 2.

Immunoelectron microscopic identification of hepatitis C virus (HCV) particles in the plasma of individuals with clinical sustained virological response (SVR) as visualized by immunogold staining with anti-E2 mAb. (a–c) HCV particles in unfractionated plasma of 3–46/F obtained at 54 months after achieving SVR (d–f). HCV RNA-reactive particles recovered from fractions 3 and 4 after ultracentrifugation over a 30% sucrose cushion of 16–45/M plasma collected at 66 months after SVR (g–i). HCV virions in unfractionated plasma of 18–54/F obtained at 36 months following clinical SVR. (j) The unfractionated plasma from 3 to 46/F exposed to the isotype mAb control instead of anti-E2 mAb. Preparations were counterstained with 1% phosphotungstic acid. Bars indicate 50 nm.

Occult Hepatitis C Virus Infection in Patients With Clinical Sustained Virological Response and Past Exposure to Hepatitis B Virus

In the first instance, HBV DNA was identified, using our highly sensitive PCR/NAH assay,[11,34] in 17 of 22 (78%) plasma, 9 of 18 (50%) PBMC and 2 of 2 (100%) liver samples tested (Table 1 and Fig. 3), giving a total case positivity of 8 of 9 for HBV (Table 2). However, the lack of available material prevented us from determining the status of HBV replication in these cases.

Figure 3.

Detection of dual occult infection with hepatitis C virus (HCV) and hepatitis B virus (HBV) in individuals with clinically apparent sustained virological response (SVR) to anti-HCV therapy and past exposure to HBV. HCV RNA and HBV DNA were detected in plasma (Pl), peripheral blood mononuclear cells (P) and liver (L) by nested RT-PCR/NAH (for HCV RNA) or nested PCR/NAH (for HBV DNA) as described in Patients, Clinical Samples and Methods. Total RNA or DNA extracted from a patient with symptomatic chronic hepatitis C or serum hepatitis B surface antigen-positive chronic hepatitis B, respectively, was used as a positive control (PC). Contamination controls included water added instead of cDNA (HCV RNA analysis) or DNA (HBV DNA analysis) and amplified by direct (DW) and nested (NW) reactions and mock (M) treated as test RNA or DNA.

Using the same approach to virus detection as discussed in previous section, HCV RNA was ultimately identified in 83% plasma, 54.5% PBMC and 100% liver samples of patients with past exposure to HBV. As in the case of individuals without a past history with HBV, there was also evidence for the presence of HCV-negative strand (Table 1), circulating HCV-like particles (cases 16–45M and 18–54/F; Fig. 2) and single-nucleotide polymorphisms in PBMC (case 24–34F; Supplementary Table S2). Overall, the data as a whole lend support to the conclusion that past HBV exposure seems to have no identifiable influence on the frequency and virological properties of occult HCV persistence.

Liver Histology in Individuals With Occult Hepatitis C Virus Infection and With or Without Past Hepatitis B Virus Exposure

Histological examination of liver biopsies obtained before and after treatment (post-SVR) (Table 3; Supplementary Fig. S1) revealed (i) a decrease in necroinflammatory activity after SVR in five cases; (ii) no remarkable changes between pre- and post-treatment in two patients; and (iii) increased disease activity after SVR in two cases (1–61/F, 3–46/F). It was evident that despite histological improvement in the majority of cases investigated, most biopsies still displayed some degree (predominantly grades 1 and 2) of necroinflammation with piecemeal necrosis and lymphocytic infiltrations, as well as minimal to mild fibrosis (mainly stages 1 and 2) after SVR (Supplementary Fig. 1). But in the end, no remarkable differences were observed in liver histology between individuals with or without past exposure to HBV.

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