Fibrosis stage was higher at FU in four patients. For two of them, fibrosis stage had increased from F1 to F2 (TE values 9.3 and 7.3 kPa, respectively, at FU); one of them had a detectable virus with highly sensitive PCR methods (OCI). The other two patients had very high TE values (>75 and 42.2 kPa) and both were diagnosed with large HCCs, with extrahepatic spread.
Occult HCV infection
Among the 52 patients with negative HCV-RNA by the routine method, OCI was detected in two patients (Fig. 3). Both were men and had normal laboratory tests (ALT, AST, platelets, INR, albumin, and hemoglobin) at FU, but TE values corresponded to F2. They showed no signs of HCC, although radiological imaging was not performed and they were not under HCC surveillance.
For one of them, both baseline biopsy and TE at FU showed F2 (TE median: 8.3 kPa) 8 years after EOT. This patient had low alcohol consumption, no comorbidity, and was on no medication, whereas data on BMI are missing. A second and third highly sensitive HCV-RNA test were obtained 5 and 9 months after the first test, where the second sample was negative, but the third was again HCV-RNA positive. The genotype was the same before treatment and at FU (genotype 1) and the RNA sequences were identical in both samples at FU.
The other patient had F1 at baseline, whereas the TE value at FU was median 7.3 kPa (F2) 9 years after EOT. He had a BMI of 24, no comorbidity, was receiving no medication and did not consume any alcohol. Second and third highly sensitive HCV-RNA tests were obtained 7 and 11 months after the first test and were both negative. This patient had genotype 2 before treatment but genotype 1 at FU. The RNA sequence was different from the one obtained from the first patient.
In total, three patients (all men) were diagnosed with HCC. One had died before and two were found in relation to the FU visit because of unexpectedly high TE values (probably measuring the elasticity of HCC) and were examined by ultrasound and computed tomography scan, revealing large HCCs (Table 3). The diagnosis of HCC was established at 8.5, 8.5, and 11 years after EOT, respectively, at ages 55–63 years. None of them had cirrhosis before treatment and thus were not under HCC surveillance. The tumors were found at a very late stage, and all three patients died within a year from diagnosis. Two patients had HCV genotype 3a and one patient had genotype 2b before SVR. None of them had diabetes and none were obese. In the deceased patient, a liver biopsy (to follow fibrosis) had been performed 8 years after SVR, showing regression of fibrosis to F1, and this was only 3 years before HCC diagnosis. The two patients who were examined at FU had no biochemical signs of cirrhosis, with normal ALT, platelets, INR, albumin and hemoglobin values, except that one of them had an elevated AST, around four times the upper level of normal, resulting in an AST to platelet index score of 1.96. One patient reported low and the other patient reported moderate alcohol consumption. The patient who died before FU had moderate alcohol consumption according to medical records.
There were three patients with HCC in our cohort, which yields a standardized incidence ratio of 82.0 compared with the 0.037 expected HCCs in an age-adjusted and sex-adjusted general population cohort. The incidence rate of HCC in our cohort was 280 per 100 000 PY (95% confidence interval: 60–830). The annual incidence rate for men between 55 and 59 years in the general population is 10–14 per 100 000.
All 54 patients completed the questionnaire. In total, 12 (22%) patients answered that they were careful with alcohol because of their former HCV infection to avoid further liver damage (Table 4). No or very low consumption was reported by 52% of the patients, and 44% reported low consumption. The difference between the mean TE value for patients with no or very low alcohol consumption (5.1 kPa, SD: 1.9) and patients with low to moderate consumption (5.3 kPa, SD: 1.8) was not statistically significant when excluding patients with HCC (probably erroneous TE values) or HCV reinfection (data not shown).
The long-term FU (median: 10.5 years after EOT) in this study shows that SVR after HCV treatment generally leads to normal liver function tests, undetectable HCV-RNA with standard method tests and regression of fibrosis. We could, however, observe the presence of OCI in two patients: one with detectable HCV-RNA at two time points. This was not associated with serious liver damage or development of HCC during this long-term FU. Whether the OCI was the cause for the remaining F2 liver fibrosis in these patients needs to be further investigated. Importantly, three patients with SVR, all without pretreatment cirrhosis, developed HCC at age 55–63, 8–11 years after EOT, and about 20–35 years after the primary infection.
Among the patients with SVR in this cohort, two (4%) had detectable HCV-RNA in whole blood with a highly sensitive method 8 and 9 years, respectively, after SVR, indicative of OCI. Both patients were negative in a second sample, but one patient had detectable HCV-RNA in a third sample (identical sequence in the first and third samples) 9 months after the first sample was obtained. This is in line with previous studies of OCI where the appearance of RNA sequences has fluctuated over time, often to disappear some years after SVR 24. One of the patients had another HCV genotype at FU than before treatment, which may be explained by coinfection with two genotypes before treatment. We could not analyze pretreatment samples to look for the genotype 1 strain. We presume that OCI has been prevalent in both patients from EOT until FU. There might have been more patients with OCI at an earlier time point in this cohort.
Previous studies of OCI, with median FU time from 1 to 10 years after SVR, have found detection rates of HCV-RNA in PBMCs that vary from 0 to 50% and detection rates in liver biopsy specimens from 0 to 83% 4,5. The large variations are partly because of the lack of a standardized universal sensitive method for detecting HCV-RNA 5. Residual HCV-RNA seems to become negative if several consecutive tests are taken, indicating that HCV eventually is cleared 4,9,25–27. An association between OCI and progression of liver disease has not been proven and there is no reported human case proving transmission of HCV from a patient with OCI 25,28,29. It is speculated that the prevalence of OCI could increase in the future as a result of the shorter duration of therapy with DAAs and in the absence of IFN 5.
In the current study, we found no conclusive evidence of the progress of the liver disease in patients with OCI. However, and unlike the majority of patients in this study, both patients had fibrosis stage two at FU and no signs of regression of fibrosis. As the changes were very small after a long FU time, the clinical importance of these observations is probably low or at least unclear. None of the patients with HCC had OCI at FU.
A major observation in this study is that HCC could be diagnosed many years after SVR. The patients who developed HCC were diagnosed at age 55 years or older. Surprisingly, none of these patients had cirrhosis before treatment and no indication of cirrhosis at FU, except an elevated AST in one patient, which may have been associated with the large tumor. This patient had fibrosis stage 1 in pretreatment biopsy. The tumors were detected at a very late stage, without curative treatment options. It was not possible to calculate the exact incidence of HCC in this cohort as the study was not designed to look for HCC, and ultrasound of the liver was only performed in the patients with unexpectedly high TE values at FU. However, at least three of 97 patients with SVR developed HCC, representing an 82 times higher incidence than expected in a similar cohort in the general population. This indicates that noncirrhotic patients also have an increased risk of HCC a long time after cure from chronic HCV infection. One of the largest studies of the persistent risk of HCC long term after SVR was carried out in the USA using data from the Veterans Affairs HCV Clinical Case Registry. Among 10 800 patients with SVR and a mean FU time of 2.8 years, the overall annual HCC risk was 0.33%. Patients with cirrhosis before treatment had an annual risk of 1.39%, and for noncirrhotic patients, the risk was 0.16%. The annual risk for patients treated after the age of 65 was 0.95%, irrespective of cirrhosis 15.
Previously shown risk factors for HCC in HCV-infected individuals are liver cirrhosis, male sex, heavy alcohol consumption, diabetes mellitus and older age at the time of SVR 16,18–20,30. In the HCC patients in this study, the possible common risk factors were alcohol consumption (low or moderate consumption were reported), male sex and age older than 55 years; however, these patients achieved SVR at age 45–53 years. None of them were obese or had diabetes. The association between obesity and HCC is not clear; diverse results have been found in previous reports 31,32.
Our findings raise the question of whether HCC surveillance should also include patients without cirrhosis, especially those with risk factors or after a certain age. In patients with chronic hepatitis B, HCC surveillance is recommended when the incidence is higher than 0.2 per 100 PY 33,34. More studies are needed to investigate the long-term risk of HCC in both noncirrhotic and cirrhotic patients with SVR.
A history of alcohol abuse is common among patients with HCV infection in Sweden 35. In this study, the reported alcohol consumption after SVR was low in most patients. Many patients reported that they were sober alcoholics at FU and probably had higher alcohol consumption earlier in life. However, patients with heavy alcohol consumption were usually not eligible for treatment with IFN in 1990–2005. This, together with the self-reported alcohol consumption, indicates that a heavy drinking problem during treatment and after SVR was rare among these patients. Only one patient reported high alcohol consumption at FU. There was no significant difference in liver stiffness between those with no/very low consumption and those with low/moderate consumption.
In this study, the rate of reinfection was low (6%) after a long FU time, despite the fact that the majority of patients had contracted HCV through IDU. In a Norwegian study, 94 individuals with a history of IDU before treatment were followed for a median of seven years after SVR to assess HCV reinfection. Thirty-nine percent had relapsed into IDU, and 27% of these patients were reinfected with HCV 36. In our study, 5.6% of 54 patients had relapsed into IDU, all of them reinfected. It is possible that relapse into IDU was more common in the group that did not attend the FU study. For the 38 patients not available for FU visit, register data showed a diagnosis related to illicit drugs in five (13%) patients and an HCV diagnosis in one patient after 2007.
The advantages of this study are the long FU time of patients with SVR, and that all patients who had achieved SVR in the predetermined time period were invited to participate, without exclusions. A majority of these patients had FU visits and the remaining patients were followed through national registers, resulting in no persons lost to FU after a median of 10 years. The FU visits enabled blood analyses for OCI in patients with long FU duration, up to 21.5 years after EOT, and in a rather large number of patients, compared with previous studies of OCI.
In contrast with the rather large study of OCI, a potential limitation of the study of liver disease before and after SVR is the somewhat small size of the study cohort. However, the patients were thoroughly examined at FU visits and the FU time is longer than that in most studies. The surprisingly high number of HCC diagnoses would probably not have been detected with a shorter FU. The primary aim of the study was not to assess the risk for HCC and we did not have any control group for comparison. However, the finding of three HCCs in this small group of noncirrhotic patients in the long term after SVR was higher than expected, and a surprise, which raises questions about the long-term risk of HCC in an aging patient group long time after SVR. The patients in the study are those who were treated first of all in the 1990s and there are limited data on the long-term outcome after SVR in this population. We did not include patients treated after 2005 to solely include those with long-term FU.
Another limitation is that the fibrosis stage was estimated with TE at FU and compared with liver biopsy results at baseline. It has been suggested that TE overestimates the regression of fibrosis after SVR 37. However, TE is a noninvasive, rapid method without risk for the patient, and has become the preferred method for fibrosis staging in HCV-infected patients, and therefore the most suitable method for this study.
In this study of patients with SVR, genotypes 2 and 3 dominated and the patients were mostly noncirrhotic before treatment. This indicates that the IFN-based therapy almost only cured those with mild disease and genotypes 2 or 3 and that patients with genotype 1 (the most common in Sweden) and cirrhotic patients were more likely to fail treatment 38. A long-term FU after DAA therapy may show different results, both for the risk of HCC and for the occurrence of OCI. It is not yet known how effective DAAs are at clearing OCI.
SVR is associated with clinical cure and regression of liver fibrosis in most patients after 10 years of FU. However, occult HCV infection can be detected in some patients many years after achievement of SVR; this may have a negative effect on the regression of liver fibrosis, but more studies are needed. Future studies are planned to determine whether the viable virus can be recovered from PBMC. If so, this raises the question of whether those repetitively positive for HCV in PBMC should be retreated. Further, the risk of HCC is not eliminated in all noncirrhotic patients with SVR. Studies of the long-term outcome more than 10 years after IFN therapy and eventually after DAA therapy are needed.
This study was supported by the Research Committee of Region Örebro County Council (Grant numbers: OLL-522501 and OLL-590001).
M.S. received research support from the Stockholm County Council, The Swedish Science Council and The Swedish Cancer Foundation.
Conflicts of interest
A.-S. D. has served as a speaker and/or consultant for AbbVie, BMS, Gilead, Janssen, Medivir, MSD and Roche. S.A. has served as a speaker and/or consultant for AbbVie, BMS, Gilead, Janssen, MSD/Merck, Medivir, Roche, and Tillotts Pharma, and has received research grants from AbbVie and Gilead. E.D.B. is currently an employee of Medivir AB (Stockholm, Sweden), although not at the time when the study was conducted. For the remaining authors, there are no conflicts of interest.
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