Introduction
It is believed that 185 million people worldwide have antibodies to the hepatitis C virus (HCV)¹. After initial infection 20% to 40% of patients spontaneously clear the virus. Many of these patients subsequently come to medical attention when they are found to have antibodies to hepatitis C virus but are negative for HCV RNA. It is likely that these patients do not require further follow-up, provided they have no evidence of chronic liver disease. Given the numbers involved, 37 – 74 million people world-wide, the management of these patients has significant economic and social implications. The main question is whether virological relapse occurs in these patients, and if so how frequently? We decided to look at this question using data collected from the Irish anti-D cohort². This cohort of patients has been carefully followed up since discovery – including those patients who were RNA negative at diagnosis. This cohort is unusual for hepatitis C patients in that few have other risk factors for liver disease and the chances of re-infection from injecting drug use or blood transfusion are very low. Thus, this cohort is an excellent group in which to study re-activation rate of hepatitis C virus following spontaneous clearance.

Methods
Information was obtained from a national HCV database which was set up in 2004. For the purpose of this database, hepatitis C infection is defined as the detection of hepatitis C specific antibodies or RNA. It records data on an ongoing basis from the hospital medical records, in seven specialist hepatology units nationally, of patients infected with HCV through blood or blood products, including anti-D immunoglobulin³. The database also includes patients who have died. The background to the anti-D cohort is that in February 1994, batches of anti-D immunoglobulin used in Ireland during 1977-1979 to prevent Rh iso-immunization were found to be contaminated with hepatitis C virus (HCV) from a single infected donor. Contaminated batches from a second HCV infected donor were used during 1991-1994. In March 1994, a national screening programme was initiated for all women who had received anti-D immunoglobulin between 1970 and 1994. A total of 64,907 patients were screened by 1998⁴. It is estimated that 863 patients had evidence of viral transmission in the first outbreak (1977-9), and 120 in the second outbreak (1991-4). Patients were infected with HCV genotype 1b in the first and genotype 3a in the second outbreak. The median ages at infection for each outbreak were 28 and 30 years, respectively. Most patients had their first HCV antibody test in 1994-1995 and initial RNA tests were subsequently carried out on the majority of these patients between 1994 and 1998.

Patients were offered the option to have their data included in the national HCV database. To date, the participation rates are 682/863 (79%) and 73/120 (61%) from the first and second anti-D cohorts, respectively. Data were collected from the date of diagnosis up to 31^st Dec 2013. The results of serial HCV RNA tests were analysed. We specifically looked at patients who were HCV RNA negative on diagnosis and investigated whether there were any subsequent positive HCV RNA results.

Results
In the database 347/755 patients tested positive for HCV antibodies, but negative for HCV RNA when first tested, without prior antiviral therapy. Ninety three percent (321/347) of these patients had subsequent HCV RNA tests performed. The number of HCV RNA tests per patient ranging from 2 to 31 with a mean of 10.2 per patient and a median of 10. For 98% of these patients, subsequent RNA tests were performed at least one year after the first negative test and 85% had one or more RNA tests at least five years after the first negative test. The interval from the first negative test to the latest RNA test ranged from 0-19 years with a mean of 12 years and a median of 13 years.

A total of 9 patients, whose first HCV RNA test was negative, had at least one subsequent positive HCV RNA test. Details of these patients are summarised in table 1. All of the discrepant results occurred in the years 1995-98 and most likely represent methodological issues with early generation hepatitis C assays. Seven patients were from outbreak 1 (1977-9) and two from outbreak 2 (1991-4). Six patients with initial negative HCV RNA tests had one subsequent positive RNA result in the years 1996-8. All had repeatedly negative RNA tests subsequently, without antiviral therapy, and therefore the single positive test results were most likely false positives. Two patients whose first RNA test was negative, had multiple subsequent positive RNA results, thus their first tests were likely to have been false negatives. One patient whose first RNA test was negative in 1996 had 18 subsequent positive RNA results between 1996 and 2013, with two intervening negative RNA results, in 2005 and 2011. This finding probably reflects a low level persisting viraemia. None of the patients with discrepant results had known risk factors for re-infection.

Discussion
In this study, we demonstrate that spontaneous viral clearance is equivalent to cure of the virus in the Irish anti-D cohort. These patients were infected by the administration of blood products (anti-D immunoglobulin), and have a very low prevalence of high-risk behaviour (0.8% of the anti-D infected patients in the national hepatitis C database had a history of current or former injecting drug use (personal communication, Niamh Murphy, HPSC), February 2014). Thus they have a very low risk of re-infection from other sources and are a good group in which to study durability of viral clearance. We identified a number of patients with discrepant RNA results and have explained why we interpreted these as false positive or false negative results. These results occurred in the early days of HCV PCR viral testing 1995-98 and probably reflect the limitations of the PCR assays at the time. One patient with fluctuating negative and positive RNA results probably had a low level persisting viraemia. This study supports the concept that infection with the hepatitis C virus can be resolved permanently. This is in contrast to hepatitis B virus clearance, where there is always the risk of re-activation if the patient becomes immune-suppressed⁵.

These results are important as concern has been expressed that hepatitis C virus may persist in patients who appear to have cleared the virus or that significant liver damage may occur in patients who are HCV antibody positive but RNA negative. Hoare et al reported abnormal liver biopsies with inflammation and fibrosis in antibody positive, RNA negative patients⁶. They postulated that HCV may persist in the liver, causing progressive disease, despite serological clearance. Carreno et al and Dries et al described finding HCV RNA in the liver tissue of patients who were HCV antibody positive but persistently HCV RNA negative by PCR in serum^7,8. Occult HCV is a term used to describe the presence of HCV RNA in liver cells or peripheral blood mononuclear cells but negative HCV RNA by PCR in serum. It appears to be a relatively common but most likely a benign phenomenon, although the natural history is not completely elucidated⁹. The finding of progressive liver disease in HCV RNA negative patients by Hoare et al remains controversial and awaits replication¹⁰. A number of careful studies have detected minute quantities of possibly infectious HCV RNA in patients following SVR^11,12. It is possible that some patients may intermittently express HCV RNA for up to 8 years after SVR but not thereafter¹². One school of thought suggests that the reappearance of minute quantities of HCV RNA may induce specific HCV T-Cell responses and thus help to maintain immunity¹². However it is important to note that other investigators have not been able to demonstrate residual HCV RNA in patients with presumed viral clearance¹³. The reasons for these discrepant results are not clear¹⁴. Despite theoretical concerns, in the national hepatitis C database there is very little evidence of liver disease in the patients infected through anti-D who spontaneously cleared the virus. Thirty-five years after infection, none had developed HCC, one patient had developed cirrhosis and one patient had died from liver-related causes. Of the patients who remained chronically infected, 19% had developed cirrhosis, two percent had developed HCC and 6% died from liver-related causes¹⁵.

Our study supports the concept that patients who have spontaneously cleared hepatitis C virus and are HCV RNA negative have viral cure. One limitation of our findings is that in each of the two transmission events described here, the patients were infected by a unique HCV virus and not a range of different viruses. In spite of methodological issues with early assays it appears that two sequential negative results for HCV RNA are sufficient to confirm spontaneous viral clearance. We suggest that if patients have two negative HCV RNA tests 6 months apart, and no evidence of chronic liver disease, they may be discharged from specialist care with no requirement for further follow-up. This would result in significant cost savings for the health service and almost certainly have psychological benefits for patients.

Acknowledgements and Disclosures
We wish to thank the women who are participants in the national hepatitis C database project, the staff of the participating hepatology units, and the national hepatitis C database steering committee; Ajay Oza, Health Protection Surveillance Centre; Dr Louise Pomeroy, Irish Blood Transfusion Service.

Conflicts of interest: Prof McCormick has been an investigator in antiviral hepatitis C virus drug trials sponsored by Abbvie Ltd and has participated in Advisory Boards organised by Janssen, MSD, Abbvie and BMS. There are no conflicts of interest related to this study.

Correspondence:
Dr Lelia Thornton, Specialist in Public Health Medicine, HSE - Health Protection Surveillance Centre, 25-27 Middle Gardiner Street, Dublin 1, D01 A4A3, Ireland
Email: [email protected]
Phone: 35318765300
Mobile phone: 353866042155

References

1. Hanafiah KM, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: new estimates of age-specific antibody to HCV seroprevalence. Hepatology. 2013; 57: 1333-42.
2. Kenny-Walsh E, for the Irish Hepatology Research Group. Clinical outcomes after hepatitis C infection from contaminated anti-D immune globulin. Irish Hepatology Research Group. N. Engl J Med. 1999; 340: 1228-33.
3. Health Protection Surveillance Centre. National Hepatitis C Database. Baseline Report 2007. www.hcvdatabase.ie; 2007.
4. Lawlor E, Power J,Garson JA, Yap PL, Davisdon F, Columb G, Smith D, Pomeroy L, O’Riordan J, Simmonds P, Tedder RS. Transmission rates of hepatitis C virus by different batches of a contaminated anti-D immunoglobulin preparation. Vox Sanguinis. 1999; 76: 138-43.
5. Markovic S, Drozina G, Vovk M, Fidler-Jenko M. Reactivation of hepatitis B but not hepatitis C in patients with malignant lymphoma and immunosuppressive therapy. A prospective study in 305 patients. Hepatogastroenterology. 1999; 46: 2925-30.
6. Hoare M, Gelson WTH, Rushbrook SM, Curran MD, Woodall T, Coleman N, Davies SE, Alexander GJ. Histological changes in HCV anti-positive, HCV RNA-negative subjects suggest persistent virus infection. Hepatology. 2008; 48: 1737-45.
7. Carreno V, Pardo M, Lopez-Alcorocho JM, Rodrigues-Inigo E, Bartolome J, Castillo I. Detection of hepatitis C virus (HCV) RNA in the liver of healthy anti-HCV antibody-positive, serum HCV-RNA negative patients with normal alanine aminotransferase levels. J Infect Dis. 2006; 194: 53-60.
8. Dries V, von Both I, Muller M, Gerken G, Schirmacher P, Odenthal M, Berten Schlager R, Drebber U, Meyer zum Buschenfeld KH, Dienes HP. Detection of hepatitis Cvirus in paraffin-embedded liver biopsies of patients negative for viral RNA in serum. Hepatology. 1999; 29: 223-9.
9. De Marco L, Manzini P, Trevisan M, Gillio-Tos A, Danielle F, Balloco C, Pizzi A, De Filippo E, D’antico S, Violante B, Valfre A, Curti F, Merietti F, Rishiardi L. Prevalence and follow up of occult HCV infection in an Italian population free of clinically detectable infectious liver disease. PLoS One. 2012 1371; 7(8): 22.
10. Wiesner RH. Is there disease progression in patients who are hepatitis C virus antibody-positive and hepatitis C virus RNA-seronegative? Hepatology. 2008; 48: 1734-6.
11. 11. MacPartland SA, Pham TNQ, Guy CS, Michalak TI. Hepatitis C persisting after clinically apparent sustained virological response to antiviral theraphy retains infectivity in vitro. Hepatology. 2009; 49: 1431-41.
12. Veerapu NS, Raghuraman S, Liang TJ, Heller T, Rehermann B. Sporadic reappearance of munite amounts of hepatitis C virus RNA after successful therapy stimulates cellular immune responses. Gastroenterology. 2011; 140: 676-85.
13. Fujiwara K, Alter HJ. Investigation of residual hepatitis C virus in presumed recovered subjects. Hepatology. 2013; 57: 483-91.
14. Seeff LB. Sustained virologic response: is this equivalent to cure of chronic hepatitis C? Hepatology. 2013; 57: 438-40.
15. Health Protection Surveillance Centre. National Hepatitis C Database 2015 Report. www.hcvdatabase.ie; 2015. Dublin: Health Service Executive, Ireland, 2015.

p582