T. N. Q. Pham; C. S. Coffin; N. D. Churchill; S. J. Urbanski; S. S. Lee; T. I. Michalak
Posted: 02/28/2012; J Viral Hepat. 2012;19(2):103-111. © 2012 Blackwell Publishing
Abstract and Introduction
Abstract
Hepatitis C virus (HCV) and hepatitis B virus (HBV) frequently coinfect and persist long after clinical resolution. We assessed the incidence of low-level (occult) HCV infection (OCI) after sustained virological response (SVR) to standard anti-HCV therapy in individuals with or without past exposure to HBV to recognize whether HBV could influence the prevalence of OCI, HCV level and hepatic histology. Plasma and peripheral blood mononuclear cells (PBMC) were collected from 24 individuals at 6- to 12-month intervals for up to 72 months after SVR. Liver histology was available for nine patients. HCV and HBV genomes were detected with sensitivity <10 genome copies/mL. In individuals without HBV exposure (n = 15), comprehensive analyses of sequential plasma and PBMC samples revealed HCV RNA in all 15 cases (75% plasma and 61% PBMC). In the group with HBV exposure (n = 9), evidenced by circulating anti-HBc and/or HBV DNA detection by a highly sensitive assay, HCV RNA was identified in all cases (83% plasma and 59% PBMC), at levels similar to those in HBV nonexposed individuals. In both groups of patients, most liver biopsies included those reactive for viral genomes displayed low-grade inflammation (8 of 9) and fibrosis (7 of 9). Sequence polymorphisms at the 5`-UTR between PBMC and liver or plasma, as well as circulating HCV virion-like particles, were observed in patients with or without HBV exposure. In conclusion, the prevalence of OCI after SVR is comparable in individuals with or without past exposure to HBV. HCV loads and liver alterations in OCI appear to be unaffected by low-level HBV DNA carriage.
Introduction
There are at least 370 million people chronically infected with hepatitis B virus (HBV) and 170 million of those with chronic hepatitis C virus (HCV) infection worldwide. The chronic hepatitis induced by the viruses can lead to fibrosis, cirrhosis and hepatocellular carcinoma (HCC).[1,2] However, while 90–95% of adults infected with HBV spontaneously resolve acute hepatitis, up to 85% of those infected with HCV develop chronic hepatitis C (CHC).[3,4]
Although hepatocytes are major targets of HBV and HCV, both pathogens can also propagate in the cells of the immune system, as evidenced by the presence of their genomes and respective replicative intermediates and proteins in the lymphatic organs and peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B (CHB) and C.[5–10] The existence of small amounts of HBV and HCV genomes in plasma, PBMC or liver after clinical resolution of hepatitis highlighted the natural propensity for these two viruses to induce a persistent infection.[11–15] Occult HBV infection (OBI) is defined as detection of HBV DNA in liver, plasma and/or PBMC in the absence of hepatitis B surface antigen (HBsAg) in serum. Occult HCV infection (OCI) is characterized by low-level HCV RNA persisting in plasma, liver or PBMC in anti-HCV antibody-reactive individuals who resolved hepatitis C either spontaneously or after antiviral treatment or seemingly anti-HCV antibody-negative individuals with infection of unknown aetiology.[14–17] Although the existence of OBI has been generally accepted, that of OCI remains controversial in that HCV persisting in plasma, liver and/or immune cells after resolution of hepatitis C has not been observed by all investigators. This inconsistency is likely related to variations in the sensitivity of HCV RNA detection assays employed, processing of clinical samples, the number of samples tested and the amount of material used for analysis.[18] Because of their shared modes of transmission, coinfection with HCV and HBV is common, particularly in high-risk populations and in areas considered to be endemic for both viruses. Molecular and pathological consequences of interactions between HCV and HBV in coinfected patients are not fully elucidated given the relatively contradicting data that are available. On one hand, acute HCV infection in the context of CHB and vice versa acute HBV infection or OBI in CHC have been associated with a more active liver disease and a greater likelihood of developing cirrhosis and HCC.[19–24] Along this line, HBV superinfection has been correlated with an assumed complete clearance of serum HCV RNA, or even both HCV RNA and HBV DNA, in patients with CHC.[24–26] The reverse was also true for HCV superinfection.[22,24] In cell culture studies, HCV core or nonstructural proteins (e.g. NS5A) were found to repress HBV DNA synthesis.[27–29] On the other hand, in arguing against the negative influence of one virus over the other, other investigations demonstrated not only a coexistence of hepatic HBV and HCV in coinfected patients,[30,31] but also a simultaneous replication of HCV and HBV within the same hepatoma cell.[32,33]
Nevertheless, given the lymphotropic propensity of HCV and HBV, what remained undefined was whether HCV and HBV could affect each other's ability to propagate in immune cells, especially in the context of occult infection. Such recognition would be important in furthering our understanding of the full extent of viral persistence. With this in mind, the current study examined whether HBV exposure would influence the prevalence of HCV occult infection, the level of persisting HCV and liver pathology in patients with clinical resolution of CHC.
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]
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.
Discussion
In this study, we examined the prevalence of OCI in patients with clinically resolved hepatitis C who had or did not have past exposure to HBV. Our findings overall suggest that low-level HBV DNA carriage in plasma, PBMC or liver does not have a noticeable effect on the prevalence, virological characteristics and, most likely, features of liver histology encountered in the course of OCI. The data also indicate that regardless of the existence of past HBV exposure, the levels of HCV RNA, especially in plasma, could vary significantly over time in OCI. Given that both HCV and HBV genomes were detectable in the same compartments in many of the cases examined, it can be proposed that the reciprocal inhibition of HBV and HCV replication observed in highly viraemic hepatitis patients[22,24,26,41] is not evident in low-level coinfection with these viruses.
Since its discovery in recent years,[14] OCI continuing after clinical resolution of hepatitis C has been a subject of investigations by many groups. Although we and others have collectively documented the presence of low-level HCV persistence in plasma, PBMC and/or liver in individuals with spontaneous or treatment-induced recovery from CHC,[13–15] studies from other groups came to rather opposite conclusions.[42–44] As evidenced in this study, HCV RNA, in both plasma and PBMC, could fluctuate considerably over time in low-level HCV infection. This, in consequence, highlights the importance of testing multiple samples and, if required, using a larger amount of starting material for nucleic acid extraction. In addition, as ex vivo stimulation of lymphoid cells with mitogens could greatly enhance virus detection,[14,39] we routinely adopt this approach to our investigation of OCI in PBMC. Nevertheless, this methodology was not used by most, if not all, studies that had refuted the existence of occult HCV persistence.[42–44] Perhaps, a combination of the differences mentioned elsewhere, together with other factors discussed elsewhere,[17,18] could help reconcile the discrepancies between the data that argued for[13–15] and those that were against[42–44] the notion of OCI persisting after clinically diagnosed recovery. Further, it is important to note that in this study, as well in those published elsewhere [8,13–15,36], low-level occult HCV persistence was not just about mere detection of HCV RNA. Indeed, not only the persistent expression of HCV replicative strand in PBMC and liver tissue was documented, but also HCV sequence polymorphisms in PBMC compared with plasma or liver, circulating virion-like particles and their replication competence were demonstrated.[36]
In most cases evaluated, liver histology showed improvement after otherwise successful treatment. Nevertheless, there was also an indication of persistent low-grade liver inflammation and fibrosis (see Table 3), which was seemingly irrespective of whether the individuals were concurrently positive or not for HBV DNA. The question of whether OBI could contribute to such alterations after achieving SVR could not be conclusively addressed in this study and will require a similar investigation with a larger number of cases. At this point, our finding of hepatic changes after SVR is consistent with that reported by others.[13,15,45]
Taken together, the current study provides new insights into characteristics of occult HCV persistence in general and in individuals with past exposure to HBV in particular. It also offers a standardized approach with greater uniformity and sensitivity in the identification of OCI. Our data indicate that a past encounter with HBV may not negatively influence the prevalence and characteristics of low-level HCV persistence continuing after resolution of CHC achieved following antiviral therapy.
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