Occult hepatitis C: How convincing are the current data?
Authors: Martin-Walter Welker, Stefan Zeuzem
Journal: Hepatology (2008)

Sustained virologic response (SVR), defined as undetectable hepatitis C virus (HCV) RNA in serum 24 weeks after withdrawal of (pegylated)-interferon-alfa (PEG-IFN)-based antiviral therapy, has become the primary goal for therapy of patients infected with HCV. Recent reports about "occult" persistence of HCV infection despite constantly negative serum HCV RNA1-14 have led to uncertainty in patients and physicians. This review gives a summary of available data regarding "occult" HCV infection. "Occult" HCV infection has been defined as detection of HCV RNA in liver tissue alone or in liver tissue as well as in peripheral blood mononuclear cells (PBMCs) despite constantly undetectable levels of HCV RNA in serum.15 Two forms have been distinguished: (1) detection of HCV RNA in liver tissue of patients with liver disease of unknown origin (anti-HCV negative, serum HCV RNA negative) and (2) detection of HCV RNA in liver tissue in patients with spontaneous or treatment-induced HCV RNA clearance from serum (anti-HCV positive, serum HCV RNA negative).8, 10 HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; NAH, nucleic acid hybridization; PBMC, peripheral blood mononuclear cell; RBV, ribavirin; RT-PCR, reverse transcription-polymerase chain reaction; TMA, transcription-mediated assay; PEG-IFN, pegylated interferon-alfa; SVR, sustained virologic response; vge, virus genome equivalents. HCV is a plus-strand RNA virus, which replicates via a complementary RNA minus-strand.16 The hepatocyte is the primary target cell, but HCV is potentially also lymphotropic.17-23 Because HCV has no known ability to integrate its nucleotide sequence into the host genome and due to the short half-life of HCV of at most 3 hours,24, 25 uninterrupted viral replication is required to maintain infection. Detection of the antigenomic strand is considered an indicator of viral replication. The studies describing "occult" HCV infection in different clinical settings are based on detection of genomic and antigenomic HCV RNA in various compartments.3, 5, 7, 8, 10, 26-28 Testing for HCV RNA in liver specimens is considered the gold standard. Because liver biopsy is an invasive procedure, HCV RNA detectability was investigated only in PBMCs within some studies.15 The required sensitive methods are sophisticated and challenging molecular biological techniques.3, 5, 7, 8, 10, 26-28 In addition to commercially available test systems, in-house nested reverse transcription–polymerase chain reaction (RT-PCR) protocols and nucleic acid hybridization (NAH) RT-PCR were used with sensitivities ranging from 1-100 IU/mL. Furthermore, PBMCs were mitogen-stimulated in cell culture within some studies in order to increase the quantity of potential viral particles.27, 29, 30 In approximately 10% of patients with abnormal liver function tests, the etiology is unknown, but detection of hepatitis B virus (HBV) DNA as well as HCV RNA in serum or liver has been described.31-33 Only few studies focus on "occult" HCV infection as a possible cause of cryptogenic liver disease (Table 1), and data are conflicting.2, 10, 34-40 In the analysis by Castillo et al., which first described "occult" HCV infection in patients with liver disease of unknown origin,15 100 patients with persistently abnormal aminotransferase and gamma glutamyl transpeptidase values were investigated.10 All known causes of liver disease were excluded, such as viral (HBV, HCV) and autoimmune hepatitis as well as alcoholic, drug-induced, metabolic, and genetic liver disorders. Strategies to avoid cross-contamination and to provide optimized testing conditions, including use of several negative controls and enhanced sample processing, were applied according to the respective method section. An unexpectedly high number of 57 of 100 liver samples (57%) tested positive for genomic HCV RNA by RT-PCR. Antigenomic HCV RNA was detectable in 48 of these 57 samples (84%) by in situ hybridization. In addition, PBMCs from 40 of the 57 patients (70%) were found positive for HCV RNA.10 Genotyping, a test which typically requires the presence of at least 20,000 IU/mL,41, 42 was successful. Core antigen, however, was not detected in liver samples, possibly due to low expression levels. Infectious virus particles were also not isolated, and therefore replication was not ultimately proven. Within a subsequent study, 10 of the 57 patients were treated with PEG-IFN plus ribavirin (RBV) for 24 weeks.13 Primary inclusion criteria were detectability of HCV RNA in liver and PBMCs, absence of anti-HCV and HCV RNA in serum, elevation of alanine aminotransferase level, and proof of necroinflammatory activity in liver biopsy. Twenty-four weeks after the end of treatment, PBMCs tested negative for HCV RNA in 7 of 10 patients (70%) and a sustained biochemical response was achieved in 6 of 10 patients (60%). A complete biochemical and virologic response in PBMCs was achieved in 3 of 10 patients (30%). However, HCV RNA remained detectable in five of five liver biopsy samples (100%). The high prevalence of "occult" HCV infection in patients with cryptogenic liver disease described by Castillo et al.10 has not been confirmed by other groups. Tagiuri et al. investigated 26 patients with cryptogenic liver disease as well as 57 patients with alcoholic and nonalcoholic fatty liver disease. HCV RNA was undetectable in both serum and PBMCs within all 26 and 57 patients, respectively.37 Two clinical situations can be distinguished: (1) spontaneously resolved hepatitis C and (2) (PEG-)IFN–based treatment-induced SVR. More data is available on the latter, and patients with spontaneously resolved HCV infection are sometimes analyzed together with treated patients. Fluctuating serum HCV RNA concentrations in patients with acute hepatitis C infection are well known, leading to temporarily undetectable levels in individual constellations. Most data indicate that circulating HCV RNA in serum closely reflects intrahepatic HCV RNA kinetics and that (repeated) testing of serum is effective to differentiate between persistent and cured HCV infection.25, 43-49 Data regarding detection of HCV RNA in compartments (PBMCs, liver) other than serum in populations of patients with spontaneously resolved hepatitis C (anti-HCV positive, serum HCV RNA negative, no former antiviral treatment) are conflicting.7, 47, 50-54 The rates of detectability of HCV RNA in PBMCs range from 0%-50%.7, 50, 54 Within the largest study (n = 69, including six patients with SVR after IFN-based antiviral therapy), HCV RNA could neither be detected by cell-associated transcription-mediated assay (TMA; sensitivity, 2-50 HCV RNA copies in 5 × 106 PBMCs) nor by nested RT-PCR (sensitivity, 15-150 HCV RNA molecules in 5 × 106 PBMCs), respectively.50 Within two smaller studies, HCV RNA was detected in 6 of 12 and 3 of 30 PBMC samples (50% and 10%), respectively.7, 54 The rates of detectability of HCV RNA in liver biopsies range from 0%-83%. Four studies reported a high percentage (27 of 44 [61%], 10 of 12 [83%], 10 of 12 [83%], one of two [50%]) of intrahepatic detection of HCV RNA.7, 51-53 In contrast, within a cohort of 33 women, who had received HCV contaminated anti-D immunoglobulin, none was found to be intrahepatic HCV RNA positive.47 In patients with chronic hepatitis C and (PEG-)IFN–induced SVR, late relapse is rare (Tables 2 and 3).55, 56 Similar results have been obtained for patients with IFN-induced SVR after acute hepatitis C infection.57 Generally, these patients are considered to be cured, whereas the clinical significance of residual detection of HCV RNA fragments in various compartments remains unclear. Data about persistent detection of HCV RNA in PBMCs in patients with treatment-induced SVR are conflicting, because some studies found genomic and antigenomic HCV RNA in PBMCs3, 7, 9, 10, 27, 58-68 while others did not.50, 69-76 Detection of HCV RNA in PBMCs in patients with SVR could reflect prolonged clearance of viral residues from this compartment, as indicated by longitudinal testing of 10 patients with SVR.77 In liver specimens, most studies show only a small percentage of persistent detection of HCV RNA after (PEG-)IFN–induced SVR (Table 4). A higher percentage of intrahepatic positive cases was reported by Castillo et al.8 They found antigenomic HCV RNA in 15 of 20 liver samples and genomic HCV RNA in 19 of 20 liver samples (75% and 95%) obtained 35.4 months (mean ± standard deviation, 35.0 months) after withdrawal of (PEG-)IFN–based antiviral therapy as well as in 12 of 20 and 13 of 20 PBMC samples (60% and 65%), respectively.8 The HCV core region was amplified, cloned, and sequenced. Although the phylogenetic tree showed an apparent clustering between the clones of the core sequences compared to GenBank sequences of genotype 1 to 6, there was no difference compared to the known pretreatment genotype. Also, the genetic distances among clones were lower than among patients, which argues against contamination. The question of why late relapse is so rare (Tables 2 and 3) remains unanswered when infectious HCV RNA is present in liver tissue within the majority of patients with SVR as indicated by the study by Castillo et al.8 Moreover, these results were not confirmed within recent and larger studies, characterized by appropriate sample processing and use of highly sensitive tests (Table 4).64, 70, 76, 78 "Absence" of HCV RNA in serum could be explained by very low level viremia, not detectable by tests even with a lower detection limit of 50 IU/mL. Four articles report detection of HCV RNA in serum or whole blood samples in the constellation of "occult" HCV infection type A or B under circumstances of enhanced sensitivity. Methods applied comprise enrichment of suspected virus particles by ultracentrifugation of serum, use of whole blood samples instead of serum or plasma, or use of more sensitive tests, e.g., nested real-time RT-PCR (lower detection limit, <5 IU/mL) or NAH RT-PCR (lower detection limit, <10 virus genome equivalents [vge]/mL).1, 5, 6, 27 Within one article, samples from patients already described by Castillo et al. were analyzed, which limits the number of patients actually investigated.6, 10 Within the largest study comprising 106 patients with "occult" HCV infection (serum HCV RNA negative, liver HCV RNA positive) by Bartolomé et al., sensitivity was enhanced by ultracentrifugation of serum samples before performance of real-time RT-PCR.1 Unfortunately, no information was provided from which a population of this large number of patients with "occult" HCV infection was derived. After ultracentrifugation, 62 of 106 serum samples (58%) tested positive for HCV RNA. TMA was performed in a subpopulation of 10 patients with detectable HCV RNA in serum after ultracentrifugation. The viral load of these specimens was reported to be within a range from 60-160 vge/mL.1 Surprisingly, TMA, a highly sensitive molecular assay (sensitivity, <10 IU/mL, ≈50 vge/mL),79, 80 tested negative in all 10 samples. Furthermore, the data were not confirmed within a recent study investigating 80 patients with (PEG-)IFN ± RBV–induced SVR. In this study, a very sensitive PCR hybridization assay (sensitivity, 1-5 IU/mL) was used and all serum (mean follow-up, 50.3 months) and all liver (follow-up at least 42.0 months after SVR) samples tested negative for HCV RNA.64 Adaptive cellular response mechanisms, especially specific T cell responses, are believed to play a key role in recovery from HCV infection as well as in chronic hepatitis C.81-90 Nevertheless, HCV can escape from specific humoral and cellular responses.91, 92 Antibodies to HCV proteins can decrease in patients who have cleared HCV RNA from their serum despite persistence of a specific cellular response.85 HCV-specific lymphocytes can be the only evidence of former HCV infection in otherwise seronegative patients.93, 94 Quiroga et al. analyzed specific T cell responses in patients with detection of HCV RNA in liver tissue despite repeatedly negative tests for HCV RNA and anti-HCV antibodies in serum.14 They identified functional virus-specific memory CD4(+) and CD8(+) T cells and reported that magnitudes of T cell responses were inversely correlated with the extent of HCV RNA in liver tissue.14 On one hand, this could reflect ongoing interaction of the specific cellular immune system with persistent, but immunologically well-controlled viral replication. On the contrary, the results could be explained by coexistence of memory cells with viral residuals of unknown clinical significance after resolved HCV infection. Chronic hepatitis C is well known for its association with autoantibodies and autoimmune disorders.95-97 Quiroga et al. found anti-GOR immunglobulin G antibodies in 22 of 110 anti-HCV negative, liver HCV RNA positive patients (20%) in comparison with 70 of 110 patients with chronic hepatitis C (64%), and none within a control group of 120 patients with liver diseases unrelated to HCV.98 In patients with chronic hepatitis C, the clinical significance of non organ-specific autoantibody titers, e.g., low titer antinuclear antibodies, is controversial.96, 99 The clinical significance of autoantibodies in patients with "occult" HCV infection remains even more undefined. Mixed cryoglobulinemia is considered an extrahepatic manifestation of HCV infection. A possible association of "occult" HCV infection and cryoglobulinemia was suggested by case report series with limited numbers of patients.100, 101 In three of three patients who were negative for anti-HCV and serum HCV RNA with essential cryoglobulinemia, HCV RNA was detected in cryoprecipitates.100 In patients with SVR after antiviral therapy due to chronic hepatitis C who were suffering from mixed cryoglobulinemia, genomic and antigenomic HCV RNA sequences were detected in five of nine PBMC samples (56%).101 On the other hand, a recent study failed to demonstrate "occult" HCV infection in a heterogeneous cohort of patients with resolved HCV infection and vasculitis (n = 8) as well as in patients without HCV infection who had connective tissue disease (n = 23).35 Although HCV RNA was detected in PBMC samples from a heterogeneous control group of serum HCV RNA–positive patients, no HCV RNA was detected in PBMCs from patients with resolved or without HCV infection (zero of eight and 0 of 23, respectively).35 More studies on the possible association of "occult" HCV infection with mixed cryoglobulinemia and other autoimmune disorders are needed, especially regarding the potential therapeutic benefits of (PEG) IFN–based therapy. Acquired impairment of the immune system may result from human immunodeficiency virus (HIV) infection, severe underlying illness, e.g., patients with end-stage renal disease, and immunosuppressive therapy, e.g., after organ transplantation. Reactivation of HCV in patients with compromised immunity has been reported within case reports and small studies.26, 102-105 Late relapse, however, in immunocompromised patients achieving SVR after (PEG-)IFN–based antiviral therapy is rare (Table 3). Presence of HCV RNA in serum of HIV-infected patients without detectable anti-HCV antibodies has been described.106, 107 HCV replication despite lack of measurable humoral response is sometimes misnamed "occult" hepatitis C. It should be clearly distinguished from "occult" HCV infection defined by detection of HCV RNA in PBMCs or liver despite undetectable HCV RNA in serum. In patients suffering from end-stage renal disease in chronic hemodialysis programs, persistence of HCV RNA in PBMCs in 2 of 11 patients (18%) despite transiently negative HCV RNA in serum was reported.108 In the largest study focusing on "occult" HCV infection in patients with end-stage renal disease, HCV RNA was detected in 49 of 109 PBMC samples (45%).36 Patients undergoing liver transplantation with SVR after (PEG-)IFN–based antiviral therapy have shown long-term histological improvement and clearance of serum and intrahepatic HCV RNA.49 Available data at this time argue against frequent persistence of infectious HCV particles in PBMCs or liver in patients achieving SVR. Clinically most important, late virologic relapse rates are comparable between patients with and without compromised immune systems (Tables 2 and 3). The crucial question remains whether detection of HCV RNA fragments, even from the antigenomic strand, should be interpreted as ongoing viral replication or molecular biological residuals of a resolved virus infection. Even more uncertainty arises about the clinical significance of these findings. One study compared "occult" HCV infection and overt chronic hepatitis C. Aminotransferase values and histological scores were lower in the group classified as having "occult" HCV infection, but cholesterol and triglyceride levels were higher. However, this study lacked a control group of patients with cryptogenic, intrahepatic HCV RNA–negative liver disease.12 Castillo et al. compared the histological changes in 76 patients without serum markers for HBV or HCV infection, but intrahepatic detection of only HBV DNA (n = 17), only HCV RNA (n = 35), or detection of both HBV DNA and HCV RNA (n = 24). No histopathological differences were observed within these groups.11 Incidence of hepatocellular carcinoma (HCC) rises with progression of the disease to liver cirrhosis and is low in patients without cirrhosis with chronic hepatitis C. However, even low serum HCV RNA concentrations were reported to be associated with hepatocarcinogenesis in patients with cirrhosis.109 Detection of HCV RNA has been described in both nontumoral and tumoral liver tissue of patients with HCC, despite negative serum HCV RNA.4, 110 "Occult" HCV infection has been associated with HCC, but a causal relationship between detection of HCV RNA fragments in tissue samples from patients with HCC and liver carcinogenesis has not yet been proven. Detection of HCV RNA sequences in PBMCs or liver specimens despite undetectable levels of HCV RNA in serum has been described. However, the significance of these findings for the clinical course for the majority of patients is uncertain. Data are conflicting in patients with idiopathic liver disease. Some groups report detection of HCV RNA sequences in various compartments while other groups failed to detect HCV RNA. Replication of HCV was not shown by virus isolation or confirmed by detection of viral proteins in any study. Patients achieving SVR after (PEG-)IFN–based antiviral therapy have a low risk of late virologic relapse.55, 56 It is actually unknown how many of these patients with reported late relapse have "true" relapse and how many were reinfected. SVR has been linked with constant improvement of quality of life,111, 112 histological improvement,48, 113 decreased risk of development of liver cancer,114, 115 and reduced liver-related mortality.116, 117 Data about persistent detection of HCV RNA in liver samples or PBMCs are conflicting, but the majority of data indicate that patients with SVR also clear HCV RNA from liver.48, 118 Articles on "occult" HCV infection that report very high rates of detectability of HCV RNA in patients with cryptogenic liver disease or former hepatitis C were published by yet only five groups.1-8, 10-15, 68, 98, 100 Their reports about detection of HCV RNA in liver despite constantly negative HCV RNA in serum in a high percentage were not confirmed by recent studies, but more results must be awaited before a final conclusion can be drawn. Studies investigating potential infectiousness of subjects with "occult" HCV infection have not been conducted. Yet, there is no reported case proving transmission of HCV from a patient with "occult" HCV infection. In conclusion, HCV-infected patients with no detectable HCV RNA in serum 24 weeks after the end of treatment should remain to be considered noninfectious and cured of their infection. Further follow-up data are needed regarding spontaneous relapse and recurrence of infection in these patients. Therefore, in patients with SVR, annual surveillance including HCV RNA testing seems clinically reasonable despite a lack of formal cost-effectiveness analyses.