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Occult hepatitis Cvirus infection:what does it mean?
Tram N. Q. Pham1, Carla S. Coffin2 and Tomasz I. Michalak1
1 Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial
University, St John’s, Newfoundland and Labrador, Canada
2 University of Calgary Liver Unit, Division of Gastroenterology, University of Calgary, Calgary, AB, Canada
consequences of OCI – HCV genome detection – hepatitis C virus – immune system
– low-level HCV infection – occult HCV persistence
Prof. Tomasz I. Michalak, Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John’s, Newfoundland and Labrador, Canada
Tel: 1709 777 7301
Fax: 1709 777 8279
Received 2 July 2009
Accepted 23 November 2009
Occult hepatitis C virus infection (OCI) is a recently identified entity of which the existence became evident when nucleic acid amplification assays of enhanced sensitivity were introduced for the detection of hepatitis C virus (HCV) genome and its replication. This form of HCV infection has been found to persist in the presence of antibodies against HCV and normal levels of liver enzymes for years after spontaneous or antiviral therapy-induced resolution of hepatitis C and, therefore, can be termed as secondary OCI. HCV RNA in OCI circulate at fluctuating levels normally not exceeding 200 genome copies per millilitre of serum or plasma, while low levels of virus genome and its replicative intermediate RNA-negative strand are detectable in the liver and, importantly, immune cells, which provide an opportunity to detect active virus replication without the need for acquiring a liver biopsy. In addition to secondary OCI, a form of OCI accompanied by persistently moderately elevated serum liver enzymes in the absence of antibodies to HCV, which can be termed as cryptogenic OCI, has also been described. The current understanding of the nature and characteristics of OCI, methods and pitfalls of its detection, as well as the documented and expected pathological consequences of OCI will be summarized in this review.
At least 170 million people worldwide are currently symptomatically infected with hepatitis C virus (HCV). Depending on certain not yet well-recognized host and viral characteristics, up to 35% of the infected individuals can spontaneously resolve the acute infection within 6 months, but the others develop chronic hepatitis C (CHC) and replicate the virus seemingly indefinitely.
With time, the induced CHC can lead to liver fibrosis, cirrhosis and development of hepatocellular carcinoma (HCC)
(1). In this regard, end-stage liver disease because of chronic HCV infection is currently the number one reason for liver transplantation (LT) in many parts of the world. With the absence of a prophylactic vaccine and the failure of current therapy to suppress HCV replication in nearly 50% of patients with CHC, it is unlikely that the
socioeconomic burden brought on by HCV will subside in the foreseeable future.
Hepatitis C virus is a member of the Flaviviridae family and occurs in six known genotypes. At present, patients with CHC carrying genotypes 2 and 3 typically receive 24 weeks of a combination of pegylated a-interferon and ribavirin (P-IFN/RBV), whereas those infected with the other strains may be treated for 48 weeks with the same treatment regimen. In current clinical practice, patients whose HCV RNA is undetectable by standard clinical assays at the end of therapy and continues to be so for at least 6 months thereafter are considered to have achieved a sustained virological response (SVR) and to have cleared HCV infection. By this definition, it is estimated that SVR is achieved in 45–55% of patients infected with genotype 1and up to 90% of those with genotype 2 or 3 (2, 3). In recent studies, as much as 60% of patients infected with HCV genotype 4 have been shown to achieve such clinically defined SVR (4).
Although HCV is conventionally known to target hepatocytes, evidence exists clearly demonstrating that HCV also invades and propagates in cells of the immune system, albeit at a lower per cell level than in hepatic tissue (5–10). Indeed, HCV RNA positive and negative (replicative) strands, as well as virus proteins have been identified in peripheral blood mononuclear cells (PBMC) in patients with CHC [reviewed in Blackard et al. (9)]. In addition, cellular genes known conventionally to be involved in the host’s antiviral responses have been shown to be differentially expressed in PBMC of patients with CHC relative to those in healthy individuals and that such expression profiles are correlated with HCV RNA loads in PBMC (11). A more comprehensive Liver International (2010) 502 _c 2010 John Wiley & Sons A/S
Liver International ISSN 1478-3223 analysis of HCV compartmentalization in CHC patients revealed the presence of replicating virus in T and B lymphocytes, as well as in monocytes (12, 13). Similarly, the ability of lymphocytes to support replication of wildtype HCV leading to a release of infectious viral particles have also been demonstrated in vitro (14, 15). The notion of HCV lymphotropism is further supported by the presence of HCV variants in immune cells which differ from those in serum or liver, as shown by clonal sequencing and single-stranded conformational polymorphism (12, 13, 16–18). On this note, certain sequence polymorphisms within the internal ribosomal entry site (IRES) of the 50-untranslated region (50-UTR) of the
HCV genomes found in lymphoid cells have been shown able to alter the IRES activity, such that it would promote HCV replication in immune cells but not in nonhaematopoetic cells (19, 20).
The natural propensity of HCV to infect cells of the immune system is consistent with the observations of a greater prevalence of HCV infection in mixed cryoglobulinaemia and non-Hodgkin lymphoma (NHL) (21, 22).
Indeed, an in depth analysis of HCV infection in immune
cells in CHC patients with markers of NHL has revealed a significantly higher frequency of HCV RNA in B-cell NHL than in non-B-cell NHL (22, 23). Furthermore, remission of mixed cryoglobulinaemia has been associated with reduction of HCV load and, conversely, progression of disease appears to be correlated with a rebound in viraemia (24).
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Identification and definition of occult hepatitis C virus infection
As alluded to previously, clinical resolution of hepatitis C, either spontaneous or therapy-induced, has conventionally been deemed a reflection of complete eradication of HCV. However, the past 5 years has seen an emergence of works from different groups documenting the persistence of HCV at low levels in plasma [usually below
100–200 virus genome equivalents (vge) or genome copies/ml], PBMC and hepatic tissue (typically between 10 and 100 vge/mg total RNA at both locations), and of replicating HCV genomes in PBMC and liver in individuals for many years after having been considered clinically cured of hepatitis C (25–28). As in the case of
CHC, different immune cell subsets were found infected with HCV and, in certain situations, the major, if not exclusive, reservoir of replicating HCV resided within a particular immune cell subtype (12). Further, sequencing analyses of HCV variants found in OCI have indicated that in many of these patients, the virus from lymphoid cells can be different from that in the circulation (12, 13). As in the case of CHC, PBMC in individuals with OCI continuing after clinically apparent SVR exhibit a pattern of cytokine expression which is clearly distinct from that of healthy individuals. Also, the gene expression profiles in OCI are not identical to those accompanying CHC, implying that the low-level persistent infection is not an immunologically neutral event (11, 29).
In parallel with the identification of OCI following spontaneous or antiviral therapy-induced resolution of hepatitis C, which normally occurs in the context of detectable antibodies against HCV (anti-HCV) and normal serum levels of liver enzymes, OCI has also been identified in anti-HCV non-reactive patients with persistently elevated liver enzymes (30). In contrast to the OCI that continues after termination of clinically evident hepatitis C and can be termed as secondary OCI, the aetiology of the anti-HCV non-reactive OCI is unknown.
Table 1 summarizes the similarities and distinctive characteristics of the secondary and the cryptogenic forms of OCI.
The identification of OCI was possible after introduction in research laboratories of highly sensitive nucleic acid amplification assays, which involve a reverse transcription (RT) step, where viral RNA is first transcribed to cDNA, and then cDNA amplified by usually two rounds of polymerase chain reaction (PCR). In our laboratory, this is followed by hybridization of the amplicons generated with an HCV probe, which further increases sensitivity of the signal detection. The sensitivity achieved is consistently _ 10 vge/ml (_ 3 IU/ml) or _ 5 vge/mg (_ 1.5 IU/mg) total RNA. Although OCI discovery was facilitated by development and application of highly sensitive research assays, recent evidence indicates that employment of clinical molecular tests of Table 1.
Occult hepatitis C virus infection improved sensitivities also allows the detection of OCI (16, 26).
Occult hepatitis C virus infection: the controversy
Because of its relatively recent identification, OCI has been the subject of research by many investigators using samples collected from patients in different geographical regions. Works from several laboratories (16, 26–28, 31, 32) have since substantiated the original finding of OCI (25) and also correlated the existence of OCI with the presence of prolonged HCV-specific T-cell responses and with the specific cytokine expression profile in circulating lymphoid cells (11, 29, 33). In some reports, the association between OCI and protracted alterations in liver histology continuing after clinical resolution of hepatitis C has been uncovered (26, 27, 32). Nevertheless, data reported by a few others do not support the notion of OCI existence (34–39). Thus, in contrast to the high rate of HCV persistence among individuals with clinical resolution of hepatitis C documented in earlier studies in which high-sensitivity research assays were applied, the recent works reported a seemingly sustained HCV RNA negativity in a vast majority of the individuals with SVR using the commercially available transcription-mediated assay (TMA) with sensitivity 5.3 IU/ml (38, 39). The question of whether HCV does persist as OCI in individuals who clinically resolved hepatitis C is undoubtedly of tremendous epidemiological and pathogenic importance and deserves a thorough investigation. This may confirm not only a new view on the natural history of HCV infection but may also warrant considerations of new preventive and therapeutic practices against HCV infection, which continues beyond a clinically apparent recovery.
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Factors influencing detection of occult hepatitis C virus infection
Because levels of circulating virus in OCI are normally below 100–200 vge/ml and they tend to fluctuate both in plasma and at the sites of virus replication, it is conceivable that differences in the sensitivity of detection assays, the procedures of how samples are handled and processed, and whether singular plasma sample or serial samples collected from different compartments of virus occurrence (i.e. plasma, PBMC and liver) are analysed could collectively result in highly different outcomes with respect to detection of low levels of HCV genomes (40–42).
Firstly, in contrast to nearly all clinical assays currently used for HCV RNA detection, which typically involve only one round (direct) amplification of viral RNA by RT-PCR, the research assays applied in the discovery of OCI consisted of nested RT-PCR followed by detection of amplicons via nucleic acid hybridization (NAH), i.e. Southern blot hybridization. As long as a complete transfer of the amplicons from a gel to a membrane matrix is ensured, the Southern blotting analysis, which makes use of the hybridization between amplified products and a labelled HCV gene-specific probe, not only validates the specificity of the identified signals but also enhances the sensitivity of the detection by as much as 10-fold. Indeed, in certain cases, HCV RNA signals can be only detected after NAH analysis, as illustrated in Figure 1. Nevertheless, because these research assays are not of a high output, are time consuming and require great attention to details, calibration and control procedures, their application in clinical laboratory setting for routine HCV RNA evaluation is limited. Moreover, the fact that the sensitivity of HCV RNA detection assays used by different groups and those supplied by Plasma PBMC Liver commercial providers is reported in different ways, i.e. international units (IU)/ml vs. genome copies or vge/ml, inadvertently creates further uncertainty. Since 1 IU could range from 2 to 7 vge, depending on assays (43), it is certain that an assay with a limit of detection (LOD) of 10 IU/ml would not have the same sensitivity as that with 10 vge/ml. Overall, given the sensitivity of the research assays used in recent years for OCI detection is as low as _ 3 IU/ml or _ 1.5 IU/mg total RNA, which would be more sensitive than that of the TMA (sensitivity 5.3 IU/ml) used in the clinical studies recently reported (e.g. 38, 39), this may represent one of the crucial elements why differences in the rates of HCV genome identification after SVR and diagnosis of low-level HCV persistence are observed.
Secondly, it has been shown that ex vivo mitogen stimulation of PBMC significantly augments HCV replication leading to detection of virus genomes in apparently HCV non-reactive cells (12, 25, 44). Specifically, although HCV RNA on average is detectable in naive (uncultured) PBMC in approximately 30% of persons with clinically resolved hepatitis C when tested by RT-PCR/NAH (25), up to 75% of these individuals can in fact be positive for HCV RNA in PBMC by the same assay if the cells were stimulated ex vivo with mitogen/s before analysis (12, 41). This effectively
means that screening of untreated PBMC would likely result in an underestimation of the true extent of OCI occurrence, as it may have been the case in the studies which did not support the existence of OCI (34–39). It is worth mentioning that the finding of mitogen-induced upregulation of virus replication is not unique to HCV because such an enhancement has also been observed for other viruses such as measles virus, human immunodeficiency virus (HIV) and herpesvirus, which are also capable of inducing persistent infections (45–47).
Thirdly, it is very important to indicate that the quality and the amount of recovered RNA is of paramount significance when identification of low levels of HCV genome is attempted. Inappropriate handing of blood, PBMC and tissue samples and suboptimal extraction procedures may diminish or destroy fragile viral RNA occurring at trace quantities which is predestined for RTPCR analysis (42).
Fourthly, in a recent study, we found that because the levels of HCV RNA in OCI are generally low and inevitably fluctuate at times, using one concentration of RNA from plasma or cells for testing may occasionally be insufficient to accurately assess the incidence of OCI.
Indeed, the data have indicated that detection of OCI could be enhanced by 10–15% if RNA from non-standard amounts of plasma (1–4 ml) or cells (5_105, 1_106 or 4_106 cells) were used for testing instead of standard concentrations recovered from 250 ml of plasma and 2_106 cells (T. N. Q. Pham and T. I. Michalak, unpublished observations). It should be pointed out that the usefulness of analysing greater volumes of plasma in regard to identification of OCI has also been reported by others (26, 48).
Fifthly, screening serial samples obtained from the same individual at different time points of follow-up may be necessitated. The collective data from our studies completed in recent years, which totalled 480 patients from different geographical regions who were followed for up to 9 years after achieving clinical SVR, revealed consistent detection of HCV RNA in plasma and PBMC in only a minority of cases [(12, 25, 29); TNQ Pham et al., unpublished data]. It should be mentioned that these approaches were not employed by most investigators whose data did not support the existence of OCI (34–37).
Sixthly, as alluded to earlier, in certain cases of OCI, we found that despite apparent absence of HCV RNA in total PBMC, the viral genomes were readily identifiable in certain immune subsets, including T cells, B cells or monocytes (12). This indicates that screening of only PBMC in these situations would lead to a false-negative result. The practical use of analysing the different subsets of immune cells for HCV RNA in clinical laboratories would be highly restricted, if not impossible. Nonetheless, this well illustrates how the data on HCV genome detection because of unavoidable limitations posed on the clinical assays need to be taken under consideration when the final conclusion on the absence of HCV RNA is made in a person with past history of hepatitis C.
Taken together, the experience accumulated in recent years indicates that the detection of small amounts of HCV RNA, as it is the case in OCI, is a highly complex task which, because of the high sensitivity of the molecular approaches used and usually limited availability of viral genomic material, requires extreme care in preparation of samples, strict contamination-controlled conditions, and highly skilful personnel paying diligent attention to procedural details.
-- 15 апр 2010, 23:48 --
Potential clinical consequences of occult hepatitis C virus infection
The bulk of published data indicate that a SVR following P-IFN/RBV therapy improves clinical outcomes and slows down, if not inhibits, progression of liver disease.
Overall, histological studies showed improved liver inflammation and fibrosis scores in patients with CHC and advanced fibrosis or cirrhosis (49–52). Although the length of follow-up varies in different studies, patients who have normal serum alanine aminotransferase (ALT) levels and undetectable HCV RNA by standard laboratory assays for at least 6 months are unlikely to develop a clinically significant disease on a longer follow-up. The data available from a meta-analysis of pertinent clinical trials and prospective studies indicate that the pooled probability of late relapse among such responders is 8.7% (ranging from 5.8 to 11.6%, with 95% confidence interval) (53).
However, more recent data indicate that the risk of relapse is even lower. In the largest and longest Liver International (2010) _c 2010 John Wiley & Sons A/S 505
Pham et al. Occult hepatitis C virus infection study of successfully treated CHC to date, George et al. (39) followed 150 patients for 5 years after a SVR. Over 90% of patients had no evidence of serum virological relapse by clinical assays (i.e. RT-PCR, sensitivity 100 copies/ml or 29 IU/ml and TMA, sensitivity 5.3 IU/ml) and in 60 patients from which long-term biopsies were obtained, 82% showed decreased fibrosis.
Occult hepatitis C virus infection reactivation
It is theoretically possible that OCI predisposes to reactivation of clinically evident HCV infection, especially in situations where the immune system is compromised because of comorbid disease or suppressive treatment. Indeed, most case reports that detail relapse after SVR have been linked to the risk of immune suppression. For example, although successful treatment of HCV infection before LT can prevent clinically significant re-infection after LT in most cases, there are several notable reports of recurrent HCV post-LT despite an apparent SVR (54–56). Iatrogenic immunosuppression secondary to steroids or chemotherapy has also been associated with a risk of recurrent HCV infection. Lin et al. (57) reported on two patients that relapsed when immune suppressive therapy was given within a few weeks of achieving clinically apparent SVR. One patient received prednisone for bronchitis and the second relapsed soon after immune suppression was started following kidney transplantation. Similarly, HCV recurrence of the same genotype following a successful completion of chemotherapy for NHL has also been reported in a patient with SVR (58). These data suggest that SVR does not lead to full restoration of antiviral immunity and that sterilizing immunity with complete elimination of virus is unlikely. The cases also caution against the use of immunosuppressive therapy in the immediate aftermath of SVR. Similarly, Lee et al. (59), described a patient with hypogammaglobulinaemia whose acute HCV infection appeared to resolve after IFN therapy, although the patient relapsed immediately and then appeared to clear virus spontaneously. The patient relapsed again after receipt of corticosteroid therapy and seemingly cleared virus again 8.5 years later. Thus, although considered to be a sustained responder, this individual continued to carry virus at levels not detectable by clinical assays. The virus was likely kept under control by the immune system until the corticosteroid-induced suppression led to the relapse. Interestingly, in one study, a cohort of HIVinfected individuals that had been previously infected and apparently completely cleared HCV was found to have transiently low levels of HCV compared with HIVnegative individuals exposed to HCV (60). The persistence of HCV at greater levels in HIV-positive persons than in those negative for HIV may imply that individuals who apparently cleared HCV in fact carried the virus at low levels and the infection relapsed after HIVinduced immunosuppression (60). Taken together, these studies suggest that our clinical practice may need to be modified in temporarily or prolonged immunocompromised patients with a history of HCV infection and SVR, as it is the case for patients with a previous exposure to hepatitis B virus (HBV) and persistent occult HBV infection.
Nonetheless, it would be prudent to confirm the findings from the case reports mentioned above in comprehensive investigations of larger cohorts of relevant patients using highly sensitive assays before making such recommendations.
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The role of occult hepatitis C virus infection in progressive liver disease
Although it is well documented that liver histology is markedly improved in a significant proportion of patients with SVR, the potential influence of OCI in individuals with persistent transaminitis and risk of progressive liver disease remains unclear. Also undefined is whether OCI may be a contributing factor to protracted
liver inflammation in persons who do not improve after SVR. Intriguing histological data has been reported with regard to the possible association of OCI with the development of chronic liver disease. For example, Berasain et al. (61) reviewed 1075 consecutive liver biopsies from anti-HCV, HBsAg-negative patients with unexplained ALT elevations (41.5 upper limit of normal on two occasions). In this study, 27% of all cases with chronic hypertransaminasaemia were positive for either HBV DNA or HCV RNA. Overall, the presence of viral genomes in the circulation, either HBV DNA or HCV RNA, appeared to predict a more severe disease.
Thus, patients with cryptogenic liver disease who tested positive for the viral genomes had a higher prevalence of cirrhosis than those who tested negative for HBV DNA and HCV RNA. Similarly, in 100 patients with abnormal liver function tests of unknown aetiology, Castillo et al. (30) found HCV RNA in liver biopsy specimens from 57 of 100 patients who were negative for anti-HCV and serum HCV RNA. Of the 57 patients with OCI, 20 (35%) had necroinflammatory lesions, a frequency which is significantly higher than that found in patients without intrahepatic HCV RNA (14%). In addition, the number of cases with fibrosis was markedly greater in patients with OCI (17.5%) than in those without (2.3%). Also, liver cirrhosis was seen in three patients with OCI, but not in those without (30).
Occult hepatitis C virus infection and hepatocellular carcinoma development
There is evidence to suggest that HCC continues to develop in certain patients with SVR. Veldt et al. (62, 63) followed 142 patients with SVR and advanced fibrosis diagnosed before treatment and found a 2% risk of developing HCC. Makiyami and colleagues reported that among 1197 sustained responders 27 (2.3%) developed HCC and that despite the apparent HCV RNA nonreactivity by standard clinical assays during follow-up HCV RNA was detected in one non-cancerous tissue specimen by nested RT-PCR (64). In another multicentre study of 1124 patients with SVR, Kobayashi et al. (65) reported that HCC had developed in 3.5% of patients who achieved SVR after IFN therapy, although whether HCC was associated with the presence of OCI was not addressed. Compared with sustained responders who did
not develop hepatoma, patients who have HCC were more often male, older and had a more histologically advanced disease before antiviral therapy. In very recent reports, the development of a well-differentiated HCC was diagnosed 13 years after achieving clinically evident SVR (66) or HCC still relapsed even if patients with SVR received curative hepatectomy (67). While it is conceivable that sustained HCV-related liver injury had initiated the carcinogenesis process, the contribution of OCI to the persistence of this injury and the relevance of low level HCV replication to the oncogenic transformation remain to be established. In general, patients with a past history of HCV infection and coinciding fibrosis should have continuing HCC surveillance even if virus elimination has been apparently successful (64, 65, 68).
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Extrahepatic diseases and occult hepatitis C virus infection
Several extrahepatic diseases have been associated with chronic HCV infection and, in most cases, their existence appears to be directly related to this viral infection. These include haematological diseases, such as cryoglobulinaemia and lymphoma (as noted above), autoimmune disorders, such as thyroiditis, kidney diseases and dermatological conditions, such as lichen planus and porphyria cutanea tardae (69–72). The role of OCI in the pathogenesis of these diseases is not defined. In a prospective evaluation of the prevalence of cryoglobulins in 226 patients with chronic liver disease, 127 (56.2%) were found to have chronic HCV infection while cryoglobulins were found in 69 (54%) of them, frequently with anti-HCV and HCV RNA concentrated in the cryoprecipitates (73). Given that the incorporation of HCV and antibody into the cryoprecipitate may reduce serum levels of anti-HCV and HCV RNA to below detectable levels, the existence of HCV infection, especially OCI, in these patients should be carefully evaluated taking this fact into consideration.
Multiple reports have described an association between chronic HCV infection and B-cell NHL. A meta-analysis of 48 studies concluded that the prevalence of HCV infection in patients with B-cell NHL was 15%, which is much greater than in the general population (_1.5%) and in patients with other haematological malignancies (2.9%), suggesting a potential aetiological role of HCV (74).
The strongest association of HCV infection with lymphoma is in the subset of patients with immunocytoma, a low-grade malignancy that has previously been associated with cryoglobulinaemia (75). Primary hepatic lymphoma has also been reported in association with HCV infection (76). One of the largest studies with that focus on this issue included 501 HCV-infected patients who had received IFN treatment and who were compared with 2078 consecutive patients not receiving therapy. The annual incidence of lymphoma in patients with HCV was estimated to be 0.23% based on clinical laboratory tests.
Relative to those who did not achieve SVR, the risk of lymphoma was reduced significantly, but not entirely eliminated, in those who did (77). The incidence of OCI in this disorder has not yet been evaluated.
Occult hepatitis C virus infection has also been implicated in the development of renal disease. The most common manifestations of kidney disease in patients with HCV are membranoproliferative and membranous glomerulonephritis (GLN). The pathogenesis is likely related to deposition of HCVantigen–anti-HCV immune complexes in the basement membrane of glomeruli (78).
Fowell et al. (79) reported a case where HCV RNA was identified in renal tissue from an SVR patient with membranoproliferative GLN, despite the absence of detectable virus genomes in the serum and liver by COBAS Amplicor qualitative assay (lower LOD 50 IU/ml; Roche Diagnostics Limited, Burgess Hill, UK). In patients with chronic kidney disease, Barril et al. (80) described the incidence of HCV RNA in PBMC in 49 of 109 (45%) serum anti-HCV and HCV RNA-negative haemodialysed patients with abnormal liver enzymes.
HCV RNA-negative strand was detected in PBMC in 53% of these patients, a finding that could have a potential impact on the management of haemodialysis patients (80).
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Do individuals with occult hepatitis C virus infection require antiviral therapy?
The issue of antiviral therapy in patients with OCI is controversial, although only very limited data have been published. Nonetheless, there are reports indicating that it might be of benefit. For example, in one series of three individuals with mixed cryoglobulinaemia who were repeatedly negative for anti-HCV and HCV RNA by
clinical assays in serum and in concentrated cryoglobulins, two of them were treated with IFN (75). Both patients demonstrated excellent clinical and laboratory responses but the cryoglobulinaemia eventually relapsed following withdrawal of therapy. While HCV RNA was detected in the cryoprecipitates of both patients at the time of relapse, the virus genomes were found for the first time in the third case during a flare of cryoglobulinaemia coincident with varicella infection. Of note is that in all three patients, anti-HCV remained negative throughout follow-up. It was concluded that some essential forms of mixed cryoglobulinaemia can be caused by OCI and treatment with IFN is a valid option in such cases (81).
In one interesting study (82), 10 patients with OCI accompanied by elevated serum ALT were treated with P-IFN/RBV for 6 months and followed for 24 weeks.
Although eight patients had normalized ALT levels and Occult hepatitis C virus infection cleared HCV RNA from PBMC at the end of therapy, only three remained HCV RNA negative in PBMC and displayed normal ALT levels after the 6-month follow-up.
Five patients underwent a second liver biopsy after P-IFN/RBV treatment and, although none of them lost HCV RNA from the liver, a significant decrease was observed in the amount of intrahepatic viral RNA and in three patients liver necroinflammation and fibrosis scores had decreased in comparison with the pretreatment lesions. Thus, treatment of OCI may be indicated in patients with a more clinically apparent liver disease (82).
Occult hepatitis C virus infection and hepatitis C virus infectivity
The in vitro infectivity of HCV persisting at low levels following SVR has not been investigated until recently (83). In a recent study, de novo HCV infection in human normal T cells was employed to assess whether traces of the virus found in plasma and in supernatant from cultured PBMC derived from individuals with SVR following P-IFN/RBV treatment could establish productive HCV replication in vitro (14). The transmission of the infection was evidenced by: (I) detection of HCV RNA negative strand and HCV NS5a protein; (II) production of virus with different biophysical properties from those of the plasma-derived virus; (III) emergence of unique HCV variants in de novo infected T cells which were distinct from those in the original plasma inoculum; (IV) prevention of HCV infection following viral neutralization with anti-HCV E2 antibody; (V) blocking of infection with anti-CD81 antibody (CD81 is the prototype virus co-receptor) and (VI) inhibition of infection by pre-treatment of the cells with recombinant human IFN-a. Also, HCV virions specifically recognized by anti-E2 antibody were uncovered by immunoelectron microscopy in plasma of individuals with SVR and in the culture supernatant from de novo infected T cells exposed to the plasma. Overall, trace HCV carried in three of the nine investigated individuals for up to 72 months after clinically apparent SVR could elicit a productive infection in vitro (83).
In relevant studies in chimpanzees, diluted plasma from a patient with acute post-transfusion hepatitis which contained approximately 10 virions was capable of inducing an infection in a chimpanzee, as characterized by elevated serum ALT and liver inflammation (84).
Similarly, as few as 20 copies of HCV RNA prepared by dilution of serum collected during the pre-acute phase of hepatitis C of an infected chimpanzee has been shown to
induce HCV RNA-positive infection in the absence of ALTelevation (85). In comparison, 20–50 copies of HCV circulating in individuals with SVR were found able to establish productive infection in the T-cell infection system mentioned above (83). Certainly, it would be highly valuable to assess infectivity of OCI in a chimpanzee model by using samples directly derived from appropriate individuals and by applying assays of enhanced sensitivity for detection of both HCV genome and its replication in different compartments of virus occurrence, as well as by utilizing other approaches identifying active low-level virus replication which applicability has been ascertained in recent years.
At present, reports on OCI infectivity for immunocompetent, healthy individuals are very sparce, however, in one relevant report, the prevalence of HCV infection among family members of patients with OCI was similar to that found among family members of patients with CHC (86). This could be indicative of the importance of
measures preventing intrafamilial transmission of HCV independent of whether infection is symptomatic or progresses silently.
Occult hepatitis C virus infection appears to be a common, if not natural, consequence of either spontaneous or therapy-induced resolution of clinically evident hepatitis C, although it cannot be excluded that it might also be an outcome of asymptomatic exposure to the virus. The availability of clinical laboratory assays with sensitivities compatible to the current research tests and implementation of standardized methods of highly efficient recovery and transcription of small amounts of HCV RNA would help improve the precision with which OCI is identified among patients and the general population.
Also, the introduction of consistent diagnostic protocols, including testing of multiple samples from more than one compartment of virus occurrence, would be worth considering. It is hoped that the data obtained would be able to identify the true scope of clinical and epidemiological problems associated with silent forms of HCV persistence. In consequence, this knowledge should not only advance our understanding of the nature and pathological consequences of OCI, but also recognize a need for therapy and establish more stringent criteria for the assessment of therapeutic outcomes. l 2009; 50: 256–63.