Conceived and designed the experiments: LDM AGT LR. Performed the experiments: LDM VF. Analyzed the data: GR FM LR CS. Contributed reagents/materials/analysis tools: GR VK DP SP RT PV. Wrote the paper: LDM AGT.
The authors have declared that no competing interests exist.
Occult Hepatitis C virus (HCV) infection is a new pathological entity characterized by presence of liver disease and absence or very low levels of detectable HCV-RNA in serum. Abnormal values of liver enzymes and presence of replicative HCV-RNA in peripheral blood mononuclear cells are also observed. Aim of the study was to evaluate occult HCV occurrence in a population unselected for hepatic disease.
We chose from previous epidemiological studies three series of subjects (n = 276, age range 40–65 years) unselected for hepatic disease. These subjects were tested for the presence of HCV antibodies and HCV-RNA in plasma and in the peripheral blood mononuclear cells (PBMCs) by using commercial systems. All subjects tested negative for HCV antibodies and plasma HCV-RNA and showed normal levels of liver enzymes; 9/276 patients (3.3%) were positive for HCV-RNA in PBMCs, identifying a subset of subjects with potential occult HCV infection. We could determine the HCV type for 8 of the 9 patients finding type 1a (3 patients), type 1b (2 patients), and type 2a (3 patients).
The results of this study show evidence that occult HCV infection may occur in a population unselected for hepatic disease. A potential risk of HCV infection spread by subjects harbouring occult HCV infection should be considered. Design of prospective studies focusing on the frequency of infection in the general population and on the clinical evolution of occult HCV infection will be needed to verify this unexpected finding.
Occult Hepatitis C virus (HCV) infection has been defined
In the frame of a case-control study nested in the EPIC (European Prospective Investigation into Cancer and Nutrition) Italy cohort
To support the results obtained in control samples from the EPIC Italy cohort study (Richiardi et al. submitted), we analysed two additional independent series for detection of HCV antibodies and HCV-RNA both in plasma and in PBMCs.
All subjects in the study had normal levels of ALT (range from 5 to 17 IU/L) and AST (range from 5 to 25 IU/L) and resulted negative for HCV antibodies and viremia. All RNA extracted samples resulted positive at the b-actin amplification, confirming adequacy for the HCV PCR analyses. Considering all study series (“EPIC Italy”, “Turin Case-Control Bladder Cancer”, “Cervical Cancer Screening”), we found that 9 out of the 276 (3.3%; 95% CI: 1.5–6%) subjects testing negative for HCV antibodies and viremia were positive for HCV-RNA in their PBMCs. HCV-RNA positivity was confirmed by direct sequencing matching the 5′-UTR region of HCV genome according to the Blast sequence. As summarized in
Subjects | Number of subjects | HCV antibodies (%) | HCV –RNA plasma (%) | HCV –RNA PBMCs (%) |
All subjects | 276 | 0 (0) | 0 (0) | 9 (3.3%) |
Men (age range: 36–65 years) | 111 | 0 (0) | 0 (0) | 3 (2.7%) |
Women (age range: 39–76 years) | 165 | 0 (0) | 0 (0) | 6 (3.6%) |
All subjects | 176 | 0 (0) | 0 (0) | 7 (4.0%) |
Men (age range: 36–65 years) | 71 | 0 (0) | 0 (0) | 3 (4.2%) |
Women (age range: 39–76 years) | 105 | 0 (0) | 0 (0) | 4 (3.8%) |
Men (age range: 42–66 years) | 40 | 0 (0) | 0 (0) | 0 (0%) |
Women (age range: 46–61 years) | 60 | 0 (0) | 0 (0) | 2 (3.3%) |
PBMCs, peripheral blood mononuclear cells.
Patient | Sex | Age | ALT/AST |
HCV antibody | HCV-RNA plasma | HCV-RNA PBMCs | HCV Genotyping |
1 | M | 51 | −/− | − | − | + | 1a |
2 | M | 60 | −/− | − | − | + | n.d. |
3 | F | 46 | −/− | − | − | + | 1b |
4 | F | 52 | −/− | − | − | + | 1b |
5 | F | 56 | −/− | − | − | + | 2a |
6 | M | 41 | −/− | − | − | + | 2a |
7 | F | 54 | −/− | − | − | + | 2a |
8 | F | 55 | −/− | − | − | + | 1a |
9 | F | 47 | −/− | − | − | + | 1a |
: −/− indicates normal values below 50 IU/L and 35 IU/L for men and women respectively for ALT, and below 40 IU/L and 32 IU/L for men and women respectively for AST determination.
n.d.: the amplification resulting in a very weak fragment and sequence analysis did not succeed.
At present, occult HCV infection has been described in relation to hepatic diseases, and data on the frequency of HCV-RNA in the PBMCs of the general population are unknown.
We identified subjects apparently free of liver disease, since testing normally for liver enzymes and negative for serological HCV markers, which unexpectedly showed potential occult HCV infection features.
Negative serological results were unlikely due to low sensitivity, as the tests employed to detect HCV antibodies and circulating virus have been extensively reported to have high reliability, with a 98.8% sensitivity and a 100% specificity
Despite the most prevalent HCV genotype in Italy
The unexpected result of this study raises the issue of occult HCV infection frequency in the general population and its potential implications on the safety of medical procedures and the onset of liver disease. Subjects with occult HCV infection may not harbor detectable circulating virions, but they could be still potentially infectious, as PBMCs have been demonstrated to be permissive for viral replication
The potential risk for HCV spread from occult HCV subjects through blood transfusions and hemodialysis units should be considered
Moreover, since it has been observed that very low-level of HCV-RNA may persist in the PBMCs for many years representing a potential risk for the recurrence of infection, the clinical potential in hepatocarcinogenesis should be evaluated
The results of this study show evidence that occult HCV infection may occur in a population unselected for hepatic disease. A potential risk of HCV infection spread by subjects who unknowingly may carry occult HCV infection and of liver disease onset should be considered.
Design of prospective studies focusing on the prevalence and on the clinical evolution of occult HCV infection in the general population will be needed to verify this unexpected finding, as well as the analysis of HCV-RNA replication potential, the identification of HCV genotypes involved in occult HCV infection, and the evolution of liver disease.
Ethic statement was not required for this specific study. All participants recruited, controls included, donated blood samples in previous epidemiological studies, following written informed consent given to principal investigators. These blood samples were re-used, since previous ethical approval included enabling other non-specified tests to be carried out on the biological samples supplied.
The present study was performed including a total of 276 subjects (111 men and 165 women) as sample of the general population. All subjects aged between 40 and 65, were apparently liver disease free at recruitment, and were enrolled in the frame of three different epidemiologic studies previously approved by local ethic committees: EPIC Italy cohort (protocol number: 96/01)
All participants in the studies, following written informed consent given to principal investigators, donated a blood sample, which was collected and fractioned (red blood cells, buffy-coat, serum and plasma) on the day of enrolment and snap frozen in liquid nitrogen or at −80°C.
EPIC Italy cohort series: The European Prospective Investigation into Cancer and Nutrition (EPIC) included healthy subjects enrolled in 10 European countries. The EPIC Italy cohort involved 5 centres (Turin, Varese, Florence, Naples, and Ragusa) with a total of 47,000 subjects enrolled between 1992 and 1998. In the present study, we included 176 subjects randomly selected as controls for the Non-Hodgkin's Lymphoma study (Richiardi et al. submitted) from the eligible members of the EPIC Italy cohort: 39 from Turin, 74 from Varese, 42 from Florence, 11 from Naples, and 10 from Ragusa. These controls were matched to the corresponding lymphoma case on sex, centre, age (within 5 years), and period of collection of the blood sample (within 90 days). They were a sample of the general population, although blood donors and attendants to screening programs were over-sampled. Detailed information for each volunteer about diet and life-style habits, anthropometric measurements and residential and occupational history was collected.
Turin Case-Control Bladder Cancer Study series: Hospital-based case-control investigations at two urology departments in S. Giovanni Battista Hospital in Turin, Italy. Subjects with newly diagnosed bladder cancer in the Turin metropolitan area were recruited as cases. Subjects with benign diseases, mainly prostatic hyperplasia and cystitis (all newly diagnosed), and patients treated at the medical and surgical departments for hernias, vasculopathies, diabetes, heart failure, asthma or other benign diseases (none was represented in >10%), were recruited from the medical and surgical departments of the same hospital as controls. Patients with cancer other than bladder, liver or renal disease, and smoking-related conditions were excluded. In the present study were included 40 men (aged between 40 and 65) consecutively recruited as controls between 2001 and 2003.
Cervical Cancer Screening series: Italian project designed in the frame of Cervical Cancer Screening Programmes to study papillomavirus infection, frequency, prevalence, and implication in cervical lesions progression. In the present study, we included 60 women (aged between 40 and 65) consecutively recruited in Turin between 2002 and 2004 which gave consent to donate a blood sample.
Quantitative levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma were evaluated by using an UV test on Roche automated clinical chemistry analyzer (Cobas, Roche/Hitachi Modular analyzers, North America, USA). This method has been standardized according to the original International Federation of Clinical Chemistry (IFCC) formulation. The expected normal values according to IFCC were up to 50 IU/L and up to 35 IU/L for men and women respectively for ALT, and up to 40 IU/L and up to 32 IU/L for men and women respectively for AST determination. The analytical sensitivity is 4 IU/L for both the determinations, and the measuring range is 4–600 IU/L and 4–800 IU/L for ALT and AST respectively.
Detection of HCV antibodies was performed by a commercial enzyme immunoassay test of third-generation (ORTHO HCV 3.0 – commercialized by Dia Sorin, Saluggia, Italy) following the manufacturer instructions. Samples showing optical densities above the cut off value were considered positive. The sensitivity and specificity of the test were estimated of 98.8% and 100% respectively
Total RNA was isolated from 150 µL of buffy-coat and 150 µL plasma using SV total RNA Isolation system (Promega, Madison, Wi, USA) and EXTRAgene (Amplimedical, Turin, Italy) respectively, following the manufacturer instructions. In 150 µL of buffy-coat there was a range between 106 and 107 cells: they were washed three times by using SV RNA Red Blood Cell Lysis Solution (Promega, Madison, Wi, USA) in order to avoid contaminants carry over. Adequacy of RNA extraction was evaluated by retro-transcription of 5 µL by the Reverse Transcription System (Promega) to cDNA and amplification of a 171 bp fragment of the b-actin gene (primer forward
We used plasma rather than serum because prior RT-PCR studies showed a higher sensitivity when the analyses were performed with plasma
The positive PCR products of the 5′UTR region were excised from the gel and purified with the “PCR Clean-up Gel Extraction Kit” (Macherey-Nagel, Dueren, Germany). Direct sequencing was carried out using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applera, Monza, Italy), and sequence analysis was performed with the ABI 310 automated capillary system (Applera, Monza, Italy), following the manufacturer instructions. Sequences were analyzed with the BLAST Software (
We thank all the volunteers who generously enrolled in our studies and the dedicated clinical research staff and nurses who collaborated in recruitment. We thank Alfa Wasserman company that kindly provided the primer for sequencing assay. We thank dr. Fabrizio Tondat of the Molecular Oncology Laboratory, C.E.R.M.S., Turin, who kindly performed the sequencing analyses.