Investigation of Residual Hepatitis C Virus in Presumed Recovered Subjects

Abstract

Recent studies have found hepatitis C virus (HCV) RNA in peripheral blood mononuclear cells (PBMCs) of the majority of presumed recovered subjects. We investigated this unexpected finding using samples from patients whose HCV RNA and anti-HCV status had been serially confirmed. HCV RNA was detected in PBMCs from 66/67 chronic HCV carriers. Subpopulation analysis revealed that the viral load (log copies/10⁶ cells) in B cells (4.14 ± 0.71) was higher than in total PBMCs (3.62 ± 0.71, p<0.05), T cells (1.67 ± 0.88, p<0.05), and non-B/T cells (2.48 ± 1.15, p<0.05). HCV negative-strand RNA was not detected in PBMCs from any of 25 chronically infected patients. No residual viral RNA was detected in total PBMCs or plasma of 59 presumed recovered subjects (11 spontaneous, 48 treatment-induced) using nested real-time PCR with a detection limit of 2 copies/μg RNA (from ~1×10⁶ cells). PBMCs from two healthy HCV-negative blood donors became HCV RNA positive, with B-cell predominance, when mixed in vitro with HCV RNA positive plasma, thus passively mimicking cells from chronic HCV carriers. No residual HCV was detected in liver or other tissues from two spontaneously recovered chimpanzees. Conclusion: 1) HCV RNA was detected in PBMCs of most chronic HCV carriers and was predominant in the B cell subpopulation; 2) HCV detected in PBMCs was in a non-replicative form; 3) HCV passively adsorbed to PBMCs of healthy controls in vitro becoming indistinguishable from PBMCs of chronic HCV carriers; 4) Residual HCV was not detected in the plasma or PBMCs of any spontaneous or treatment recovered subjects or in chimpanzee liver suggesting that the classic pattern of recovery from HCV infection is generally equivalent to viral eradication.

Introduction

It is estimated that of the 170 million individuals are infected with hepatitis C virus (HCV) worldwide (1), approximately 20% will develop cirrhosis, and 1% to 5% of patients with cirrhosis will progress to hepatocellular carcinoma (HCC) (2). B cell abnormalities, including mixed cryoglobulinemia and non-Hodgkin lymphoma may also occur (3, 4). Spontaneous recovery from acute HCV infection, or treatment-induced recovery from chronic HCV infection has been considered to represent probable cure (5, 6). However, other studies have reported finding residual HCV RNA in the serum or PBMC of recovered subjects (7, 8) and have cast doubt on the complete eradication of virus in presumed recovered subjects. This is a critical issue because if residual HCV exists, there is the potential for reactivation in the setting of organ transplantation, chemotherapy, or other acquired immunosuppressive states, as reported in occult HBV infection (9). Although HCV reactivation is limited to a few case reports (10–12), the potential for residual HCV in persons whose serum tests HCV RNA negative raises concern regarding the use of organs from presumed HCV recovered subjects in the transplant setting and raises the potential that such individuals would remain at risk for the subsequent development of HCC. It is thus essential to determine whether residual HCV infection exists in persons whose serum markers suggest full recovery.

We sought to investigate the presence of residual HCV in PBMCs and plasma by: 1) measuring HCV RNA in the PBMCs and mononuclear cell subsets of chronically infected and recovered subjects using a sensitive nested real-time detection (n-RTD) polymerase chain reaction (PCR) with a detection limit of 2 copies/μgRNA; 2) determining if PBMC-associated HCV RNA is in a replicative form by negative-strand PCR and cell culture ; and 3) determining if HCV might bind to PBMCs without infecting them. In addition, we examined tissue samples from the liver and other organs of two spontaneously recovered chimpanzees for the presence of HCV RNA.

Patients

Consenting blood donors identified as anti-HCV positive by enzyme immunoassay at the time of routine blood donation at the Department of Transfusion Medicine NIH or the Greater Chesapeake and Potomac Region of the American Red Cross were enrolled in an NIH prospective study of the natural history of HCV infection (13). Approximately 750 participants were enrolled. Of these, 67 chronic HCV carriers and 36 presumed recovered subjects (11 spontaneously recovered and 25 sustained virologic treatment responders) were randomly selected for the study. In addition, 23 treatment-recovered patients from Japan were included in the study bringing the total number of recovered subjects to 59. From the total population, 43 chronic carriers, 3 spontaneously recovered and 13 treatment-recovered subjects had their PBMCs studied in cell culture. The selection of donors for PBMC studies depended on the availability of fresh PBMCs from donors in active follow-up; the small number of spontaneously recovered subjects providing fresh PBMC reflected the low proportion of recovered subjects who remained in active clinical follow-up. Demographic and clinical characteristics of the patients are shown in Table 1. The chronic group and the recovered group had similar characteristics except for viral load and serum transaminase levels. The study protocols were reviewed and approved by the appropriate institutional review boards, and all subjects gave written informed consent to participate in the study.

Table 1.

Demographic and clinical characteristics of patients

a) Patients for HCV RNA negative strand detection and PBMC subsets studies
	Chronic (n=29)	Recovered (n=26)
Age (yrs)	52.8 ± 10.7	52.1 ± 9.4	NS
Sex (M/F)	16/13	12/14	NS
HCV viral load (log(IU/ml))	6.1 ± 0.7	ND
ALT (U/L)	64.2 ± 44.4	22.7 ± 10.9	P<0.001
AST (U/L)	48.4 ± 27.9	24.3 ± 5.3	P<0.001
T-Bil (mg/dL)	0.8 ± 0.4	0.8 ± 0.3	NS
Alb (g/dL)	3.8 ± 0.3	4.1 ± 0.3	NS
Platelet (K/μL)	225.5 ±58.8	229.0 ± 53.8	NS
PT (sec)	12.6 ± 0.7	12.6 ± 0.4	NS

b) Patients for cell culture studies
	Chronic (n=43)	Recovered (n=16)
Age (yrs)	57 ± 7	55 ± 10	NS
Sex (M/F)	26/17	8/8	NS
HCV viral load (log(IU/ml))	6.3 ± 0.7 (range 4.4 to 7.6)	ND
ALT (U/L)	62 ± 30	29.4 ± 9.3	P<0.0001
AST (U/L)	40 ± 22	17.4 ± 7.4	P<0.0001
T-Bil (mg/dL)	0.53 ± 0.23	0.58 ± 0.34	NS
Alb (g/dL)	4 ± 0.3	4.1 ± 0.3	NS
Platelet (K/μL)	226 ± 66	228 ± 66	NS
PT (sec)	13.3 ± 0.8	13.5 ± 0.6	NS

c) Group of recovered patients from Japan
	Recovered (n=23)
Age (yrs)	52 ± 13
Sex (M/F)	11/12
HCV viral load (log(IU/ml))	ND
ALT (U/L)	26.4 ± 41.4
AST (U/L)	21.4 ± 5.7
T-Bil (mg/dL)	0.71± 0.2
Alb (g/dL)	4.54± 0.4
Platelet (K/μL)	183 ± 48
PT (sec)	11.8 ± 0.5

ALT, alanine aminotransferase; AST, aspartate aminotransferase; T-Bil, total bilirubin; Alb, albumin; PT, prothrombin time; ND, not detectable

Methods

Serologic and Molecular Assays

A detailed description of methods used for HCV detection, including serologic assays for anti-HCV, molecular assays for HCV RNA and assays for HCV negative –strand detection, and HCV RNA isolation can be found in Supporting Information.

PBMCs and PBMC subset separation

After separation of plasma from whole blood, PBMCs were isolated by Ficoll-Paque, washed twice in Dulbecco’s phosphate-buffered saline and two times in complete RPMI medium. The B cell subset was purified by positive selection using MACS magnetic beads coated with anti-CD19 antibodies (Miltenyi Biotec Inc., Auburn, CA) and the T cell subset was purified from the non-B cell fraction by using beads coated with anti-CD3 antibodies. The efficiency of separation was > 85% for B cells and >95 % for T cells as confirmed by FACS analysis (BD Biosciences, San Diego, CA).

PBMC purification for cell culture studies

PBMC were isolated by Ficoll-Paque PLUS density gradient from ACD anticoagulated blood. The cells were washed with 1×PBS and complete RPMI 1640 medium and suspended in culture medium at 1×10⁶ cells/ml. Two culture methods were followed. Twelve patients’ samples (3 recovered and 9 chronic) were cultured without exogenous stimuli according to the previously described method (14). Briefly, two million cells were suspended in 2 ml complete RPMI 1640 medium using round-bottom 5 ml polystyrene tubes and left undisturbed in a 5% CO₂ incubator. For the rest of the patients, after isolation and cell washes, 4 ml of culture medium plus 5 μg/ml of phytohemagglutinin (PHA-L) was added to 4 × 10⁶ cells and incubated at 37°C in a 5% CO₂ incubator. At day 3 or 4, 3 ml of the medium was replaced by complete RPMI plus 1 μg/ml of PHA-L and 20 U/ml IL-2. Subsequently, for both techniques, half of the supernatant was replaced twice a week with fresh medium. Supernatants (SN) and cell samples were collected once or twice weekly during 3 weeks of culture.

HCV detection

We followed the RTD-PCR method designed by Takeuchi et al. (15); however, to obtain better sensitivity, nested-RTD PCR was performed, as reported previously for the detection of HCV (16, 17, see Supporting Information for details).

HCV negative strand detection

For negative strand HCV RNA detection, the rTth based method (8, 18) was used with minor modification as described in detail in Supporting Information.

In vitro HCV particle attachment to PBMCs from healthy donors

PBMCs from 2 healthy donors and plasma from 4 chronic carriers were mixed in four separate experiments. In each experiment, healthy cells and RNA positive plasma were incubated for 2 hours at room temperature and the cells then washed three times in medium. Washed PBMCs and their fractions were tested for the presence of HCV RNA by nested RT-PCR. CD19 separation was performed by positive selection and total PBMCs were separated into B cell, and non-B, cell fractions.

Statistical analysis

Student t test and Mann-Whitney U test were used for comparison of continuous variables. Fisher’s exact probability test was used for frequency comparisons. Spearman’s rank-order correlation was used to evaluate the correlation between HCV viral load in serum and HCV viral load in PBMCs. A P value of 0.05 or less was considered significant. Statistical analyses were performed using STATA (version 7.0, Stata corp, College station, TX). Data analysis and graphs from the cell culture section were performed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA).

RESULTS

Relationship between HCV viral load in serum and uncultured PBMCs

A comparison between serum HCV viral load and the viral load in PBMCs was performed on samples from 28 chronically infected patients. Viral loads in these blood compartments showed moderate correlation (P < 0.001, r² = 0.51) (Figure 1). A more comprehensive assessment was performed in 8 patients in whom HCV viral load was measured in total PBMCs, CD19 positive B cells, CD3 positive T cells, and CD19, CD3 negative subsets. In 7 of 8 patients, viral load (log copies/10⁶ cells) in the B cell subset (4.14 ± 0.71) was significantly higher than in total PBMC (3.62 ± 0.71, p < 0.05), T cells (1.67 ± 0.88, p < 0.05), and non-B, non-T cells (2.48 ± 1.15, p < 0.05) (Figure 2). In one patient, virus was not detected in any cell fraction.

Figure 1.

Correlation between serum and PBMC HCV viral load.

Moderate correlation was observed between serum HCV viral load and the viral load in PBMCs. HCV viral load from plasma and PBMCs was performed using 28 chronic patients.

Figure 2.

HCV viral load in PBMC subpopulations: B cells [CD19 (+)], T cells [CD3 (+)], and non-B/T cells [CD19 (−) CD3 (−)]. Results of 7/8 patients for whom HCV RNA was detectable.

Detection of negative-strand HCV RNA in PBMCs and PBMC subpopulations

Using the negative-strand specific nested-RTD PCR, HCV negative strand was assayed in total PBMCs of 25 HCV-infected chronic carriers and in the B, T, and non-B, non-T cell subsets from 8 of these carriers. No negative-strand HCV RNA was detected in any of 25 total PBMC samples or 8 PBMC subsets (see Table 2). As a control, negative strand HCV RNA was detected (3400 copies per μg total cellular RNA) in liver tissue from one chronically infected chimpanzee.

Table 2.

Summary of the results obtained from chronic carriers and presumed recovered subjects

	Number of HCV+ results out of total samples analyzed
Tests performed	Chronic carriers	Presumed recovered subjects
HCV RNA in plasma	67/67	0/59
HCV RNA in total PBMC	66/67	0/59
HCV RNA in PBMC subsets
CD19+	7/8	0/21
CD3+	7/8	0/21
CD19− CD3−	7/8	0/21
HCV negative strand in total PBMC	0/25	na
HCV negative strand in PBMC subsets
CD19+	0/8	na
CD3+	0/8
CD19− CD3−	0/8
PBMC cultures	34/43	0/16

na: not applicable

In vitro incubation of the plasma from chronic HCV carriers and PBMCs from healthy donors

To investigate whether the higher viral load in the B cell subpopulation reflected the specific attachment of HCV virions to B cells, plasma from 4 chronically infected patients were mixed with PBMCs from 2 HCV-negative healthy donors and incubated for 2 hours. PBMC subsets were then separated and washed. After this in vitro incubation, the PBMCs and PBMC subsets of the normal donor became HCV RNA positive and the distribution of RNA was similar to that shown in chronically infected patients being greatest in the B cell fraction (Figure 3). Hence, it appeared that HCV RNA could passively adsorb to normal PBMCs and preferentially adsorb to B cells.

Figure 3.

HCV particle binds to surface of PBMCs. PBMCs from two healthy donors were mixed with plasma from four chronic HCV carriers (P1 to P4), and incubated for 2 hours. Then, cells were washed and CD19 separation was performed. HCV viral loads in total PBMC, CD19 positive, and CD19 negative cells were quantified. ***: p<0.001; **: p<0.01.

Residual virus in recovered subjects

A total of 59 samples from recovered patients, including 36 from the US and 23 from Japan were included in the study. The samples were first confirmed as HCV RNA negative by qualitative COBAS AMPLICOR assays (Roche Diagnostic Systems). Then residual HCV in plasma from 26 of the 36 US recovered patients was measured with our more sensitive method using RNA extraction-purification and nested-RTD PCR. Even though the detection limit of the method was as little as 10 IU/ml, as confirmed by diluted WHO International HCV control, no HCV RNA was detected in any of these plasma samples, each tested in four replicates. In addition, residual HCV was sought in total PBMCs from 59 patients and from the B cell subpopulations of 21 patients. No HCV RNA was detected in any of the PBMC samples or B cell subset samples from these patients whose serum tested anti-HCV positive, HCV RNA negative (presumed recovered subjects) as shown in Table 2.

HCV detection in PBMC cultures

Since previous reports described the finding of HCV replicative intermediates in stimulated cultured PBMC of sustained virologic responders or chronic carriers (7, 8,19), we cultured PBMCs from 59 patients, including 43 chronic carriers and 16 recovered patients. The culture supernatants (SNs) and cells were evaluated at different time points during 3 weeks of culture. An average of 5 culture SNs and 4 cell samples were analyzed per patient.

None of the 16 recovered patients demonstrated the presence of HCV genome in their SN or cultured cells. Conversely, 34 of 43 (79%) chronic carriers had detectable HCV RNA in their PBMC cultures (cells and/or SN) (Table 2). Calculating the number of HCV+ SN out of the total SN analyzed per culture, the frequency of HCV+ detection had a median value of 33% and mean of 35%. In culture, the HCV viral load diminished over time and the majority of HCV positive cultures were observed during the first week (Table 3, Fig. 4). The maximum viral load reached during the 1^st week of culture was 490 copies/ml for SN fractions and 196 copies/μg total RNA for cells (Table 3). Viral levels decreased rapidly and progressively in culture, reaching very low levels by week 3 at which time there was only one positive SN with a viral load of 7 copies/ml (Figure 4, Table 3). However, cell fractions maintained their positivity with 61% (24 of 39 patients) of cell samples still positive at week 3, though the mean copy number diminished from 15.5 at week 1 to 4.2 at week 3 (Table 3). No correlation was found between the viral load in plasma or uncultured PBMC and the HCV RNA level observed during cell cultures. Overall, it appeared that HCV+ PBMCs lost reactivity in culture over time and showed no evidence for in-vitro replication.

Table 3.

HCV viral load and percentage of chronic carriers with HCV+ cultures from cells and cell free supernatants along the PBMC culture period.

			Day 0	Week 1	Week 2	Week 3
PBMC	% patients with HCV+ cultures (n)		100 (38/38)	76 (19/25)	78 (25/32)	61 (23/38)
	VL1	Mean ± SD	2898 ± 4029	15.5 ± 36	5 ± 10	4.2 ± 10
		Range	52 – 17,025	0 – 196	0 – 48	0 – 73
Culture SN	% patients with HCV+ cultures		na	81 (30/37)	14 (5/37)	3 (1/39)
	VL2	Mean ± SD	na	38 ± 79	2 ± 7	0.1 ± 0.8
		Range	na	0 – 490	0 – 45	0 – 7

na: not applicable

VL₁: viral load in cultured cells expressed in copies/μg total RNA

VL₂: viral load in culture SN expressed in copies/ml SN

Figure 4.

HCV viral loads in cultured PBMCs and cell free supernatant from HCV chronic carriers during three weeks in vitro culture period. Decreasing viral levels were observed as the cultures progressed.

Residual HCV in tissue samples from recovered chimpanzees

Tissue samples from two chimpanzees who had recovered from acute HCV infection were available for study. The first chimpanzee (4X417) recovered from a single episode of acute HCV infection and the second chimpanzee (4X355) recovered from two separate episodes of acute HCV infection. Since chimpanzee 4X355 died of unrelated causes after recovery from the second HCV infection, necropsy samples of spleen, kidney, digestive organs, lymph nodes and liver were available for assay. No residual HCV RNA was detected in the liver or other tissue samples from this chimpanzee and no HCV RNA was detected in the liver biopsy sample from recovered chimpanzee 4×417.

Discussion

The assumption that persons who recover from HCV infection, either spontaneously or following treatment, have eradicated the virus has been challenged (7, 8), but data are conflicting and the persistence of virus in presumed recovered subjects remains controversial (5, 20). Reports of finding HCV RNA in the leukocytes of the majority of recovered subjects has raised the possibility that HCV infection generally is incurable and that it might exacerbate from residual foci in the setting of malignancy, transplantation or other states of immune suppression. This is a vital issue because the number of presumed recovered subjects is large and the lifetime likelihood that some of these individuals will become immunosuppressed by virtue of age, disease or medication is high. In the United States, where it has been estimated that four million persons have been infected with HCV (21), it can be estimated further that approximately one million (25%) have spontaneously recovered or recovered by virtue of treatment. These are probably underestimates of both the number of infected and recovered subjects because they are based on select counties in the US and not on the highest risk populations, such as drug addicts and prisoners. Whatever the exact number of subjects who meet the serologic and molecular criteria for HCV recovery, it is critical to resolve whether such individuals are truly cured or whether they harbor residual virus that could later reactivate under specific circumstances. Studies from Canada (7, 20) suggest that 75%–86% of presumed recovered subjects have HCV RNA in their PBMCs even in the absence of HCV RNA in plasma. Furthermore, some case reports of HCV reemergence and relapse after SVR demonstrated identical sequences during viral reappearance suggesting that the original infection had persisted at undetectable serum levels rather than HCV reinfection (10, 12). These reports support the existence of viral reservoirs from which HCV could reemerge and suggest that PBMCs may be an important viral reservoir. In contrast, other investigators have failed to detect HCV RNA in the PBMCs of presumed recovered subjects using sensitive transcription mediated amplification (TMA) and nested PCR assays optimized for detection of virus in mononuclear cells (16, 17, 22, 23). Overall, although there are case-reports of HCV reappearance in recovered patients who are subsequently immunosuppressed (10, 11), such occurrences are unusual, suggesting full eradication of the virus in most patients who achieve a spontaneous or treatment-induced recovery. New light has been shed on this subject by Veerapu, et al (24) who studied 117 subjects who had recovered from HCV infection as assessed by standard criteria. They found no evidence of residual virus in the 19 subjects who had spontaneously recovered and in only 15% of 98 patients who had a sustained virologic response (SVR) to anti-viral therapy. In these 15%, HCV RNA was intermittently detected in serial samples using a sensitive nested PCR, but not detected by commercial real-time PCR. Thus, the level of virus was very low, was detected only sporadically and, in all subjects, disappeared over time, usually in the first years after cessation of therapy. Phylogenetic analysis showed that the emergent virus was identical to the virus present prior to treatment. The authors conclude that a small minority of patients classified as SVR continue to replicate HCV and they postulate that replication resides in the liver and not in PBMCs. Interestingly, the reemergence of virus was accompanied by the reappearance of an HCV-specific T-cell response that presumably would control this low-level infection, consistent with the absence of clinical exacerbation and the transient nature of the viremia. The study of Veerapu, et al is consistent with the findings from several groups (25, 26), which show that about 6% of SVRs have transient residual virus when tested by a highly sensitive TMA or nested RT-PCR. The controversy in the literature could be the consequence of patient selection that differed as to the source of infection, the type of treatment and especially the interval since a sustained virologic response was achieved. Another important element of the disparity could reside in the sensitivity and specificity of the assays used to detect positive- and negative- strand RNA in plasma and PBMC samples. The technologic challenges of demonstrating HCV replication in extra-hepatic sites are well summarized by Blackard, et al (27).

On this background of uncertainty, we conclude that in our patient cohort, there is no evidence for ongoing HCV replication in presumed recovered subjects and no evidence that HCV replicates in PBMCs of chronically infected patients. Rather, we propose that in chronically infected patients, HCV binds to the surface of PBMC subsets, particularly B cells, but does not replicate within these cells. These conclusions are supported by the following findings. First, although HCV RNA was found in the PBMCs of 66 out of 67 chronic HCV carriers tested, it was not found to be present in a replicative form in 25 consecutive patients tested using an rTth-based assay for negative strand HCV RNA capable of detecting 50 copies of negative strand RNA and readily detecting negative-strand RNA in the liver of a chronically infected chimpanzee. Second, in PBMC culture, virtually all the HCV RNA present in PBMCs at time zero was lost within the first week of culture (Table 3) and the minute residual RNA (0.6%) further decayed over time, suggesting that HCV in PBMCs is not in a replicative form, but rather follows a pattern of rapid and then asymptotic decay in culture. However, other studies, particularly in HCV-HIV coinfected patients (14), have provided evidence for HCV replication in PBMC culture, including detection of viral genotypes that were not present in the patient’s plasma (28). Third, we confirmed that, HCV RNA in the PBMCs of chronic carriers was differentially distributed among leukocyte subsets, being highly concentrated on CD19+ B cells as reported previously (29). In a unique aspect of this study, we demonstrated that HCV-RNA negative PBMCs, derived from healthy blood donors, became HCV RNA positive when mixed with plasma from chronic HCV carriers and that, in this in-vitro setting, HCV RNA was also concentrated on B cells. Hence, normal PBMCs and their subsets could be made to mimic PBMCs from HCV carriers by mixing them with RNA positive plasma, suggesting simple adsorption rather than intracellular replication. Thus, the finding of HCV RNA in PBMCs or their subsets in HCV carriers does not necessarily imply viral replication or an occult site of infection but rather PMBCs may be a passive carrier of HCV that is replicated in other sites. There is a logical explanation for this observation in that most HCV in chronic carriers circulates as immune complexes (30) and that PBMCs, particularly B cells, are rich in immunoglobulin receptors. Alternatively, unbound HCV could attach to known HCV receptors present on PBMCs such as CD81, SBR1 and claudin-1 without truly supporting HCV replication in vitro (31). Determining the precise mechanism for HCV attachment to PBMCs will require additional study.

Full recovery from HCV infection is indicated by several observations: 1) In this study, we were unable to detect HCV RNA in the PBMCs from 59 presumed recovered subjects using a sensitive nested PCR that measured down to 10 IU. In addition, none of the recovered patients had HCV positive cultures even after activation making it unlikely they harbored an occult reservoir of HCV in their mononuclear cells; 2) Bernardin, et al (16), using a highly sensitive TMA assay and a relatively large input volume (0.5 ml of plasma and 2.5 × 10⁶ PBMC), failed to detect HCV RNA in the plasma or PBMCs of 69 aviremic blood donors who were anti-HCV and RIBA positive and presumed to have spontaneously recovered; 3) Operkalski, et al (32) demonstrated that of 12 recipients of blood from donors who had the pattern of HCV recovery (anti-HCV+, HCV RNA-negative by TMA), only one (8%) seroconverted for anti-HCV compared to 83 of 85 (98%) who received an HCV RNA-positive blood unit; 4) we did not detect HCV RNA in the liver tissue of two chimpanzees who showed the serologic and molecular pattern of HCV recovery, nor was HCV RNA found in any non-hepatic tissue of one recovered animal that was necropsied; 5) several studies that examined liver biopsies after treatment-induced recovery showed the general absence of HCV RNA in liver tissue and only very low levels in those who had not yet totally cleared the virus (22, 33).

Thus, the bulk of evidence suggests that HCV can be cleared not only from plasma, but also from tissue and that PBMCs are an unlikely source of residual replicating virus. The disparity between finding HCV RNA in the PBMCs of the vast majority of recovered subjects (7, 8, 19) and not finding HCV RNA in any subjects in this study and others (16, 17, 22, 23) cannot be fully explained at this time. Nonetheless, we feel the data presented herein strongly support the concept of absolute HCV clearance in most patients with spontaneous or treatment-induced recovery. Further, this study demonstrates that the finding of HCV RNA in the PBMCs of chronically infected patients does not necessarily imply replication within PBMCs. Rather, our data suggest that PBMCs are passive carriers of HCV that is derived from other replication sites, predominantly, if not exclusively, the liver. These findings and the general absence of documented HCV reactivation in presumed recovered subjects provide support for the concept of “curing” HCV infection. Nonetheless, our study is not sufficiently large to exclude the possibility that a minority of patients who are classified as “recovered” by standard criteria may still harbor small amounts of virus for a limited time as documented by Veerapu, et al (24). While, in any given patient, documentation of viral eradication will require serial testing over time, presumed recovered patients can be reassured that the reproducible loss of detectable HCV RNA in plasma generally denotes viral eradication and that any low-level residual virus can probably be contained by their reinvigorated cellular immune response to HCV that is no longer stunted by an overwhelming viral load.

List of Abbreviations

HCV

hepatitis C virus

PBMCs

peripheral blood mononuclear cells

HCC

hepatocellular carcinoma

PCR

polymerase chain reaction

n-RTD

nested real-time detection

RIBA

recombinant immunoblot assay

PBS

phosphate-buffered saline

PHA

phytohemagglutinin

SN/ SNs

supernatant/s

TMA

transcription mediated amplification

SVR

sustained virological response