Journal of Clinical Virology 61 (2014) 315–320
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Review
Seronegative occult hepatitis C virus infection: Clinical implications ˜ ∗ Vicente Carreno Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain
a r t i c l e
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Article history: Received 3 July 2014 Received in revised form 29 August 2014 Accepted 20 September 2014 Keywords: HCV-RNA Liver Occult HCV PBMC
a b s t r a c t Occult hepatitis C virus infection (OCI) was first described in anti-HCV and serum HCV-RNA negative patients with abnormal values of liver enzymes but who presented HCV-RNA in liver and in peripheral blood mononuclear cells. Up to now, two types of OCI are recognized: seronegative OCI (anti-HCV and serum HCV-RNA negative) and seropositive OCI (anti-HCV positive and serum HCV-RNA negative). The concept of OCI is still a matter of debate, probably because both types of OCI are not considered as different entities. This review focuses on seronegative OCI. The existence of seronegative OCI has been documented all around the world with the implication of different HCV genotypes (1–4). Seronegative OCI is associated with cryptogenic chronic hepatitis and liver cirrhosis and it may be involved in the appearance of hepatocellular carcinoma. Also seronegative OCI may increase the histological liver damage in chronic hepatitis B and in HIV-infected patients. It may have a negative influence in the natural history of hemodialysis patients and in immune-mediated glomerulonephritis. Seronegative OCI has been detected also in patients with haematological diseases, among healthy subjects and in drug users. Other publications indicate the potential infectivity of seronegative OCI in the setting of family members, sexual partners and liver transplantation. In summary, seronegative OCI may play a role in liver diseases and other human pathologies and may be present in healthy people but larger studies are needed to confirm these findings. © 2014 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4.
5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Worldwide distribution and genotypes of OCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Impact of OCI in liver diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Influence of OCI in other diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OCI among healthy population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. OCI among drug users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The possible infectivity of OCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Discordant studies on seronegative OCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection Of OCI: HCV-RNA fragments or complete virions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: OCI, occult hepatitis C virus infection; anti-HCV, antibodies against hepatitis C virus; HCV-RNA, hepatitis C virus RNA; PBMC, peripheral blood mononuclear cells; HCC, hepatocellular carcinoma; ALT, alanine aminotransferase; HIV, human immunodeficiency virus; NAFLD, non-alcoholic fatty liver disease; PCR, polymerase chain reaction. ∗ Correspondence to: Fundación para el Estudio de las Hepatitis Virales, Guzmán el Bueno 72, 28015 Madrid, Spain. Tel.: +34 91 544 6013; fax: +34 91 544 9228. E-mail address: [email protected] http://dx.doi.org/10.1016/j.jcv.2014.09.007 1386-6532/© 2014 Elsevier B.V. All rights reserved.
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1. Introduction Occult hepatitis C virus infection (OCI) was first described among patients with cryptogenic chronic hepatitis and abnormal liver function tests who were anti-HCV negative by different commercial assays and serum HCV-RNA negative by standard PCR but presented HCV-RNA in liver and could have viral RNA in peripheral blood mononuclear cells (PBMC) [1]. Later studies demonstrated that OCI may be also diagnosed by concentrating 2 ml of serum by ultracentrifugation followed by HCV-RNA detection by real-time PCR [2]. This procedure increases the sensitivity of HCV-RNA detection eightfold. Also, an in-house immunoassay for HCVcore-specific antibodies allows identification of OCI [3]. Finally, HCV specific CD4+ and CD8+ T-cell responses have also been detected in patients with OCI and the maintenance of such immune responses indicates that HCV persists and is replicating in the liver and in the PBMC of the patients [4]. For diagnosis of OCI, the most sensitive technique is HCV-RNA detection in liver (100%), followed by the detection in PBMC (up to 70%), in serum after ultracentrifugation (up to 58%) and anti-HCVcore detection (nearly 40%) [1,5]. Further reports have evidenced the existence of two types of OCI: seronegative OCI (anti-HCV and serum HCV-RNA negative) and seropositive OCI (anti-HCV positive, serum HCV-RNA negative). In both types of infections, patients have HCV-RNA in liver and viral RNA may be detected in PBMC and/or in serum after ultracentrifugation. The seropositive OCI includes patients who presumably have resolved HCV infection (spontaneously or after antiviral therapy) and asymptomatic HCV carriers [6–9], but other publications (the majority of them in sustained virological responders after antiviral therapy) have not found this seropositive OCI, thus questioning its existence [10–13]. Regarding seronegative OCI there are many papers that confirm its existence [14–33], while only a few ones failed to detect it [34–36]. The majority of the papers that have detected seronegative OCI, confirm the specificity of HCV-RNA detection by sequencing. There are several differences between seronegative and seropositive OCI patients. The former are anti-HCV negative, have abnormal or normal values of liver enzymes (see later) and have not been treated with antiviral therapy. The seropositive OCI cases are anti-HCV positive, have generally normal liver enzymes and the majority of the reported cases have received antiviral therapy achieving a sustained response. So, both types of OCI should be considered as separate entities to avoid misinterpretation of the results. The present review is focused only in the clinical implications of seronegative OCI, hereafter referred as OCI. It must be remarked that all the articles commented in this review include exclusively anti-HCV and serum HCV-RNA negative cases. 2. Worldwide distribution and genotypes of OCI In the first report of OCI in Spanish patients, only HCV genotype 1b was identified [1]. Later studies performed in different countries (Australia, Colombia, Egypt, Georgia, Iran, Italy, Pakistan, and USA) have identified (in most of them by sequencing) the implication of other HCV genotypes (1a, 2a, 2c, 3a, 3b, 4a) in OCI [17,18,20–23,25,27–32]. 2.1. Impact of OCI in liver diseases Several authors have studied the presence and possible implications of OCI in cryptogenic chronic hepatitis using different approaches [1,19,21,23,26,30]. The prevalence of OCI in cryptogenic chronic hepatitis when testing HCV-RNA in PBMC is 10% (Table 1). As expected, when testing for HCV-RNA in liver, this prevalence increases substantially, ranging form 25% to 74%. Overall, in these
studies a large number of patients with cryptogenic chronic hepatitis has been included (n = 393). Liver cirrhosis was diagnosed (by histological examination or by ultrasonography) in 7% of patients with OCI. So, it should be concluded that OCI has a pathological role in cryptogenic chronic hepatitis and may be responsible of at least 7% of cryptogenic liver cirrhosis. Two studies reported detection of OCI in non-alcoholic fatty liver disease (NAFLD). Granieri et al. [22] included 25 patients with NAFLD and found OCI in the liver of 8/25 (32%) patients. Saad et al. [20] studied 27 NAFLD patients and detected HCV-RNA in the biopsies of 11 patients (41%). Although it seems that there is a high prevalence of OCI in NAFLD more studies are needed to confirm this finding and to determine its possible contribution to liver damage in these patients. OCI was detected in two reports among patients with chronic hepatitis B. Thus, De Marco et al. [27] found OCI in 2/7 patients by testing for HCV-RNA in PBMC. Also, Castillo et al. [37] detected OCI in the liver of 21/52 (40%) patients with chronic hepatitis B and antiHIV negative. When comparing patients with or without OCI they found that the former had significantly more lobular inflammation (1.6 ± 1.5 vs. 1.1 ± 1.6, p = 0.033), a higher fibrosis index (1.3 ± 0.3 vs. 0.7 ± 0.2, p = 0.028), and more presence of fibrosis (16/21: 76% vs. 15/31: 48% p = 0.045) than the later. In conclusion, it seems that OCI may increase the histological liver damage in chronic hepatitis B but this finding has to be further investigated in a larger cohort of patients. The possible link between OCI and hepatocellular carcinoma (HCC) of unknown origin has been also studied. Esaki et al. [14] found HCV-RNA in the non-tumour liver tissue of a patient with HCC. Comar et al. [15] studied the presence of HCV-RNA in paraffin embedded liver biopsies from five patients with HCC by in situ PCR. They found that 2/5 patients had HCV-RNA in the tumour liver tissue. Finally, Hernandez et al. [31] published a study analysing liver tissues from 42 patients with HCC and found that three of them (7%) had viral RNA. These findings suggest that OCI may have a role in the development of HCC although studies with more cases are necessary to definitively establish the relation of OCI and HCC.
3. Influence of OCI in other diseases The incidence of HCV infection among hemodialysis patients is high and a concern about viral transmission is well recognized. Barril et al. [38] in a multicenter study performed in Spain determined the possible presence of OCI in hemodialysis. They enrolled 109 patients with abnormal values of liver function tests (alanine aminotransferase [ALT] and/or gamma-glutamyl transpeptidase). They found that 49/109 (45%) of the patients had HCV-RNA in PBMC. HCV-RNA negative-strand was detected in 26/49 (53%) of the cases with OCI, indicating viral replication. The specificity of the results was demonstrated by cloning, sequencing and phylogenetic analysis. Hemodialysis patients with OCI had significantly higher ALT values (p = 0.04) and had been on hemodialysis for a longer period of time (p = 0.03). In a cholecystectomy performed by laparoscopy in one of the patients with OCI, liver cirrhosis was visualized. Finally, deaths were more frequent among hemodialysis patients with than in those without OCI (39% vs 20%; p = 0.031). Recently, Baid-Agrawal et al. [33] have reported the presence of OCI in 0.25% of hemodialysis and 0.5% of kidney transplant patients in Germany. The difference in the OCI prevalence between both studies may be explained not only by the different HCV prevalence in the general population in Spain (2%) and in Germany (0.3%) but also because in the Spanish study all patients had abnormal values of liver function tests while in the German cohort only 15.3% and 12.5% of the hemodialysis and kidney transplant patients, respectively, presented altered transaminases.
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Table 1 Occult hepatitis C virus in cryptogenic chronic hepatitis. Author
Patients N
Zaghloul et al. [19] Bokharaei-Salim et al. [21] Keyvani et al. [30] Castillo et al. [1] Idrees et al. [23] Gad et al. [26]
40 69 45a 100 31 108
Total
393
OCI N (%)
4 (10%) 7 (10%) 4 (9%) 57 (57%) 23 (74%) 27 (25%) 122
Liver cirrhosis in patients with OCI N (%)
HCV-RNA detection
Not available 4 (57%) 4 (100%) 3 (5%) 0 1 (4%)
PBMC PBMC PBMC Liver Liver Liver
12 (7%)
OCI: occult hepatitis C virus infection; PBMC: peripheral blood mononuclear cells. a All patients had cryptogenic liver cirrhosis.
As the prevalence of serological markers of HCV among patients with glomerular nephropathies is also relatively high, Castillo et al. [39] determined the possible existence of OCI in 87 patients with primary and secondary immune-mediated glomerulonephritis and in 26 patients with hereditary glomerulonephritis (controls). Diagnosis of OCI was done by detecting HCV-RNA in PBMC or in serum after ultracentrifugation. Patients with immune-mediated glomerulonephritis had significantly (p = 0.001) more OCI (34/87: 39%) than patients with hereditary glomerulonephritis (1/26: 3.8%). By means of cloning, sequencing and phylogenetic analysis it was shown the specificity of the results. Among patients with immune-mediated glomerulonephritis serum creatinine levels were significantly higher in those with OCI than in the negative ones (p = 0.015). In a multivariate analysis it was found that patients with immune-mediated glomerulonephritis had 13 times more risk of having an OCI than patients with hereditary glomerulonephropathy (95% confidence interval: 1.695–104.229; p = 0.014). After three years of follow-up, patients with OCI presented significantly lower creatinine clearance than those without OCI (p = 0.032) and the frequency of progression to end-stage renal disease was higher (although with no significance) in patients with OCI than in the negative ones. Finally, HCV-RNA was retested again after 3 years in seven patients with OCI and all of them remained positive. In summary, these studies suggest that OCI may have a role in the progression of renal diseases and since HCV replication has been observed in these patients, concerns about uncontrolled HCV transmission in hemodialysis units have arisen. Also, the possible presence of OCI among patients with lymphoproliferative disorders has been studied taking into account the reported association between HCV infection and these diseases. Farahani et al. [28] included 104 patients with lymphoproliferative disorders and found HCV-RNA in PBMC in two patients (1.9%), one with non-Hodgkin’s lymphoma and the other with chronic lymphocytic leukaemia. El-Sayed et al. [16] studied 21 patients with non-Hodgkin’s lymphoma and found that four (19%) had HCV-RNA in PBMC. Finally, Youssef et al. [25] also detected HCV-RNA in PBMC of 10/37 (27%) patients with non-Hodgkin’s lymphoma. So, these studies that included a substantial number of patients (N = 162) with lymphoproliferative disorders have found that 10% of them had an OCI. However, the possible influence of OCI in the course of these diseases has not been evaluated. Recently, Gatserelia et al. [32] from Georgia, have reported the frequency of OCI among patients with HIV infection. They recruited 161 HIV-infected patients and tested HCV-RNA in their PBMC. Among 98 HIV patients with no evidence of liver disease, two (2%) had OCI. Another 34 HIV positive patients had a cryptogenic chronic hepatitis and four (12%) were diagnosed of OCI. Finally, 29 HIV infected patients had a chronic hepatitis B and nine of them (31%) had OCI. This percentage is slightly lower than the 40% reported by Castillo et al. [37] in anti-HIV negative patients with chronic hepatitis B but in the latter study the diagnosis of OCI was performed
by detecting HCV-RNA in liver biopsies. Gatserelia et al. [32] also found that the genotypes of OCI were 1b, 2a/2c and 3a and that HIVinfected patients with OCI had more frequently liver fibrosis (by FibroScan) and higher fibrosis score than patients without OCI. In conclusion, it seems that OCI has a high prevalence in HIV infected patients and that this infection may induce a more aggressive liver damage, but these findings require confirmation in a larger number of patients.
4. OCI among healthy population The presence of OCI has also been described in healthy population without evidence of liver disease. De Marco et al. [17] performed a study in healthy subjects that were enrolled in three different epidemiological studies on cancer in Italy. Among the 276 healthy individuals with normal values of liver enzymes and without serological markers of HCV infection, nine (3.3%) had HCV-RNA in PBMC. Taking into account that the prevalence of anti-HCV in the Italian general population is around 2.7% [19], the total number of patients with HCV infection may be underestimated. Youssef et al. [25] also studied 50 healthy persons and they found OCI in two of them (4%) by testing for viral RNA in PBMC. In summary, OCI may be present in the general healthy population and this is a potential risk for HCV transmission as it is currently not diagnosed. So, there is a need for performing more studies with a greater number of healthy subjects to confirm and clarify the role of OCI infection in this population. In this setting, although HCV transmission by blood donations in developed countries is rare, it can not be discarded the asymptomatic transmission of OCI. Therefore, it is important to investigate the prevalence and significance of OCI in a large multicenter study in blood donors in order to modify screening procedures if necessary.
4.1. OCI among drug users Recently, the presence of OCI has been described among injecting drug users. Sugden et al. [29] studied 20 injecting drug users in a significantly solid work. They analyzed the presence of HCV-RNA in PBMC of drug addicts from Australia. After obtaining blood samples, an aliquot was analyzed in a centre in Australia and the other was sent to a laboratory in Canada. The criterium for a positive result was the simultaneous detection of HCV-RNA in PBMC with identical sequencing results. Of the 20 injecting drug users, two of them (10%) had HCV-RNA in PBMC and HCV genotype was 1a. In another two cases, there were discordant results between both laboratories and these samples were considered negative. This paper indicates the existence of OCI in injecting drug users, although studies including more subjects are needed to determine the real prevalence of OCI among this population.
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4.2. The possible infectivity of OCI Several works have suggested the possible transmission capacity of OCI. Bartolomé et al. [2] studied the physico-chemical characteristics of OCI in comparison to classical hepatitis C. They performed iodixanol density gradient studies in serum of five patients with OCI and five patients with chronic hepatitis C and found that the density of the viral particles in OCI was similar to that isolated from patients with chronic hepatitis C, suggesting that serum from patients with OCI may contain infectious HCV particles. Cortés-Mancera et al. [19] reported the transmission and persistence of OCI in a patient with cryptogenic liver cirrhosis who underwent liver transplantation. Ten month later the patient was retransplantated due to a chronic cholestasis of unknown origin. HCV-RNA was detected in the native liver and in the first liver graft. Sequencing and phylogenetic analysis discarded crosscontamination and demonstrated that the HCV infecting both livers was the same (genotype 1a). This report proves that OCI is able to infect the liver graft, persisting 10 months and probably inducing a liver failure that required retransplantation. Further studies should be done to investigate the role of OCI in the setting of liver transplantation. Also Roque-Cuellar et al. [24] reported the spread of OCI among seronegative heterosexual partners of patients with chronic HCV infection as they detected OCI (HCV-RNA positive in PBMC) in 13% of the partners. In another study, the intrafamilial transmission of OCI was studied in 102 family members of 50 patients with OCI and in 118 family members of 59 patients with chronic hepatitis C by testing for anti-HCV and serum HCV-RNA [40]. The prevalence of anti-HCV among family members of patients with OCI was 9.8% (10/102) and 3.4% (4/118) among family members of patients with chronic hepatitis C. In addition, there were 14 family members (seven household contacts of OCI cases and the other seven of chronic hepatitis C patients) who presented abnormal values of liver enzymes without serological markers of HBV or HCV infection. Only two of them (both were relatives of two different OCI patients) agreed to be studied for OCI and HCV-RNA was detected in their liver biopsy. These results suggest that OCI may be transmissible among heterosexual partners and family members. Another study [41] reported the response of OCI to antiviral therapy. Ten patients with OCI (all cases with HCV genotype 1) and who presented HCV-RNA in PBMC (persistently detectable), abnormal ALT values and chronic hepatitis in the liver biopsy were treated for 6 months with Pegylated-Interferon plus Ribavirin and were followed for another 6 months. At the end of the follow-up, six (60%) patients remained with normal ALT values and HCV-RNA in PBMC was undetectable in seven patients. The results of this study suggest that the response pattern to antiviral therapy in OCI is similar to that of chronic hepatitis C, indicating that the virus is replicating in OCI and intact viral particles are present in these patients. Also the persistence of OCI over time was investigated in 37 patients followed during 4.6 years [42]. Serum HCV-RNA after ultracentrifugation was found (with fluctuations) over the whole follow-up period. So, OCI may persist and replicate for a long period of time. 4.3. Discordant studies on seronegative OCI Three studies failed to detect seronegative OCI. In the first one, Halfon et al. [34] included 22 patients with cryptogenic chronic hepatitis and 23 patients with systemic connective tissue disease. None of these patients tested HCV-RNA positive in PBMC and the authors questioned the existence of OCI. However, this publication has several limitations. (i) Determination of anti-HCVcore or detection of HCV-RNA in serum after ultracentrifugation or in liver biopsy
was not performed and so, an important percentage of patients who in fact may have an OCI could be missed [5]. (ii) No description on preservation of PBMC was given and this may be critical since deterioration of cells would lead to false negative results. (iii) There is not information of the purity and integrity of the RNA extracted from cells and these data are essential when results are critically dependent on sampling procedures, sample properties, template quality, etc. [43]. Pisaturo et al. [35] tested for HCV-RNA in PBMC of 60 HIVinfected patients with no positive results and authors ruled out the existence of OCI. The same group studied the presence of OCI in 28 patients with oncohematological disorders under immunosuppressive therapy (by testing HCV-RNA in PBMC) with negative results [36]. However, in both studies the existence of OCI cannot be excluded since detection of anti-HCVcore, or viral RNA in serum after ultracentrifugation or in liver biopsies was not performed. 5. Detection Of OCI: HCV-RNA fragments or complete virions? It has been hypothesized that OCI is the result of detecting fragments of HCV-RNA and does not reflect the presence of HCV virions [44]. The findings of the studies on OCI indicate that this is not the case for several reasons: 1. HCV-RNA strand of negative polarity has been detected in PBMC and liver of patients with OCI indicating an active viral replication, which could not be possible if these patients present only HCV-RNA fragments [45]. 2. HCV particles isolated from patients with OCI and from patients with chronic HCV infection have similar buoyant densities [2]. 3. The behaviour of OCI under antiviral therapy is identical to that of patients with chronic hepatitis C [41]. 4. Reinfection of a liver graft by occult HCV and its persistence for 10 months has been described [18]. 5. The persistence of OCI during a prolonged period (years) has been reported [42]. 6. In HuH7 cells transfected with HCV subgenomic replicon, the HCV-RNA half-life is around 17 h when replication is suppressed. As HCV-RNA has been detected for months or years in PBMC and liver, the HCV-RNA detected in OCI cannot be fragments [46,47]. So, it should be concluded that detection of HCV-RNA in liver and in PBMC of patients with OCI reflects the presence of complete and active viral genomes and not residual HCV-RNA fragments. 6. Conclusions Occult HCV infection is found worldwide and all HCV genotypes studied up to now are involved in this infection. At least, 7% of cryptogenic liver cirrhosis may be caused by OCI and it seems that it could be responsible of hepatocellular carcinoma development. OCI may worse the histological liver damage in patients with chronic hepatitis B and the natural history of patients with chronic renal diseases. OCI is also detected in patients with lymphoproliferative disorders but its role in the evolution of these diseases must be studied In addition, OCI infection has been described in HIV-infected patients (inducing a more aggressive liver lesion), in injecting drug-users and even in healthy populations and there are also several data indicating that OCI may be infectious. Also, as OCI seems to affect many different clinical settings, it would be advisable to study its prevalence and significance in other populations (blood donors, healthy people, health-care workers, pregnant women, etc.).
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Funding The writing of this manuscript has been supported in part by the Fundación Investigaciones Biomédicas, Madrid, Spain.
Competing interests None declared.
Ethical approval Not applicable.
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Occurrence of occult HCV infection among HIV infected patients in Georgia. Georgian Med News 2014;226:37–41. [33] Baid-Agrawal S, Schindler R, Reinke P, Staedtler A, Rimpler S, Malik B, et al. Prevalence of occult hepatitis C infection in chronic hemodialysis and kidney transplant patients. J Hepatol 2014;60:928–33, http://dx.doi.org/10.1016/ j.jhep.2014.01.012. [34] Halfon P, Bourlière M, Ouzan D, Sène D, Saadoun D, Khiri H, et al. Occult hepatitis C virus infection revisited with ultrasensitive real-time PCR assay. J Clin Microbiol 2008;46:2106–8, http://dx.doi.org/10.1128/JCM.00345-08. [35] Pisaturo M, Guastafierro S, Filippini P, Tonziello G, Sica A, Di Martino F, et al. Absence of occult HCV infection in patients experiencing an immunodepression condition. Infez Med 2013;21:296–301. [36] Coppola N, Pisaturo M, Guastafierro S, Tonziello G, Sica A, Sagnelli C, et al. 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Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Review
Seronegative occult hepatitis C virus infection: Clinical implications ˜ ∗ Vicente Carreno Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 3 July 2014 Received in revised form 29 August 2014 Accepted 20 September 2014 Keywords: HCV-RNA Liver Occult HCV PBMC
a b s t r a c t Occult hepatitis C virus infection (OCI) was first described in anti-HCV and serum HCV-RNA negative patients with abnormal values of liver enzymes but who presented HCV-RNA in liver and in peripheral blood mononuclear cells. Up to now, two types of OCI are recognized: seronegative OCI (anti-HCV and serum HCV-RNA negative) and seropositive OCI (anti-HCV positive and serum HCV-RNA negative). The concept of OCI is still a matter of debate, probably because both types of OCI are not considered as different entities. This review focuses on seronegative OCI. The existence of seronegative OCI has been documented all around the world with the implication of different HCV genotypes (1–4). Seronegative OCI is associated with cryptogenic chronic hepatitis and liver cirrhosis and it may be involved in the appearance of hepatocellular carcinoma. Also seronegative OCI may increase the histological liver damage in chronic hepatitis B and in HIV-infected patients. It may have a negative influence in the natural history of hemodialysis patients and in immune-mediated glomerulonephritis. Seronegative OCI has been detected also in patients with haematological diseases, among healthy subjects and in drug users. Other publications indicate the potential infectivity of seronegative OCI in the setting of family members, sexual partners and liver transplantation. In summary, seronegative OCI may play a role in liver diseases and other human pathologies and may be present in healthy people but larger studies are needed to confirm these findings. © 2014 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4.
5. 6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Worldwide distribution and genotypes of OCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Impact of OCI in liver diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Influence of OCI in other diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OCI among healthy population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. OCI among drug users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The possible infectivity of OCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Discordant studies on seronegative OCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection Of OCI: HCV-RNA fragments or complete virions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: OCI, occult hepatitis C virus infection; anti-HCV, antibodies against hepatitis C virus; HCV-RNA, hepatitis C virus RNA; PBMC, peripheral blood mononuclear cells; HCC, hepatocellular carcinoma; ALT, alanine aminotransferase; HIV, human immunodeficiency virus; NAFLD, non-alcoholic fatty liver disease; PCR, polymerase chain reaction. ∗ Correspondence to: Fundación para el Estudio de las Hepatitis Virales, Guzmán el Bueno 72, 28015 Madrid, Spain. Tel.: +34 91 544 6013; fax: +34 91 544 9228. E-mail address: [email protected] http://dx.doi.org/10.1016/j.jcv.2014.09.007 1386-6532/© 2014 Elsevier B.V. All rights reserved.
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1. Introduction Occult hepatitis C virus infection (OCI) was first described among patients with cryptogenic chronic hepatitis and abnormal liver function tests who were anti-HCV negative by different commercial assays and serum HCV-RNA negative by standard PCR but presented HCV-RNA in liver and could have viral RNA in peripheral blood mononuclear cells (PBMC) [1]. Later studies demonstrated that OCI may be also diagnosed by concentrating 2 ml of serum by ultracentrifugation followed by HCV-RNA detection by real-time PCR [2]. This procedure increases the sensitivity of HCV-RNA detection eightfold. Also, an in-house immunoassay for HCVcore-specific antibodies allows identification of OCI [3]. Finally, HCV specific CD4+ and CD8+ T-cell responses have also been detected in patients with OCI and the maintenance of such immune responses indicates that HCV persists and is replicating in the liver and in the PBMC of the patients [4]. For diagnosis of OCI, the most sensitive technique is HCV-RNA detection in liver (100%), followed by the detection in PBMC (up to 70%), in serum after ultracentrifugation (up to 58%) and anti-HCVcore detection (nearly 40%) [1,5]. Further reports have evidenced the existence of two types of OCI: seronegative OCI (anti-HCV and serum HCV-RNA negative) and seropositive OCI (anti-HCV positive, serum HCV-RNA negative). In both types of infections, patients have HCV-RNA in liver and viral RNA may be detected in PBMC and/or in serum after ultracentrifugation. The seropositive OCI includes patients who presumably have resolved HCV infection (spontaneously or after antiviral therapy) and asymptomatic HCV carriers [6–9], but other publications (the majority of them in sustained virological responders after antiviral therapy) have not found this seropositive OCI, thus questioning its existence [10–13]. Regarding seronegative OCI there are many papers that confirm its existence [14–33], while only a few ones failed to detect it [34–36]. The majority of the papers that have detected seronegative OCI, confirm the specificity of HCV-RNA detection by sequencing. There are several differences between seronegative and seropositive OCI patients. The former are anti-HCV negative, have abnormal or normal values of liver enzymes (see later) and have not been treated with antiviral therapy. The seropositive OCI cases are anti-HCV positive, have generally normal liver enzymes and the majority of the reported cases have received antiviral therapy achieving a sustained response. So, both types of OCI should be considered as separate entities to avoid misinterpretation of the results. The present review is focused only in the clinical implications of seronegative OCI, hereafter referred as OCI. It must be remarked that all the articles commented in this review include exclusively anti-HCV and serum HCV-RNA negative cases. 2. Worldwide distribution and genotypes of OCI In the first report of OCI in Spanish patients, only HCV genotype 1b was identified [1]. Later studies performed in different countries (Australia, Colombia, Egypt, Georgia, Iran, Italy, Pakistan, and USA) have identified (in most of them by sequencing) the implication of other HCV genotypes (1a, 2a, 2c, 3a, 3b, 4a) in OCI [17,18,20–23,25,27–32]. 2.1. Impact of OCI in liver diseases Several authors have studied the presence and possible implications of OCI in cryptogenic chronic hepatitis using different approaches [1,19,21,23,26,30]. The prevalence of OCI in cryptogenic chronic hepatitis when testing HCV-RNA in PBMC is 10% (Table 1). As expected, when testing for HCV-RNA in liver, this prevalence increases substantially, ranging form 25% to 74%. Overall, in these
studies a large number of patients with cryptogenic chronic hepatitis has been included (n = 393). Liver cirrhosis was diagnosed (by histological examination or by ultrasonography) in 7% of patients with OCI. So, it should be concluded that OCI has a pathological role in cryptogenic chronic hepatitis and may be responsible of at least 7% of cryptogenic liver cirrhosis. Two studies reported detection of OCI in non-alcoholic fatty liver disease (NAFLD). Granieri et al. [22] included 25 patients with NAFLD and found OCI in the liver of 8/25 (32%) patients. Saad et al. [20] studied 27 NAFLD patients and detected HCV-RNA in the biopsies of 11 patients (41%). Although it seems that there is a high prevalence of OCI in NAFLD more studies are needed to confirm this finding and to determine its possible contribution to liver damage in these patients. OCI was detected in two reports among patients with chronic hepatitis B. Thus, De Marco et al. [27] found OCI in 2/7 patients by testing for HCV-RNA in PBMC. Also, Castillo et al. [37] detected OCI in the liver of 21/52 (40%) patients with chronic hepatitis B and antiHIV negative. When comparing patients with or without OCI they found that the former had significantly more lobular inflammation (1.6 ± 1.5 vs. 1.1 ± 1.6, p = 0.033), a higher fibrosis index (1.3 ± 0.3 vs. 0.7 ± 0.2, p = 0.028), and more presence of fibrosis (16/21: 76% vs. 15/31: 48% p = 0.045) than the later. In conclusion, it seems that OCI may increase the histological liver damage in chronic hepatitis B but this finding has to be further investigated in a larger cohort of patients. The possible link between OCI and hepatocellular carcinoma (HCC) of unknown origin has been also studied. Esaki et al. [14] found HCV-RNA in the non-tumour liver tissue of a patient with HCC. Comar et al. [15] studied the presence of HCV-RNA in paraffin embedded liver biopsies from five patients with HCC by in situ PCR. They found that 2/5 patients had HCV-RNA in the tumour liver tissue. Finally, Hernandez et al. [31] published a study analysing liver tissues from 42 patients with HCC and found that three of them (7%) had viral RNA. These findings suggest that OCI may have a role in the development of HCC although studies with more cases are necessary to definitively establish the relation of OCI and HCC.
3. Influence of OCI in other diseases The incidence of HCV infection among hemodialysis patients is high and a concern about viral transmission is well recognized. Barril et al. [38] in a multicenter study performed in Spain determined the possible presence of OCI in hemodialysis. They enrolled 109 patients with abnormal values of liver function tests (alanine aminotransferase [ALT] and/or gamma-glutamyl transpeptidase). They found that 49/109 (45%) of the patients had HCV-RNA in PBMC. HCV-RNA negative-strand was detected in 26/49 (53%) of the cases with OCI, indicating viral replication. The specificity of the results was demonstrated by cloning, sequencing and phylogenetic analysis. Hemodialysis patients with OCI had significantly higher ALT values (p = 0.04) and had been on hemodialysis for a longer period of time (p = 0.03). In a cholecystectomy performed by laparoscopy in one of the patients with OCI, liver cirrhosis was visualized. Finally, deaths were more frequent among hemodialysis patients with than in those without OCI (39% vs 20%; p = 0.031). Recently, Baid-Agrawal et al. [33] have reported the presence of OCI in 0.25% of hemodialysis and 0.5% of kidney transplant patients in Germany. The difference in the OCI prevalence between both studies may be explained not only by the different HCV prevalence in the general population in Spain (2%) and in Germany (0.3%) but also because in the Spanish study all patients had abnormal values of liver function tests while in the German cohort only 15.3% and 12.5% of the hemodialysis and kidney transplant patients, respectively, presented altered transaminases.
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Table 1 Occult hepatitis C virus in cryptogenic chronic hepatitis. Author
Patients N
Zaghloul et al. [19] Bokharaei-Salim et al. [21] Keyvani et al. [30] Castillo et al. [1] Idrees et al. [23] Gad et al. [26]
40 69 45a 100 31 108
Total
393
OCI N (%)
4 (10%) 7 (10%) 4 (9%) 57 (57%) 23 (74%) 27 (25%) 122
Liver cirrhosis in patients with OCI N (%)
HCV-RNA detection
Not available 4 (57%) 4 (100%) 3 (5%) 0 1 (4%)
PBMC PBMC PBMC Liver Liver Liver
12 (7%)
OCI: occult hepatitis C virus infection; PBMC: peripheral blood mononuclear cells. a All patients had cryptogenic liver cirrhosis.
As the prevalence of serological markers of HCV among patients with glomerular nephropathies is also relatively high, Castillo et al. [39] determined the possible existence of OCI in 87 patients with primary and secondary immune-mediated glomerulonephritis and in 26 patients with hereditary glomerulonephritis (controls). Diagnosis of OCI was done by detecting HCV-RNA in PBMC or in serum after ultracentrifugation. Patients with immune-mediated glomerulonephritis had significantly (p = 0.001) more OCI (34/87: 39%) than patients with hereditary glomerulonephritis (1/26: 3.8%). By means of cloning, sequencing and phylogenetic analysis it was shown the specificity of the results. Among patients with immune-mediated glomerulonephritis serum creatinine levels were significantly higher in those with OCI than in the negative ones (p = 0.015). In a multivariate analysis it was found that patients with immune-mediated glomerulonephritis had 13 times more risk of having an OCI than patients with hereditary glomerulonephropathy (95% confidence interval: 1.695–104.229; p = 0.014). After three years of follow-up, patients with OCI presented significantly lower creatinine clearance than those without OCI (p = 0.032) and the frequency of progression to end-stage renal disease was higher (although with no significance) in patients with OCI than in the negative ones. Finally, HCV-RNA was retested again after 3 years in seven patients with OCI and all of them remained positive. In summary, these studies suggest that OCI may have a role in the progression of renal diseases and since HCV replication has been observed in these patients, concerns about uncontrolled HCV transmission in hemodialysis units have arisen. Also, the possible presence of OCI among patients with lymphoproliferative disorders has been studied taking into account the reported association between HCV infection and these diseases. Farahani et al. [28] included 104 patients with lymphoproliferative disorders and found HCV-RNA in PBMC in two patients (1.9%), one with non-Hodgkin’s lymphoma and the other with chronic lymphocytic leukaemia. El-Sayed et al. [16] studied 21 patients with non-Hodgkin’s lymphoma and found that four (19%) had HCV-RNA in PBMC. Finally, Youssef et al. [25] also detected HCV-RNA in PBMC of 10/37 (27%) patients with non-Hodgkin’s lymphoma. So, these studies that included a substantial number of patients (N = 162) with lymphoproliferative disorders have found that 10% of them had an OCI. However, the possible influence of OCI in the course of these diseases has not been evaluated. Recently, Gatserelia et al. [32] from Georgia, have reported the frequency of OCI among patients with HIV infection. They recruited 161 HIV-infected patients and tested HCV-RNA in their PBMC. Among 98 HIV patients with no evidence of liver disease, two (2%) had OCI. Another 34 HIV positive patients had a cryptogenic chronic hepatitis and four (12%) were diagnosed of OCI. Finally, 29 HIV infected patients had a chronic hepatitis B and nine of them (31%) had OCI. This percentage is slightly lower than the 40% reported by Castillo et al. [37] in anti-HIV negative patients with chronic hepatitis B but in the latter study the diagnosis of OCI was performed
by detecting HCV-RNA in liver biopsies. Gatserelia et al. [32] also found that the genotypes of OCI were 1b, 2a/2c and 3a and that HIVinfected patients with OCI had more frequently liver fibrosis (by FibroScan) and higher fibrosis score than patients without OCI. In conclusion, it seems that OCI has a high prevalence in HIV infected patients and that this infection may induce a more aggressive liver damage, but these findings require confirmation in a larger number of patients.
4. OCI among healthy population The presence of OCI has also been described in healthy population without evidence of liver disease. De Marco et al. [17] performed a study in healthy subjects that were enrolled in three different epidemiological studies on cancer in Italy. Among the 276 healthy individuals with normal values of liver enzymes and without serological markers of HCV infection, nine (3.3%) had HCV-RNA in PBMC. Taking into account that the prevalence of anti-HCV in the Italian general population is around 2.7% [19], the total number of patients with HCV infection may be underestimated. Youssef et al. [25] also studied 50 healthy persons and they found OCI in two of them (4%) by testing for viral RNA in PBMC. In summary, OCI may be present in the general healthy population and this is a potential risk for HCV transmission as it is currently not diagnosed. So, there is a need for performing more studies with a greater number of healthy subjects to confirm and clarify the role of OCI infection in this population. In this setting, although HCV transmission by blood donations in developed countries is rare, it can not be discarded the asymptomatic transmission of OCI. Therefore, it is important to investigate the prevalence and significance of OCI in a large multicenter study in blood donors in order to modify screening procedures if necessary.
4.1. OCI among drug users Recently, the presence of OCI has been described among injecting drug users. Sugden et al. [29] studied 20 injecting drug users in a significantly solid work. They analyzed the presence of HCV-RNA in PBMC of drug addicts from Australia. After obtaining blood samples, an aliquot was analyzed in a centre in Australia and the other was sent to a laboratory in Canada. The criterium for a positive result was the simultaneous detection of HCV-RNA in PBMC with identical sequencing results. Of the 20 injecting drug users, two of them (10%) had HCV-RNA in PBMC and HCV genotype was 1a. In another two cases, there were discordant results between both laboratories and these samples were considered negative. This paper indicates the existence of OCI in injecting drug users, although studies including more subjects are needed to determine the real prevalence of OCI among this population.
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4.2. The possible infectivity of OCI Several works have suggested the possible transmission capacity of OCI. Bartolomé et al. [2] studied the physico-chemical characteristics of OCI in comparison to classical hepatitis C. They performed iodixanol density gradient studies in serum of five patients with OCI and five patients with chronic hepatitis C and found that the density of the viral particles in OCI was similar to that isolated from patients with chronic hepatitis C, suggesting that serum from patients with OCI may contain infectious HCV particles. Cortés-Mancera et al. [19] reported the transmission and persistence of OCI in a patient with cryptogenic liver cirrhosis who underwent liver transplantation. Ten month later the patient was retransplantated due to a chronic cholestasis of unknown origin. HCV-RNA was detected in the native liver and in the first liver graft. Sequencing and phylogenetic analysis discarded crosscontamination and demonstrated that the HCV infecting both livers was the same (genotype 1a). This report proves that OCI is able to infect the liver graft, persisting 10 months and probably inducing a liver failure that required retransplantation. Further studies should be done to investigate the role of OCI in the setting of liver transplantation. Also Roque-Cuellar et al. [24] reported the spread of OCI among seronegative heterosexual partners of patients with chronic HCV infection as they detected OCI (HCV-RNA positive in PBMC) in 13% of the partners. In another study, the intrafamilial transmission of OCI was studied in 102 family members of 50 patients with OCI and in 118 family members of 59 patients with chronic hepatitis C by testing for anti-HCV and serum HCV-RNA [40]. The prevalence of anti-HCV among family members of patients with OCI was 9.8% (10/102) and 3.4% (4/118) among family members of patients with chronic hepatitis C. In addition, there were 14 family members (seven household contacts of OCI cases and the other seven of chronic hepatitis C patients) who presented abnormal values of liver enzymes without serological markers of HBV or HCV infection. Only two of them (both were relatives of two different OCI patients) agreed to be studied for OCI and HCV-RNA was detected in their liver biopsy. These results suggest that OCI may be transmissible among heterosexual partners and family members. Another study [41] reported the response of OCI to antiviral therapy. Ten patients with OCI (all cases with HCV genotype 1) and who presented HCV-RNA in PBMC (persistently detectable), abnormal ALT values and chronic hepatitis in the liver biopsy were treated for 6 months with Pegylated-Interferon plus Ribavirin and were followed for another 6 months. At the end of the follow-up, six (60%) patients remained with normal ALT values and HCV-RNA in PBMC was undetectable in seven patients. The results of this study suggest that the response pattern to antiviral therapy in OCI is similar to that of chronic hepatitis C, indicating that the virus is replicating in OCI and intact viral particles are present in these patients. Also the persistence of OCI over time was investigated in 37 patients followed during 4.6 years [42]. Serum HCV-RNA after ultracentrifugation was found (with fluctuations) over the whole follow-up period. So, OCI may persist and replicate for a long period of time. 4.3. Discordant studies on seronegative OCI Three studies failed to detect seronegative OCI. In the first one, Halfon et al. [34] included 22 patients with cryptogenic chronic hepatitis and 23 patients with systemic connective tissue disease. None of these patients tested HCV-RNA positive in PBMC and the authors questioned the existence of OCI. However, this publication has several limitations. (i) Determination of anti-HCVcore or detection of HCV-RNA in serum after ultracentrifugation or in liver biopsy
was not performed and so, an important percentage of patients who in fact may have an OCI could be missed [5]. (ii) No description on preservation of PBMC was given and this may be critical since deterioration of cells would lead to false negative results. (iii) There is not information of the purity and integrity of the RNA extracted from cells and these data are essential when results are critically dependent on sampling procedures, sample properties, template quality, etc. [43]. Pisaturo et al. [35] tested for HCV-RNA in PBMC of 60 HIVinfected patients with no positive results and authors ruled out the existence of OCI. The same group studied the presence of OCI in 28 patients with oncohematological disorders under immunosuppressive therapy (by testing HCV-RNA in PBMC) with negative results [36]. However, in both studies the existence of OCI cannot be excluded since detection of anti-HCVcore, or viral RNA in serum after ultracentrifugation or in liver biopsies was not performed. 5. Detection Of OCI: HCV-RNA fragments or complete virions? It has been hypothesized that OCI is the result of detecting fragments of HCV-RNA and does not reflect the presence of HCV virions [44]. The findings of the studies on OCI indicate that this is not the case for several reasons: 1. HCV-RNA strand of negative polarity has been detected in PBMC and liver of patients with OCI indicating an active viral replication, which could not be possible if these patients present only HCV-RNA fragments [45]. 2. HCV particles isolated from patients with OCI and from patients with chronic HCV infection have similar buoyant densities [2]. 3. The behaviour of OCI under antiviral therapy is identical to that of patients with chronic hepatitis C [41]. 4. Reinfection of a liver graft by occult HCV and its persistence for 10 months has been described [18]. 5. The persistence of OCI during a prolonged period (years) has been reported [42]. 6. In HuH7 cells transfected with HCV subgenomic replicon, the HCV-RNA half-life is around 17 h when replication is suppressed. As HCV-RNA has been detected for months or years in PBMC and liver, the HCV-RNA detected in OCI cannot be fragments [46,47]. So, it should be concluded that detection of HCV-RNA in liver and in PBMC of patients with OCI reflects the presence of complete and active viral genomes and not residual HCV-RNA fragments. 6. Conclusions Occult HCV infection is found worldwide and all HCV genotypes studied up to now are involved in this infection. At least, 7% of cryptogenic liver cirrhosis may be caused by OCI and it seems that it could be responsible of hepatocellular carcinoma development. OCI may worse the histological liver damage in patients with chronic hepatitis B and the natural history of patients with chronic renal diseases. OCI is also detected in patients with lymphoproliferative disorders but its role in the evolution of these diseases must be studied In addition, OCI infection has been described in HIV-infected patients (inducing a more aggressive liver lesion), in injecting drug-users and even in healthy populations and there are also several data indicating that OCI may be infectious. Also, as OCI seems to affect many different clinical settings, it would be advisable to study its prevalence and significance in other populations (blood donors, healthy people, health-care workers, pregnant women, etc.).
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Funding The writing of this manuscript has been supported in part by the Fundación Investigaciones Biomédicas, Madrid, Spain.
Competing interests None declared.
Ethical approval Not applicable.
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